Sample records for acid lipase activity

  1. Acid Lipase Disease

    MedlinePlus

    ... of Neurological Disorders and Stroke conducts and supports research to understand lipid storage diseases such as acid lipase deficiency and ... of Neurological Disorders and Stroke conducts and supports research to understand lipid storage diseases such as acid lipase deficiency and ...

  2. Identification and characterization of a triacylglycerol lipase in Arabidopsis homologous to mammalian acid lipases.

    PubMed

    El-Kouhen, Karim; Blangy, Stéphanie; Ortiz, Emilia; Gardies, Anne-Marie; Ferté, Natalie; Arondel, Vincent

    2005-11-07

    Triacylglycerol (TAG) lipases have been thoroughly characterized in mammals and microorganisms. By contrast, very little is known on plant TAG lipases. An Arabidopsis cDNA called AtLip1 (At2g15230), which exhibits strong homology to lysosomal acid lipase, was found to drive the synthesis of an active TAG lipase when expressed in the baculovirus system. The lipase had a maximal activity at pH 6 and the specific activity was estimated to be about 45 micromol min(-1) mg(-1) protein using triolein as a substrate. Knock-out mutant analysis showed no phenotype during germination indicating that this enzyme is fully dispensable for TAG storage breakdown during germination. Northern blot analyses indicated that the transcript is present in all tissues tested.

  3. Inhibition of Microbial Lipases by Fatty Acids

    PubMed Central

    Smith, J. L.; Alford, John A.

    1966-01-01

    Addition of lard or sodium oleate to the medium used for lipase production by Pseudomonas fragi resulted in a decreased accumulation of lipase in the culture supernatant fluid without affecting cell growth. The production and activity of lipase was inhibited by lard, sodium oleate, and the salts of other unsaturated fatty acids. Some divalent cations, Tweens, lecithin, and bovine serum prevented oleate inhibition, but did not reverse it. Similar inhibitory actions were observed with Geotrichum candidum lipase, but not with a staphylococcal lipase or pancreatic lipase. A protective effect by protein in crude enzyme preparations is indicated. The ability of oleate to lower surface tension does not appear to be related to its ability to inhibit lipase. PMID:5970458

  4. Acid Lipase from Candida viswanathii: Production, Biochemical Properties, and Potential Application

    PubMed Central

    de Almeida, Alex Fernando; Carmona, Eleonora Cano

    2013-01-01

    Influences of environmental variables and emulsifiers on lipase production of a Candida viswanathii strain were investigated. The highest lipase activity (101.1 U) was observed at 210 rpm, pH 6.0, and 27.5°C. Other fermentation parameters analyzed showed considerable rates of biomass yield (Y L/S = 1.381 g/g), lipase yield (Y L/S = 6.892 U/g), and biomass productivity (P X = 0.282 g/h). Addition of soybean lecithin increased lipase production in 1.45-fold, presenting lipase yield (Y L/S) of 10.061 U/g. Crude lipase presented optimal activity at acid pH of 3.5, suggesting a new lipolytic enzyme for this genus and yeast in general. In addition, crude lipase presented high stability in acid conditions and temperature between 40 and 45°C, after 24 h of incubation in these temperatures. Lipase remained active in the presence of organic solvents maintaining above 80% activity in DMSO, methanol, acetonitrile, ethanol, acetone, 1-propanol, isopropanol, and 2-propanol. Effectiveness for the hydrolysis of a wide range of natural triglycerides suggests that this new acid lipase has high potential application in the oleochemical and food industries for hydrolysis and/or modification of triacylglycerols to improve the nutritional properties. PMID:24350270

  5. Enhancement of lipase catalyzed-fatty acid methyl esters production from waste activated bleaching earth by nullification of lipase inhibitors.

    PubMed

    Dwiarti, Lies; Ali, Ehsan; Park, Enoch Y

    2010-01-01

    This study sought to identify inhibitory factors of lipase catalyzed-fatty acid methyl esters (FAME) production from waste activated bleaching earth (wABE). During the vegetable oil refinery process, activated bleaching earth (ABE) is used for removing the impure compounds, but adsorbs vegetable oil up to 35-40% as on a weight basis, and then the wABE is discarded as waste material. The impurities were extracted from the wABE with methanol and evaluated by infra-red (IR) spectroscopy, which revealed that some were chlorophyll-plant pigments. The chlorophylls inhibited the lipase during FAME conversion from wABE. The inhibition by a mixture of chlorophyll a and b was found to be competitive. The inhibition of the enzymatic hydrolysis of waste vegetable oil contained in wABE by chlorophyll a alone was competitive, while the inhibition by chlorophyll b alone was non-competitive. Furthermore, the addition of a small amount of alkali nullified this inhibitory effect and accelerated the FAME production rate. When 0.9% KOH (w/w wABE) was added to the transesterification reaction with only 0.05% lipase (w/w wABE), the maximum FAME production rate improved 120-fold, as compared to that without the addition of KOH. The alkali-combined lipase significantly enhanced the FAME production rate from wABE, in spite of the presence of the plant pigments, and even when a lower amount of lipase was used as a catalyst.

  6. Acid lipase inhibitor in chicken plasma identified as apolipoprotein A-I.

    PubMed

    Fujii, M; Higuchi, T; Mukai, S; Yonekura, M; Yano, T; Kawaguchi, H; Nonaka, K; Fukunaga, T; Sugimoto, Y; Yamada, S

    1996-10-01

    We have reported a inhibitor of acid lipases in liver lysosomes and erythrocytes from chickens [M. Fujii et al., Int. J. Biochem., 22, 895-898 (1990)]. In this paper, the properties of the inhibitor were described in comparison with those of apo A-I of chicken. The purified inhibitor migrated with the same mobility on SDS-PAGE as apo A-I, and had a molecular weight of 27,000. The peptide map from the lipase inhibitor was similar to that of apo A-I. Antibodies to the acid lipase inhibitor also reacted with apo A-I. Apo A-I inhibited the acid lipase activities of liver lysosomes and erythrocytes from chickens as strongly as the lipase inhibitor. The N-terminal amino acid sequence of lipase inhibitor was identical to that of apo A-I as far as residue 20. The amino acid sequence of peptides obtained from the inhibitor by cleavage with CNBr corresponded to internal sequence of apo A-I, and so the CNBr-peptides were derived by cleavage after the methionine residues in apo A-I. The findings showed that the inhibitor of the acid lipases in liver lysosomes and erythrocytes from chickens was identical to apo A-I.

  7. Lingual lipase activity in the orosensory detection of fat by humans

    PubMed Central

    Kulkarni, Bhushan V.

    2014-01-01

    Lingual lipase generates nonesterified fatty acids (NEFA) from dietary fats during oral processing by lipolysis. Lingual lipase in rodents has strong lipolytic activity and plays a critical role in oral detection of fats. The functional activity of lingual lipase during oral processing of high-fat foods in humans remains poorly characterized. Five commonly consumed high-fat foods varying in physical states and fatty acid composition (almond, almond butter, olive oil, walnut, and coconut) were masticated by 15 healthy human subjects at the rate of one chew per second with and without lipase inhibitor orlistat. Salivary NEFA concentrations were measured. To determine the role of lingual lipase in oral fat detection, sensory ratings were obtained from the same 15 human subjects for almond butter with and without orlistat. Lingual lipase was active during oral processing of almond and coconut. No activity of lingual lipase was detected during processing of almond butter. There was only weak evidence lingual lipase is a determinant of oral fat detection. Lingual lipase may only contribute to NEFA generation and oral fat detection of fatty foods that require stronger oral processing effort. PMID:24694384

  8. Lipase activities in castor bean endosperm during germinaion. [Ricinus communis; glyoxysomes

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Muto, S.; Beevers, H.

    1974-01-01

    Two lipases were found in extracts from castor bean (Ricinus communis L.) endosperm. One, with optimal activity at pH 5.0 (acid lipase), was present in dry seeds and displayed high activity during the first 2 days of germination. The second, with an alkaline pH optimum (alkaline lipase), was particularly active during days 3 to 5. When total homogenates of endosperm were fractionated into fat layer, supernatant, and particulate fractions, the acid lipase was recovered in the fat layer, and the alkaline lipase was located primarily in the particulate fraction. Sucrose density gradient centrifugation showed that the alkaline lipase was locatedmore » mainly in glyoxysomes, with some 30 percent of the activity in the endoplasmic reticulum. When glyoxysomes were broken by osmotic shock and exposed to KCl, which solubilizes most of the enzymes, the alkaline lipase remained particulate and was recovered with the glyoxysomal ''ghosts'' at equilibrium density 1.21 g/cm/sup 3/ on the sucrose gradient. Association of the lipase with the glyoxysomal membrane was supported by the responses to detergents and to butanol. The alkaline lipase hydrolyzed only monosubstituted glycerols. The roles of the two lipases in lipid utilization during germination of castor bean are discussed.« less

  9. Radical scavenging activity of lipophilized products from lipase-catalyzed transesterification of triolein with cinnamic and ferulic acids.

    PubMed

    Choo, Wee-Sim; Birch, Edward John

    2009-02-01

    Lipase-catalyzed transesterification of triolein with cinnamic and ferulic acids using an immobilized lipase from Candida antarctica (E.C. 3.1.1.3) was conducted to evaluate the antioxidant activity of the lipophilized products as model systems for enhanced protection of unsaturated oil. The lipophilized products were identified using ESI-MS. Free radical scavenging activity was determined using the DPPH radical method. The polarity of the solvents proved important in determining the radical scavenging activity of the substrates. Ferulic acid showed much higher radical scavenging activity than cinnamic acid, which has limited activity. The esterification of cinnamic acid and ferulic acid with triolein resulted in significant increase and decrease in the radical scavenging activity, respectively. These opposite effects were due to the effect of addition of electron-donating alkyl groups on the predominant mechanism of reaction (hydrogen atom transfer or electron transfer) of a species with DPPH. The effect of esterification of cinnamic acid was confirmed using ethyl cinnamate which greatly enhances the radical scavenging activity. Although, compared to the lipophilized cinnamic acid product, the activity was lower. The radical scavenging activity of the main component isolated from lipophilized cinnamic acid product using solid phase extraction, monocinnamoyl dioleoyl glycerol, was as good as the unseparated mixture of lipophilized product. Based on the ratio of a substrate to DPPH concentration, lipophilized ferulic acid was a much more efficient radical scavenger than lipophilized cinnamic acid.

  10. Synthesis of structured triacylglycerols enriched in n-3 fatty acids by immobilized microbial lipase.

    PubMed

    Araújo, Maria Elisa Melo Branco de; Campos, Paula Renata Bueno; Alberto, Thiago Grando; Contesini, Fabiano Jares; Carvalho, Patrícia de Oliveira

    The search for new biocatalysts has aroused great interest due to the variety of micro-organisms and their role as enzyme producers. Native lipases from Aspergillus niger and Rhizopus javanicus were used to enrich the n-3 long-chain polyunsaturated fatty acids content in the triacylglycerols of soybean oil by acidolysis with free fatty acids from sardine oil in solvent-free media. For the immobilization process, the best lipase/support ratios were 1:3 (w/w) for Aspergillus niger lipase and 1:5 (w/w) for Rhizopus javanicus lipase using Amberlite MB-1. Both lipases maintained constant activity for 6 months at 4°C. Reaction time, sardine-free fatty acids:soybean oil mole ratio and initial water content of the lipase were investigated to determine their effects on n-3 long-chain polyunsaturated fatty acids incorporation into soybean oil. Structured triacylglycerols with 11.7 and 7.2% of eicosapentaenoic acid+docosahexaenoic acid were obtained using Aspergillus niger lipase and Rhizopus javanicus lipase, decreasing the n-6/n-3 fatty acids ratio of soybean oil (11:1 to 3.5:1 and 4.7:1, respectively). The best reaction conditions were: initial water content of lipase of 0.86% (w/w), sardine-free faty acids:soybean oil mole ratio of 3:1 and reaction time of 36h, at 40°C. The significant factors for the acidolysis reaction were the sardine-free fatty acids:soybean oil mole ratio and reaction time. The characterization of structured triacylglycerols was obtained using easy ambient sonic-spray ionization mass spectrometry. The enzymatic reaction led to the formation of many structured triacylglycerols containing eicosapentaenoic acid, docosahexaenoic acid or both polyunsaturated fatty acids. Copyright © 2016 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. All rights reserved.

  11. Nitrogen dioxide induced changes in level of free fatty acids, triglyceride, esterified fatty acid, ganglioside and lipase activity in the guinea pig brain

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Farahani, H.; Hasan, M.

    1992-02-01

    The biochemical response to controlled inhalation of nitrogen dioxide (NO2) was studied in 18 male guinea pigs. Animals were exposed to 2.5, 5.0, and 10 ppm NO2 for 2h daily for 35 consecutive days, and the results compared with six control animals exposed to filtered air for 2h daily for same period. Five biochemical parameters, including triglyceride, free fatty acids, esterified fatty acid, ganglioside and lipase activity were measured immediately after the last day of exposure. At 2.5 ppm NO2 inhalation no significant changes occurred in any region of the central nervous system (CNS). While as the dose concentration wasmore » increased to 5 and 10 ppm nitrogen dioxide, significant dose-related alteration were observed in the levels of triglyceride, free fatty acid, esterified fatty acid, ganglioside and lipase activity in the different regions of the guinea pig CNS.« less

  12. Different Covalent Immobilizations Modulate Lipase Activities of Hypocrea pseudokoningii.

    PubMed

    Pereira, Marita G; Velasco-Lozano, Susana; Moreno-Perez, Sonia; Polizeli, Aline M; Heinen, Paulo R; Facchini, Fernanda D A; Vici, Ana C; Cereia, Mariana; Pessela, Benevides C; Fernandez-Lorente, Gloria; Guisan, Jose M; Jorge, João A; Polizeli, Maria de Lourdes T M

    2017-09-04

    Enzyme immobilization can promote several advantages for their industrial application. In this work, a lipase from Hypocrea pseudokoningii was efficiently linked to four chemical supports: agarose activated with cyanogen bromide (CNBr), glyoxyl-agarose (GX), MANAE-agarose activated with glutaraldehyde (GA) and GA-crosslinked with glutaraldehyde. Results showed a more stable lipase with both the GA-crosslinked and GA derivatives, compared to the control (CNBr), at 50 °C, 60 °C and 70 °C. Moreover, all derivatives were stabilized when incubated with organic solvents at 50%, such as ethanol, methanol, n -propanol and cyclohexane. Furthermore, lipase was highly activated (4-fold) in the presence of cyclohexane. GA-crosslinked and GA derivatives were more stable than the CNBr one in the presence of organic solvents. All derivatives were able to hydrolyze sardine, açaí ( Euterpe oleracea ), cotton seed and grape seed oils. However, during the hydrolysis of sardine oil, GX derivative showed to be 2.3-fold more selectivity (eicosapentaenoic acid (EPA)/docosahexaenoic acid (DHA) ratio) than the control. Additionally, the types of immobilization interfered with the lipase enantiomeric preference. Unlike the control, the other three derivatives preferably hydrolyzed the R -isomer of 2-hydroxy-4-phenylbutanoic acid ethyl ester and the S -isomer of 1-phenylethanol acetate racemic mixtures. On the other hand, GX and CNBr derivatives preferably hydrolyzed the S -isomer of butyryl-2-phenylacetic acid racemic mixture while the GA and GA-crosslink derivatives preferably hydrolyzed the R -isomer. However, all derivatives, including the control, preferably hydrolyzed the methyl mandelate S -isomer. Moreover, the derivatives could be used for eight consecutive cycles retaining more than 50% of their residual activity. This work shows the importance of immobilization as a tool to increase the lipase stability to temperature and organic solvents, thus enabling the possibility of

  13. Effect of prolonged refrigeration on the lipid profile, lipase activity, and oxidative status of human milk.

    PubMed

    Bertino, Enrico; Giribaldi, Marzia; Baro, Cristina; Giancotti, Valeria; Pazzi, Marco; Peila, Chiara; Tonetto, Paola; Arslanoglu, Sertac; Moro, Guido E; Cavallarin, Laura; Gastaldi, Daniela

    2013-04-01

    The study was aimed at evaluating the effect of prolonged refrigeration of fresh human milk (HM) on its fatty acid profile, free fatty acid content, lipase activities, and oxidative status. HM from mothers of preterm newborns was collected, pooled, and placed in the neonatal intensive care unit (NICU) refrigerator. Pooled milk was aliquoted and analyzed within 3 hours of collection, and after 24, 48, 72, and 96 hours of storage. The milk samples were analyzed for pH, total and free fatty acid profile, lipase activity at room temperature and at 4°C, lipase activity at room temperature in presence of sodium cholate (bile salt-dependent lipase), total antioxidant capacity, thiobarbituric acid reactive species, malondialdehyde, and conjugated diene concentration. The experiment was replicated in 3 independent trials. Prolonged refrigeration did not affect the fatty acid composition of breast milk, and preserved both its overall oxidative status and the activity of HM lipolytic enzymes. In particular, bile salt-dependent lipase activity, long-chain polyunsaturated fatty acids, and medium-chain saturated fatty acid concentrations were unaffected for up to 96 hours of refrigerated storage. Prolonged refrigeration of fresh HM for 96 hours maintained its overall lipid composition. The limited lipolysis during storage should be ascribed to the activity of lipoprotein lipase, responsible for the decrease in pH. Our study demonstrates that infants who receive expressed milk stored for up to 96 hours receive essentially the same supply of fatty acids and active lipases as do infants fed directly at the breast.

  14. Active-site titration analysis of surface influence on immobilized Candida antarctica Lipase B activity

    USDA-ARS?s Scientific Manuscript database

    Matrix morphology and surface polarity effects were investigated for Candida antarctica lipase B immobilization. Measurements of the amount of lipase immobilized (bicinchoninic acid method) and the catalyst’s tributyrin hydrolysis activity, coupled with a determination of the lipase’s functional fr...

  15. Collagen-Immobilized Lipases Show Good Activity and Reusability for Butyl Butyrate Synthesis.

    PubMed

    Dewei, Song; Min, Chen; Haiming, Cheng

    2016-11-01

    Candida rugosa lipases were immobilized onto collagen fibers through glutaraldehyde cross-linking method. The immobilization process has been optimized. Under the optimal immobilization conditions, the activity of the collagen-immobilized lipase reached 340 U/g. The activity was recovered of 28.3 % by immobilization. The operational stability of the obtained collagen-immobilized lipase for hydrolysis of olive oil emulsion was determined. The collagen-immobilized lipase showed good tolerance to temperature and pH variations in comparison to free lipase. The collagen-immobilized lipase was also applied as biocatalyst for synthesis of butyl butyrate from butyric acid and 1-butanol in n-hexane. The conversion yield was 94 % at the optimal conditions. Of its initial activity, 64 % was retained after 5 cycles for synthesizing butyl butyrate in n-hexane.

  16. Production of a Novel Cold-Active Lipase from Pichia lynferdii Y-7723

    USDA-ARS?s Scientific Manuscript database

    Lipase (triacylglycerol acylhydrolases, E.C. 3.1.1.3.) is one of the most important enzymes applied to the broad range of industrial application field. Especially, lipases with abnormal functionality such as thermo stability, alkaline, acidic, cold-activity gain special attention because of their a...

  17. Differences in the intramolecular structure of structured oils do not affect pancreatic lipase activity in vitro or the absorption by rats of (n-3) fatty acids.

    PubMed

    Porsgaard, Trine; Xu, Xuebing; Göttsche, Jesper; Mu, Huiling

    2005-07-01

    The fatty acid composition and intramolecular structure of dietary triacylglycerols (TAGs) influence their absorption. We compared the in vitro pancreatic lipase activity and the lymphatic transport in rats of fish oil and 2 enzymatically interesterified oils containing 10:0 and (n-3) PUFAs of marine origin to investigate whether the positional distribution of fatty acids influenced the overall bioavailability of (n-3) PUFAs in the body. The structured oils had the (n-3) PUFA either mainly at the sn-1,3 position (LML, M = medium-chain fatty acid, L = long-chain fatty acid) or mainly at the sn-2 position (MLM). Oils were administered to lymph-cannulated rats and lymph was collected for 24 h. The fatty acid composition as well as the lipid class distribution of lymph samples was determined. In vitro pancreatic lipase activity was greater when fish oil was the substrate than when the structured oils were the substrates (P < 0.001 at 40 min). This was consistent with a greater 8-h recovery of total fatty acids from fish oil compared with the 2 structured oils (P < 0.05). The absorption profiles of MLM and LML in rats and their in vitro rates of lipase activity did not differ. This indicates that the absorption rate is highly influenced by the lipase activity, which in turn is affected by the fatty acid composition and intramolecular structure. The lipid class distribution in lymph collected from the 3 groups of rats did not differ. In conclusion, the intramolecular structure did not affect the overall absorption of (n-3) PUFAs.

  18. Microwave-Assisted Resolution of α-Lipoic Acid Catalyzed by an Ionic Liquid Co-Lyophilized Lipase.

    PubMed

    Liu, Ning; Wang, Lei; Wang, Zhi; Jiang, Liyan; Wu, Zhuofu; Yue, Hong; Xie, Xiaona

    2015-05-29

    The combination of the ionic liquid co-lyophilized lipase and microwave irradiation was used to improve enzyme performance in enantioselective esterification of α-lipoic acid. Effects of various reaction conditions on enzyme activity and enantioselectivity were investigated. Under optimal condition, the highest enantioselectivity (E = 41.2) was observed with a high enzyme activity (178.1 μmol/h/mg) when using the ionic liquid co-lyophilized lipase with microwave assistance. Furthermore, the ionic liquid co-lyophilized lipase exhibited excellent reusability under low power microwave.

  19. Oral lipase activities and fat-taste receptors for fat-taste sensing in chickens.

    PubMed

    Kawabata, Yuko; Kawabata, Fuminori; Nishimura, Shotaro; Tabata, Shoji

    2018-01-01

    It has been reported that a functional fat-taste receptor, GPR120, is present in chicken oral tissues, and that chickens can detect fat taste in a behavioral test. However, although triglycerides need to be digested to free fatty acids to be recognized by fat-taste receptors such as GPR120, it remains unknown whether lipase activities exist in chicken oral tissues. To examine this question, we first cloned another fat-taste receptor candidate gene, CD36, from the chicken palate. Then, using RT-PCR, we determined that GPR120 and CD36 were broadly expressed in chicken oral and gastrointestinal tissues. Also by RT-PCR, we confirmed that several lipase genes were expressed in both oral and gastrointestinal tissues. Finally, we analyzed the lipase activities of oral tissues by using a fluorogenic triglyceride analog as a lipase substrate. We found there are functional lipases in oral tissues as well as in the stomach and pancreas. These results suggested that chickens have a basic fat-taste reception system that incorporates a triglycerides/oral-lipases/free fatty acids/GPR120 axis and CD36 axis. Copyright © 2017 Elsevier Inc. All rights reserved.

  20. Enzymatic Synthesis of Structured Lipids using a Novel Cold-Active Lipase from Pichia lynferdii NRRL Y-7723

    USDA-ARS?s Scientific Manuscript database

    Structured lipids (SL) were synthesized by the acidolysis of borage oil with caprylic acid using lipases. Six commercial lipases from different sources and a novel lipase from Pichia lynferdii NRRL Y-7723 were screened for their acidolysis activities and Lipozyme RM IM and NRRL Y-7723 lipase were s...

  1. Use of a fluorescent radiolabeled triacylglycerol as a substrate for lipoprotein lipase and hepatic triglyceride lipase

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Dousset, N.; Negre, A.; Salvayre, R.

    1988-06-01

    A fluorescent radiolabeled triacylglycerol has been synthesized by using a fluorescent fatty acid (pyrene decanoic acid) and a radiolabeled oleic acid. This analog of the natural substrate, 1(3)pyrene decanoic-2,3 (1,2)-dioleoyl-sn-glycerol, has been tested as substrate for determining lipoprotein lipase and hepatic triacylglycerol lipase activities in post-heparin plasma. Optimal conditions for the determination of the two post-heparin plasma lipases were similar to those using radiolabeled triolein. Using this substrate, both post-heparin lipases exhibited their characteristic properties (pH optimum and effect of inhibitors) and attacked external ester bonds (1 or 3) containing pyrene decanoic and oleic acids at a similar rate.

  2. Role of lipase in the regulation of postprandial gastric acid secretion and emptying of fat in humans: a study with orlistat, a highly specific lipase inhibitor

    PubMed Central

    Borovicka, J; Schwizer, W; Guttmann, G; Hartmann, D; Kosinski, M; Wastiel, C; Bischof-Delaloye, A; Fried, M

    2000-01-01

    BACKGROUND AND AIMS—To investigate the importance of lipase on gastric functions, we studied the effects of orlistat, a potent and specific inhibitor of lipase, on postprandial gastric acidity and gastric emptying of fat.
METHODS—Fourteen healthy volunteers participated in a double blind, placebo controlled, randomised study. In a two way cross over study with two test periods of five days, separated by at least 14 days, orlistat 120 mg three times daily or placebo was given with standardised daily meals. In previous experiments we found that this dose almost completely inhibited postprandial duodenal lipase activity. Subjects underwent 28 hour intragastric pH-metry on day 4, and a gastric emptying study with a mixed meal (800 kcal) labelled with 999mTc sulphur colloid (solids) and 111Inthiocyanate (fat) on day 5. Gastric pH data were analysed for three postprandial hours and the interdigestive periods.
RESULTS—Orlistat inhibited almost completely (by 75%) lipase activity and accelerated gastric emptying of both the solid (by 52%) and fat (by 44%) phases of the mixed meal (p<0.03). Orlistat increased postprandial gastric acidity (from a median pH of 3.3 to 2.7; p<0.01). Postprandial cholecystokinin release was lower with orlistat (p<0.03).
CONCLUSION—Lipase has an important role in the regulation of postprandial gastric acid secretion and fat emptying in humans. These effects might be explained by lipolysis induced release of cholecystokinin.


Keywords: lipase; orlistat; gastric secretion; gastric emptying; pH-metry PMID:10807887

  3. Immobilization of lipases in hydrophobic chitosan for selective hydrolysis of fish oil: The impact of support functionalization on lipase activity, selectivity and stability.

    PubMed

    Urrutia, P; Arrieta, R; Alvarez, L; Cardenas, C; Mesa, M; Wilson, L

    2018-03-01

    The objective of this paper was to carry out an integral study of the use of hydrophobic chitosan as a low-cost support for immobilizing lipases and their further application in the selective hydrolysis of fish oil. Chitosan functionalized with different alkyl chains (C4, C8, C12) were characterized by FTIR, TGA, SEM, and Rose Bengal adsorption. Lipase B from Candida antarctica (CalB) and lipase from Rhizomucor miehei (RML) were immobilized obtaining a higher expressed activity at a longer alkyl chain length of support. Biocatalyst thermal stability showed that the impact of the alkyl chain length on enzyme stabilization varied according to the lipase source. The biocatalysts were applied in menhaden oil hydrolysis. Total polyunsaturated fatty acids released after 30 h of reaction with lipases immobilized in butyl, octyl and dodecyl-chitosan was 60, 107, and 90 mM for CalB biocatalysts, and 560, 392, and 50 mM for RML biocatalysts, respectively. Selectivity of CalB was not affected by the alkyl chain, while in the case of RML, a higher selectivity to cis-4,7,10,13,16,19-docohexaenoic acid release was obtained with dodecyl-chitosan. In conclusion, the adequate functionalization of chitosan varied according to lipase source, affecting their activity, stability and performance in the hydrolysis of fish oil. Copyright © 2017 Elsevier B.V. All rights reserved.

  4. Lipase-specific foldases.

    PubMed

    Rosenau, Frank; Tommassen, Jan; Jaeger, Karl-Erich

    2004-02-06

    Lipases represent the most important class of enzymes used in biotechnology. Many bacteria produce and secrete lipases but the enzymes originating from Pseudomonas and Burkholderia species seem to be particularly useful for a wide variety of different biocatalytic applications. These enzymes are usually encoded in an operon together with a second gene which codes for a lipase-specific foldase, Lif, which is necessary to obtain enzymatically active lipase. A detailed analysis based on amino acid homology has suggested the classification of Lif proteins into four different families and also revealed the presence of a conserved motif, Rx1x2FDY(F/C)L(S/T)A. Recent experimental evidence suggests that Lifs are so-called steric chaperones, which exert their physiological function by lowering energetic barriers during the folding of their cognate lipases, thereby providing essential steric information needed to fold lipases into their enzymatically active conformation.

  5. How gastric lipase, an interfacial enzyme with a Ser-His-Asp catalytic triad, acts optimally at acidic pH.

    PubMed

    Chahinian, Henri; Snabe, Torben; Attias, Coralie; Fojan, Peter; Petersen, Steffen B; Carrière, Frédéric

    2006-01-24

    Gastric lipase is active under acidic conditions and shows optimum activity on insoluble triglycerides at pH 4. The present results show that gastric lipase also acts in solution on vinyl butyrate, with an optimum activity above pH 7, which suggests that gastric lipase is able to hydrolyze ester bonds via the classical mechanism of serine hydrolases. These results support previous structural studies in which the catalytic triad of gastric lipase was reported to show no specific features. The optimum activity of gastric lipase shifted toward lower pH values, however, when the vinyl butyrate concentration was greater than the solubility limit. Experiments performed with long-chain triglycerides showed that gastric lipase binds optimally to the oil-water interface at low pH values. To study the effects of the pH on the adsorption step independently from substrate hydrolysis, gastric lipase adsorption on solid hydrophobic surfaces was monitored by total internal reflection fluorescence (TIRF), as well as using a quartz crystal microbalance. Both techniques showed a pH-dependent reversible gastric lipase adsorption process, which was optimum at pH 5 (Kd = 6.5 nM). Lipase adsorption and desorption constants (ka = 147,860 M(-1) s(-1) and kd = 139 x 10(-4) s(-1) at pH 6) were estimated from TIRF experiments. These results indicate that the optimum activity of gastric lipase at acidic pH is only "apparent" and results from the fact that lipase adsorption at lipid-water interfaces is the pH-dependent limiting step in the overall process of insoluble substrate hydrolysis. This specific kinetic feature of interfacial enzymology should be taken into account when studying any soluble enzyme acting on an insoluble substrate.

  6. α-Eleostearic acid-containing triglycerides for a continuous assay to determine lipase sn-1 and sn-3 regio-preference.

    PubMed

    El Alaoui, Meddy; Soulère, Laurent; Noiriel, Alexandre; Queneau, Yves; Abousalham, Abdelkarim

    2017-08-01

    Lipases are essentially described as sn-1 and sn-3 regio-selective. Actually few methods are available to measure this lipase regio-selectivity, moreover they require chiral chromatography analysis or specific derivations which are discontinuous and time consuming. In this study we describe a new, convenient, sensitive and continuous spectrophotometric method to screen lipases regio-selectivity using synthetic triglycerides (TG) containing α-eleostearic acid (9Z, 11E, 13E-octadecatrienoic acid) either at the sn-1 position [1-α-eleostearoyl-2,3-octadecyl-sn-glycerol (sn-EOO)] or at the sn-3 position [1,2-octadecyl-3-α-eleostearoyl-sn-glycerol (sn-OOE)] and coated onto the wells of microtiter plates. A non-hydrolysable ether bond, with a non UV-absorbing alkyl chain, was introduced at the other sn positions to prevent acyl chain migration during TG synthesis or lipolysis. The synthesis of TG containing α-eleostearic acid was performed from S-glycidol in six steps to obtain sn-EOO and in five steps to sn-OOE. The α-eleostearic acid conjugated triene constitutes an intrinsic chromophore and, consequently, confers the strong UV absorption properties of this free fatty acid as well as of the TG harboring it. The lipase activity on coated sn-EOO or sn-OOE was measured by the increase in the absorbance at 272nm due to the transition of α-eleostearic acid from the adsorbed to the soluble state. Human and porcine pancreatic lipases, guinea pig pancreatic lipase related protein 2, Thermomyces lanuginosus lipase, Candida antarctica lipase A and Candida antarctica lipase B were all used to validate the assay. This continuous high-throughput screening method could determine directly without any processes after lipolysis the regio-selectivity of various lipases. Copyright © 2017 Elsevier B.V. All rights reserved.

  7. Lipase-mediated lipid removal from propolis extract and its antiradical and antimicrobial activity.

    PubMed

    Park, Hyein; Bae, Song Hwan; Park, Yooheon; Choi, Hyeon-Son; Suh, Hyung Joo

    2015-06-01

    Propolis contains many antioxidants such as polyphenols and flavonoids. However, propolis-derived lipid components interrupt an efficient isolation of antioxidants from propolis extract. We examined the effectiveness of various lipase treatments for the removal of lipids from propolis extract and evaluated the biological features of the extract. Lipase OF and Novozyme 435 treatments did not reduce fatty acid level in propolis extract. However, Lipozyme TL IM-treated propolis extract showed a significant decrease in fatty acid level, suggesting the removal of lipids. Lipozyme RM IM also significantly decreased the fatty acid level of the extract, but was accompanied by the reduction of polyphenols and flavonoids, which are antioxidants. In Lipozyme TL IM treatment, an increase in active flavonoids, such as Artepillin C and kaempferide, was observed, with a slight increase of ferric reducing/antioxidant power (FRAP) radical-scavenging activity. In addition, antimicrobial activity towards skin health-related bacteria such as Staphylococcus epidermidis and Propionibacterium acnes was enhanced by Lipozyme TL IM treatment. Lipozyme TL IM treatment effectively removes lipids from propolis extract and enhances antibacterial activity. Therefore, we suggest that Lipozyme TL IM is a useful lipase for lipid removal of propolis extract. © 2014 Society of Chemical Industry.

  8. Lipase activity in stallion seminal plasma and the effect of lipase on stallion spermatozoa during storage at 5 degrees C.

    PubMed

    Carver, D A; Ball, B A

    2002-11-01

    Previous studies have demonstrated a detrimental effect of seminal plasma on the maintenance of motility of cooled equine spermatozoa; however, the mechanism for the adverse effect of seminal plasma during cooled storage remains undetermined. In goats, a glycoprotein component of bulbourethral gland secretion contains lipase activity that is detrimental to sperm motility when stored in skim milk-based extenders. The objective of the current study was to determine the amount of lipase activity in stallion seminal plasma and to determine the effect of added lipase on spermatozoal motility during cooled semen storage. In the first experiment, seminal plasma (1.0 ml) was assayed for lipase activity based upon hydrolysis of triglycerides (olive oil substrate) into free fatty acids and subsequent titration of pH change (SigmaDiagnostic Lipase Kit). Lipase activity in stallion seminal plasma was 0.36 +/- 0.02 Sigma units/ml, (mean + S.E.M.; n = 16 ejaculates from six stallions). In the second experiment, equine semen (three ejaculates from each of four stallions) was divided into five treatment aliquots. In Treatment 1, semen was extended 1:3 with nonfat dried skim milk extender (NFDSM). In treatment groups 2 through 5, spermatozoa were washed by centrifugation (300 x g for 15 min) and resuspended in NFDSM to a final concentration of 25 x 10(6) spermatozoa/ml. Porcine pancreatic lipase (pPL) was added to Treatment 3 (10 pPL units/ml), Treatment 4 (100 pPL units/ml) and Treatment 5 (100 pPL units/ml, heat inactivated at 100 degrees C for 5 min) while Treatment 2 had no pancreatic lipase added and served as the control. Samples were cooled slowly to 5 degrees C, and stored at 5 degrees C until evaluation. Sperm motility was evaluated at time 0, 24, 48 and 72 h by computerized semen analysis, and data were analyzed via repeated measures ANOVA. The addition of 100 units/ml but not 10 units/ml of pPL decreased (P < 0.01) total and progressive motility of stored sperm. Heat

  9. Synthesis of Rosin Acid Starch Catalyzed by Lipase

    PubMed Central

    Lin, Rihui; Li, He; Long, Han; Su, Jiating; Huang, Wenqin

    2014-01-01

    Rosin, an abundant raw material from pine trees, was used as a starting material directly for the synthesis of rosin acid starch. The esterification reaction was catalyzed by lipase (Novozym 435) under mild conditions. Based on single factor experimentation, the optimal esterification conditions were obtained as follows: rosin acid/anhydrous glucose unit in the molar ratio 2 : 1, reaction time 4 h at 45°C, and 15% of lipase dosage. The degree of substitution (DS) reaches 0.098. Product from esterification of cassava starch with rosin acid was confirmed by FTIR spectroscopy and iodine coloration analysis. Scanning electron microscopy and X-ray diffraction analysis showed that the morphology and crystallinity of the cassava starch were largely destroyed. Thermogravimetric analysis indicated that thermal stability of rosin acid starch decreased compared with native starch. PMID:24977156

  10. Lipase-catalyzed synthesis of fatty acid amide (erucamide) using fatty acid and urea.

    PubMed

    Awasthi, Neeraj Praphulla; Singh, R P

    2007-01-01

    Ammonolysis of fatty acids to the corresponding fatty acid amides is efficiently catalysed by Candida antartica lipase (Novozym 435). In the present paper lipase-catalysed synthesis of erucamide by ammonolysis of erucic acid and urea in organic solvent medium was studied and optimal conditions for fatty amides synthesis were established. In this process erucic acid gave 88.74 % pure erucamide after 48 hour and 250 rpm at 60 degrees C with 1:4 molar ratio of erucic acid and urea, the organic solvent media is 50 ml tert-butyl alcohol (2-methyl-2-propanol). This process for synthesis is economical as we used urea in place of ammonia or other amidation reactant at atmospheric pressure. The amount of catalyst used is 3 %.

  11. Acidolysis and glyceride synthesis reactions using fatty acids with two Pseudomonas lipases having different substrate specificities.

    PubMed

    Kojima, Yuzo; Sakuradani, Eiji; Shimizu, Sakayu

    2006-09-01

    Enzymatic acidolysis and glyceride synthesis using polyunsaturated fatty acids (PUFAs) with lipases from Pseudomonas fluorescens HU380 (HU-lipase), P. fluorescens AK102 (AK-lipase), and Candida rugosa (CR-lipase) were studied. The acidolysis of triolein with eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA) in n-hexane was evaluated with lipases immobilized on Celite 545. HU-lipase showed the highest incorporation rate at a low temperature (10 degrees C) with either EPA or DHA as the acyl donor, and the rate decreased with increasing reaction temperature. At 45 degrees C, the rates for EPA and DHA were 7.1 and 0.5 relative to those at 10 degrees C, respectively. The EPA incorporation rate was even higher at a low temperature (10 degrees C), and the DHA incorporation rate increased with decreasing temperature. Although AK-lipase showed the reverse tendency for incorporation rate, the DHA incorporation rate increased with increasing reaction temperature with both PUFAs. HU-lipase reacted well with PUFAs such as DHA, EPA, arachidonic acid (AA), mead acid (MA), and dihomo-gamma-linolenic acid (DGLA) on acidolysis and glyceride synthesis. The reactivities of AK-lipase toward these PUFAs except for DGLA, i.e., MA, AA, EPA, and DHA, were low for both reactions. The unique substrate specificities of the lipases from the Pseudomonas strains will enable us to use these lipases for the modification of fats and oils containing PUFAs such as fish oil.

  12. Can the fatty acid selectivity of plant lipases be predicted from the composition of the seed triglyceride?

    PubMed

    Hellyer, S A; Chandler, I C; Bosley, J A

    1999-09-22

    To address the question can the fatty acid selectivity of plant lipases be predicted from the composition of the seed triglyceride, we have characterised the selectivity of lipases from a wide range of oilseeds with diverse fatty acid compositions. For this study, a novel hydrolysis assay using a fully randomised oil, was developed. From some seed sources (e.g. Cinnamomum camphora), lipases show high preference for particular fatty acids, whilst from others (e.g. Brassica napus, Theobroma cacao80% saturated or 'unusual' fatty acids may contain lipases which exhibit selectivity. It therefore follows that since the majority of seeds are composed of unsaturated fatty acids, that highly selective lipases will be unusual in nature. However lipases from some species of the Cuphea genera show exceptionally high preference for particular fatty acids. For example, lipase from seeds of Cuphea procumbans has over 20-fold selectivity for C10:0.

  13. Genetics Home Reference: lysosomal acid lipase deficiency

    MedlinePlus

    ... Cegielska J, Whitley CB, Eckert S, Valayannopoulos V, Quinn AG. Clinical Features of Lysosomal Acid Lipase Deficiency. J ... qualified healthcare professional . About Selection Criteria for Links Data Files & API Site Map Subscribe Customer Support USA. ...

  14. Optimization of culture conditions for production of a novel cold-active lipase from Pichia lynferdii NRRL Y-7723

    USDA-ARS?s Scientific Manuscript database

    Lipases with abnormal properties such as thermo stability, alkalinity, acidity and cold-activity receive industrial attention because of their usability under restricted reaction conditions. Most microbial cold-active lipases originate from psychrotrophic and psychrophilic microorganisms found in An...

  15. [Influence of tobacco smoking on lipase activity in patients with pancreatitis].

    PubMed

    Sliwińska-Mossoń, Mariola; Milnerowicz, Halina

    2005-01-01

    The aim of this study is to prove the influence of tobacco smoking on lipase activity in the blood of smoking and non-smoking health persons and in smoking and non-smoking patients with diagnosed acute (AP), chronic exaggerated (CEP) and chronic pancreatitis (CP). The blood has been collected from 28 healthy persons and 55 patients with AP, CEP and CP. The enzyme activity has been determined using the colorimetric method with substrate 1,2-odilauryl-rac-glycero-3-glutaric acid -(6-methylresorufin) ester. The exposures to tobacco smoke have been examined on the basic of concentration of cotinine in the serum of patients. The highest lipase activity has been found in smoking patients with CEP. It has been noted that the serum lipase activity is significantly higher in smoking and healthy persons (p<0,05) then in non-smoking and healthy patients. However no significant differences have been found between the lipase activity in smoking patients with CP and non-smoking patients with CP. Smoking patients with AP and CEP have been found to have a significantly increased enzyme activity (p>0.01; p>0.05 respectively) when compared to non-smoking patients. Results of examination indicate that tobacco smoking has a significant influence on exocrine function of pancreas.

  16. Development of a high-throughput assay for measuring lipase activity using natural triacylglycerols coated on microtiter plates.

    PubMed

    Serveau-Avesque, Carole; Verger, Robert; Rodriguez, Jorge A; Abousalham, Abdelkarim

    2013-09-21

    We have designed a convenient, specific, sensitive and continuous lipase assay based on the use of natural triacylglycerols (TAGs) from the Aleurites fordii seed oil which contains α-eleostearic acid (9,11,13,cis,trans,trans-octadecatrienoic acid) and which was coated in the wells of microtiter plates. The coated TAG film cannot be desorbed by the various buffers used during the lipase assay. Upon lipase action, α-eleostearic acid is liberated and desorbed from the interface and then solubilized into the micellar phase. Consequently, the UV absorbance of the α-eleostearic acid is considerably enhanced due to the transformation from an adsorbed to a water soluble state. The lipase activity can be measured continuously by recording the variations with time of the UV absorption spectra. The rate of lipolysis was monitored by measuring the increase of OD at 272 nm, which was found to be linear with time and directly proportional to the amount of added lipase. This microtiter plate lipase assay, based on coated TAGs, presents various advantages as compared to the classical systems: (i) coated TAGs on the microtiter plates could be stored for a long-time at 4 °C, (ii) higher sensitivity in lipase detection, (iii) good reproducibility, and (iv) increase of signal to noise ratio due to high UV absorption after transfer of α-eleostearic acid from an adsorbed to a soluble state. Low concentrations, down to 1 pg mL(-1) of pure Thermomyces lanuginosus or human pancreatic lipase, could be detected under standard assay conditions. The detection sensitivity of this coated method is around 1000 times higher as compared to those obtained with the classical emulsified systems. This continuous high throughput lipase assay could be used to screen new lipases and/or lipase inhibitors present in various biological samples.

  17. New lipase assay using Pomegranate oil coating in microtiter plates.

    PubMed

    Ülker, Serdar; Placidi, Camille; Point, Vanessa; Gadenne, Benoît; Serveau-Avesque, Carole; Canaan, Stéphane; Carrière, Frédéric; Cavalier, Jean-François

    2016-01-01

    Lipases play various roles in fat digestion, lipoprotein metabolism, and in the mobilization of fat stored in lipid bodies in animals, plants and microorganisms. In association with these physiological functions, there is an important field of research for discovering lipase inhibitors and developing new treatments of diseases such as obesity, atherosclerosis, diabetes and tuberculosis. In this context, the development of convenient, specific and sensitive analytical methods for the detection and assay of lipases and/or lipase inhibitors is of major importance. It is shown here that purified triacylglycerols (TAGs) from Punica granatum (Pomegranate) seed oil coated on microtiter plates can be used for the continuous assay of lipase activity by recording the variations with time of the UV absorption spectra at 275 nm. UV absorption is due the release of punicic acid (9Z,11E,13Z-octadeca-9,11,13-trienoic acid), a conjugated triene contained in Pomegranate oil. This new microtiter plate assay allows to accurately measure the activity of a wider range of lipases compared to the similar assay previously developed with Tung oil containing α-eleostearic acid (9Z,11E,13E-octadeca-9,11,13-trienoic acid), including the LipY lipase from Mycobacterium tuberculosis. Although punicic acid is a diastereoisomer of α-eleostearic acid, the Δ(13)cis double bound found in punicic acid gives a different structure to the acyl chain that probably favours the interaction of Pomegranate TAGs with the lipase active site. The microplate lipase assay using Pomegranate TAGs shows high sensitivity, reproducibility and remarkable relevance for the high-speed screening of lipases and/or lipase inhibitors directly from raw culture media without any purification step. Copyright © 2015 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

  18. Contribution of single amino acid and codon substitutions to the production and secretion of a lipase by Bacillus subtilis.

    PubMed

    Skoczinski, Pia; Volkenborn, Kristina; Fulton, Alexander; Bhadauriya, Anuseema; Nutschel, Christina; Gohlke, Holger; Knapp, Andreas; Jaeger, Karl-Erich

    2017-09-25

    Bacillus subtilis produces and secretes proteins in amounts of up to 20 g/l under optimal conditions. However, protein production can be challenging if transcription and cotranslational secretion are negatively affected, or the target protein is degraded by extracellular proteases. This study aims at elucidating the influence of a target protein on its own production by a systematic mutational analysis of the homologous B. subtilis model protein lipase A (LipA). We have covered the full natural diversity of single amino acid substitutions at 155 positions of LipA by site saturation mutagenesis excluding only highly conserved residues and qualitatively and quantitatively screened about 30,000 clones for extracellular LipA production. Identified variants with beneficial effects on production were sequenced and analyzed regarding B. subtilis growth behavior, extracellular lipase activity and amount as well as changes in lipase transcript levels. In total, 26 LipA variants were identified showing an up to twofold increase in either amount or activity of extracellular lipase. These variants harbor single amino acid or codon substitutions that did not substantially affect B. subtilis growth. Subsequent exemplary combination of beneficial single amino acid substitutions revealed an additive effect solely at the level of extracellular lipase amount; however, lipase amount and activity could not be increased simultaneously. Single amino acid and codon substitutions can affect LipA secretion and production by B. subtilis. Several codon-related effects were observed that either enhance lipA transcription or promote a more efficient folding of LipA. Single amino acid substitutions could improve LipA production by increasing its secretion or stability in the culture supernatant. Our findings indicate that optimization of the expression system is not sufficient for efficient protein production in B. subtilis. The sequence of the target protein should also be considered as an

  19. Lipase catalyzed epoxidation of fatty acid methyl esters derived from unsaturated vegetable oils in absence of carboxylic acid.

    PubMed

    Sustaita-Rodríguez, Alejandro; Ramos-Sánchez, Víctor H; Camacho-Dávila, Alejandro A; Zaragoza-Galán, Gerardo; Espinoza-Hicks, José C; Chávez-Flores, David

    2018-04-11

    Nowadays the industrial chemistry reactions rely on green technologies. Enzymes as lipases are increasing its use in diverse chemical processes. Epoxidized fatty acid methyl esters obtained from transesterification of vegetable oils have recently found applications as polymer plasticizer, agrochemical, cosmetics, pharmaceuticals and food additives. In this research article, grapeseed, avocado and olive oils naturally containing high percents of mono and poly unsaturations were used as starting materials for the production of unsaturated fatty acid methyl esters. The effect of lauric acid as an active oxygen carrier was studied on epoxidation reactions where unsaturated fatty acid methyl esters were converted to epoxy fatty acid methyl esters using immobilized Candida antarctica Lipase type B as catalyst and hydrogen peroxide as oxygen donor at mild temperature and pressure conditions. After this study it was confirmed by 1 H NMR, 13 C NMR and GC-MS that the addition of lauric acid to the enzymatic reaction is unnecessary to transform the alkenes in to epoxides. It was found that quantitative conversions were possible in despite of a carboxylic acid absence.

  20. Pancreatic lipase-related protein 2 digests fats in human milk and formula in concert with gastric lipase and carboxyl ester lipase

    PubMed Central

    Johnson, Karin; Ross, Leah; Miller, Rita; Xiao, Xunjun; Lowe, Mark E.

    2013-01-01

    INTRODUCTION Dietary fats must be digested into fatty acids and monoacylglycerols prior to absorption. In adults, colipase-dependent pancreatic triglyceride lipase (PTL) contributes significantly to fat digestion. In newborn rodents and humans, the pancreas expresses low levels of PTL. In rodents, a homologue of PTL, pancreatic lipase related protein 2 (PLRP2) and carboxyl ester lipase (CEL) compensate for the lack of PTL. In human newborns, the role for PLRP2 in dietary fat digestion is unclear. To clarify the potential of human PLRP2 to influence dietary fat digestion in newborns, we determined PLRP2 activity against human milk and infant formula. METHODS The activity of purified recombinant PLRP2, gastric lipase and CEL against fats in human milk and formula was measured with each lipase alone and in combination with a standard pH-stat assay. RESULTS Colipase added to human milk stimulated fat digestion. PLRP2 and CEL had activity against human milk and formula. Pre-digestion with gastric lipase increased PLRP2 activity against both substrates. Together, CEL and PLRP2 activity was additive with formula and synergistic with human milk. CONCLUSIONS PLRP2 can digest fats in human milk and formula. PLRP2 acts in concert with CEL and gastric lipase to digest fats in human milk in vitro. PMID:23732775

  1. Production of fatty acid butyl esters using the low cost naturally immobilized Carica papaya lipase.

    PubMed

    Su, Erzheng; Wei, Dongzhi

    2014-07-09

    In this work, the low cost naturally immobilized Carica papaya lipase (CPL) was investigated for production of fatty acid butyl esters (FABE) to fulfill the aim of reducing the lipase cost in the enzymatic butyl-biodiesel process. The CPL showed specificities to different alcohol acyl acceptors. Alcohols with more than three carbon atoms did not have negative effects on the CPL activity. The CPL catalyzed butanolysis for FABE production was systematically investigated. The reaction solvent, alcohol/oil molar ratio, enzyme amount, reaction temperature, and water activity all affected the butanolysis process. Under the optimized conditions, the highest conversion of 96% could be attained in 24 h. These optimal conditions were further applied to CPL catalyzed butanolysis of other vegetable oils. All of them showed very high conversion. The CPL packed-bed reactor was further developed, and could be operated continuously for more than 150 h. All of these results showed that the low cost Carica papaya lipase can be used as a promising lipase for biodiesel production.

  2. Electrospun polylactic acid and polyvinyl alcohol fibers as efficient and stable nanomaterials for immobilization of lipases.

    PubMed

    Sóti, Péter Lajos; Weiser, Diana; Vigh, Tamás; Nagy, Zsombor Kristóf; Poppe, László; Marosi, György

    2016-03-01

    Electrospinning was applied to create easy-to-handle and high-surface-area membranes from continuous nanofibers of polyvinyl alcohol (PVA) or polylactic acid (PLA). Lipase PS from Burkholderia cepacia and Lipase B from Candida antarctica (CaLB) could be immobilized effectively by adsorption onto the fibrous material as well as by entrapment within the electrospun nanofibers. The biocatalytic performance of the resulting membrane biocatalysts was evaluated in the kinetic resolution of racemic 1-phenylethanol (rac-1) and 1-phenylethyl acetate (rac-2). Fine dispersion of the enzymes in the polymer matrix and large surface area of the nanofibers resulted in an enormous increase in the activity of the membrane biocatalyst compared to the non-immobilized crude powder forms of the lipases. PLA as fiber-forming polymer for lipase immobilization performed better than PVA in all aspects. Recycling studies with the various forms of electrospun membrane biocatalysts in ten cycles of the acylation and hydrolysis reactions indicated excellent stability of this forms of immobilized lipases. PLA-entrapped lipases could preserve lipase activity and enantiomer selectivity much better than the PVA-entrapped forms. The electrospun membrane forms of CaLB showed high mechanical stability in the repeated acylations and hydrolyses than commercial forms of CaLB immobilized on polyacrylamide beads (Novozyme 435 and IMMCALB-T2-150).

  3. Production of an acidic and thermostable lipase of the mesophilic fungus Penicillium simplicissimum by solid-state fermentation.

    PubMed

    Gutarra, Melissa L E; Godoy, Mateus G; Maugeri, Francisco; Rodrigues, Maria Isabel; Freire, Denise M G; Castilho, Leda R

    2009-11-01

    The production of a lipase by a wild-type Brazilian strain of Penicillium simplicissimum in solid-state fermentation of babassu cake, an abundant residue of the oil industry, was studied. The enzyme production reached about 90 U/g in 72 h, with a specific activity of 4.5 U/mg of total proteins. The crude lipase showed high activities at 35-60 degrees C and pH 4.0-6.0, with a maximum activity at 50 degrees C and pH 4.0-5.0. Enzyme stability was enhanced at pH 5.0 and 6.0, with a maximum half-life of 5.02 h at 50 degrees C and pH 5.0. Thus, this lipase shows a thermophilic and thermostable behavior, what is not common among lipases from mesophilic filamentous fungi. The crude enzyme catalysed the hydrolysis of triglycerides and p-nitrophenyl esters (C4:0-C18:0), preferably acting on substrates with medium-chain fatty acids. This non-purified lipase in addition to interesting properties showed a reduced production cost making feasible its applicability in many fields.

  4. Lipase and its modulator from Pseudomonas sp. strain KFCC 10818: proline-to-glutamine substitution at position 112 induces formation of enzymatically active lipase in the absence of the modulator.

    PubMed

    Kim, E K; Jang, W H; Ko, J H; Kang, J S; Noh, M J; Yoo, O J

    2001-10-01

    A lipase gene, lipK, and a lipase modulator gene, limK, of Pseudomonas sp. strain KFCC 10818 have been cloned, sequenced, and expressed in Escherichia coli. The limK gene is located immediately downstream of the lipK gene. Enzymatically active lipase was produced only in the presence of the limK gene. The effect of the lipase modulator LimK on the expression of active lipase was similar to those of the Pseudomonas subfamily I.1 and I.2 lipase-specific foldases (Lifs). The deduced amino acid sequence of LimK shares low homology (17 to 19%) with the known Pseudomonas Lifs, suggesting that Pseudomonas sp. strain KFCC 10818 is only distantly related to the subfamily I.1 and I.2 Pseudomonas species. Surprisingly, a lipase variant that does not require LimK for its correct folding was isolated in the study to investigate the functional interaction between LipK and LimK. When expressed in the absence of LimK, the P112Q variant of LipK formed an active enzyme and displayed 63% of the activity of wild-type LipK expressed in the presence of LimK. These results suggest that the Pro(112) residue of LipK is involved in a key step of lipase folding. We expect that the novel finding of this study may contribute to future research on efficient expression or refolding of industrially important lipases and on the mechanism of lipase folding.

  5. Lipase and Its Modulator from Pseudomonas sp. Strain KFCC 10818: Proline-to-Glutamine Substitution at Position 112 Induces Formation of Enzymatically Active Lipase in the Absence of the Modulator

    PubMed Central

    Kim, Eun Kyung; Jang, Won Hee; Ko, Jung Ho; Kang, Jong Seok; Noh, Moon Jong; Yoo, Ook Joon

    2001-01-01

    A lipase gene, lipK, and a lipase modulator gene, limK, of Pseudomonas sp. strain KFCC 10818 have been cloned, sequenced, and expressed in Escherichia coli. The limK gene is located immediately downstream of the lipK gene. Enzymatically active lipase was produced only in the presence of the limK gene. The effect of the lipase modulator LimK on the expression of active lipase was similar to those of the Pseudomonas subfamily I.1 and I.2 lipase-specific foldases (Lifs). The deduced amino acid sequence of LimK shares low homology (17 to 19%) with the known Pseudomonas Lifs, suggesting that Pseudomonas sp. strain KFCC 10818 is only distantly related to the subfamily I.1 and I.2 Pseudomonas species. Surprisingly, a lipase variant that does not require LimK for its correct folding was isolated in the study to investigate the functional interaction between LipK and LimK. When expressed in the absence of LimK, the P112Q variant of LipK formed an active enzyme and displayed 63% of the activity of wild-type LipK expressed in the presence of LimK. These results suggest that the Pro112 residue of LipK is involved in a key step of lipase folding. We expect that the novel finding of this study may contribute to future research on efficient expression or refolding of industrially important lipases and on the mechanism of lipase folding. PMID:11566993

  6. A computational search for lipases that can preferentially hydrolyze long-chain omega-3 fatty acids from fish oil triacylglycerols.

    PubMed

    Kamal, Md Zahid; Barrow, Colin J; Rao, Nalam Madhusudhana

    2015-04-15

    Consumption of long-chain omega-3 fatty acids is known to decrease the risk of major cardiovascular events. Lipases, a class of triacylglycerol hydrolases, have been extensively tested to concentrate omega-3 fatty acids from fish oils, under mild enzymatic conditions. However, no lipases with preference for omega-3 fatty acids selectivity have yet been discovered or developed. In this study we performed an exhaustive computational study of substrate-lipase interactions by docking, both covalent and non-covalent, for 38 lipases with a large number of structured triacylglycerols containing omega-3 fatty acids. We identified some lipases that have potential to preferentially hydrolyze omega-3 fatty acids from structured triacylglycerols. However omega-3 fatty acid preferences were found to be modest. Our study provides an explanation for absence of reports of lipases with omega-3 fatty acid hydrolyzing ability and suggests methods for developing these selective lipases. Copyright © 2014 Elsevier Ltd. All rights reserved.

  7. Hydrolysis Activity of Virgin Coconut Oil Using Lipase from Different Sources.

    PubMed

    Nguyen, T A V; Le, Truong D; Phan, Hoa N; Tran, Lam B

    2018-01-01

    Two types of lipase, Candida rugosa lipase (CRL) and porcine pancreas lipase (PPL), were used to hydrolyze virgin coconut oil (VCO). The hydrolysis process was carried out under four parameters, VCO to buffer ratio, lipase concentration, pH, and temperature, which have a significant effect on hydrolysis of lipase. CRL obtained the best hydrolysis condition at 1 : 5 of VCO to buffer ratio, 1.5% of CRL concentration, pH 7, and temperature of 40°C. Meanwhile, PPL gave different results at 1 : 4 of VCO to buffer ratio, 2% of lipase concentration, pH 7.5, and 40°C. The highest hydrolysis degree of CRL and PPL was obtained after 16 hours and 26 hours, reaching 79.64% and 27.94%, respectively. Besides, the hydrolysis process was controlled at different time course (every half an hour) at the first 4 hours of reaction to compare the initial hydrolysis degree of these two lipase types. FFAs from hydrolyzed products were isolated and determined the percentage of each fatty acid which contributes to the FFAs mixture. As a result, medium chain fatty acids (MCFAs) made up the main contribution in composition of FFAs and lauric acid (C12) was the largest segment (47.23% for CRL and 44.23% for PPL).

  8. Hydrolysis Activity of Virgin Coconut Oil Using Lipase from Different Sources

    PubMed Central

    Phan, Hoa N.; Tran, Lam B.

    2018-01-01

    Two types of lipase, Candida rugosa lipase (CRL) and porcine pancreas lipase (PPL), were used to hydrolyze virgin coconut oil (VCO). The hydrolysis process was carried out under four parameters, VCO to buffer ratio, lipase concentration, pH, and temperature, which have a significant effect on hydrolysis of lipase. CRL obtained the best hydrolysis condition at 1 : 5 of VCO to buffer ratio, 1.5% of CRL concentration, pH 7, and temperature of 40°C. Meanwhile, PPL gave different results at 1 : 4 of VCO to buffer ratio, 2% of lipase concentration, pH 7.5, and 40°C. The highest hydrolysis degree of CRL and PPL was obtained after 16 hours and 26 hours, reaching 79.64% and 27.94%, respectively. Besides, the hydrolysis process was controlled at different time course (every half an hour) at the first 4 hours of reaction to compare the initial hydrolysis degree of these two lipase types. FFAs from hydrolyzed products were isolated and determined the percentage of each fatty acid which contributes to the FFAs mixture. As a result, medium chain fatty acids (MCFAs) made up the main contribution in composition of FFAs and lauric acid (C12) was the largest segment (47.23% for CRL and 44.23% for PPL). PMID:29623233

  9. Utilization of acid pre-treated coconut dregs as a substrate for production of detergent compatible lipase by Bacillus stratosphericus.

    PubMed

    Mohd Zin, Nur Bainun; Mohamad Yusof, Busyra; Oslan, Siti Nurbaya; Wasoh, Helmi; Tan, Joo Shun; Ariff, Arbakariya B; Halim, Murni

    2017-12-01

    In recent years, many efforts have been directed to explore the methods to reduce the production costs of industrial lipase by improving the yield and the use of low-cost agricultural wastes. Coconut dregs, which is a lignocellulosic by-product from coconut oil and milk processing plants, is rich in cellulose (36%) and crude fat (9%). A newly isolated Bacillus stratosphericus has been demonstrated to perform cellulose hydrolysis on coconut dregs producing fermentable sugars. The highest extracellular lipase activity of 140 U/mL has been achieved in submerged fermentation with acid pre-treated coconut dregs. The lipase was found to be active over a wide range of temperatures and pHs. The activity of lipase can be generally increased by the presence of detergent ingredients such as Tween-80, cetyltrimethylammonium bromide, hydrogen peroxide and phosphate per sulphate. The great compatibility of lipase in commercial detergents has also underlined its potential as an additive ingredient in biodetergent formulations.

  10. Discovery and characterizaton of a novel lipase with transesterification activity from hot spring metagenomic library.

    PubMed

    Yan, Wei; Li, Furong; Wang, Li; Zhu, Yaxin; Dong, Zhiyang; Bai, Linhan

    2017-03-01

    A new gene encoding a lipase (designated as Lip-1 ) was identified from a metagenomic bacterial artificial chromosome(BAC) library prepared from a concentrated water sample collected from a hot spring field in Niujie, Eryuan of Yunnan province in China. The open reading frame of this gene encoded 622 amino acid residues. It was cloned, fused with the oleosin gene and over expressed in Escherichia coli to prepare immobilized lipase artificial oil body AOB-sole-lip-1. The monomeric Sole-lip-1 fusion protein presented a molecular mass of 102.4 kDa. Enzyme assays using olive oil and methanol as the substrates in petroleum ether confirmed its transesterification activity. Hexadecanoic acid methyl ester, 8,11-Octadecadienoic acid methyl ester, 8-Octadecenoic acid methyl ester, and Octadecanoic acid methyl ester were detected. It showed favorable transesterification activity with optimal temperature 45 °C. Besides, the maximal biodiesel yield was obtained when the petroleum ether system as the organic solvent and the substrate methanol in 350 mmol/L (at a molar ratio of methanol of 10.5:1) and the water content was 1%. In light of these advantages, this lipase presents a promising resource for biodiesel production.

  11. Liquid lipases for enzymatic concentration of n-3 polyunsaturated fatty acids in monoacylglycerols via ethanolysis: Catalytic specificity and parameterization.

    PubMed

    He, Yongjin; Li, Jingbo; Kodali, Sitharam; Balle, Thomas; Chen, Bilian; Guo, Zheng

    2017-01-01

    This work examined catalytic specificity and fatty acid selectivity of five liquid lipases C. antarctica lipase A and B (CAL-A/B), and lipase TL (T. lanuginosus), Eversa Transfrom and NS in ethanolysis of fish oil with the aim to concentrate n-3 PUFAs into monoacylglycerols (MAGs) products. Lipase TL, Eversa Transform & NS entail a much faster reaction and produce higher MAGs yield (>30%); whereas CAL-A obtains the highest concentration of n-3 PUFAs/DHA/EPA into MAGs products (88.30%); followed by lipase NS (81.02%). 13 C NMR analysis indicates that CAL-B and lipase TL are sn-1,3 specific; but CAL-A and lipase Eversa Transform are non-regiospecific or weak sn-2 specific; which plausibly explains high enrichment effect of the latter two lipases. All liquid lipases are observed reusable for a certain times (lipase Eversa Transform up to 12 times), demonstrating their competitive advantage over immobilized form for industrial application because of their higher activity and cheaper operation cost. Copyright © 2016 Elsevier Ltd. All rights reserved.

  12. Strain improvement of Aspergillus niger for enhanced lipase production.

    PubMed

    Sandana Mala, John Geraldine; Kamini, Numbi R.; Puvanakrishnan, Rengarajulu

    2001-08-01

    The enhancement of lipase production from Aspergillus niger was attempted by ultraviolet (UV) and nitrous acid mutagenesis, and the mutants were selected on media containing bile salts. Nitrous acid mutants exhibited increased efficiency for lipase production when compared with UV mutants in submerged fermentation. The hyperproducing UV and nitrous acid mutants were further subjected to a second step of mutagenesis to devise an economical and ecofriendly technique for lipase production by the effective use of hydrocarbons. One percent kerosene was found to be optimal for lipase production, and one of the mutant strains NAII exhibited 2.53 times more increased lipase activity than the parental strain did. This investigation indicates a possible role for the A. niger mutant strains in the biodegradation of oil-polluted environments for the development of ecofriendly technologies.

  13. Lipase-Catalyzed Production of 6-O-cinnamoyl-sorbitol from D-sorbitol and Cinnamic Acid Esters.

    PubMed

    Kim, Jung-Ho; Bhatia, Shashi Kant; Yoo, Dongwon; Seo, Hyung Min; Yi, Da-Hye; Kim, Hyun Joong; Lee, Ju Hee; Choi, Kwon-Young; Kim, Kwang Jin; Lee, Yoo Kyung; Yang, Yung-Hun

    2015-05-01

    To overcome the poor properties of solubility and stability of cinnamic acid, cinnamate derivatives with sugar alcohols were produced using the immobilized Candida antarctica lipase with vinyl cinnamate and D-sorbitol as substrate at 45 °C. Immobilized C. antarctica lipase was found to synthesize 6-O-cinnamoyl-sorbitol and confirmed by HPLC and (1)H-NMR and had a preference for vinyl cinnamate over other esters such as allyl-, ethyl-, and isobutyl cinnamate as co-substrate with D-sorbitol. Contrary to D-sorbitol, vinyl cinnamate, and cinnamic acid, the final product 6-O-cinnamoyl-sorbitol was found to have radical scavenging activity. This would be the first report on the biosynthesis of 6-O-cinnamoyl-sorbitol with immobilized enzyme from C. antarctica.

  14. Lipase production in lipolytic yeast from Wonorejo mangrove area

    NASA Astrophysics Data System (ADS)

    Alami, Nur Hidayatul; Nasihah, Liziyatin; Umar, Rurin Luswidya Artaty; Kuswytasari, Nengah Dwianita; Zulaika, Enny; Shovitri, Maya

    2017-06-01

    Lipase is an enzyme that is often used in industry and become a commercial enzyme. One group of microorganisms capable of producing lipase is a yeast. This study aims to screen yeast from Wonorejo mangrove that potential to produce lipase and to optimize the production of these enzymes. Screening test include the measurement of lipolytic index and value of fatty acid. Yeast with the best value of fatty acid will be continued to the measurement of lipase activity. It is affected by several environmental factors, such as pH, temperature, and incubation time. This research was conducted to observe the optimization variation on environmental factors combination to produce lipase. Lipase activity was tested by using p-Nitrophenyl Palmitate (pNPP). Absorbency was measured by spectrofotometer on wavelength of 410 nm. Measurement of the enzyme activity was done by interpolating the absorbance values on the p-nitrophenol standard curve then calculated by the formula. All data were analyzed by using descriptive quantitative method. The results show that the highest lypolityc index was 2.08. The highest value of fatty acid was 0.49 that was reached on 168 hours of incubation. Candida W3.8 expressed the highest lypolylitic potential. The optimum environment to produce lipase by Candida W 3.8 was on 120 hours of incubation time, in temperature range of 27°C - 45°C and pH range of 4,5 - 7.

  15. Engineering Lipases: walking the fine line between activity and stability

    NASA Astrophysics Data System (ADS)

    Dasetty, Siva; Blenner, Mark A.; Sarupria, Sapna

    2017-11-01

    Lipases are enzymes that hydrolyze lipids and have several industrial applications. There is a tremendous effort in engineering the activity, specificity and stability of lipases to render them functional in a variety of environmental conditions. In this review, we discuss the recent experimental and simulation studies focused on engineering lipases. Experimentally, mutagenesis studies have demonstrated that the activity, stability, and specificity of lipases can be modulated by mutations. It has been particularly challenging however, to elucidate the underlying mechanisms through which these mutations affect the lipase properties. We summarize results from experiments and molecular simulations highlighting the emerging picture to this end. We end the review with suggestions for future research which underscores the delicate balance of various facets in the lipase that affect their activity and stability necessitating the consideration of the enzyme as a network of interactions.

  16. The short form of the recombinant CAL-A-type lipase UM03410 from the smut fungus Ustilago maydis exhibits an inherent trans-fatty acid selectivity.

    PubMed

    Brundiek, Henrike; Saß, Stefan; Evitt, Andrew; Kourist, Robert; Bornscheuer, Uwe T

    2012-04-01

    The Ustilago maydis lipase UM03410 belongs to the mostly unexplored Candida antarctica lipase (CAL-A) subfamily. The two lipases with [corrected] the highest identity are a lipase from Sporisorium reilianum and the prototypic CAL-A. In contrast to the other CAL-A-type lipases, this hypothetical U. maydis lipase is annotated to possess a prolonged N-terminus of unknown function. Here, we show for the first time the recombinant expression of two versions of lipase UM03410: the full-length form (lipUMf) and an Nterminally truncated form (lipUMs). For comparison to the prototype, the expression of recombinant CAL-A in E. coli was investigated. Although both forms of lipase UM03410 could be expressed functionally in E. coli, the N-terminally truncated form (lipUMs) demonstrated significantly higher activities towards p-nitrophenyl esters. The functional expression of the N-terminally truncated lipase was further optimized by the appropriate choice of the E. coli strain, lowering the cultivation temperature to 20 °C and enrichment of the cultivation medium with glucose. Primary characteristics of the recombinant lipase are its pH optimum in the range of 6.5-7.0 and its temperature optimum at 55 °C. As is typical for lipases, lipUM03410 shows preference for long chain fatty acid esters with myristic acid ester (C14:0 ester) being the most preferred one.More importantly, lipUMs exhibits an inherent preference for C18:1Δ9 trans and C18:1Δ11 trans-fatty acid esters similar to CAL-A. Therefore, the short form of this U. maydis lipase is the only other currently known lipase with a distinct trans-fatty acid selectivity.

  17. Egg Yolk Factor of Staphylococcus aureus II. Characterization of the Lipase Activity

    PubMed Central

    Shah, D. B.; Wilson, J. B.

    1965-01-01

    Shah, D. B. (University of Wisconsin, Madison), and J. B. Wilson. Egg yolk factor of Staphylococcus aureus. II. Characterization of the lipase activity. J. Bacteriol. 89:949–953. 1965.—The staphylococcal egg yolk factor was characterized as a lipase. The enzyme had an optimal pH of 7.8, but the optimal pH of stability was 7. Substrate specificity data showed that the relative rate of hydrolysis was lowest with triacetin as substrate, was maximal with tributyrin, and decreased as the chain length of the acyl moieties increased. The enzyme showed an absolute requirement for a fatty acid acceptor like calcium, when the acyl moiety of triglyceride was water-insoluble. Magnesium, strontium, and barium functioned equally well as fatty acid acceptors. The enzyme was able to hydrolyze coconut oil, peanut oil, olive oil, and egg yolk oil. PMID:14276120

  18. A Novel Cold-Active Lipase from Candida albicans: Cloning, Expression and Characterization of the Recombinant Enzyme

    PubMed Central

    Lan, Dong-Ming; Yang, Ning; Wang, Wen-Kai; Shen, Yan-Fei; Yang, Bo; Wang, Yong-Hua

    2011-01-01

    A novel lipase gene lip5 from the yeast Candida albicans was cloned and sequenced. Alignment of amino acid sequences revealed that 86–34% identity exists with lipases from other Candida species. The lipase and its mutants were expressed in the yeast Pichia pastoris, where alternative codon usage caused the mistranslation of 154-Ser and 293-Ser as leucine. 154-Ser to leucine resulted in loss of expression of Lip5, and 293-Ser to leucine caused a marked reduction in the lipase activity. Lip5-DM, which has double mutations that revert 154 and 293 to serine residues, showed good lipase activity, and was overexpressed and purified by (NH4)2SO4 precipitation and ion-exchange chromatography. The pure Lip5-DM was stable at low temperatures ranging from 15–35 °C and pH 5–9, with the optimal conditions being 15–25 °C and pH 5–6. The activation energy of recombinant lipase was 8.5 Kcal/mol between 5 and 25 °C, suggesting that Lip5-DM was a cold–active lipase. Its activity was found to increase in the presence of Zn2+, but it was strongly inhibited by Fe2+, Fe3+, Hg2+ and some surfactants. In addition, the Lip5-DM could not tolerate water-miscible organic solvents. Lip5-DM exhibited a preference for the short-and medium-chain length p-nitrophenyl (C4 and C8 acyl group) esters rather than the long chain length p-nitrophenyl esters (C12, C16 and C18 acyl group) with highest activity observed with the C8 derivatives. The recombinant enzyme displayed activity toward triacylglycerols, such as olive oil and safflower oil. PMID:21747717

  19. Acetylsalicylic acid (aspirin) reduces damage to reconstituted human tissues infected with Candida species by inhibiting extracellular fungal lipases

    PubMed Central

    Trofa, David; Agovino, Mariangela; Stehr, Frank; Schäfer, Wilhelm; Rykunov, Dmitry; Fiser, András; Hamari, Zsuzsanna; Nosanchuk, Joshua D.; Gácser, Attila

    2009-01-01

    A reconstituted human tissue model was used to mimic Candida albicans and Candida parapsilosis infection in order to investigate the protective effects of acetylsalicylic acid (aspirin, ASA). We found that therapeutic concentrations of ASA reduced tissue damage in the in vitro infection model. We further evaluated the lipase inhibitory effects of ASA by investigating the growth of C. albicans, C. parapsilosis and C. parapsilosis lipase negative (Δcplip1-2/Δcplip1-2) mutants in a lipid rich minimal medium supplemented with olive oil and found that a therapeutic concentration of ASA inhibited the growth of wild type fungi. The lipase inhibitors quinine and ebelactone B were also shown to reduce growth and protect against tissue damage from Candida species, respectively. A lipolytic activity assay also showed that therapeutic concentrations of ASA inhibited C. antarctica and C. cylindracea purified lipases obtained through a commercial kit. The relationship between ASA and lipase was characterized through a computed structural model of the Lipase-2 protein from C. parapsilosis in complex with ASA. The results suggest that development of inhibitors of fungal lipases could result in broad-spectrum therapeutics, especially since fungal lipases are not homologous to their human analogues. PMID:19703582

  20. A monolithic lipase reactor for biodiesel production by transesterification of triacylglycerides into fatty acid methyl esters

    PubMed Central

    Urban, Jiri; Svec, Frantisek; Fréchet, Jean M.J.

    2011-01-01

    An enzymatic reactor with lipase immobilized on a monolithic polymer support has been prepared and used to catalyze the transesterification of triacylglycerides into the fatty acid methyl esters commonly used for biodiesel. A design of experiments procedure was used to optimize the monolithic reactor with variables including control of the surface polarity of the monolith via variations in the length of the hydrocarbon chain in alkyl methacrylate monomer, time of grafting of 1-vinyl-4,4-dimethylazlactone used to activate the monolith, and time used for the immobilization of porcine lipase. Optimal conditions involved the use of a poly(stearyl methacrylate-co-ethylene dimethacrylate) monolith, grafted first with vinylazlactone, then treated with lipase for 2 h to carry out the immobilization of the enzyme. Best conditions for the transesterification of glyceryl tributyrate included a temperature of 37°C and a 10 min residence time of the substrate in the bioreactor. The reactor did not lose its activity even after pumping through it a solution of substrate equaling 1,000 reactor volumes. This enzymatic reactor was also used for the transesterification of triacylglycerides from soybean oil to fatty acid methyl esters thus demonstrating the ability of the reactor to produce biodiesel. PMID:21915852

  1. A monolithic lipase reactor for biodiesel production by transesterification of triacylglycerides into fatty acid methyl esters.

    PubMed

    Urban, Jiri; Svec, Frantisek; Fréchet, Jean M J

    2012-02-01

    An enzymatic reactor with lipase immobilized on a monolithic polymer support has been prepared and used to catalyze the transesterification of triacylglycerides into the fatty acid methyl esters commonly used for biodiesel. A design of experiments procedure was used to optimize the monolithic reactor with variables including control of the surface polarity of the monolith via variations in the length of the hydrocarbon chain in alkyl methacrylate monomer, time of grafting of 1-vinyl-4,4-dimethylazlactone used to activate the monolith, and time used for the immobilization of porcine lipase. Optimal conditions involved the use of a poly(stearyl methacrylate-co-ethylene dimethacrylate) monolith, grafted first with vinylazlactone, then treated with lipase for 2 h to carry out the immobilization of the enzyme. Best conditions for the transesterification of glyceryl tributyrate included a temperature of 37°C and a 10 min residence time of the substrate in the bioreactor. The reactor did not lose its activity even after pumping through it a solution of substrate equaling 1,000 reactor volumes. This enzymatic reactor was also used for the transesterification of triacylglycerides from soybean oil to fatty acid methyl esters thus demonstrating the ability of the reactor to produce biodiesel. Copyright © 2011 Wiley Periodicals, Inc.

  2. Novel Metagenome-Derived, Cold-Adapted Alkaline Phospholipase with Superior Lipase Activity as an Intermediate between Phospholipase and Lipase

    PubMed Central

    Lee, Mi-Hwa; Oh, Ki-Hoon; Kang, Chul-Hyung; Kim, Ji-Hoon; Oh, Tae-Kwang; Ryu, Choong-Min

    2012-01-01

    A novel lipolytic enzyme was isolated from a metagenomic library obtained from tidal flat sediments on the Korean west coast. Its putative functional domain, designated MPlaG, showed the highest similarity to phospholipase A from Grimontia hollisae CIP 101886, though it was screened from an emulsified tricaprylin plate. Phylogenetic analysis showed that MPlaG is far from family I.6 lipases, including Staphylococcus hyicus lipase, a unique lipase which can hydrolyze phospholipids, and is more evolutionarily related to the bacterial phospholipase A1 family. The specific activities of MPlaG against olive oil and phosphatidylcholine were determined to be 2,957 ± 144 and 1,735 ± 147 U mg−1, respectively, which means that MPlaG is a lipid-preferred phospholipase. Among different synthetic esters, triglycerides, and phosphatidylcholine, purified MPlaG exhibited the highest activity toward p-nitrophenyl palmitate (C16), tributyrin (C4), and 1,2-dihexanoyl-phosphatidylcholine (C8). Finally, MPlaG was identified as a phospholipase A1 with lipase activity by cleavage of the sn-1 position of OPPC, interfacial activity, and triolein hydrolysis. These findings suggest that MPlaG is the first experimentally characterized phospholipase A1 with lipase activity obtained from a metagenomic library. Our study provides an opportunity to improve our insight into the evolution of lipases and phospholipases. PMID:22544255

  3. Biodiesel production with immobilized lipase: A review.

    PubMed

    Tan, Tianwei; Lu, Jike; Nie, Kaili; Deng, Li; Wang, Fang

    2010-01-01

    Fatty acid alkyl esters, also called biodiesel, are environmentally friendly and show great potential as an alternative liquid fuel. Biodiesel is produced by transesterification of oils or fats with chemical catalysts or lipase. Immobilized lipase as the biocatalyst draws high attention because that process is "greener". This article reviews the current status of biodiesel production with immobilized lipase, including various lipases, immobilization methods, various feedstocks, lipase inactivation caused by short chain alcohols and large scale industrialization. Adsorption is still the most widely employed method for lipase immobilization. There are two kinds of lipase used most frequently especially for large scale industrialization. One is Candida antartica lipase immobilized on acrylic resin, and the other is Candida sp. 99-125 lipase immobilized on inexpensive textile membranes. However, to further reduce the cost of biodiesel production, new immobilization techniques with higher activity and stability still need to be explored. Copyright 2010 Elsevier Inc. All rights reserved.

  4. Wolman's disease and cholesteryl ester storage disorder: the phenotypic spectrum of lysosomal acid lipase deficiency.

    PubMed

    Pericleous, Marinos; Kelly, Claire; Wang, Tim; Livingstone, Callum; Ala, Aftab

    2017-09-01

    Lysosomal acid lipase deficiency is a rare, autosomal recessive condition caused by mutations in the gene encoding lysosomal acid lipase (LIPA) that result in reduced or absent activity of this essential enzyme. The severity of the resulting disease depends on the nature of the underlying mutation and magnitude of its effect on enzymatic function. Wolman's disease is a severe disorder that presents during infancy, resulting in failure to thrive, hepatomegaly, and hepatic failure, and an average life expectancy of less than 4 months. Cholesteryl ester storage disorder arises later in life and is less severe, although the two diseases share many common features, including dyslipidaemia and transaminitis. The prevalence of these diseases has been estimated at one in 40 000 to 300 000, but many cases are undiagnosed and unreported, and awareness among clinicians is low. Lysosomal acid lipase deficiency-which can be diagnosed using dry blood spot testing-is often misdiagnosed as non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), hereditary dyslipidaemia, or cryptogenic cirrhosis. There are no formal guidelines for treatment of these patients, and treatment options are limited. In this Review we appraise the existing literature on Wolman's disease and cholesteryl ester storage disease, and discuss available treatments, including enzyme replacement therapy, oral lipid-lowering therapy, stem-cell transplantation, and liver transplantation. Copyright © 2017 Elsevier Ltd. All rights reserved.

  5. Bacillus sp. PS35 Lipase-Immobilization on Styrene-Divinyl Benzene Resin and Application in Fatty Acid Methyl Ester Synthesis.

    PubMed

    Palanisamy, Kanmani; Kuppamuthu, Kumaresan; Jeyaseelan, Aravind

    2015-09-01

    Lipase is an enzyme with immense application potential. Ester synthesis by lipase catalysis in organic media is an area of key industrial relevance. Enzymatic preparations with traits that cater to the needs of this function are hence being intensely researched. The objectives of the study were to immobilize the lipase from Bacillus sp. PS35 by cross-linking and adsorption onto styrene-divinyl benzene (Sty-Dvb) hydrophobic resin and to comparatively characterize the free and immobilized lipase preparations. The work also aimed to apply the immobilized lipase for catalysing the fatty acid methyl ester (FAME) synthesis from palm oil and optimize the process parameters for maximizing the yield. In this study, the purified lipase from Bacillus sp. PS35 was immobilized by adsorption onto styrene-divinyl benzene hydrophobic resin with gluteraldehyde cross-linking. The immobilized enzyme showed better pH and temperature stabilities than the free lipase. Organic solvent stability was also enhanced, with the relative activity in the presence of methanol being shifted from 53% to 81%, thereby facilitating the enzyme's application in fatty acid methyl ester synthesis. It exhibited remarkable storage stability over a 30-day period and after 20 repetitive uses. Cross-linking also reduced enzyme leakage by 49%. The immobilized lipase was then applied for biodiesel production from palm oil. Methanol and oil molar ratio of 5:1, three step methanol additions, and an incubation temperature of 50°C were established to be the ideal conditions favoring the transesterification reaction, resulting in 97% methyl ester yield. These promising results offer scope for further investigation and process scale up, permitting the enzyme's commercial application in a practically feasible and economically agreeable manner.

  6. In situ localization of the genetic locus encoding the lysosomal acid lipase/cholesteryl esterase (LIPA) deficient in wolman disease to chromosome 10q23. 2-q23. 3

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Anderson, R.A.; Rao, N.; Byrum, R.S.

    1993-01-01

    Human acid lipase/cholesteryl esterase (EC 3.1.1.13) is a 46-kDa glycoprotein required for the lysosomal hydrolysis of cholesteryl esters and triglycerides that cells acquire through the receptor-mediated endocytosis of low-density lipoproteins. This activity is essential in the provision of free cholesterol for cell metabolism as well as for the feedback signal that modulates endogenous cellular cholesterol production. The extremely low level of lysosomal acid lipase in patients afflicted with the hereditary, allelic lysosomal storage disorders Woman disease (WD) and cholesteryl ester storage disease (CESD) (MIM Number 278000 (6)) is associated with the massive intralysosomal lipid storage and derangements in the regulationmore » of cellular cholesterol production (10). Both WD and CESD cells lack a specific acid lipase isoenzyme and it is thought that the different mutations associated with WD and CESD are in the structural gene for this isoenzyme, LIPA. Analysis of the activity of the acid lipase isoenzyme in cell extracts from human-Chinese hamster somatic cell hybrids (4, 11) demonstrated the concordant segregation of the gene locus for lysosomal acid lipase with the glutamate oxaloacetate transaminase-1 (GOT1) enzyme marker for human chromosome 10 which was subsequently localized to 10q24.1 q25.1 (8). 11 refs., 1 figs.« less

  7. Lipase immobilization on epoxy-activated poly(vinyl acetate-acrylamide) microspheres.

    PubMed

    Zhang, Dong-Hao; Peng, Li-Juan; Wang, Yun; Li, Ya-Qiong

    2015-05-01

    Poly(vinyl acetate-acrylamide) microspheres with an average diameter of 2-4μm were successfully prepared and characterized via SEM and FTIR. Then the microspheres were modified with epoxy groups through reacting with epichlorohydrin and used as carriers to covalently immobilize Candida rugosa lipase. The results revealed that agitation played an important role on epoxy activation and the immobilization ratio increased with the increase of the epoxy density. On the other hand, the specific activity of the immobilized lipase as well as the activity recovery declined gradually with the increase in the immobilization ratio from 72% to 93%, which were attributed to the steric hindrance effects caused by enzyme overloading. When epoxy density was 76μmol/g microsphere, the activity recovery reached the maximum at 47.5%, and the activity of the immobilized lipase was 261.3U/g microsphere. Moreover, the thermal stability of the immobilized lipase was much better than that of the free one, which indicated potential applications of the immobilized lipase. Copyright © 2015 Elsevier B.V. All rights reserved.

  8. Lipase-catalyzed synthesis of palmitanilide: Kinetic model and antimicrobial activity study.

    PubMed

    Liu, Kuan-Miao; Liu, Kuan-Ju

    2016-01-01

    Enzymatic syntheses of fatty acid anilides are important owing to their wide range of industrial applications in detergents, shampoo, cosmetics, and surfactant formulations. The amidation reaction of Mucor miehei lipase Lipozyme IM20 was investigated for direct amidation of triacylglycerol in organic solvents. The process parameters (reaction temperature, substrate molar ratio, enzyme amount) were optimized to achieve the highest yield of anilide. The maximum yield of palmitanilide (88.9%) was achieved after 24 h of reaction at 40 °C at an enzyme concentration of 1.4% (70 mg). Kinetics of lipase-catalyzed amidation of aniline with tripalmitin has been investigated. The reaction rate could be described in terms of the Michaelis-Menten equation with a Ping-Pong Bi-Bi mechanism and competitive inhibition by both the substrates. The kinetic constants were estimated by using non-linear regression method using enzyme kinetic modules. The enzyme operational stability study showed that Lipozyme IM20 retained 38.1% of the initial activity for the synthesis of palmitanilide (even after repeated use for 48 h). Palmitanilide, a fatty acid amide, exhibited potent antimicrobial activity toward Bacillus cereus. Copyright © 2015 Elsevier Inc. All rights reserved.

  9. The Effects of One Amino Acid Substitutions at the C-Terminal Region of Thermostable L2 Lipase by Computational and Experimental Approach.

    PubMed

    Sani, Hartini Ahmad; Shariff, Fairolniza Mohd; Rahman, Raja Noor Zaliha Raja Abd; Leow, Thean Chor; Salleh, Abu Bakar

    2018-01-01

    The substitutions of the amino acid at the predetermined critical point at the C-terminal of L2 lipase may increase its thermostability and enzymatic activity, or even otherwise speed up the unfolding of the protein structure. The C-terminal of most proteins is often flexible and disordered. However, some protein functions are directly related to flexibility and play significant role in enzyme reaction. The critical point for mutation of L2 lipase structure was predicted at the position 385 of the L2 sequence, and the best three mutants were determined based on I-Mutant2.0 software. The best three mutants were S385E, S385I and S385V. The effects of the substitution of the amino acids at the critical point were analysed with molecular dynamics simulation by using Yet Another Scientific Artificial Reality Application software. The predicted mutant L2 lipases were found to have lower root mean square deviation value as compared to L2 lipase. It was indicated that all the three mutants had higher compactness in the structure, consequently enhanced the stability. Root mean square fluctuation analysis showed that the flexibility of L2 lipase was reduced by mutations. Purified S385E lipase had an optimum temperature of 80 °C in Tris-HCl pH 8. The highest enzymatic activity of purified S385E lipase was obtained at 80 °C temperature in Tris-HCl pH 8, while for L2 lipase it was at 70 °C in Glycine-NaOH pH 9. The thermal stability of S385V lipase was enhanced as compared to other protein since that the melting point (T m ) value was at 85.96 °C. S385I lipase was more thermostable compared to recombinant L2 lipase and other mutants at temperature 60 °C within 16 h preincubation.

  10. Enzymatic synthesis of eugenol benzoate by immobilized Staphylococcus aureus lipase: optimization using response surface methodology and determination of antioxidant activity.

    PubMed

    Horchani, Habib; Ben Salem, Nadia; Zarai, Zied; Sayari, Adel; Gargouri, Youssef; Chaâbouni, Moncef

    2010-04-01

    The ability of a non-commercial immobilized Staphylococcus aureus lipase to catalyze the esterification of eugenol with benzoic acid was checked and the antioxidant power of the ester formed was evaluated. Response surface methodology based on four variables (the reaction temperature, the amount of lipase, the benzoic acid/eugenol molar ratio and the volume of solvent) was used to optimize the experimental conditions of eugenol benzoate synthesis. The maximum conversion yield (75%) was obtained using 240 IU of immobilized lipase, a benzoic acid/eugenol molar ratio of 1.22 dissolved in 4.6 ml chloroform at 41 degrees Celsius. The antioxidant activities of eugenol and its ester were evaluated. Compared to BHT, used as a model synthetic antioxidant, the eugenol benzoate showed a higher antioxidative activity. The IC(50) value for 1,1-diphenyl-2-picrylhydrazyl was found to be 18.2 microg/ml versus 20.2 microg/ml for eugenol and eugenol benzoate. Copyright 2009 Elsevier Ltd. All rights reserved.

  11. Gallic acid-based alkyl esters synthesis in a water-free system by celite-bound lipase of Bacillus licheniformis SCD11501.

    PubMed

    Sharma, Shivika; Kanwar, Shamsher S; Dogra, Priyanka; Chauhan, Ghanshyam S

    2015-01-01

    Gallic acid (3, 4, 5- trihydroxybenzoic acid) is an important antioxidant, anti-inflammatory, and radical scavenging agent. In the present study, a purified thermo-tolerant extra-cellular lipase of Bacillus licheniformis SCD11501 was successfully immobilized by adsorption on Celite 545 gel matrix followed by treatment with a cross-linking agent, glutaraldehyde. The celite-bound lipase treated with glutaraldehyde showed 94.8% binding/retention of enzyme activity (36 U/g; specific activity 16.8 U/g matrix; relative increase in enzyme activity 64.7%) while untreated matrix resulted in 88.1% binding/retention (28.0 U/g matrix; specific activity 8.5 U/g matrix) of lipase. The celite-bound lipase was successfully used to synthesis methyl gallate (58.2%), ethyl gallate (66.9%), n-propyl gallate (72.1%), and n-butyl gallate (63.8%) at 55(o) C in 10 h under shaking (150 g) in a water-free system by sequentially optimizing various reaction parameters. The low conversion of more polar alcohols such as methanol and ethanol into their respective gallate esters might be due to the ability of these alcohols to severely remove water from the protein hydration shell, leading to enzyme inactivation. Molecular sieves added to the reaction mixture resulted in enhanced yield of the alkyl ester(s). The characterization of synthesised esters was done through fourier transform infrared (FTIR) spectroscopy and (1) H NMR spectrum analysis. © 2015 American Institute of Chemical Engineers.

  12. Biotechnological applications of halophilic lipases and thioesterases.

    PubMed

    Schreck, Steven D; Grunden, Amy M

    2014-02-01

    Lipases and esterases are enzymes which hydrolyze ester bonds between a fatty acid moiety and an esterified conjugate, such as a glycerol or phosphate. These enzymes have a wide spectrum of use in industrial applications where their high activity, broad substrate specificity, and stability under harsh conditions have made them integral in biofuel production, textile processing, waste treatment, and as detergent additives. To date, these industrial applications have mainly leveraged enzymes from mesophilic and thermophilic organisms. However, increasingly, attention has turned to halophilic enzymes as catalysts in environments where high salt stability is desired. This review provides a brief overview of lipases and esterases and examines specific structural motifs and evolutionary adaptations of halophilic lipases. Finally, we examine the state of research involving these enzymes and provide an in-depth look at an exciting algal-based biofuel production system. This system uses a recombinant halophilic lipase to increase oil production efficiency by cleaving algal fatty acids from the acyl carrier protein, which eliminates feedback inhibition of fatty acid synthesis.

  13. Properties of a membrane-bound triglyceride lipase of rapeseed (Brassica napus L.) cotyledons.

    PubMed

    Rosnitschek, I; Theimer, R R

    1980-04-01

    The properties of the alkaline lipase activity (EC 3.1.1.3) that was recovered almost completely from a microsomal membrane fraction of 4-d-old rapeseed (Brassica napus L.) cotyledons were studied employing a titrimetric test procedure. The apparent KM was 6.5 mmol l(-1), with emulgated sunflower oil as the substrate. The products of triglyceride hydrolysis in vitro were glycerol, free fatty acids, and minor amounts of mono- and diglycerides. Maximum lipase activity depended on the preincubation of the lipolytic membrane fraction in 0.15 mol l(-1) NaCl and on the presence of at least 0.1 mol l(-1) NaCl in the test mixture. Desoxycholate and up to 0.1 mol l(-1) CaCl2 also activated the enzyme while EDTA and detergents such as trito x-100, digitonin, tween 85, and sodium dodecylsulfate were inhibitory. The rapeseed lipase displayed a conspicuous substrate selectivity among different plant triglycerides; the activity was inversely correlated with the oleic acid content of the oils. Water-soluble triacetin and the phospholipid lecithin were not hydrolyzed. Increasing amounts of free fatty acids reduced lipase activity; erucic acid, a major component of rapeseed oil, exhibited the strongest effect, suggesting a possible role in the regulation of lipase activity in vivo. The data demonstrate that the lipolytic membrane fraction houses a triglyceride lipase with properties similar to other plant and animal lipases. It can both qualitatively and quantitatively account for the fat degradation in rapeseed cotyledons. The evidence that provides further reason to acknowledge the membranous appendices of the spherosomes as the intracellular site of lipolysis is discussed.

  14. Inhibition of dog and human gastric lipases by enantiomeric phosphonate inhibitors: a structure-activity study.

    PubMed

    Miled, Nabil; Roussel, Alain; Bussetta, Cécile; Berti-Dupuis, Liliane; Rivière, Mireille; Buono, Gérard; Verger, Robert; Cambillau, Christian; Canaan, Stéphane

    2003-10-14

    The crystal structures of gastric lipases in the apo form [Roussel, A., et al. (1999) J. Biol. Chem. 274, 16995-17002] or in complex with the (R(P))-undecyl butyl phosphonate [C(11)Y(4)(+)] [Roussel, A., et al. (2002) J. Biol. Chem. 277, 2266-2274] have improved our understanding of the structure-activity relationships of acid lipases. In this report, we have performed a kinetic study with dog and human gastric lipases (DGL and HGL, respectively) using several phosphonate inhibitors by varying the absolute configuration of the phosphorus atom and the chain length of the alkyl/alkoxy substituents. Using the two previously determined structures and that of a new crystal structure obtained with the other (S(P))-phosphonate enantiomer [C(11)Y(4)(-)], we constructed models of phosphonate inhibitors fitting into the active site crevices of DGL and HGL. All inhibitors with a chain length of fewer than 12 carbon atoms were found to be completely buried in the catalytic crevice, whereas longer alkyl/alkoxy chains were found to point out of the cavity. The main stereospecific determinant explaining the stronger inhibition of the S(P) enantiomers is the presence of a hydrogen bond involving the catalytic histidine as found in the DGL-C(11)Y(4)(-) complex. On the basis of these results, we have built a model of the first tetrahedral intermediate corresponding to the tristearoyl-lipase complex. The triglyceride molecule completely fills the active site crevice of DGL, in contrast with what is observed with other lipases such as pancreatic lipases which have a shallower and narrower active site. For substrate hydrolysis, the supply of water molecules to the active site might be achieved through a lateral channel identified in the protein core.

  15. Bacillus sp. PS35 Lipase-Immobilization on Styrene-Divinyl Benzene Resin and Application in Fatty Acid Methyl Ester Synthesis

    PubMed Central

    Palanisamy, Kanmani; Kuppamuthu, Kumaresan; Jeyaseelan, Aravind

    2015-01-01

    Background Lipase is an enzyme with immense application potential. Ester synthesis by lipase catalysis in organic media is an area of key industrial relevance. Enzymatic preparations with traits that cater to the needs of this function are hence being intensely researched. Objective The objectives of the study were to immobilize the lipase from Bacillus sp. PS35 by cross-linking and adsorption onto styrene-divinyl benzene (Sty-Dvb) hydrophobic resin and to comparatively characterize the free and immobilized lipase preparations. The work also aimed to apply the immobilized lipase for catalysing the fatty acid methyl ester (FAME) synthesis from palm oil and optimize the process parameters for maximizing the yield. Materials and Methods In this study, the purified lipase from Bacillus sp. PS35 was immobilized by adsorption onto styrene-divinyl benzene hydrophobic resin with gluteraldehyde cross-linking. Results The immobilized enzyme showed better pH and temperature stabilities than the free lipase. Organic solvent stability was also enhanced, with the relative activity in the presence of methanol being shifted from 53% to 81%, thereby facilitating the enzyme’s application in fatty acid methyl ester synthesis. It exhibited remarkable storage stability over a 30-day period and after 20 repetitive uses. Cross-linking also reduced enzyme leakage by 49%. The immobilized lipase was then applied for biodiesel production from palm oil. Methanol and oil molar ratio of 5:1, three step methanol additions, and an incubation temperature of 50°C were established to be the ideal conditions favoring the transesterification reaction, resulting in 97% methyl ester yield. Conclusions These promising results offer scope for further investigation and process scale up, permitting the enzyme’s commercial application in a practically feasible and economically agreeable manner. PMID:28959298

  16. Perkinsus marinus, a protozoan parasite of the Eastern oyster (Crassostrea virginica): effects of temperature on the uptake and metabolism of fluorescent lipid analogs and lipase activities.

    PubMed

    Chu, Fu-Lin E; Soudant, P; Lund, E D

    2003-10-01

    The effects of temperature on the uptake and metabolism of fluorescent labeled palmitic acid (FLC16) and phosphatidylcholine (FLPC) and lipase activities in the oyster protozoan parasite, Perkinsus marinus, meront stage were tested at 10, 18, and 28 degrees C. Temperature significantly affected not only the uptake, assimilation, and metabolism of both FLC16 and FLPC in P. marinus, but also its triacylglycerol (TAG) lipase activities. The incorporation of both FLC16 and FLPC increased with temperature and paralleled the increase in the amount of total fatty acids in P. marinus meront cultures. The incorporation of FLC16 was higher than FLPC at all temperatures. The percentage of FLC16 metabolized to TAG was significantly higher at higher temperatures. Trace amounts of incorporated FLC16 were detected in monoacylglycerol (MAG) and PC at 18 and 28 degrees C. P. marinus meronts metabolized FLPC to TAG, diacylglycerol (DAG), monoacylglycerol (MAG), free fatty acids (FFA), phosphatidylethanolamine (PE), and cardiolipin (CL). The conversion of FLPC to TAG and PE was highest at 28 degrees C. The relative proportions of individual fatty acids and total saturated, monounsaturated and polyunsaturated fatty acids changed with temperatures. While total saturated fatty acids (SAFAs) increased with temperature, total monounsaturated fatty acids (MUFAs) decreased with temperature. Total polyunsaturated fatty acids (PUFAs) increased from 28 to 18 degrees C. The findings of increase of total SAFAs and decrease of total MUFAs with the increase of temperatures and upward shift of total PUFAs from 28 to 18 degrees C suggest that, as in other organisms, P. marinus is capable of adapting to changes in environmental temperatures by modifying its lipid metabolism. Generally, higher lipase activities were noted at higher cultivation temperatures. Both TAG lipase and phospholipase activities were detected in P. marinus cells and their extra cellular products (ECP), but phospholipase

  17. Anti- and pro-lipase activity of selected medicinal, herbal and aquatic plants, and structure elucidation of an anti-lipase compound.

    PubMed

    Ado, Muhammad Abubakar; Abas, Faridah; Mohammed, Abdulkarim Sabo; Ghazali, Hasanah M

    2013-11-26

    Plants that help in slowing down the digestion of triacylglycerols (TAGs) in the pancreas and small intestine of humans play an important role in the reduction of obesity. On the other hand, there may be plants or plant parts that stimulate intestinal lipolytic activity, thus contributing to greater TAG assimilation. The aim of this study was to evaluate the aqueous methanolic extracts of ninety eight (98) medicinal, herbal and aquatic plant materials from Malaysia for their effect on porcine pancreatic lipase (PPL) activity and to identify the structure of an anti-lipase compound from one of the sources. The degree of inhibition was also quantified as relative to orlistat activity against PPL (orlistat equivalents). Results revealed that while 19.4% of the extracts were found to have anti-lipase activity ≥80%, 12% were actually found to promote PPL activity. Twenty two percent (22.4%) exhibited moderate inhibition (41%-80%) and 2% were neutral toward PPL activity. The ripe fruit of Averrhoa carambola and the leaves of Archidendron jiringa (Jack) I.C Nielsen L. (jering), Cynometra cauliflora (nam-nam) and Aleurites moluccana (L.) Willd (candle nut/buah keras) had the highest (100%) anti-lipase activity and are equivalent to 0.11 µg orlistat/mL. Plants that stimulated lipase activity included Pimpinella anisum L. (aniseed/jintan manis), activating the enzyme by 186.5%. Kaempferol 3-O-rhamnoside was isolated from the ethyl acetate fraction of C. cauliflora leaves and found to be an active lipase inhibitor. The structure was elucidated using 1H-NMR, 13C-NMR and 2D-NMR analyses.

  18. Synthesis, characterization and application of lipase-conjugated citric acid-coated magnetic nanoparticles for ester synthesis using waste frying oil.

    PubMed

    Patel, Unisha; Chauhan, Kishor; Gupte, Shilpa

    2018-04-01

    In the present work, magnetic nanoparticles (MNPs) were prepared by chemical precipitation of trivalent and divalent iron ions which were functionalized using citric acid. The bacterial isolate Staphylococcus epidermidis KX781317 was isolated from oil-contaminated site. The isolate produced lipase, which was purified and immobilized on magnetic nanoparticles (MNPs) for ester synthesis from waste frying oil (WFO). The characterization of MNPs employed conventional TEM, XRD and FTIR techniques. TEM analysis of MNPs showed the particle size in the range of 20-50 nm. FTIR spectra revealed the binding of citric acid to Fe 3 O 4 and lipase on citric acid-coated MNPs. The citric acid-coated MNPs and lipase-conjugated citric acid-coated MNPs had similar XRD patterns which indicate MNPs could preserve their magnetic properties. The maximum immobilization efficiency 98.21% of lipase-containing citric acid-coated MNPs was observed at ratio 10:1 of Cit-MNPs:lipase. The pH and temperature optima for lipase conjugated with Cit-MNPs were 7 and 35 °C, respectively. Isobutanol was found to be an effective solvent for ester synthesis and 1:2 ratio of oil:alcohol observed significant for ester formation. The ester formation was determined using TLC and the % yield of ester conversion was calculated. The rate of ester formation is directly proportional to the enzyme load. Formed esters were identified as isobutyl laurate ester and isobutyl myristate ester through GC-MS analysis.

  19. Effects of surfactants on lipase structure, activity, and inhibition.

    PubMed

    Delorme, Vincent; Dhouib, Rabeb; Canaan, Stéphane; Fotiadu, Frédéric; Carrière, Frédéric; Cavalier, Jean-François

    2011-08-01

    Lipase inhibitors are the main anti-obesity drugs prescribed these days, but the complexity of their mechanism of action is making it difficult to develop new molecules for this purpose. The efficacy of these drugs is known to depend closely on the physico-chemistry of the lipid-water interfaces involved and on the unconventional behavior of the lipases which are their target enzymes. The lipolysis reaction which occurs at an oil-water interface involves complex equilibria between adsorption-desorption processes, conformational changes and catalytic mechanisms. In this context, surfactants can induce significant changes in the partitioning of the enzyme and the inhibitor between the water phase and lipid-water interfaces. Surfactants can be found at the oil-water interface where they compete with lipases for adsorption, but also in solution in the form of micellar aggregates and monomers that may interact with hydrophobic parts of lipases in solution. These various interactions, combined with the emulsification and dispersion of insoluble substrates and inhibitors, can either promote or decrease the activity and the inhibition of lipases. Here, we review some examples of the various effects of surfactants on lipase structure, activity and inhibition, which show how complex the various equilibria involved in the lipolysis reaction tend to be.

  20. Rhizomucor miehei triglyceride lipase is processed and secreted from transformed Aspergillus oryzae.

    PubMed

    Huge-Jensen, B; Andreasen, F; Christensen, T; Christensen, M; Thim, L; Boel, E

    1989-09-01

    The cDNA encoding the precursor of the Rhizomucor miehei triglyceride lipase was inserted in an Aspergillus oryzae expression vector. In this vector the expression of the lipase cDNA is under control of the Aspergillus oryzae alpha-amylase gene promoter and the Aspergillus niger glucoamylase gene terminator. The recombinant plasmid was introduced into Aspergillus oryzae, and transformed colonies were selected and screened for lipase expression. Lipase-positive transformants were grown in a small fermentor, and recombinant triglyceride lipase was purified from the culture broth. The purified enzymatically active recombinant lipase (rRML) secreted from A. oryzae was shown to have the same characteristics with respect to mobility on reducing SDS-gels and amino acid composition as the native enzyme. N-terminal amino acid sequencing indicated that approximately 70% of the secreted rRML had the same N-terminal sequence as the native Rhizomucor miehei enzyme, whereas 30% of the secreted rRML was one amino acid residue shorter in the N-terminal. The recombinant lipase precursor, which has a 70 amino acid propeptide, is thus processed in and secreted from Aspergillus oryzae. We have hereby demonstrated the utility of this organism as a host for the production of recombinant triglyceride lipases.

  1. Biochemical characterization of a lipase from olive fruit (Olea europaea L.).

    PubMed

    Panzanaro, S; Nutricati, E; Miceli, A; De Bellis, L

    2010-09-01

    Lipase (triacylglycerol acylhydrolase; EC 3.1.1.3) is the first enzyme of the degradation path of stored triacylglycerols (TAGs). In olive fruits, lipase may determine the increase of free fatty acids (FFAs) which level is an important index of virgin olive oil quality. However, despite the importance of virgin olive oil for nutrition and human health, few studies have been realized on lipase activity in Olea europaea fruits. In order to characterize olive lipase, fruits of the cv. Ogliarola, widely diffused in Salento area (Puglia, Italy), were harvested at four stages of ripening according to their skin colour (green, spotted I, spotted II, purple). Lipase activity was detected in the fatty layer obtained after centrifugation of the olive mesocarp homogenate. The enzyme exhibited a maximum activity at pH 5.0. The addition of calcium in the lipase assay medium leads to an increment of activity, whereas in the presence of copper the activity was reduced by 75%. Furthermore, mesocarp lipase activity increases during olive development but declined at maturity (purple stage). The data represent the first contribution to the biochemical characterization of an olive fruit lipase associated to oil bodies. 2010 Elsevier Masson SAS. All rights reserved.

  2. Characterization of Purified Staphylococcal Lipase1

    PubMed Central

    Vadehra, D. V.; Harmon, L. G.

    1967-01-01

    Purified staphylococcal lipase had an optimal pH of 8.3 for activity at 37 C, and an optimal temperature of 45 C at pH 8.0. During storage, the enzyme lost less than 10% of the activity over a period of 21 days at 4 and -23 C. The enzyme retained 93% of the activity when heated for 30 min at 50 C and was 95% destroyed in 30 min at 70 C. The purified lipase was capable of hydrolyzing a variety of natural fats and oils. However, the enzyme was three times more active on nonhydrogenated soybean oil than on hydrogenated soybean oil with an iodine value of <3.0. The enzyme was also capable of hydrolyzing fatty acids on the α, β, and α′ positions of a synthetic mixed triglyceride. In general, the presence of oxidizing agents increased the activity and the presence of reducing agents decreased the activity of the lipase enzyme. PMID:6035042

  3. Antibacterial Activity of Free Fatty Acids from Hydrolyzed Virgin Coconut Oil Using Lipase from Candida rugosa.

    PubMed

    Nguyen, Van Thi Ai; Le, Truong Dang; Phan, Hoa Ngoc; Tran, Lam Bich

    2017-01-01

    Free fatty acids (FFAs) were obtained from hydrolyzed virgin coconut oil (VCO) by Candida rugosa lipase (CRL). Four factors' influence on hydrolysis degree (HD) was examined. The best hydrolysis conditions in order to get the highest HD value were determined at VCO to buffer ratio 1 : 5 (w/w), CRL concentration 1.5% (w/w oil), pH 7, and temperature 40°C. After 16 hours' reaction, the HD value achieved 79.64%. FFAs and residual hydrolyzed virgin coconut oil (HVCO) were isolated from the hydrolysis products. They were tested for their antibacterial activity against Gram-negative and Gram-positive bacteria, which can be found in contaminated food and cause food poisoning. FFAs showed their inhibition against Bacillus subtilis (ATCC 11774), Escherichia coli (ATCC 25922), Salmonella enteritidis (ATCC 13076), and Staphylococcus aureus (ATCC 25923) at minimum inhibitory concentration (MIC) of 50%, 60%, 20%, and 40%, respectively. However, VCO and HVCO did not show their antibacterial activity against these tested bacteria.

  4. Antibacterial Activity of Free Fatty Acids from Hydrolyzed Virgin Coconut Oil Using Lipase from Candida rugosa

    PubMed Central

    Phan, Hoa Ngoc; Tran, Lam Bich

    2017-01-01

    Free fatty acids (FFAs) were obtained from hydrolyzed virgin coconut oil (VCO) by Candida rugosa lipase (CRL). Four factors' influence on hydrolysis degree (HD) was examined. The best hydrolysis conditions in order to get the highest HD value were determined at VCO to buffer ratio 1 : 5 (w/w), CRL concentration 1.5% (w/w oil), pH 7, and temperature 40°C. After 16 hours' reaction, the HD value achieved 79.64%. FFAs and residual hydrolyzed virgin coconut oil (HVCO) were isolated from the hydrolysis products. They were tested for their antibacterial activity against Gram-negative and Gram-positive bacteria, which can be found in contaminated food and cause food poisoning. FFAs showed their inhibition against Bacillus subtilis (ATCC 11774), Escherichia coli (ATCC 25922), Salmonella enteritidis (ATCC 13076), and Staphylococcus aureus (ATCC 25923) at minimum inhibitory concentration (MIC) of 50%, 60%, 20%, and 40%, respectively. However, VCO and HVCO did not show their antibacterial activity against these tested bacteria. PMID:29259829

  5. Covalent immobilization of Candida rugosa lipase on aldehyde functionalized hydrophobic support and the application for synthesis of oleic acid ester.

    PubMed

    Temoçin, Zülfikar

    2013-01-01

    This study focuses on Candida rugosa lipase (CRL) immobilization by covalent attachment on poly(ethylene terephthalate)-grafted glycidyl methacrylate (PET-g-GMA) fiber. The immobilization yielded a protein loading of 2.38 mg g(-1) of PET-g-GMA fiber. The performances of the immobilized and free CRLs were evaluated with regard to hydrolysis of olive oil and esterification of oleic acid. The optimum activity pH of the CRL was changed by immobilization to neutral range. The maximum activity of the free and immobilized CRLs occurred at 40 and 45 °C respectively. The immobilized lipase retained 65% of its original activity at 50 °C for 2 h. It was found that the immobilized lipase stored at 4 °C retained 90% of its original activity after 35 days, whereas the free lipase stored at 4 °C retained 69% of its original activity after the same period. In the esterification experiments, the immobilized CRL could maintain a high activity at a water content range from 1.5 to 6% (v/v), while the activity of free CRL showed a clear dependence on water content and decreased rapidly at above 3% (v/v) water content. In addition, after five reuses, the esterification percent yield of the immobilized CRL slightly decreased from 29 to 27%.

  6. Purification and characterization of an extracellular lipase from a thermophilic Rhizopus oryzae strain isolated from palm fruit.

    PubMed

    Hiol; Jonzo; Rugani; Druet; Sarda; Comeau

    2000-03-01

    We have isolated a lipolytic strain from palm fruit that was identified as a Rhizopus oryzae. Culture conditions were optimized and highest lipase production amounting to 120 U/ml was achieved after 4 days of cultivation. The extracellular lipase was purified 1200-fold by ammonium sulfate precipitation, sulphopropyl-Sepharose chromatography, Sephadex G 75 gel filtration and a second sulphopropyl-Sepharose chromatography. The specific activity of the purified enzyme was 8800 U/mg. The lipolytic enzyme has a molecular mass of 32 kDa by SDS-polyacrylamide gel electrophoresis and gel filtration. The enzyme exhibited a single band in active polyacrylamide gel electrophoresis and its isoelectric point was 7.6. Analysis of Rhizopus oryzae lipase by RP-HPLC confirmed the homogeneity of the enzyme preparation. Determination of the N-terminal sequence over 19 amino acid residues showed a high homology with lipases of the same genus. The optimum pH for enzyme activity was 7.5. Lipase was stable in the pH range from 4.5 to 7.5. The optimum temperature for lipase activity was 35 degrees C and about 65% of its activity was retained after incubation at 45 degrees C for 30 min. The lipolytic enzyme was inhibited by Triton X100, SDS, and metal ions such as Fe(3+), Cu(2+), Hg(2+) and Fe(2+). Lipase activity against triolein was enhanced by sodium cholate or taurocholate. The purified lipase had a preference for the hydrolysis of saturated fatty acid chains (C(8)-C(18)) and a 1, 3-position specificity. It showed a good stability in organic solvents and especially in long chain-fatty alcohol. The enzyme poorly hydrolyzed triacylglycerols containing n-3 polyunsaturated fatty acids, and appeared as a suitable biocatalyst for selective esterification of sardine free fatty acids with hexanol as substrate. About 76% of sardine free fatty acids were esterified after 30 h reaction whereas 90% of docosahexaenoic acid (DHA) was recovered in the unesterified fatty acids.

  7. A novel glucan-binding protein with lipase activity from the oral pathogen Streptococcus mutans.

    PubMed

    Shah, Deepan S H; Russell, Roy R B

    2004-06-01

    Streptococcus mutans produces extracellular glucosyltransferases (GTFs) that synthesize glucans from sucrose. These glucans are important in determining the permeability properties and adhesiveness of dental plaque. GTFs and the GbpA glucan-binding protein are characterized by a binding domain containing a series of 33-amino-acid repeats, called 'A' repeats. The S. mutans genome sequence was searched for ORFs containing 'A' repeats, and one novel gene, gbpD, which appears to be unique to the mutans group of streptococci, was identified. The GbpD sequence revealed the presence of three 'A' repeats, in the middle of the protein, and a novel glucan-binding assay showed that GbpD binds to dextran with a K(D) of 2-3 nM. Construction of truncated derivatives of GbpD confirmed that the 'A' repeat region was essential for binding. Furthermore, a gbpD knockout mutant was modified in the extent of aggregation induced by polymers derived from sucrose. The N-terminus of GbpD has a signal sequence, followed by a region with no homologues in the public databases, while the C-terminus has homology to the alpha/beta hydrolase family (including lipases and carboxylesterases). GbpD contains the two regions typical of these enzymes: a GxSxG active site 'lipase box' and an 'oxyanion hole'. GbpD released free fatty acids (FFAs) from a range of triglycerides in the presence of calcium, indicating a lipase activity. The glucan binding/lipase bifunctionality suggested the natural substrate for the enzyme may be a surface macromolecule consisting of carbohydrate linked to lipid. The gbpD mutant was less hydrophobic than wild-type and pure recombinant GbpD reduced the hydrophobicity of S. mutans and another plaque bacterium, Streptococcus sanguinis. GbpD bound to and released FFA from lipoteichoic acid (LTA) of S. sanguinis, but had no effect on LTA from S. mutans. These results raise the intriguing possibility that GbpD may be involved in direct interspecies competition within the plaque

  8. Role of Lipase from Community-Associated Methicillin-Resistant Staphylococcus aureus Strain USA300 in Hydrolyzing Triglycerides into Growth-Inhibitory Free Fatty Acids

    PubMed Central

    Cadieux, Brigitte; Vijayakumaran, Vithooshan; Bernards, Mark A.; McGavin, Martin J.

    2014-01-01

    Part of the human host innate immune response involves the secretion of bactericidal lipids on the skin and delivery of triglycerides into abscesses to control invading pathogens. Two Staphylococcus aureus lipases, named SAL1 and SAL2, were identified in the community-associated methicillin-resistant S. aureus strain USA300, which, presumably, are produced and function to degrade triglycerides to release free fatty acids. We show that the SAL2 lipase is one of the most abundant proteins secreted by USA300 and is proteolytically processed from the 72-kDa proSAL2 to the 44-kDa mature SAL2 by the metalloprotease aureolysin. We show that spent culture supernatants had lipase activity on both short- and long-chain fatty acid substrates and that deletion of gehB, encoding SAL2, resulted in the complete loss of these activities. With the use of gas chromatography-mass spectrometry, we show that SAL2 hydrolyzed trilinolein to linoleic acid, a fatty acid with known antistaphylococcal properties. When added to cultures of USA300, trilinolein and, to a lesser extent, triolein inhibited growth in a SAL2-dependent manner. This effect was shown to be due to the enzymatic activity of SAL2 on these triglycerides, since the catalytically inactive SAL2 Ser412Ala mutant was incapable of hydrolyzing the triglycerides or yielding delayed growth in their presence. Overall, these results reveal that SAL2 hydrolyzes triglycerides of both short- and long-chain fatty acids and that the released free fatty acids have the potential to cause significant delays in growth, depending on the chemical nature of the free fatty acid. PMID:25225262

  9. Utilization of agroindustrial residues for lipase production by solid-state fermentation

    PubMed Central

    Damaso, Mônica Caramez Triches; Passianoto, Moisés Augusto; de Freitas, Sidinéa Cordeiro; Freire, Denise Maria Guimarães; Lago, Regina Celi Araujo; Couri, Sonia

    2008-01-01

    The aim of this work was to produce lipases by solid-state fermentation (SSF) using, as substrate, agroindustrial residue supplemented with by-products from corn oil refining process or olive oil. For a group of ten fungi strains selected in the first steps, the lipase activity obtained by SSF varied from 7.7 to 58.6 U/g of dry substrate (gds). Among the evaluated strains, the Aspergillus niger mutant 11T53A14 was selected by presenting the best enzymatic production. For the fermentation tests, two substrates were also investigated: wheat bran and corn cob, both supplemented with olive oil. The best results were obtained with wheat bran. Additionally, three industrial by-products from corn oil refining (soapstock, stearin and fatty acids) were evaluated as substitutes to the olive oil in the function of lipases production inducer. Among them, soapstock and stearin were the best inducers, whereas fatty acids presented an inhibitor effect. The highest lipase activities using soapstock, stearin and fatty acids were 62.7 U/gds, 37.7 U/gds and 4.1 U/gds, respectively. PMID:24031288

  10. Synthesis of α-linolenic acid-rich triacylglycerol using a newly prepared immobilized lipase.

    PubMed

    Kim, Heejin; Choi, Nakyung; Oh, Se-Wook; Kim, Yangha; Hee Kim, Byung; Kim, In-Hwan

    2017-12-15

    An α-linolenic acid (ALA)-rich triacylglycerol (TAG) was synthesized from an ALA-rich fatty acid (FA) from perilla oil and glycerol, using a newly prepared immobilized lipase under vacuum. The ALA-rich FA (purity >90wt%) used as the substrate was prepared by urea complexation from perilla oil FAs. Liquid Lipozyme TL 100L lipase from Thermomyces lanuginosus was used for immobilization. Nine different hydrophilic and hydrophobic carriers for immobilization were tested, and Duolite A568, which is a hydrophilic resin, was selected as the best carrier. This immobilized lipase was used to synthesize TAG by direct esterification under vacuum. The parameters investigated were temperature, enzyme loading, and vacuum level. The optimum reaction conditions were a temperature of 60°C, an enzyme loading of 15% (based on the total weight of the substrate), and a vacuum of 0.7kPa, respectively. The maximum conversion to TAG of ca. 88wt% was obtained in 12h under the optimum conditions. Copyright © 2017 Elsevier Ltd. All rights reserved.

  11. Dependence of PERT endpoint on endogenous lipase activity.

    PubMed

    Gao, Wen-Yi; Mulberg, Andrew E

    2014-11-01

    To clarify and to understand the potential for misinterpretation of change in fecal fat quantitation during pancreatic enzyme replacement therapy (PERT) trials for treatment of exocrine pancreatic insufficiency. Analysis of clinical trials submitted to the U.S. Food and Drug Administration (FDA) for approval of PERT that enrolled 123 cystic fibrosis adult and pediatric patients treated with Creon, Pertzye, Ultresa, and Zenpep. The CFA% defines lipase activity as a percentage of converting substrate of "Total Daily Dietary Fat Intake." PERT trials performed to date have modified the definition to converting the "Shared Daily Fat Intake," generating "Partial CFA" for the exogenous lipase: the higher the activity of coexisting endogenous lipase, the lower the "Partial CFA" of exogenous measured. This review shows that "Partial CFA" is not CFA. Enrollment of patients with low HPLA during treatment may improve the interpretability of "Partial CFA" measured by PERT trials.

  12. Encapsulation of lipase within metal-organic framework (MOF) with enhanced activity intensified under ultrasound.

    PubMed

    Nadar, Shamraja S; Rathod, Virendra K

    2018-01-01

    The enzyme under lower-intensity ultrasonic irradiation leads to favorable conformational changes, thereby enhancing its activity. In this study, lipase activity was augmented upto 1.6-folds after ultrasonic treatment at 22kHz and 11.38Wcm -2 for 25min. This highly activated lipase was encapsulated within zeolite imidazolate framework-8 (ZIF-8) as a metal-organic framework (MOF) material via facile one-step biomineralization method by simply mixing aqueous solution of 2-methylimidazole (13.3mmol) and zinc acetate (1.33mmol) along with sonicated lipase within 10min at room temperature (28±2°C). The prepared lipase-MOF was characterized by using FT-IR, FT-Raman, XRD, BET, confocal scanning laser microscopy, TGA and SEM. Further, the thermal stability of lipase embedded MOF was evaluated in the range of 55-75°C on the basis of half-life which showed 3.2 folds increment as against free lipase. In Michaelis-Menten kinetics studies, sonicated lipase entrapped MOF showed nearly same K m and V max values as that of sonicated free lipase. Moreover, the immobilized lipase exhibited up to 54% of residual activity after seven successive cycles of reuse, whereas it retained 90% of residual activity till twenty-five days of storage. Finally, the conformational changes occurred in lipase after sonication treatment and encapsulation within MOF were analyzed by using FT-IR data analysis tools and fluorescent spectroscopy. Copyright © 2017 Elsevier Inc. All rights reserved.

  13. Harnessing biodiesel-producing microbes: from genetic engineering of lipase to metabolic engineering of fatty acid biosynthetic pathway.

    PubMed

    Yan, Jinyong; Yan, Yunjun; Madzak, Catherine; Han, Bingnan

    2017-02-01

    Microbial production routes, notably whole-cell lipase-mediated biotransformation and fatty-acids-derived biosynthesis, offer new opportunities for synthesizing biodiesel. They compare favorably to immobilized lipase and chemically catalyzed processes. Genetically modified whole-cell lipase-mediated in vitro route, together with in vivo and ex vivo microbial biosynthesis routes, constitutes emerging and rapidly developing research areas for effective production of biodiesel. This review presents recent advances in customizing microorganisms for producing biodiesel, via genetic engineering of lipases and metabolic engineering (including system regulation) of fatty-acids-derived pathways. Microbial hosts used include Escherichia coli, Saccharomyces cerevisiae, Pichia pastoris and Aspergillus oryzae. These microbial cells can be genetically modified to produce lipases under different forms: intracellularly expressed, secreted or surface-displayed. They can be metabolically redesigned and systematically regulated to obtain balanced biodiesel-producing cells, as highlighted in this study. Such genetically or metabolically modified microbial cells can support not only in vitro biotransformation of various common oil feedstocks to biodiesel, but also de novo biosynthesis of biodiesel from glucose, glycerol or even cellulosic biomass. We believe that the genetically tractable oleaginous yeast Yarrowia lipolytica could be developed to an effective biodiesel-producing microbial cell factory. For this purpose, we propose several engineered pathways, based on lipase and wax ester synthase, in this promising oleaginous host.

  14. Studies on activity, distribution, and zymogram of protease, α-amylase, and lipase in the paddlefish Polyodon spathula.

    PubMed

    Ji, H; Sun, H T; Xiong, D M

    2012-06-01

    A series of biochemical determination and electrophoretic observations have been conducted to analyze the activities and characteristics of protease, α-amylase, and lipase of paddlefish Polyodon spathula. The results obtained have been compared with those of bighead carp (Aristichthys nobilis) and hybrid sturgeon (Huso dauricus ♀ × Acipenser schrenki Brandt ♂), in order to increase available knowledge of the physiological characteristics of this sturgeon species and to gain information with regard to its nutrition. Further, a comparative study of enzymatic activity, distribution, and characterization between commercial feed-reared paddlefish (CG) and natural live food-reared (NG) paddlefish was conducted. Results showed that higher proteolytic activity was observed in the pH range 2.5-3.0 and at a pH of 7.0 for paddlefish. Levels of acid protease activity of paddlefish were similar to that of hybrid sturgeon, and significantly higher than that of bighead carp. The inhibition assay of paddlefish showed that the rate of inhibition of tosyl-phenylalanine chloromethyl ketone was approximately 2.6-fold that of tosyl-lysine chloromethyl ketone. There was no significant difference observed for acid protease activity between PG and CG groups, whereas the activity of alkaline protease, α-amylase, and lipase in the PG group were significantly lower than those in the CG group. The substrate sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis further showed that there were certain types of enzymes, especially α-amylase, with similar molecular mass in the paddlefish and hybrid sturgeon. It can be inferred that acid digestion was main mechanism for protein hydrolysis in paddlefish, as reported for other fishes with a stomach. This indicates that the paddlefish requires higher alkaline protease, α-amylase, and lipase activity to digest natural live food.

  15. Biochemical characterization of Yarrowia lipolytica LIP8, a secreted lipase with a cleavable C-terminal region.

    PubMed

    Kamoun, Jannet; Schué, Mathieu; Messaoud, Wala; Baignol, Justine; Point, Vanessa; Mateos-Diaz, Eduardo; Mansuelle, Pascal; Gargouri, Youssef; Parsiegla, Goetz; Cavalier, Jean-François; Carrière, Frédéric; Aloulou, Ahmed

    2015-02-01

    Yarrowia lipolytica is a lipolytic yeast possessing 16 paralog genes coding for lipases. Little information on these lipases has been obtained and only the major secreted lipase, namely YLLIP2, had been biochemically and structurally characterized. Another secreted lipase, YLLIP8, was isolated from Y. lipolytica culture medium and compared with the recombinant enzyme produced in Pichia pastoris. N-terminal sequencing showed that YLLIP8 is produced in its active form after the cleavage of a signal peptide. Mass spectrometry analysis revealed that YLLIP8 recovered from culture medium lacks a C-terminal part of 33 amino acids which are present in the coding sequence. A 3D model of YLLIP8 built from the X-ray structure of the homologous YLLIP2 lipase shows that these truncated amino acids in YLLIP8 belong to an additional C-terminal region predicted to be mainly helical. Western blot analysis shows that YLLIP8 C-tail is rapidly cleaved upon enzyme secretion since both cell-bound and culture supernatant lipases lack this extension. Mature recombinant YLLIP8 displays a true lipase activity on short-, medium- and long-chain triacylglycerols (TAG), with an optimum activity at alkaline pH on medium chain TAG. It has no apparent regioselectivity in TAG hydrolysis, thus generating glycerol and FFAs as final lipolysis products. YLLIP8 properties are distinct from those of the 1,3-regioselective YLLIP2, acting optimally at acidic pH. These lipases are tailored for complementary roles in fatty acid uptake by Y. lipolytica. Copyright © 2014 Elsevier B.V. All rights reserved.

  16. Role of lipase from community-associated methicillin-resistant Staphylococcus aureus strain USA300 in hydrolyzing triglycerides into growth-inhibitory free fatty acids.

    PubMed

    Cadieux, Brigitte; Vijayakumaran, Vithooshan; Bernards, Mark A; McGavin, Martin J; Heinrichs, David E

    2014-12-01

    Part of the human host innate immune response involves the secretion of bactericidal lipids on the skin and delivery of triglycerides into abscesses to control invading pathogens. Two Staphylococcus aureus lipases, named SAL1 and SAL2, were identified in the community-associated methicillin-resistant S. aureus strain USA300, which, presumably, are produced and function to degrade triglycerides to release free fatty acids. We show that the SAL2 lipase is one of the most abundant proteins secreted by USA300 and is proteolytically processed from the 72-kDa proSAL2 to the 44-kDa mature SAL2 by the metalloprotease aureolysin. We show that spent culture supernatants had lipase activity on both short- and long-chain fatty acid substrates and that deletion of gehB, encoding SAL2, resulted in the complete loss of these activities. With the use of gas chromatography-mass spectrometry, we show that SAL2 hydrolyzed trilinolein to linoleic acid, a fatty acid with known antistaphylococcal properties. When added to cultures of USA300, trilinolein and, to a lesser extent, triolein inhibited growth in a SAL2-dependent manner. This effect was shown to be due to the enzymatic activity of SAL2 on these triglycerides, since the catalytically inactive SAL2 Ser412Ala mutant was incapable of hydrolyzing the triglycerides or yielding delayed growth in their presence. Overall, these results reveal that SAL2 hydrolyzes triglycerides of both short- and long-chain fatty acids and that the released free fatty acids have the potential to cause significant delays in growth, depending on the chemical nature of the free fatty acid. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  17. Lipase Activity in Insect Oral Secretions Mediates Defense Responses in Arabidopsis1[C][W][OA

    PubMed Central

    Schäfer, Martin; Fischer, Christine; Meldau, Stefan; Seebald, Eileen; Oelmüller, Ralf; Baldwin, Ian T.

    2011-01-01

    How plants perceive herbivory is not yet well understood. We investigated early responses of the model plant Arabidopsis (Arabidopsis thaliana) to attack from the generalist grasshopper herbivore, Schistocerca gregaria (Caelifera). When compared with wounding alone, S. gregaria attack and the application of grasshopper oral secretions (GS) to puncture wounds elicited a rapid accumulation of various oxylipins, including 13-hydroperoxy octadecatrienoic acid, 12-oxo-phytodienoic acid (OPDA), jasmonic acid, and jasmonic acid-isoleucine. Additionally, GS increased cytosolic calcium levels, mitogen-activated protein kinase (MPK3 and MPK6) activity, and ethylene emission but not the accumulation of hydrogen peroxide. Although GS contain caeliferin A16:0, a putative elicitor of caeliferan herbivores, treatment with pure, synthetic caeliferin A16:0 did not induce any of the observed responses. With mutant plants, we demonstrate that the observed changes in oxylipin levels are independent of MPK3 and MPK6 activity but that MPK6 is important for the GS-induced ethylene release. Biochemical and pharmacological analyses revealed that the lipase activity of GS plays a central role in the GS-induced accumulation of oxylipins, especially OPDA, which could be fully mimicked by treating puncture wounds only with a lipase from Rhizopus arrhizus. GS elicitation increased the levels of OPDA-responsive transcripts. Because the oral secretions of most insects used to study herbivory-induced responses in Arabidopsis rapidly elicit similar accumulations of OPDA, we suggest that lipids containing OPDA (arabidopsides) play an important role in the activation of herbivory-induced responses. PMID:21546453

  18. Interfacial Activation of Candida antarctica Lipase B: Combined Evidence from Experiment and Simulation.

    PubMed

    Zisis, Themistoklis; Freddolino, Peter L; Turunen, Petri; van Teeseling, Muriel C F; Rowan, Alan E; Blank, Kerstin G

    2015-09-29

    Lipase immobilization is frequently used for altering the catalytic properties of these industrially used enzymes. Many lipases bind strongly to hydrophobic surfaces where they undergo interfacial activation. Candida antarctica lipase B (CalB), one of the most commonly used biocatalysts, is frequently discussed as an atypical lipase lacking interfacial activation. Here we show that CalB displays an enhanced catalytic rate for large, bulky substrates when adsorbed to a hydrophobic interface composed of densely packed alkyl chains. We attribute this increased activity of more than 7-fold to a conformational change that yields a more open active site. This hypothesis is supported by molecular dynamics simulations that show a high mobility for a small "lid" (helix α5) close to the active site. Molecular docking calculations confirm that a highly open conformation of this helix is required for binding large, bulky substrates and that this conformation is favored in a hydrophobic environment. Taken together, our combined approach provides clear evidence for the interfacial activation of CalB on highly hydrophobic surfaces. In contrast to other lipases, however, the conformational change only affects large, bulky substrates, leading to the conclusion that CalB acts like an esterase for small substrates and as a lipase for substrates with large alcohol substituents.

  19. Characterization of an extracellular lipase by Pseudomonas koreensis BK-L07 isolated from soil.

    PubMed

    Anbu, Periasamy

    2014-01-01

    Screening using spirit blue agar revealed that strain BK-L07 had the highest lipase activity. Furthermore, the isolated strain was identified as Pseudomonas sp. based on morphological, physiological, biochemical, and molecular analyses. The 16S rRNA gene sequence of strain BK-L07 shared a high similarity with that of Pseudomonas koreensis (99%). The nutritional conditions and physicochemical properties were influenced by P. koreensis BK-L07. The maximum lipase production was obtained in tryptic soy broth medium at pH 8.0 and a temperature of 25°C after 36 hr of incubation. In addition, the lipase activity was determined using different carbon sources and lipase inducers. The lipase production was greatest when 1% maltose was used as the carbon source and olive oil was used as the lipase inducer. The lipase production was significantly increased approximately threefold in the optimized medium when compared with the original medium. Further, the lipase was purified by ammonium sulfate precipitation and gel filtration chromatography with a purification yield of 10.8%. The molecular mass of lipase was 45 kDa. The optimum temperature and pH were 40°C and 8.0, respectively. The enzyme was stable up to 50°C and at pH from 7 to 9. In addition, the enzyme activity was stimulated by MgSO4 and completely inhibited by ethylenediamine tetraacetic acid (EDTA), indicating the metalloenzyme type. The lipase activity was toward medium to long chain length of fatty acids (C10 to C18). Supplemental materials are available for this article. Go to the publisher's online edition of Preparative Biochemistry and Biotechnology to view the supplemental file.

  20. Bioremediation of cooking oil waste using lipases from wastes

    PubMed Central

    do Prado, Débora Zanoni; Facanali, Roselaine; Marques, Márcia Mayo Ortiz; Nascimento, Augusto Santana; Fernandes, Célio Junior da Costa; Zambuzzi, William Fernando

    2017-01-01

    Cooking oil waste leads to well-known environmental impacts and its bioremediation by lipase-based enzymatic activity can minimize the high cytotoxic potential. In addition, they are among the biocatalysts most commercialized worldwide due to the versatility of reactions and substrates. However, although lipases are able to process cooking oil wastes, the products generated from this process do not necessarily become less toxic. Thus, the aim of the current study is to analyze the bioremediation of lipase-catalyzed cooking oil wastes, as well as their effect on the cytotoxicity of both the oil and its waste before and after enzymatic treatment. Thus, assessed the post-frying modification in soybean oil and in its waste, which was caused by hydrolysis reaction catalyzed by commercial and home-made lipases. The presence of lipases in the extracts obtained from orange wastes was identified by zymography. The profile of the fatty acid esters formed after these reactions was detected and quantified through gas chromatography and fatty acids profile compared through multivariate statistical analyses. Finally, the soybean oil and its waste, with and without enzymatic treatment, were assessed for toxicity in cytotoxicity assays conducted in vitro using fibroblast cell culture. The soybean oil wastes treated with core and frit lipases through transesterification reaction were less toxic than the untreated oils, thus confirming that cooking oil wastes can be bioremediated using orange lipases. PMID:29073166

  1. Bioremediation of cooking oil waste using lipases from wastes.

    PubMed

    Okino-Delgado, Clarissa Hamaio; Prado, Débora Zanoni do; Facanali, Roselaine; Marques, Márcia Mayo Ortiz; Nascimento, Augusto Santana; Fernandes, Célio Junior da Costa; Zambuzzi, William Fernando; Fleuri, Luciana Francisco

    2017-01-01

    Cooking oil waste leads to well-known environmental impacts and its bioremediation by lipase-based enzymatic activity can minimize the high cytotoxic potential. In addition, they are among the biocatalysts most commercialized worldwide due to the versatility of reactions and substrates. However, although lipases are able to process cooking oil wastes, the products generated from this process do not necessarily become less toxic. Thus, the aim of the current study is to analyze the bioremediation of lipase-catalyzed cooking oil wastes, as well as their effect on the cytotoxicity of both the oil and its waste before and after enzymatic treatment. Thus, assessed the post-frying modification in soybean oil and in its waste, which was caused by hydrolysis reaction catalyzed by commercial and home-made lipases. The presence of lipases in the extracts obtained from orange wastes was identified by zymography. The profile of the fatty acid esters formed after these reactions was detected and quantified through gas chromatography and fatty acids profile compared through multivariate statistical analyses. Finally, the soybean oil and its waste, with and without enzymatic treatment, were assessed for toxicity in cytotoxicity assays conducted in vitro using fibroblast cell culture. The soybean oil wastes treated with core and frit lipases through transesterification reaction were less toxic than the untreated oils, thus confirming that cooking oil wastes can be bioremediated using orange lipases.

  2. Effect of membranes with various hydrophobic/hydrophilic properties on lipase immobilized activity and stability.

    PubMed

    Chen, Guan-Jie; Kuo, Chia-Hung; Chen, Chih-I; Yu, Chung-Cheng; Shieh, Chwen-Jen; Liu, Yung-Chuan

    2012-02-01

    In this study, three membranes: regenerated cellulose (RC), glass fiber (GF) and polyvinylidene fluoride (PVDF), were grafted with 1,4-diaminobutane (DA) and activated with glutaraldehyde (GA) for lipase covalent immobilization. The efficiencies of lipases immobilized on these membranes with different hydrophobic/hydrophilic properties were compared. The lipase immobilized on hydrophobic PVDF-DA-GA membrane exhibited more than an 11-fold increase in activity compared to its immobilization on a hydrophilic RC-DA-GA membrane. The relationship between surface hydrophobicity and immobilized efficiencies was investigated using hydrophobic/hydrophilic GF membranes which were prepared by grafting a different ratio of n-butylamine/1,4-diaminobutane (BA/DA). The immobilized lipase activity on the GF membrane increased with the increased BA/DA ratio. This means that lipase activity was exhibited more on the hydrophobic surface. Moreover, the modified PVDF-DA membrane was grafted with GA, epichlorohydrin (EPI) and cyanuric chloride (CC), respectively. The lipase immobilized on the PVDF-DA-EPI membrane displayed the highest specific activity compared to other membranes. This immobilized lipase exhibited more significant stability on pH, thermal, reuse, and storage than did the free enzyme. The results exhibited that the EPI modified PVDF is a promising support for lipase immobilization. Copyright © 2011 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  3. Endothelial lipase is a major determinant of HDL level

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Ishida, Tatsuro; Choi, Sungshin; Kundu, Ramendra K.

    2003-01-30

    lipoprotein lipase (44 percent identity) and hepatic lipase (41 percent identity), two well-characterized lipases that function at vascular endothelial surfaces. Critical motifs associated with lipase activity (GXSXG and the catalytic triad S169, D193, H274), and with heparin binding were strongly conserved. Interestingly, in contrast to both lipoprotein lipase and hepatic lipase, endothelial lipase has little triglyceride hydrolase activity in vitro but instead cleaves fatty acids from the sn-1 position of phosphatidylcho-line. In in vitro assays the enzyme is most active on lipids presented in HDL, although it will release fatty acids from all classes of lipoproteins. Consistent with this finding, adenovirus-mediated overexpression of endothelial lipase in LDL receptor-deficient mice reduced plasma concentrations of VLDL and LDL cholesterol by about 50 percent, whereas HDL-C decreased to almost zero in these animals. These data suggested that endothelial lipase may play a role in HDL catabolism.« less

  4. Evaluation of cellulose-binding domain fused to a lipase for the lipase immobilization.

    PubMed

    Hwang, Sangpill; Ahn, Jungoh; Lee, Sumin; Lee, Tai Gyu; Haam, Seungjoo; Lee, Kangtaek; Ahn, Ik-Sung; Jung, Joon-Ki

    2004-04-01

    A cellulose-binding domain (CBD) fragment of a cellulase gene of Trichoderma hazianum was fused to a lipase gene of Bacillus stearothermophilus L1 to make a gene cluster for CBD-BSL lipase. The specific activity of CBD-BSL lipase for oil hydrolysis increased by 33% after being immobilized on Avicel (microcrystalline cellulose), whereas those of CBD-BSL lipase and BSL lipase decreased by 16% and 54%, respectively, after being immobilized on silica gel. Although the loss of activity of an enzyme immobilized by adsorption has been reported previously, the loss of activity of the CBD-BSL lipase immobilized on Avicel was less than 3% after 12 h due to the irreversible binding of CBD to Avicel.

  5. Biodiesel production by transesterification using immobilized lipase.

    PubMed

    Narwal, Sunil Kumar; Gupta, Reena

    2013-04-01

    Biodiesel can be produced by transesterification of vegetable or waste oil catalysed by lipases. Biodiesel is an alternative energy source to conventional fuel. It combines environmental friendliness with biodegradability, low toxicity and renewability. Biodiesel transesterification reactions can be broadly classified into two categories: chemical and enzymatic. The production of biodiesel using the enzymatic route eliminates the reactions catalysed under acid or alkali conditions by yielding product of very high purity. The modification of lipases can improve their stability, activity and tolerance to alcohol. The cost of lipases and the relatively slower reaction rate remain the major obstacles for enzymatic production of biodiesel. However, this problem can be solved by immobilizing the enzyme on a suitable matrix or support, which increases the chances of re-usability. The main factors affecting biodiesel production are composition of fatty acids, catalyst, solvents, molar ratio of alcohol and oil, temperature, water content, type of alcohol and reactor configuration. Optimization of these parameters is necessary to reduce the cost of biodiesel production.

  6. QCM-D and ToF-SIMS Investigation to Deconvolute the Relationship between Lipid Adsorption and Orientation on Lipase Activity.

    PubMed

    Joyce, Paul; Kempson, Ivan; Prestidge, Clive A

    2015-09-22

    Quartz crystal microbalance with dissipation (QCM-D) and time-of-flight secondary ion mass spectrometry (ToF-SIMS) were used to provide insights into the relationship between lipid adsorption kinetics and molecular behavior in porous silica particles of varying hydrophobicities on lipase activity. Lipase (an interfacial enzyme that cleaves ester bonds to break down lipids to fatty acids and monoglycerides) activity was controlled by loading triglycerides at different surface coverages in hydrophilic and hydrophobic porous silica particles. The rate of lipid adsorption increased 2-fold for the hydrophobic surface compared to the hydrophilic surface. However, for submonolayer lipid coverage, the hydrophilic surface enhanced lipase activity 4-fold, whereas the hydrophobic surface inhibited lipase activity 16-fold, compared to lipid droplets in water. A difference in lipid orientation for low surface coverage, evidenced by ToF-SIMS, indicated that lipid adsorbs to hydrophilic silica in a conformation promoting hydrolysis. Multilayer coverage on hydrophobic and hydrophilic surfaces was indistinguishable with ToF-SIMS analysis. Increased lipid adsorption for both substrates facilitated digestion kinetics comparable to a conventional emulsion. Improved understanding of the interfacial adsorption and orientation of lipid and its digestibility in porous silica has implications in improving the uptake of pharmaceuticals and nutrients from lipid-based delivery systems.

  7. In vitro Inhibition of Pancreatic Lipase by Polyphenols:
A Kinetic, Fluorescence Spectroscopy and Molecular Docking Study

    PubMed Central

    2017-01-01

    Summary The inhibitory activity and binding characteristics of caffeic acid, p-coumaric acid, quercetin and capsaicin, four phenolic compounds found in hot pepper, against porcine pancreatic lipase activity were studied and compared to hot pepper extract. Quercetin was the strongest inhibitor (IC50=(6.1±2.4) µM), followed by p-coumaric acid ((170.2±20.6) µM) and caffeic acid ((401.5±32.1) µM), while capsaicin and a hot pepper extract had very low inhibitory activity. All polyphenolic compounds showed a mixed-type inhibition. Fluorescence spectroscopy studies showed that polyphenolic compounds had the ability to quench the intrinsic fluorescence of pancreatic lipase by a static mechanism. The sequence of Stern-Volmer constant was quercetin, followed by caffeic and p-coumaric acids. Molecular docking studies showed that caffeic acid, quercetin and p-coumaric acid bound near the active site, while capsaicin bound far away from the active site. Hydrogen bonds and π-stacking hydrophobic interactions are the main pancreatic lipase-polyphenolic compound interactions observed. PMID:29540986

  8. Action of Lipases of Staphylococcus aureus on Milk Fat1

    PubMed Central

    Vadehra, D. V.; Harmon, L. G.

    1965-01-01

    The activity of the lipase(s) of two strains of coagulase-positive Staphylococcus aureus was determined in milk fat incubated at 15, 22, and 30 C for 8 days. Total fat hydrolysis was measured by acid degree values (ADV). Neutral lipids were separated into component groups on a Florisil column. Free fatty acids were determined by temperature-programmed gas-liquid chromatography. The ADV were 25 to 50% greater at 22 than at 15 C and 4 to 7 times greater at 30 than at 22 C. The lipases liberated as much as 0.48 g of fatty acids per gram of fat during 8 days at 30 C. The enzyme showed a predilection for the palmitic acid-glycerol bond. Addition of fatty acids C14 to C18 inclusive to inoculated sterile skim milk caused inhibition of S. aureus as follows: (i) complete at 0.05 and 0.10% concentration of C10 and (ii) partial at 0.05 and complete at 0.10% concentration of C8. The samples showing inhibition were negative for peptonization, coagulase, and change in pH. Addition of oleic and stearic acid to sterile skim milk inoculated with S. aureus resulted in an increase in nonprotein nitrogen, and the C4 to C12 acids caused a decrease in protease activity. PMID:14325270

  9. Lysosomal acid lipase deficiency--an under-recognized cause of dyslipidaemia and liver dysfunction.

    PubMed

    Reiner, Željko; Guardamagna, Ornella; Nair, Devaki; Soran, Handrean; Hovingh, Kees; Bertolini, Stefano; Jones, Simon; Ćorić, Marijana; Calandra, Sebastiano; Hamilton, John; Eagleton, Terence; Ros, Emilio

    2014-07-01

    Lysosomal acid lipase deficiency (LAL-D) is a rare autosomal recessive lysosomal storage disease caused by deleterious mutations in the LIPA gene. The age at onset and rate of progression vary greatly and this may relate to the nature of the underlying mutations. Patients presenting in infancy have the most rapidly progressive disease, developing signs and symptoms in the first weeks of life and rarely surviving beyond 6 months of age. Children and adults typically present with some combination of dyslipidaemia, hepatomegaly, elevated transaminases, and microvesicular hepatosteatosis on biopsy. Liver damage with progression to fibrosis, cirrhosis and liver failure occurs in a large proportion of patients. Elevated low-density lipoprotein cholesterol levels and decreased high-density lipoprotein cholesterol levels are common features, and cardiovascular disease may manifest as early as childhood. Given that these clinical manifestations are shared with other cardiovascular, liver and metabolic diseases, it is not surprising that LAL-D is under-recognized in clinical practice. This article provides practical guidance to lipidologists, endocrinologists, cardiologists and hepatologists on how to recognize individuals with this life-limiting disease. A diagnostic algorithm is proposed with a view to achieving definitive diagnosis using a recently developed blood test for lysosomal acid lipase. Finally, current management options are reviewed in light of the ongoing development of enzyme replacement therapy with sebelipase alfa (Synageva BioPharma Corp., Lexington, MA, USA), a recombinant human lysosomal acid lipase enzyme. Copyright © 2014 The Authors. Published by Elsevier Ireland Ltd.. All rights reserved.

  10. UHPLC/HR-ESI-MS/MS Profiling of Phenolics from Tunisian Lycium arabicum Boiss. Antioxidant and Anti-lipase Activities' Evaluation.

    PubMed

    Affes, Marwa; Fakhfakh, Jawhar; Daoud, Ines; Brieudes, Vincent; Halabalaki, Maria; El Feki, Abdelfattah; Allouche, Noureddine

    2017-09-01

    This study was performed in the aim to evaluate nine different extracts from Tunisian Lycium arabicum for their total phenolic and total flavonoid contents, phytochemical analyses as well as their antioxidant and anti-lipase activities. The in vitro antioxidant property was investigated using three complementary methods (DPPH, ferric reducing antioxidant power (FRAP), and β-carotene-linoleic acid bleaching assays) while anti-lipase activity was evaluated using 4-methylumbelliferyl oleate method. From all of the tested extracts the most potent found to be the polar MeOH extracts especially those of stems and leaves. In order to investigate the chemical composition of these extracts and possible correlation of their constituents with the observed activities, an UHPLC/HR-ESI-MS/MS analysis was performed. Several compounds belonging to different chemical classes were tentatively identified such as rutin and kampferol rutinoside, the major constituents of the leaves, and N-caffeoyltyramine, lyciumide A, N-dihydrocaffeoyltyramine as well as fatty acids: trihydroxyoctadecadienoic acid and hydroxyoctadecadienoic acid isomers were detected abundantly in the stems. These results showed that the MeOH extracts of stems and leaves of L. arabicum can be considered as a potential source of biological active compounds. © 2017 Wiley-VHCA AG, Zurich, Switzerland.

  11. Lipase-catalyzed synthesis of fattythioic acids from palm oil.

    PubMed

    Al-Mulla, Emad A Jaffar

    2011-01-01

    The present work focuses on the synthesis of fattythioic acids (FTAs) by a one-step lipase catalyzed reaction of palm oil with carbonothioic S,S-acid using Lipozyme. The product was characterized using Fourier transform infrared (FTIR) spectroscopy, proton nuclear magnetic resonance ((1)H NMR) technique and elemental analysis. The effects of various reaction parameters such as reaction time, temperature, amount of enzyme, molar ratio of substrates, and various organic solvents of the reaction system were investigated. The optimum conditions to produce FTAs were respectively, incubation time, 20 h, temperature, 40°C, amount of enzyme, 0.05 g and molar ratio of carbonothioic S,S-acid to palm oil, 5.0:1.0. Hexane was the best solvent for this reaction. The conversion of the products at optimum conditions was around 91%.

  12. Purification and substrate specificity of Staphylococcus hyicus lipase.

    PubMed

    van Oort, M G; Deveer, A M; Dijkman, R; Tjeenk, M L; Verheij, H M; de Haas, G H; Wenzig, E; Götz, F

    1989-11-28

    The Staphylococcus hyicus lipase gene has been cloned and expressed in Staphylococcus carnosus. From the latter organism the enzyme was secreted into the medium as a protein with an apparent molecular mass of 86 kDa. This protein was purified, and the amino-terminal sequence showed that the primary gene product was indeed cleaved at the proposed signal peptide cleavage site. The protein was purified from large-scale preparations after tryptic digestion. This limited proteolysis reduced the molecular mass to 46 kDa and increased the specific activity about 3-fold. Although the enzyme had a low specific activity in the absence of divalent cations, the activity increased about 40-fold in the presence of Sr2+ or Ca2+ ions. The purified lipase has a broad substrate specificity. The acyl chains were removed from the primary and secondary positions of natural neutral glycerides and from a variety of synthetic glyceride analogues. Thus triglycerides were fully hydrolyzed to free fatty acid and glycerol. The enzyme hydrolyzed naturally occurring phosphatidylcholines, their synthetic short-chain analogues, and lysophospholipids to free fatty acids and water-soluble products. The enzyme had a 2-fold higher activity on micelles of short-chain D-lecithins than on micelles composed of the L-isomers. Thus the enzyme from S. hyicus has lipase activity and also high phospholipase A and lysophospholipase activity.

  13. Lipoprotein lipase variants interact with polyunsaturated fatty acids to modulate obesity traits in Puerto Ricans

    USDA-ARS?s Scientific Manuscript database

    Lipoprotein lipase (LPL) is a candidate gene for obesity based on its role in triglyceride hydrolysis and the partitioning of fatty acids towards storage or oxidation. Whether dietary fatty acids modify LPL associated obesity risk is unknown. We examined five single nucleotide polymorphisms (SNPs) (...

  14. Pancreatic lipase inhibitory activity of taraxacum officinale in vitro and in vivo

    PubMed Central

    Zhang, Jian; Kang, Min-Jung; Kim, Myung-Jin; Kim, Mi-Eun; Song, Ji-Hyun; Lee, Young-Min

    2008-01-01

    Obesity has become a worldwide health problem. Orlistat, an inhibitor of pancreatic lipase, is currently approved as an anti-obesity drug. However, gastrointestinal side effects caused by Orlistat may limit its use. In this study the inhibitory activities of dandelion (Taraxacum officinale) against pancreatic lipase in vitro and in vivo were measured to determine its possible use as a natural anti-obesity agent. The inhibitory activities of the 95% ethanol extract of T. officinale and Orlistat were measured using 4-methylumbelliferyl oleate (4-MU oleate) as a substrate at concentrations of 250, 125, 100, 25, 12.5 and 4 µg/ml. To determine pancreatic lipase inhibitory activity in vivo, mice (n=16) were orally administered with corn oil emulsion (5 ml/kg) alone or with the 95% ethanol extract of T. officinale (400 mg/kg) following an overnight fast. Plasma triglyceride levels were measured at 0, 90, 180, and 240 min after treatment and incremental areas under the response curves (AUC) were calculated. The 95% ethanol extract of T. officinale and Orlistat, inhibited, porcine pancreatic lipase activity by 86.3% and 95.7% at a concentration of 250 µg/ml, respectively. T. officinale extract showed dose-dependent inhibition with the IC50 of 78.2 µg/ml. A single oral dose of the extract significantly inhibited increases in plasma triglyceride levels at 90 and 180 min and reduced AUC of plasma triglyceride response curve (p<0.05). The results indicate that T. officinale exhibits inhibitory activities against pancreatic lipase in vitro and in vivo. Further studies to elucidate anti-obesity effects of chronic consumption of T. officinale and to identify the active components responsible for inhibitory activity against pancreatic lipase are necessary. PMID:20016719

  15. Simultaneous conversion of free fatty acids and triglycerides to biodiesel by immobilized Aspergillus oryzae expressing Fusarium heterosporum lipase.

    PubMed

    Amoah, Jerome; Quayson, Emmanuel; Hama, Shinji; Yoshida, Ayumi; Hasunuma, Tomohisa; Ogino, Chiaki; Kondo, Akihiko

    2017-03-01

    The presence of high levels of free fatty acids (FFA) in oil is a barrier to one-step biodiesel production. Undesirable soaps are formed during conventional chemical methods, and enzyme deactivation occurs when enzymatic methods are used. This work investigates an efficient technique to simultaneously convert a mixture of free fatty acids and triglycerides (TAG). A partial soybean hydrolysate containing 73.04% free fatty acids and 24.81% triglycerides was used as a substrate for the enzymatic production of fatty acid methyl ester (FAME). Whole-cell Candida antarctica lipase B-expressing Aspergillus oryzae, and Novozym 435 produced only 75.2 and 73.5% FAME, respectively. Fusarium heterosporum lipase-expressing A. oryzae produced more than 93% FAME in 72 h using three molar equivalents of methanol. FFA and TAG were converted simultaneously in the presence of increasing water content that resulted from esterification. Therefore, F. heterosporum lipase with a noted high level of tolerance of water could be useful in the industrial production of biodiesel from feedstock that has high proportion of free fatty acids. Copyright © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  16. Cloning and seasonal secretion of the pancreatic lipase-related protein 2 present in goat seminal plasma.

    PubMed

    Sias, Barbara; Ferrato, Francine; Pellicer-Rubio, Maria-Teresa; Forgerit, Yvonick; Guillouet, Philippe; Leboeuf, Bernard; Carrière, Frédéric

    2005-01-05

    The storage of frozen semen for artificial insemination is usually performed in the presence of egg yolk or skimmed milk as protective agents. In goats, the use of skimmed milk extenders requires, however, that most of the seminal plasma is removed before dilution of spermatozoa because it is deleterious for their survival. It has been previously demonstrated that a lipase (BUSgp60) secreted by the accessory bulbourethral gland was responsible for the cellular death of goat spermatozoa, through the lipolysis of residual milk lipids and the release of toxic free fatty acids. This lipase was purified from the whole seminal plasma of goat and was found to display both lipase and phospholipase A activities, this latter activity representing the main phospholipase activity detected in goat seminal plasma. Based on its N-terminal amino acid sequence, identical to that of BUSgP60 purified from bulbourethral gland secretion, and the design of degenerated oligonucleotides, the lipase was cloned from total mRNA isolated from bulbourethral gland. DNA sequencing confirmed it was the goat pancreatic-lipase-related protein 2 (GoPLRP2). The physiological role of GoPLRP2 is still unknown but this enzyme might be associated with the reproductive activity of goats. A significant increase in lipase secretion was observed every year in August and the level of lipase activity in the semen remained high till December, i.e., during the breeding season. A parallel increase in the plasmatic levels of testosterone suggested that GoPLRP2 expression might be regulated by sexual hormones. The lipase activity level measured in goat seminal plasma, which could reach 1000 U/ml during the breeding season, was one of the highest lipase activity measured in natural sources, including gastric and pancreatic juices.

  17. Lipase in aqueous-polar organic solvents: Activity, structure, and stability

    PubMed Central

    Kamal, Md Zahid; Yedavalli, Poornima; Deshmukh, Mandar V; Rao, Nalam Madhusudhana

    2013-01-01

    Studying alterations in biophysical and biochemical behavior of enzymes in the presence of organic solvents and the underlying cause(s) has important implications in biotechnology. We investigated the effects of aqueous solutions of polar organic solvents on ester hydrolytic activity, structure and stability of a lipase. Relative activity of the lipase monotonically decreased with increasing concentration of acetone, acetonitrile, and DMF but increased at lower concentrations (upto ∼20% v/v) of dimethylsulfoxide, isopropanol, and methanol. None of the organic solvents caused any appreciable structural change as evident from circular dichorism and NMR studies, thus do not support any significant role of enzyme denaturation in activity change. Change in 2D [15N, 1H]-HSQC chemical shifts suggested that all the organic solvents preferentially localize to a hydrophobic patch in the active-site vicinity and no chemical shift perturbation was observed for residues present in protein's core. This suggests that activity alteration might be directly linked to change in active site environment only. All organic solvents decreased the apparent binding of substrate to the enzyme (increased Km); however significantly enhanced the kcat. Melting temperature (Tm) of lipase, measured by circular dichroism and differential scanning calorimetry, altered in all solvents, albeit to a variable extent. Interestingly, although the effect of all organic solvents on various properties on lipase is qualitatively similar, our study suggest that magnitudes of effects do not appear to follow bulk solvent properties like polarity and the solvent effects are apparently dictated by specific and local interactions of solvent molecule(s) with the protein. PMID:23625694

  18. Effect of metal ions on the hydrolytic and transesterification activities of Candida rugosa lipase.

    PubMed

    Katiyar, Madhu; Ali, Amjad

    2013-01-01

    In order to study the effect of metal ions on lipase activity, hydrolytic and transesterification activities of Candida rugosa lipase were investigated in presence of alkali (Na⁺ and K⁺), alkaline earth (Ca⁺² and Ba⁺²) and transition (Cr⁺³, Fe⁺³, Co⁺², Cu⁺² and Ni⁺²) metal ions. Maximum enhancement in hydrolytic activity of lipase was observed by Ca⁺², and in transesterification activity by Cr⁺³ and Co⁺². The kinetics of the lipase catalyzed transesterification (methanolysis and ethanolysis) reactions were also studied, and the activation energies of methanolysis and ethanolysis were reduced from 10.16 and 10.24 kcal mol⁻¹, respectively, to 5.41 and 7.55 kcal mol⁻¹, respectively, when reactions were performed in presence of Co⁺². Thus, in lipase catalyzed transesterification Cr⁺³ or Co⁺² could be added to the assay in order to produce the biodiesel in relatively shorter reaction duration.

  19. Role of active site rigidity in activity: MD simulation and fluorescence study on a lipase mutant.

    PubMed

    Kamal, Md Zahid; Mohammad, Tabrez Anwar Shamim; Krishnamoorthy, G; Rao, Nalam Madhusudhana

    2012-01-01

    Relationship between stability and activity of enzymes is maintained by underlying conformational flexibility. In thermophilic enzymes, a decrease in flexibility causes low enzyme activity while in less stable proteins such as mesophiles and psychrophiles, an increase in flexibility is associated with enhanced enzyme activity. Recently, we identified a mutant of a lipase whose stability and activity were enhanced simultaneously. In this work, we probed the conformational dynamics of the mutant and the wild type lipase, particularly flexibility of their active site using molecular dynamic simulations and time-resolved fluorescence techniques. In contrast to the earlier observations, our data show that active site of the mutant is more rigid than wild type enzyme. Further investigation suggests that this lipase needs minimal reorganization/flexibility of active site residues during its catalytic cycle. Molecular dynamic simulations suggest that catalytically competent active site geometry of the mutant is relatively more preserved than wild type lipase, which might have led to its higher enzyme activity. Our study implies that widely accepted positive correlation between conformation flexibility and enzyme activity need not be stringent and draws attention to the possibility that high enzyme activity can still be accomplished in a rigid active site and stable protein structures. This finding has a significant implication towards better understanding of involvement of dynamic motions in enzyme catalysis and enzyme engineering through mutations in active site.

  20. Activation of milk lipase by serum proteins: possible role in the occurrence of lipolysis in raw bovine milk.

    PubMed

    Clegg, R A

    1980-02-01

    The increase in unesterified fatty acid content of unpasteurized bulk milk in storage at 4 degrees C in the presence of known effectors of bovine milk lipoprotein lipase originating from bovine serum was studied. Bovine serum and high density lipoprotein (HDL) caused an increase in developed unesterified fatty acid levels whilst lipoprotein-free serum, apo-HDL, all individual apo-HDL tested, and the unfractionated C-peptide fraction were without lipolytic effect. In the presence of HDL-lipids, 2 C-peptides stimulated considerable lipolysis, as did the combination of HDL-lipid with unfractionated C-peptides. These characteristics of unesterified fatty acid development could be duplicated in milk whose endogenous lipolytic activity had been destroyed by heat treatment (75 degrees C for 5 min) and then restored by addition of purified bovine milk lipoprotein lipase. Radioactively labelled glycerol trioleate in milk was not hydrolysed in the same way as milk fat on the addition of serum liproproteins.

  1. Medicinal Plants and Their Inhibitory Activities against Pancreatic Lipase: A Review

    PubMed Central

    Seyedan, Atefehalsadat; Alshawsh, Mohammed Abdullah; Alshagga, Mustafa Ahmed; Koosha, Sanaz

    2015-01-01

    Obesity is recognized as a major life style disorder especially in developing countries and it is prevailing at an alarming speed in new world countries due to fast food intake, industrialization, and reduction of physical activity. Furthermore, it is associated with a vast number of chronic diseases and disabilities. To date, relatively effective drugs, from either natural or synthetic sources, are generally associated with serious side effects, often leading to cessation of clinical trials or even withdrawal from the market. In order to find new compounds which are more effective or with less adverse effects compared to orlistat, the drug that has been approved for obesity, new compounds isolated from natural products are being identified and screened for antiobesity effects, in particular, for their pancreatic lipase inhibitory effect. Pancreatic lipase inhibitory activity has been extensively used for the determination of potential efficacy of natural products as antiobesity agents. In attempts to identify natural products for overcoming obesity, more researches have been focused on the identification of newer pancreatic lipase inhibitors with less unpleasant adverse effects. In this review, we consider the potential role of plants that have been investigated for their pancreatic lipase inhibitory activity. PMID:26640503

  2. Studies on the substrate and stereo/regioselectivity of adipose triglyceride lipase, hormone-sensitive lipase, and diacylglycerol-O-acyltransferases.

    PubMed

    Eichmann, Thomas O; Kumari, Manju; Haas, Joel T; Farese, Robert V; Zimmermann, Robert; Lass, Achim; Zechner, Rudolf

    2012-11-30

    Adipose triglyceride lipase (ATGL) is rate-limiting for the initial step of triacylglycerol (TAG) hydrolysis, generating diacylglycerol (DAG) and fatty acids. DAG exists in three stereochemical isoforms. Here we show that ATGL exhibits a strong preference for the hydrolysis of long-chain fatty acid esters at the sn-2 position of the glycerol backbone. The selectivity of ATGL broadens to the sn-1 position upon stimulation of the enzyme by its co-activator CGI-58. sn-1,3 DAG is the preferred substrate for the consecutive hydrolysis by hormone-sensitive lipase. Interestingly, diacylglycerol-O-acyltransferase 2, present at the endoplasmic reticulum and on lipid droplets, preferentially esterifies sn-1,3 DAG. This suggests that ATGL and diacylglycerol-O-acyltransferase 2 act coordinately in the hydrolysis/re-esterification cycle of TAGs on lipid droplets. Because ATGL preferentially generates sn-1,3 and sn-2,3, it suggests that TAG-derived DAG cannot directly enter phospholipid synthesis or activate protein kinase C without prior isomerization.

  3. Design of Heterogeneous Hoveyda-Grubbs Second-Generation Catalyst-Lipase Conjugates.

    PubMed

    Neville, Anthony; Iniesta, Javier; Palomo, Jose M

    2016-12-06

    Heterogeneous catalysts have been synthesi zed by the conjugation of Hoveyda-Grubbs second-generation catalyst with a lipase. The catalytic properties of the organometallic compound in solution were firstly optimized, evaluating the activity of Ru in the ring-closing metathesis of diethyldiallymalonate at 25 °C at different solvents and in the presence of different additives. The best result was found using tetrahydrofuran as a solvent. Some additives such as phenylboronic acid or polyetheneglycol slightly improved the activity of the Ru catalyst whereas others, such as pyridine or dipeptides affected it negatively. The organometallic compound immobilized on functionalized-surface materials activated with boronic acid or epoxy groups (around 50-60 µg per mg support) and showed 50% conversion at 24 h in the ring-closing metathesis. Cross-linked enzyme aggregates (CLEA's) of the Hoveyda-Grubbs second-generation catalyst with Candida antarctica lipase (CAL-B) were prepared, although low Ru catalyst was found to be translated in low conversion. Therefore, a sol-gel preparation of the Hoveyda-Grubbs second-generation and CAL-B was performed. This catalyst exhibited good activity in the metathesis of diethyldiallymalonate in toluene and in aqueous media. Finally, a new sustainable approach was used by the conjugation lipase-Grubbs in solid phase in aqueous media. Two strategies were used: one using lipase previously covalently immobilized on an epoxy-Sepharose support (hydrophilic matrix) and then conjugated with grubbs; and in the second, the free lipase was incubated with organometallic in aqueous solution and then immobilized on epoxy-Sepharose. The different catalysts showed excellent conversion values in the ring-closing metathesis of diethyldiallymalonate in aqueous media at 25 °C.

  4. Purification and characterization of a cold-active lipase from Pichia lynferdii Y-7723: pH-dependant activity deviation

    USDA-ARS?s Scientific Manuscript database

    Lipases with abnormal functionalities such as high thermostability and optimal activity at extreme conditions gain special attentions because of their applicability in the restricted reaction conditions. In particular, cold-active lipases have gained special attentions in various industrial fields s...

  5. Secoiridoids from the stem barks of Fraxinus rhynchophylla with pancreatic lipase inhibitory activity.

    PubMed

    Ahn, Jong Hoon; Shin, Eunjin; Liu, Qing; Kim, Seon Beom; Choi, Kyeong-Mi; Yoo, Hwan-Soo; Hwang, Bang Yeon; Lee, Mi Kyeong

    2013-01-01

    Pancreatic lipase digests dietary fats by hydrolysis, which is a key enzyme for lipid absorption. Therefore, reduction of fat absorption by the inhibition of pancreatic lipase is suggested to be a therapeutic strategy for obesity. From the EtOAc-soluble fraction of the stem barks of Fraxinus rhynchophylla (Oleaceae), four secoiridoids such as ligstroside (1), oleuropein (2), 2"-hydroxyoleuropein (3) and hydroxyframoside B (4) were isolated. The inhibitory activity of these compounds on pancreatic lipase was assessed using porcine pancreatic lipase as an in vitro assay system. Compound 4 showed the strongest inhibition on pancreatic lipase, which followed by compounds 1-3. In addition, compound 4 exerted inhibitory effect on pancreatic lipase in a mixed mechanism of competitive and noncompetitive manner. Taken together, F. rhynchophylla and its constituents might be beneficial to obesity.

  6. Carbon nanotube-lipase hybrid nanoflowers with enhanced enzyme activity and enantioselectivity.

    PubMed

    Li, Kai; Wang, Jianhua; He, Yaojia; Abdulrazaq, Miaad Adnan; Yan, Yunjun

    2018-06-19

    Various nanoflowers are synthesized as supports for different methods of enzyme immobilization; however, the activities of these immobilized enzymes are limited because of their confinement in the nanoflowers. In order to increase the performance of nanoflowers, in this study, different protein-phosphate hybrid nanostructures were successfully synthesized and further enhanced by carbon nanotubes (CNTs) under the same conditions. Only Cu 3 (PO 4 ) 2 complex nanostructures exhibited flower-like structures and showed excellent results after enhancement with CNTs in this framework. An esterification reaction between lauric acid and 1-dodecanol was used to test enzyme activity during immobilization, revealing that the Cu 3 (PO 4 ) 2 /CNT/protein complex exhibited 68-fold higher activity relative to free lipase and 51-fold higher than that of Cu 3 (PO 4 ) 2 /Burkholderia cepacia lipase hybrid nanoflowers in the absence of CNTs. All three hybrid nanostructures showed good performance and exhibited excellent reusability in resolution reactions between 1-phenylethanol and vinyl acetate. Additionally, the substrate enantiomeric excess (ee s ) reached 98% in only 10 min, and the corresponding Cu 3 (PO 4 ) 2 /CNT/protein complex could be recycled eight times without obvious loss of activity. This approach involving nanoflowers enhanced with CNTs will be highly beneficial for decreasing mass-transfer resistance and providing enhanced enzyme loading along with promising potential for industrial application. Copyright © 2018 Elsevier B.V. All rights reserved.

  7. A simplified method for active-site titration of lipases immobilised on hydrophobic supports.

    PubMed

    Nalder, Tim D; Kurtovic, Ivan; Barrow, Colin J; Marshall, Susan N

    2018-06-01

    The aim of this work was to develop a simple and accurate protocol to measure the functional active site concentration of lipases immobilised on highly hydrophobic supports. We used the potent lipase inhibitor methyl 4-methylumbelliferyl hexylphosphonate to titrate the active sites of Candida rugosa lipase (CrL) bound to three highly hydrophobic supports: octadecyl methacrylate (C18), divinylbenzene crosslinked methacrylate (DVB) and styrene. The method uses correction curves to take into account the binding of the fluorophore (4-methylumbelliferone, 4-MU) by the support materials. We showed that the uptake of the detection agent by the three supports is not linear relative to the weight of the resin, and that the uptake occurs in an equilibrium that is independent of the total fluorophore concentration. Furthermore, the percentage of bound fluorophore varied among the supports, with 50 mg of C18 and styrene resins binding approximately 64 and 94%, respectively. When the uptake of 4-MU was calculated and corrected for, the total 4-MU released via inhibition (i.e. the concentration of functional lipase active sites) could be determined via a linear relationship between immobilised lipase weight and total inhibition. It was found that the functional active site concentration of immobilised CrL varied greatly among different hydrophobic supports, with 56% for C18, compared with 14% for DVB. The described method is a simple and robust approach to measuring functional active site concentration in immobilised lipase samples. Copyright © 2018 Elsevier Inc. All rights reserved.

  8. Effect of the physicochemical properties of binary ionic liquids on lipase activity and stability.

    PubMed

    Yao, Peipei; Yu, Xinxin; Huang, Xirong

    2015-01-01

    In the present study, the lipase-catalyzed hydrolysis of p-nitrophenyl butyrate is used as a model reaction to determine the activity and stability of Candida rugosa lipase in binary ionic liquids (ILs). The binary ILs consist of hydrophobic 1-butyl-3-methylimidazolium hexafluorophosphate ([Bmim]PF6) and a small amount of hydrophilic 1-butyl-3-methylimidazolium nitrate ([Bmim]NO3) or 1-butyl-3-methylimidazolium trifluoromethanesulfonate ([Bmim]CF3SO3) or 1-butyl-3-methylimidazolium tetrafluoroborate ([Bmim]BF4). The activity and the stability of lipase are first correlated with the physicochemical properties of the binary ILs. In the three binary IL systems, both the hydrophilicity and the polarity of the systems increase with the increase of the content of hydrophilic ILs (HILs). At a fixed concentration of HIL, they vary in a descending order of [Bmim]PF6/[Bmim]NO3>[Bmim]PF6/[Bmim]CF3SO3>[Bmim]PF6/[Bmim]BF4. This order is in contrast with the order of the lipase conformation stability, i.e., the higher the polarity of ILs, the more unstable the lipase conformation. However, both the activity and the stability of lipase depend on the type and the content of the HIL in binary ILs, showing a complex dependency. Analysis shows that the catalytic performance of lipase in the binary ILs is affected not only by the direct influence of the ILs on lipase conformation, but also through their indirect influence on the physicochemical properties of water. The present study helps to explore binary IL mixtures suitable for lipase-based biocatalysis. Copyright © 2015 Elsevier B.V. All rights reserved.

  9. Transesterification of waste cooking oil by an organic solvent-tolerant alkaline lipase from Streptomyces sp. CS273.

    PubMed

    Mander, Poonam; Yoo, Hah-Young; Kim, Seung Wook; Choi, Yun Hee; Cho, Seung Sik; Yoo, Jin Cheol

    2014-02-01

    The aim of this present study was to produce a microbial enzyme that can potentially be utilized for the enzymatic transesterification of waste cooking oil. To that end, an extracellular lipase was isolated and purified from the culture broth of Streptomyces sp. CS273. The molecular mass of purified lipase was estimated to be 36.55 kDa by SDS PAGE. The optimum lipolytic activity was obtained at alkaline pH 8.0 to 8.5 and temperature 40 °C, while the enzyme was stable in the pH range 7.0 ∼ 9.0 and at temperature ≤40 °C. The lipase showed highest hydrolytic activity towards p-nitrophenyl myristate (C14). The lipase activity was enhanced by several salts and detergents including NaCl, MnSo₄, and deoxy cholic acid, while phenylmethylsulfonyl fluoride at concentration 10 mM inhibited the activity. The lipase showed tolerance towards different organic solvents including ethanol and methanol which are commonly used in transesterification reactions to displace alcohol from triglycerides (ester) contained in renewable resources to yield fatty acid alkyl esters known as biodiesel. Applicability of the lipase in transesterification of waste cooking oil was confirmed by gas chromatography mass spectrometry analysis.

  10. Biological Activities of Oleanolic Acid Derivatives from Calendula officinalis Seeds.

    PubMed

    Zaki, Ahmed; Ashour, Ahmed; Mira, Amira; Kishikawa, Asuka; Nakagawa, Toshinori; Zhu, Qinchang; Shimizu, Kuniyoshi

    2016-05-01

    Phytochemical examination of butanol fraction of Calendula officinalis seeds led to the isolation of two compounds identified as 28-O-β-D-glucopyranosyl-oleanolic acid 3-O-β-D-glucopyranosyl (1→3)-β-D-glucopyranosiduronic acid (CS1) and oleanolic acid 3-O-β-D-glucopyranosyl (1→3)-β-D-glucopyranosiduronic acid (CS2). Biological evaluation was carried out for these two compounds such as melanin biosynthesis inhibitory, hyaluronic acid production activities, anti obesity using lipase inhibition and adipocyte differentiation as well as evaluation of the protective effect against hydrogen peroxide induced neurotoxicity in neuro-2A cells. The results showed that, compound CS2 has a melanin biosynthesis stimulatory activity; however, compound CS1 has a potent stimulatory effect for the production of hyaluronic acid on normal human dermal fibroblast from adult (NHDF-Ad). Both compounds did not show any inhibitory effect on both lipase and adipocyte differentiation. Compound CS2 could protect neuro-2A cells and increased cell viability against H2 O2 . These activities (melanin biosynthesis stimulatory and protective effect against H2 O2 of CS2 and hyaluronic acid productive activities of these triterpene derivatives) have been reported for the first time. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

  11. Biodegradable products by lipase biocatalysis.

    PubMed

    Linko, Y Y; Lämsä, M; Wu, X; Uosukainen, E; Seppälä, J; Linko, P

    1998-11-18

    The interest in the applications of biocatalysis in organic syntheses has rapidly increased. In this context, lipases have recently become one of the most studied groups of enzymes. We have demonstrated that lipases can be used as biocatalyst in the production of useful biodegradable compounds. A number of examples are given. 1-Butyl oleate was produced by direct esterification of butanol and oleic acid to decrease the viscosity of biodiesel in winter use. Enzymic alcoholysis of vegetable oils without additional organic solvent has been little investigated. We have shown that a mixture of 2-ethyl-1-hexyl esters can be obtained in a good yield by enzymic transesterification from rapeseed oil fatty acids for use as a solvent. Trimethylolpropane esters were also similarly synthesized as lubricants. Finally, the discovery that lipases can also catalyze ester syntheses and transesterification reactions in organic solvent systems has opened up the possibility of enzyme catalyzed production of biodegradable polyesters. In direct polyesterification of 1,4-butanediol and sebacic acid, polyesters with a mass average molar mass of the order of 56,000 g mol-1 or higher, and a maximum molar mass of about 130,000 g mol-1 were also obtained by using lipase as biocatalyst. Finally, we have demonstrated that also aromatic polyesters can be synthesized by lipase biocatalysis, a higher than 50,000 g mol-1 mass average molar mass of poly(1,6-hexanediyl isophthalate) as an example.

  12. Characterization of biotechnologically relevant extracellular lipase produced by Aspergillus terreus NCFT 4269.10

    PubMed Central

    Sethi, Bijay Kumar; Nanda, Prativa Kumari; Sahoo, Santilata

    2016-01-01

    Enzyme production by Aspergillus terreus NCFT 4269.10 was studied under liquid static surface and solid-state fermentation using mustard oil cake as a substrate. The maximum lipase biosynthesis was observed after incubation at 30 °C for 96 h. Among the domestic oils tested, the maximum lipase biosynthesis was achieved using palm oil. The crude lipase was purified 2.56-fold to electrophoretic homogeneity, with a yield of 8.44%, and the protein had a molecular weight of 46.3 kDa as determined by SDS-PAGE. Enzyme characterization confirmed that the purified lipase was most active at pH 6.0, temperature of 50 °C, and substrate concentration of 1.5%. The enzyme was thermostable at 60 °C for 1 h, and the optimum enzyme–substrate reaction time was 30 min. Sodium dodecyl sulfate and commercial detergents did not significantly affect lipase activity during 30-min incubation at 30 °C. Among the metal ions tested, the maximum lipase activity was attained in the presence of Zn2+, followed by Mg2+ and Fe2+. Lipase activity was not significantly affected in the presence of ethylenediaminetetraacetic acid, sodium lauryl sulfate and Triton X-100. Phenylmethylsulfonyl fluoride (1 mM) and the reducing, β-mercaptoethanol significantly inhibited lipase activity. The remarkable stability in the presence of detergents, additives, inhibitors and metal ions makes this lipase unique and a potential candidate for significant biotechnological exploitation. PMID:26887237

  13. The inhibitory effect of some drugs on Candida rugosa Lipase and Pancreatic Human Lipase: In vitro and in silico studies.

    PubMed

    Serseg, Talia; Benarous, Khedidja

    2018-03-18

    Side effects of some drugs may be useful in certain cases. In this work, we studied the inhibitory effects of some medications as: Folic Acid which is taken by pregnant women, Colchicine and Febuxostat which is used as treatment of gout disease. These cases are linked to obesity, where women (BMI ≥ 30) have twice higher odds of having an NTD-affected pregnancy than the normal weight women, and the Gout disease frequently occurs in combination of a Metabolic syndrome. The risk of gout increases with the increase of the mass index. In the first part of this study, we studied the inhibition activity of these medications on lipase activity of Candida rugosa in vitro, the results show that these drugs have an important inhibition activity with IC50 values 0.64 mg/ml for Folic acid and 0.66 mg/ml for Febuxostat. İn silico studies were aimed to determine the mechanism of inhibition and different interactions for two enzymes: Candida rugosa lipase and human pancreatic lipase. Autodock vina was used for molecular docking with 50 runs and 1000 obtained solutions. The results show competitive, Non-competitive and uncompetitive inhibition for folic acid, febuxostat and colchicine respectively for two enzymes with different repetition ratios of hydrogen bonds. The saved interactions were with His449 and Ser209 for the three molecules. These observations support a higher intake of dietary folate, and febuxostat for losing weight to decreased NTD risk and prevent hyperuricemia and recurrent gout attacks. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  14. Identification of a putative triacylglycerol lipase from papaya latex by functional proteomics.

    PubMed

    Dhouib, R; Laroche-Traineau, J; Shaha, R; Lapaillerie, D; Solier, E; Rualès, J; Pina, M; Villeneuve, P; Carrière, F; Bonneu, M; Arondel, V

    2011-01-01

    Latex from Caricaceae has been known since 1925 to contain strong lipase activity. However, attempts to purify and identify the enzyme were not successful, mainly because of the lack of solubility of the enzyme. Here, we describe the characterization of lipase activity of the latex of Vasconcellea heilbornii and the identification of a putative homologous lipase from Carica papaya. Triacylglycerol lipase activity was enriched 74-fold from crude latex of Vasconcellea heilbornii to a specific activity (SA) of 57 μmol·min(-1)·mg(-1) on long-chain triacylglycerol (olive oil). The extract was also active on trioctanoin (SA = 655 μmol·min(-1)·mg(-1) ), tributyrin (SA = 1107 μmol·min(-1)·mg(-1) ) and phosphatidylcholine (SA = 923 μmol·min(-1)·mg(-1) ). The optimum pH ranged from 8.0 to 9.0. The protein content of the insoluble fraction of latex was analyzed by electrophoresis followed by mass spectrometry, and 28 different proteins were identified. The protein fraction was incubated with the lipase inhibitor [(14) C]tetrahydrolipstatin, and a 45 kDa protein radiolabeled by the inhibitor was identified as being a putative lipase. A C. papaya cDNA encoding a 55 kDa protein was further cloned, and its deduced sequence had 83.7% similarity with peptides from the 45 kDa protein, with a coverage of 25.6%. The protein encoded by this cDNA had 35% sequence identity and 51% similarity to castor bean acid lipase, suggesting that it is the lipase responsible for the important lipolytic activities detected in papaya latex. © 2010 The Authors Journal compilation © 2010 FEBS.

  15. Requirement of lid2 for interfacial activation of a family I.3 lipase with unique two lid structures.

    PubMed

    Cheng, Maria; Angkawidjaja, Clement; Koga, Yuichi; Kanaya, Shigenori

    2012-10-01

    A family I.3 lipase from Pseudomonas sp. MIS38 (PML) is characterized by the presence of two lids (lid1 and lid2) that greatly change conformation upon substrate binding. While lid1 represents the commonly known lid in lipases, lid2 is unique to PML and other family I.3 lipases. To clarify the role of lid2 in PML, a lid2 deletion mutant (ΔL2-PML) was constructed by deleting residues 35-64 of PML. ΔL2-PML requires calcium ions for both lipase and esterase activities as does PML, suggesting that it exhibits activity only when lid1 is fully open and anchored by the catalytically essential calcium ion, as does PML. However, when the enzymatic activity was determined using triacetin, the activity of PML exponentially increased as the substrate concentration reached and increased beyond the critical micellar concentration, while that of ΔL2-PML did not. These results indicate that PML undergoes interfacial activation, while ΔL2-PML does not. The activities of ΔL2-PML for long-chain triglycerides significantly decreased while its activity for fatty acid ethyl esters increased, compared with those of PML. Comparison of the tertiary models of ΔL2-PML in a closed and open conformation, which are optimized by molecular dynamics simulation, with the crystal structures of PML suggests that the hydrophobic surface area provided by lid1 and lid2 in an open conformation is considerably decreased by the deletion of lid2. We propose that the hydrophobic surface area provided by these lids is necessary to hold the micellar substrates firmly to the active site and therefore lid2 is required for interfacial activation of PML. © 2012 The Authors Journal compilation © 2012 FEBS.

  16. Sodium chloride effects on lipase activity in germinating rape seeds.

    PubMed

    Ben Miled, D D; Zarrouk, M; Chérif, A

    2000-12-01

    Seeds of rape (Brassica napus L.) were germinated at various NaCl concentrations up to 200 mM. Germinating levels, seedling growth, triacylglycerol mobilization and lipase activity were investigated. High salt concentrations resulted in retardation of seed germination. Seedling growth as measured by radicle length was severely reduced by NaCl doses higher than 50 mM. Moreover, the mobilization of storage oil in control rapeseed seedlings, started about 24 h after imbibition. As for germination and growth, elevated salt concentrations are found to delay triacylglycerol degradation. Experiments using triolein as substrate indicated clearly that lipase activity was inhibited by salt treatment.

  17. Biodiesel Production by Aspergillus niger Lipase Immobilized on Barium Ferrite Magnetic Nanoparticles

    PubMed Central

    El-Batal, Ahmed I.; Farrag, Ayman A.; Elsayed, Mohamed A.; El-Khawaga, Ahmed M.

    2016-01-01

    In this study, Aspergillus niger ADM110 fungi was gamma irradiated to produce lipase enzyme and then immobilized onto magnetic barium ferrite nanoparticles (BFN) for biodiesel production. BFN were prepared by the citrate sol-gel auto-combustion method and characterized by transmission electron microscopy (TEM), X-ray diffraction (XRD), Fourier transform infrared (FTIR) and scanning electron microscopy with energy dispersive analysis of X-ray (SEM/EDAX) analysis. The activities of free and immobilized lipase were measured at various pH and temperature values. The results indicate that BFN–Lipase (5%) can be reused in biodiesel production without any treatment with 17% loss of activity after five cycles and 66% loss in activity in the sixth cycle. The optimum reaction conditions for biodiesel production from waste cooking oil (WCO) using lipase immobilized onto BFN as a catalyst were 45 °C, 4 h and 400 rpm. Acid values of WCO and fatty acid methyl esters (FAMEs) were 1.90 and 0.182 (mg KOH/g oil), respectively. The measured flash point, calorific value and cetane number were 188 °C, 43.1 MJ/Kg and 59.5, respectively. The cloud point (−3 °C), pour point (−9 °C), water content (0.091%) and sulfur content (0.050%), were estimated as well. PMID:28952576

  18. Biodiesel Production by Aspergillus niger Lipase Immobilized on Barium Ferrite Magnetic Nanoparticles.

    PubMed

    El-Batal, Ahmed I; Farrag, Ayman A; Elsayed, Mohamed A; El-Khawaga, Ahmed M

    2016-05-12

    In this study, Aspergillus niger ADM110 fungi was gamma irradiated to produce lipase enzyme and then immobilized onto magnetic barium ferrite nanoparticles (BFN) for biodiesel production. BFN were prepared by the citrate sol-gel auto-combustion method and characterized by transmission electron microscopy (TEM), X-ray diffraction (XRD), Fourier transform infrared (FTIR) and scanning electron microscopy with energy dispersive analysis of X-ray (SEM/EDAX) analysis. The activities of free and immobilized lipase were measured at various pH and temperature values. The results indicate that BFN-Lipase (5%) can be reused in biodiesel production without any treatment with 17% loss of activity after five cycles and 66% loss in activity in the sixth cycle. The optimum reaction conditions for biodiesel production from waste cooking oil (WCO) using lipase immobilized onto BFN as a catalyst were 45 °C, 4 h and 400 rpm. Acid values of WCO and fatty acid methyl esters (FAMEs) were 1.90 and 0.182 (mg KOH/g oil), respectively. The measured flash point, calorific value and cetane number were 188 °C, 43.1 MJ/Kg and 59.5, respectively. The cloud point (-3 °C), pour point (-9 °C), water content (0.091%) and sulfur content (0.050%), were estimated as well.

  19. Preliminary studies on immobilization of lipase using chicken eggshell

    NASA Astrophysics Data System (ADS)

    Salleh, S.; Serri, N. A.; Hena, S.; Tajarudin, H. A.

    2016-06-01

    A few advantages of enzyme immobilization are reusability of expensive enzyme, improvement of stability and activity compared to crude enzyme. Various organic components can be used as carrier for enzyme immobilization such as chicken eggshell. It can be used as a carrier for immobilization as its mineral component mostly contains of calcium carbonate. In the present study, Tributyrin method was used to test enzyme activity of Rhizomucour Miehei, Candida Antarctica and Candida Rugosa. Rhizomucour Miehei shows the highest enzyme activity (360.8 mol/min/mL lipase) and was used in further experiment. Experiment was continued to study incubation time for lipase immobilization on eggshell (1-4 hours) and reaction time of esterification of sugar ester (0-72 hours). Two hours incubation time for lipase immobilization was observed and gives the highest yield of sugar ester (78.13%). Fructose and stearic acid as substrate was used for the production of sugar ester. The highest percentage of sugar ester production was shown at 36 hours of reaction time.

  20. Inhibition of phospholipase A1, lipase and galactolipase activities of pancreatic lipase-related protein 2 by methyl arachidonyl fluorophosphonate (MAFP).

    PubMed

    Amara, Sawsan; Delorme, Vincent; Record, Michel; Carrière, Frédéric

    2012-11-01

    Methyl arachidonyl fluorophosphonate (MAFP) is a known inhibitor of cytosolic phospholipase A2 and some other serine enzymes. MAFP was found here to be an irreversible inhibitor of human pancreatic lipase-related protein 2 (HPLRP2), an enzyme displaying lipase, phospholipase A1 and galactolipase activities. In the presence of MAFP, mass spectrometry analysis of HPLRP2 revealed a mass increase of 351Da, suggesting a covalent binding of MAFP to the active site serine residue. When HPLRP2 was pre-incubated with MAFP before measuring residual activity, a direct inhibition of HPLRP2 occurred, confirming that HPLRP2 has an active site freely accessible to solvent and differs from most lipases in solution. HPLRP2 activities on tributyrin (TC4), phosphatidylcholine (PC) and monogalactosyl dioctanoylglycerol (C8-MGDG) were equally inhibited under these conditions. Bile salts were not required to trigger the inhibition, but they significantly increased the rate of HPLRP2 inhibition, probably because of MAFP micellar solubilization. Since HPLRP2 is active on various substrates that self-organize differently in the presence of water, HPLRP2 inhibition by MAFP was tested in the presence of these substrates after adding MAFP in the course of the lipolysis reaction. In this case, the rates of inhibition of lipase, phospholipase A1 and galactolipase activities were not equivalent (triglycerides>PC>MGDG), suggesting different enzyme/inhibitor partitioning between the aqueous phase and lipid aggregates. The inhibition by MAFP of a well identified phospholipase A1 (HPLRP2), present in pancreatic juice and also in human monocytes, indicates that MAFP cannot be used for discriminating phospholipase A2 from A1 activities at the cellular level. Copyright © 2012 Elsevier B.V. All rights reserved.

  1. Purification and biochemical characterization of a cold-active lipase from Antarctic sea ice bacteria Pseudoalteromonas sp. NJ 70.

    PubMed

    Wang, Quanfu; Hou, Yanhua; Ding, Yu; Yan, Peisheng

    2012-09-01

    An extracellular cold-active lipase from Antarctic sea ice bacteria Pseudoalteromonas sp. NJ 70 was purified and characterized. The overall purification based on lipase activity was 27.5-fold with a yield of 25.4 %. The purified lipase showed as a single band on SDS-PAGE with an apparent molecular weight of 37 kDa. The optimum temperature and pH were 35 °C and 7.0, respectively. The lipase activity was enhanced by Ca(2+) and Mg(2+), while was partially inhibited by other metals such as Cu(2+), Zn(2+), Ba(2+), Pb(2+), Fe(2+) and Mn(2+). The lipase had high tolerance to a wide range of NaCl concentrations (0-2 M NaCl). It exhibited high levels of activity in the presence of DTT, Thiourea, H(2)O(2) as well as in the presence of various detergents such as Span 60, Tween-80, Triton X-100. In addition, the lipase showed a preference for long-chain p-nitrophenyl esters (C(12)-C(18)). These results indicated that this lipase could be a novel cold-active lipase.

  2. Medicinal plant phytochemicals and their inhibitory activities against pancreatic lipase: molecular docking combined with molecular dynamics simulation approach.

    PubMed

    Ahmed, Bilal; Ali Ashfaq, Usman; Usman Mirza, Muhammad

    2018-05-01

    Obesity is the worst health risk worldwide, which is linked to a number of diseases. Pancreatic lipase is considered as an affective cause of obesity and can be a major target for controlling the obesity. The present study was designed to find out best phytochemicals against pancreatic lipase through molecular docking combined with molecular dynamics (MD) simulation. For this purpose, a total of 3770 phytochemicals were docked against pancreatic lipase and ranked them on the basis of binding affinity. Finally, 10 molecules (Kushenol K, Rosmarinic acid, Reserpic acid, Munjistin, Leachianone G, Cephamycin C, Arctigenin, 3-O-acetylpadmatin, Geniposide and Obtusin) were selected that showed strong bonding with the pancreatic lipase. MD simulations were performed on top five compounds using AMBER16. The simulated complexes revealed stability and ligands remained inside the binding pocket. This study concluded that these finalised molecules can be used as drug candidate to control obesity.

  3. Lipase assay in soils by copper soap colorimetry.

    PubMed

    Saisuburamaniyan, N; Krithika, L; Dileena, K P; Sivasubramanian, S; Puvanakrishnan, R

    2004-07-01

    A simple and sensitive method for the estimation of lipase activity in soils is reported. In this method, 50mg of soil is incubated with emulsified substrate, the fatty acids liberated are treated with cupric acetate-pyridine reagent, and the color developed is measured at 715 nm. Use of olive oil in this protocol leads to an estimation of true lipase activity in soils. The problem of released fatty acids getting adsorbed onto the soil colloids is obviated by the use of isooctane, and separate standards for different soils need not be developed. Among the various surfactants used for emulsification, polyvinyl alcohol is found to be the most effective. Incubation time of 20 min, soil concentration of 50 mg, pH 6.5, and incubation temperature of 37 degrees C were found to be the most suitable conditions for this assay. During the process of enrichment of the soils with oil, interference by the added oil is avoided by the maintenance of a suitable control, wherein 50 mg of soil is added after stopping the reaction. This assay is sensitive and it could be adopted to screen for lipase producers from enriched soils and oil-contaminated soils before resorting to isolation of the microbes by classical screening methods.

  4. Fluorescent microplate assay method for high-throughput detection of lipase transesterification activity.

    PubMed

    Zheng, Jianyong; Wei, Wei; Lan, Xing; Zhang, Yinjun; Wang, Zhao

    2018-05-15

    This study describes a sensitive and fluorescent microplate assay method to detect lipase transesterification activity. Lipase-catalyzed transesterification between butyryl 4-methyl umbelliferone (Bu-4-Mu) and methanol in tert-butanol was selected as the model reaction. The release of 4-methylumbelliferone (4-Mu) in the reaction was determined by detecting the fluorescence intensity at λ ex 330 nm and λ em 390 nm. Several lipases were used to investigate the accuracy and efficiency of the proposed method. Apparent Michaelis constant (Km) was calculated for transesterification between Bu-4-Mu and methanol by the lipases. The main advantages of the assay method include high sensitivity, inexpensive reagents, and simple detection process. Copyright © 2018 Elsevier Inc. All rights reserved.

  5. The galactolipase activity of Fusarium solani (phospho)lipase.

    PubMed

    Jallouli, Raida; Othman, Houcemeddine; Amara, Sawsan; Parsiegla, Goetz; Carriere, Frédéric; Srairi-Abid, Najet; Gargouri, Youssef; Bezzine, Sofiane

    2015-03-01

    The purified (phospho)lipase of Fusarium solani (FSL), was known to be active on both triglycerides and phospholipids. This study aimed at assessing the potential of this enzyme in hydrolyzing galactolipids. FSL was found to hydrolyze at high rates of synthetic medium chains monogalactosyldiacylglycerol (4658±146U/mg on DiC8-MGDG) and digalactosyldiacylglycerol (3785±83U/mg on DiC8-DGDG) and natural long chain monogalactosyldiacylglycerol extracted from leek leaves (991±85U/mg). It is the microbial enzyme with the highest activity on galactolipids identified so far with a level of activity comparable to that of pancreatic lipase-related protein 2. FSL maximum activity on galactolipids was measured at pH8. The analysis of the hydrolysis product of natural MGDG from leek showed that FSL hydrolyzes preferentially the ester bond at the sn-1 position of galactolipids. To investigate the structure-activity relationships of FSL, a 3D model of this enzyme was built. In silico docking of medium chains MGDG and DGDG and phospholipid in the active site of FSL reveals structural solutions which are in concordance with in vitro tests. Copyright © 2015 Elsevier B.V. All rights reserved.

  6. Effect of variation of trans-fatty acid in lactating rats' diet on lipoprotein lipase activity in mammary gland, liver, and adipose tissue.

    PubMed

    Assumpção, Renata Pereira; dos Santos, Flávia Duarte; de Mattos Machado Andrade, Priscila; Barreto, Giselle Freire; das Graças Tavares do Carmo, Maria

    2004-09-01

    Lactation is associated with an increase in lipoprotein lipase (LPL) activity in the mammary gland (MG) and a decrease in adipose tissue because lactation redirects circulating substrates to the MG for milk synthesis. We investigated the effects of different dietary contents of trans-fatty acid (TFA) on LPL activity in maternal tissues and fatty acid composition in milk. Lactating rats were fed semisynthetic isocaloric diets containing 7% soy oil (control), 7% partially hydrogenated vegetable oil (7%-PHVO), 5% PHVO plus 2% soy oil (5%-PHVO), or 3.5% PHVO plus 3.5% soy oil (3.5%-PHVO). On lactation day 14, animals were decapitated and MG, liver, and parametrial adipose tissue were extracted to determine total lipid contents, percentages of TFA, and LPL activity. Milk lipid composition was examined by gas chromatographic analysis of the gastric content of 14-d-old suckling pups. Maternal consumption of TFA increased dietary TFA incorporation in MG and liver and decreased it in parametrial adipose tissue. Diets with higher trans concentrations (7%-PHVO) significantly increased lipid content in the MG, and all groups fed trans-based diets showed significant increases in LPL activity in the MG. Although LPL increased in the MG, milk of rats fed TFA-based diets had significant decreases in the percentage of essential fatty acids. TFA intake during lactation alters maternal lipid metabolism and the percentage of essential fatty acids in milk; therefore, it is important to alert the population to avoid excessive intake of TFAs during lactation.

  7. Cloning and characterization of newly isolated lipase from Enterobacter sp. Bn12.

    PubMed

    Farrokh, Parisa; Yakhchali, Bagher; Karkhane, Ali Asghar

    2014-01-01

    A mesophilic Enterobacter sp. Bn12 producing an alkaline thermostable lipase was isolated from soil in Tehran, Iran. The lipase gene (ELBn12) was identified from a genomic library. Sequence analysis of the DNA fragment revealed an open reading frame of 879 bp encoding a lipase with a molecular mass of 31.3 kDa. The deduced amino acid sequence showed 96% identity with a lipase of Enterobacter sp. Ag1 and the identity of their DNA sequences was 88.9%. ELBn12 belongs to the lipase subfamily I.1 and its catalytic triad consists of Ser82, Asp237 and His259. The lipase was expressed in Escherichia coli (BL21) pLysS and partially purified by anion exchange chromatography. The maximum activity of ELBn12 was obtained at temperature of 60 °C and pH 8.0 towards tricaprylin (C8) and its specific activity was around 2900 U/mg. ELBn12 was stable within a broad pH range from 6.0 to 11.0. The enzyme showed high stability in both polar and nonpolar organic solvents at 50% (v/v). The lipase activity was enhanced in the presence of 10 mM of Ca(2+), Mg(2+) and K(+), while heavy metals (Fe(3+) and Zn(2+)) had strong inhibitory effect. ELBn12 showed high activity in the presence of 1% (w/v) nonionic surfactants, however ionic surfactants inhibited the lipolytic activity. ELBn12 characteristics show that it has a potential to be used in various industrial processes.

  8. Cloning and characterization of newly isolated lipase from Enterobacter sp. Bn12

    PubMed Central

    Farrokh, Parisa; Yakhchali, Bagher; Karkhane, Ali Asghar

    2014-01-01

    A mesophilic Enterobacter sp. Bn12 producing an alkaline thermostable lipase was isolated from soil in Tehran, Iran. The lipase gene (ELBn12) was identified from a genomic library. Sequence analysis of the DNA fragment revealed an open reading frame of 879 bp encoding a lipase with a molecular mass of 31.3 kDa. The deduced amino acid sequence showed 96% identity with a lipase of Enterobacter sp. Ag1 and the identity of their DNA sequences was 88.9%. ELBn12 belongs to the lipase subfamily I.1 and its catalytic triad consists of Ser82, Asp237 and His259. The lipase was expressed in Escherichia coli (BL21) pLysS and partially purified by anion exchange chromatography. The maximum activity of ELBn12 was obtained at temperature of 60 °C and pH 8.0 towards tricaprylin (C8) and its specific activity was around 2900 U/mg. ELBn12 was stable within a broad pH range from 6.0 to 11.0. The enzyme showed high stability in both polar and nonpolar organic solvents at 50% (v/v). The lipase activity was enhanced in the presence of 10 mM of Ca2+, Mg2+ and K+, while heavy metals (Fe3+ and Zn2+) had strong inhibitory effect. ELBn12 showed high activity in the presence of 1% (w/v) nonionic surfactants, however ionic surfactants inhibited the lipolytic activity. ELBn12 characteristics show that it has a potential to be used in various industrial processes. PMID:25242958

  9. Lipase of Aspergillus niger NCIM 1207: A Potential Biocatalyst for Synthesis of Isoamyl Acetate.

    PubMed

    Mhetras, Nutan; Patil, Sonal; Gokhale, Digambar

    2010-10-01

    Commercial lipase preparations and mycelium bound lipase from Aspergillus niger NCIM 1207 were used for esterification of acetic acid with isoamyl alcohol to obtain isoamyl acetate. The esterification reaction was carried out at 30°C in n-hexane with shaking at 120 rpm. Initial reaction rates, conversion efficiency and isoamyl acetate concentration obtained using Novozyme 435 were the highest. Mycelium bound lipase of A. niger NCIM 1207 produced maximal isoamyl acetate formation at an alcohol/acid ratio of 1.6. Acetic acid at higher concentrations than required for the critical alcohol/acid ratio lower than 1.3 and higher than 1.6 resulted in decreased yields of isoamyl acetate probably owing to lowering of micro-aqueous environmental pH around the enzyme leading to inhibition of enzyme activity. Mycelium bound A. niger lipase produced 80 g/l of isoamyl acetate within 96 h even though extremely less amount of enzyme activity was used for esterification. The presence of sodium sulphate during esterification reaction at higher substrate concentration resulted in increased conversion efficiency when we used mycelium bound enzyme preparations of A. niger NCIM 1207. This could be due to removal of excess water released during esterification reaction by sodium sulphate. High ester concentration (286.5 g/l) and conversion (73.5%) were obtained within 24 h using Novozyme 435 under these conditions.

  10. Enhanced expression of lipase I from Galactomyces geotrichum by codon optimisation in Pichia pastoris.

    PubMed

    Qiao, Hanzhen; Zhang, Wenfei; Guan, Wutai; Chen, Fang; Zhang, Shihai; Deng, Zixiao

    2017-10-01

    Relatively poor heterologous protein yields have limited the commerical applications of Galactomyces geotrichum lipase I (GGl I) efficacy trials. To address this, we have redesigned the GGl I gene to preferentially match codon frequencies of Pichia pastoris (P. pastoris) while retaining the same amino acid sequence. The wild type and codon optimised GGl I (GGl I-wt and GGl I-op) were synthesised and cloned into pPICZαA with an N-terminal 6 × His tag sequence and expressed in P. pastoris X 33. The hydrolytic activity of GGl I-op was 150 U/mL, whereas the activity of the GGl I-wt could not be detected. GGl I-op recombinant proteins were purified by Ni-affinity chromatography and then characterised. The identity and purity of GGl I were confirmed by SDS-PAGE, MALDI-TOF mass spectrometry and Western blot analysis. Enzymatic deglycosylation was used to show that the lipase is a glycosylated protein, containing ∼10% sugar. The molecular weight (MW) of the GGl I secreted by recombinant P. pastoris was approximated at 63 kDa. The optimum pH and temperature of the recombinant lipase were 8.0 and 35 °C, respectively. The enzyme was active over a broad pH range (7.0-9.0) and temperature range (20 °C-45 °C). The lipase showed high activity toward medium- and long-chain fatty acid methyl esters (C8-C16) and retained much of its activity in the presence of Tween-80 and Trition X-100. Lipase activity was stimulated by Mg 2+ , Ca 2+ , Mn 2+ and Cu 2+ and inhibited by Fe 2+ , Fe 3+ , Zn 2+ and Co 2+ . This lipase may prove useful to the detergent industry and in organic synthesis reactions. Copyright © 2017. Published by Elsevier Inc.

  11. Purification and characterization of extracellular lipase from a new strain: Pseudomonas aeruginosa SRT 9

    PubMed Central

    Borkar, Prita S.; Bodade, Ragini G.; Rao, Srinivasa R.; Khobragade, C.N.

    2009-01-01

    An extra cellular lipase was isolated and purified from the culture broth of Pseudomonas aeruginosa SRT 9 to apparent homogeneity using ammonium sulfate precipitation followed by chromatographic techniques on phenyl Sepharose CL- 4B and Mono Q HR 5/5 column, resulting in a purification factor of 98 fold with specific activity of 12307.8 U/mg. The molecular weight of the purified lipase was estimated by SDS-PAGE to be 29 kDa with isoelectric point of 4.5. Maximum lipase activity was observed in a wide range of temperature and pH values with optimum temperature of 55ºC and pH 6.9. The lipase preferably acted on triacylglycerols of long chain (C14-C16) fatty acids. The lipase was inhibited strongly by EDTA suggesting the enzyme might be metalloprotein. SDS and metal ions such as Hg2+, Zn2+, Cu2+, Ag2+ and Fe2+ decreased the lipase activity remarkedly. Its marked stability and activity in organic solvents suggest that this lipase is highly suitable as a biotechnological tool with a variety of applications including organo synthetic reactions and preparation of enantiomerically pure pharmaceuticals. The Km and Vmax value of the purified enzyme for triolein hydrolysis were calculated to be 1.11 mmol/L and 0.05 mmol/L/min respectively. PMID:24031373

  12. Deciphering the toxicity of bisphenol a to Candida rugosa lipase through spectrophotometric methods.

    PubMed

    Zhang, Rui; Zhao, Lining; Liu, Rutao

    2016-10-01

    Bisphenol A is widely used in the manufacture of food packaging and beverage containers and can invade our food and cause contamination. Candida rugose lipase has been a versatile enzyme for biocatalysis and biotransformations to produce useful materials for food, pharmaceutical and flavor. The interactions between bisphenol A and Candida rugosa lipase in vitro were studied by UV-vis, steady-state fluorescence, circular dichroism, synchronous fluorescence, light scattering spectra, molecular docking and enzyme activity assay to better understand the toxicity and toxic mechanisms of bisphenol A. The intrinsic fluorescence of the tryptophan amino acid residue and the secondary structure of the globular protein candida rugose lipase were made use of to thoroughly investigate the structural changes caused by bisphenol A. The results of the fluorescence indicated that bisphenol A interacted with candida rugose lipase and made tryptophan be exposed to a hydrophobic environment. Multi-spectroscopic measurements showed that the addition of bisphenol A increased the intrinsic fluorescence of Candida rugosa lipase, loosened its skeleton structure and changed its secondary structure. Also, the increased activity of Candida rugosa lipase revealed that the position or the structure of the catalytic triad of Candida rugosa lipase may be changed. The molecular docking results showed that bisphenol A bound with the residue Serine 209 which could be another reason for the increased activity of Candida rugosa lipase. Moreover, as can be seen from the results of resonance light scattering and dynamic light scattering, the volume of the Candida rugosa lipase was decreased and the lid may be stripped. Copyright © 2016 Elsevier B.V. All rights reserved.

  13. Isolation and analysis of lipase-overproducing mutants of Serratia marcescens.

    PubMed

    Kawai, E; Akatsuka, H; Sakurai, N; Idei, A; Matsumae, H; Shibatani, T; Komatsubara, S; Omori, K

    2001-01-01

    We have isolated a lipase-overproducing mutant, GE14, from Serratia marcescens 8000 after three rounds of N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis. The mutant GE14 produced 95 kU/ml of extracellular lipase in the lipase medium, which was about threefold higher than that of produced by the original strain 8000. Enzymatic characteristics including specific activity of purified lipases from culture supernatants of GE14 and 8000 were almost same. The lipase gene (lipA) of GE14 contained two base substitutions; one in the promoter region and another in the N-terminal region of the lipA gene without an amino acid substitution. Promoter analysis using lipA-lacZ fusion plasmids revealed that these substitutions were responsible for the increase in the lipA expression level, independently. In contrast, no base substitution was found in the genes encoding the lipase secretion device, the Lip system. In addition, the genes coding for metalloprotease and the cell surface layer protein which are both secreted through the Lip system and associated with extracellular lipase production, also contained no base substitution. The strain GE14 carrying a high-copy-number lipA plasmid produced a larger amount of the extracellular lipase than the recombinant strains of 8000 and other mutants also did, indicating that GE14 was not only a lipase-overproducing strain, but also an advantageous host strain for overproducing the lipase by a recombinant DNA technique. These results suggest that the lipase-overproducing mutant GE14 and its recombinant strains are promising candidates for the industrial production of the S. marcescens lipase.

  14. Lipase-catalyzed enantioselective synthesis of (R,R)-lactide from alkyl lactate to produce PDLA (poly D-lactic acid) and stereocomplex PLA (poly lactic acid).

    PubMed

    Jeon, Byoung Wook; Lee, Jumin; Kim, Hyun Sook; Cho, Dae Haeng; Lee, Hyuk; Chang, Rakwoo; Kim, Yong Hwan

    2013-10-20

    R-lactide, a pivotal monomer for the production of poly (D-lactic acid) (PDLA) or stereocomplex poly (lactic acid) (PLA) was synthesized from alkyl (R)-lactate through a lipase-catalyzed reaction without racemization. From among several types of lipase, only lipase B from Candida antarctica (Novozym 435; CAL-B) was effective in the reaction that synthesized (R,R)-lactide. Enantiopure (R,R)-lactide, which consisted of over 99% enantiomeric excess, was synthesized from methyl (R)-lactate through CAL-B catalysis. Removal of the methanol by-product was critical to obtain a high level of lactide conversion. The (R,R)-lactide yield was 56% in a reaction containing 100 mg of Novozym 435, 10 mM methyl (R)-lactate and 1500 mg of molecular sieve 5A in methyl tert-butyl ether (MTBE). The important monomer (R,R)-lactide that is required for the production of the widely recognized bio-plastic PDLA and the PLA stereocomplex can be obtained using this novel synthetic method. Copyright © 2013 Elsevier B.V. All rights reserved.

  15. A broad pH range indicator-based spectrophotometric assay for true lipases using tributyrin and tricaprylin[S

    PubMed Central

    Camacho-Ruiz, María de los Angeles; Mateos-Díaz, Juan Carlos; Carrière, Frédéric; Rodriguez, Jorge A.

    2015-01-01

    A continuous assay is proposed for the screening of acidic, neutral, or alkaline lipases using microtiter plates, emulsified short- and medium-chain TGs, and a pH indicator. The lipase activity measurement is based on the decrease of the pH indicator optical density due to protonation which is caused by the release of FFAs during the hydrolysis of TGs and thus acidification. Purified lipases with distinct pH optima and an esterase were used to validate the method. The rate of lipolysis was found to be linear with time and proportional to the amount of enzyme added in each case. Specific activities measured with this microplate assay method were lower than those obtained by the pH-stat technique. Nevertheless, the pH-dependent profiles of enzymatic activity were similar with both assays. In addition, the substrate preference of each enzyme tested was not modified and this allowed discriminating lipase and esterase activities using tributyrin (low water solubility) and tricaprylin (not water soluble) as substrates. This continuous lipase assay is compatible with a high sample throughput and can be applied for the screening of lipases and lipase inhibitors from biological samples. PMID:25748441

  16. A plasmonic nanosensor for lipase activity based on enzyme-controlled gold nanoparticles growth in situ

    NASA Astrophysics Data System (ADS)

    Tang, Yan; Zhang, Wei; Liu, Jia; Zhang, Lei; Huang, Wei; Huo, Fengwei; Tian, Danbi

    2015-03-01

    A plasmonic nanosensor for lipase activity was developed based on one-pot nanoparticle growth. Tween 80 was selected not only as the substrate for lipase recognition but also as the reducing and stabilizing agent for the sensor fabrication. The different molecular groups in Tween 80 could have different roles in the fabrication procedure; the H2O2 produced by the autoxidation of the ethylene oxide subunits in Tween 80 could reduce the AuCl4- ions to Au atoms, meanwhile, the lipase could hydrolyze its carboxyl ester bond, which could, in turn, control the rate of nucleation of the gold nanoparticles (AuNPs) and tailor the localized surface plasmon resonance (LSPR) of the AuNP transducers. The color changes, which depend on the absence or presence of the lipase, could be used to sense the lipase activity. A linear response ranging from 0.025 to 4 mg mL-1 and a detection limit of the lipase as low as 3.47 μg mL-1 were achieved. This strategy circumvents the problems encountered by general enzyme assays that require sophisticated instruments and complicated assembling steps. The methodology can benefit the assays of heterogeneous-catalyzed enzymes.A plasmonic nanosensor for lipase activity was developed based on one-pot nanoparticle growth. Tween 80 was selected not only as the substrate for lipase recognition but also as the reducing and stabilizing agent for the sensor fabrication. The different molecular groups in Tween 80 could have different roles in the fabrication procedure; the H2O2 produced by the autoxidation of the ethylene oxide subunits in Tween 80 could reduce the AuCl4- ions to Au atoms, meanwhile, the lipase could hydrolyze its carboxyl ester bond, which could, in turn, control the rate of nucleation of the gold nanoparticles (AuNPs) and tailor the localized surface plasmon resonance (LSPR) of the AuNP transducers. The color changes, which depend on the absence or presence of the lipase, could be used to sense the lipase activity. A linear response

  17. Comparative study on digestive lipase activities on the self emulsifying excipient Labrasol, medium chain glycerides and PEG esters.

    PubMed

    Fernandez, Sylvie; Jannin, Vincent; Rodier, Jean-David; Ritter, Nicolas; Mahler, Bruno; Carrière, Frédéric

    2007-05-01

    Labrasol is a lipid-based self-emulsifying excipient used in the preparation of lipophilic drugs intended for oral delivery. It is mainly composed of PEG esters and glycerides with medium acyl chains, which are potential substrates for digestive lipases. The hydrolysis of Labrasol by porcine pancreatic extracts, human pancreatic juice and several purified digestive lipases was investigated in the present study. Classical human pancreatic lipase (HPL) and porcine pancreatic lipase, which are the main lipases involved in the digestion of dietary triglycerides, showed very low levels of activity on the entire Labrasol excipient as well as on separated fractions of glycerides and PEG esters. On the other hand, gastric lipase, pancreatic lipase-related protein 2 (PLRP2) and carboxyl ester hydrolase (CEH) showed high specific activities on Labrasol. These lipases were found to hydrolyze the main components of Labrasol (PEG esters and monoglycerides) used as individual substrates, whereas these esters were found to be poor substrates for HPL. The lipolytic activity of pancreatic extracts and human pancreatic juice on Labrasol(R) is therefore mainly due to the combined action of CEH and PLRP2. These two pancreatic enzymes, together with gastric lipase, are probably the main enzymes involved in the in vivo lipolysis of Labrasol taken orally.

  18. 21 CFR 862.1465 - Lipase test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... Lipase test system. (a) Identification. A lipase test system is a device intended to measure the activity of the enzymes lipase in serum. Lipase measurements are used in diagnosis and treatment of diseases...

  19. 21 CFR 862.1465 - Lipase test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... Lipase test system. (a) Identification. A lipase test system is a device intended to measure the activity of the enzymes lipase in serum. Lipase measurements are used in diagnosis and treatment of diseases...

  20. One-pot conjugated linoleic acid production from castor oil by Rhizopus oryzae lipase and resting cells of Lactobacillus plantarum.

    PubMed

    Khaskheli, Abid Ali; Talpur, Farah Naz; Cebeci Aydin, Aysun; Jawaid, Sana; Surhio, Muhammad Ali; Afridi, Hassan Imran

    2017-10-01

    Conjugated linoleic acid (CLA) has attracted as novel type of fatty acids having unusual health-promoting properties such as anticarcinogenic and antiobesitic effects. The present work employed castor oil as substrate for one-pot production of CLA using washed cells of Lactobacillus plantarum (L. plantarum) and lipases as catalysts. Among the screened lipases, the lipase Rhizopus oryzae (ROL) greatly assisted resting cells to produce CLA. Mass spectral analysis of the product showed that two major isomers of CLA were produced in the reaction mixture i.e. cis-9, trans-11 56.55% and trans-10, cis-12 43.45%. Optimum factors for CLA synthesis were found as substrate concentration (8 mg/mL), pH (6.5), washed cell concentration (12% w/v), and incubation time of 20 h. Hence, the combination of ROL with L. plantarum offers one pot production of CLA selectively using castor oil as a cost-effective substrate.

  1. A broad pH range indicator-based spectrophotometric assay for true lipases using tributyrin and tricaprylin.

    PubMed

    Camacho-Ruiz, María de Los Angeles; Mateos-Díaz, Juan Carlos; Carrière, Frédéric; Rodriguez, Jorge A

    2015-05-01

    A continuous assay is proposed for the screening of acidic, neutral, or alkaline lipases using microtiter plates, emulsified short- and medium-chain TGs, and a pH indicator. The lipase activity measurement is based on the decrease of the pH indicator optical density due to protonation which is caused by the release of FFAs during the hydrolysis of TGs and thus acidification. Purified lipases with distinct pH optima and an esterase were used to validate the method. The rate of lipolysis was found to be linear with time and proportional to the amount of enzyme added in each case. Specific activities measured with this microplate assay method were lower than those obtained by the pH-stat technique. Nevertheless, the pH-dependent profiles of enzymatic activity were similar with both assays. In addition, the substrate preference of each enzyme tested was not modified and this allowed discriminating lipase and esterase activities using tributyrin (low water solubility) and tricaprylin (not water soluble) as substrates. This continuous lipase assay is compatible with a high sample throughput and can be applied for the screening of lipases and lipase inhibitors from biological samples. Copyright © 2015 by the American Society for Biochemistry and Molecular Biology, Inc.

  2. Optimization of Lipase-Mediated Synthesis of 1-Nonene Oxide Using Phenylacetic Acid and Hydrogen Peroxide

    PubMed Central

    Abdulmalek, Emilia; Arumugam, Mahashanon; Basri, Mahiran; Rahman, Mohd Basyaruddin Abdul

    2012-01-01

    Herein, an efficient epoxidation of 1-nonene is described. In a simple epoxidation system, commercially available Novozym 435, an immobilized Candida antarctica lipase B, and hydrogen peroxide (H2O2) were utilized to facilitate the in situ oxidation of phenylacetic acid to the corresponding peroxy acid which then reacted with 1-nonene to give 1-nonene oxide with high yield and selectivity. The aliphatic terminal alkene was epoxidised efficiently in chloroform to give an excellent yield (97%–99%) under the optimum reaction conditions, including temperature (35 °C), initial H2O2 concentration (30%), H2O2 amount (4.4 mmol), H2O2 addition rate (one step), acid amount (8.8 mmol), and stirring speed (250 rpm). Interestingly, the enzyme was stable under the single-step addition of H2O2 with a catalytic activity of 190.0 Ug−1. The entire epoxidation process was carried out within 12 h using a conventional water bath shaker. PMID:23202943

  3. Fasting upregulates adipose triglyceride lipase and hormone-sensitive lipase levels and phosphorylation in mouse kidney.

    PubMed

    Marvyn, Phillip M; Bradley, Ryan M; Button, Emily B; Mardian, Emily B; Duncan, Robin E

    2015-06-01

    Circulating non-esterified fatty acids (NEFA) rise during fasting and are taken up by the kidneys, either directly from the plasma or during re-uptake of albumin from glomerular filtrate, and are stored as triacylglycerol (TAG). Subsequent utilization of stored fatty acids requires their hydrolytic release from cellular lipid droplets, but relatively little is known about renal lipolysis. We found that total [(3)H]triolein hydrolase activity of kidney lysates was significantly increased by 15% in the fasted state. Adipose triglyceride lipase (Atgl) and hormone-sensitive lipase (Hsl) mRNA expression was time-dependently increased by fasting, along with other fatty acid metabolism genes (Pparα, Cd36, and Aox). ATGL and HSL protein levels were also significantly induced (by 239 ± 7% and 322 ± 8%, respectively). Concomitant with changes in total protein levels, there was an increase in ATGL phosphorylation at the AMPK-regulated serine 406 site in the 14-3-3 binding motif, and an increase in HSL phosphorylation at serines 565 and 660 that are regulated by AMPK and PKA, respectively. Using immunofluorescence, we further demonstrate nearly ubiquitous expression of ATGL in the renal cortex with a concentration on the apical/lumenal surface of some cortical tubules. Our findings suggest a role for ATGL and HSL in kidney lipolysis.

  4. Oyster mushroom’s lipase enzyme entrapment on calcium alginate as biocatalyst in the synthesis of lauryl diethanolamide

    NASA Astrophysics Data System (ADS)

    Wijayati, N.; Masubah, K.; Supartono

    2017-02-01

    Lipase is an enzyme with large biotechnology applications, such as hydrolysis in the food industry, applications in chemical industry, synthesis of polymers and surfactants. Lipase was isolated from oyster mushroom with activity 0,93 U/mg and protein content 1,1234 mg/mL. Lipase was immobilized by entrapment method in a matrix of Ca-alginate. This report describes that we have developed for the synthesis of lauryl diethanolamide The result showed that the optimum condition of lipase immobilization was achieved on 3% Na-alginate solution with protein content 0,84 mg/mL and the activity 3,33 U/mg. An amide (22.911%) formed from the amidation of lauric acid and diethanolamine.

  5. Spleen dimensions are inversely associated with lysosomal acid lipase activity in patients with non-alcoholic fatty liver disease.

    PubMed

    Polimeni, Licia; Pastori, Daniele; Baratta, Francesco; Tozzi, Giulia; Novo, Marta; Vicinanza, Roberto; Troisi, Giovanni; Pannitteri, Gaetano; Ceci, Fabrizio; Scardella, Laura; Violi, Francesco; Angelico, Francesco; Del Ben, Maria

    2017-12-01

    Fatty liver and splenomegaly are typical features of genetic lysosomal acid lipase (LAL) deficiency. No data in adult patients with non-genetic reduction of LAL activity are available. We investigate the association between spleen dimensions and LAL activity in non-alcoholic fatty liver disease (NAFLD) patients, in whom a reduced LAL activity has been reported. We include 425 consecutive patients who underwent abdominal ultrasound to evaluate hepatic steatosis and spleen dimensions. LAL activity was measured with dried blood spot method (Lalistat2). NAFLD was present in 74.1% of screened patients. Higher median spleen longitudinal diameter (10.6 vs. 9.9 cm; p < 0.001) and spleen area (SA) (32.7 vs. 27.7 cm 2 ; p < 0.001), together with a higher and proportion of splenomegaly (17.8 vs. 5.5%, p = 0.001), are present in patients with NAFLD compared to those without. In NAFLD patients, median LAL activity is 0.9 nmol/spot/h. LAL activity is lower in 56 patients with splenomegaly, as compared to those without (p = 0.009). At multivariable logistic regression analysis, age (above median, OR 0.344; p = 0.003), LAL activity (below median, OR 2.206, p = 0.028), and platelets (OR 0.101, p = 0.002) are significantly associated with splenomegaly. NAFLD patients disclose a relatively high prevalence of spleen enlargement and splenomegaly, which are significantly associated with a reduced LAL activity, suggesting that LAL may contribute to spleen enlargement in this setting.

  6. The Effect of Storage at Three Different Temperatures on the Activity of Lipase Solution.

    ERIC Educational Resources Information Center

    Bradley, Karen; Mathewman, David

    1984-01-01

    Presented are procedures used to assay the activity of lipase during storage at three different temperatures. Since lipase solutions can decay even when refrigerated, it is recommended that the enzyme be freshly prepared prior to laboratory sessions in which they are used. (JN)

  7. Implication of substrate-assisted catalysis on improving lipase activity or enantioselectivity in organic solvents.

    PubMed

    Tsai, Shau-Wei; Chen, Chun-Chi; Yang, Hung-Shien; Ng, I-Son; Chen, Teh-Liang

    2006-08-01

    In comparison with the biocatalyst engineering and medium engineering approaches, very few examples have been reported on using the substrate engineering approach such as substrate-assisted catalysis (SAC) for naturally occurring or engineered lipases and serine proteases to improve the enzyme activity and enantioselectivity. By employing lipase-catalyzed hydrolysis of (R,S)-naproxen esters in water-saturated isooctane as the model system, we demonstrate the proton shuttle device to the leaving alcohol of the substrate as a new means of SAC to effectively improve the lipase activity or enantioselectivity. The result cannot only provide a strong evidence for the rate-limiting proton transfer for the bond-breaking of tetrahedron intermediate of the acylation step, but also sheds light for performing the hydrolysis, transesterification or aminolysis in organic solvents for the ester substrate that originally lipases cannot catalyze, but now can after introducing the device.

  8. Characterization of fatty acid modifying enzyme activity in staphylococcal mastitis isolates and other bacteria

    PubMed Central

    2012-01-01

    Background Fatty acid modifying enzyme (FAME) has been shown to modify free fatty acids to alleviate their bactericidal effect by esterifying fatty acids to cholesterol or alcohols. Although it has been shown in previous studies that FAME is required for Staphylococcus aureus survival in skin abscesses, FAME is poorly studied compared to other virulence factors. FAME activity had also been detected in coagulase-negative staphylococci (CNS). However, FAME activity was only surveyed after a bacterial culture was grown for 24 h. Therefore if FAME activity was earlier in the growth phase, it would not have been detected by the assay and those strains would have been labeled as FAME negative. Results Fifty CNS bovine mastitis isolates and several S. aureus, Escherichia coli, and Streptococcus uberis strains were assayed for FAME activity over 24 h. FAME activity was detected in 54% of CNS and 80% S. aureus strains surveyed but none in E. coli or S. uberis. While some CNS strains produced FAME activity comparable to the lab strain of S. aureus, the pattern of FAME activity varied among strains and across species of staphylococci. All CNS that produced FAME activity also exhibited lipase activity. Lipase activity relative to colony forming units of these CNS decreased over the 24 h growth period. No relationship was observed between somatic cell count in the milk and FAME activity in CNS. Conclusions Some staphylococcal species surveyed produced FAME activity, but E. coli and S. uberis strains did not. All FAME producing CNS exhibited lipase activity which may indicate that both these enzymes work in concert to alter fatty acids in the bacterial environment. PMID:22726316

  9. Influence of gold nanoparticles of varying size in improving the lipase activity within cationic reverse micelles.

    PubMed

    Maiti, Subhabrata; Das, Dibyendu; Shome, Anshupriya; Das, Prasanta Kumar

    2010-02-08

    Herein, we report the effect of gold nanoparticles (GNPs) in enhancing lipase activity in reverse micelles of cetyltrimethylammonium bromide (CTAB)/water/isooctane/n-hexanol. The size and concentration of the nanoparticles were varied and their specific roles were assessed in detail. An overall enhancement of activity was observed in the GNP-doped CTAB reverse micelles. The improvement in activity becomes more prominent with increasing concentration and size of the GNPs (0-52 microM and ca. 3-30 nm, respectively). The observed highest lipase activity (k(2)=1070+/-12 cm(3) g(-1) s(-1)) in GNP-doped CTAB reverse micelles ([GNP]: 52 microm, ca. 20 nm) is 2.5-fold higher than in CTAB reverse micelles without GNPs. Improvement in the lipase activity is only specific to the GNP-doped reverse micellar media, whereas GNP deactivates and structurally deforms the enzyme in aqueous media. The reason for this activation is probably due to the formation of larger-sized reverse micelles in which the GNP acts as a polar core and the surfactants aggregate around the nanoparticle ('GNP pool') instead of only water. Lipase at the augmented interface of the GNP-doped reverse micelle showed improved activity because of enhancement in both the substrate and enzyme concentrations and increased flexibility in the lipase conformation. The extent of the activation is greater in the case of the larger-sized GNPs. A correlation has been established between the activity of lipase and its secondary structure by using circular dichroism and FTIR spectroscopic analysis. The generalized influence of GNP is verified in the reverse micelles of another surfactant, namely, cetyltripropylammonium bromide (CTPAB). TEM, dynamic light scattering (DLS), and UV/Vis spectroscopic analysis were utilized to characterize the GNPs and the organized aggregates. For the first time, CTAB-based reverse micelles have been found to be an excellent host for lipase simply by doping with appropriately sized GNPs.

  10. Neutrophil chemotaxis by Propionibacterium acnes lipase and its inhibition.

    PubMed Central

    Lee, W L; Shalita, A R; Suntharalingam, K; Fikrig, S M

    1982-01-01

    The chemoattraction of Propionibacterium acnes lipase for neutrophils and the effect of lipase inhibitor and two antibiotic agents on the chemotaxis were evaluated. Of the various fractions tested, partially purified lipase (fraction 2c) was the most active cytotaxin produced by P. acnes. Serum mediators were not required for the generation of chemotaxis by lipase in vitro. Diisopropyl phosphofluoridate at low concentration (10(-4) mM) completely inhibited lipase activity as well as polymorphonuclear leukocyte chemotaxis generated by lipase. Tetracycline hydrochloride and erythromycin base at concentrations of 10(-1) mM and 1 mM, respectively, caused 100% inhibition of PMN migration toward lipase or zymosan-activated serum. The inhibiting activity of the antibiotics was directed against cells independently of any effect on lipase. Chemotaxis by P. acnes lipase suggests a wider role for this enzyme in the inflammatory process and the pathogenesis of acne vulgaris. Images PMID:7054130

  11. Lipase-catalyzed production of short-chain acids terpenyl esters of interest to the food industry.

    PubMed

    Laboret, F; Perraud, R

    1999-12-01

    The production of low molecular weight esters as flavor compounds by biotechnological processes has a potential interest for the food industry. The use of natural available substrates and enzymes is an essential part of the process design, because the products may obtain natural label. In this study, direct esterification of citronellol and geraniol with short-chain fatty acids catalyzed by free lipase from Mucor miehei was performed with high yields in n-hexane. The effects of the acid:alcohol ratio on the bioconversion rate of increasing chain length esters was investigated. To reach the optimum yield, substrates and enzyme concentration were determined. The inhibiting effects of acid are strongly attenuated by reducing the quantity of acid and increasing the amount of enzyme in media following the optimum values. Improvements have been made to increase the ester purity. The consumption of excess substrate by adding calculated amounts of acid gives a 10% yield enhancement, and leads to 100% pure terpenyl esters. The first steps to a scale-up application were attempted using a reactor that allowed us to produce ester quantities up to 100 cm3. Separation and purification of the products were treated with success, underlining the lipase stability and efficiency under the conditions of this study. The ability to recover the enzyme, and reusing it in bioconversions, plays a major role in reducing the cost of the overall process.

  12. Mechanism of acetaldehyde-induced deactivation of microbial lipases

    PubMed Central

    2011-01-01

    Background Microbial lipases represent the most important class of biocatalysts used for a wealth of applications in organic synthesis. An often applied reaction is the lipase-catalyzed transesterification of vinyl esters and alcohols resulting in the formation of acetaldehyde which is known to deactivate microbial lipases, presumably by structural changes caused by initial Schiff-base formation at solvent accessible lysine residues. Previous studies showed that several lipases were sensitive toward acetaldehyde deactivation whereas others were insensitive; however, a general explanation of the acetaldehyde-induced inactivation mechanism is missing. Results Based on five microbial lipases from Candida rugosa, Rhizopus oryzae, Pseudomonas fluorescens and Bacillus subtilis we demonstrate that the protonation state of lysine ε-amino groups is decisive for their sensitivity toward acetaldehyde. Analysis of the diverse modification products of Bacillus subtilis lipases in the presence of acetaldehyde revealed several stable products such as α,β-unsaturated polyenals, which result from base and/or amino acid catalyzed aldol condensation of acetaldehyde. Our studies indicate that these products induce the formation of stable Michael-adducts at solvent-accessible amino acids and thus lead to enzyme deactivation. Further, our results indicate Schiff-base formation with acetaldehyde to be involved in crosslinking of lipase molecules. Conclusions Differences in stability observed with various commercially available microbial lipases most probably result from different purification procedures carried out by the respective manufacturers. We observed that the pH of the buffer used prior to lyophilization of the enzyme sample is of utmost importance. The mechanism of acetaldehyde-induced deactivation of microbial lipases involves the generation of α,β-unsaturated polyenals from acetaldehyde which subsequently form stable Michael-adducts with the enzymes. Lyophilization of

  13. Production and Characterization of Lipases by Two New Isolates of Aspergillus through Solid-State and Submerged Fermentation

    PubMed Central

    Colla, Luciane Maria; Ficanha, Aline M. M.; Rizzardi, Juliana; Bertolin, Telma Elita; Reinehr, Christian Oliveira; Costa, Jorge Alberto Vieira

    2015-01-01

    Due to the numerous applications of lipases in industry, there is a need to study their characteristics, because lipases obtained from different sources may present different properties. The aim of this work was to accomplish the partial characterization of lipases obtained through submerged fermentation and solid-state fermentation by two species of Aspergillus. Fungal strains were isolated from a diesel-contaminated soil and selected as good lipases producers. Lipases obtained through submerged fermentation presented optimal activities at 37°C and pH 7.2 and those obtained through solid-state fermentation at 35°C and pH 6.0. The enzymes produced by submerged fermentation were more temperature-stable than those obtained by solid-state fermentation, presenting 72% of residual activity after one hour of exposition at 90°C. Lipases obtained through submerged fermentation had 80% of stability in acidic pH and those obtained through solid-state fermentation had stability greater than 60% in alkaline pH. PMID:26180809

  14. Microwave assisted synthesis and anti-lipase activity of some new fluorine-containing benzimidazoles.

    PubMed

    Menteşe, E; Yilmaz, F; Ülker, S; Kahveci, B

    2015-01-01

    In this study, a new series of fluorine containing benzimidazoles (4a-l) and bisbenzimidazoles (6a-c, 8) were synthesized by the reaction of o-phenylenediamines with iminoester hydrochlorides (3a-l, 7) in methanol under microwave irradiation. The structures of these newly synthesized compounds were identified by IR, (1)H-NMR, (13)C-NMR, mass spectroscopy and elemental analysis data. The synthesized compounds were screened for their pancreatic lipase activities. Our results indicate that the compounds 6a, 6b and 6c can serve as an anti-lipase agent. The compounds 6b and 6c inhibited pancreatic lipase activity by 84.03% and 97.49% at a concentration of 3 µg/mL, respectively. © Georg Thieme Verlag KG Stuttgart · New York.

  15. A Phase 3 Trial of Sebelipase Alfa in Lysosomal Acid Lipase Deficiency.

    PubMed

    Burton, Barbara K; Balwani, Manisha; Feillet, François; Barić, Ivo; Burrow, T Andrew; Camarena Grande, Carmen; Coker, Mahmut; Consuelo-Sánchez, Alejandra; Deegan, Patrick; Di Rocco, Maja; Enns, Gregory M; Erbe, Richard; Ezgu, Fatih; Ficicioglu, Can; Furuya, Katryn N; Kane, John; Laukaitis, Christina; Mengel, Eugen; Neilan, Edward G; Nightingale, Scott; Peters, Heidi; Scarpa, Maurizio; Schwab, K Otfried; Smolka, Vratislav; Valayannopoulos, Vassili; Wood, Marnie; Goodman, Zachary; Yang, Yijun; Eckert, Stephen; Rojas-Caro, Sandra; Quinn, Anthony G

    2015-09-10

    Lysosomal acid lipase is an essential lipid-metabolizing enzyme that breaks down endocytosed lipid particles and regulates lipid metabolism. We conducted a phase 3 trial of enzyme-replacement therapy in children and adults with lysosomal acid lipase deficiency, an underappreciated cause of cirrhosis and severe dyslipidemia. In this multicenter, randomized, double-blind, placebo-controlled study involving 66 patients, we evaluated the safety and effectiveness of enzyme-replacement therapy with sebelipase alfa (administered intravenously at a dose of 1 mg per kilogram of body weight every other week); the placebo-controlled phase of the study was 20 weeks long and was followed by open-label treatment for all patients. The primary end point was normalization of the alanine aminotransferase level. Secondary end points included additional disease-related efficacy assessments, safety, and side-effect profile. Substantial disease burden at baseline included a very high level of low-density lipoprotein cholesterol (≥190 mg per deciliter) in 38 of 66 patients (58%) and cirrhosis in 10 of 32 patients (31%) who underwent biopsy. A total of 65 of the 66 patients who underwent randomization completed the double-blind portion of the trial and continued with open-label treatment. At 20 weeks, the alanine aminotransferase level was normal in 11 of 36 patients (31%) in the sebelipase alfa group and in 2 of 30 (7%) in the placebo group (P=0.03), with mean changes from baseline of -58 U per liter versus -7 U per liter (P<0.001). With respect to prespecified key secondary efficacy end points, we observed improvements in lipid levels and reduction in hepatic fat content (P<0.001 for all comparisons, except P=0.04 for triglycerides). The number of patients with adverse events was similar in the two groups; most events were mild and were considered by the investigator to be unrelated to treatment. Sebelipase alfa therapy resulted in a reduction in multiple disease-related hepatic and

  16. Validation of a commercial 1,2-o-dilauryl-rac-glycero glutaric acid-(6'-methylresorufin) ester lipase assay for diagnosis of canine pancreatitis.

    PubMed

    Goodband, Emily L; Serrano, Gonçalo; Constantino-Casas, Fernando; Archer, Joy; Watson, Penny J; Williams, Tim L

    2018-01-01

    The objectives of this study were fourfold: technical validation of a commercial canine 1,2-o-dilauryl-rac-glycero glutaric acid-(6'-methylresorufin) ester (DGGR) lipase assay, to calculate a reference interval for DGGR lipase by the indirect a posteriori method, to establish biological validity of the assay, and to assess agreement between DGGR lipase and specific canine pancreatic lipase (Spec cPL) assays. Dogs with histologically confirmed acute pancreatitis (n=3), chronic pancreatitis (n=8) and normal pancreatic tissue (n=7) with stored (-80°C) serum samples were identified. Relevant controls were selected. Precision, reproducibility and linearity of DGGR lipase, and the effect of sample haemolysis and freezing, were assessed. Sensitivity and specificity of DGGR lipase and Spec cPL were determined. Agreement between these two parameters was calculated using Cohen's kappa coefficient (κ). The DGGR lipase assay demonstrated excellent precision, reproducibility and linearity. Sample haemolysis and storage at -80°C for 12 months did not influence the assay. DGGR lipase (>245IU/l) and Spec cPL (>400µg/l) both showed poor sensitivity but excellent specificity for acute pancreatitis, and poor to moderate sensitivity but excellent specificity for chronic pancreatitis. Substantial agreement (κ=0.679) was found between DGGR lipase and Spec cPL. The validated DGGR lipase assay had similar sensitivity and specificity for the diagnosis of acute and chronic pancreatitis to Spec cPL. DGGR lipase is a reliable alternative to Spec cPL for the diagnosis of pancreatitis.

  17. Structure-guided modification of Rhizomucor miehei lipase for production of structured lipids.

    PubMed

    Zhang, Jun-Hui; Jiang, Yu-Yan; Lin, Ying; Sun, Yu-Fei; Zheng, Sui-Ping; Han, Shuang-Yan

    2013-01-01

    To improve the performance of yeast surface-displayed Rhizomucor miehei lipase (RML) in the production of human milk fat substitute (HMFS), we mutated amino acids in the lipase substrate-binding pocket based on protein hydrophobicity, to improve esterification activity. Five mutants: Asn87Ile, Asn87Ile/Asp91Val, His108Leu/Lys109Ile, Asp256Ile/His257Leu, and His108Leu/Lys109Ile/Asp256Ile/His257Leu were obtained and their hydrolytic and esterification activities were assayed. Using Discovery Studio 3.1 to build models and calculate the binding energy between lipase and substrates, compared to wild-type, the mutant Asp256Ile/His257Leu was found to have significantly lower energy when oleic acid (3.97 KJ/mol decrease) and tripalmitin (7.55 KJ/mol decrease) were substrates. This result was in accordance with the esterification activity of Asp256Ile/His257Leu (2.37-fold of wild-type). The four mutants were also evaluated for the production of HMFS in organic solvent and in a solvent-free system. Asp256Ile/His257Leu had an oleic acid incorporation of 28.27% for catalyzing tripalmitin and oleic acid, and 53.18% for the reaction of palm oil with oleic acid. The efficiency of Asp256Ile/His257Leu was 1.82-fold and 1.65-fold that of the wild-type enzyme for the two reactions. The oleic acid incorporation of Asp256Ile/His257Leu was similar to commercial Lipozyme RM IM for palm oil acidolysis with oleic acid. Yeast surface-displayed RML mutant Asp256Ile/His257Leu is a potential, economically feasible catalyst for the production of structured lipids.

  18. Biodegradation of waste greases and biochemical properties of a novel lipase from Pseudomonas synxantha PS1.

    PubMed

    Cai, Xianghai; Chen, Siqi; Yang, Hong; Wang, Wei; Lin, Lin; Shen, Yaling; Wei, Wei; Wei, Dong-Zhi

    2016-07-01

    A lipase-producing bacterial strain was isolated from oil-well-produced water in Shengli oilfield (Shandong province, China) and was identified as Pseudomonas synxantha by 16S rDNA sequence analysis (named Pseudomonas synxantha PS1). Strain PS1 showed a maximum lipase activity of 10.8 U/mL after culturing for 48 h at 30 °C, with lactose (4 g/L) as carbon source, tryptone (8 g/L) as nitrogen source, olive oil (0.5%, v/v) as inductor, and the initial pH 8.0. Meanwhile, the lipase gene from P. synxantha PS1 was cloned and expressed in Escherichia coli BL21 with the vector pET28a. The novel gene (lipPS1) has an open reading frame of 1425 bp and encodes a 474 aa lipase (LipPS1) sharing the most identity (87%) with the lipase in Pseudomonas fluorescens. LipPS1 preferably acted on substrates with a long chain (C10-C18) of fatty acids. The optimum pH and temperature of the recombinant enzyme were 8.0 and 40 °C, respectively, towards the optimum substrate p-nitrophenyl palmitate. The LipPS1 showed remarkable stability under alkaline conditions and was stable at pH 7.0-10.0 (retaining more than 60% activity). From the organic solvents tests, the lipase was activated by 15% (v/v) methanol (112%), 15% ethanol (127%), and 15% n-butyl alcohol (116%). LipPS1 presented strong biodegradability of waste grease; 93% of waste grease was hydrolyzed into fatty acid after 12 h at 30 °C. This is the first report of the lipase activity and lipase gene obtained from P. synxantha (including wild strain and recombinant strain) and of the recombinant LipPS1 with the detailed enzymatic properties. Also a preliminary study of the biodegradability of waste greases shows the potential value in industry applications.

  19. Cell-bound lipases from Burkholderia sp. ZYB002: gene sequence analysis, expression, enzymatic characterization, and 3D structural model.

    PubMed

    Shu, Zhengyu; Lin, Hong; Shi, Shaolei; Mu, Xiangduo; Liu, Yanru; Huang, Jianzhong

    2016-05-03

    The whole-cell lipase from Burkholderia cepacia has been used as a biocatalyst in organic synthesis. However, there is no report in the literature on the component or the gene sequence of the cell-bound lipase from this species. Qualitative analysis of the cell-bound lipase would help to illuminate the regulation mechanism of gene expression and further improve the yield of the cell-bound lipase by gene engineering. Three predictive cell-bound lipases, lipA, lipC21 and lipC24, from Burkholderia sp. ZYB002 were cloned and expressed in E. coli. Both LipA and LipC24 displayed the lipase activity. LipC24 was a novel mesophilic enzyme and displayed preference for medium-chain-length acyl groups (C10-C14). The 3D structural model of LipC24 revealed the open Y-type active site. LipA displayed 96 % amino acid sequence identity with the known extracellular lipase. lipA-inactivation and lipC24-inactivation decreased the total cell-bound lipase activity of Burkholderia sp. ZYB002 by 42 % and 14 %, respectively. The cell-bound lipase activity from Burkholderia sp. ZYB002 originated from a multi-enzyme mixture with LipA as the main component. LipC24 was a novel lipase and displayed different enzymatic characteristics and structural model with LipA. Besides LipA and LipC24, other type of the cell-bound lipases (or esterases) should exist.

  20. Pseudomonas sp. BUP6 produces a thermotolerant alkaline lipase with trans-esterification efficiency in producing biodiesel.

    PubMed

    Priji, Prakasan; Sajith, Sreedharan; Faisal, Panichikkal Abdul; Benjamin, Sailas

    2017-12-01

    The present study describes the characteristics of a thermotolerant and alkaline lipase secreted by Pseudomonas sp. BUP6, a novel rumen bacterium isolated from Malabari goat, and its trans -esterification efficiency in producing biodiesel from used cooking oil (UCO). The extracellular lipase was purified to homogeneity (35.8 times purified with 14.8% yield) employing (NH 4 ) 2 SO 4 salt precipitation and Sephadex G-100 chromatography. The apparent molecular weight of this lipase on SDS-PAGE was 35 kDa, the identity of which was further confirmed by MALDI-TOF/MS. The purified lipase was found stable at a pH range of 7-9 with the maximum activity (707 U/ml) at pH 8.2; and was active at the temperature ranging from 35 to 50 °C with the optimum at 45 °C (891 U/ml). Triton X-100 and EDTA had no effect on the activity of lipase; whereas SDS, Tween-80 and β-mercaptoethanol inhibited its activity significantly. Moreover, Ca 2+ (1.0 mM) enhanced the activity of lipase (1428 U/ml) by 206% vis-à-vis initial activity; while Zn 2+ , Fe 2+ and Cu 2+ decreased the activity significantly. Using para -nitrophenyl palmitate as substrate, the K m (11.6 mM) and V max [668.9 μmol/(min/mg)] of the purified lipase were also determined. Crude lipase was used for analyzing its trans -esterification efficiency with used cooking oil and methanol which resulted in the worthy yield of fatty acid methyl esters, FAME (45%) at 37 °C, indicating its prospects in biodiesel industry. Thus, the lipase secreted by the rumen bacterium, Pseudomonas sp. BUP6, offers great potentials to be used in various industries including the production of biodiesel by trans -esterification.

  1. Inhibition of Propionibacterium acnes lipase activity by the antifungal agent ketoconazole.

    PubMed

    Unno, Mizuki; Cho, Otomi; Sugita, Takashi

    2017-01-01

    The common skin disease acne vulgaris is caused by Propionibacterium acnes. A lipase secreted by this microorganism metabolizes sebum and the resulting metabolites evoke inflammation in human skin. The antifungal drug ketoconazole inhibits P. acnes lipase activity. We previously showed that the drug also inhibits the growth of P. acnes. Thus, ketoconazole may serve as an alternative treatment for acne vulgaris, which is important because the number of antibiotic-resistant P. acnes strains has been increasing. © 2017 The Societies and John Wiley & Sons Australia, Ltd.

  2. Synthesis of retinyl palmitate catalyzed by Candida sp.99-125 lipase immobilized on fiber-like SBA-15 mesoporous material.

    PubMed

    Kai, Zhu; Jianqiang, Wang; Yan-hua, Wang; Hui, Liu; Ping-fang, Han; Ping, Wei

    2011-09-01

    Candida sp.99-125 lipase was suitable for transesterification of fats and oils to produce fatty acid methyl ester. The adsorption of Candida sp.99-125 lipase onto the fiber-like SBA-15 mesoporous material has been studied. The unaltered structural order of the fiber-like SBA-15 before and after the adsorption has been confirmed by FT-IR, SEM and N2 adsorption. The amount of adsorbed Candida sp.99-125 lipase depends both on the solution pH and reaction time. Good adsorption capacity of Candida sp.99-125 lipase on fiber-like SBA-15 may be due to solution pH from 5.0 to 9.0 especially at 7.0 (93.99 mg enzyme per gram silica is obtained and the activity recovery is 281.05%). A high lipase loading (135.9 mg enzyme per gram silica) was obtained, but it did not produce a proportionate level of catalytic activity. The immobilized Candida sp.99-125 lipase showed increased adaptability in the hydrolysis of p-nitrophenyl acetate compared to free Candida sp.99-125 lipase at pH 5.0-9.0. Meanwhile, the immobilized Candida sp.99-125 lipase showed higher thermal stability than that of free Candida sp.99-125 lipase. And the synthesis of retinyl palmitate in organic solvent with the immobilized Candida sp.99-125 lipase was investigated. The influence factors, such as: the solvent used, the molar ratio and concentrations of substrates, the reaction time and the amount of lipase were studied and optimized. In the conditions of transesterificating 0.164 g retinyl acetate and 0.32 g palmitic acid, 10 mL of solvent hexane, 1:4 of mass ratio of lipase to retinyl acetate, and 6 hours of reaction time, 74.6% of retinyl acetate was converted into retinyl plamitate.

  3. Efficient biocatalyst by encapsulating lipase into nanoporous gold

    PubMed Central

    2013-01-01

    Lipases are one of the most important biocatalysts for biotechnological applications. Immobilization is an efficient method to increase the stability and reusability of lipases. In this study, nanoporous gold (NPG), a new kind of nanoporous material with tunable porosity and excellent biocompatibility, was employed as an effective support for lipase immobilization. The pore size of NPG and adsorption time played key roles in the construction of lipase-NPG biocomposites. The morphology and composition of NPG before and after lipase loading are verified using a scanning electron microscope, equipped with an energy-dispersive X-ray spectrometer. The resulting lipase-NPG biocomposites exhibited excellent catalytic activity and remarkable reusability. The catalytic activity of the lipase-NPG biocomposite with a pore size of 35 nm had no decrease after ten recycles. Besides, the lipase-NPG biocomposite exhibited high catalytic activity in a broader pH range and higher temperature than that of free lipase. In addition, the leaching of lipase from NPG could be prevented by matching the protein’s diameter and pore size. Thus, the encapsulation of enzymes within NPG is quite useful for establishing new functions and will have wide applications for different chemical processes. PMID:23601503

  4. Influence of ammonium salts on the lipase/esterase activity assay using p-nitrophenyl esters as substrates.

    PubMed

    De Yan, Hong; Zhang, Yin Jun; Liu, Hong Cai; Zheng, Jian Yong; Wang, Zhao

    2013-01-01

    p-Nitrophenyl esters with a short-chain carboxylic group, such as p-nitrophenyl acetate (p-NPA) and p-nitrophenyl butyrate (p-NPB), could be effectively hydrolyzed by ammonium salts. p-Nitrophenyl esters were usually used as substrates to assay the lipase/esterase activity. Ammonium sulfate precipitation was often used to purify proteins, and some ammonium salts were usually used as nitrogen sources or inorganic salts for the lipase/esterase production. To study the effect of ammonium salts on the assay of the lipase/esterase activity, the contributing factors of hydrolysis of p-NPA/p-NPB catalyzed by ammonium salts were investigated. The lipase activities were compared in the presence and absence of ammonium sulfate. The hydrolysis reaction could be catalyzed under neutral and alkaline circumstances. The hydrolysis rate increased with the increase in the reaction temperature or the concentration of ammonium ion. When p-NPA was employed as the substrate for the analysis of the lipase/esterase activity, the effect of ammonium sulfate on the analysis could be neutralized by setting a control when the concentration of ammonium sulfate was less than 40% saturation. However, when the concentration of ammonium sulfate increased from 40% to 100% saturation, the enzyme activities decreased about 13-40%, which could not be ignored for accurate analysis of the enzyme activity. © 2013 International Union of Biochemistry and Molecular Biology, Inc.

  5. Covalent attachment of microbial lipase onto microporous styrene-divinylbenzene copolymer by means of polyglutaraldehyde.

    PubMed

    Dizge, Nadir; Keskinler, Bülent; Tanriseven, Aziz

    2008-10-01

    A novel method for immobilization of Thermomyces lanuginosus lipase onto polyglutaraldehyde-activated poly(styrene-divinylbenzene) (STY-DVB), which is a hydrophobic microporous support has been successfully developed. The copolymer was prepared by the polymerization of the continuous phase of a high internal phase emulsion (polyHIPE). The concentrated emulsion consists of a mixture of styrene and divinylbenzene containing a suitable surfactant and an initiator as the continuous phase and water as the dispersed phase. Lipase from T. lanuginosus was immobilized covalently with 85% yield on the internal surface of the hydrophobic microporous poly(styrene-divinylbenzene) copolymer and used as a biocatalyst for the transesterification reaction. The immobilized enzyme has been fully active 30 days in storage and retained the activity during the 15 repeated batch reactions. The properties of free and immobilized lipase were studied. The effects of protein concentration, pH, temperature, and time on the immobilization, activity, and stability of the immobilized lipase were also studied. The newly synthesized microporous poly(styrene-divinylbenzene) copolymer constitutes excellent support for lipase. It given rise to high immobilization yield, retains enzymatic activity for 30 days, stable in structure and allows for the immobilization of large amount of protein (11.4mg/g support). Since immobilization is simple yet effective, the newly immobilized lipase could be used in several application including oil hydrolysis, production of modified oils, biodiesel synthesis, and removal of fatty acids from oils.

  6. Hydrolysis of triolein in phospholipid vesicles and microemulsions by a purified rat liver acid lipase.

    PubMed

    Burrier, R E; Brecher, P

    1983-10-10

    An acid lipase was purified from rat liver lysosomes. Lipase purification involved affinity chromatography, gel filtration, and stabilization of the purified preparation using ethylene glycol and Triton X-100. A molecular weight of 67,000-69,000 was determined independently using density gradient centrifugation, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and gel filtration. To study enzyme action, model substrates were prepared by incorporating radiolabeled triolein into either unilamellar vesicles or microemulsions. Substrates were prepared by cosonicating aqueous dispersions of lecithin and triolein. Formation of vesicles or emulsions depended on the relative amount of each lipid and on sonication conditions. Vesicles were prepared at molar ratios between 70:1 and 26:1 (lecithin:triolein) and the microemulsion preparation at a molar ratio of 1:1. The substrate particles were of similar size (220-250 A) as determined by Bio-Gel A-15m chromatography. Hydrolysis of triolein contained in vesicles or emulsions was similar with respect to pH, temperature, and reaction products. Kinetic studies on vesicles with increasing triolein content showed progressively greater Vmax values (0-0.6 mumol/min/mg), and Vmax for the emulsion was 3.1 mumol/min/mg. Addition of human very low or low density lipoprotein produced a dose-dependent inhibition with both substrates. The results show that synthetically prepared microemulsions are stable and effective substrates for the acid lipase and indicate that surface-oriented triolein is hydrolyzed in both preparations.

  7. SITES OF LIPOPROTEIN LIPASE ACTIVITY IN ADIPOSE TISSUE PERFUSED WITH CHYLOMICRONS

    PubMed Central

    Blanchette-Mackie, E. Joan; Scow, Robert O.

    1971-01-01

    Lipoprotein lipase activity was studied in rat parametrial adipose tissue perfused with chylomicrons and in gelatin blocks containing postheparin plasma and chylomicrons. The tissues and blocks were fixed in glutaraldehyde and incubated in 0.035 M CaCl2-0.1 M Tris medium (pH 8.3) at 38°C. The doubly labeled chylomicron triglycerides (glycerol-3H and palmitate-14C) in the tissues and blocks were hydrolyzed during incubation to free fatty acids (FFA) and the FFA remained in the specimens; hydrolysis was inhibited by 0.004 M diethyl paranitrophenyl phosphate (E-600). Incubated blocks and tissue were treated with 0.05 M Pb(NO3)2, postfixed in OsO4, dehydrated with acetone, embedded in Epon, and examined by electron microscopy. The incubated blocks contained electronlucent areas and granular and laminar precipitates at sites of hydrolysis. Similar precipitates were found in incubated tissue, within vacuoles and microvesicles of capillary endothelium, and in the subendothelial space (between the endothelium and pericytes), but not in the capillary lumen or in or near fat cells. The cytochemical reaction was greatly reduced, in blocks and tissues incubated with E-600. It is concluded that plasma glycerides are hydrolyzed by lipoprotein lipase in capillary endothelial cells and in the subendothelial space of adipose tissue and that glycerides across the endothelial cells within a membrane-bounded system. PMID:4329521

  8. The effect of diet on the expression of lipase genes in the midgut of the lightbrown apple moth (Epiphyas postvittana Walker; Tortricidae).

    PubMed

    Christeller, J T; Poulton, J; Markwick, N M; Simpson, R M

    2010-02-01

    We have identified lipase-like genes from an Epiphyas postvittana larval midgut EST library. Of the 10 pancreatic lipase family genes, six appear to encode active lipases and four encode inactive lipases, based on the presence/absence of essential catalytic residues. The four gastric lipase family genes appear to encode active proteins. Phylogenetic analysis of 54 lepidopteran pancreatic lipase proteins resolved the clade into five groups of midgut origin and a sixth of non-midgut lipases. The inactive proteins formed two separate groups with highly conserved mutations. The lepidopteran midgut lipases formed a ninth subfamily of pancreatic lipases. Eighteen insect and human gastric lipases were analysed phylogenetically with only very weak support for any groupings. Gene expression was measured in the larval midgut following feeding on five artificial diets and on apple leaves. The artificial diets contained different levels of triacylglycerol, linoleic acid and cholesterol. Significant changes in gene expression (more than 100-fold for active pancreatic lipases) were observed. All the inactive lipases were also highly expressed. The gastric lipase genes were expressed at lower levels and suppressed in larvae feeding on leaves. Together, protein motif analysis and the gene expression data suggest that, in phytophagous lepidopteran larvae, the pancreatic lipases may function in vivo as galactolipases and phospholipases whereas the gastric lipases may function as triacylglycerol hydrolases.

  9. Lipase immobilized on the hydrophobic polytetrafluoroethene membrane with nonwoven fabric and its application in intensifying synthesis of butyl oleate.

    PubMed

    Wang, Shu-Guang; Zhang, Wei-Dong; Li, Zheng; Ren, Zhong-Qi; Liu, Hong-Xia

    2010-11-01

    The synthesis of butyl oleate was studied in this paper with immobilized lipase. Five types of membrane were used as support to immobilize Rhizopus arrhizus lipase by following a procedure combining filtration and protein cross-linking. Results showed that hydrophobic polytetrafluoroethene membrane with nonwoven fabric (HO-PTFE-NF) was the favorite choice in terms of higher protein loading, activity, and specific activity of immobilized lipase. The factors including solvent polarity, lipase dosage, concentration, and molar ratio of substrate and temperature were found to have significant influence on conversion. Results showed that hexane (logP = 3.53) was a favorable solvent for the biosynthesis of butyl oleate in our studies. The optimal conditions were experimentally determined of 50 U immobilized lipase, molar ratio of oleic acid to butanol of 1.0, substrate concentration of 0.12 mol/L, temperature of 37 °C, and reaction time of 2 h. The conversion was beyond 91% and decreased slightly after 18 cycles. Lipase immobilization can improve the conversion and the repeated use of immobilized lipase relative to free lipase.

  10. Triacylglycerol kinetics in endotoxic rats with suppressed lipoprotein lipase activity

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Bagby, G.J.; Corll, C.B.; Martinez, R.R.

    1987-07-01

    Hypertriglyceridemia observed in animals after bacterial endotoxin administration and some forms of sepsis can result from increased hepatic triacylglycerol (TG) output or decreased TG clearance by extrahepatic tissues. To differentiate between these two possibilities, TG and free fatty acid (FFA) kinetics were determined in control and endotoxin-injected rats 18 h after treatment. Plasma TG and FFA kinetics were assessed by a constant intravenous infusion with (9,10-/sup 3/H)palmitate-labeled very low-density lipoprotein and (1-/sup 14/C)palmitate bound to albumin, respectively. In addition, lipoprotein lipase (LPL) activity was determined in heart, skeletal muscle, and adipose tissue as well as in postheparin plasma of functionallymore » hepatectomized, adrenalectomized, and gonadectomized rats. Plasma FFA acid concentrations were slightly increased in endotoxin-treated rats but their turnover did not differ from control. Endotoxin-treated rats had a threefold increase in plasma TG concentrations and decreased heart, skeletal muscle, and post-heparin plasma LPL activity. Plasma TG turnover was decreased, indicating that hypertriglyceridemia was not due to an increased TG output by the liver. Instead, the endotoxin-induced increase in plasma TG concentration was consequence of the 80% reduction in TG metabolic clearance rate. Thus, suppression of LPL activity in endotoxic animals impairs TG clearance resulting in hypertriglyceridemia. Furthermore, endotoxin administration reduced the delivery of TG-FFA to extrahepatic tissues because hepatic synthesis and secretion of TG from plasma FFA was decreased and LPL activity was suppressed.« less

  11. Effect of preduodenal lipase inhibition in suckling rats on dietary octanoic acid (C8:0) gastric absorption and plasma octanoylated ghrelin concentration.

    PubMed

    Lemarié, F; Cavalier, J-F; Garcia, C; Boissel, F; Point, V; Catheline, D; Legrand, P; Carrière, F; Rioux, V

    2016-09-01

    Part of medium chain fatty acids (MCFAs) coming from dietary triglycerides (TGs) can be directly absorbed through the gastric mucosa after the action of preduodenal lipase (lingual lipase in the rat). MCFA gastric absorption, particularly that of octanoic acid (C8:0), may have a physiological importance in the octanoylation of ghrelin, the orexigenic gastric peptide acting as an endogenous ligand of the hypothalamic growth hormone secretagogue receptor 1a (GHSR-1a). However, the amount of C8:0 absorbed in the stomach and its metabolic fate still haven't been clearly characterized. The purpose of the present study was to further characterize and quantify the importance of preduodenal lipase activity on the release and gastric absorption of dietary C8:0 and on the subsequent ghrelin octanoylation in the stomach mucosa. Fifteen days old rats received fat emulsions containing triolein or [1,1,1-(13)C]-Tri-C8:0 and a specific inhibitor of preduodenal lipase, 5-(2-(benzyloxy)ethoxy)-3-(3-phenoxyphenyl)-1,3,4-oxadiazol-2(3H)-one or BemPPOX. The fate of the (13)C-C8:0 was followed in rat tissues after 30 and 120min of digestion and octanoylated ghrelin was measured in the plasma. This work (1) demonstrates that part of C8:0 coming from Tri-C8:0 is directly absorbed at the gastric level, (2) allows the estimation of C8:0 gastric absorption level (1.3% of the (13)C-C8:0 in sn-3 position after 30min of digestion), as well as (3) the contribution of rat lingual lipase to total lipolysis and to duodenal absorption of dietary FAs (at least 30%), (4) shows no short-term effect of dietary Tri-C8:0 consumption and subsequent increase of C8:0 gastric tissue content on plasma octanoylated ghrelin concentration. Copyright © 2016 Elsevier B.V. All rights reserved.

  12. Removing the Active-Site Flap in Lipase A from Candida antarctica Produces a Functional Enzyme without Interfacial Activation.

    PubMed

    Wikmark, Ylva; Engelmark Cassimjee, Karim; Lihammar, Richard; Bäckvall, Jan-E

    2016-01-01

    A mobile region is proposed to be a flap that covers the active site of Candida antarctica lipase A. Removal of the mobile region retains the functional properties of the enzyme. Interestingly interfacial activation, required for the wild-type enzyme, was not observed for the truncated variant, although stability, activity, and stereoselectivity were very similar for the wild-type and variant enzymes. The variant followed classical Michaelis-Menten kinetics, unlike the wild type. Both gave the same relative specificity in the transacylation of a primary and a secondary alcohol in organic solvent. Furthermore, both showed the same enantioselectivity in transacylation of alcohols and the hydrolysis of alcohol esters, as well as in the hydrolysis of esters chiral at the acid part. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  13. Effects of organic solvent, water activity, and salt hydrate pair on the sn-1,3 selectivity and activity of whole-cell lipase from Aspergillus niger GZUF36.

    PubMed

    Li, Cuiqin; Zhang, Fuhao; Gao, Zexin; He, Laping; Zeng, Xuefeng; Zhu, Qiujin; Yu, Lijuan

    2018-01-01

    We previously screened a whole-cell lipase EC 3.1.1.3 from the novel strain Aspergillus niger GZUF36, which exhibited 1,3-selectivity in the synthesis of 1,3-diacylglycerol via glycerolysis. However, the mechanism of lipase selectively in catalyzing the sn-1,3 position remains ambiguous. This work was performed to investigate the 1,3-selective mechanism of lipase using glycerolysis to synthesize 1,3-diacylglycerol (1,3-DG) as a model reaction by changing solvent(s) and water activity (a w ), and addition of salt hydrate pair. The measured diacylglycerol yield was also used to examine lipase activity. Results indicated that not only organic solvent and a w have strong effect on the sn-1,3 selectivity, but also ions of salt hydrate pair also affected selectivity. Lipase conformation was altered by hydrophobic interactions of the solvent, a w , or ions of salt hydrate, resulting in distinct sn-1,3 selectivity of the lipase. The salt hydrate pair changed the lipase conformation and selectivity not only by a w but also by static interactions, which was rarely reported. These parameters also affected lipase activity. The lipase displayed the highest selectivity (about 88%) and activity in solvents of t-butanol and n-hexane (1:29, v/v) at a w 0.43. The results demonstrated that the sn-1,3 selectivity and activity of the lipase from A. niger GZUF36 may be improved by control of some crucial factors. This work laid a foundation for the application of lipase in the synthesis of 1,3-DG and other structural and functional lipids.

  14. Isolation and characterization of novel lipases/esterases from a bovine rumen metagenome.

    PubMed

    Privé, Florence; Newbold, C Jamie; Kaderbhai, Naheed N; Girdwood, Susan G; Golyshina, Olga V; Golyshin, Peter N; Scollan, Nigel D; Huws, Sharon A

    2015-07-01

    Improving the health beneficial fatty acid content of meat and milk is a major challenge requiring an increased understanding of rumen lipid metabolism. In this study, we isolated and characterized rumen bacterial lipases/esterases using functional metagenomics. Metagenomic libraries were constructed from DNA extracted from strained rumen fluid (SRF), solid-attached bacteria (SAB) and liquid-associated rumen bacteria (LAB), ligated into a fosmid vector and subsequently transformed into an Escherichia coli host. Fosmid libraries consisted of 7,744; 8,448; and 7,680 clones with an average insert size of 30 to 35 kbp for SRF, SAB and LAB, respectively. Transformants were screened on spirit blue agar plates containing tributyrin for lipase/esterase activity. Five SAB and four LAB clones exhibited lipolytic activity, and no positive clones were found in the SRF library. Fosmids from positive clones were pyrosequenced and twelve putative lipase/esterase genes and two phospholipase genes retrieved. Although the derived proteins clustered into diverse esterase and lipase families, a degree of novelty was seen, with homology ranging from 40 to 78% following BlastP searches. Isolated lipases/esterases exhibited activity against mostly short- to medium-chain substrates across a range of temperatures and pH. The function of these novel enzymes recovered in ruminal metabolism needs further investigation, alongside their potential industrial uses.

  15. Increasing the activity and enantioselectivity of lipases by sol-gel immobilization: further advancements of practical interest

    NASA Astrophysics Data System (ADS)

    Tielmann, Patrick; Kierkels, Hans; Zonta, Albin; Ilie, Adriana; Reetz, Manfred T.

    2014-05-01

    The entrapment of lipases in hydrophobic silicate matrices formed by sol-gel mediated hydrolysis of RSi(OCH3)3/Si(OCH3)4 as originally reported in 1996 has been improved over the years by a number of modifications. In the production of second-generation sol-gel lipase immobilizates, a variety of additives during the sol-gel process leads to increased activity and enhanced stereoselectivity in esterifying kinetic resolution. Recent advances in this type of lipase immobilization are reviewed here, in addition to new results regarding the sol-gel entrapment of the lipase from Burkholderia cepacia. It constitutes an excellent heterogeneous biocatalyst in the acylating kinetic resolution of two synthetically and industrially important chiral alcohols, rac-sulcatol and rac-trans-2-methoxycyclohexanol. The observation that the catalyst can be used 10 times in recycling experiments without losing its significant activity or enantioselectivity demonstrates the practical viability of the sol-gel approach.The entrapment of lipases in hydrophobic silicate matrices formed by sol-gel mediated hydrolysis of RSi(OCH3)3/Si(OCH3)4 as originally reported in 1996 has been improved over the years by a number of modifications. In the production of second-generation sol-gel lipase immobilizates, a variety of additives during the sol-gel process leads to increased activity and enhanced stereoselectivity in esterifying kinetic resolution. Recent advances in this type of lipase immobilization are reviewed here, in addition to new results regarding the sol-gel entrapment of the lipase from Burkholderia cepacia. It constitutes an excellent heterogeneous biocatalyst in the acylating kinetic resolution of two synthetically and industrially important chiral alcohols, rac-sulcatol and rac-trans-2-methoxycyclohexanol. The observation that the catalyst can be used 10 times in recycling experiments without losing its significant activity or enantioselectivity demonstrates the practical viability of

  16. Protease and lipase activities of fungal and bacterial strains derived from an artisanal raw ewe's milk cheese.

    PubMed

    Ozturkoglu-Budak, Sebnem; Wiebenga, Ad; Bron, Peter A; de Vries, Ronald P

    2016-11-21

    We previously identified the microbiota present during cheese ripening and observed high protease and lipase activity in Divle Cave cheese. To determine the contribution of individual isolates to enzyme activities, we investigated a range of species representing this microbiota for their proteolytic and lipolytic ability. In total, 17 fungal, 5 yeast and 18 bacterial strains, previously isolated from Divle Cave cheese, were assessed. Qualitative protease and lipase activities were performed on skim-milk agar and spirit-blue lipase agar, respectively, and resulted in a selection of strains for quantitative assays. For the quantitative assays, the strains were grown on minimal medium containing irradiated Divle Cave cheese, obtained from the first day of ripening. Out of 16 selected filamentous fungi, Penicillium brevicompactum, Penicillium cavernicola and Penicillium olsonii showed the highest protease activity, while Mucor racemosus was the best lipase producer. Yarrowia lipolytica was the best performing yeast with respect to protease and lipase activity. From the 18 bacterial strains, 14 and 11 strains, respectively showed protease and lipase activity in agar plates. Micrococcus luteus, Bacillus stratosphericus, Brevibacterium antiquum, Psychrobacter glacincola and Pseudomonas proteolytica displayed the highest protease and lipase activity. The proteases of yeast and filamentous fungi were identified as mainly aspartic protease by specific inhibition with Pepstatin A, whereas inhibition by PMSF (phenylmethylsulfonyl fluoride) indicated that most bacterial enzymes belong to serine type protease. Our results demonstrate that aspartic proteases, which usually have high milk clotting activity, are predominantly derived from fungal strains, and therefore fungal enzymes appear to be more suitable for use in the cheese industry. Microbial enzymes studied in this research might be alternatives for rennin (chymosin) from animal source because of their low cost and stable

  17. Lipases as catalysts in synthesis of fine chemicals.

    PubMed

    Baldessari, Alicia

    2012-01-01

    The application of lipases as catalysts in the synthesis of an intermediate of alfuzosin and lapyrium chloride is described. In the first case, the one-pot procedure to obtain the intermediate involves the treatment of tetrahydrofuroic acid with ethanol in the presence of Candida antarctica lipase followed by the addition of N-methyl-1,3-diaminopropane. In the second part of the chapter, an efficient route for large-scale preparation of lapyrium chloride is developed from chloroacetic acid in four steps, three of them enzymatic. Due to the chemoselective behavior of the lipases, both products described in the present chapter were obtained in a high degree of purity and yield, applying mild reaction conditions, and following a low environmental impact methodology.

  18. In vitro investigation of the potential health benefits of wild Mediterranean dietary plants as anti-obesity agents with α-amylase and pancreatic lipase inhibitory activities.

    PubMed

    Marrelli, Mariangela; Loizzo, Monica Rosa; Nicoletti, Marcello; Menichini, Francesco; Conforti, Filomena

    2014-08-01

    Inhibition of digestive enzymes is one of the most widely studied mechanisms used to determine the potential efficacy of natural products as anti-obesity agents. In vitro studies reported here were performed to evaluate the inhibitory activity of formulations of edible plants from Italy on amylase and lipase by monitoring the hydrolysis of nitrophenyl caprilate and the hydrolysis of glycoside bonds in digestible carbohydrate foods. The formulation obtained from Capparis sicula exhibited the strongest inhibitory effect on pancreatic lipase (IC50 = 0.53 mg mL(-1) ) while the Borago officinalis formulation exhibited the strongest inhibitory effect on α-amylase (IC50 = 31.61 µg mL(-1) ). In order to characterise the extracts, high-performance thin-layer chromatography analysis of the formulations was performed, revealing the predominance of (±)-catechin in Mentha aquatica formulation, rutin in C. sicula, and caffeic acid and chlorogenic acid in Echium vulgare. The results obtained indicated that the extracts of C. sicula and B. officinalis could be good candidates for further studies to isolate pancreatic lipase and α-amylase inhibitors, respectively. © 2013 Society of Chemical Industry.

  19. Molecular and enzymatic characterization of a subfamily I.4 lipase from an edible oil-degrader Bacillus sp. HH-01.

    PubMed

    Kamijo, Takashi; Saito, Akihiro; Ema, Sadaharu; Yoh, Inchi; Hayashi, Hiroko; Nagata, Ryo; Nagata, Yoshiho; Ando, Akikazu

    2011-02-01

    An edible-oil degrading bacterial strain HH-01 was isolated from oil plant gummy matter and was classified as a member of the genus Bacillus on the basis of the nucleotide sequence of the 16S rRNA gene. A putative lipase gene and its flanking regions were cloned from the strain based on its similarity to lipase genes from other Bacillus spp. The deduced product was composed of 214 amino acids and the putative mature protein, consisting of 182 amino acids, exhibited 82% amino acid sequence identity with the subfamily I.4 lipase LipA of Bacillus subtilis 168. The recombinant product was successfully overproduced as a soluble form in Escherichia coli and showed lipase activity. The gene was, therefore, designated as lipA of HH-01. HH-01 LipA was stable at pH 4-11 and up to 30°C, and its optimum pH and temperature were 8-9 and 30°C, respectively. The enzyme showed preferential hydrolysis of the 1(3)-position ester bond in trilinolein. The activity was, interestingly, enhanced by supplementing with 1 mM CoCl(2), in contrast to other Bacillus lipases. The lipA gene seemed to be constitutively transcribed during the exponential growth phase, regardless of the presence of edible oil.

  20. Process optimization for production and purification of a thermostable, organic solvent tolerant lipase from Acinetobacter sp. AU07.

    PubMed

    Gururaj, P; Ramalingam, Subramanian; Nandhini Devi, Ganesan; Gautam, Pennathur

    2016-01-01

    The purpose of this study was to isolate, purify and optimize the production conditions of an organic solvent tolerant and thermostable lipase from Acinetobacter sp. AU07 isolated from distillery waste. The lipase production was optimized by response surface methodology, and a maximum production of 14.5U/mL was observed at 30°C and pH 7, using a 0.5% (v/v) inoculum, 2% (v/v) castor oil (inducer), and agitation 150rpm. The optimized conditions from the shake flask experiments were validated in a 3L lab scale bioreactor, and the lipase production increased to 48U/mL. The enzyme was purified by ammonium sulfate precipitation and ion exchange chromatography and the overall yield was 36%. SDS-PAGE indicated a molecular weight of 45kDa for the purified protein, and Matrix assisted laser desorption/ionization time of flight analysis of the purified lipase showed sequence similarity with GDSL family of lipases. The optimum temperature and pH for activity of the enzyme was found to be 50°C and 8.0, respectively. The lipase was completely inhibited by phenylmethylsulfonyl fluoride but minimal inhibition was observed when incubated with ethylenediaminetetraacetic acid and dithiothreitol. The enzyme was stable in the presence of non-polar hydrophobic solvents. Detergents like SDS inhibited enzyme activity; however, there was minimal loss of enzyme activity when incubated with hydrogen peroxide, Tween 80 and Triton X-100. The kinetic constants (Km and Vmax) revealed that the hydrolytic activity of the lipase was specific to moderate chain fatty acid esters. The Vmax, Km and Vmax/Km ratio of the enzyme were 16.98U/mg, 0.51mM, and 33.29, respectively when 4-nitrophenyl palmitate was used as a substrate. Copyright © 2016 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. All rights reserved.

  1. Ionic liquids as a reaction medium for lipase-catalyzed methanolysis of sunflower oil.

    PubMed

    Sunitha, S; Kanjilal, S; Reddy, P S; Prasad, R B N

    2007-12-01

    Ionic liquids, 1-butyl-3-methyl imidazolium hexafluorophosphate ([BMIm][PF(6)]) and 1-ethyl-3-methyl imidazolium hexafluorophosphate ([EMIm][PF(6)]), were used for the methanolysis of sunflower oil using Candida antarctica lipase (Novozyme 435) and gave yields of fatty acid methyl esters at 98-99% within 10 h. The optimum conditions of methanolysis in hydrophobic ionic liquids are 2% (w/w) lipase, 1:1 (w/w) oil/ionic liquid and 1:8 (mol/mol) oil/methanol at 58-60 degrees C. Methanolysis using hydrophilic ionic liquids, 3-methyl imidazolium tetrafluoroborate ([HMIm][BF(4)]) and 1-butyl-3-methyl imidazolium tetrafluoroborate ([BMIm][BF(4)]), gave very poor yields. A hydrophobic ionic liquid thus protects the lipase from methanol. Recovered ionic liquids and lipase were used for four successive reaction cycles without any significant loss of activity.

  2. Lipase Maturation Factor 1 (Lmf1): Structure and Role in Lipase Folding and Assembly

    PubMed Central

    Ehrhardt, Nicole

    2014-01-01

    Purpose of review Lipase maturation factor 1 (LMF1) is a membrane-bound protein located in the endoplasmic reticulum (ER). It is essential to the folding and assembly (i.e., maturation) of a select group of lipases that include lipoprotein lipase (LPL), hepatic lipase (HL) and endothelial lipase (EL). The purpose of this review is to examine recent studies that have begun to elucidate the structure and function of LMF1, and to place it in the context of lipase folding and assembly. Recent findings Recent studies identified mutations in LMF1 that cause combined lipase deficiency and hypertriglyceridemia in humans. These mutations result in the truncation of a large, evolutionarily conserved domain called DUF1222, which is essential for interaction with lipases and their attainment of enzymatic activity. The structural complexity of LMF1 has been further characterized by solving its topology in the ER membrane. Recent studies indicate that in addition to LPL and HL, the maturation of EL is also dependent on LMF1. Based on its apparent specificity for dimeric lipases, LMF1 is proposed to play an essential role in the assembly and/or stabilization of head-to-tail lipase homodimers. Summary LMF1 functions in the maturation of a select group of secreted lipases that assemble into homodimers in the ER. These dimeric lipases include LPL, HL and EL, all of which contribute significantly to plasma triglyceride and HDL cholesterol levels in human populations. Future studies involving genetically engineered mouse models will be required to fully elucidate the role of LMF1 in normal physiology and disease. PMID:20224398

  3. Sebelipase alfa improves atherogenic biomarkers in adults and children with lysosomal acid lipase deficiency.

    PubMed

    Wilson, Don P; Friedman, Mark; Marulkar, Sachin; Hamby, Tyler; Bruckert, Eric

    Measures of atherogenic cholesterol, with and without concomitant use of lipid-lowering medications (LLMs), are reported with up to 52 weeks of sebelipase alfa treatment in children and adults with lysosomal acid lipase deficiency (LAL-D) participating in the phase 3 Acid Lipase Replacement Investigating Safety and Efficacy study (NCT01757184). To examine the effects of sebelipase alfa on levels of atherogenic biomarkers in the Acid Lipase Replacement Investigating Safety and Efficacy study. Data were prospectively collected for LDL particle (LDL-P) number, LDL-C, HDL-C, apolipoprotein B (apoB), apolipoprotein A1 (apoA1), and LDL-P size. Differences at week 20 between the sebelipase alfa and placebo groups were assessed for the overall LAL-D cohort and for patients receiving and not receiving LLMs. Changes from baseline after up to 52 weeks of treatment were also calculated for the overall cohort and separately for patients receiving and not receiving LLMs. Baseline values for LDL-C, LDL-P number, and apoB were elevated while HDL-C and apoA1 were low. Treatment with sebelipase alfa for 20 weeks significantly improved atherogenic measures compared with placebo irrespective of LLM usage. The reduction in LDL-C with sebelipase alfa was associated with a reduction in the LDL-P number. Treatment for up to 52 weeks was associated with sustained improvements of LDL-P, LDL-C, HDL-C, apoB, and apoA1, regardless of LLM use. Patients with LAL-D have high atherogenic risk. It is essential to address the underlying LAL deficiency to restore cholesterol homeostasis in LAL-D patients, as treatment with sebelipase alfa improves atherogenic measures regardless of LLM use and for a sustained period. Sebelipase alfa appears to reduce LDL-C by decreasing the LDL-P number, suggesting improvement in cardiovascular disease risk in LAL-D patients. Copyright © 2018 National Lipid Association. Published by Elsevier Inc. All rights reserved.

  4. Fat digestion in the stomach: stability of lingual lipase in the gastric environment.

    PubMed

    Fink, C S; Hamosh, P; Hamosh, M

    1984-03-01

    Digestion of dietary fat starts in the stomach, where lingual lipase hydrolyzes triglycerides to free fatty acids and partial glycerides at pH 3.0-6.0. Lingual lipase is secreted continuously from lingual serous glands and accumulates in the stomach between meals, when gastric pH is less than 3.0. We have, therefore, examined the resistance of lingual lipase to low pH and its possible protection by dietary components present in the stomach contents. Partially purified rat lingual lipase (7-15 micrograms enzyme protein) was preincubated at 37 degrees C for 10-60 min at pH 1.0-6.0 before incubation for assay of lipolytic activity, hydrolysis of tri-[3H]olein at pH 5.4. The data show that partially purified rat lingual lipase preparations are stable at 37 degrees C in the pH range of 2.5-6.0. Enzyme activity, however, is rapidly and irreversibly lost during preincubation at pH 1.0-2.4 for 10-30 min. Protein (gelatin 1% or albumin 1% or 2.5%) cannot prevent the inactivation of lingual lipase at low pH. The large molecular species (molecular weight greater than 500,000) of lingual lipase (thought to be an aggregate of enzyme with lipids) is slightly more resistant to inactivation than the 46,000 dalton preparation, suggesting that lipids might protect the enzyme from inactivation. Indeed, about 60% of the initial lipase activity is preserved during incubation at pH 2.0 in the presence of 50 mM lecithin or 10 mM triolein. The data indicate that triglycerides which are hydrolyzed by this enzyme as well as phospholipids that are not hydrolyzed can prevent the inactivation of the enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

  5. Efficient heterologous expression of Fusarium solani lipase, FSL2, in Pichia pastoris, functional characterization of the recombinant enzyme and molecular modeling.

    PubMed

    Jallouli, Raida; Parsiegla, Goetz; Carrière, Frédéric; Gargouri, Youssef; Bezzine, Sofiane

    2017-01-01

    The gene coding for a lipase of Fusarium solani, designated as FSL2, shows an open reading frame of 906bp encoding a 301-amino acid polypeptide with a molecular mass of 30kDa. Based on sequence similarity with other fungal lipases, FSL2 contains a catalytic triad, consisting of Ser144, Asp198, and His256. FSL2 cDNA was subcloned into the pGAPZαA vector containing the Saccharomyces cerevisiae α-factor signal sequence and this construct was used to transform Pichia pastoris and achieve a high-level extracellular production of a FSL2 lipase. Maximum lipase activity was observed after 48h. The optimum activity of the purified recombinant enzyme was measured at pH 8.0-9.0 and 37°C. FSL2 is remarkably stable at alkaline pH values up to 12 and at temperatures below 40°C. It has high catalytic efficiency towards triglycerides with short to long chain fatty acids but with a marked preference for medium and long chain fatty acids. FSL2 activity is decreased at sodium taurodeoxycholate concentrations above the Critical Micelle Concentration (CMC) of this anionic detergent. However, lipase activity is enhanced by Ca 2+ and inhibited by EDTA or Cu 2+ and partially by Mg 2+ or K + . In silico docking of medium chain triglycerides, monogalctolipids (MGDG), digalactolipids (DGDG) and long chain phospholipids in the active site of FSL2 reveals structural solutions. Copyright © 2016 Elsevier B.V. All rights reserved.

  6. Hierarchical ZIF-8 toward Immobilizing Burkholderia cepacia Lipase for Application in Biodiesel Preparation.

    PubMed

    Adnan, Miaad; Li, Kai; Wang, Jianhua; Xu, Li; Yan, Yunjun

    2018-05-10

    A hierarchical mesoporous zeolitic imidazolate framework (ZIF-8) was processed based on cetyltrimethylammonium bromide (CTAB) as a morphological regulating agent and amino acid (l-histidine) as assisting template agent. Burkholderia cepacia lipase (BCL) was successfully immobilized by ZIF-8 as the carrier via an adsorption method (BCL-ZIF-8). The immobilized lipase (BCL) showed utmost activity recovery up to 1279%, a 12-fold boost in its free counterpart. BCL-ZIF-8 was used as a biocatalyst in the transesterification reaction for the production of biodiesel with 93.4% yield. There was no significant lowering of conversion yield relative to original activity for BCL-ZIF-8 when continuously reused for eight cycles. This work provides a new outlook for biotechnological importance by immobilizing lipase on the hybrid catalyst (ZIF-8) and opens the door for its uses in the industrial field.

  7. Identification and sequence analyses of novel lipase encoding novel thermophillic bacilli isolated from Armenian geothermal springs.

    PubMed

    Shahinyan, Grigor; Margaryan, Armine; Panosyan, Hovik; Trchounian, Armen

    2017-05-02

    Among the huge diversity of thermophilic bacteria mainly bacilli have been reported as active thermostable lipase producers. Geothermal springs serve as the main source for isolation of thermostable lipase producing bacilli. Thermostable lipolytic enzymes, functioning in the harsh conditions, have promising applications in processing of organic chemicals, detergent formulation, synthesis of biosurfactants, pharmaceutical processing etc. In order to study the distribution of lipase-producing thermophilic bacilli and their specific lipase protein primary structures, three lipase producers from different genera were isolated from mesothermal (27.5-70 °C) springs distributed on the territory of Armenia and Nagorno Karabakh. Based on phenotypic characteristics and 16S rRNA gene sequencing the isolates were identified as Geobacillus sp., Bacillus licheniformis and Anoxibacillus flavithermus strains. The lipase genes of isolates were sequenced by using initially designed primer sets. Multiple alignments generated from primary structures of the lipase proteins and annotated lipase protein sequences, conserved regions analysis and amino acid composition have illustrated the similarity (98-99%) of the lipases with true lipases (family I) and GDSL esterase family (family II). A conserved sequence block that determines the thermostability has been identified in the multiple alignments of the lipase proteins. The results are spreading light on the lipase producing bacilli distribution in geothermal springs in Armenia and Nagorno Karabakh. Newly isolated bacilli strains could be prospective source for thermostable lipases and their genes.

  8. An interfacial and comparative in vitro study of gastrointestinal lipases and Yarrowia lipolytica LIP2 lipase, a candidate for enzyme replacement therapy.

    PubMed

    Bénarouche, Anaïs; Point, Vanessa; Carrière, Frédéric; Cavalier, Jean-François

    2014-07-01

    Lipolytic activities of Yarrowia lipolytica LIP2 lipase (YLLIP2), human pancreatic (HPL) and dog gastric (DGL) lipases were first compared using lecithin-stabilized triacylglycerol (TAG) emulsions (Intralipid) at various pH and bile salt concentrations. Like DGL, YLLIP2 was able to hydrolyze TAG droplets covered by a lecithin monolayer, while HPL was not directly active on that substrate. These results were in good agreement with the respective kinetics of adsorption on phosphatidylcholine (PC) monomolecular films of the same three lipases, YLLIP2 being the most tensioactive lipase. YLLIP2 adsorption onto a PC monolayer spread at the air/water interface was influenced by pH-dependent changes in the enzyme/lipid interfacial association constant (KAds) which was optimum at pH 6.0 on long-chain egg PC monolayer, and at pH 5.0 on medium chain dilauroylphosphatidylcholine film. Using substrate monolayers (1,2-dicaprin, trioctanoin), YLLIP2 displayed the highest lipolytic activities on both substrates in the 25-35 mN m(-1) surface pressure range. YLLIP2 was active in a large pH range and displayed a pH-dependent activity profile combining DGL and HPL features at pH values found in the stomach (pH 3-5) and in the intestine (pH 6-7), respectively. The apparent maximum activity of YLLIP2 was observed at acidic pH 4-6 and was therefore well correlated with an efficient interfacial binding at these pH levels, whatever the type of interfaces (Intralipid emulsions, substrate or PC monolayers). All these findings support the use of YLLIP2 in enzyme replacement therapy for the treatment of pancreatic exocrine insufficiency, a pathological situation in which an acidification of intestinal contents occurs. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

  9. Lipase-catalyzed biodiesel production from waste activated bleaching earth as raw material in a pilot plant.

    PubMed

    Park, Enoch Y; Sato, Masayasu; Kojima, Seiji

    2008-05-01

    The production of fatty acid methyl esters (FAMEs) from waste activated bleaching earth (ABE) discarded by the crude oil refining industry using lipase from Candida cylindracea was investigated in a 50-L pilot plant. Diesel oil or kerosene was used as an organic solvent for the transesterification of triglycerides embedded in the waste ABE. When 1% (w/w) lipase was added to waste ABE, the FAME content reached 97% (w/w) after reaction for 12 h at 25 degrees C with an agitation rate of 30 rpm. The FAME production rate was strongly dependent upon the amount of enzyme added. Mixtures of FAME and diesel oil at ratios of 45:55 (BDF-45) and 35:65 (BDF-35) were assessed and compared with the European specifications for biodiesel as automotive diesel fuel, as defined by pr EN 14214. The biodiesel quality of BDF-45 met the EN 14214 standard. BDF-45 was used as generator fuel, and the exhaust emissions were compared with those of diesel oil. The CO and SO2 contents were reduced, but nitrogen oxide emission increased by 10%. This is the first report of a pilot plant study of lipase-catalyzed FAME production using waste ABE as a raw material. This result demonstrates a promising reutilization method for the production of FAME from industrial waste resources containing vegetable oils for use as a biodiesel fuel.

  10. Immobilization of Moniliella spathulata R25L270 Lipase on Ionic, Hydrophobic and Covalent Supports: Functional Properties and Hydrolysis of Sardine Oil.

    PubMed

    Souza, Lívia T de A; Moreno-Perez, Sonia; Fernández Lorente, Gloria; Cipolatti, Eliane P; de Oliveira, Débora; Resende, Rodrigo R; Pessela, Benevides C

    2017-09-25

    The oleaginous yeast Moniliella spathulata R25L270 was the first yeast able to grow and produce extracellular lipase using Macaúba ( Acrocomia aculeate ) cake as substrate. The novel lipase was recently identified, and presented promising features for biotechnological applications. The M. spathulata R25L270 lipase efficiently hydrolyzed vegetable and animal oils, and showed selectivity for generating cis -5,8,11,15,17-eicosapentaenoic acid from sardine oil. The enzyme can act in a wide range of temperatures (25-48 °C) and pH (6.5-8.4). The present study deals with the immobilization of M. spathulata R25L270 lipase on hydrophobic, covalent and ionic supports to select the most active biocatalyst capable to obtain omega-3 fatty acids (PUFA) from sardine oil. Nine immobilized agarose derivatives were prepared and biochemically characterized for thermostability, pH stability and catalytic properties (K M and V max ). Ionic supports improved the enzyme-substrate affinity; however, it was not an effective strategy to increase the M. spathulata R25L270 lipase stability against pH and temperature. Covalent support resulted in a biocatalyst with decreased activity, but high thermostability. The enzyme was most stabilized when immobilized on hydrophobic supports, especially Octyl-Sepharose. Compared with the free enzyme, the half-life of the Octyl-Sepharose derivative at 60 °C increased 10-fold, and lipase stability under acidic conditions was achieved. The Octyl-Sepharose derivative was selected to obtain omega-3 fatty acids from sardine oil, and the maximal enzyme selectivity was achieved at pH 5.0.

  11. Immobilization of a Commercial Lipase from Penicillium camembertii (Lipase G) by Different Strategies

    PubMed Central

    Mendes, Adriano A.; Freitas, Larissa; de Carvalho, Ana Karine F.; de Oliveira, Pedro C.; de Castro, Heizir F.

    2011-01-01

    The objective of this work was to select the most suitable procedure to immobilize lipase from Penicillium camembertii (Lipase G). Different techniques and supports were evaluated, including physical adsorption on hydrophobic supports octyl-agarose, poly(hydroxybutyrate) and Amberlite resin XAD-4; ionic adsorption on the anionic exchange resin MANAE-agarose and covalent attachment on glyoxyl-agarose, MANAE-agarose cross-linked with glutaraldehyde, MANAE-agarose-glutaraldehyde, and epoxy-silica-polyvinyl alcohol composite. Among the tested protocols, the highest hydrolytic activity (128.2 ± 8.10 IU·g−1 of support) was achieved when the lipase was immobilized on epoxy-SiO2-PVA using hexane as coupling medium. Lipase immobilized by ionic adsorption on MANAE-agarose also gave satisfactory result, attaining 55.6 ± 2.60 IU·g−1 of support. In this procedure, the maximum loading of immobilized enzyme was 9.3 mg·g−1 of gel, and the highest activity (68.8 ± 2.70 IU·g−1 of support) was obtained when 20 mg of protein·g−1 was offered. Immobilization carried out in aqueous medium by physical adsorption on hydrophobic supports and covalent attachment on MANAE-agarose-glutaraldehyde and glyoxyl-agarose was shown to be unfeasible for Lipase G. Thermal stability tests revealed that the immobilized derivative on epoxy-SiO2-PVA composite using hexane as coupling medium had a slight higher thermal stability than the free lipase. PMID:21811674

  12. Hypocholesterolemic Effect and In Vitro Pancreatic Lipase Inhibitory Activity of an Opuntia ficus-indica Extract.

    PubMed

    Padilla-Camberos, Eduardo; Flores-Fernandez, Jose Miguel; Fernandez-Flores, Ofelia; Gutierrez-Mercado, Yanet; Carmona-de la Luz, Joel; Sandoval-Salas, Fabiola; Mendez-Carreto, Carlos; Allen, Kirk

    2015-01-01

    Cholesterol control is fundamental for prevention of cardiovascular disorders. In this work, the hypocholesterolemic activity of an aqueous Opuntia ficus-indica extract (AOE) was tested in triton-induced mice. The inhibitory activity on pancreatic lipase enzyme was evaluated in vitro by the same extract. Furthermore, polyphenol content of the extract was evaluated. Hypercholesterolemia was induced in three groups of mice by intraperitoneal administration of Triton WR-1339. After induction of hypercholesterolemia, the groups were treated with an AOE (500 mg/kg) and saline solution and the positive control group with orlistat, respectively. Cholesterol levels were measured 24 h later in peripheral blood. The levels of blood cholesterol after administration of AOE significantly decreased compared to negative control. The inhibitory activity of AOE on pancreatic lipase enzyme was evaluated at concentrations from 60 to 1000 μg/mL. The AOE inhibited the pancreatic lipase with an IC50 = 588.5 μg/mL. The AOE had a high content of polyphenolic compounds. These results show that AOE is able to prevent hypercholesterolemia by pancreatic lipase inhibition, in part due to its polyphenolic compounds.

  13. Hypocholesterolemic Effect and In Vitro Pancreatic Lipase Inhibitory Activity of an Opuntia ficus-indica Extract

    PubMed Central

    Flores-Fernandez, Jose Miguel; Fernandez-Flores, Ofelia; Gutierrez-Mercado, Yanet; Carmona-de la Luz, Joel; Sandoval-Salas, Fabiola; Mendez-Carreto, Carlos

    2015-01-01

    Cholesterol control is fundamental for prevention of cardiovascular disorders. In this work, the hypocholesterolemic activity of an aqueous Opuntia ficus-indica extract (AOE) was tested in triton-induced mice. The inhibitory activity on pancreatic lipase enzyme was evaluated in vitro by the same extract. Furthermore, polyphenol content of the extract was evaluated. Hypercholesterolemia was induced in three groups of mice by intraperitoneal administration of Triton WR-1339. After induction of hypercholesterolemia, the groups were treated with an AOE (500 mg/kg) and saline solution and the positive control group with orlistat, respectively. Cholesterol levels were measured 24 h later in peripheral blood. The levels of blood cholesterol after administration of AOE significantly decreased compared to negative control. The inhibitory activity of AOE on pancreatic lipase enzyme was evaluated at concentrations from 60 to 1000 μg/mL. The AOE inhibited the pancreatic lipase with an IC50 = 588.5 μg/mL. The AOE had a high content of polyphenolic compounds. These results show that AOE is able to prevent hypercholesterolemia by pancreatic lipase inhibition, in part due to its polyphenolic compounds. PMID:26078966

  14. Eco-friendly methodology for efficient synthesis and scale-up of 2-ethylhexyl-p-methoxycinnamate using Rhizopus oryzae lipase and its biological evaluation.

    PubMed

    Kumar, Vinod; Jahan, Firdaus; Kameswaran, Karthikeya; Mahajan, Richi V; Saxena, Rajendra Kumar

    2014-06-01

    Lipase-mediated synthesis of phenolic acid esters is a green and economical alternative to current chemical methods. Octyl methoxycinnamate, an important UVB-absorbing compound, was synthesized by the esterification of p-methoxycinnamic acid with 2-ethyl hexanol using Rhizopus oryzae lipase. A molar ratio of 1:2 of p-methoxycinnamic acid and 2-ethyl hexanol was found to give an optimum yield using cyclo-octane (50 ml) as reaction solvent, at a temperature of 45 °C, and 750 U of lipase, resulting in a yield of 91.3 % in 96 h. This reaction was successfully scaled up to 400-ml reaction size where 88.6 %bioconversion was achieved. The synthesized compound was found to have superior antioxidant activity as compared to ascorbic acid. The synthesized compound also exhibited good antimicrobial activity against Escherichia coli, Klebsiella pneumonia, Salmonella typhi, Staphylococcus aures, Candida albicans (yeast), Aspergillus niger, Alternaria solani, and Fussarium oxysporum by well diffusion method in terms of zone of inhibitions (in mm).

  15. Inhibition of Gastric Lipase as a Mechanism for Body Weight and Plasma Lipids Reduction in Zucker Rats Fed a Rosemary Extract Rich in Carnosic Acid

    PubMed Central

    Romo Vaquero, María; Yáñez-Gascón, María-Josefa; García Villalba, Rocío; Larrosa, Mar; Fromentin, Emilie; Ibarra, Alvin; Roller, Marc; Tomás-Barberán, Francisco; Espín de Gea, Juan Carlos; García-Conesa, María-Teresa

    2012-01-01

    Background Rosemary (Rosmarinus officinalis L.) extracts (REs) exhibit hepatoprotective, anti-obesity and anti-inflammatory properties and are widely used in the food industry. REs are rich in carnosic acid (CA) and carnosol which may be responsible for some of the biological activities of REs. The aim of this study was to investigate whether inhibition of lipase activity in the gut may be a mechanism by which a RE enriched in CA (40%) modulates body weight and lipids levels in a rat model of metabolic disorders and obesity. Methods and Principal Findings RE was administered for 64 days to lean (fa/+) and obese (fa/fa) female Zucker rats and body weight, food intake, feces weight and blood biochemical parameters were monitored throughout the study. Lipase activity (hydrolysis of p-nitrophenylbutyrate) was measured in the gastrointestinal tract at the end of the study and the contents of CA, carnosol and methyl carnosate were also determined. Sub-chronic administration of RE moderately reduced body weight gain in both lean and obese animals but did not affect food intake. Serum triglycerides, cholesterol and insulin levels were also markedly decreased in the lean animals supplemented with RE. Importantly, lipase activity was significantly inhibited in the stomach of the RE-supplemented animals where the highest content of intact CA and carnosol was detected. Conclusions Our results confirm that long-term administration of RE enriched in CA moderates weight gain and improves the plasma lipids profile, primarily in the lean animals. Our data also suggest that these effects may be caused, at least in part, by a significant inhibition of gastric lipase and subsequent reduction in fat absorption. PMID:22745826

  16. Postprandial serum triacylglycerols and oxidative stress in mice after consumption of fish oil, soy oil or olive oil: possible role for paraoxonase-1 triacylglycerol lipase-like activity.

    PubMed

    Fuhrman, Bianca; Volkova, Nina; Aviram, Michael

    2006-09-01

    Postprandial triacylglycerols and oxidative stress responses are influenced by the type of fat consumed. We investigated the effect of individual unsaturated fatty acids or oils (fish, soy, or olive) on postprandial triglyceridemia response in association with serum resistance to oxidation and paraoxonase-1 (PON1) activity. Balb/C mice were supplemented with phosphate buffered saline (control), docosahexaenoic acid (omega-3), linoleic acid (omega-6), or oleic acid (omega-9; 500 microg/300 microL of phosphate buffered saline) and with fish, soy, or olive oil (300 microL); blood samples were collected 2 h after feeding. Serum triacylglycerol and oxidative stress responses increased after intake of all unsaturated fatty acids and oil supplements. However, ingestion of fish oil or its major fatty acid, docosahexaenoic acid, induced the most remarkable increase in postprandial serum triacylglycerols and in the susceptibility of serum to in vitro oxidation. Serum PON1 activity was decreased by 24% after fish oil ingestion. The increase in postprandial serum susceptibility to oxidation was lower after soy oil supplementation to PON1-transgenic mice in comparison with Balb/C mice, showing that PON1 attenuates the postprandial serum oxidative response. In parallel, in PON1-transgenic mice, a decreased postprandial triacylglycerol response was noted, suggesting PON1 involvement in triacylglycerol metabolism. PON1 exhibited a triacylglycerol lipase-like activity on chylomicrons. PON1 attenuates the postprandial oxidative stress response, and this could have resulted from PON1 lipase-like activity on chylomicron triacylglycerols.

  17. Rat lingual lipase: partial purification, hydrolytic properties, and comparison with pancreatic lipase.

    PubMed

    Roberts, I M; Montgomery, R K; Carey, M C

    1984-10-01

    We have partially purified lingual lipase from the serous glands of rat tongue. With a combination of Triton X-100 extraction or Triton X-114 phase-separation techniques, Bio-Bead SM-2 treatment, dialysis, and gel filtration on Sephadex G-200 or Sephacryl S-300, we obtained a sparingly soluble lipid-free protein demonstrating hydrolytic activity against triglycerides and negligible phospholipase or cholesteryl esterase activities. Compared with homogenate, specific activities of the enzyme were enriched 3- to 5-fold prior to gel filtration and 10-fold after gel filtration. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration under denaturing conditions (6 M guanidine X HCl or 0.1% sodium dodecyl sulfate) revealed one major glycoprotein band with Mr approximately 50,000. Gel filtration of the active enzyme in 0.1% Triton X-100 gave an Mr approximately 270,000-300,000, suggesting extensive self-aggregation. With both tributyrin and triolein, the pH optimum of the purified enzyme was 4.0 and activity extended from pH 2.0 to 8.0. In contrast to purified human pancreatic lipase, lingual lipase hydrolyzed triglyceride emulsions and mixed micelles stabilized with both short-chain (dihexanoyl) and long-chain (egg) lecithin and were inhibited only slightly (18-25%) by micellar concentrations of two common bile salts, taurodeoxycholate and taurocholate. Our results suggest that the hydrolysis of dietary fat by lingual lipase may extend from the pharynx through the esophagus and stomach and into the upper small intestine.

  18. Hydrolysis products generated by lipoprotein lipase and endothelial lipase differentially impact THP-1 macrophage cell signalling pathways.

    PubMed

    Essaji, Yasmin; Yang, Yanbo; Albert, Carolyn J; Ford, David A; Brown, Robert J

    2013-08-01

    Macrophages express lipoprotein lipase (LPL) and endothelial lipase (EL) within atherosclerotic plaques; however, little is known about how lipoprotein hydrolysis products generated by these lipases might affect macrophage cell signalling pathways. We hypothesized that hydrolysis products affect macrophage cell signalling pathways associated with atherosclerosis. To test our hypothesis, we incubated differentiated THP-1 macrophages with products from total lipoprotein hydrolysis by recombinant LPL or EL. Using antibody arrays, we found that the phosphorylation of six receptor tyrosine kinases and three signalling nodes--most associated with atherosclerotic processes--was increased by LPL derived hydrolysis products. EL derived hydrolysis products only increased the phosphorylation of tropomyosin-related kinase A, which is also implicated in playing a role in atherosclerosis. Using electrospray ionization-mass spectrometry, we identified the species of triacylglycerols and phosphatidylcholines that were hydrolyzed by LPL and EL, and we identified the fatty acids liberated by gas chromatography-mass spectrometry. To determine if the total liberated fatty acids influenced signalling pathways, we incubated differentiated THP-1 macrophages with a mixture of the fatty acids that matched the concentrations of liberated fatty acids from total lipoproteins by LPL, and we subjected cell lysates to antibody array analyses. The analyses showed that only the phosphorylation of Akt was significantly increased in response to fatty acid treatment. Overall, our study shows that macrophages display potentially pro-atherogenic signalling responses following acute treatments with LPL and EL lipoprotein hydrolysis products.

  19. Production of FAME and FAEE via Alcoholysis of Sunflower Oil by Eversa Lipases Immobilized on Hydrophobic Supports.

    PubMed

    Remonatto, Daniela; de Oliveira, J Vladimir; Manuel Guisan, J; de Oliveira, Débora; Ninow, Jorge; Fernandez-Lorente, Gloria

    2018-01-03

    The performance of two new commercial low-cost lipases Eversa® Transform and Eversa® Transform 2.0 immobilized in different supports was investigated. The two lipases were adsorbed on four different hydrophobic supports. Interesting results were obtained for both lipases and for the four supports. However, the most active derivative was prepared by immobilization of Eversa® Transform 2.0 on Sepabeads C-18. Ninety-nine percent of fatty acid ethyl ester was obtained, in 3 h at 40 °C, by using hexane as solvent, a molar ratio of 4:1 (ethanol/oil), and 10 wt% of immobilized biocatalyst. The final reaction mixture contained traces of monoacylglycerols but was completely free of diacylglycerols. After four reaction cycles, the immobilized biocatalyst preserved 75% of activity. Both lipases immobilized in Sepabeads C-18 were very active with ethanol and methanol as acceptors, but they were much more stable in the presence of ethanol.

  20. The polycyclic aromatic hydrocarbons benzo[a]pyrene and phenanthrene inhibit intestinal lipase activity in rainbow trout (Oncorhynchus mykiss).

    PubMed

    de Gelder, Stefan; Sæle, Øystein; de Veen, Bas T H; Vos, Joëlle; Flik, Gert; Berntssen, Marc H G; Klaren, Peter H M

    2017-08-01

    Elevated levels of polycyclic aromatic hydrocarbons (PAHs) are detected in aquafeeds where fish oils are (partially) replaced by vegetable oils. The highly lipophilic PAHs solubilize readily in oil droplets and micelles in the intestinal lumen that can affect enzymatic lipid digestion by altering lipase activity. We therefore investigated the effect of two PAHs, benzo[a]pyrene (BaP) and phenanthrene (PHE), on bile salt-activated lipase (BAL) activity in desalted luminal extracts of the proximal intestine of rainbow trout (Oncorhynchus mykiss) using the triacylglycerides rapeseed oil and fish oil as substrates. The hydrolysis of rapeseed oil and fish oil measured at a calculated substrate concentration of 2.2mM, increased linearly up to 30min at 15°C. Substrate dependency under initial velocity conditions was described by simple Michaelis-Menten kinetics with a K m value of 1.2mM for rapeseed and fish oil. Rapeseed oil hydrolysis was inhibited by 1nM BaP and 10nM PHE. The hydrolysis of fish oil was only inhibited by 10μM BaP. The in vitro lipase activity data were corroborated by TLC/HPLC analysis of the reaction products, showing that in the presence of BaP and PHE, 46-80% less free fatty acids (FFA) were hydrolysed from rapeseed and fish oil triacylglycerides. The presence of low concentrations of BaP and PHE decreased rapeseed oil hydrolysis by BAL whereas fish oil hydrolysis was not affected. The replacement of fish oil by rapeseed oil in aquafeeds introduces PAHs that could affect lipid digestion. Copyright © 2017 Elsevier Inc. All rights reserved.

  1. Differential effect of combined lipase deficiency (cld/cld) on human hepatic lipase and lipoprotein lipase secretion.

    PubMed

    Boedeker, J C; Doolittle, M H; White, A L

    2001-11-01

    Combined lipase deficiency (cld) is a recessively inherited disorder in mice associated with a deficiency of LPL and hepatic lipase (HL) activity. LPL is synthesized in cld tissues but is retained in the endoplasmic reticulum (ER), whereas mouse HL (mHL) is secreted but inactive. In this study we investigated the effect of cld on the secretion of human HL (hHL) protein mass and activity. Differentiated liver cell lines were derived from cld mice and their normal heterozygous (het) littermates by transformation of hepatocytes with SV40 large T antigen. After transient transfection with lipase expression constructs, secretion of hLPL activity from cld cells was only 12% of that from het cells. In contrast, the rate of secretion of hHL activity and protein mass per unit of expressed hHL mRNA was identical for the two cell lines. An intermediate effect was observed for mHL, with a 46% reduction in secretion of activity from cld cells. The ER glucosidase inhibitor, castanospermine, decreased secretion of both hLPL and hHL from het cells by approximately 70%, but by only approximately 45% from cld cells. This is consistent with data suggesting that cld may result from a reduced concentration of the ER chaperone calnexin. In conclusion, our results demonstrate a differential effect of cld on hLPL, mHL, and hHL secretion, suggesting differential requirements for activation and exit of the enzymes from the ER.

  2. Requirement of catalytic-triad and related amino acids for the acyltransferase activity of Tanacetum cinerariifolium GDSL lipase/esterase TcGLIP for ester-bond formation in pyrethrin biosynthesis.

    PubMed

    Kikuta, Yukio; Yamada, Gen; Mitsumori, Tomonori; Takeuchi, Takayuki; Nakayama, Koji; Katsuda, Yoshio; Hatanaka, Akikazu; Matsuda, Kazuhiko

    2013-01-01

    We have recently discovered that a GDSL lipase/esterase (TcGLIP) in Tanacetum cinerariifolium catalyzed acyltransferase activity to form an ester bond in the natural insecticide, pyrethrin. TcGLIP contained Ser40 in Block I, Gly64 in Block II, Asn168 in Block III and Asp318 and His321 in Block V, suggesting underlying hydrolase activity, although little is known about their role in acyltransferase activity. We expressed TcGLIP here in Esherichia coli as a fusion with maltose-binding protein (MBP), part of the fusion being cleaved with a protease to obtain MBP-free TcGLIP. A kinetic analysis revealed that the MBP moiety scarcely influenced the kinetic parameters. The effects on acyltransferase activity of mutations of Gly64, Asn168, Asp318 and His321 were investigated by using MBP-fused TcGLIP. Mutations of these amino acids markedly reduced the acyltransferase activity, suggesting their critical role in the production of pyrethrins.

  3. Smoking is associated with increased hepatic lipase activity, insulin resistance, dyslipidaemia and early atherosclerosis in Type 2 diabetes.

    PubMed

    Kong, C; Nimmo, L; Elatrozy, T; Anyaoku, V; Hughes, C; Robinson, S; Richmond, W; Elkeles, R S

    2001-06-01

    We have studied the relationships between hepatic lipase activity, smoking, dyslipidaemia insulin resistance, and early atherosclerosis in 67 Type 2 diabetic subjects, 47 non-smokers and 20 smokers. Insulin resistance was measured using an insulin modified frequently sampled intravenous glucose tolerance test. Early atherosclerosis was assessed using high-resolution ultrasound to measure carotid intima media thickness (IMT) and an arterial ultrasonic score (AUS). Smokers had higher serum cholesterol and triglyceride, lower HDL and HDL2 cholesterol as well as increased hepatic lipase activity. They were also more insulin resistant than non-smokers. Smokers also had higher patient AUS scores. On multiple regression analysis, hepatic lipase activity emerged as the most significant variable affecting patient AUS. We suggest that smoking accentuates the dyslipidaemia of Type 2 diabetic subjects and this is associated with increased hepatic lipase activity. This may be one mechanism whereby smoking further increases the risk of cardiovascular disease in Type 2 diabetes.

  4. Monoglyceride lipase as a drug target: At the crossroads of arachidonic acid metabolism and endocannabinoid signaling.

    PubMed

    Grabner, Gernot F; Zimmermann, Robert; Schicho, Rudolf; Taschler, Ulrike

    2017-07-01

    Monoglyerides (MGs) are short-lived, intermediary lipids deriving from the degradation of phospho- and neutral lipids, and monoglyceride lipase (MGL), also designated as monoacylglycerol lipase (MAGL), is the major enzyme catalyzing the hydrolysis of MGs into glycerol and fatty acids. This distinct function enables MGL to regulate a number of physiological and pathophysiological processes since both MGs and fatty acids can act as signaling lipids or precursors thereof. The most prominent MG species acting as signaling lipid is 2-arachidonoyl glycerol (2-AG) which is the most abundant endogenous agonist of cannabinoid receptors in the body. Importantly, recent observations demonstrate that 2-AG represents a quantitatively important source for arachidonic acid, the precursor of prostaglandins and other inflammatory mediators. Accordingly, MGL-mediated 2-AG degradation affects lipid signaling by cannabinoid receptor-dependent and independent mechanisms. Recent genetic and pharmacological studies gave important insights into MGL's role in (patho-)physiological processes, and the enzyme is now considered as a promising drug target for a number of disorders including cancer, neurodegenerative and inflammatory diseases. This review summarizes the basics of MG (2-AG) metabolism and provides an overview on the therapeutic potential of MGL. Copyright © 2017 Elsevier Inc. All rights reserved.

  5. Stability studies of immobilized lipase on rice husk and eggshell membrane

    NASA Astrophysics Data System (ADS)

    Abdulla, R.; Sanny, S. A.; Derman, E.

    2017-06-01

    Lipase immobilization for biodiesel production is gaining importance day by day. In this study, lipase from Burkholderia cepacia was immobilized on activated support materials namely rice husk and egg shell membrane. Both rice husk and eggshell membrane are natural wastes that holds a lot of potential as immobilization matrix. Rice husk and eggshell membrane were activated with glutaraldehyde. Lipase was immobilized on the glutaraldehyde-activated support material through adsorption. Immobilization efficiency together with enzyme activity was observed to choose the highest enzyme loading for further stability studies. Immobilization efficiency of lipase on rice husk was 81 as compared to an immobilization efficiency of 87 on eggshell membrane. Immobilized lipase on eggshell membrane exhibited higher enzyme activity as compared to immobilized lipase on rice husk. Eggshell membrane also reported higher stability than rice husk as immobilization matrix. Both types of immobilized lipase retatined its activity after ten cycles of reuse. In short, eggshell membrane showed to be a better immobilization platform for lipase as compared to rice husk. However, with further improvement in technique of immobilization, the stability of both types of immobilized lipase can be improved to a greater extent.

  6. Optimization of Lipase production from a novel strain Thalassospira permensis M35-15 using Response Surface Methodology

    PubMed Central

    Kai, Wang; Peisheng, Yan

    2016-01-01

    ABSTRACT Lipases can catalyze the hydrolysis of glycerol, esters and long chain fatty acids. A lipase producing isolate M35-15 was screened and identified as Thalassospira permensis using 16S rRNA gene sequence analysis. To our knowledge this is the first report on Thalassospira permensis producing lipases. In this paper the optimization of medium composition for the increase in bacterial lipase was achieved using statistical methods. Firstly the key ingredients were selected by Plackett-Burman experimental design, then the levels of the ingredients were optimized using central composite design of Response Surface Methodology. The predicted optimal lipase activity was 11.49 U under the conditions that medium composition were 5.15 g/l glucose, 11.74 g/l peptone, 6.74 g/l yeast powder and 22.90 g/l olive oil emulsifier. PMID:27285376

  7. Optimization of Lipase production from a novel strain Thalassospira permensis M35-15 using Response Surface Methodology.

    PubMed

    Kai, Wang; Peisheng, Yan

    2016-09-02

    Lipases can catalyze the hydrolysis of glycerol, esters and long chain fatty acids. A lipase producing isolate M35-15 was screened and identified as Thalassospira permensis using 16S rRNA gene sequence analysis. To our knowledge this is the first report on Thalassospira permensis producing lipases. In this paper the optimization of medium composition for the increase in bacterial lipase was achieved using statistical methods. Firstly the key ingredients were selected by Plackett-Burman experimental design, then the levels of the ingredients were optimized using central composite design of Response Surface Methodology. The predicted optimal lipase activity was 11.49 U under the conditions that medium composition were 5.15 g/l glucose, 11.74 g/l peptone, 6.74 g/l yeast powder and 22.90 g/l olive oil emulsifier.

  8. Isolation and characterization of novel lipases from a metagenomic library of the microbial community in the pitcher fluid of the carnivorous plant Nepenthes hybrida.

    PubMed

    Morohoshi, Tomohiro; Oikawa, Manabu; Sato, Shoko; Kikuchi, Noriko; Kato, Norihiro; Ikeda, Tsukasa

    2011-10-01

    Members of the genus Nepenthes are carnivorous plants that use the pitfall method of insect capture as a supplementary nutritional source. We extracted metagenomic DNA from the microbial community found in the pitcher fluid of Nepenthes and constructed a plasmid-based metagenomic library. An activity-based screening method enabled the isolation of two lipase genes, lip1 and lip2. Both Lip1 and Lip2 belong to a novel family or subfamily of lipases and show lipase activities in acidic conditions, such as those found in pitcher fluid. This study was conducted under the assumption that the secreted Lip1 and Lip2 were capable of enzymatic activity in the acidic pitcher fluid. Copyright © 2011 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  9. Immobilization, Regiospecificity Characterization and Application of Aspergillus oryzae Lipase in the Enzymatic Synthesis of the Structured Lipid 1,3-Dioleoyl-2-Palmitoylglycerol.

    PubMed

    Cai, Haiying; Li, Yang; Zhao, Minjie; Fu, Guanwen; Lai, Jia; Feng, Fengqin

    2015-01-01

    The enzymatic synthesis of 1,3-dioleoyl-2-palmitoylglycerol (OPO), one of the main components of human milk fats, has been hindered by the relatively high cost of sn-1,3-specific lipases and the deficiency in biocatalyst stability. The sn-1,3-specific lipase from Aspergillus oryzae (AOL) is highly and efficiently immobilized with the polystyrene-based hydrophobic resin D3520, with a significant 49.54-fold increase in specific lipase activity compared with the AOL powder in catalyzing the synthesis of OPO through the acidolysis between palm stearin and oleic acid (OA). The optimal immobilization conditions were investigated, including time course, initial protein concentration and solution pH. The sn-1,3 specificity of lipases under different immobilization conditions was evaluated and identified as positively associated with the lipase activity, and the pH of the immobilization solution influenced the regiospecificity and synthetic activity of these lipases. Immobilized AOL D3520, as the biocatalyst, was used for the enzymatic synthesis of the structured lipid OPO through the acidolysis between palm stearin and OA. The following conditions were optimized for the synthesis of structured lipid OPO: 65 °C temperature; 1:8 substrate molar ratio between palm stearin and OA; 8% (w/w) enzyme load; 3.5% water content of the immobilized lipase; and 1 h reaction time. Under these conditions, highly efficient C52 production (45.65%) was achieved, with a tripalmitin content of 2.75% and a sn-2 palmitic acid (PA) proportion of 55.08% in the system.

  10. Metal-Chelate Immobilization of Lipase onto Polyethylenimine Coated MCM-41 for Apple Flavor Synthesis.

    PubMed

    Sadighi, Armin; Motevalizadeh, Seyed Farshad; Hosseini, Morteza; Ramazani, Ali; Gorgannezhad, Lena; Nadri, Hamid; Deiham, Behnaz; Ganjali, Mohammad Reza; Shafiee, Abbas; Faramarzi, Mohammad Ali; Khoobi, Mehdi

    2017-08-01

    An enzyme immobilized on a mesoporous silica nanoparticle can serve as a multiple catalyst for the synthesis of industrially useful chemicals. In this work, MCM-41 nanoparticles were coated with polyethylenimine (MCM-41@PEI) and further modified by chelation of divalent metal ions (M = Co 2+ , Cu 2+ , or Pd 2+ ) to produce metal-chelated silica nanoparticles (MCM-41@PEI-M). Thermomyces lanuginosa lipase (TLL) was immobilized onto MCM-41, MCM-41@PEI, and MCM-41@PEI-M by physical adsorption. Maximum immobilization yield and efficiency of 75 ± 3.5 and 65 ± 2.7% were obtained for MCM@PEI-Co, respectively. The highest biocatalytic activity at extremely acidic and basic pH (pH = 3 and 10) values were achieved for MCM-PEI-Co and MCM-PEI-Cu, respectively. Optimum enzymatic activity was observed for MCM-41@PEI-Co at 75 °C, while immobilized lipase on the Co-chelated support retained 70% of its initial activity after 14 days of storage at room temperature. Due to its efficient catalytic performance, MCM-41@PEI-Co was selected for the synthesis of ethyl valerate in the presence of valeric acid and ethanol. The enzymatic esterification yield for immobilized lipase onto MCM-41@PEI-Co was 60 and 53%, respectively, after 24 h of incubation in n-hexane and dimethyl sulfoxide media. Graphical Abstract Divalent metal chelated polyethylenimine coated MCM-41 (MCM-41@PEI-M) was used for immobilization of Thermomyces lanuginosa lipase catalyzing green apple flavor preparation.

  11. An ultraviolet spectrophotometric assay for the screening of sn-2-specific lipases using 1,3-O-dioleoyl-2-O-α-eleostearoyl-sn-glycerol as substrate

    PubMed Central

    Mendoza, Lilia D.; Rodriguez, Jorge A.; Leclaire, Julien; Buono, Gerard; Fotiadu, Frédéric; Carrière, Frédéric; Abousalham, Abdelkarim

    2012-01-01

    In the present study, we propose a continuous assay for the screening of sn-2 lipases by using triacylglycerols (TAGs) from Aleurites fordii seed (tung oil) and a synthetic TAG containing the α-eleostearic acid at the sn-2 position and the oleic acid (OA) at the sn-1 and sn-3 positions [1,3-O-dioleoyl-2-O-α-eleostearoyl-sn-glycerol (sn-OEO)]. Each TAG was coated into a microplate well, and the lipase activity was measured by optical density increase at 272 nm due to transition of α-eleostearic acid from the adsorbed to the soluble state. The sn-1,3-regioselective lipases human pancreatic lipase (HPL), LIP2 lipase from Yarrowia lipolytica (YLLIP2), and a known sn-2 lipase, Candida antarctica lipase A (CALA) were used to validate this method. TLC analysis of lipolysis products showed that the lipases tested were able to hydrolyze the sn-OEO and the tung oil TAGs, but only CALA hydrolyzed the sn-2 position. The ratio of initial velocities on sn-OEO and tung oil TAGs was used to estimate the sn-2 preference of lipases. CALA was the enzyme with the highest ratio (0.22 ± 0.015), whereas HPL and YLLIP2 showed much lower ratios (0.072 ± 0.026 and 0.038 ± 0.016, respectively). This continuous sn-2 lipase assay is compatible with a high sample throughput and thus can be applied to the screening of sn-2 lipases. PMID:22114038

  12. Monitoring lipase/esterase activity by stopped flow in a sequential injection analysis system using p-nitrophenyl butyrate.

    PubMed

    Pliego, Jorge; Mateos, Juan Carlos; Rodriguez, Jorge; Valero, Francisco; Baeza, Mireia; Femat, Ricardo; Camacho, Rosa; Sandoval, Georgina; Herrera-López, Enrique J

    2015-01-27

    Lipases and esterases are biocatalysts used at the laboratory and industrial level. To obtain the maximum yield in a bioprocess, it is important to measure key variables, such as enzymatic activity. The conventional method for monitoring hydrolytic activity is to take out a sample from the bioreactor to be analyzed off-line at the laboratory. The disadvantage of this approach is the long time required to recover the information from the process, hindering the possibility to develop control systems. New strategies to monitor lipase/esterase activity are necessary. In this context and in the first approach, we proposed a lab-made sequential injection analysis system to analyze off-line samples from shake flasks. Lipase/esterase activity was determined using p-nitrophenyl butyrate as the substrate. The sequential injection analysis allowed us to measure the hydrolytic activity from a sample without dilution in a linear range from 0.05-1.60 U/mL, with the capability to reach sample dilutions up to 1000 times, a sampling frequency of five samples/h, with a kinetic reaction of 5 min and a relative standard deviation of 8.75%. The results are promising to monitor lipase/esterase activity in real time, in which optimization and control strategies can be designed.

  13. Monitoring Lipase/Esterase Activity by Stopped Flow in a Sequential Injection Analysis System Using p-Nitrophenyl Butyrate

    PubMed Central

    Pliego, Jorge; Mateos, Juan Carlos; Rodriguez, Jorge; Valero, Francisco; Baeza, Mireia; Femat, Ricardo; Camacho, Rosa; Sandoval, Georgina; Herrera-López, Enrique J.

    2015-01-01

    Lipases and esterases are biocatalysts used at the laboratory and industrial level. To obtain the maximum yield in a bioprocess, it is important to measure key variables, such as enzymatic activity. The conventional method for monitoring hydrolytic activity is to take out a sample from the bioreactor to be analyzed off-line at the laboratory. The disadvantage of this approach is the long time required to recover the information from the process, hindering the possibility to develop control systems. New strategies to monitor lipase/esterase activity are necessary. In this context and in the first approach, we proposed a lab-made sequential injection analysis system to analyze off-line samples from shake flasks. Lipase/esterase activity was determined using p-nitrophenyl butyrate as the substrate. The sequential injection analysis allowed us to measure the hydrolytic activity from a sample without dilution in a linear range from 0.05–1.60 U/mL, with the capability to reach sample dilutions up to 1000 times, a sampling frequency of five samples/h, with a kinetic reaction of 5 min and a relative standard deviation of 8.75%. The results are promising to monitor lipase/esterase activity in real time, in which optimization and control strategies can be designed. PMID:25633600

  14. Crystal structure of a triacylglycerol lipase from Penicillium expansum at 1.3 A determined by sulfur SAD

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Bian, Chuanbing; Yuan, Cai; Chen, Liqing

    2010-04-05

    Triacylglycerol lipases (EC 3.1.1.3) are present in many different organisms including animals, plants, and microbes. Lipases catalyze the hydrolysis of long-chain triglycerides into fatty acids and glycerol at the interface between the water insoluble substrate and the aqueous phase. Lipases can also catalyze the reverse esterification reaction to form glycerides under certain conditions. Lipases of microbial origin are of considerable commercial interest for wide variety of biotechnological applications in industries, including detergent, food, cosmetic, pharmaceutical, fine chemicals, and biodiesel. Nowadays, microbial lipases have become one of the most important industrial enzymes. PEL (Penicillium expansum lipase) is a fungal lipase frommore » Penicillium expansum strain PF898 isolated from Chinese soil that has been subjected to several generations of mutagenesis to increase its enzymatic activity. PEL belongs to the triacylglycerol lipases family, and its catalytic characteristics have been studied. The enzyme has been used in Chinese laundry detergent industry for several years (http://www.leveking.com). However, the poor thermal stability of the enzyme limits its application. To further study and improve this enzyme, PEL was cloned and sequenced. Furthermore, it was overexpressed in Pichia pastoris. PEL contains GHSLG sequence, which is the lipase consensus sequence Gly-X1-Ser-X2-Gly, but has a low amino acid sequence identities to other lipases. The most similar lipases are Rhizomucor miehei (PML) and Rhizopus niveus (PNL) with a 21% and 20% sequence identities to PEL, respectively. Interestingly, the similarity of PEL with the known esterases is somewhat higher with 24% sequence identity to feruloyl esterase A. Here, we report the 1.3 {angstrom} resolution crystal structure of PEL determined by sulfur SAD phasing. This structure not only presents a new lipase structure at high resolution, but also provides a structural platform to analyze the

  15. Pancreatic lipase inhibitory constituents from Morus alba leaves and optimization for extraction conditions.

    PubMed

    Jeong, Ji Yeon; Jo, Yang Hee; Kim, Seon Beom; Liu, Qing; Lee, Jin Woo; Mo, Eun Jin; Lee, Ki Yong; Hwang, Bang Yeon; Lee, Mi Kyeong

    2015-06-01

    The leaves of Morus alba (Moraceae) have been traditionally used for the treatment of metabolic diseases including diabetes and hyperlipidemia. Thus, inhibitory effect of M. alba leaves on pancreatic lipase and their active constituents were investigated in this study. Twenty phenolic compounds including ten flavonoids, eight benzofurans, one stilbene and one chalcones were isolated from the leaves of M. alba. Among the isolated compounds, morachalcone A (20) exerted strong pancreatic lipase inhibition with IC50 value of 6.2 μM. Other phenolic compounds containing a prenyl group showed moderate pancreatic lipase inhibition with IC50 value of <50 μM. Next, extraction conditions with maximum pancreatic lipase inhibition and phenolic content were optimized using response surface methodology with three-level-three-factor Box-Behnken design. Our results suggested the optimized extraction condition for maximum pancreatic lipase inhibition and phenolic content as ethanol concentration of 74.9%; temperature 57.4 °C and sample/solvent ratio, 1/10. The pancreatic lipase inhibition and total phenolic content under optimized condition were found to be 58.5% and 26.2 μg GAE (gallic acid equivalent)/mg extract, respectively, which were well matched with the predicted value. Copyright © 2015 Elsevier Ltd. All rights reserved.

  16. Lipase in biphasic alginate beads as a biocatalyst for esterification of butyric acid and butanol in aqueous media.

    PubMed

    Ng, Choong Hey; Yang, Kun-Lin

    2016-01-01

    Esterification of organic acids and alcohols in aqueous media is very inefficient due to thermodynamic constraints. However, fermentation processes used to produce organic acids and alcohols are often conducted in aqueous media. To produce esters in aqueous media, biphasic alginate beads with immobilized lipase are developed for in situ esterification of butanol and butyric acid. The biphasic beads contain a solid matrix of calcium alginate and hexadecane together with 5 mg/mL of lipase as the biocatalyst. Hexadecane in the biphasic beads serves as an organic phase to facilitate the esterification reaction. Under optimized conditions, the beads are able to catalyze the production of 0.16 mmol of butyl butyrate from 0.5 mmol of butyric acid and 1.5 mmol of butanol. In contrast, when monophasic beads (without hexadecane) are used, only trace amount of butyl butyrate is produced. One main application of biphasic beads is in simultaneous fermentation and esterification (SFE) because the organic phase inside the beads is very stable and does not leach out into the culture medium. SFE is successfully conducted with an esterification yield of 6.32% using biphasic beads containing iso-octane even though the solvent is proven toxic to the butanol-producing Clostridium spp. Copyright © 2015 Elsevier Inc. All rights reserved.

  17. Label-free measurement of the yeast short chain TAG lipase activity by ESI-MS after one-step esterification.

    PubMed

    Ham, Hye Jin; Seo, Jongcheol; Yoon, Hye-Joo; Shin, Seung Koo

    2017-03-01

    Triacylglycerol (TAG) lipases hydrolyze ester bonds in TAG and release diacylglycerol (DAG), monoacylglycerol (MAG), and FA. We present a one-step chemical derivatization method for label-free quantification of a mixture of TAG, DAG, and MAG following lipase assay by ESI-MS. Because the ionization efficiencies of TAG, DAG, and MAG are not identical, lipase reaction products, DAG and MAG, are derivatized to TAG species by esterifying their hydroxyl groups using acyl chloride, whose acyl chain contains one less (or one more) -CH 2 group than that of substrate TAG. This resulted in three TAG species that were separated by 14 Da from one another and exhibited similar ion responses representing their molar amounts in the mass spectra. A good linear correlation was observed between peak intensity ratios and molar ratios in calibration curve. This method enables simultaneous quantification of TAG, DAG, and MAG in lipase assay and, in turn, allows stoichiometric determination of the concentrations of FAs released from TAG and DAG separately. By applying this strategy to measure both TAG and DAG lipolytic activities of the yeast Tgl2 lipase, we demonstrated its usefulness in studying enzymatic catalysis, as lipase enzymes often show dissimilar activities toward these lipids. Copyright © 2017 by the American Society for Biochemistry and Molecular Biology, Inc.

  18. Efficient and low-cost alternative of lipase concentration aiming at the application in the treatment of waste cooking oils.

    PubMed

    Preczeski, Karina P; Kamanski, Angela B; Scapini, Thamarys; Camargo, Aline F; Modkoski, Tatiani A; Rossetto, Vanusa; Venturin, Bruno; Mulinari, Jéssica; Golunski, Simone M; Mossi, Altemir J; Treichel, Helen

    2018-06-01

    In this study, we evaluated the concentration of lipases from Aspergillus niger using efficient and low-cost methods aiming at application in the treatment of waste cooking oils. The change in ionic strength of the medium by the addition of salt and precipitation with ethanol increased the specific activity from 2.90 to 28.50 U/mg, resulting in a purification factor of 9.82-fold. The use of acetone resulted in a specific activity of 33.63 U/mg, resulting in a purification factor of 11.60-fold. After that, the concentrated lipase was used in the hydrolysis of waste cooking oil and 753.07 and 421.60 µmol/mL of free fatty acids were obtained for the enzyme precipitated with ethanol and acetone, respectively. The hydrolysis of waste cooking oil catalyzed by homemade purified lipase in ultrasonic media can be considered a pretreatment of oil by converting a significant amount of triglycerides into free fatty acids.

  19. Site-directed mutagenesis studies of the aromatic residues at the active site of a lipase from Malassezia globosa.

    PubMed

    Gao, Chongliang; Lan, Dongming; Liu, Lu; Zhang, Houjin; Yang, Bo; Wang, Yonghua

    2014-07-01

    The lipase from Malassezia globosa (SMG1) has specific activity on mono- and diacylglycerol but not on triacylglycerol. The structural analysis of SMG1 structure shows that two bulky aromatic residues, W116 and W229, lie at the entrance of the active site. To study the functions of these two residues in the substrate recognition and the catalytic reaction, they were mutated to a series of amino acids. Subsequently, biochemical properties of these mutants were investigated. Although the activities decrease, W229L and W116A show a significant shift in substrate preference. W229L has an increased preference for short-chain substrates whereas W116A has preference for long-chain substrates. Besides, the half-lives of W116A and W116H at 45 °C are 346.6 min and 115.5 min respectively, which improve significantly compared to that of native enzyme. Moreover, the optimum substrate of W116A, W116F and W229F mutants shifted from p-nitrophenyl caprylate to p-nitrophenyl myristate. These findings not only shed light onto the lipase structure/function relationship but also lay the framework for the potential industrial applications. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

  20. Estolides Synthesis Catalyzed by Immobilized Lipases

    PubMed Central

    Aguieiras, Erika C. G.; Veloso, Cláudia O.; Bevilaqua, Juliana V.; Rosas, Danielle O.; da Silva, Mônica A. P.; Langone, Marta A. P.

    2011-01-01

    Estolides are vegetable-oil-based lubricants obtained from oleic acid or any source of hydroxy fatty acids. In this work, the estolides synthesis from oleic acid and methyl ricinoleate (biodiesel from castor oil), using immobilized commercial lipases (Novozym 435, Lipozyme RM-IM, and Lipozyme TL-IM) in a solvent-free medium was investigated. Acid value was used to monitor the reaction progress by determining the consumption of acid present in the medium. Novozym 435 showed the best performance. Water removal improved the conversion. Novozym 435 was more active at atmospheric pressure. Novozym 435 was reused four times with conversion reaching 15% after the fourth reaction at 80°C. Estolides produced under the reaction conditions used in this work presented good properties, such as, low temperature properties as pour point (−24°C), viscosity (23.9 cSt at 40°C and 5.2 cSt at 100°C), and viscosity index (153). PMID:21755040

  1. Orientating lipase molecules through surface chemical control for enhanced activity: A QCM-D and ToF-SIMS investigation.

    PubMed

    Joyce, Paul; Kempson, Ivan; Prestidge, Clive A

    2016-06-01

    Bio-active materials consisting of lipase encapsulated within porous silica particles were engineered to control the adsorption kinetics and molecular orientation of lipase, which play critical roles in the digestion kinetics of triglycerides. The adsorption kinetics of Candida antartica lipase A (CalA) was monitored using quartz crystal microbalance with dissipation (QCM-D) and controlled by altering the hydrophobicity of a silica binding support. The extent of adsorption was 2-fold greater when CalA was adsorbed onto hydrophobic silica compared to hydrophilic silica. Time-of-flight secondary ion mass spectrometry (ToF-SIMS) fragmentation patterns, in conjunction with multivariate statistics, demonstrated enhanced exposure of the lipase's catalytic domain, specifically the histidine group responsible for activity, when CalA was adsorbed on hydrophilic silica. Consequently, lipid digestion kinetics were enhanced when CalA was loaded in hydrophilic porous silica particles, i.e., a 2-fold increase in the pseudo-first-order rate constant for digestion when compared to free lipase. In contrast, digestion kinetics were inhibited when CalA was hosted in hydrophobic porous silica, i.e., a 5-fold decrease in pseudo-first-order rate constant for digestion when compared to free lipase. These findings provide valuable insights into the mechanism of lipase action which can be exploited to develop smarter food and drug delivery systems consisting of porous lipid-based materials. Copyright © 2016 Elsevier B.V. All rights reserved.

  2. Cloning, sequencing and characterization of lipase from a polyhydroxyalkanoate- (PHA-) synthesizing Pseudomonas resinovorans

    USDA-ARS?s Scientific Manuscript database

    Lipase gene (lip) of a biodegradable polyhydroxyalkanoate- (PHA-) synthesizing bacterium P. resinovorans NRRL B-2649 was cloned, sequenced and characterized by using consensus primers and PCR-based genome walking method. The ORF of the putative Lip (314 amino acids) and its active site (Ser111, Asp...

  3. Immobilization of lipase and keratinase on functionalized SBA-15 nanostructured materials

    NASA Astrophysics Data System (ADS)

    Le, Hy G.; Vu, Tuan A.; Tran, Hoa T. K.; Dang, Phuong T.

    2013-12-01

    SBA-15 nanostructured materials were synthesized via hydrothermal treatment and were functionalized with 3- aminopropyltriethoxysilane (APTES). The obtained samples were characterized by different techniques such as XRD, BET, TEM, IR and DTA. After functionalization, it showed that these nanostrucrured materials still maintained the hexagonal pore structure of the parent SBA-15. The model enzyms chosen in this study were lipase and keratinase. Lipase was a biocatalyst for hydrolyzation of long chain triglycerides or methyl esters of long chain alcohols and fatty acids; keratinase is a proteolytic enzyme that catalyzes the cleavage of keratin. The functionalized SBA-15 materials were used to immobilize lipase and keratinase, exhibiting higher activity than that of the unfunctionalized pure silica SBA-15 ones. This might be due to the enhancing of surface hydrophobicity upon functionalization. The surface functionalization of the nanostructured silicas with organic groups can favor the interaction between enzyme and the supports and consequently increasing the operational stability of the immobilized enzymes. The loading of lipase on functionalized SBA-15 materials was higher than that of keratinase. This might be rationalized by the difference in size of enzyms.

  4. Extracts from the edible seaweed, Ascophyllum nodosum, inhibit lipase activity in vitro: contributions of phenolic and polysaccharide components.

    PubMed

    Austin, Ceri; Stewart, Derek; Allwood, J William; McDougall, Gordon J

    2018-01-24

    A polyphenol-rich extract (PRE) from the edible seaweed, Ascophyllum nodosum, inhibited pancreatic lipase activity in an oil-based turbidimetric assay with an IC 50 of 200 μg gallic acid equivalents (GAE) perassay) [∼230 μg DW] whereas the known inhibitor, Orlistat, gave an IC 50 at 0.4 μg per assay. A phlorotannin-enriched fraction (TRF) purified from the PRE was more potent with an IC 50 = 60 μg GAE per assay (∼65 μg DW). When the assay was started by the addition of lipase, both Orlistat and TRF were much less effective which suggests that pre-incubation of enzyme and inhibitor improved inhibition. Based on phenol content, water extracts from Ascophyllum were more potent lipase inhibitors than PRE (IC 50 ∼ 150 μg GAE per assay). However, this was equivalent to ∼580 μg DW and these extracts contained polysaccharides (e.g. alginate content = 110 μg mL -1 ) which may also contribute to inhibition. Indeed, a polysaccharide-enriched fraction obtained by ethanol precipitation gave an IC 50 of 1000 μg DW which was equivalent to 130 μg GAE and 420 μg alginate per assay. Therefore a >3 fold increase in alginate content did not markedly improve inhibition. Re-precipitation increased alginate content and reduced polyphenol content but lipase inhibition was markedly reduced (i.e. IC 50 at ∼1100 μg DW per assay, 700 μg alginate and 25 μg GAE). Purifying the polysaccharide fraction by ion exchange removed all phenolics but the IC 50 increased to >2500 μg DW, equivalent to >1970 μg alginate per assay. In conclusion, polysaccharides and phlorotannins may inhibit lipase in an additive fashion, with phlorotannins apparently more effective in vitro. However, interactions between these components may be important when food products containing this edible seaweed are consumed.

  5. Evaluation of Expression of Lipases and Phospholipases of Malassezia restricta in Patients with Seborrheic Dermatitis

    PubMed Central

    Lee, Yang Won; Lee, Shin Yung; Lee, Younghoon

    2013-01-01

    Background Malassezia species (spp.) are cutaneous opportunistic pathogens and associated with various dermatological diseases including seborrheic dermatitis, dandruff and atopic dermatitis. Almost all Malassezia spp. are obligatorily lipid-dependent, which might be caused by lack of the myristic acid synthesis. Recent genome analysis of M. restricta and M. globosa suggested that the absence of a gene encoding fatty acid synthesis might be compensated by abundant genes encoding hydrolases, which produce fatty acids, and that lipases and phospholipases may play a role in virulence of the fungus. Objective The current study aimed to investigate the contribution of lipases and phospholipases in virulence of the M. restricta as being the most frequently isolated Malassezia spp. from the human skin. Methods Swap samples of two different body sites of at least 18 patients with seborrheic dermatitis were obtained and in vivo expression of lipases and phospholipases of M. restricta was analyzed by the gene specific two-step nested RT-PCR. Results The results of the current study suggest that majority of the patients display expression of lipase RES_0242. Conclusion These data imply a possible role of lipase in the host environment to produce free fatty acids for the fungus. PMID:24003273

  6. Evaluation of Expression of Lipases and Phospholipases of Malassezia restricta in Patients with Seborrheic Dermatitis.

    PubMed

    Lee, Yang Won; Lee, Shin Yung; Lee, Younghoon; Jung, Won Hee

    2013-08-01

    Malassezia species (spp.) are cutaneous opportunistic pathogens and associated with various dermatological diseases including seborrheic dermatitis, dandruff and atopic dermatitis. Almost all Malassezia spp. are obligatorily lipid-dependent, which might be caused by lack of the myristic acid synthesis. Recent genome analysis of M. restricta and M. globosa suggested that the absence of a gene encoding fatty acid synthesis might be compensated by abundant genes encoding hydrolases, which produce fatty acids, and that lipases and phospholipases may play a role in virulence of the fungus. The current study aimed to investigate the contribution of lipases and phospholipases in virulence of the M. restricta as being the most frequently isolated Malassezia spp. from the human skin. Swap samples of two different body sites of at least 18 patients with seborrheic dermatitis were obtained and in vivo expression of lipases and phospholipases of M. restricta was analyzed by the gene specific two-step nested RT-PCR. The results of the current study suggest that majority of the patients display expression of lipase RES_0242. These data imply a possible role of lipase in the host environment to produce free fatty acids for the fungus.

  7. Transesterification of plant oils using Staphylococcus haemolyticus L62 lipase displayed on Escherichia coli cell surface using the OmpA signal peptide and EstAβ8 anchoring motif.

    PubMed

    Jo, Jin Chul; Kim, Soon-Ja; Kim, Hyung Kwoun

    2014-12-01

    Staphylococcus haemolyticus L62 (SHL62) lipase was displayed on the outer membrane of Escherichia coli using the OmpA signal peptide and the autotransporter EstAβ8 protein. Localization of SHL62 lipase on the outer membrane of E. coli was confirmed using immunofluorescence microscopy and flow cytometry analysis. Lipase activity of the displayed SHL62 lipase was also measured using spectrophotometry and pH titration. SHL62 lipase activity of whole cells reached 2.0U/ml culture (OD600nm of 10) when it was measured by the p-nitrophenyl caprylate assay after being induced with 1mM IPTG for 24h. The optimum temperature and pH for the lipase was 45°C and 10, respectively. Furthermore, it maintained more than 90% of maximum lipase activity at up to 50°C and in a pH range of 5-9. The hydrolytic activity assay conduted with various substrates confirmed that p-nitrophenyl caprylate and corn oil were preferred substrates among various synthetic and natural substrates, respectively. The displayed SHL62 lipase produced fatty acid esters from various alcohols and plant oils through transesterification. Copyright © 2014 Elsevier Inc. All rights reserved.

  8. Mechanisms of lipase maturation

    PubMed Central

    Péterfy, Miklós

    2010-01-01

    Lipases are acyl hydrolases that represent a diverse group of enzymes present in organisms ranging from prokaryotes to humans. This article focuses on an evolutionarily related family of extracellular lipases that include lipoprotein lipase, hepatic lipase and endothelial lipase. As newly synthesized proteins, these lipases undergo a series of co- and post-translational maturation steps occurring in the endoplasmic reticulum, including glycosylation and glycan processing, and protein folding and subunit assembly. This article identifies and discusses mechanisms that direct early and late events in lipase folding and assembly. Lipase maturation employs the two general chaperone systems operating in the endoplasmic reticulum, as well as a recently identified lipase-specific chaperone termed lipase maturation factor 1. We propose that the two general chaperone systems act in a coordinated manner early in lipase maturation in order to help create partially folded monomers; lipase maturation factor 1 then facilitates final monomer folding and subunit assembly into fully functional homodimers. Once maturation is complete, the lipases exit the endoplasmic reticulum and are secreted to extracellular sites, where they carry out a number of functions related to lipoprotein and lipid metabolism. PMID:20543905

  9. Chemical composition and inhibitory activities on dipeptidyl peptidase IV and pancreatic lipase of two underutilized species from the Brazilian Savannah: Oxalis cordata A.St.-Hil. and Xylopia aromatica (Lam.) Mart.

    PubMed

    Oliveira, Verena B; Araújo, Raquel L B; Eidenberger, Thomas; Brandão, Maria G L

    2018-03-01

    Brazil has the greatest vegetal biodiversity in the world, but products derived from native species are not optimally utilized. Oxalis cordata and Xylopia aromatica are two underutilized species whose leaves and fruits, respectively, have been used as food in the 19th century. In this study, we used chemical and in vitro assays to evaluate the potential of these species as functional foods. The inhibitory activity on pancreatic lipase and DPP-IV were evaluated using the crude extracts and fractions ethyl acetate, butanol and water of these two species. For polyphenols determination, samples were prepared with different solvents and these were analysed by chromatographic and spectroscopic methods. Finally, fatty acids profile was determinated by gas chromatography. The crude extract (IC 50 =0.84mg/ml), ethyl acetate extract (IC 50 =0.88mg/ml) an aqueous fraction (IC 50 =0.63mg/ml) of C. cordata were inhibitory on pancreatic lipase but inactive against dipeptidyl peptidase IV (DPP-IV). Extracts from X. aromatica were inactive against the lipase pancreatic enzyme, but a butanolic fraction inhibited DPP-IV (IC 50 =0.71±0.05mg/ml). The phenolic acids orientin/isorientin, chlorogenic acid (0.32g/100g) and the flavonoid derivatives rutin (0.27g/100g), quercetin and luteolin were observed in all products. Additionally, fatty acid quantification showed that oleic (7.5g/100g) and linoleic acid (6.5g/100g) were predominant in X. aromatica fruit. This study confirms the potential for the use of both plants as functional foods due to their nutritional value, biological activity and important phytochemical content. Copyright © 2017 Elsevier Ltd. All rights reserved.

  10. SOA genes encode proteins controlling lipase expression in response to triacylglycerol utilization in the yeast Yarrowia lipolytica.

    PubMed

    Desfougères, Thomas; Haddouche, Ramdane; Fudalej, Franck; Neuvéglise, Cécile; Nicaud, Jean-Marc

    2010-02-01

    The oleaginous yeast Yarrowia lipolytica efficiently metabolizes hydrophobic substrates such as alkanes, fatty acids or triacylglycerol. This yeast has been identified in oil-polluted water and in lipid-rich food. The enzymes involved in lipid breakdown, for use as a carbon source, are known, but the molecular mechanisms controlling the expression of the genes encoding these enzymes are still poorly understood. The study of mRNAs obtained from cells grown on oleic acid identified a new group of genes called SOA genes (specific for oleic acid). SOA1 and SOA2 are two small genes coding for proteins with no known homologs. Single- and double-disrupted strains were constructed. Wild-type and mutant strains were grown on dextrose, oleic acid and triacylglycerols. The double mutant presents a clear phenotype consisting of a growth defect on tributyrin and triolein, but not on dextrose or oleic acid media. Lipase activity was 50-fold lower in this mutant than in the wild-type strain. The impact of SOA deletion on the expression of the main extracellular lipase gene (LIP2) was monitored using a LIP2-beta-galactosidase promoter fusion protein. These data suggest that Soa proteins are components of a molecular mechanism controlling lipase gene expression in response to extracellular triacylglycerol.

  11. Response surface methodology as an approach to determine optimal activities of lipase entrapped in sol-gel matrix using different vegetable oils.

    PubMed

    Pinheiro, Rubiane C; Soares, Cleide M F; de Castro, Heizir F; Moraes, Flavio F; Zanin, Gisella M

    2008-03-01

    The conditions for maximization of the enzymatic activity of lipase entrapped in sol-gel matrix were determined for different vegetable oils using an experimental design. The effects of pH, temperature, and biocatalyst loading on lipase activity were verified using a central composite experimental design leading to a set of 13 assays and the surface response analysis. For canola oil and entrapped lipase, statistical analyses showed significant effects for pH and temperature and also the interactions between pH and temperature and temperature and biocatalyst loading. For the olive oil and entrapped lipase, it was verified that the pH was the only variable statistically significant. This study demonstrated that response surface analysis is a methodology appropriate for the maximization of the percentage of hydrolysis, as a function of pH, temperature, and lipase loading.

  12. Monoacylglycerol lipase – a target for drug development?

    PubMed Central

    Fowler, CJ

    2012-01-01

    The endocannabinoid (eCB) system is involved in processes as diverse as control of appetite, perception of pain and the limitation of cancer cell growth and invasion. The enzymes responsible for eCB breakdown are attractive pharmacological targets, and fatty acid amide hydrolase inhibitors, which potentiate the levels of the eCB anandamide, are now undergoing pharmaceutical development. ‘Drugable’ selective inhibitors of monoacylglycerol lipase, a key enzyme regulating the levels of the other main eCB, 2-arachidonoylglycerol, were however not identified until very recently. Their availability has resulted in a large expansion of our knowledge concerning the pharmacological consequences of monoacylglycerol lipase inhibition and hence the role(s) played by the enzyme in the body. In this review, the pharmacology of monoacylglycerol lipase will be discussed, together with an analysis of the therapeutic potential of monoacylglycerol lipase inhibitors as analgesics and anticancer agents. PMID:22428756

  13. Quantitative study of lipase secretion, extracellular lipolysis, and lipid storage in the yeast Yarrowia lipolytica grown in the presence of olive oil: analogies with lipolysis in humans.

    PubMed

    Najjar, Amal; Robert, Sylvie; Guérin, Clémence; Violet-Asther, Michèle; Carrière, Frédéric

    2011-03-01

    Lipase secretion, extracellular lipolysis, and fatty acid uptake were quantified in the yeast Yarrowia lipolytica grown in the presence of olive oil and/or glucose. Specific lipase assays, Western blot analysis, and ELISA indicated that most of the lipase activity measured in Y. lipolytica cultures resulted from the YLLIP2 lipase. Lipase production was triggered by olive oil and, during the first hours of culture, most of the lipase activity and YLLIP2 immunodetection remained associated with the yeast cells. YLLIP2 was then released in the culture medium before it was totally degraded by proteases. Olive oil triglycerides were largely degraded when the lipase was still attached to the cell wall. The fate of lipolysis products in the culture medium and inside the yeast cell, as well as lipid storage, was investigated simultaneously by quantitative TLC-FID and GC analysis. The intracellular levels of free fatty acids (FFA) and triglycerides increased transiently and were dependent on the carbon sources. A maximum fat storage of 37.8% w/w of yeast dry mass was observed with olive oil alone. A transient accumulation of saturated FFA was observed whereas intracellular triglycerides became enriched in unsaturated fatty acids. So far, yeasts have been mainly used for studying the intracellular synthesis, storage, and mobilization of neutral lipids. The present study shows that yeasts are also interesting models for studying extracellular lipolysis and fat uptake by the cell. The quantitative data obtained here allow for the first time to establish interesting analogies with gastrointestinal and vascular lipolysis in humans.

  14. [Immobilization of Candida sp. lipase on resin D301].

    PubMed

    Wang, Yanhua; Zhu, Kai; Liu, Hui; Han, Pingfang; Wei, Ping

    2009-12-01

    We immobilized Candida sp. lipase onto seven kinds of industrial adsorption and ion exchange resins. By determining the activity of each immobilized enzyme, the weakly basic anionic exchange resin of D301 showed the best results for the immobilization of Candida sp. lipase. Comparing the scanning electron micrographs of D301 with Novozym 435 (immobilized Candida antarctica lipase B from Novo Nordisk Corp.), we selected D301 as a carrier for the immobilization of Candida sp. lipase. And we pretreated the resin D301 with the bifunctional agent glutaraldehyde and crosslinked it with Candida sp. lipase. The optimal conditions for the immobilization of Candida sp. lipase were as follows: 8 mL of the amount of 5% glutaraldehyde solution, five hours of the time pretreated D301 with glutaraldehyde, 1.0 g/L the concentration of Candida sp. lipase used, pH of the phosphate buffered, 6.0 and 10 hours of time for immobilization, respectively. The activity of immobilized enzyme was over 35 U/mg and the efficiency of immobilization was around 3.5 Ul(mg x h).

  15. Sulforaphane induced adipolysis via hormone sensitive lipase activation, regulated by AMPK signaling pathway.

    PubMed

    Lee, Ju-Hee; Moon, Myung-Hee; Jeong, Jae-Kyo; Park, Yang-Gyu; Lee, You-Jin; Seol, Jae-Won; Park, Sang-Youel

    2012-10-05

    Sulforaphane, an aliphatic isothiocyanate derived from cruciferous vegetables, is known for its antidiabetic properties. The effects of sulforaphane on lipid metabolism in adipocytes are not clearly understood. Here, we investigated whether sulforaphane stimulates lipolysis. Mature adipocytes were incubated with sulforaphane for 24h and analyzed using a lipolysis assay which quantified glycerol released into the medium. We investigated gene expression of hormone-sensitive lipase (HSL), and levels of HSL phosphorylation and AMP-activated protein kinase on sulforaphane-mediated lipolysis in adipocytes. Sulforaphane promoted lipolysis and increased both HSL gene expression and HSL activation. Sulforaphane suppressed AMPK phosphorylation at Thr-172 in a dose-dependent manner, which was associated with a decrease in HSL phosphorylation at Ser-565, enhancing the phosphorylation of HSL Ser-563. Taken together, these results suggest that sulforaphane promotes lipolysis via hormone sensitive lipase activation mediated by decreasing AMPK signal activation in adipocytes. Copyright © 2012 Elsevier Inc. All rights reserved.

  16. Importance of the lid and cap domains for the catalytic activity of gastric lipases.

    PubMed

    Miled, N; Bussetta, C; De caro, A; Rivière, M; Berti, L; Canaan, S

    2003-09-01

    Human gastric lipase (HGL) is an enzyme secreted by the stomach, which is stable and active despite the highly acidic environment. It has been clearly established that this enzyme is responsible for 30% of the fat digestion processes occurring in human. This globular protein belongs to the alpha/beta hydrolase fold family and its catalytic serine is deeply buried under a domain called the extrusion domain, which is composed of a 'cap' domain and a segment consisting of 58 residues, which can be defined as a lid. The exact roles played by the cap and the lid domains during the catalytic step have not yet been elucidated. We have recently solved the crystal structure of the open form of the dog gastric lipase in complex with a covalent inhibitor. The detergent molecule and the inhibitor were mimicking a triglyceride substrate that would interact with residues belonging to both the cap and the lid domains. In this study, we have investigated the role of the cap and the lid domains, using site-directed mutagenesis procedures. We have produced truncated mutants lacking the lid and the cap. After expressing these mutants and purifying them, their activity was found to have decreased drastically in comparison with the wild type HGL. The lid and the cap domains play an important role in the catalytic reaction mechanism. Based on these results and the structural data (open form of DGL), we have pointed out the cap and the lid residues involved in the binding with the lipidic substrate.

  17. Survival in infants treated with sebelipase Alfa for lysosomal acid lipase deficiency: an open-label, multicenter, dose-escalation study.

    PubMed

    Jones, Simon A; Rojas-Caro, Sandra; Quinn, Anthony G; Friedman, Mark; Marulkar, Sachin; Ezgu, Fatih; Zaki, Osama; Gargus, J Jay; Hughes, Joanne; Plantaz, Dominique; Vara, Roshni; Eckert, Stephen; Arnoux, Jean-Baptiste; Brassier, Anais; Le Quan Sang, Kim-Hanh; Valayannopoulos, Vassili

    2017-02-08

    Infants presenting with lysosomal acid lipase deficiency have marked failure to thrive, diarrhea, massive hepatosplenomegaly, anemia, rapidly progressive liver disease, and death typically in the first 6 months of life; the only available potential treatment has been hematopoietic stem cell transplantation, which is associated with high morbidity and mortality in this population. The study objective was to evaluate safety and efficacy (including survival) of enzyme replacement with sebelipase alfa in infants with lysosomal acid lipase deficiency. This is an ongoing multicenter, open-label, phase 2/3 study conducted in nine countries. The study enrolled infants with growth failure prior to 6 months of age with rapidly progressive lysosomal acid lipase deficiency; they received once-weekly doses of sebelipase alfa initiated at 0.35 mg/kg with intrapatient dose escalation up to 5 mg/kg. The main outcome of interest is survival to 12 months and survival beyond 24 months of age. Nine patients were enrolled; median age at baseline was 3.0 months (range 1.1-5.8 months). Sixty-seven percent (exact 95% CI 30%-93%) of sebelipase alfa-treated infants survived to 12 months of age compared with 0% (exact 95% CI 0%-16%) for a historical control group (n = 21). Patients who survived to age 12 months exhibited improvements in weight-for-age, reductions in markers of liver dysfunction and hepatosplenomegaly, and improvements in anemia and gastrointestinal symptoms. Three deaths occurred early (first few months of life), two patients died because of advanced disease, and a third patient died following complications of non-protocol-specified abdominal paracentesis. A fourth death occurred at 15 months of age and was related to other clinical conditions. The five surviving patients have survived to age ≥24 months with continued sebelipase alfa treatment; all have displayed marked improvement in growth parameters and liver function. Serious adverse events considered

  18. Enzyme immobilization on ultrafine cellulose fibers via poly(acrylic acid) electrolyte grafts.

    PubMed

    Chen, Hong; Hsieh, You-Lo

    2005-05-20

    Ultrafine cellulose fiber (diameter 200-400 nm) surfaces were grafted with polyacrylic acid (PAA) via either ceric ion initiated polymerization or methacrylation of cellulose with methacrylate chloride (MACl) and subsequent free-radical polymerization of acrylic acid. PAA grafts by ceric ion initiated polymerization increased with increasing reaction time (2-24 h), monomer (0.3-2.4 M), and initiator (1-10 mM) concentrations, and spanned a broad range from 5.5-850%. PAA grafts on the methacrylated cellulose fibers also increased with increasing molar ratios of MACl to cellulosic hydroxyl groups (MACl/OH, 2-6.4) and monomer acrylic acid (AA) to initiator potassium persulfate (KPS) ratios ([AA]/[KPS], 1.5-6), and were in a much narrower range between 12.8% and 29.4%. The adsorption of lipase (at 1 mg/ml lipase and pH 7) and the activity of adsorbed lipase (pH 8.5, 30 degrees C), in both cases decreased with increasing PAA grafts. The highest adsorption and activity of the lipase on the ceric ion initiated grafted fibers were 1.28 g/g PAA and 4.3 U/mg lipase, respectively, at the lowest grafting level of 5.5% PAA, whereas they were 0.33 g/g PAA and 7.1 U/mg lipase, respectively, at 12.8% PAA grafts on the methacrylated and grafted fibers. The properties of the grafted fibers and the absorption behavior and activity of lipase suggest that the PAA grafts are gel-like by ceric-initiated reaction and brush-like by methacrylation and polymerization. The adsorbed lipase on the ceric ion-initiated grafted surface possessed greatly improved organic solvent stability over the crude lipase. The adsorbed lipases exhibited 0.5 and 0.3 of the initial activity in the second and third assay cycles, respectively. (c) 2004 Wiley Periodicals, Inc.

  19. Immobilized lipase from Candida sp. 99-125 on hydrophobic silicate: characterization and applications.

    PubMed

    Zhao, Bin; Liu, Xinlong; Jiang, Yanjun; Zhou, Liya; He, Ying; Gao, Jing

    2014-08-01

    Lipase Candida sp. 99-125 has been proved to be quite effective in catalyzing organic synthesis reactions and is much cheaper than commercial lipases. Mesoporous silicates are attractive materials for the immobilization of enzymes due to their unique structures. The present research designed a hydrophobic silicate with uniform pore size suitable for the comfort of lipase Candida sp. 99-125 for improving its activity and stability. The resulting immobilized lipase (LP@PMO) by adsorption was employed to catalyze hydrolysis, esterification, and transesterification reactions, and the performances were compared with the lipase immobilized on hydrophilic silicate (LP@PMS) and native lipase. The LP@PMO showed as high activity as that of native lipase in hydrolysis and much increased catalytic activity and reusability in the reactions for biodiesel production. Besides, LP@PMO also possessed better organic stability. Such results demonstrate that immobilization of lipase onto hydrophobic supports is a promising strategy to fabricate highly active and stable biocatalysts for applications.

  20. Enzymatic synthesis of capric acid-rich structured lipids (MUM type) using Candida antarctica lipase.

    PubMed

    SilRoy, Sumita; Ghosh, Mahua

    2011-01-01

    The objective of the work was to produce capric acid rich structured lipids starting from various Indian indigenous vegetable oils, such as rice bran, ground nut and mustard oils. Acidolysis reaction between individual vegetable oils and capric acid in one is to three molar ratios at 45 degree centigrade temperature was carried out using position specific Candida antarctica lipase so as to protect the Sn-2 position of the oils which are rich in unsaturated fatty acids. The incorporation of capric acid depended on the reaction time showing 6 % within 6 h and 30.8 % in 72 h with rice bran oil. Similarly, in ground nut oil incorporation of capric acid was 34.2 % in 72 h compared to 5.3 % in 6 h. Thus mustard oil showed much lower incorporation than the other two oils, with 3.3 % and 19.5 % in 6 and 72 h respectively. The incorporation of capric acid was influenced by the nature of the fatty acids present in the original oil. The fatty acid composition of Sn-2 position of the structured triacylglycerols of the three oils revealed that capric acid was mainly replacing the fatty acids occupying the Sn-1 and 3 positions of the triglyceride molecule.

  1. Immobilization of Yarrowia lipolytica Lipase on Macroporous Resin Using Different Methods: Characterization of the Biocatalysts in Hydrolysis Reaction.

    PubMed

    Sun, Jingjing; Chen, Yiling; Sheng, Jun; Sun, Mi

    2015-01-01

    To improve the reusability and organic solvent tolerance of microbial lipase and expand the application of lipase (hydrolysis, esterification, and transesterification), we immobilized marine microbial lipase using different methods and determined the properties of immobilized lipases. Considering the activity and cost of immobilized lipase, the concentration of lipase was fixed at 2 mg/mL. The optimal temperature of immobilized lipases was 40°C and 5°C higher than free lipase. The activities of immobilized lipases were much higher than free lipase at alkaline pH (more than 50% at pH 12). The free lipase lost most activity (35.3%) and immobilized lipases retained more than 46.4% of their initial activity after 3 h heat treatment at 70°C. At alkaline pH, immobilized lipases were more stable than free lipase (more than 60% residue activity at pH 11 for 3 h). Immobilized lipases retained 80% of their activity after 5 cycles and increased enzyme activity (more than 108.7%) after 3 h treatment in tert-butanol. Immobilization of lipase which improved reusability of lipase and provided a chance to expand the application of marine microbial lipase in organic system expanded the application range of lipase to catalyze hydrolysis and esterification in harsh condition.

  2. Smart conjugated polymer nanocarrier for healthy weight loss by negative feedback regulation of lipase activity

    NASA Astrophysics Data System (ADS)

    Chen, Yu-Lei; Zhu, Sha; Zhang, Lei; Feng, Pei-Jian; Yao, Xi-Kuang; Qian, Cheng-Gen; Zhang, Can; Jiang, Xi-Qun; Shen, Qun-Dong

    2016-02-01

    Healthy weight loss represents a real challenge when obesity is increasing in prevalence. Herein, we report a conjugated polymer nanocarrier for smart deactivation of lipase and thus balancing calorie intake. After oral administration, the nanocarrier is sensitive to lipase in the digestive tract and releases orlistat, which deactivates the enzyme and inhibits fat digestion. It also creates negative feedback to control the release of itself. The nanocarrier smartly regulates activity of the lipase cyclically varied between high and low levels. In spite of high fat diet intervention, obese mice receiving a single dose of the nanocarrier lose weight over eight days, whereas a control group continues the tendency to gain weight. Daily intragastric administration of the nanocarrier leads to lower weight of livers or fat pads, smaller adipocyte size, and lower total cholesterol level than that of the control group. Near-infrared fluorescence of the nanocarrier reveals its biodistribution.Healthy weight loss represents a real challenge when obesity is increasing in prevalence. Herein, we report a conjugated polymer nanocarrier for smart deactivation of lipase and thus balancing calorie intake. After oral administration, the nanocarrier is sensitive to lipase in the digestive tract and releases orlistat, which deactivates the enzyme and inhibits fat digestion. It also creates negative feedback to control the release of itself. The nanocarrier smartly regulates activity of the lipase cyclically varied between high and low levels. In spite of high fat diet intervention, obese mice receiving a single dose of the nanocarrier lose weight over eight days, whereas a control group continues the tendency to gain weight. Daily intragastric administration of the nanocarrier leads to lower weight of livers or fat pads, smaller adipocyte size, and lower total cholesterol level than that of the control group. Near-infrared fluorescence of the nanocarrier reveals its biodistribution

  3. Expression of a Deschampsia antarctica Desv. Polypeptide with Lipase Activity in a Pichia pastoris Vector

    PubMed Central

    Rabert, Claudia; Gutiérrez-Moraga, Ana; Navarrete-Gallegos, Alejandro; Navarrete-Campos, Darío; Bravo, León A.; Gidekel, Manuel

    2014-01-01

    The current study isolated and characterized the Lip3F9 polypeptide sequence of Deschampsia antarctica Desv. (GeneBank Accession Number JX846628), which was found to be comprised of 291 base pairs and was, moreover, expressed in Pichia pastoris X-33 cells. The enzyme was secreted after 24 h of P. pastoris culture incubation and through induction with methanol. The expressed protein showed maximum lipase activity (35 U/L) with an optimal temperature of 37 °C. The lipase-expressed enzyme lost 50% of its specific activity at 25 °C, a behavior characteristic of a psychrotolerant enzyme. Recombinant enzyme activity was measured in the presence of ionic and non-ionic detergents, and a decrease in enzyme activity was detected for all concentrations of ionic and non-ionic detergents assessed. PMID:24514564

  4. Specificity of the lipase-specific foldases of gram-negative bacteria and the role of the membrane anchor.

    PubMed

    El Khattabi, M; Ockhuijsen, C; Bitter, W; Jaeger, K E; Tommassen, J

    1999-06-01

    Folding of lipases that are secreted by Pseudomonads and other gram-negative bacteria via the type II secretion pathway is facilitated by dedicated chaperones, called lipase-specific foldases (Lifs). Lifs are membrane-anchored proteins with a large periplasmic domain. The functional interaction between the Lif and its cognate lipase is specific, since the Pseudomonas aeruginosa Lif was found not to substitute for Lifs from Burkholderia glumae or Acinetobacter calcoaceticus. However, the P. aeruginosa Lif was able to activate the lipase from the closely related species P. alcaligenes. Hybrid proteins constructed from parts of the P. aeruginosa and B. glumae Lifs revealed that the C-terminal 138 amino acids of the B. glumae Lif determine the specificity of the interaction with the cognate lipase. Furthermore, the periplasmic domain of the B. glumae Lif was functional when cloned in frame with a cleavable signal sequence, which demonstrates that the membrane anchor is not essential for Lif function in vivo. However, the recombinant Lif was released into the medium, indicating that the function of the membrane anchor is to prevent secretion of the Lif together with the lipase.

  5. A Cutinase from Trichoderma reesei with a lid-covered active site and kinetic properties of true lipases.

    PubMed

    Roussel, Alain; Amara, Sawsan; Nyyssölä, Antti; Mateos-Diaz, Eduardo; Blangy, Stéphanie; Kontkanen, Hanna; Westerholm-Parvinen, Ann; Carrière, Frédéric; Cambillau, Christian

    2014-11-11

    Cutinases belong to the α/β-hydrolase fold family of enzymes and degrade cutin and various esters, including triglycerides, phospholipids and galactolipids. Cutinases are able to degrade aggregated and soluble substrates because, in contrast with true lipases, they do not have a lid covering their catalytic machinery. We report here the structure of a cutinase from the fungus Trichoderma reesei (Tr) in native and inhibitor-bound conformations, along with its enzymatic characterization. A rare characteristic of Tr cutinase is its optimal activity at acidic pH. Furthermore, Tr cutinase, in contrast with classical cutinases, possesses a lid covering its active site and requires the presence of detergents for activity. In addition to the presence of the lid, the core of the Tr enzyme is very similar to other cutinase cores, with a central five-stranded β-sheet covered by helices on either side. The catalytic residues form a catalytic triad involving Ser164, His229 and Asp216 that is covered by the two N-terminal helices, which form the lid. This lid opens in the presence of surfactants, such as β-octylglucoside, and uncovers the catalytic crevice, allowing a C11Y4 phosphonate inhibitor to bind to the catalytic serine. Taken together, these results reveal Tr cutinase to be a member of a new group of lipolytic enzymes resembling cutinases but with kinetic and structural features of true lipases and a heightened specificity for long-chain triglycerides. Copyright © 2014 Elsevier Ltd. All rights reserved.

  6. Amplification of thermostable lipase genes fragment from thermogenic phase of domestic waste composting process

    NASA Astrophysics Data System (ADS)

    Nurhasanah, Nurbaiti, Santi; Madayanti, Fida; Akhmaloka

    2015-09-01

    Lipases are lipolytic enzymes, catalyze the hydrolysis of fatty acid ester bonds of triglycerides to produce free fatty acids and glycerol. The enzyme is widely used in various fields of biotechnological industry. Hence, lipases with unique properties (e.g.thermostable lipase) are still being explored by variation methods. One of the strategy is by using metagenomic approach to amplify the gene directly from environmental sample. This research was focused on amplification of lipase gene fragment directly from the thermogenic phase of domestic waste composting in aerated trenches. We used domestic waste compost from waste treatment at SABUGA, ITB for the sample. Total chromosomal DNA were directly extracted from several stages at thermogenic phase of compost. The DNA was then directly used as a template for amplification of thermostable lipase gene fragments using a set of internal primers namely Flip-1a and Rlip-1a that has been affixed with a GC clamp in reverse primer. The results showed that the primers amplified the gene from four stages of thermogenic phase with the size of lipase gene fragment of approximately 570 base pairs (bp). These results were further used for Denaturing Gradient Gel Electrophoresis (DGGE) analysis to determine diversity of thermostable lipase gene fragments.

  7. Sempervivum davisii: phytochemical composition, antioxidant and lipase-inhibitory activities.

    PubMed

    Uzun, Yusuf; Dalar, Abdullah; Konczak, Izabela

    2017-12-01

    Sempervivum davisii Muirhead (Crassulaceae) is a traditional medicinal herb from Eastern Anatolia. To date the composition of phytochemicals and physiological properties of this herb were not subjected to any research. This study identifies compounds in S. davisii hydrophilic extracts and evaluates their potential biological properties. Ethanol-based lyophilized extracts were obtained from aerial parts of plant (10 g of ground dry plant material in 200 mL of acidified aqueous ethanol, shaken for 2 h at 22 °C with supernatant collected and freeze-dried under vacuum). Phytochemical composition was investigated by liquid chromatography mass spectrometry (LC-MS/MS, phenolics) and gas chromatography mass spectrometry (GC-MS, volatiles). Phenolic compounds were quantified by high-performance liquid chromatography (HPLC) and the Folin-Ciocalteu assay. Subsequently, antioxidant capacity [ferric reducing antioxidant power (FRAP), oxygen radical absorbance capacity (ORAC) assays] and enzyme inhibitory properties (isolated porcine pancreatic lipase) of the extracts were determined. Polyphenolic compounds were the main constituents of lyophilized extracts, among which kaempferol glycosides and quercetin hexoside dominated. The extracts exhibited potent antioxidant (FRAP values of 1925.2-5973.3 μM Fe 2+ /g DW; ORAC values of 1858.5-4208.7 μM Trolox Eq./g DW) and moderate lipase inhibitory (IC 50 : 11.6-2.96 mg/mL) activities. Volatile compounds (nonanal, dehydroxylinalool oxide isomers, 2-decenal, 2-undecenal, 2,6-di-tetr-butylphenol) were also found. Phenolic compounds with the dominating kaempferol and quercetin derivatives are the sources of potent antioxidant properties of S. davisii hydrophilic extracts. The extracts exhibit moderate inhibitory properties towards isolated pancreatic lipase.

  8. Characterization of an extracellular lipase from the biocontrol fungus, Nomuraea rileyi MJ, and its toxicity toward Spodoptera litura.

    PubMed

    Supakdamrongkul, Piyaporn; Bhumiratana, Amaret; Wiwat, Chanpen

    2010-11-01

    An extracellular lipase from Nomuraea rileyi MJ was purified 23.9-fold with 1.69% yield by ammonium sulfate precipitation followed by Sephacryl S-100 HR column chromatography. By mass spectrometry and SDS-polyacrylamide gel electrophoresis, the molecular weight of the homogenous lipase was 81kDa. The N-terminal sequence was determined as LeuSerValGluGlnThrLysLeuSerLysLeuAlaTyrAsnAsp and it showed no homology to sequences of known lipases. The optimum pH and temperature for activity were 8.0 and 35°C, respectively. The enzyme was stable in the pH range 7.0-9.0 and at 15-35°C for 1h. Higher activity was observed in the presence of surfactants, Na(+), NH(4)(+) ions, NaN(3) and ethylenediaminetetraacetic acid (EDTA), while Co(2+) and Cu(2+) ions, cysteine and dithiothreitol (DTT) strongly inhibited activity. The purified lipase hydrolyzed both synthetic and natural triglycerides with maximum activity for trilaurin and coconut oil, respectively. It also hydrolyzed esters of p-nitrophenol (pNP) with highest activity for p-nitrophenyl caprate (pNPCA). The purified lipase was found to promote N. rileyi spore germination in vitro in that germination reached 98% in conidial suspensions containing purified lipase at 2.75 U. Moreover, it enhanced toxicity of N. rileyi toward Spodoptera litura larvae with mortality via topical application reaching 63.3% at 4-10days post-treatment which calculated to be 2.7 times higher than the mortality obtained using conidial suspensions alone. Copyright © 2010 Elsevier Inc. All rights reserved.

  9. Kinetic studies of Chromobacterium viscosum lipase in AOT water in oil microemulsions and gelatin microemulsion-based organogels.

    PubMed

    Jenta, T R; Batts, G; Rees, G D; Robinson, B H

    1997-06-05

    Kinetic studies have shown that octyl decanoate synthesis by Chromobacterium viscosum (CV) lipase in sodium bis-2-(ethylhexyl) sulfosuccinate (AOT) water in oil (w/o) microemulsions occurs via the nonsequential (ping-pong) bi bi mechanism. There was evidence of single substrate inhibition by decanoic acid at high concentrations. Initial rate data yielded estimates for acid and alcohol Michaelis constants of ca. 10(-1) mol dm(-3) and a maximum rate under saturation conditions of ca. 10(-3) mol dm(-3) s(-1) for a lipase concentration of 0.36 mg cm(-3). CV lipase immobilized in AOT microemulsion-based organogels (MBGs) was also found to catalyze the synthesis of octyl decanoate according to the ping-pong bi bi mechanism. Reaction rates were similar in the free and immobilized systems under comparable conditions. Initial rates at saturating (but noninhibiting) substrate concentrations were first order with respect to CV lipase concentration in both w/o microemulsions and the MBG/oil systems. Gradients yielded an apparent k(cat) = 4.4 x 10(-4) mol g(-1) s(-1) in the case of w/o microemulsions, and 6.1 x 10(-4) mol g(-1) s(-1) for CV lipase immobilized in the MBGs. A third system comprising w/o microemulsions containing substrates and gelatin at concentrations comparable to those employed in the MBG formulations, provided a useful link between the conventional liquid microemulsion medium and the solid organogels. The nongelation of these intermediate systems stems from the early inclusion of substrate during a modified preparative protocol. The presence of substrate appears to prevent the development of a percolated microstructure that is thought to be a prerequisite for MBG formation. FT-NMR was employed as a semicontinuous in situ assay procedure. The apparent activity expressed by CV lipase in compositionally equivalent liquid and solid phase gelatin-containing systems was similar. An apparent activation energy of 24 +/- 2 kJ mol(-1) was determined by (1)H-NMR for

  10. PPARγ regulates exocrine pancreas lipase.

    PubMed

    Danino, Hila; Naor, Ronny Peri-; Fogel, Chen; Ben-Harosh, Yael; Kadir, Rotem; Salem, Hagit; Birk, Ruth

    2016-12-01

    Pancreatic lipase (triacylglycerol lipase EC 3.1.1.3) is an essential enzyme in hydrolysis of dietary fat. Dietary fat, especially polyunsaturated fatty acids (PUFA), regulate pancreatic lipase (PNLIP); however, the molecular mechanism underlying this regulation is mostly unknown. As PUFA are known to regulate expression of proliferator-activated receptor gamma (PPARγ), and as we identified in-silico putative PPARγ binding sites within the putative PNLIP promoter sequence, we hypothesized that PUFA regulation of PNLIP might be mediated by PPARγ. We used in silico bioinformatics tools, reporter luciferase assay, PPARγ agonists and antagonists, PPARγ overexpression in exocrine pancreas AR42J and primary cells to study PPARγ regulation of PNLIP. Using in silico bioinformatics tools we mapped PPARγ binding sites (PPRE) to the putative promoter region of PNLIP. Reporter luciferase assay in AR42J rat exocrine pancreas acinar cells transfected with various constructs of the putative PNLIP promoter showed that PNLIP transcription is significantly enhanced by PPARγ dose-dependently, reaching maximal levels with multi PPRE sites. This effect was significantly augmented in the presence of PPARγ agonists and reduced by PPARγ antagonists or mutagenesis abrogating PPRE sites. Over-expression of PPARγ significantly elevated PNLIP transcript and protein levels in AR42J cells and in primary pancreas cells. Moreover, PNLIP expression was up-regulated by PPARγ agonists (pioglitazone and 15dPGJ2) and significantly down-regulated by PPARγ antagonists in non-transfected rat exocrine pancreas AR42J cell line cells. PPARγ transcriptionally regulates PNLIP gene expression. This transcript regulation resolves part of the missing link between dietary PUFA direct regulation of PNLIP. Copyright © 2016 Elsevier B.V. All rights reserved.

  11. Enhanced biocatalytic esterification with lipase-immobilized chitosan/graphene oxide beads.

    PubMed

    Lau, Siaw Cheng; Lim, Hong Ngee; Basri, Mahiran; Fard Masoumi, Hamid Reza; Ahmad Tajudin, Asilah; Huang, Nay Ming; Pandikumar, Alagarsamy; Chia, Chin Hua; Chia, Chi Hua; Andou, Yoshito

    2014-01-01

    In this work, lipase from Candida rugosa was immobilized onto chitosan/graphene oxide beads. This was to provide an enzyme-immobilizing carrier with excellent enzyme immobilization activity for an enzyme group requiring hydrophilicity on the immobilizing carrier. In addition, this work involved a process for the preparation of an enzymatically active product insoluble in a reaction medium consisting of lauric acid and oleyl alcohol as reactants and hexane as a solvent. This product enabled the stability of the enzyme under the working conditions and allowed the enzyme to be readily isolated from the support. In particular, this meant that an enzymatic reaction could be stopped by the simple mechanical separation of the "insoluble" enzyme from the reaction medium. Chitosan was incorporated with graphene oxide because the latter was able to enhance the physical strength of the chitosan beads by its superior mechanical integrity and low thermal conductivity. The X-ray diffraction pattern showed that the graphene oxide was successfully embedded within the structure of the chitosan. Further, the lipase incorporation on the beads was confirmed by a thermo-gravimetric analysis. The lipase immobilization on the beads involved the functionalization with coupling agents, N-hydroxysulfosuccinimide sodium (NHS) and 1-ethyl-(3-dimethylaminopropyl) carbodiimide (EDC), and it possessed a high enzyme activity of 64 U. The overall esterification conversion of the prepared product was 78% at 60 °C, and it attained conversions of 98% and 88% with commercially available lipozyme and novozyme, respectively, under similar experimental conditions.

  12. Functional cell-surface display of a lipase-specific chaperone.

    PubMed

    Wilhelm, Susanne; Rosenau, Frank; Becker, Stefan; Buest, Sebastian; Hausmann, Sascha; Kolmar, Harald; Jaeger, Karl-Erich

    2007-01-02

    Lipases are important enzymes in biotechnology. Extracellular bacterial lipases from Pseudomonads and related species require the assistance of specific chaperones, designated "Lif" proteins (lipase specific foldases). Lifs, a unique family of steric chaperones, are anchored to the periplasmic side of the inner membrane where they convert lipases into their active conformation. We have previously shown that the autotransporter protein EstA from P. aeruginosa can be used to direct a variety of proteins to the cell surface of Escherichia coli. Here we demonstrate for the first time the functional cell-surface display of the Lif chaperone and FACS (fluorescence-activated cell sorting)-based analysis of bacterial cells that carried foldase-lipase complexes. The model Lif protein, LipH from P. aeruginosa, was displayed at the surface of E. coli cells. Surface exposed LipH was functional and efficiently refolded chemically denatured lipase. The foldase autodisplay system reported here can be used for a variety of applications including the ultrahigh-throughput screening of large libraries of foldase variants generated by directed evolution.

  13. Enhanced thermostability of silica-immobilized lipase from Bacillus coagulans BTS-3 and synthesis of ethyl propionate.

    PubMed

    Kumar, Satyendra; Pahujani, Shweta; Ola, R P; Kanwar, S S; Gupta, Reena

    2006-06-01

    A lipase from the thermophilic isolate Bacillus coagulans BTS-3 was produced and purified. The enzyme was purified 40-fold to homogeneity by ammonium sulfate precipitation and DEAE-Sepharose column chromatography. Its molecular weight was 31 kDa on SDS-PAGE. The purified lipase was immobilized on silica and its binding efficiency was found to be 60%. The enzyme took 60 min to bind maximally onto the support. The pH and temperature optima of immobilized lipase were same as those of the free enzyme, i.e. 8.5 and 55 degrees C, respectively. The immobilized enzyme had shown marked thermostability on the elevated temperatures of 55, 60, 65 and 70 degrees C. The immobilized enzyme was reused for eigth cycles as it retained almost 80% of its activity. The catalytic activity of immobilized enzyme was enhanced in n-hexane and ethanol. The immobilized enzyme when used for esterification of ethanol and propionic acid showed 96% conversion in n-hexane in 12 h at 55 degrees C.

  14. Genetic and Structure-Function Studies of Missense Mutations in Human Endothelial Lipase

    PubMed Central

    Razzaghi, Hamid; Tempczyk-Russell, Anna; Haubold, Kurt; Santorico, Stephanie A.; Shokati, Touraj; Christians, Uwe; Churchill, Mair E. A.

    2013-01-01

    Endothelial lipase (EL) plays a pivotal role in HDL metabolism. We sought to characterize EL and its interaction with HDL as well as its natural variants genetically, functionally and structurally. We screened our biethnic population sample (n = 802) for selected missense mutations (n = 5) and identified T111I as the only common variant. Multiple linear regression analyses in Hispanic subjects revealed an unexpected association between T111I and elevated LDL-C (p-value = 0.012) and total cholesterol (p-value = 0.004). We examined lipase activity of selected missense mutants (n = 10) and found different impacts on EL function, ranging from normal to complete loss of activity. EL-HDL lipidomic analyses indicated that EL has a defined remodeling of HDL without exhaustion of the substrate and a distinct and preference for several fatty acids that are lipid mediators and known for their potent pro- and anti-inflammatory properties. Structural studies using homology modeling revealed a novel α/β motif in the C-domain, unique to EL. The EL dimer was found to have the flexibility to expand and to bind various sizes of HDL particles. The likely impact of the all known missense mutations (n = 18) on the structure of EL was examined using molecular modeling and the impact they may have on EL lipase activity using a novel structure-function slope based on their structural free energy differences. The results of this multidisciplinary approach delineated the impact of EL and its variants on HDL. Moreover, the results suggested EL to have the capacity to modulate vascular health through its role in fatty acid-based signaling pathways. PMID:23536757

  15. Monoacylglycerol lipase exerts dual control over endocannabinoid and fatty acid pathways to support prostate cancer.

    PubMed

    Nomura, Daniel K; Lombardi, Donald P; Chang, Jae Won; Niessen, Sherry; Ward, Anna M; Long, Jonathan Z; Hoover, Heather H; Cravatt, Benjamin F

    2011-07-29

    Cancer cells couple heightened lipogenesis with lipolysis to produce fatty acid networks that support malignancy. Monoacylglycerol lipase (MAGL) plays a principal role in this process by converting monoglycerides, including the endocannabinoid 2-arachidonoylglycerol (2-AG), to free fatty acids. Here, we show that MAGL is elevated in androgen-independent versus androgen-dependent human prostate cancer cell lines, and that pharmacological or RNA-interference disruption of this enzyme impairs prostate cancer aggressiveness. These effects were partially reversed by treatment with fatty acids or a cannabinoid receptor-1 (CB1) antagonist, and fully reversed by cotreatment with both agents. We further show that MAGL is part of a gene signature correlated with epithelial-to-mesenchymal transition and the stem-like properties of cancer cells, supporting a role for this enzyme in protumorigenic metabolism that, for prostate cancer, involves the dual control of endocannabinoid and fatty acid pathways. Copyright © 2011 Elsevier Ltd. All rights reserved.

  16. Screening for hydrolytic enzymes reveals Ayr1p as a novel triacylglycerol lipase in Saccharomyces cerevisiae.

    PubMed

    Ploier, Birgit; Scharwey, Melanie; Koch, Barbara; Schmidt, Claudia; Schatte, Jessica; Rechberger, Gerald; Kollroser, Manfred; Hermetter, Albin; Daum, Günther

    2013-12-13

    Saccharomyces cerevisiae, as well as other eukaryotes, preserves fatty acids and sterols in a biologically inert form, as triacylglycerols and steryl esters. The major triacylglycerol lipases of the yeast S. cerevisiae identified so far are Tgl3p, Tgl4p, and Tgl5p (Athenstaedt, K., and Daum, G. (2003) YMR313c/TGL3 encodes a novel triacylglycerol lipase located in lipid particles of Saccharomyces cerevisiae. J. Biol. Chem. 278, 23317-23323; Athenstaedt, K., and Daum, G. (2005) Tgl4p and Tgl5p, two triacylglycerol lipases of the yeast Saccharomyces cerevisiae, are localized to lipid particles. J. Biol. Chem. 280, 37301-37309). We observed that upon cultivation on oleic acid, triacylglycerol mobilization did not come to a halt in a yeast strain deficient in all currently known triacylglycerol lipases, indicating the presence of additional not yet characterized lipases/esterases. Functional proteome analysis using lipase and esterase inhibitors revealed a subset of candidate genes for yet unknown hydrolytic enzymes on peroxisomes and lipid droplets. Based on the conserved GXSXG lipase motif, putative functions, and subcellular localizations, a selected number of candidates were characterized by enzyme assays in vitro, gene expression analysis, non-polar lipid analysis, and in vivo triacylglycerol mobilization assays. These investigations led to the identification of Ayr1p as a novel triacylglycerol lipase of yeast lipid droplets and confirmed the hydrolytic potential of the peroxisomal Lpx1p in vivo. Based on these results, we discuss a possible link between lipid storage, lipid mobilization, and peroxisomal utilization of fatty acids as a carbon source.

  17. The Metagenome-Derived Enzymes LipS and LipT Increase the Diversity of Known Lipases

    PubMed Central

    Chow, Jennifer; Kovacic, Filip; Dall Antonia, Yuliya; Krauss, Ulrich; Fersini, Francesco; Schmeisser, Christel; Lauinger, Benjamin; Bongen, Patrick; Pietruszka, Joerg; Schmidt, Marlen; Menyes, Ina; Bornscheuer, Uwe T.; Eckstein, Marrit; Thum, Oliver; Liese, Andreas; Mueller-Dieckmann, Jochen; Jaeger, Karl-Erich; Streit, Wolfgang R.

    2012-01-01

    Triacylglycerol lipases (EC 3.1.1.3) catalyze both hydrolysis and synthesis reactions with a broad spectrum of substrates rendering them especially suitable for many biotechnological applications. Most lipases used today originate from mesophilic organisms and are susceptible to thermal denaturation whereas only few possess high thermotolerance. Here, we report on the identification and characterization of two novel thermostable bacterial lipases identified by functional metagenomic screenings. Metagenomic libraries were constructed from enrichment cultures maintained at 65 to 75°C and screened resulting in the identification of initially 10 clones with lipolytic activities. Subsequently, two ORFs were identified encoding lipases, LipS and LipT. Comparative sequence analyses suggested that both enzymes are members of novel lipase families. LipS is a 30.2 kDa protein and revealed a half-life of 48 h at 70°C. The lipT gene encoded for a multimeric enzyme with a half-life of 3 h at 70°C. LipS had an optimum temperature at 70°C and LipT at 75°C. Both enzymes catalyzed hydrolysis of long-chain (C12 and C14) fatty acid esters and additionally hydrolyzed a number of industry-relevant substrates. LipS was highly specific for (R)-ibuprofen-phenyl ester with an enantiomeric excess (ee) of 99%. Furthermore, LipS was able to synthesize 1-propyl laurate and 1-tetradecyl myristate at 70°C with rates similar to those of the lipase CalB from Candida antarctica. LipS represents the first example of a thermostable metagenome-derived lipase with significant synthesis activities. Its X-ray structure was solved with a resolution of 1.99 Å revealing an unusually compact lid structure. PMID:23112831

  18. Application of a statistically enhanced, novel, organic solvent stable lipase from Bacillus safensis DVL-43.

    PubMed

    Kumar, Davender; Parshad, Rajinder; Gupta, Vijay Kumar

    2014-05-01

    This paper presents the molecular identification of a newly isolated bacterial strain producing a novel and organic solvent stable lipase, statistical optimization of fermentation medium, and its application in the synthesis of ethyl laurate. On the basis of nucleotide homology and phylogenetic analysis of 16S rDNA sequence, the strain was identified as Bacillus safensis DVL-43 (Gen-bank accession number KC156603). Optimization of fermentation medium using Plackett-Burman design and response surface methodology led to 11.4-fold increase in lipase production. The lipase from B. safensis DVL-43 exhibited excellent stability in various organic solvents. The enzyme retained 100% activity after 24h incubation in xylene, DMSO and toluene, each solvent being used at a concentration of 25% (v/v). The use of partially purified DVL-43 lipase as catalyst in the synthesis of ethyl laurate, an esterification product of lauric acid and ethanol, resulted in 80% esterification in 12h under optimized conditions. The formation of ethyl laurate was confirmed using TLC and (1)H NMR. Organic solvent stable lipases exhibiting potential application in enzymatic esterification are in great demand in flavor, fine chemicals and pharma industries. We could not find any report on lipase production from B. safensis strain and its application in esterification. Copyright © 2014 Elsevier B.V. All rights reserved.

  19. Improved catalytic properties of Penicillium notatum lipase immobilized in nanoscale silicone polymeric films.

    PubMed

    Rehman, Saima; Wang, Ping; Bhatti, Haq Nawaz; Bilal, Muhammad; Asgher, Muhammad

    2017-04-01

    Lipases are one of the most proficient biocatalysts having enormous biotechnological prospective. Immobilization offers a potential solution to improve the stability and recycling characteristics of lipases. An extracellular lipase from Penicillium notatum (PNL) was immobilized in silicon polymers (SiP) through entrapment, and subsequently coated this matrix on the network of fibers in the sponges. The silicone polymers-immobilized lipase (SiP-lipase) displayed highest apparent activity and entrapment efficiency of 1.19Ug -1 polymers and 92.3%, respectively. It also exhibited greater catalytic activity in broad-working pHs and higher temperature than equivalent free-state of enzyme. Immobilization caused an improvement in thermo-stability of the lipase with an increase in energy of activation. The recycling potential of SiP-lipase was investigated. After reusing the sponge pieces for ten reaction cycles, the SiP preserved its structure without leakage of enzyme, and retained around 90% of its original activity. The SiP surface analysis was envisaged by scanning electron microscopy that further confirmed the recycling efficiency of SiP-lipase. Overall, SiP-lipase displayed a number of useful properties that make it a promising candidate for future applications in different chemical processes. Copyright © 2017 Elsevier B.V. All rights reserved.

  20. Selective Enrichment of Omega-3 Fatty Acids in Oils by Phospholipase A1

    PubMed Central

    Puri, Munish; Barrow, Colin; Rao, Nalam Madhusudhana

    2016-01-01

    Omega fatty acids are recognized as key nutrients for healthier ageing. Lipases are used to release ω-3 fatty acids from oils for preparing enriched ω-3 fatty acid supplements. However, use of lipases in enrichment of ω-3 fatty acids is limited due to their insufficient specificity for ω-3 fatty acids. In this study use of phospholipase A1 (PLA1), which possesses both sn-1 specific activity on phospholipids and lipase activity, was explored for hydrolysis of ω-3 fatty acids from anchovy oil. Substrate specificity of PLA1 from Thermomyces lenuginosus was initially tested with synthetic p-nitrophenyl esters along with a lipase from Bacillus subtilis (BSL), as a lipase control. Gas chromatographic characterization of the hydrolysate obtained upon treatment of anchovy oil with these enzymes indicated a selective retention of ω-3 fatty acids in the triglyceride fraction by PLA1 and not by BSL. 13C NMR spectroscopy based position analysis of fatty acids in enzyme treated and untreated samples indicated that PLA1 preferably retained ω-3 fatty acids in oil, while saturated fatty acids were hydrolysed irrespective of their position. Hydrolysis of structured triglyceride,1,3-dioleoyl-2-palmitoylglycerol, suggested that both the enzymes hydrolyse the fatty acids at both the positions. The observed discrimination against ω-3 fatty acids by PLA1 appears to be due to its fatty acid selectivity rather than positional specificity. These studies suggest that PLA1 could be used as a potential enzyme for selective concentrationof ω-3 fatty acids. PMID:26978518

  1. Selective Enrichment of Omega-3 Fatty Acids in Oils by Phospholipase A1.

    PubMed

    Ranjan Moharana, Tushar; Byreddy, Avinesh R; Puri, Munish; Barrow, Colin; Rao, Nalam Madhusudhana

    2016-01-01

    Omega fatty acids are recognized as key nutrients for healthier ageing. Lipases are used to release ω-3 fatty acids from oils for preparing enriched ω-3 fatty acid supplements. However, use of lipases in enrichment of ω-3 fatty acids is limited due to their insufficient specificity for ω-3 fatty acids. In this study use of phospholipase A1 (PLA1), which possesses both sn-1 specific activity on phospholipids and lipase activity, was explored for hydrolysis of ω-3 fatty acids from anchovy oil. Substrate specificity of PLA1 from Thermomyces lenuginosus was initially tested with synthetic p-nitrophenyl esters along with a lipase from Bacillus subtilis (BSL), as a lipase control. Gas chromatographic characterization of the hydrolysate obtained upon treatment of anchovy oil with these enzymes indicated a selective retention of ω-3 fatty acids in the triglyceride fraction by PLA1 and not by BSL. 13C NMR spectroscopy based position analysis of fatty acids in enzyme treated and untreated samples indicated that PLA1 preferably retained ω-3 fatty acids in oil, while saturated fatty acids were hydrolysed irrespective of their position. Hydrolysis of structured triglyceride,1,3-dioleoyl-2-palmitoylglycerol, suggested that both the enzymes hydrolyse the fatty acids at both the positions. The observed discrimination against ω-3 fatty acids by PLA1 appears to be due to its fatty acid selectivity rather than positional specificity. These studies suggest that PLA1 could be used as a potential enzyme for selective concentrationof ω-3 fatty acids.

  2. Valorization of Palm Oil Industrial Waste as Feedstock for Lipase Production.

    PubMed

    Silveira, Erick A; Tardioli, Paulo W; Farinas, Cristiane S

    2016-06-01

    The use of residues from the industrial processing of palm oil as carbon source and inducer for microbial lipase production can be a way to add value to such residues and to contribute to reduced enzyme costs. The aim of this work was to investigate the feasibility of using palm oil industrial waste as feedstock for lipase production in different cultivation systems. Evaluation was made of lipase production by a selected strain of Aspergillus niger cultivated under solid-state (SSF) and submerged fermentation (SmF). Lipase activity levels up to 15.41 IU/mL were achieved under SSF. The effects of pH and temperature on the lipase activity of the SSF extract were evaluated using statistical design methodology, and maximum activities were obtained between pH 4.0 and 6.5 and at temperatures between 37 and 55 °C. This lipase presented good thermal stability up to 60 °C and higher specificity towards long carbon chain substrates. The results demonstrate the potential application of palm oil industrial residues for lipase production and contribute to the technological advances needed to develop processes for industrial enzymes production.

  3. Efficient water removal in lipase-catalyzed esterifications using a low-boiling-point azeotrope.

    PubMed

    Yan, Youchun; Bornscheuer, Uwe T; Schmid, Rolf D

    2002-04-05

    High conversions in lipase-catalyzed syntheses of esters from free acyl donors and an alcohol requires efficient removal of water preferentially at temperatures compatible to enzyme activity. Using a lipase B from Candida antarctica (CAL-B)-mediated synthesis of sugar fatty-acid esters, we show that a mixture of ethyl methylketone (EMK) and hexane (best ratio: 4:1, vo/vo) allows efficient removal of water generated during esterification. Azeotropic distillation of the solvent mixture (composition: 26% EMK, 55% hexane, 19% water) takes place at 59 degrees C, which closely matches the optimum temperature reported for CAL-B. Water is then removed from the azeotrope by membrane vapor permeation. In case of glucose stearate, 93% yield was achieved after 48 h using an equimolar ratio of glucose and stearic acid. CAL-B could be reused for seven reaction cycles, with 86% residual activity after 14 d total reaction time at 59 degrees C. A decrease in fatty-acid chain length as well as increasing temperatures (75 degrees C) resulted in lower conversions. In addition, immobilization of CAL-B on a magnetic polypropylene carrier (EP 100) facilitated separation of the biocatalyst. Copyright 2002 Wiley Periodicals, Inc. Biotechnol Bioeng 78: 31--34, 2002; DOI 10.1002/bit.10084

  4. Adipose Triglyceride Lipase Is Implicated in Fuel- and Non-fuel-stimulated Insulin Secretion*

    PubMed Central

    Peyot, Marie-Line; Guay, Claudiane; Latour, Martin G.; Lamontagne, Julien; Lussier, Roxane; Pineda, Marco; Ruderman, Neil B.; Haemmerle, Guenter; Zechner, Rudolf; Joly, Érik; Madiraju, S. R. Murthy; Poitout, Vincent; Prentki, Marc

    2009-01-01

    Reduced lipolysis in hormone-sensitive lipase-deficient mice is associated with impaired glucose-stimulated insulin secretion (GSIS), suggesting that endogenous β-cell lipid stores provide signaling molecules for insulin release. Measurements of lipolysis and triglyceride (TG) lipase activity in islets from HSL−/− mice indicated the presence of other TG lipase(s) in the β-cell. Using real time-quantitative PCR, adipose triglyceride lipase (ATGL) was found to be the most abundant TG lipase in rat islets and INS832/13 cells. To assess its role in insulin secretion, ATGL expression was decreased in INS832/13 cells (ATGL-knockdown (KD)) by small hairpin RNA. ATGL-KD increased the esterification of free fatty acid (FFA) into TG. ATGL-KD cells showed decreased glucose- or Gln + Leu-induced insulin release, as well as reduced response to KCl or palmitate at high, but not low, glucose. The KATP-independent/amplification pathway of GSIS was considerably reduced in ATGL-KD cells. ATGL−/− mice were hypoinsulinemic and hypoglycemic and showed decreased plasma TG and FFAs. A hyperglycemic clamp revealed increased insulin sensitivity and decreased GSIS and arginine-induced insulin secretion in ATGL−/− mice. Accordingly, isolated islets from ATGL−/− mice showed reduced insulin secretion in response to glucose, glucose + palmitate, and KCl. Islet TG content and FFA esterification into TG were increased by 2-fold in ATGL−/− islets, but glucose usage and oxidation were unaltered. The results demonstrate the importance of ATGL and intracellular lipid signaling for fuel- and non-fuel-induced insulin secretion. PMID:19389712

  5. Bound Phenolics of Quinoa Seeds Released by Acid, Alkaline, and Enzymatic Treatments and Their Antioxidant and α-Glucosidase and Pancreatic Lipase Inhibitory Effects.

    PubMed

    Tang, Yao; Zhang, Bing; Li, Xihong; Chen, Peter X; Zhang, Hua; Liu, Ronghua; Tsao, Rong

    2016-03-02

    Unextractable phenolics from plant foods and their role in health benefits have become increasingly important. Meal residues of three quinoa seeds free of fat and extractable phenolics were subjected to acid, alkaline, and enzymatic hydrolyses. The total and individual phenolic compounds released were analyzed, and 19 phenolics, predominantly phenolic acids and several flavonoids, were identified. The concentration of bound phenolics was highest in black quinoa followed by red and white, regardless of the hydrolysis method. Higher phenolic contents also showed stronger antioxidant activities and inhibition of α-glucosidase and pancreatic lipase activities. Carbohydrases, that is, pectinase, xylanase and feruloyl esterase, which effectively liberated bound phenolics are known to be secreted by colonic bacteria, suggesting potential antioxidant and anti-inflammatory effects by these compounds in the large intestine during colonic fermentation. These results can also be applied to treat foods high in bound phenolics to enhance bioaccessibility.

  6. The heterozygous N291S mutation in the lipoprotein lipase gene impairs whole-body insulin sensitivity and affects a distinct set of plasma metabolites in humans.

    PubMed

    Berg, Sofia Mikkelsen; Havelund, Jesper; Hasler-Sheetal, Harald; Kruse, Vibeke; Pedersen, Andreas James Thestrup; Hansen, Aleksander Bill; Nybo, Mads; Beck-Nielsen, Henning; Højlund, Kurt; Færgeman, Nils Joakim

    Mutations in the lipoprotein lipase gene causing decreased lipoprotein lipase activity are associated with surrogate markers of insulin resistance and the metabolic syndrome in humans. We investigated the hypothesis that a heterozygous lipoprotein lipase mutation (N291S) induces whole-body insulin resistance and alterations in the plasma metabolome. In 6 carriers of a heterozygous lipoprotein lipase mutation (N291S) and 11 age-matched and weight-matched healthy controls, we examined insulin sensitivity and substrate metabolism by euglycemic-hyperinsulinemic clamps combined with indirect calorimetry. Plasma samples were taken before and after the clamp (4 hours of physiological hyperinsulinemia), and metabolites were measured enzymatically or by gas chromatography-mass spectrometry. Compared with healthy controls, heterozygous carriers of a defective lipoprotein lipase allele had elevated fasting plasma levels triglycerides (P < .006), and markedly impaired insulin-stimulated glucose disposal rates (P < .024) and nonoxidative glucose metabolism (P < .015). Plasma metabolite profiling demonstrated lower circulating levels of pyruvic acid and α-tocopherol in the N291S carriers than in controls both before and after stimulation with insulin (all >1.5-fold change and P < .05). Heterozygous carriers with a defective lipoprotein lipase allele are less insulin sensitive and have increased plasma levels of nonesterified fatty acids and triglycerides. The heterozygous N291S carriers also have a distinct plasma metabolomic signature, which may serve as a diagnostic tool for deficient lipoprotein lipase activity and as a marker of lipid-induced insulin resistance. Copyright © 2017 National Lipid Association. Published by Elsevier Inc. All rights reserved.

  7. Screening for Hydrolytic Enzymes Reveals Ayr1p as a Novel Triacylglycerol Lipase in Saccharomyces cerevisiae*

    PubMed Central

    Ploier, Birgit; Scharwey, Melanie; Koch, Barbara; Schmidt, Claudia; Schatte, Jessica; Rechberger, Gerald; Kollroser, Manfred; Hermetter, Albin; Daum, Günther

    2013-01-01

    Saccharomyces cerevisiae, as well as other eukaryotes, preserves fatty acids and sterols in a biologically inert form, as triacylglycerols and steryl esters. The major triacylglycerol lipases of the yeast S. cerevisiae identified so far are Tgl3p, Tgl4p, and Tgl5p (Athenstaedt, K., and Daum, G. (2003) YMR313c/TGL3 encodes a novel triacylglycerol lipase located in lipid particles of Saccharomyces cerevisiae. J. Biol. Chem. 278, 23317–23323; Athenstaedt, K., and Daum, G. (2005) Tgl4p and Tgl5p, two triacylglycerol lipases of the yeast Saccharomyces cerevisiae, are localized to lipid particles. J. Biol. Chem. 280, 37301–37309). We observed that upon cultivation on oleic acid, triacylglycerol mobilization did not come to a halt in a yeast strain deficient in all currently known triacylglycerol lipases, indicating the presence of additional not yet characterized lipases/esterases. Functional proteome analysis using lipase and esterase inhibitors revealed a subset of candidate genes for yet unknown hydrolytic enzymes on peroxisomes and lipid droplets. Based on the conserved GXSXG lipase motif, putative functions, and subcellular localizations, a selected number of candidates were characterized by enzyme assays in vitro, gene expression analysis, non-polar lipid analysis, and in vivo triacylglycerol mobilization assays. These investigations led to the identification of Ayr1p as a novel triacylglycerol lipase of yeast lipid droplets and confirmed the hydrolytic potential of the peroxisomal Lpx1p in vivo. Based on these results, we discuss a possible link between lipid storage, lipid mobilization, and peroxisomal utilization of fatty acids as a carbon source. PMID:24187129

  8. Sebelipase alfa over 52 weeks reduces serum transaminases, liver volume and improves serum lipids in patients with lysosomal acid lipase deficiency

    PubMed Central

    Valayannopoulos, Vassili; Malinova, Vera; Honzík, Tomas; Balwani, Manisha; Breen, Catherine; Deegan, Patrick B.; Enns, Gregory M.; Jones, Simon A.; Kane, John P.; Stock, Eveline O.; Tripuraneni, Radhika; Eckert, Stephen; Schneider, Eugene; Hamilton, Gavin; Middleton, Michael S.; Sirlin, Claude; Kessler, Bruce; Bourdon, Christopher; Boyadjiev, Simeon A.; Sharma, Reena; Twelves, Chris; Whitley, Chester B.; Quinn, Anthony G.

    2014-01-01

    Background and aims Lysosomal Acid Lipase Deficiency is an autosomal recessive enzyme deficiency resulting in lysosomal accumulation of cholesteryl esters and triglycerides. LAL-CL04, an ongoing extension study, investigates the long-term effects of sebelipase alfa, a recombinant human lysosomal acid lipase. Methods Sebelipase alfa (1 mg/kg or 3 mg/kg) was infused every-other-week to eligible subjects. Safety and tolerability assessments, including liver function, lipid profiles and liver volume assessment, were carried out at regular intervals. Results 216 infusions were administered to eight adult subjects through Week 52 during LAL-CL04. At Week 52, mean alanine aminotransferase and aspartate aminotransferase were normal with mean change from baseline of −58% and −40%. Mean change for low density lipoprotein, total cholesterol, triglyceride and high-density lipoprotein were −60%, −39%, −36%, and +29%, respectively. Mean liver volume by magnetic resonance imaging and hepatic proton density fat fraction decreased (12% and 55%, respectively). Adverse events were mainly mild and unrelated to sebelipase alfa. Infusion-related reactions were uncommon: three events of moderate severity were reported in two subjects; one patient's event was suggestive of hypersensitivity-like reaction, but additional testing did not confirm this, and the subject has successfully re-started sebelipase alfa. Of samples tested to date, no anti-drug antibodies have been detected. Conclusions Long-term dosing with sebelipase alfa in Lysosomal Acid Lipase-Deficient patients is well tolerated and produces sustained reductions in transaminases, improvements in serum lipid profile and reduction in hepatic fat fraction. A randomized, placebo-controlled phase 3 trial in children and adults is underway (ARISE: NCT01757184). PMID:24993530

  9. Liver disease and dyslipidemia as a manifestation of lysosomal acid lipase deficiency (LAL-D). Clinical and diagnostic aspects, and a new treatment. An update.

    PubMed

    Bay, Luisa; Canero Velasco, Cristina; Ciocca, Mirta; Cotti, Andrea; Cuarterolo, Miriam; Fainboim, Alejandro; Fassio, Eduardo; Galoppo, Marcela; Pinero, Federico; Rozenfeld, Paula

    2017-06-01

    Lysosomal acid lipase deficiency (LAL-D) is still a little recognized genetic disease with significant morbidity and mortality in children and adults. This document provides guidance on when to suspect LAL-D and how to diagnose it. It is recommended to add lysosomal acid lipase deficiency to the List of differential diagnoses of sepsis, oncological diseases, storage diseases, persistent diarrhea, chronic malnutrition, and hemophagocytic lymphohistiocytosis. It should also be considered in young patients with dyslipidemia and atherosclerosis as well as diseases associated with fatty liver and/or hepatomegaly. LAL-D should be suspected in patients with hepatomegaly, hyperlipidemia and /or elevated transaminases found during routine checks or testing for other conditions, and in patients with cryptogenic cirrhosis. At present, there is the option of a specific enzyme replacement treatment. Sociedad Argentina de Pediatría.

  10. Glycerol acyl-transfer kinetics of a circular permutated Candida antarctica Lipase B

    USDA-ARS?s Scientific Manuscript database

    Triacylglycerols containing a high abundance of unusual fatty acids, such as y-linolenic acid, or novel arylaliphatic acids, such as ferulic acid, are useful in pharmaceutical and cosmeceutical applications. Candida antarctica lipase B (CALB) is quite often used for non-aqueous synthesis, although ...

  11. The genotypic diversity and lipase production of some thermophilic bacilli from different genera

    PubMed Central

    Koc, Melih; Cokmus, Cumhur; Cihan, Arzu Coleri

    2015-01-01

    Abstract Thermophilic 32 isolates and 20 reference bacilli were subjected to Rep-PCR and ITS-PCR fingerprinting for determination of their genotypic diversity, before screening lipase activities. By these methods, all the isolates and references could easily be differentiated up to subspecies level from each other. In screening assay, 11 isolates and 7 references were found to be lipase producing. Their extracellular lipase activities were measured quantitatively by incubating in both tributyrin and olive oil broths at 60 °C and pH 7.0. During the 24, 48 and 72-h period of incubation, the changes in the lipase activities, culture absorbance, wet weight of biomass and pH were all measured. The activity was determined by using pNPB in 50 mM phosphate buffer at pH 7.0 at 60 °C. The lipase production of the isolates in olive oil broths varied between 0.008 and 0.052, whereas these values were found to be 0.002-0.019 (U/mL) in the case of tyributyrin. For comparison, an index was established by dividing the lipase activities to cell biomass (U/mg). The maximum thermostable lipase production was achieved by the isolates F84a, F84b, and G. thermodenitrificans DSM 465T (0.009, 0.008 and 0.008 U/mg) within olive oil broth, whereas G. stearothermophilus A113 displayed the highest lipase activity than its type strain in tyributyrin. Therefore, as some of these isolates displayed higher activities in comparison to references, new lipase producing bacilli were determined by presenting their genotypic diversity with DNA fingerprinting techniques. PMID:26691464

  12. Discrimination between closed and open forms of lipases using electrophoretic techniques.

    PubMed

    Miled, N; Riviere, M; Cavalier, J F; Buono, G; Berti, L; Verger, R

    2005-03-15

    The enhanced catalytic activity of lipases is often associated with structural changes. The three-dimensional (3D) structures showed that the covalently inhibited lipases exist under their open conformations, in contrast to their native closed forms. We studied the inhibition of various lipases--human and dog gastric lipases, human pancreatic lipase, and Humicola lanuginosa lipase--by the octyl-undecyl phosphonate inhibitor, and we measured the subsequent modifications of their respective electrophoretic mobility. Furthermore, the experimental values of the isoelectric points found for the native (closed) and inhibited (open) lipases are in agreement with theoretical calculations based on the electrostatic potential. We concluded that there is a significant difference in the isoelectric points between the closed (native) and open (inhibited) conformations of the four lipases investigated. Thus, analysis of the electrophoretic pattern is proposed as an easy experimental tool to differentiate between a closed and an open form of a given lipase.

  13. The smooth-hound lipolytic system: Biochemical characterization of a purified digestive lipase, lipid profile and in vitro oil digestibility.

    PubMed

    Achouri, Neila; Smichi, Nabil; Gargouri, Youssef; Miled, Nabil; Fendri, Ahmed

    2017-09-01

    In order to identify fish enzymes displaying novel biochemical properties, we choose the common smooth-hound (Mustelus mustelus) as a starting biological material to characterize the digestive lipid hydrolyzing enzyme. A smooth-hound digestive lipase (SmDL) was purified from a delipidated pancreatic powder. The SmDL molecular weight was around 50kDa. Specific activities of 2200 and 500U/mg were measured at pH 9 and 40°C using tributyrin and olive oil emulsion as substrates, respectively. Unlike known mammal pancreatic lipases, the SmDL was stable at 50°C and it retained 90% of its initial activity after 15min of incubation at 60°C. Interestingly, bile salts act as an activator of the SmDL. It's worth to notice that the SmDL was also salt-tolerant since it was active in the presence of high salt concentrations reaching 0.8M. Fatty acid (FA) analysis of oil from the smooth-hound viscera showed a dominance of unsaturated ones (UFAs). Interestingly, the major n-3 fatty acids were DHA and EPA with contents of 18.07% and 6.14%, respectively. In vitro digestibility model showed that the smooth hound oil was efficiently hydrolyzed by pancreatic lipases, which suggests the higher assimilation of fish oils by consumers. Copyright © 2017 Elsevier B.V. All rights reserved.

  14. Gene organization and primary structure of human hormone-sensitive lipase: possible significance of a sequence homology with a lipase of Moraxella TA144, an antarctic bacterium.

    PubMed Central

    Langin, D; Laurell, H; Holst, L S; Belfrage, P; Holm, C

    1993-01-01

    The human hormone-sensitive lipase (HSL) gene encodes a 786-aa polypeptide (85.5 kDa). It is composed of nine exons spanning approximately 11 kb, with exons 2-5 clustered in a 1.1-kb region. The putative catalytic site (Ser423) and a possible lipid-binding region in the C-terminal part are encoded by exons 6 and 9, respectively. Exon 8 encodes the phosphorylation site (Ser551) that controls cAMP-mediated activity and a second site (Ser553) that is phosphorylated by 5'-AMP-activated protein kinase. Human HSL showed 83% identity with the rat enzyme and contained a 12-aa deletion immediately upstream of the phosphorylation sites with an unknown effect on the activity control. Besides the catalytic site motif (Gly-Xaa-Ser-Xaa-Gly) found in most lipases, HSL shows no homology with other known lipases or proteins, except for a recently reported unexpected homology between the region surrounding its catalytic site and that of the lipase 2 of Moraxella TA144, an antarctic psychrotrophic bacterium. The gene of lipase 2, which catalyses lipolysis below 4 degrees C, was absent in the genomic DNA of five other Moraxella strains living at 37 degrees C. The lipase 2-like sequence in HSL may reflect an evolutionarily conserved cold adaptability that might be of critical survival value when low-temperature-mobilized endogenous lipids are the primary energy source (e.g., in poikilotherms or hibernators). The finding that HSL at 10 degrees C retained 3- to 5-fold more of its 37 degrees C catalytic activity than lipoprotein lipase or carboxyl ester lipase is consistent with this hypothesis. Images Fig. 5 PMID:8506334

  15. Purification and Characterization of a Thermostable Lipase from Geobacillus thermodenitrificans IBRL-nra

    PubMed Central

    Balan, Anuradha; Ibrahim, Darah; Abdul Rahim, Rashidah; Ahmad Rashid, Fatimah Azzahra

    2012-01-01

    Thermostable lipase from Geobacillus thermodenitrificans IBRL-nra was purified and characterized. The production of thermostable lipase from Geobacillus thermodenitrificans IBRL-nra was carried out in a shake-flask system at 65°C in cultivation medium containing; glucose 1.0% (w/v); yeast extract 1.25% (w/v); NaCl 0.45% (w/v) olive oil 0.1% (v/v) with agitation of 200 rpm for 24 hours. The extracted extracellular crude thermostable lipase was purified to homogeneity by using ultrafiltration, Heparin-affinity chromatography, and Sephadex G-100 gel-filtration chromatography by 34 times with a final yield of 9%. The molecular weight of the purified enzyme was estimated to be 30 kDa after SDS-PAGE analysis. The optimal temperature for thermostable lipase was 65°C and it retained its initial activity for 3 hours. Thermostable lipase activity was highest at pH 7.0 and stable for 16 hours at this pH at 65°C. Thermostable lipase showed elevated activity when pretreated with BaCl2, CaCl2, and KCl with 112%, 108%, and 106%, respectively. Lipase hydrolyzed tripalmitin (C16) and olive oil with optimal activity (100%) compared to other substrates. PMID:23198138

  16. Glycerol Monolaurate Inhibits Lipase Production by Clinical Ocular Isolates Without Affecting Bacterial Cell Viability.

    PubMed

    Flanagan, Judith Louise; Khandekar, Neeta; Zhu, Hua; Watanabe, Keizo; Markoulli, Maria; Flanagan, John Terence; Papas, Eric

    2016-02-01

    We sought to determine the relative lipase production of a range of ocular bacterial isolates and to assess the efficacy of glycerol monolaurate (GML) in inhibiting this lipase production in high lipase-producing bacteria without affecting bacterial cell growth. Staphylococcus aureus,Staphylococcus epidermidis,Propionibacterium acnes, and Corynebacterium spp. were inoculated at a density of 10(6)/mL in varying concentrations of GML up to 25 μg/mL for 24 hours at 37 °C with constant shaking. Bacterial suspensions were centrifuged, bacterial cell density was determined, and production of bacterial lipase was quantified using a commercial lipase assay kit. Staphylococcus spp. produced high levels of lipase activity compared with P. acnes and Corynebacterium spp. GML inhibited lipase production by Staphylococcal spp. in a dose-dependent manner, with S. epidermidis lipase production consistently more sensitive to GML than S. aureus. Glycerol monolaurate showed significant (P < 0.05) lipase inhibition above concentrations of 15 μg/mL in S. aureus and was not cytotoxic up to 25 μg/mL. For S. epidermidis, GML showed significant (P < 0.05) lipase inhibition above 7.5 μg/mL. Lipase activity varied between species and between strains. Staphylococcal spp. produced higher lipase activity compared with P. acnes and Corynebacterium spp. Glycerol monolaurate inhibited lipase production by S. aureus and S. epidermidis at concentrations that did not adversely affect bacterial cell growth. GML can be used to inhibit ocular bacterial lipase production without proving detrimental to commensal bacteria viability.

  17. Enhanced Biocatalytic Esterification with Lipase-Immobilized Chitosan/Graphene Oxide Beads

    PubMed Central

    Lau, Siaw Cheng; Lim, Hong Ngee; Basri, Mahiran; Fard Masoumi, Hamid Reza; Ahmad Tajudin, Asilah; Huang, Nay Ming; Pandikumar, Alagarsamy; Chia, Chi Hua; Andou, Yoshito

    2014-01-01

    In this work, lipase from Candida rugosa was immobilized onto chitosan/graphene oxide beads. This was to provide an enzyme-immobilizing carrier with excellent enzyme immobilization activity for an enzyme group requiring hydrophilicity on the immobilizing carrier. In addition, this work involved a process for the preparation of an enzymatically active product insoluble in a reaction medium consisting of lauric acid and oleyl alcohol as reactants and hexane as a solvent. This product enabled the stability of the enzyme under the working conditions and allowed the enzyme to be readily isolated from the support. In particular, this meant that an enzymatic reaction could be stopped by the simple mechanical separation of the “insoluble” enzyme from the reaction medium. Chitosan was incorporated with graphene oxide because the latter was able to enhance the physical strength of the chitosan beads by its superior mechanical integrity and low thermal conductivity. The X-ray diffraction pattern showed that the graphene oxide was successfully embedded within the structure of the chitosan. Further, the lipase incorporation on the beads was confirmed by a thermo-gravimetric analysis. The lipase immobilization on the beads involved the functionalization with coupling agents, N-hydroxysulfosuccinimide sodium (NHS) and 1-ethyl-(3-dimethylaminopropyl) carbodiimide (EDC), and it possessed a high enzyme activity of 64 U. The overall esterification conversion of the prepared product was 78% at 60°C, and it attained conversions of 98% and 88% with commercially available lipozyme and novozyme, respectively, under similar experimental conditions. PMID:25127038

  18. Cold-adapted organic solvent tolerant alkalophilic family I.3 lipase from an Antarctic Pseudomonas.

    PubMed

    Ganasen, Menega; Yaacob, Norhayati; Rahman, Raja Noor Zaliha Raja Abd; Leow, Adam Thean Chor; Basri, Mahiran; Salleh, Abu Bakar; Ali, Mohd Shukuri Mohamad

    2016-11-01

    Lipolytic enzymes with cold adaptation are gaining increasing interest due to their biotechnological prospective. Previously, a cold adapted family I.3 lipase (AMS8 lipase) was isolated from an Antarctic Pseudomonas. AMS8 lipase was largely expressed in insoluble form. The refolded His-tagged recombinant AMS8 lipase was purified with 23.0% total recovery and purification factor of 9.7. The purified AMS8 lipase migrated as a single band with a molecular weight approximately 65kDa via electrophoresis. AMS8 lipase was highly active at 30°C at pH 10. The half-life of AMS8 lipase was reported at 4 and 2h under the incubation of 30 and 40°C, respectively. The lipase was stable over a broad range of pH. It showed enhancement effect in its relative activity under the presence of Li + , Na + , K + , Rb + and Cs + after 30min treatment. Heavy metal ions such as Cu 2+ , Fe 3+ and Zn 2+ inhibited AMS8 activity. This cold adapted alkalophilic AMS lipase was also active in various organic solvent of different polarity. These unique properties of this biological macromolecule will provide considerable potential for many biotechnological applications and organic synthesis at low temperature. Copyright © 2016 Elsevier B.V. All rights reserved.

  19. Co-expression, purification and characterization of the lipase and foldase of Burkholderia contaminans LTEB11.

    PubMed

    Alnoch, Robson Carlos; Stefanello, Adriano Alves; Paula Martini, Viviane; Richter, Jeferson Luiz; Mateo, Cesar; Souza, Emanuel Maltempi de; Mitchell, David Alexander; Muller-Santos, Marcelo; Krieger, Nadia

    2018-05-30

    Genes encoding lipase LipBC (lipA) and foldase LifBC (lipB) were identified in the genome of Burkholderia contaminans LTEB11. Analysis of the predicted amino acid sequence of lipA showed its high identity with lipases from Pseudomonas luteola (91%), Burkholderia cepacia (96%) and Burkholderia lata (97%), and classified LipBC lipase in the lipase subfamily I.2. The genes lipA and lipB were amplified and cloned into expression vectors pET28a(+) and pT7-7, respectively. His-tagged LipBC and native LifBC were co-expressed in Escherichia coli and purified. LipBC and LifBC have molecular weights of 35.9 kDa and 37 kDa, respectively, and remain complexed after purification. The Lip-LifBC complex was active and stable over a wide range of pH values (6.5-10) and temperatures (25-45 °C), with the highest specific activity (1426 U mg -1 ) being against tributyrin. The Lip-LifBC complex immobilized on Sepabeads was able to catalyze the synthesis of ethyl-oleate in n‑hexane with an activity of 4 U g -1 , maintaining high conversion (>80%) over 5 reaction cycles of 6 h at 45 °C. The results obtained in this work provide a basis for the development of applications of recombinant LipBC in biocatalysis. Copyright © 2018 Elsevier B.V. All rights reserved.

  20. Bioreactors with immobilized lipases: state of the art.

    PubMed

    Balcão, V M; Paiva, A L; Malcata, F X

    1996-05-01

    This review attempts to provide an updated compilation of studies reported in the literature pertaining to reactors containing lipases in immobilized forms, in a way that helps the reader direct a bibliographic search and develop an integrated perspective of the subject. Highlights are given to industrial applications of lipases (including control and economic considerations), as well as to methods of immobilization and configurations of reactors in which lipases are used. Features associated with immobilized lipase kinetics such as enzyme activities, adsorption properties, optimum operating conditions, and estimates of the lumped parameters in classical kinetic formulations (Michaelis-Menten model for enzyme action and first-order model for enzyme decay) are presented in the text in a systematic tabular form.

  1. Cholesterol efflux from THP-1 macrophages is impaired by the fatty acid component from lipoprotein hydrolysis by lipoprotein lipase.

    PubMed

    Yang, Yanbo; Thyagarajan, Narmadaa; Coady, Breanne M; Brown, Robert J

    2014-09-05

    Lipoprotein lipase (LPL) is an extracellular lipase that primarily hydrolyzes triglycerides within circulating lipoproteins. Macrophage LPL contributes to atherogenesis, but the mechanisms behind it are poorly understood. We hypothesized that the products of lipoprotein hydrolysis generated by LPL promote atherogenesis by inhibiting the cholesterol efflux ability by macrophages. To test this hypothesis, we treated human THP-1 macrophages with total lipoproteins that were hydrolyzed by LPL and we found significantly reduced transcript levels for the cholesterol transporters ATP binding cassette transporter A1 (ABCA1), ABCG1, and scavenger receptor BI. These decreases were likely due to significant reductions for the nuclear receptors liver-X-receptor-α, peroxisome proliferator activated receptor (PPAR)-α, and PPAR-γ. We prepared a mixture of free fatty acids (FFA) that represented the ratios of FFA species within lipoprotein hydrolysis products, and we found that the FFA mixture also significantly reduced cholesterol transporters and nuclear receptors. Finally, we tested the efflux of cholesterol from THP-1 macrophages to apolipoprotein A-I, and we found that the treatment of THP-1 macrophages with the FFA mixture significantly attenuated cholesterol efflux. Overall, these data show that the FFA component of lipoprotein hydrolysis products generated by LPL may promote atherogenesis by inhibiting cholesterol efflux, which partially explains the pro-atherogenic role of macrophage LPL. Copyright © 2014 Elsevier Inc. All rights reserved.

  2. Probing Conformational Changes and Interfacial Recognition Site of Lipases With Surfactants and Inhibitors.

    PubMed

    Mateos-Diaz, E; Amara, S; Roussel, A; Longhi, S; Cambillau, C; Carrière, F

    2017-01-01

    Structural studies on lipases by X-ray crystallography have revealed conformational changes occurring in the presence of surfactants/inhibitors and the pivotal role played by a molecular "lid" of variable size and structure depending on the enzyme. Besides controlling the access to the enzyme active site, the lid is involved in lipase activation, formation of the interfacial recognition site (IRS), and substrate docking within the active site. The combined use of surfactants and inhibitors has been critical for a better understanding of lipase structure-function relationships. An overview of crystal structures of lipases in complex with surfactants and inhibitors reveals common structural features and shows how surfactants monomers interact with the lid in its open conformation. The location of surfactants, inhibitors, and hydrophobic residues exposed upon lid opening provides insights into the IRS of lipases. The mechanism by which surfactants promote the lid opening can be further investigated in solution by site-directed spin labeling of lipase coupled to electron paramagnetic resonance spectroscopy. These experimental approaches are illustrated here by results obtained with mammalian digestive lipases, fungal lipases, and cutinases. © 2017 Elsevier Inc. All rights reserved.

  3. Replacement of soybean oil by fish oil increases cytosolic lipases activities in liver and adipose tissue from rats fed a high-carbohydrate diets.

    PubMed

    Rodrigues, Angélica Heringer; Moreira, Carolina Campos Lima; Neves, Maria José; Botion, Leida Maria; Chaves, Valéria Ernestânia

    2018-06-01

    Several studies have demonstrated that fish oil consumption improves metabolic syndrome and comorbidities, as insulin resistance, nonalcoholic fatty liver disease, dyslipidaemia and hypertension induced by high-fat diet ingestion. Previously, we demonstrated that administration of a fructose-rich diet to rats induces liver lipid accumulation, accompanied by a decrease in liver cytosolic lipases activities. In this study, the effect of replacement of soybean oil by fish oil in a high-fructose diet (FRUC, 60% fructose) for 8 weeks on lipid metabolism in liver and epididymal adipose tissue from rats was investigated. The interaction between fish oil and FRUC diet increased glucose tolerance and decreased serum levels of triacylglycerol (TAG), VLDL-TAG secretion and lipid droplet volume of hepatocytes. In addition, the fish oil supplementation increased the liver cytosolic lipases activities, independently of the type of carbohydrate ingested. Our results firmly establish the physiological regulation of liver cytosolic lipases to maintain lipid homeostasis in hepatocytes. In epididymal adipose tissue, the replacement of soybean oil by fish oil in FRUC diet did not change the tissue weight and lipoprotein lipase activity; however, there was increased basal and insulin-stimulated de novo lipogenesis and glucose uptake. Increased cytosolic lipases activities were observed, despite the decreased basal and isoproterenol-stimulated glycerol release to the incubation medium. These findings suggest that fish oil increases the glycerokinase activity and glycerol phosphorylation from endogenous TAG hydrolysis. Our findings are the first to show that the fish oil ingestion increases cytosolic lipases activities in liver and adipose tissue from rats treated with high-carbohydrate diets. Copyright © 2018. Published by Elsevier Inc.

  4. Biodiesel production using lipase immobilized on epoxychloropropane-modified Fe3O4 sub-microspheres.

    PubMed

    Zhang, Qian; Zheng, Zhong; Liu, Changxia; Liu, Chunqiao; Tan, Tianwei

    2016-04-01

    Superparamagnetic Fe3O4 sub-microspheres with diameters of approximately 200 nm were prepared via a solvothermal method, and then modified with epoxychloropropane. Lipase was immobilized on the modified sub-microspheres. The immobilized lipase was used in the production of biodiesel fatty acid methyl esters (FAMEs) from acidified waste cooking oil (AWCO). The effects of the reaction conditions on the biodiesel yield were investigated using a combination of response surface methodology and three-level/three-factor Box-Behnken design (BBD). The optimum synthetic conditions, which were identified using Ridge max analysis, were as follows: immobilized lipase:AWCO mass ratio 0.02:1, fatty acid:methanol molar ratio 1:1.10, hexane:AWCO ratio 1.33:1 (mL/g), and temperature 40 °C. A 97.11% yield was obtained under these conditions. The BBD and experimental data showed that the immobilized lipase could generate biodiesel over a wide temperature range, from 0 to 40 °C. Consistently high FAME yields, in excess of 80%, were obtained when the immobilized lipase was reused in six replicate trials at 10 and 20 °C. Copyright © 2016 Elsevier B.V. All rights reserved.

  5. Covalent immobilization of lipase onto aminopropyl-functionalized hydroxyapatite-encapsulated-γ-Fe2O3 nanoparticles: A magnetic biocatalyst for interesterification of soybean oil.

    PubMed

    Xie, Wenlei; Zang, Xuezhen

    2017-07-15

    Hydroxyapatite-encapsulated γ-Fe 2 O 3 nanoparticles were prepared, and lipase from Candida rugosa was then covalently bound onto the magnetic materials via covalent linkages. The magnetic carrier and immobilized lipase were characterized by enzyme activity assays, XRD, FT-IR, TEM, VSM and N 2 adsorption-desorption techniques. Results demonstrated that γ-Fe 2 O 3 nanoparticles were coated with the hydroxyapatite, and the lipase was indeed tethered to the magnetic carriers without damage to their structure. The immobilized lipase showed a strong magnetic responsiveness and displayed high catalytic activities towards the interesterification of soybean oil. The interesterified products were evaluated for their total fatty acid (FA) composition, slip melting point (SMP), iodine value, triacylglycerols (TAGs) profile and FA composition at sn-2 position in TAGs. The FA positional distributions and TAG species significantly changed after the enzymatic interesterification. Besides this, the interesterified products showed an obvious reduction in their SMP in comparison with the physical blends. Copyright © 2017 Elsevier Ltd. All rights reserved.

  6. Strategy to Overcome Effect of Raw Materials on Enzymatic Process of Biodiesel from Non-edible Oils Using Candida sp. 99-125 Lipase.

    PubMed

    Nie, Kaili; Wang, Fang; Tan, Tianwei; Liu, Luo

    2015-11-01

    Non-edible oils are preferred raw materials for biodiesel production. However, the properties of raw materials significantly affect the synthesis process, leading to difficulties to design one process suitable for any kind of raw material. In this study, the composition of five typical non-edible oils was analyzed. The major difference was the content of free fatty acids, reflected from their acid values. The influence of different oils was investigated by using lipase from Candida sp. 99-125. At low lipase dosage and low water content, the conversion was found proportional to the acid value. However, by increasing the water content or lipase dosage, we observed that the conversions for all kinds of oils used in this study could exceed 80%. Time course analysis indicates that the lipase used in this study catalyzed hydrolysis followed by esterification, rather than direct transesterification. Accumulation of free fatty acids at the very beginning was necessary. A high water content facilitated the hydrolysis of oils with low acid value. This lipase showed capability to transform all the oils by controlling the water content.

  7. Lipolysis of Visceral Adipocyte Triglyceride by Pancreatic Lipases Converts Mild Acute Pancreatitis to Severe Pancreatitis Independent of Necrosis and Inflammation

    PubMed Central

    Patel, Krutika; Trivedi, Ram N.; Durgampudi, Chandra; Noel, Pawan; Cline, Rachel A.; DeLany, James P.; Navina, Sarah; Singh, Vijay P.

    2016-01-01

    Visceral fat necrosis has been associated with severe acute pancreatitis (SAP) for over 100 years; however, its pathogenesis and role in SAP outcomes are poorly understood. Based on recent work suggesting that pancreatic fat lipolysis plays an important role in SAP, we evaluated the role of pancreatic lipases in SAP-associated visceral fat necrosis, the inflammatory response, local injury, and outcomes of acute pancreatitis (AP). For this, cerulein pancreatitis was induced in lean and obese mice, alone or with the lipase inhibitor orlistat and parameters of AP induction (serum amylase and lipase), fat necrosis, pancreatic necrosis, and multisystem organ failure, and inflammatory response were assessed. Pancreatic lipases were measured in fat necrosis and were overexpressed in 3T3-L1 cells. We noted obesity to convert mild cerulein AP to SAP with greater cytokines, unsaturated fatty acids (UFAs), and multisystem organ failure, and 100% mortality without affecting AP induction or pancreatic necrosis. Increased pancreatic lipase amounts and activity were noted in the extensive visceral fat necrosis of dying obese mice. Lipase inhibition reduced fat necrosis, UFAs, organ failure, and mortality but not the parameters of AP induction. Pancreatic lipase expression increased lipolysis in 3T3-L1 cells. We conclude that UFAs generated via lipolysis of visceral fat by pancreatic lipases convert mild AP to SAP independent of pancreatic necrosis and the inflammatory response. PMID:25579844

  8. Medium chain and behenic acid incorporated structured lipids from sal, mango and kokum fats by lipase acidolysis.

    PubMed

    Bebarta, Biranchi; M, Jhansi; Kotasthane, Pranitha; Sunkireddy, Yella Reddy

    2013-01-15

    Medium chain (MC) and behenic fatty acids were incorporated into kokum, sal and mango fats using 1,3-specific lipase catalysed acidolysis. The incorporation of fatty acids increased with increase in concentration of fatty acids and duration of reaction. The order of incorporation of fatty acids was C22:0>C10:0>C8:0, to the extent of 53%, 42.5%, 35.8%, respectively, after 16 h, using kokum as substrate. The same trend was observed with sal or mango fats as substrates though the percentages incorporated were different. The modified products with higher contents of MC were liquids with no solid fats, even at 0°C, and which showed low cloud point due to an increase in triacylglycerols containing lower chain fatty acids. The modified products after incorporating both MC and C22:0 showed long melting ranges and were suitable for use in bakery, confectionery, etc. as vanaspati substitutes. Copyright © 2012 Elsevier Ltd. All rights reserved.

  9. A newly high alkaline lipase: an ideal choice for application in detergent formulations

    PubMed Central

    2011-01-01

    Background Bacterial lipases received much attention for their substrate specificity and their ability to function in extreme environments (pH, temperature...). Many staphylococci produced lipases which were released into the culture medium. Reports of thermostable lipases from Staphylococcus sp. and active in alkaline conditions are not previously described. Results A newly soil-isolated Staphylococcus sp. strain ESW secretes an induced lipase in the culture medium. The effects of temperature, pH and various components in a detergent on the activity and stability of Staphylococcus sp. lipase (SL1) were studied in a preliminary evaluation for use in detergent formulation solutions. The enzyme was highly active over a wide range of pH from 9.0 to 13.0, with an optimum at pH 12.0. The relative activity at pH 13.0 was about 60% of that obtained at pH 12.0. It exhibited maximal activity at 60°C. This novel lipase, showed extreme stability towards non-ionic and anionic surfactants after pre-incubation for 1 h at 40°C, and relative stability towards oxidizing agents. Additionally, the crude enzyme showed excellent stability and compatibility with various commercial solid and liquid detergents. Conclusions These properties added to the high activity in high alkaline pH make this novel lipase an ideal choice for application in detergent formulations. PMID:22123072

  10. FoxO1 controls lysosomal acid lipase in adipocytes: implication of lipophagy during nutrient restriction and metformin treatment

    PubMed Central

    Lettieri Barbato, D; Tatulli, G; Aquilano, K; Ciriolo, M R

    2013-01-01

    Finding new molecular pathways and strategies modulating lipolysis in adipocytes is an attractive goal of the current research. Indeed, it is becoming clear that several human age-related pathologies are caused by adipose tissue expansion and altered lipid metabolism. In the present work, we show that transcription factor forkhead homeobox type protein O1 (FoxO1) is upregulated by nutrient restriction (NR) in adipocytes and exerts the transcriptional control of lipid catabolism via the induction of lysosomal acid lipase (Lipa). An increased autophagy and colocalization of lipid droplets (LDs) with lysosomes was observed implying lipophagy in Lipa-mediated LDs degradation. Interestingly, we found that metformin (Metf), a biguanide drug commonly used to treat type-2 diabetes, exerts effects comparable to that of NR. Actually, it was able to elicit FoxO1-dependent Lipa induction as well as LDs degradation through lipophagy. Moreover, we demonstrate that, during NR or Metf treatment, free fatty acids released by Lipa are directed toward AMP-activated protein kinase-mediated mitochondrial oxidation, thus maintaining energetic homeostasis in adipocytes. In conclusion, our data show that lysosomal-mediated lipid catabolism is activated by NR in adipocytes and give further support to the use of Metf as a NR mimetic to combat age-related diseases associated with altered lipid metabolism. PMID:24136225

  11. Hypolipidemic mechanism of oryzanol components- ferulic acid and phytosterols.

    PubMed

    Bhaskaragoud, G; Rajath, S; Mahendra, V P; Kumar, G Sunil; Gopala Krishna, A G; Kumar, G Suresh

    2016-07-22

    The effect of oryzanol (well known hypolipidemic component in rice bran oil) and its chemical constituents- ferulic acid (FA) and phytosterols on hypolipidemia were investigated. Docking (in silico) studies showed that FA had a better binding ability with lipase while sterols bound well with HMG-CoA reductase. Further in vivo studies of feeding high fat (30%) to rats increased body weights, serum TC, TG, non-HDL-C and reduced HDL-C were observed, compared to normal diet fed group (ND). ORZ treated groups alleviated the lipid profile. Furthermore, increased organ weights, higher intestinal lipase activity, and liver lipid peroxidation was observed in the high-fat group (HF). These effects were ameliorated in oryzanol concentrate fed groups (ORZ). Higher fecal fat was found in ORZ groups, analysis of fecal matter by mass spectroscopy revealed the presence of FA. In vitro, a bile acid binding study supported the strong affinity of sterol towards bile acids. In conclusion, oryzanol in the intestine is cleaved into FA and sterol by intestinal lipase enzymes both lipase and HMG-CoA reductase activities were inhibited, respectively. These hydrolysates eliminated the bile acids, thus lowering lipid profiles. Copyright © 2016 Elsevier Inc. All rights reserved.

  12. Immobilization of Thermomyces lanuginosus lipase on multi-walled carbon nanotubes and its application in the hydrolysis of fish oil

    NASA Astrophysics Data System (ADS)

    Matuoog, Naeema; Li, Kai; Yan, Yunjun

    2017-12-01

    In this study, lipase from Thermomyces lanuginosus (TLL) was immobilized on carbon nanotubes (MWCNTs) by physical adsorption, and the immobilizing conditions were optimized for maximum activity. The effects of enzyme loading, pH, temperature and time on the immobilization efficiency and specific activity were evaluated. The highest enzyme activity and immobilization efficiency of 90.66% and 110.5%, respectively, were achieved when the immobilized pH was 8, and a high rate of recovery activity of 111.3% occurred at 45 °C with 30-60 min providing a good result. When the immobilization efficiency was 95.8%, the recovery activity was 112.4%. The immobilization time had little effect on the immobilization efficiency, and 6 mg g-1 of lipase provided the highest immobilization efficiency of 97.78% and recovery activity of 112.8%. When the immobilized lipase was utilized to enrich docosahexaenoic acid (DHA) from fish oil, the DHA content increased with increasing amount of lipase up to 9 mg g-1. The water content had a clear effect when of 50% water was used at 45 °C and at a pH of 7 after 10 h. The DHA contents were 4.2-fold and 2.5-fold greater than the initial content of DHA fish oil for TLL-MWCNTs and free lipase, respectively. The degrees of hydrolysis after 6 cycles of successive use were over 80% and 62% for the immobilized TLL and free TLL, respectively, indicating the system recyclability and the ease of use of the immobilized TLL in industrial applications, especially in the fields of food and medicine.

  13. Sebelipase alfa over 52 weeks reduces serum transaminases, liver volume and improves serum lipids in patients with lysosomal acid lipase deficiency.

    PubMed

    Valayannopoulos, Vassili; Malinova, Vera; Honzík, Tomas; Balwani, Manisha; Breen, Catherine; Deegan, Patrick B; Enns, Gregory M; Jones, Simon A; Kane, John P; Stock, Eveline O; Tripuraneni, Radhika; Eckert, Stephen; Schneider, Eugene; Hamilton, Gavin; Middleton, Michael S; Sirlin, Claude; Kessler, Bruce; Bourdon, Christopher; Boyadjiev, Simeon A; Sharma, Reena; Twelves, Chris; Whitley, Chester B; Quinn, Anthony G

    2014-11-01

    Lysosomal acid lipase deficiency is an autosomal recessive enzyme deficiency resulting in lysosomal accumulation of cholesteryl esters and triglycerides. LAL-CL04, an ongoing extension study, investigates the long-term effects of sebelipase alfa, a recombinant human lysosomal acid lipase. Sebelipase alfa (1mg/kg or 3mg/kg) was infused every-other-week to eligible subjects. Safety and tolerability assessments, including liver function, lipid profiles and liver volume assessment, were carried out at regular intervals. 216 infusions were administered to eight adult subjects through week 52 during LAL-CL04. At week 52, mean alanine aminotransferase and aspartate aminotransferase levels were normal with mean change from baseline of -58% and -40%. Mean changes for low-density lipoprotein, total cholesterol, triglyceride and high-density lipoprotein were -60%, -39%, -36%, and +29%, respectively. Mean liver volume by magnetic resonance imaging and hepatic proton density fat fraction decreased (12% and 55%, respectively). Adverse events were mainly mild and unrelated to sebelipase alfa. Infusion-related reactions were uncommon: three events of moderate severity were reported in two subjects; one patient's event was suggestive of a hypersensitivity-like reaction, but additional testing did not confirm this, and the subject has successfully re-started sebelipase alfa. Of samples tested to date, no anti-drug antibodies have been detected. Long-term dosing with sebelipase alfa in lysosomal acid lipase-deficient patients is well tolerated and produces sustained reductions in transaminases, improvements in serum lipid profile and reduction in the hepatic fat fraction. A randomized, placebo-controlled phase 3 trial in children and adults is underway (ARISE: NCT01757184). Copyright © 2014 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

  14. Convenient enzymatic resolution of (R,S)-2-methylbutyric acid catalyzed by immobilized lipases.

    PubMed

    Mittersteiner, Mateus; Linshalm, Bruna Luiza; Vieira, Ana Paula Furlan; Brondani, Patrícia Bulegon; Scharf, Dilamara Riva; de Jesus, Paulo Cesar

    2018-01-01

    The application of several immobilized lipases has been explored in the enantioselective esterification of (R,S)-2-methylbutyric acid, an insect pheromone precursor. With the use of Candida antarctica B, using hexane as solvent, (R)-pentyl 2-methylbutyrate was prepared in 2 h with c 40%, ee p 90%, and E = 35, while Thermomyces lanuginosus leads to c 18%, ee p 91%, and E = 26. The (S)-enantiomer was obtained by the use of Candida rugosa or Rhizopus oryzae (2-h reaction, c 34% and 35%, ee p 75 and 49%, and E = 10 and 4, respectively). Under optimal conditions, the effect of the solvent, the molar ratio, and the nucleophile were evaluated. © 2017 Wiley Periodicals, Inc.

  15. Regioselective lipase-catalyzed synthesis of 3-o-acyl derivatives of resveratrol and study of their antioxidant properties.

    PubMed

    Torres, Pamela; Poveda, Ana; Jimenez-Barbero, Jesús; Ballesteros, Antonio; Plou, Francisco J

    2010-01-27

    One of the approaches to increasing the bioavailability of resveratrol is to protect its 3-OH phenolic group. In this work, regioselective acylation of resveratrol at 3-OH was achieved by transesterification with vinyl acetate catalyzed by immobilized lipase from Alcaligenes sp. (lipase QLG). The maximum yield of 3-O-acetylresveratrol was approximately 75%, as the lipase also catalyzes its further acetylation affording the diester 3,4'-di-O-acetylresveratrol and finally the peracetylated derivative. Long saturated and unsaturated fatty acid vinyl esters were also effective as acyl donors with similar regioselectivity. In contrast, lipase B from Candida antarctica catalyzes the acylation of the phenolic group 4'-OH with 80% yield and negligible formation of higher esters. The analysis of the antioxidant properties showed that the Trolox equivalent antioxidant capability (TEAC) values for the acetyl and stearoyl derivatives at 3-OH were, respectively, 40% and 25% referred to resveratrol. The addition of an acyl chain in the 3-OH position caused a higher loss of activity compared with that at the 4'-OH.

  16. Construction of the yeast whole-cell Rhizopus oryzae lipase biocatalyst with high activity.

    PubMed

    Chen, Mei-ling; Guo, Qin; Wang, Rui-zhi; Xu, Juan; Zhou, Chen-wei; Ruan, Hui; He, Guo-qing

    2011-07-01

    Surface display is effectively utilized to construct a whole-cell biocatalyst. Codon optimization has been proven to be effective in maximizing production of heterologous proteins in yeast. Here, the cDNA sequence of Rhizopus oryzae lipase (ROL) was optimized and synthesized according to the codon bias of Saccharomyces cerevisiae, and based on the Saccharomyces cerevisiae cell surface display system with α-agglutinin as an anchor, recombinant yeast displaying fully codon-optimized ROL with high activity was successfully constructed. Compared with the wild-type ROL-displaying yeast, the activity of the codon-optimized ROL yeast whole-cell biocatalyst (25 U/g dried cells) was 12.8-fold higher in a hydrolysis reaction using p-nitrophenyl palmitate (pNPP) as the substrate. To our knowledge, this was the first attempt to combine the techniques of yeast surface display and codon optimization for whole-cell biocatalyst construction. Consequently, the yeast whole-cell ROL biocatalyst was constructed with high activity. The optimum pH and temperature for the yeast whole-cell ROL biocatalyst were pH 7.0 and 40 °C. Furthermore, this whole-cell biocatalyst was applied to the hydrolysis of tributyrin and the resulted conversion of butyric acid reached 96.91% after 144 h.

  17. Rational evolution of the unusual Y-type oxyanion hole of Rhodococcus sp. CR53 lipase LipR.

    PubMed

    Infanzón, Belén; Sotelo, Pablo H; Martínez, Josefina; Diaz, Pilar

    2018-01-01

    Rhodococcus sp CR-53 lipase LipR was the first characterized member of bacterial lipase family X. Interestingly, LipR displays some similarity with α/β-hydrolases of the C. antartica lipase A (CAL-A)-like superfamily (abH38), bearing a Y-type oxyanion hole, never found before among bacterial lipases. In order to explore this unusual Y-type oxyanion hole, and to improve LipR performance, two modification strategies based on site directed or saturation mutagenesis were addressed. Initially, a small library of mutants was designed to convert LipR Y-type oxyanion hole (YDS) into one closer to those most frequently found in bacteria (GGG(X)). However, activity was completely lost in all mutants obtained, indicating that the Y-type oxyanion hole of LipR is required for activity. A second approach was addressed to modify the two main oxyanion hole residues Tyr 110 and Asp 111 , previously described for CAL-A as the most relevant amino acids involved in stabilization of the enzyme-substrate complex. A saturation mutagenesis library was prepared for each residue (Tyr 110 and Asp 111 ), and activity of the resulting variants was assayed on different chain length substrates. No functional LipR variants could be obtained when Tyr 110 was replaced by any other amino acids, indicating that this is a crucial residue for catalysis. However, among the Asp 111 variants obtained, LipR D111G produced a functional enzyme. Interestingly, this LipR-YGS variant showed less activity than wild type LipR on short- or mid- chain substrates but displayed a 5.6-fold increased activity on long chain length substrates. Analysis of the 3D model and in silico docking studies of this enzyme variant suggest that substitution of Asp by Gly produces a wider entrance tunnel that would allow for a better and tight accommodation of larger substrates, thus justifying the experimental results obtained. Copyright © 2017 Elsevier Inc. All rights reserved.

  18. Lipase-catalyzed highly enantioselective kinetic resolution of boron-containing chiral alcohols.

    PubMed

    Andrade, Leandro H; Barcellos, Thiago

    2009-07-16

    The first application of enzymes as catalysts to obtain optically pure boron compounds is described. The kinetic resolution of boron-containing chiral alcohols via enantioselective transesterification catalyzed by lipases was studied. Aromatic, allylic, and aliphatic secondary alcohols containing a boronate ester or boronic acid group were resolved by lipase from Candida antartica (CALB), and excellent E values (E > 200) and high enantiomeric excesses (up to >99%) of both remaining substrates and acetylated product were obtained.

  19. Novel magnetic cross-linked lipase aggregates for improving the resolution of (R, S)-2-octanol.

    PubMed

    Liu, Ying; Guo, Chen; Liu, Chun-Zhao

    2015-03-01

    Novel magnetic cross-linked lipase aggregates were fabricated by immobilizing the cross-linked lipase aggregates onto magnetic particles with a high number of -NH2 terminal groups using p-benzoquinone as the cross-linking agent. At the optimal fabrication conditions, 100% of immobilization efficiency and 139% of activity recovery of the magnetic cross-linked lipase aggregates were achieved. The magnetic cross-linked lipase aggregates were able to efficiently resolve (R, S)-2-octanol, and retained 100% activity and 100% enantioselectivity after 10 cycles of reuse, whereas the cross-linked lipase aggregates only retained about 50% activity and 70% enantioselectivity due to insufficient cross-linking. These results provide a great potential for industrial applications of the magnetic cross-linked lipase aggregates. © 2014 Wiley Periodicals, Inc.

  20. A Gossypium hirsutum GDSL lipase/hydrolase gene (GhGLIP) appears to be involved in promoting seed growth in Arabidopsis.

    PubMed

    Ma, Rendi; Yuan, Hali; An, Jing; Hao, Xiaoyun; Li, Hongbin

    2018-01-01

    GDSL lipase (GLIP) plays a pivotal role in plant cell growth as a multifunctional hydrolytic enzyme. Herein, a cotton (Gossypium hirsutum L. cv Xuzhou 142) GDSL lipase gene (GhGLIP) was obtained from developing ovules and fibers. The GhGLIP cDNA contained an open reading frame (ORF) of 1,143 base pairs (bp) and encodes a putative polypeptide of 380 amino acid residues. Sequence alignment indicated that GhGLIP includes four enzyme catalytic amino acid residue sites of Ser (S), Gly (G), Asn (N) and His (H), located in four conserved blocks. Phylogenetic tree analysis showed that GhGLIP belongs to the typical class IV lipase family with potential functions in plant secondary metabolism. Subcellular distribution analysis demonstrated that GhGLIP localized to the nucleus, cytoplasm and plasma membrane. GhGLIP was expressed predominantly at 5-15 day post anthesis (dpa) in developing ovules and elongating fibers, measured as mRNA levels and enzyme activity. Ectopic overexpression of GhGLIP in Arabidopsis plants resulted in enhanced seed development, including length and fresh weight. Meanwhile, there was increased soluble sugar and protein storage in transgenic Arabidopsis plants, coupled with the promotion of lipase activity. Moreover, the expression of cotton GhGLIP is induced by ethylene (ETH) treatment in vitro. A 1,954-bp GhGLIP promoter was isolated and expressed high activity in driving green fluorescence protein (GFP) expression in tobacco leaves. Cis-acting element analysis of the GhGLIP promoter (pGhGLIP) indicated the presence of an ethylene-responsive element (ERE), and transgenic tobacco leaves with ectopic expression of pGhGLIP::GFP-GUS showed increased GUS activity after ETH treatment. In summary, these results suggest that GhGLIP is a functional enzyme involved in ovule and fiber development and performs significant roles in seed development.

  1. A Gossypium hirsutum GDSL lipase/hydrolase gene (GhGLIP) appears to be involved in promoting seed growth in Arabidopsis

    PubMed Central

    An, Jing; Hao, Xiaoyun

    2018-01-01

    GDSL lipase (GLIP) plays a pivotal role in plant cell growth as a multifunctional hydrolytic enzyme. Herein, a cotton (Gossypium hirsutum L. cv Xuzhou 142) GDSL lipase gene (GhGLIP) was obtained from developing ovules and fibers. The GhGLIP cDNA contained an open reading frame (ORF) of 1,143 base pairs (bp) and encodes a putative polypeptide of 380 amino acid residues. Sequence alignment indicated that GhGLIP includes four enzyme catalytic amino acid residue sites of Ser (S), Gly (G), Asn (N) and His (H), located in four conserved blocks. Phylogenetic tree analysis showed that GhGLIP belongs to the typical class IV lipase family with potential functions in plant secondary metabolism. Subcellular distribution analysis demonstrated that GhGLIP localized to the nucleus, cytoplasm and plasma membrane. GhGLIP was expressed predominantly at 5–15 day post anthesis (dpa) in developing ovules and elongating fibers, measured as mRNA levels and enzyme activity. Ectopic overexpression of GhGLIP in Arabidopsis plants resulted in enhanced seed development, including length and fresh weight. Meanwhile, there was increased soluble sugar and protein storage in transgenic Arabidopsis plants, coupled with the promotion of lipase activity. Moreover, the expression of cotton GhGLIP is induced by ethylene (ETH) treatment in vitro. A 1,954-bp GhGLIP promoter was isolated and expressed high activity in driving green fluorescence protein (GFP) expression in tobacco leaves. Cis-acting element analysis of the GhGLIP promoter (pGhGLIP) indicated the presence of an ethylene-responsive element (ERE), and transgenic tobacco leaves with ectopic expression of pGhGLIP::GFP-GUS showed increased GUS activity after ETH treatment. In summary, these results suggest that GhGLIP is a functional enzyme involved in ovule and fiber development and performs significant roles in seed development. PMID:29621331

  2. Fat digestion by lingual lipase: mechanism of lipolysis in the stomach and upper small intestine.

    PubMed

    Liao, T H; Hamosh, P; Hamosh, M

    1984-05-01

    Ten to 30% of dietary fat is hydrolyzed in the stomach by lingual lipase, an enzyme secreted from lingual serous glands. We investigated the substrate specificity of this enzyme as well as the potential of lingual lipase to act in the upper small intestine i.e., in the presence of bile salts and lecithin. The data presented show that partially purified preparations of rat lingual lipase and the lipase in gastric aspirates of newborn infants have identical substrate specificity: medium-chain triglycerides were hydrolyzed at rates 5-8-fold higher than long-chain triglycerides; the rat and human enzymes do not hydrolyze the ester bond of lecithin or cholesteryl-ester. In contrast to pancreatic lipase, the hydrolysis of triglycerides by lingual lipase is not inhibited by lecithin. But, similar to pancreatic lipase the activity of lingual lipase is inhibited by bile salts, the extent of inhibition varying with its nature and concentration. This inactivation is not prevented by colipase but is partially averted by lipids and protein, suggesting that lingual lipase can remain active in the duodenum. The pH optimum of the enzyme (2.2-6.5 in the rat and 3.5-6.0 in human gastric aspirates) is compatible with continued activity in the upper small intestine, especially during the neonatal period, when the luminal pH is under 6.5. The marked variation in lipase activity levels in gastric aspirates of newborn infants is probably due to individual variations in enzyme amounts. The characteristics of the lipase are however identical in infants with low, intermediate or high activity levels.(ABSTRACT TRUNCATED AT 250 WORDS)

  3. Lipase-Secreting Bacillus Species in an Oil-Contaminated Habitat: Promising Strains to Alleviate Oil Pollution.

    PubMed

    Lee, Li Pin; Karbul, Hudzaifah Mohamed; Citartan, Marimuthu; Gopinath, Subash C B; Lakshmipriya, Thangavel; Tang, Thean-Hock

    2015-01-01

    Lipases are of great interest for different industrial applications due to their diversity and versatility. Among different lipases, microbial lipases are preferable due to their broad substrate specificity, and higher stability with lower production costs compared to the lipases from plants and animals. In the past, a vast number of bacterial species have been reported as potential lipases producers. In this study, the lipases-producing bacterial species were isolated from an oil spillage area in the conventional night market. Isolated species were identified as Bacillus species by biochemical tests which indicate their predominant establishment, and further screened on the agar solid surfaces using lipid and gelatin as the substrates. Out of the ten strains tested, four potential strains were subjected to comparison analysis of the lipolytic versus proteolytic activities. Strain 10 exhibited the highest lipolytic and proteolytic activity. In all the strains, the proteolytic activity is higher than the lipolytic activity except for strain 8, suggesting the possibility for substrate-based extracellular gene induction. The simultaneous secretion of both the lipase and protease is a mean of survival. The isolated bacterial species which harbour both lipase and protease enzymes could render potential industrial-based applications and solve environmental issues.

  4. Seasonal variation in plasma lipids and lipases in young healthy humans.

    PubMed

    Cambras, Trinitat; Baena-Fustegueras, Juan A; Pardina, Eva; Ricart-Jané, David; Rossell, Joana; Díez-Noguera, Antoni; Peinado-Onsurbe, Julia

    2017-01-01

    Although intermediate metabolism is known to follow circadian rhythms, little information is available on the variation in lipase activities (lipoprotein and hepatic lipase, LPL and HL, respectively) and lipids throughout the year. In a cross-sectional study, we collected and analysed blood from 245 healthy students (110 men and 135 women) between 18 and 25 years old from the University of Barcelona throughout the annual campaign (March, May, October and December) of the blood bank. All subjects gave their written informed consent to participate. All blood samples were taken after breakfast at 8:00 and 11:00 am. Plasma glucose, total plasma protein, triacylglycerides (TAG), free fatty acids (FFA), free cholesterol and esterified cholesterol (FC and TC, respectively), cholesterol in low-density lipoproteins (cLDL), cholesterol in high-density lipoproteins (cHDL), phospholipids (PL) and lipase activities (LPL and HL) were determined. Cosinor analysis was used to evaluate the presence (significance of fit cosine curve to data and variance explained by rhythm) and characteristics of possible 12-month rhythms (acrophase, MESOR and amplitude). Statistically significant seasonal rhythms were detected for all the variables studied except proteins, with most of them peaking in the winter season. The lowest value for cLDL and the HL occurs in summer, while for cHDL and the LPL it is in winter. These findings demonstrate for the first time that in physiological conditions, plasma LPL and HL activities and lipids follow seasonal rhythms. The metabolic significance of this pattern is discussed.

  5. Factors Influencing the Digestibility of Solid Fats: Mammalian and Plant Lipases--Glyceride Structure and Solvent

    DTIC Science & Technology

    1993-05-01

    butter 7 and l-paladtcyl-2-oleoyl-3-stearin 5. Pancreatic lipase digestion of the fats extracted from desert chocolate bars 9 6. Lipase digestion of...of cocoa butter (m.p. 36° C), which consists predominantly (50%) of a TC containing palmitic, stearic, and oleic acids and of 18% of oleoyl distearin...determined. Pancreatic lipase digestion of cocoa butter and palmitoyloleovlstearin Contrary to the results obtained in vivo by Apgar et al.2, only 7% of

  6. A novel thermophilic and halophilic esterase from Janibacter sp. R02, the first member of a new lipase family (Family XVII).

    PubMed

    Castilla, Agustín; Panizza, Paola; Rodríguez, Diego; Bonino, Luis; Díaz, Pilar; Irazoqui, Gabriela; Rodríguez Giordano, Sonia

    2017-03-01

    Janibacter sp. strain R02 (BNM 560) was isolated in our laboratory from an Antarctic soil sample. A remarkable trait of the strain was its high lipolytic activity, detected in Rhodamine-olive oil supplemented plates. Supernatants of Janibacter sp. R02 displayed superb activity on transesterification of acyl glycerols, thus being a good candidate for lipase prospection. Considering the lack of information concerning lipases of the genus Janibacter, we focused on the identification, cloning, expression and characterization of the extracellular lipases of this strain. By means of sequence alignment and clustering of consensus nucleotide sequences, a DNA fragment of 1272bp was amplified, cloned and expressed in E. coli. The resulting recombinant enzyme, named LipJ2, showed preference for short to medium chain-length substrates, and displayed maximum activity at 80°C and pH 8-9, being strongly activated by a mixture of Na + and K + . The enzyme presented an outstanding stability regarding both pH and temperature. Bioinformatics analysis of the amino acid sequence of LipJ2 revealed the presence of a consensus catalytic triad and a canonical pentapeptide. However, two additional rare motifs were found in LipJ2: an SXXL β-lactamase motif and two putative Y-type oxyanion holes (YAP). Although some of the previous features could allow assigning LipJ2 to the bacterial lipase families VIII or X, the phylogenetic analysis showed that LipJ2 clusters apart from other members of known lipase families, indicating that the newly isolated Janibacter esterase LipJ2 would be the first characterized member of a new family of bacterial lipases. Published by Elsevier Inc.

  7. Lipase-nanoporous gold biocomposite modified electrode for reliable detection of triglycerides.

    PubMed

    Wu, Chao; Liu, Xueying; Li, Yufei; Du, Xiaoyu; Wang, Xia; Xu, Ping

    2014-03-15

    For triglycerides biosensor design, protein immobilization is necessary to create the interface between the enzyme and the electrode. In this study, a glassy carbon electrode (GCE) was modified with lipase-nanoporous gold (NPG) biocomposite (denoted as lipase/NPG/GCE). Due to highly conductive, porous, and biocompatible three-dimensional structure, NPG is suitable for enzyme immobilization. In cyclic voltammetry experiments, the lipase/NPG/GCE bioelectrode displayed surface-confined reaction in a phosphate buffer solution. Linear responses were obtained for tributyrin concentrations ranging from 50 to 250 mg dl(-1) and olive oil concentrations ranging from 10 to 200 mg dl(-1). The value of apparent Michaelis-Menten constant for tributyrin was 10.67 mg dl(-1) and the detection limit was 2.68 mg dl(-1). Further, the lipase/NPG/GCE bioelectrode had strong anti-interference ability against urea, glucose, cholesterol, and uric acid as well as a long shelf-life. For the detection of triglycerides in human serum, the values given by the lipase/NPG/GCE bioelectrode were in good agreement with those of an automatic biochemical analyzer. These properties along with a long self-life make the lipase/NPG/GCE bioelectrode an excellent choice for the construction of triglycerides biosensor. © 2013 Elsevier B.V. All rights reserved.

  8. Cellulose binding domain assisted immobilization of lipase (GSlip-CBD) onto cellulosic nanogel: characterization and application in organic medium.

    PubMed

    Kumar, Ashok; Zhang, Shaowei; Wu, Gaobing; Wu, Cheng Chao; Chen, JunPeng; Baskaran, R; Liu, Ziduo

    2015-12-01

    A cbd gene was cloned into the C-terminal region of a lip gene from Geobacillus stearothermophilus. The native lipase (43.5 kDa) and CBD-Lip fusion protein (60.2 kDa) were purified to homogeneity by SDS-PAGE. A highly stable cellulosic nanogel was prepared by controlled hydrolysis of microcrystalline cellulose onto which the CBD-lip fusion protein was immobilized through bio-affinity based binding. The nanogel-bound lipase showed optimum activity at 55 °C, and it remains stable and active at pH 10-10.5. Furthermore, the immobilized lipase showed an over two-fold increase of relative activity in the presence of DMSO, isopropanol, isoamyl alcohol and n-butanol, but a mild activity decrease at a low concentration of methanol and ethanol. The immobilized biocatalyst retained ~50% activity after eight repetitive hydrolytic cycles. Enzyme kinetic studies of the immobilized lipase showed a 1.24 fold increase in Vmax and 5.25 fold increase in kcat towards p-NPP hydrolysis. Additionally, the nanogel bound lipase was tested to synthesize a biodiesel ester, ethyl oleate in DMSO. Kinetic analysis showed the km 100.5 ± 4.3 mmol and Vmax 0.19 ± 0.015 mmolmin(-1) at varied oleic acid concentration. Also, the values of km and Vmax at varying concentration of ethanol were observed to be 95.9 ± 13.9 mmol and 0.22 ± 0.013 mmolmin(-1) respectively. The maximum yield of ethyl oleate 111.2 ± 1.24 mM was obtained under optimized reaction conditions in organic medium. These results suggest that this immobilized biocatalyst can be used as an efficient tool for the biotransformation reactions on an industrial scale. Copyright © 2015 Elsevier B.V. All rights reserved.

  9. Studying Gastric Lipase Adsorption Onto Phospholipid Monolayers by Surface Tensiometry, Ellipsometry, and Atomic Force Microscopy.

    PubMed

    Bénarouche, A; Sams, L; Bourlieu, C; Vié, V; Point, V; Cavalier, J F; Carrière, F

    2017-01-01

    The access to kinetic parameters of lipolytic enzyme adsorption onto lipids is essential for a better understanding of the overall catalytic process carried out by these interfacial enzymes. Gastric lipase, for instance, shows an apparent optimum activity on triglycerides (TAG) at acidic pH, which is controlled by its pH-dependent adsorption at lipid-water interfaces. Since gastric lipase acts on TAG droplets covered by phospholipids, but does not hydrolyze these lipids, phospholipid monolayers spread at the air-water interfaces can be used as biomimetic interfaces to study lipase adsorption and penetration through the phospholipid layer, independently from the catalytic activity. The adsorption of recombinant dog gastric lipase (rDGL) onto 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC) monolayers can be monitored by surface tensiometry at various enzyme concentrations, pHs, and surface pressures (Π). These experimental data and the use of Langmuir adsorption isotherm and Verger-de Haas' lipase kinetics models further allow estimating various parameters including the adsorption equilibrium constant (K Ads ), the interfacial concentration [Formula: see text] , the molar fraction [Formula: see text] (Φ E*(%) , mol%), and the molecular area [Formula: see text] of rDGL adsorbed onto the DLPC monolayer under various conditions. Additional insight into rDGL adsorption/insertion on phospholipid monolayers can be obtained by combining ellipsometry, Langmuir-Blodgett film transfer, and atomic force microscopy. When using multicomponent phospholipid monolayers with phase separation, these techniques allow to visualizing how rDGL preferentially partitions toward liquid expanded phase and at phase boundaries, gets adsorbed at various levels of insertion and impacts on the lateral organization of lipids. © 2017 Elsevier Inc. All rights reserved.

  10. Human pancreatic lipase-related protein 2 is a galactolipase.

    PubMed

    Sias, Barbara; Ferrato, Francine; Grandval, Philippe; Lafont, Dominique; Boullanger, Paul; De Caro, Alain; Leboeuf, Bernard; Verger, Robert; Carrière, Frédéric

    2004-08-10

    Human pancreatic lipase-related protein 2 (HPLRP2) was found to be expressed in the pancreas, but its biochemical properties were not investigated in detail. A recombinant HPLRP2 was produced in insect cells and the yeast Pichia pastoris and purified by cation exchange chromatography. Its substrate specificity was investigated using pH-stat and monomolecular film techniques and various lipid substrates (triglycerides, diglycerides, phospholipids, and galactolipids). Lipase activity of HPLRP2 on trioctanoin was inhibited by bile salts and poorly restored by adding colipase. In vivo, HPLRP2 therefore seems unlikely to show any lipase activity on dietary fat. In human pancreatic lipase (HPL), residues R256, D257, Y267, and K268 are involved in the stabilization of the open conformation of the lid domain, which interacts with colipase. These residues are not conserved in HPLRP2. When the corresponding mutations (R256G, D257G, Y267F, and K268E) are introduced into HPL, the effects of colipase are drastically reduced in the presence of bile salts. This may explain why colipase has such weak effects on HPLRP2. HPLRP2 displayed a very low level of activity on phospholipid micelles and monomolecular films. Its activity on monogalactosyldiglyceride monomolecular film, which was much higher, was similar to the activity of guinea pig pancreatic lipase related-protein 2, which shows the highest galactolipase activity ever measured. The physiological role of HPLRP2 suggested by the present results is the digestion of galactolipids, the most abundant lipids occurring in plant cells, and therefore, in the vegetables that are part of the human diet.

  11. Biodiesel production from crude Jatropha oil catalyzed by non-commercial immobilized heterologous Rhizopus oryzae and Carica papaya lipases.

    PubMed

    Rodrigues, J; Canet, A; Rivera, I; Osório, N M; Sandoval, G; Valero, F; Ferreira-Dias, S

    2016-08-01

    The aim of this study was to evaluate the feasibility of biodiesel production by transesterification of Jatropha oil with methanol, catalyzed by non-commercial sn-1,3-regioselective lipases. Using these lipases, fatty acid methyl esters (FAME) and monoacylglycerols are produced, avoiding the formation of glycerol as byproduct. Heterologous Rhizopus oryzae lipase (rROL) immobilized on different synthetic resins and Carica papaya lipase (rCPL) immobilized on Lewatit VP OC 1600 were tested. Reactions were performed at 30°C, with seven stepwise methanol additions. For all biocatalysts, 51-65% FAME (theoretical maximum=67%, w/w) was obtained after 4h transesterification. Stability tests were performed in 8 or 10 successive 4h-batches, either with or without rehydration of the biocatalyst between each two consecutive batches. Activity loss was much faster when biocatalysts were rehydrated. For rROL, half-life times varied from 16 to 579h. rROL on Lewatit VPOC 1600 was more stable than for rCPL on the same support. Copyright © 2016 Elsevier Ltd. All rights reserved.

  12. Protein Engineering and Homologous Expression of Serratia marcescens Lipase for Efficient Synthesis of a Pharmaceutically Relevant Chiral Epoxyester.

    PubMed

    Chen, Ke-Cai; Zheng, Ming-Min; Pan, Jiang; Li, Chun-Xiu; Xu, Jian-He

    2017-10-01

    The lipase isolated from Serratia marcescens (LipA) is a useful biocatalyst for kinetic resolution of a pharmaceutically relevant epoxyester, (±)-3-(4'-methoxyphenyl) glycidic acid methyl ester [(±)-MPGM], to afford optically pure (-)-MPGM, a key intermediate for the synthesis of diltiazem hydrochloride. Two mutants, LipA L315S and LipA S271F , were identified from the combinatorial saturation mutation library of 14 amino acid residues lining the substrate-binding pocket. LipA L315S , LipA S271F , and their combination LipA L315S/S271F showed 2.6-, 2.2-, and 4.6-fold improvements in their specific activities towards para-nitrophenyl butyrate (pNPB), respectively. Among these positive mutants, LipA S271F displayed a 3.5-fold higher specific activity towards the pharmaco substrate (±)-MPGM. Kinetic study showed that the improvement in catalytic efficiency of LipA S271F against (±)-MPGM was mainly resulted from the enhanced affinity between substrate and enzyme, as indicated by the decrease of K m . Furthermore, to address the insoluble expression issue in Escherichia coli, the homologous expression of LipA gene in S. marcescens was achieved by introducing it into an expression vector pUC18, resulting in ca. 20-fold higher lipase production. The significantly improved volumeric production and specific activity of S. marcescens lipase make it very attractive as a new-generation biocatalyst for more efficient and economical manufacturing of (-)-MPGM.

  13. Enhanced antiamyloidal activity of hydroxy cinnamic acids by enzymatic esterification with alkyl alcohols.

    PubMed

    Kondo, Hazuki; Sugiyama, Haruka; Katayama, Shigeru; Nakamura, Soichiro

    2014-01-01

    Lipophilic derivatives of hydroxyl cinnamic acids (HCAs) including caffeic acid (CA), ferulic acid, sinapic acid (SA), and chlorogenic acid were synthesized by esterification with butanol, octanol, or hexadecanol catalyzed by the lipase from Candida antarctica to investigate the effect of lipophilicity on their antiamyloidal activity assessed by the inhibitory activities toward fibrillization of amyloid β (Aβ) peptide. Among them, CA showed the highest activity at 50 μM, reducing the amyloid fibril formation of Aβ to 34.4 ± 6.8%. The antiamyloidal effects of HCAs were enhanced by esterification with alkyl alcohols, and the longer alkyl chain tended to be more effective except for SA. Aβ fibril formation was suppressed by the hexadecyl ester of CA, which was reduced to 8.8 ± 2.3%. In contrast, those of octyl and butyl esters were 19.3 ± 2.3% and 41.6 ± 6.1%, respectively. These results show that lipophilicity plays an important role in the antiamyloidal activities of esterified phenolic compounds. © 2013 International Union of Biochemistry and Molecular Biology, Inc.

  14. Adrenal and liver in normal and cld/cld mice synthesize and secrete hepatic lipase, but the lipase is inactive in cld/cld mice.

    PubMed

    Schultz, C J; Blanchette-Mackie, E J; Scow, R O

    2000-02-01

    Combined lipase deficiency (cld) is a recessive mutation in mice that causes a severe lack of lipoprotein lipase (LPL) and hepatic lipase (HL) activities, hyperlipemia, and death within 3 days after birth. Earlier studies showed that inactive LPL and HL were synthesized by cld/cld tissues and that LPL synthesized by cld/cld brown adipocytes was retained in their ER. We report here a study of HL in liver, adrenal, and plasma of normal newborn and cld/cld mice. Immunofluorescence studies showed HL was present in extracellular space, but not in cells, in liver and adrenal of both normal and cld/cld mice. When protein secretion was blocked with monensin, HL was retained intracellularly in liver cell cultures and in incubated adrenal tissues of both groups of mice. These findings demonstrated that HL was synthesized and secreted by liver and adrenal cells in normal newborn and cld/cld mice. HL activities in liver, adrenal, and plasma in cld/cld mice were very low, <8% of that in normal newborn mice, indicating that HL synthesized and secreted by cld/cld cells was inactive. Livers of both normal newborn and cld/cld mice synthesized LPL, but the level of LPL activity in cld/cld liver was very low, <9% of that in normal liver. Immunofluorescence studies showed that LPL was present intracellularly in liver of cld/cld mice, indicating that LPL was synthesized but not secreted by cld/cld liver cells. Immunofluorescent LPL was not found in normal newborn liver cells unless the cells were treated with monensin, thus demonstrating that normal liver cells synthesized and secreted LPL. Livers of both groups of mice contained an unidentified alkaline lipase activity which accounted for 34-54% of alkaline lipase activity in normal and 65% of that in cld/cld livers. Our findings indicate that liver and adrenal cells synthesized and secreted HL in both normal newborn and cld/cld mice, but the lipase was inactive in cld/cld mice. That cld/cld liver cells secreted inactive HL while

  15. Esterification of fatty acids using nylon-immobilized lipase in n-hexane: kinetic parameters and chain-length effects.

    PubMed

    Zaidi, A; Gainer, J L; Carta, G; Mrani, A; Kadiri, T; Belarbi, Y; Mir, A

    2002-02-28

    The esterification of long-chain fatty acids in n-hexane catalyzed by nylon-immobilized lipase from Candida rugosa has been investigated. Butyl oleate (22 carbon atoms), oleyl butyrate (22 carbon atoms) and oleyl oleate (36 carbon atoms) were produced at maximum reaction rates of approximately equal to 60 mmol h(-1) g(-1) immobilized enzyme when the substrates were present in equimolar proportions at an initial concentration of 0.6 mol l(-1). The observed kinetic behavior of all the esterification reactions is found to follow a ping-pong bi-bi mechanism with competitive inhibition by both substrates. The effect of the chain-length of the fatty acids and the alcohols could be correlated to some mechanistic models, in accordance with the calculated kinetic parameters.

  16. Biocatalytic Synthesis of Flavonoid Esters by Lipases and Their Biological Benefits.

    PubMed

    de Araújo, Maria Elisa M B; Franco, Yollanda E M; Messias, Marcia C F; Longato, Giovanna B; Pamphile, João A; Carvalho, Patricia de O

    2017-01-01

    Several studies have described important biological activities of flavonoids such as coronary heart disease prevention, hepatoprotective, anti-inflammatory and anticancer activities, enzyme inhibition activity, and antibacterial, antifungal, and antiviral activities. Flavonoids show promising activity as natural plant-based antioxidants due to their antioxidant and free radical scavenging properties. However, their primary applications as antioxidants in the pharmaceutical, cosmetic, and food industries are limited because of their moderately hydrophilic nature. Enzymatic acylation of natural polyphenols with fatty acids or other acyl donors has been suggested for improving the lipophilic nature of the glycosylated flavonoids. This approach increases flavonoid solubility and stability in lipophilic systems. Acylation of flavonoids with different acyl donors may also introduce beneficial properties to the molecule, such as penetration through the cell membrane and improved antioxidant, antimicrobial, anti-inflammatory, antiproliferative, cytogenetic, and enzyme inhibition activities. Chemical methods for the synthesis of flavonoid esters lead to the formation of side products and the simultaneous decomposition of the flavonoids due to harsh reaction conditions. In contrast, biocatalytic acylation of flavonoids by lipases offers advantages associated to the wide availability of these enzymes, their low cost, chemo-, regio-, and enantioselectivity, mild condition processing and non-requirement of cofactors. This article is focused on the recent development of lipase-catalyzed synthesis of flavonoid esters and the impact of the acylation reaction on their biological activities. Georg Thieme Verlag KG Stuttgart · New York.

  17. Lipase-Secreting Bacillus Species in an Oil-Contaminated Habitat: Promising Strains to Alleviate Oil Pollution

    PubMed Central

    Lee, Li Pin; Karbul, Hudzaifah Mohamed; Citartan, Marimuthu; Gopinath, Subash C. B.; Lakshmipriya, Thangavel; Tang, Thean-Hock

    2015-01-01

    Lipases are of great interest for different industrial applications due to their diversity and versatility. Among different lipases, microbial lipases are preferable due to their broad substrate specificity, and higher stability with lower production costs compared to the lipases from plants and animals. In the past, a vast number of bacterial species have been reported as potential lipases producers. In this study, the lipases-producing bacterial species were isolated from an oil spillage area in the conventional night market. Isolated species were identified as Bacillus species by biochemical tests which indicate their predominant establishment, and further screened on the agar solid surfaces using lipid and gelatin as the substrates. Out of the ten strains tested, four potential strains were subjected to comparison analysis of the lipolytic versus proteolytic activities. Strain 10 exhibited the highest lipolytic and proteolytic activity. In all the strains, the proteolytic activity is higher than the lipolytic activity except for strain 8, suggesting the possibility for substrate-based extracellular gene induction. The simultaneous secretion of both the lipase and protease is a mean of survival. The isolated bacterial species which harbour both lipase and protease enzymes could render potential industrial-based applications and solve environmental issues. PMID:26180812

  18. Hydrolysis of triacetin catalyzed by immobilized lipases: effect of the immobilization protocol and experimental conditions on diacetin yield.

    PubMed

    Hernandez, Karel; Garcia-Verdugo, Eduardo; Porcar, Raul; Fernandez-Lafuente, Roberto

    2011-05-06

    The effect of the immobilization protocol and some experimental conditions (pH value and presence of acetonitrile) on the regioselective hydrolysis of triacetin to diacetin catalyzed by lipases has been studied. Lipase B from Candida antarctica (CALB) and lipase from Rhizomucor miehei (RML) were immobilized on Sepabeads (commercial available macroporous acrylic supports) activated with glutaraldehyde (covalent immobilization) or octadecyl groups (adsorption via interfacial activation). All the biocatalysts accumulated diacetin. Covalently immobilized RML was more active towards rac-methyl mandelate than the adsorbed RML. However, this covalent RML preparation presented the lowest activity towards triacetin. For this reason, this preparation was discarded as biocatalyst for this reaction. At pH 7, acyl migration occurred giving a mixture of 1,2 and 1,3 diacetin, but at pH 5.5, only 1,2 diacetin was produced. Yields were improved at acidic pH values and in the presence of 20% acetonitrile (to over 95%). RML immobilized on octadecyl Sepabeads was proposed as optimal preparation, mainly due to its higher specific activity. Each enzyme preparation presented very different properties. Moreover, changes in the reaction conditions affected the various immobilized enzymes in a different way. Copyright © 2011 Elsevier Inc. All rights reserved.

  19. Proteinuria and lipoprotein lipase activity in Miniature schnauzer dogs with and without hypertriglyceridemia

    PubMed Central

    Furrow, E.; Jaeger, J.Q.; Parker, V.J.; Hinchcliff, K.W.; Johnson, S.E.; Murdoch, S.J.; de Boer, I.H.; Sherding, R.G.; Brunzell, J.D.

    2016-01-01

    Spontaneous hyperlipidemia in rats causes glomerular disease. Idiopathic hypertriglyceridemia (HTG) is prevalent in Miniature schnauzers, but its relationship with proteinuria is unknown. Decreased activity of major lipid metabolism enzymes, lipoprotein lipase (LPL) and hepatic lipase (HL), may play a role in the cyclic relationship between hyperlipidemia and proteinuria. These enzymes have also not been previously investigated in Miniature shnauzers. The aims of this study were to determine the relationship between HTG and proteinuria in Miniature schnauzers and to measure LPL and HL activities in a subset of dogs. Fifty-seven Miniature schnauzers were recruited (34 with and 23 without HTG). Fasting serum triglyceride concentrations and urine protein-to-creatinine ratios (UPC) were measured in all dogs, and LPL and HL activities were determined in 17 dogs (8 with and 9 without HTG). There was a strong positive correlation between triglyceride concentration and UPC (r = 0.77–0.83, P < 0.001). Proteinuria (UPC ≥ 0.5) was present in 60% of dogs with HTG and absent from all dogs without HTG (P < 0.001). Proteinuric dogs were not azotemic or hypoalbuminemic. Dogs with HTG had a 65% reduction in LPL activity relative to dogs without HTG (P < 0.001); HL activity did not differ. Proteinuria occurs with HTG in Miniature schnauzers and could be due to lipid-induced glomerular injury. Reduced LPL activity may contribute to the severity of HTG, but further assay validation is required. PMID:27256031

  20. Light harvesting amphiphiles boost the performance of lipase-based washing formulations.

    PubMed

    Díaz Blanco, Carlos; Trifonov, Anatoli; Georgiev, George; Tzanov, Tzanko

    2012-08-10

    One of the major industrial uses of lipases is as active agent in bio-based washing formulations. Current methods to improve lipase stability in detergent formulations usually entail a decrease in the enzymatic activity, thus lowering the general performance of the detergent. This work proposes an alternative approach for enzyme stabilization and activity enhancement based on the application of amphiphilic light-harvesting copolymers, called here photozymes. The biopolymer-based chitosan-Rose Bengal and the synthetic poly(SSS(0.75)-co-VBA(0.24)-co-VB/hematoporphyrin(0.01)) photozymes were used as boosting agents in washing formulations containing lipase. Organic stain removal from textiles was improved by 33% at 25°C. This cleaning enhancement was attributed to the increase of lipase activity due to interfacial activation in presence of photozymes, along with prevention of dirt re-deposition on the cleaned surfaces. Although both photozymes improved lipase activity, chitosan-Rose Bengal photozyme performed better at pHs above 9 while at pHs below this value SSS-VBA-VB/HP was the most effective. Dynamic light scattering, zeta-potential measurements, fluorescence spectroscopy and FRET experiments confirmed the pseudomicellar conformation and hosting capacity of the photozymes in aqueous media leading to improved dirt solubilization and emulsification. Moreover, the photocatalytic activity of the photozyme allowed for a de-coloration of the waste washing liquor upon UV irradiation. Copyright © 2012 Elsevier Inc. All rights reserved.

  1. A quantitative assay measuring the function of lipase maturation factor 1

    PubMed Central

    Yin, Fen; Doolittle, Mark H.; Péterfy, Miklós

    2009-01-01

    Newly synthesized lipoprotein lipase (LPL) and related members of the lipase gene family require an endoplasmic reticulum maturation factor for attainment of enzyme activity. This factor has been identified as lipase maturation factor 1 (Lmf1), and mutations affecting its function and/or expression result in combined lipase deficiency (cld) and hypertriglyceridemia. To assess the functional impact of Lmf1 sequence variations, both naturally occurring and induced, we report the development of a cell-based assay using LPL activity as a quantitative reporter of Lmf1 function. The assay uses a cell line homozygous for the cld mutation, which renders endogenous Lmf1 nonfunctional. LPL transfected into the mutant cld cell line fails to attain activity; however, cotransfection of LPL with wild-type Lmf1 restores its ability to support normal lipase maturation. In this report, we describe optimized conditions that ensure the detection of a complete range of Lmf1 function (full, partial, or complete loss of function) using LPL activity as the quantitative reporter. To illustrate the dynamic range of the assay, we tested several novel mutations in mouse Lmf1. Our results demonstrate the ability of the assay to detect and analyze Lmf1 mutations having a wide range of effects on Lmf1 function and protein expression. PMID:19471043

  2. Enzymatic Synthesis of Glyserol-Coconut Oil Fatty Acid and Glycerol-Decanoic Acis Ester as Emulsifier and Antimicrobial Agents Using Candida rugosa Lipase EC 3.1.1.3

    NASA Astrophysics Data System (ADS)

    Handayani, Sri; Putri, Ayu Tanissa Tamara; Setiasih, Siswati; Hudiyono, Sumi

    2018-01-01

    In this research, enzymatic esterification was carried out between glycerol and fatty acid from coconut oil and decanoic acid using n-hexane as solvent. In this reaction Candida rugosa lipase was used as biocatalyst. Optimization esterification reaction was carried out for parameter of the substrate ratio. The mmol ratio between fatty acid and glycerol were used are 1:1, 1:2, 1:3, and 1: 4. The highest conversion percentage obtained at the mole ratio of 1: 4 with the value of 78.5% for the glycerol-decanoic acid ester and 55.4% for the glycerol coconut oil fatty acid ester. Esterification products were characterized by FT-IR. The FT-IR spectrum showed that the ester bond was formed as indicated by the wave number 1750-1739 cm-1. The esterification products were then examined by simple emulsion test and was proved to be an emulsifier. The glycerol-coconut oil fatty acid ester produced higher stability emulsion compare with glycerol decanoic ester. The antimicrobial activity assay using disc diffusion method showed that both glycerol-coconut oil fatty acid ester and glycerol-decanoic ester had the ability inhibiting the growth of Propionibacterium acnes and Staphylococcus epidermidis. Glycerol-decanoic ester shows higher antimicrobial activity than glycerol-coconut oil fatty acid ester.

  3. Yeast Kluyveromyces lactis as host for expression of the bacterial lipase: cloning and adaptation of the new lipase gene from Serratia sp.

    PubMed

    Šiekštelė, Rimantas; Veteikytė, Aušra; Tvaska, Bronius; Matijošytė, Inga

    2015-10-01

    Many microbial lipases have been successfully expressed in yeasts, but not in industrially attractive Kluyveromyces lactis, which among other benefits can be cultivated on a medium supplemented with whey--cheap and easily available industrial waste. A new bacterial lipase from Serratia sp. was isolated and for the first time expressed into the yeast Kluyveromyces lactis by heterologous protein expression system based on a strong promoter of Kluyveromyces marxianus triosephosphate isomerase gene and signal peptide of Kluyveromyces marxianus endopolygalacturonase gene. In addition, the bacterial lipase gene was synthesized de novo by taking into account a codon usage bias optimal for K. lactis and was expressed into the yeast K. lactis also. Both resulting strains were characterized by high output level of the target protein secreted extracellularly. Secreted lipases were characterized for activity and stability.

  4. Novel surface-active oligofructose fatty acid mono-esters by enzymatic esterification.

    PubMed

    van Kempen, Silvia E H J; Boeriu, Carmen G; Schols, Henk A; de Waard, Pieter; van der Linden, Erik; Sagis, Leonard M C

    2013-06-01

    This article describes the synthesis of a series of oligofructose monoesters with fatty acids of different chain length (C8, C12, C16 and C18) to obtain food-grade surfactants with a range of amphiphilicity. Reactions were performed in a mixture of DMSO/Bu(t)OH (10/90 v/v) at 60°C and catalysed by immobilised Candida antarctica lipase B. MALDI-TOF-MS analysis showed that the crude reaction products were mixtures of unmodified oligofructose and mostly mono-esters. The conversion into mono-esters increased with the length of the fatty acid chain, reflecting the specificity of the lipase towards more lipophilic substrates. Reverse phase solid phase extraction was used to fractionate the products, which lead to sufficient purity (>93%) of the fatty acid esters for functionality testing. It was shown that derivatives of longer (C16 and C18) fatty acids were more efficient in lowering surface tension and gave a much higher dilatational modulus than derivatives of the shorter (C8 and C12) fatty acids. Copyright © 2012 Elsevier Ltd. All rights reserved.

  5. Clinical Features of Lysosomal Acid Lipase Deficiency.

    PubMed

    Burton, Barbara K; Deegan, Patrick B; Enns, Gregory M; Guardamagna, Ornella; Horslen, Simon; Hovingh, Gerard K; Lobritto, Steve J; Malinova, Vera; McLin, Valerie A; Raiman, Julian; Di Rocco, Maja; Santra, Saikat; Sharma, Reena; Sykut-Cegielska, Jolanta; Whitley, Chester B; Eckert, Stephen; Valayannopoulos, Vassili; Quinn, Anthony G

    2015-12-01

    The aim of this study was to characterize key clinical manifestations of lysosomal acid lipase deficiency (LAL D) in children and adults. Investigators reviewed medical records of LAL D patients ages ≥5 years, extracted historical data, and obtained prospective laboratory and imaging data on living patients to develop a longitudinal dataset. A total of 49 patients were enrolled; 48 had confirmed LAL D. Mean age at first disease-related abnormality was 9.0 years (range 0-42); mean age at diagnosis was 15.2 years (range 1-46). Twenty-nine (60%) were male patients, and 27 (56%) were <20 years of age at the time of consent/assent. Serum transaminases were elevated in most patients with 458 of 499 (92%) of alanine aminotransferase values and 265 of 448 (59%) of aspartate aminotransferase values above the upper limit of normal. Most patients had elevated low-density lipoprotein (64% patients) and total cholesterol (63%) at baseline despite most being on lipid-lowering therapies, and 44% had high-density lipoprotein levels below the lower limit of normal. More than half of the patients with liver biopsies (n = 31, mean age 13 years) had documented evidence of steatosis (87%) and/or fibrosis (52%). Imaging assessments revealed that the median liver volume was ∼1.15 multiples of normal (MN) and median spleen volume was ∼2.2 MN. Six (13%) patients had undergone a liver transplant (ages 9-43.5 years). This study provides the largest longitudinal case review of patients with LAL D and confirms that LAL D is predominantly a pediatric disease causing early and progressive hepatic dysfunction associated with dyslipidemia that often leads to liver failure and transplantation.

  6. Inhibition of pancreatic lipase and amylase by extracts of different spices and plants.

    PubMed

    Sellami, Mohamed; Louati, Hanen; Kamoun, Jannet; Kchaou, Ali; Damak, Mohamed; Gargouri, Youssef

    2017-05-01

    The aim of this study is to search new anti-obesity and anti-diabetic agents from plant and spices crude extracts as alternative to synthetic drugs. The inhibitory effect of 72 extracts was evaluated, in vitro, on lipase and amylase activities. Aqueous extracts of cinnamon and black tea exhibited an appreciable inhibitory effect on pancreatic amylase with IC 50 values of 18 and 87 μg, respectively. Aqueous extracts of cinnamon and mint showed strong inhibitory effects against pancreatic lipase with IC 50 of 45 and 62 μg, respectively. The presence of bile salts and colipase or an excess of interface failed to restore the lipase activity. Therefore, the inhibition of pancreatic lipase, by extracts of spices and plants, belongs to an irreversible inhibition. Crude extract of cinnamon showed the strongest anti-lipase and anti-amylase activities which offer a prospective therapeutic approach for the management of diabetes and obesity.

  7. Nine Different Chemical Species and Action Mechanisms of Pancreatic Lipase Ligands Screened Out from Forsythia suspensa Leaves All at One Time.

    PubMed

    Chen, Tinggui; Li, Yayun; Zhang, Liwei

    2017-05-12

    It is difficult to screen out as many active components as possible from natural plants all at one time. In this study, subfractions of Forsythia suspensa leaves were firstly prepared; then, their inhibitive abilities on pancreatic lipase were tested; finally, the highest inhibiting subfraction was screened by self-made immobilized pancreatic lipase. Results showed that nine ligands, including eight inhibitors and one promotor, were screened out all at one time. They were three flavonoids (rutin, IC 50 : 149 ± 6.0 μmol/L; hesperidin, 52.4 μmol/L; kaempferol-3- O -rutinoside, isolated from F. suspensa leaves for the first time, IC 50 notably reached 2.9 ± 0.5 μmol/L), two polyphenols (chlorogenic acid, 3150 ± 120 μmol/L; caffeic acid, 1394 ± 52 μmol/L), two lignans (phillyrin, promoter; arctigenin, 2129 ± 10.5 μmol/L), and two phenethyl alcohol (forsythiaside A, 2155 ± 8.5 μmol/L; its isomer). Their action mechanisms included competitive inhibition, competitive promotion, noncompetitive inhibition, and uncompetitive inhibition. In sum, using the appropriate methods, more active ingredients can be simply and quickly screened out all at one time from a complex natural product system. In addition, F. suspensa leaves contain numerous inhibitors of pancreatic lipase.

  8. Macaúba (Acrocomia aculeata) cake from biodiesel processing: a low-cost substrate to produce lipases from Moniliella spathulata R25L270 with potential application in the oleochemical industry.

    PubMed

    Souza, Lívia T A; Oliveira, Jamil S; Rodrigues, Marina Q R B; dos Santos, Vera L; Pessela, Benevides C; Resende, Rodrigo R

    2015-06-16

    Biodiesel industry wastes were evaluated as supplements for lipase production by Moniliella spathulata R25L270, which is newly identified yeast with great lipolytic potential. Macaúba cake (MC), used for the first time in this work as inducer to produce lipases, and residual oil (RO) were mixed to maximise enzyme production. The lipase secreted was biochemically characterised. The best ratio for the mixture (MC:RO) was 0.66:0.34 and the fitted values for lipase activity and total protein concentration were 0.98 U mL(-1) and 0.356 mg mL(-1), respectively. Maximum activity obtained (2.47 U mL(-1)) was achieved at 31.5°C and pH 6.7, and the enzyme was stable in this condition. A novel enzyme was purified and identified for the first time by mass spectrometry. The lipase efficiently hydrolysed different natural oils and exhibited selectivity in the production of eicosapentaenoic acid from fish oil. The use of MC and RO as a supplement to produce the new lipase from M. spathulata R25L270 may be one alternative for reducing lipase production costs and simultaneously adding value to biodiesel industry residues. The potential application of the lipase in the oleochemical industry was demonstrated by its pH and temperature stabilities and selective hydrolysis.

  9. [Industrial application of lipases].

    PubMed

    Bancerz, Renata

    2017-01-01

    The ability of lipases to perform specific reactions of transformation (biotransformation) makes these enzymes a useful tool used in many syntheses, for example: in the production of detergents, cosmetics, biosurfactants, in the oil-chemical, paper, dairy, food or pharmaceutical industries. Lipases are ubiquitous enzymes but only lipases produced by microorganisms are important for industrial applications due to their wide variety of properties such as stability in organic solvents, action under mild conditions, high substrate specificity and region- and enantioselectivity, as well as the relatively simple methods of their production in fermentors and recovery from the culture medium. This paper reviews the latest achievements in the production of lipases in the submerged fermentation and solid state fermentation using waste products from the agricultural industry. In addition, new applications of lipases were described, including those for the synthesis of biopolymers and biodiesel and for the production of enantiomeric pharmaceuticals, agrochemicals and flavoring compounds.

  10. Glycyrrhizic acid prevents high calorie diet-induced metabolic aberrations despite the suppression of peroxisome proliferator-activated receptor γ expression.

    PubMed

    Cheng, Hong Sheng; Yaw, Hui Ping; Ton, So Ha; Choy, Siew Mei; Kong, Joana Magdelene Xiao Fang; Abdul Kadir, Khalid

    2016-09-01

    To investigate the effects of glycyrrhizic acid supplementation on glucose and lipid metabolism in rodents consuming a high-fat, high-sucrose diet. Twenty-four male, 8-week old Sprague Dawley rats with an initial weight of 160 to 200 g were randomised into three groups (n = 6 for each group): groups A (standard rat chow), B (high-fat, high-sucrose diet), and C (high-fat, high-sucrose diet + 100 mg/kg/d of glycyrrhizic acid via oral administration). The rats were treated accordingly for 4 wk. Glycaemic parameters, lipid profile, stress hormones, and adiponectin levels were measured after the treatment. Relative gene expressions of peroxisome proliferator-activated receptor α and γ, lipoprotein lipase as well as gluconeogenic enzymatic activities in different tissues were also determined. Consumption of high-fat, high-sucrose diet triggered hyperglycaemia, insulin resistance, and dyslipidemia, which were effectively attenuated by supplementation with glycyrrhizic acid. Glycyrrhizic acid supplementation also effectively reduced circulating adrenaline, alleviated gluconeogenic enzymes overactivity, and promoted the upregulation of lipoprotein lipase expression in the cardiomyocytes and skeletal muscles. A high calorie diet also triggered hypoadiponectinaemia and suppression of peroxisome proliferator-activated receptor γ expression, which did not improve with glycyrrhizic acid treatment. Supplementation with glycyrrhizic acid could alleviate high calorie diet-induced glucose and lipid metabolic dysregulations by reducing circulatory stress hormones, normalizing gluconeogenic enzyme activities, and elevating muscular lipid uptake. The beneficial effects of these bioactivities outweighed the adverse effects caused by diet-induced repression of peroxisome proliferator-activated receptor γ expression, resulting in the maintenance of lipid and glucose homeostasis. Copyright © 2016 Elsevier Inc. All rights reserved.

  11. Phenolic antioxidants in some Vigna species of legumes and their distinct inhibitory effects on α-glucosidase and pancreatic lipase activities.

    PubMed

    Sreerama, Yadahally N; Takahashi, Yoko; Yamaki, Kohji

    2012-09-01

    Phenolic extracts of 4 Vigna species of legumes (mung bean, moth bean, and black and red varieties of adzuki beans) were evaluated for phenolic contents, antioxidant activities, and inhibitory properties against α-glucosidase and pancreatic lipase. Results showed that adzuki bean varieties contain higher phenolic indexes than mung bean and moth beans. Adzuki bean (black) variety was found to be the most active 2,2'-diphenyl-1-picrylhydrazyl and superoxide anion scavenger. However, the hydrogen peroxide scavenging and metal chelating abilities were significantly higher in adzuki bean (red) variety. Mung bean exhibited least antioxidant activities in all the methods tested. Phenolic extracts from these legumes also showed distinct variations in the inhibition of enzymes associated with hyperglycemia and hyperlipidemia. Inhibitory activities of all the extracts against lipase were found to be more potent than α-glucosidase. Although, α-glucosidase inhibitory activity was superior in the black variety of adzuki bean (IC(50,) 26.28 mg/mL), both adzuki bean varieties (black and red) along with moth bean showed strong inhibitory activities on lipase with no significant difference in their IC(50) values (7.32 to 9.85 mg/mL). These results suggest that Vigna species of legumes are potential source of antioxidant phenolics and also great sources of strong natural inhibitors for α-glucosidase and lipase activities. This information may help for effective utilization of these legumes as functional food ingredients for promoting health. Practical Application:  Vigna species of legumes are good sources of phenolic antioxidants and strong natural inhibitors of enzymes associated with diabetes and obesity. Therefore, utilization of these legumes in the development of functional foods with increased therapeutic value would be a significant step toward health promotion and wellness. © 2012 Institute of Food Technologists®

  12. Enzyme-assisted extraction of phenolics from winemaking by-products: Antioxidant potential and inhibition of alpha-glucosidase and lipase activities.

    PubMed

    de Camargo, Adriano Costa; Regitano-d'Arce, Marisa Aparecida Bismara; Biasoto, Aline Camarão Telles; Shahidi, Fereidoon

    2016-12-01

    Phenolics in food and agricultural processing by-products exist in the soluble and insoluble-bound forms. The ability of selected enzymes in improving the extraction of insoluble-bound phenolics from the starting material (experiment I) or the residues containing insoluble-bound phenolics (experiment II) were evaluated. Pronase and Viscozyme improved the extraction of insoluble-bound phenolics as evaluated by total phenolic content, antioxidant potential as determined by ABTS and DPPH assays, and hydroxyl radical scavenging capacity, reducing power as well as evaluation of inhibition of alpha-glucosidase and lipase activities. Viscozyme released higher amounts of gallic acid, catechin, and prodelphinidin dimer A compared to Pronase treatment. Furthermore, p-coumaric and caffeic acids, as well as procyanidin dimer B, were extracted with Viscozyme but not with Pronase treatment. Solubility plays an important role in the bioavailability of phenolic compounds, hence this study may assist in better exploitation of phenolics from winemaking by-products as functional food ingredients and/or supplements. Copyright © 2016. Published by Elsevier Ltd.

  13. Glycolipid Biosurfactants Activate, Dimerize, and Stabilize Thermomyces lanuginosus Lipase in a pH-Dependent Fashion.

    PubMed

    Madsen, Jens Kvist; Kaspersen, Jørn Døvling; Andersen, Camilla Bertel; Nedergaard Pedersen, Jannik; Andersen, Kell Kleiner; Pedersen, Jan Skov; Otzen, Daniel E

    2017-08-15

    We present a study of the interactions between the lipase from Thermomyces lanuginosus (TlL) and the two microbially produced biosurfactants (BSs), rhamnolipid (RL) and sophorolipid (SL). Both RL and SL are glycolipids; however, RL is anionic, while SL is a mixture of anionic and non-ionic species. We investigate the interactions of RL and SL with TlL at pH 6 and 8 and observe different effects at the two pH values. At pH 8, neither RL nor SL had any major effect on TlL stability or activity. At pH 6, in contrast, both surfactants increase TlL's thermal stability and fluorescence and activity measurements indicate interfacial activation of TlL, resulting in 3- and 6-fold improved activity in SL and RL, respectively. Nevertheless, isothermal titration calorimetry reveals binding of only a few BS molecules per lipase. Size-exclusion chromatography and small-angle X-ray scattering suggest formation of TlL dimers with binding of small amounts of either RL or SL at the dimeric interface, forming an elongated complex. We conclude that RL and SL are compatible with TlL and constitute promising green alternatives to traditional surfactants.

  14. Closed and open conformations of the lid domain induce different patterns of human pancreatic lipase antigenicity and immunogenicity.

    PubMed

    Halimi, Hubert; De Caro, Josiane; Carrière, Frédéric; De Caro, Alain

    2005-12-01

    Epitope mapping was performed on human pancreatic lipase (HPL) using the SPOTscan method. A set of 146 short (12 amino acid residues) synthetic overlapping peptides covering the entire amino acid sequence of HPL were used to systematically assess the immunoreactivity of antisera raised in rabbits against native HPL, HPL without a lid (HPL(-lid)) and HPL covalently inhibited by diethyl p-nitrophenyl phosphate (DP-HPL). In the latter form of HPL, the lid domain controlling the access to the active site was assumed to exist in the open conformation. All the anti-lipase sera were tested in a direct ELISA, anti-HPL serum showing the greatest antibody titer. Although from the structural point of view, the differences between the various forms of HPL were restricted to the lid domain, differences in the antigenic properties of HPL were observed with the SPOTscan method, and the anti-DP-HPL antibodies showed the strongest reactivity. Most of the peptide stretches recognized included amino acid residues which are accessible at the surface of the lipase, except for those located near the active site. Two small peptides (T173-P180, V199-A207) were identified in the vicinity of the active site, their antipeptide antibodies were produced and their reactivity towards the various forms of HPL was tested in a double sandwich ELISA. No reactivity was observed under these conditions. Two antipeptide antibodies directed against two other selected peptides, P208-V221 (belonging to the beta9 loop) and I245-F258 (belonging to the lid domain) were prepared and found to react much more strongly with DP-HPL than with HPL or HPL(-lid) in a double sandwich ELISA. These antibodies should provide useful tools for monitoring the conformational changes taking place during the opening of the HPL lid domain.

  15. Screening Brazilian Macrophomina phaseolina isolates for alkaline lipases and other extracellular hydrolases.

    PubMed

    Schinke, Claudia; Germani, José C

    2012-03-01

    Macrophomina phaseolina, phylum Ascomycota, is a phytopathogenic fungus distributed worldwide in hot dry areas. There are few studies on its secreted lipases and none on its colony radial growth rate, an indicator of fungal ability to use nutrients for growth, on media other than potato-dextrose agar. In this study, 13 M. phaseolina isolates collected in different Brazilian regions were screened for fast-growth and the production of hydrolases of industrial interest, especially alkaline lipases. Hydrolase detection and growth rate determination were done on citric pectin, gelatin, casein, soluble starch, and olive oil as substrates. Ten isolates were found to be active on all substrates tested. The most commonly detected enzymes were pectinases, amylases, and lipases. The growth rate on pectin was significantly higher (P < 0.05), while the growth rates on the different media identified CMM 2105, CMM 1091, and PEL as the fastest-growing isolates. The lipase activity of four isolates grown on olive oil was followed for 4 days by measuring the activity in the cultivation broth. The specific lipolytic activity of isolate PEL was significantly higher at 96 h (130 mU mg protein(-1)). The broth was active at 37 °C, pH 8, indicating the potential utility of the lipases of this isolate in mild alkaline detergents. There was a strong and positive correlation (0.86) between radial growth rate and specific lipolytic activity.

  16. Toward the establishment of standardized in vitro tests for lipid-based formulations. 5. Lipolysis of representative formulations by gastric lipase.

    PubMed

    Bakala-N'Goma, Jean-Claude; Williams, Hywel D; Sassene, Philip J; Kleberg, Karen; Calderone, Marilyn; Jannin, Vincent; Igonin, Annabel; Partheil, Anette; Marchaud, Delphine; Jule, Eduardo; Vertommen, Jan; Maio, Mario; Blundell, Ross; Benameur, Hassan; Müllertz, Anette; Pouton, Colin W; Porter, Christopher J H; Carrière, Frédéric

    2015-04-01

    Lipid-based formulations (LBF) are substrates for digestive lipases and digestion can significantly alter their properties and potential to support drug absorption. LBFs have been widely examined for their behaviour in the presence of pancreatic enzymes. Here, the impact of gastric lipase on the digestion of representative formulations from the Lipid Formulation Classification System has been investigated. The pHstat technique was used to measure the lipolysis by recombinant dog gastric lipase (rDGL) of eight LBFs containing either medium (MC) or long (LC) chain triglycerides and a range of surfactants, at various pH values [1.5 to 7] representative of gastric and small intestine contents under both fasting and fed conditions. All LBFs were hydrolyzed by rDGL. The highest specific activities were measured at pH 4 with the type II and IIIA MC formulations that contained Tween®85 or Cremophor EL respectively. The maximum activity on LC formulations was recorded at pH 5 for the type IIIA-LC formulation. Direct measurement of LBF lipolysis using the pHstat, however, was limited by poor LC fatty acid ionization at low pH. Since gastric lipase initiates lipid digestion in the stomach, remains active in the intestine and acts on all representative LBFs, its implementation in future standardized in vitro assays may be beneficial. At this stage, however, routine use remains technically challenging.

  17. Development of a high-throughput liquid state assay for lipase activity using natural substrates and rhodamine B.

    PubMed

    Zottig, Ximena; Meddeb-Mouelhi, Fatma; Beauregard, Marc

    2016-03-01

    A fluorescence-based assay for the determination of lipase activity using rhodamine B as an indicator, and natural substrates such as olive oil, is described. It is based on the use of a rhodamine B-natural substrate emulsion in liquid state, which is advantageous over agar plate assays. This high-throughput method is simple and rapid and can be automated, making it suitable for screening and metagenomics application. Reaction conditions such as pH and temperature can be varied and controlled. Using triolein or olive oil as a natural substrate allows monitoring of lipase activity in reaction conditions that are closer to those used in industrial settings. The described method is sensitive over a wide range of product concentrations and offers good reproducibility. Copyright © 2015 Elsevier Inc. All rights reserved.

  18. The immobilization of lipase on PVDF-co-HFP membrane

    NASA Astrophysics Data System (ADS)

    Kayhan, Naciye; Eyüpoǧlu, Volkan; Adem, Şevki

    2016-04-01

    Lipase is an enzyme having a lot of different industrial applications such as biodiesel production, biopolymer synthesis, enantiopure pharmaceutical productions, agrochemicals, etc. Its immobilized form on different substances is more conventional and useful than its free form. Supporting material was prepared using PVDF-co-HFP in laboratory conditions and attached 1,4-diaminobutane (DA) and epichlorohydrin (EPI) ligands to the membrane to immobilize lipase enzyme. The immobilization conditions such as enzyme amount, pH, the concentration of salt, thermal stability and activity were stabilized for our experimental setup. Then, biochemical characterizations were performed on immobilized lipase PVDF-co-HFP regarding optimal pH activity, temperature and thermal stability. Also, the desorption ratios of immobilized enzyme in two different pathway were investigated to confirm immobilization stability for 24 hours.

  19. Angiopoietin-like 4 mediates PPAR delta effect on lipoprotein lipase-dependent fatty acid uptake but not on beta-oxidation in myotubes.

    PubMed

    Robciuc, Marius R; Skrobuk, Paulina; Anisimov, Andrey; Olkkonen, Vesa M; Alitalo, Kari; Eckel, Robert H; Koistinen, Heikki A; Jauhiainen, Matti; Ehnholm, Christian

    2012-01-01

    Peroxisome proliferator-activated receptor (PPAR) delta is an important regulator of fatty acid (FA) metabolism. Angiopoietin-like 4 (Angptl4), a multifunctional protein, is one of the major targets of PPAR delta in skeletal muscle cells. Here we investigated the regulation of Angptl4 and its role in mediating PPAR delta functions using human, rat and mouse myotubes. Expression of Angptl4 was upregulated during myotubes differentiation and by oleic acid, insulin and PPAR delta agonist GW501516. Treatment with GW501516 or Angptl4 overexpression inhibited both lipoprotein lipase (LPL) activity and LPL-dependent uptake of FAs whereas uptake of BSA-bound FAs was not affected by either treatment. Activation of retinoic X receptor (RXR), PPAR delta functional partner, using bexarotene upregulated Angptl4 expression and inhibited LPL activity in a PPAR delta dependent fashion. Silencing of Angptl4 blocked the effect of GW501516 and bexarotene on LPL activity. Treatment with GW501516 but not Angptl4 overexpression significantly increased palmitate oxidation. Furthermore, Angptl4 overexpression did not affect the capacity of GW501516 to increase palmitate oxidation. Basal and insulin stimulated glucose uptake, glycogen synthesis and glucose oxidation were not significantly modulated by Angptl4 overexpression. Our findings suggest that FAs-PPARdelta/RXR-Angptl4 axis controls the LPL-dependent uptake of FAs in myotubes, whereas the effect of PPAR delta activation on beta-oxidation is independent of Angptl4.

  20. Angiopoietin-Like 4 Mediates PPAR Delta Effect on Lipoprotein Lipase-Dependent Fatty Acid Uptake but Not on Beta-Oxidation in Myotubes

    PubMed Central

    Robciuc, Marius R.; Skrobuk, Paulina; Anisimov, Andrey; Olkkonen, Vesa M.; Alitalo, Kari; Eckel, Robert H.; Koistinen, Heikki A.; Jauhiainen, Matti; Ehnholm, Christian

    2012-01-01

    Peroxisome proliferator-activated receptor (PPAR) delta is an important regulator of fatty acid (FA) metabolism. Angiopoietin-like 4 (Angptl4), a multifunctional protein, is one of the major targets of PPAR delta in skeletal muscle cells. Here we investigated the regulation of Angptl4 and its role in mediating PPAR delta functions using human, rat and mouse myotubes. Expression of Angptl4 was upregulated during myotubes differentiation and by oleic acid, insulin and PPAR delta agonist GW501516. Treatment with GW501516 or Angptl4 overexpression inhibited both lipoprotein lipase (LPL) activity and LPL-dependent uptake of FAs whereas uptake of BSA-bound FAs was not affected by either treatment. Activation of retinoic X receptor (RXR), PPAR delta functional partner, using bexarotene upregulated Angptl4 expression and inhibited LPL activity in a PPAR delta dependent fashion. Silencing of Angptl4 blocked the effect of GW501516 and bexarotene on LPL activity. Treatment with GW501516 but not Angptl4 overexpression significantly increased palmitate oxidation. Furthermore, Angptl4 overexpression did not affect the capacity of GW501516 to increase palmitate oxidation. Basal and insulin stimulated glucose uptake, glycogen synthesis and glucose oxidation were not significantly modulated by Angptl4 overexpression. Our findings suggest that FAs-PPARdelta/RXR-Angptl4 axis controls the LPL-dependent uptake of FAs in myotubes, whereas the effect of PPAR delta activation on beta-oxidation is independent of Angptl4. PMID:23056264

  1. Proteinuria and lipoprotein lipase activity in Miniature Schnauzer dogs with and without hypertriglyceridemia.

    PubMed

    Furrow, E; Jaeger, J Q; Parker, V J; Hinchcliff, K W; Johnson, S E; Murdoch, S J; de Boer, I H; Sherding, R G; Brunzell, J D

    2016-06-01

    Spontaneous hyperlipidemia in rats causes glomerular disease. Idiopathic hypertriglyceridemia (HTG) is prevalent in Miniature Schnauzers, but its relationship with proteinuria is unknown. Decreased activity of major lipid metabolism enzymes, lipoprotein lipase (LPL) and hepatic lipase (HL), may play a role in the cyclic relationship between hyperlipidemia and proteinuria. These enzymes have also not been previously investigated in Miniature Schnauzers. The aims of this study were to determine the relationship between HTG and proteinuria in Miniature Schnauzers and to measure LPL and HL activities in a subset of dogs. Fifty-seven Miniature Schnauzers were recruited (34 with and 23 without HTG). Fasting serum triglyceride concentrations and urine protein-to-creatinine ratios (UPC) were measured in all dogs, and LPL and HL activities were determined in 17 dogs (8 with and 9 without HTG). There was a strong positive correlation between triglyceride concentration and UPC (r = 0.77-0.83, P < 0.001). Proteinuria (UPC ≥ 0.5) was present in 60% of dogs with HTG and absent from all dogs without HTG (P < 0.001). Proteinuric dogs were not azotemic or hypoalbuminemic. Dogs with HTG had a 65% reduction in LPL activity relative to dogs without HTG (P < 0.001); HL activity did not differ. Proteinuria occurs with HTG in Miniature Schnauzers and could be due to lipid-induced glomerular injury. Reduced LPL activity may contribute to the severity of HTG, but further assay validation is required. Copyright © 2016 Elsevier Ltd. All rights reserved.

  2. Human hepatic lipase overexpression in mice induces hepatic steatosis and obesity through promoting hepatic lipogenesis and white adipose tissue lipolysis and fatty acid uptake.

    PubMed

    Cedó, Lídia; Santos, David; Roglans, Núria; Julve, Josep; Pallarès, Victor; Rivas-Urbina, Andrea; Llorente-Cortes, Vicenta; Laguna, Joan Carles; Blanco-Vaca, Francisco; Escolà-Gil, Joan Carles

    2017-01-01

    Human hepatic lipase (hHL) is mainly localized on the hepatocyte cell surface where it hydrolyzes lipids from remnant lipoproteins and high density lipoproteins and promotes their hepatic selective uptake. Furthermore, hepatic lipase (HL) is closely associated with obesity in multiple studies. Therefore, HL may play a key role on lipid homeostasis in liver and white adipose tissue (WAT). In the present study, we aimed to evaluate the effects of hHL expression on hepatic and white adipose triglyceride metabolism in vivo. Experiments were carried out in hHL transgenic and wild-type mice fed a Western-type diet. Triglyceride metabolism studies included β-oxidation and de novo lipogenesis in liver and WAT, hepatic triglyceride secretion, and adipose lipoprotein lipase (LPL)-mediated free fatty acid (FFA) lipolysis and influx. The expression of hHL promoted hepatic triglyceride accumulation and de novo lipogenesis without affecting triglyceride secretion, and this was associated with an upregulation of Srebf1 as well as the main genes controlling the synthesis of fatty acids. Transgenic mice also exhibited more adiposity and an increased LPL-mediated FFA influx into the WAT without affecting glucose tolerance. Our results demonstrate that hHL promoted hepatic steatosis in mice mainly by upregulating de novo lipogenesis. HL also upregulated WAT LPL and promoted triglyceride-rich lipoprotein hydrolysis and adipose FFA uptake. These data support the important role of hHL in regulating hepatic lipid homeostasis and confirm the broad cardiometabolic role of HL.

  3. Human hepatic lipase overexpression in mice induces hepatic steatosis and obesity through promoting hepatic lipogenesis and white adipose tissue lipolysis and fatty acid uptake

    PubMed Central

    Cedó, Lídia; Santos, David; Roglans, Núria; Julve, Josep; Pallarès, Victor; Rivas-Urbina, Andrea; Llorente-Cortes, Vicenta; Laguna, Joan Carles

    2017-01-01

    Human hepatic lipase (hHL) is mainly localized on the hepatocyte cell surface where it hydrolyzes lipids from remnant lipoproteins and high density lipoproteins and promotes their hepatic selective uptake. Furthermore, hepatic lipase (HL) is closely associated with obesity in multiple studies. Therefore, HL may play a key role on lipid homeostasis in liver and white adipose tissue (WAT). In the present study, we aimed to evaluate the effects of hHL expression on hepatic and white adipose triglyceride metabolism in vivo. Experiments were carried out in hHL transgenic and wild-type mice fed a Western-type diet. Triglyceride metabolism studies included β-oxidation and de novo lipogenesis in liver and WAT, hepatic triglyceride secretion, and adipose lipoprotein lipase (LPL)-mediated free fatty acid (FFA) lipolysis and influx. The expression of hHL promoted hepatic triglyceride accumulation and de novo lipogenesis without affecting triglyceride secretion, and this was associated with an upregulation of Srebf1 as well as the main genes controlling the synthesis of fatty acids. Transgenic mice also exhibited more adiposity and an increased LPL-mediated FFA influx into the WAT without affecting glucose tolerance. Our results demonstrate that hHL promoted hepatic steatosis in mice mainly by upregulating de novo lipogenesis. HL also upregulated WAT LPL and promoted triglyceride-rich lipoprotein hydrolysis and adipose FFA uptake. These data support the important role of hHL in regulating hepatic lipid homeostasis and confirm the broad cardiometabolic role of HL. PMID:29244870

  4. Cell-Bound Lipase and Esterase of Brevibacterium linens

    PubMed Central

    Sørhaug, Terje; Ordal, Z. John

    1974-01-01

    The activities of glycerol ester hydrolase, lipase (EC 3.1.1.3) and carboxylesterase, and esterase (EC 3.1.1.1) were determined for whole cell preparations of Brevibacterium linens by using the pH-stat assay. The culture growth liquors were inactive against the three substrates, tributyrin emulsion, triacetin, and methyl butyrate. Cells washed in water had less activity than cells washed in 5% NaCl; the ratio of activities was close to 1:2 for all strains using tributyrin emulsion as the substrate. For the esterase substrates, this relationship varied widely and was strain dependent. The ability to hydrolyze the two esterase substrates varied independently of the level of lipase activity. PMID:4824883

  5. Formation and hydrolysis of amide bonds by lipase A from Candida antarctica; exceptional features.

    PubMed

    Liljeblad, Arto; Kallio, Pauli; Vainio, Marita; Niemi, Jarmo; Kanerva, Liisa T

    2010-02-21

    Various commercial lyophilized and immobilized preparations of lipase A from Candida antarctica (CAL-A) were studied for their ability to catalyze the hydrolysis of amide bonds in N-acylated alpha-amino acids, 3-butanamidobutanoic acid (beta-amino acid) and its ethyl ester. The activity toward amide bonds is highly untypical of lipases, despite the close mechanistic analogy to amidases which normally catalyze the corresponding reactions. Most CAL-A preparations cleaved amide bonds of various substrates with high enantioselectivity, although high variations in substrate selectivity and catalytic rates were detected. The possible role of contaminant protein species on the hydrolytic activity toward these bonds was studied by fractionation and analysis of the commercial lyophilized preparation of CAL-A (Cat#ICR-112, Codexis). In addition to minor impurities, two equally abundant proteins were detected, migrating on SDS-PAGE a few kDa apart around the calculated size of CAL-A. Based on peptide fragment analysis and sequence comparison both bands shared substantial sequence coverage with CAL-A. However, peptides at the C-terminal end constituting a motile domain described as an active-site flap were not identified in the smaller fragment. Separated gel filtration fractions of the two forms of CAL-A both catalyzed the amide bond hydrolysis of ethyl 3-butanamidobutanoate as well as the N-acylation of methyl pipecolinate. Hydrolytic activity towards N-acetylmethionine was, however, solely confined to the fractions containing the truncated form of CAL-A. These fractions were also found to contain a trace enzyme impurity identified in sequence analysis as a serine carboxypeptidase. The possible role of catalytic impurities versus the function of CAL-A in amide bond hydrolysis is further discussed in the paper.

  6. Medium-chain versus long-chain triacylglycerol emulsion hydrolysis by lipoprotein lipase and hepatic lipase: Implications for the mechanisms of lipase action

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Deckelbaum, R.J.; Hamilton, J.A.; Butbul, E.

    1990-02-06

    To explore how enzyme affinities and enzyme activities regulate hydrolysis of water-insoluble substrates, the authors compared hydrolysis of phospholipid-stabilized emulsions of medium-chain (MCT) versus long-chain triacylglycerols (LCT). Because substrate solubility at the emulsion surface might modulate rates of hydrolysis, the ability of egg yolk phosphatidylcholine to solubilize MCT was examined by NMR spectroscopy. Chemical shift measurements showed that 11 mol % of ({sup 13}C)carbonyl enriched trioctanoin was incorporated into phospholipid vesicles as a surface component. Line widths of trioctanoin surface peaks were half that of LCT, and relaxation times, T{sub 1}, were also shorter for trioctanoin, showing greater mobility formore » MCT in phospholipid. In assessing the effects of these differences in solubility on lipolysis, they found that both purified bovine milk lipoprotein lipase and human hepatic lipase hydrolyzed MCT at rates at least 2-fold higher than for LCT. Differences in affinity were also demonstrated in mixed incubations where increasing amounts of LCT emulsion resulted in decreased hydrolysis of MCT emulsions. These results suggest that despite lower enzyme affinity for MCT emulsions, shorter chain triacylglycerols are more readily hydrolyzed by lipoprotein and hepatic lipases than long-chain triacylglycerols because of greater MCT solubility and mobility at the emulsion-water interface.« less

  7. High-level extracellular production and characterization of Candida antarctica lipase B in Pichia pastoris.

    PubMed

    Eom, Gyeong Tae; Lee, Seung Hwan; Song, Bong Keun; Chung, Keun-Wo; Kim, Young-Wun; Song, Jae Kwang

    2013-08-01

    The gene encoding lipase B from Candida antarctica (CalB) was expressed in Pichia pastoris after it was synthesized by the recursive PCR and cloned into the Pichia expression plasmid, pPICZαA. The CalB was successfully secreted in the recombinant P. pastoris strain X-33 with an apparent molecular weight of 34 kDa. For 140 h flask culture, the dry cell weight and the extracellular lipase activity reached at 5.4 g/l and 57.9 U/l toward p-nitrophenyl palmitate, respectively. When we performed the fed-batch fermentation using a methanol feeding strategy for 110 h, the dry cell weight and the extracellular lipase activity were increased to 135.7 g/l and 11,900 U/l; the CalB protein concentration was 1.18 g/l of culture supernatant. The characteristics of CalB recovered from the P. pastoris culture were compared with the commercial form of CalB produced in Aspergillus oryzae. The kinetic constants and specific activity, the effects of activity and stability on temperature and pH, the glycosylation extent, the degree of immobilization on macroporous resin and the yield of esterification reaction between oleic acid and n-butanol were almost identical to each other. Therefore, we successfully proved that the Pichia-based expression system for CalB in this study was industrially promising compared with one of the most efficient production systems. Copyright © 2013 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  8. Lipolysis of the semi-solid self-emulsifying excipient Gelucire 44/14 by digestive lipases.

    PubMed

    Fernandez, Sylvie; Rodier, Jean-David; Ritter, Nicolas; Mahler, Bruno; Demarne, Frédéric; Carrière, Frédéric; Jannin, Vincent

    2008-08-01

    Gelucire 44/14 is a semi-solid self-emulsifying excipient used for the oral delivery of poorly water-soluble drugs. It is composed of C8-C18 acylglycerols and PEG-32 esters, all of which are potential substrates for digestive lipases. Here we studied the lipolysis of Gelucire 44/14 by porcine pancreatic extracts, human pancreatic juice and several purified digestive lipases. Human pancreatic lipase (HPL), the main lipase involved in the digestion of triacylglycerols, did not show any significant activity on Gelucire 44/14 or on either of its individual compounds, C8-C18 acylglycerols and PEG-32 esters. Other pancreatic lipases such as human pancreatic lipase-related protein 2 (HPLRP2) showed low activity on Gelucire 44/14 although the highest activity of HPLRP2 was that observed on the C8-C18 acylglycerol fraction, which accounts for 20% (w/w) of Gelucire 44/14. In addition, HPLRP2 showed low activities on the PEG-32 esters, whether these were tested individually or mixed together. Carboxyl ester hydrolase (CEH) showed high activity on Gelucire 44/14, and the highest activities of CEH were those recorded on the total PEG-32 ester fraction and on each individual PEG-32 ester, except for PEG-32 monostearate. The highest activity of all the enzymes tested was that of dog gastric lipase (DGL) on Gelucire 44/14, although DGL showed low activity on the PEG-32 ester fraction and on each individual PEG-32 ester. We compared the lipolysis of Gelucire 44/14 with that of Labrasol, another self-emulsifying excipient, which is liquid at room temperature. Human pancreatic juice showed similar rates of activity on both Gelucire 44/14 and Labrasol. This finding means that these excipients are hydrolyzed in vivo during pancreatic digestion, mainly by CEH in the case of Gelucire 44/14 and by both HPLRP2 and CEH in that of Labrasol, whereas HPL showed very low activities on each of these two excipients. This is the first time the effects of PEG and acyl chain length on the lipolytic

  9. Lipoprotein lipase activity and mass, apolipoprotein C-II mass and polymorphisms of apolipoproteins E and A5 in subjects with prior acute hypertriglyceridaemic pancreatitis

    PubMed Central

    2009-01-01

    Background Severe hypertriglyceridaemia due to chylomicronemia may trigger an acute pancreatitis. However, the basic underlying mechanism is usually not well understood. We decided to analyze some proteins involved in the catabolism of triglyceride-rich lipoproteins in patients with severe hypertriglyceridaemia. Methods Twenty-four survivors of acute hypertriglyceridaemic pancreatitis (cases) and 31 patients with severe hypertriglyceridaemia (controls) were included. Clinical and anthropometrical data, chylomicronaemia, lipoprotein profile, postheparin lipoprotein lipase mass and activity, hepatic lipase activity, apolipoprotein C II and CIII mass, apo E and A5 polymorphisms were assessed. Results Only five cases were found to have LPL mass and activity deficiency, all of them thin and having the first episode in childhood. No cases had apolipoprotein CII deficiency. No significant differences were found between the non-deficient LPL cases and the controls in terms of obesity, diabetes, alcohol consumption, drug therapy, gender distribution, evidence of fasting chylomicronaemia, lipid levels, LPL activity and mass, hepatic lipase activity, CII and CIII mass or apo E polymorphisms. However, the SNP S19W of apo A5 tended to be more prevalent in cases than controls (40% vs. 23%, NS). Conclusion Primary defects in LPL and C-II are rare in survivors of acute hypertriglyceridaemic pancreatitis; lipase activity measurements should be restricted to those having their first episode during chilhood. PMID:19534808

  10. Cuticular fatty acids of Galleria mellonella (Lepidoptera) inhibit fungal enzymatic activities of pathogenic Conidiobolus coronatus

    PubMed Central

    Boguś, Mieczysława Irena; Włóka, Emilia; Kazek, Michalina; Kaczmarek, Agata; Zalewska, Katarzyna

    2018-01-01

    The entomopathogenic fungus Conidiobolus coronatus produces enzymes that may hydrolyze the cuticle of Galleria mellonella. Of these enzymes, elastase activity was the highest: this figure being 24 times higher than NAGase activity 553 times higher than chitinase activity and 1844 times higher than lipase activity. The present work examines the differences in the hydrolysis of cuticles taken from larvae, pupae and adults (thorax and wings), by C. coronatus enzymes. The cuticles of the larvae and adult thorax were the most susceptible to digestion by proteases and lipases. Moreover, the maximum concentration of free N-glucosamine was in the hydrolysis of G. mellonella thorax. These differences in the digestion of the various types of cuticle may result from differences in their composition. GC-MS analysis of the cuticular fatty acids isolated from pupae of G. mellonella confirmed the presence of C 8:0, C 9:0, C 12:0, C 14:0, C 15:0, C 16:1, C 16:0, C 17:0, C 18:1, C 18:0, with C 16:0 and C 18:0 being present in the highest concentrations. Additional fatty acids were found in extracts from G. mellonella imagines: C 10:0, C 13:0, C 20:0 and C 20:1, with a considerable dominance of C 16:0 and C 18:1. In larvae, C 16:0 and C 18:1 predominated. Statistically significant differences in concentration (p≤0.05) were found between the larvae, pupae and imago for each fatty acid. The qualitative and quantitative differences in the fatty acid composition of G. mellonella cuticle occurring throughout normal development might be responsible for the varied efficiency of fungal enzymes in degrading larval, pupal and adult cuticles. PMID:29518079

  11. Activation of peroxisome proliferator-activated receptor-{alpha} enhances fatty acid oxidation in human adipocytes

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Lee, Joo-Young; Hashizaki, Hikari; Goto, Tsuyoshi

    2011-04-22

    Highlights: {yields} PPAR{alpha} activation increased mRNA expression levels of adipocyte differentiation marker genes and GPDH activity in human adipocytes. {yields} PPAR{alpha} activation also increased insulin-dependent glucose uptake in human adipocytes. {yields} PPAR{alpha} activation did not affect lipid accumulation in human adipocytes. {yields} PPAR{alpha} activation increased fatty acid oxidation through induction of fatty acid oxidation-related genes in human adipocytes. -- Abstract: Peroxisome proliferator-activated receptor-{alpha} (PPAR{alpha}) is a key regulator for maintaining whole-body energy balance. However, the physiological functions of PPAR{alpha} in adipocytes have been unclarified. We examined the functions of PPAR{alpha} using human multipotent adipose tissue-derived stem cells as a humanmore » adipocyte model. Activation of PPAR{alpha} by GW7647, a potent PPAR{alpha} agonist, increased the mRNA expression levels of adipocyte differentiation marker genes such as PPAR{gamma}, adipocyte-specific fatty acid-binding protein, and lipoprotein lipase and increased both GPDH activity and insulin-dependent glucose uptake level. The findings indicate that PPAR{alpha} activation stimulates adipocyte differentiation. However, lipid accumulation was not changed, which is usually observed when PPAR{gamma} is activated. On the other hand, PPAR{alpha} activation by GW7647 treatment induced the mRNA expression of fatty acid oxidation-related genes such as CPT-1B and AOX in a PPAR{alpha}-dependent manner. Moreover, PPAR{alpha} activation increased the production of CO{sub 2} and acid soluble metabolites, which are products of fatty acid oxidation, and increased oxygen consumption rate in human adipocytes. The data indicate that activation of PPAR{alpha} stimulates both adipocyte differentiation and fatty acid oxidation in human adipocytes, suggesting that PPAR{alpha} agonists could improve insulin resistance without lipid accumulation in adipocytes. The

  12. Mexican consensus on lysosomal acid lipase deficiency diagnosis.

    PubMed

    Vázquez-Frias, R; García-Ortiz, J E; Valencia-Mayoral, P F; Castro-Narro, G E; Medina-Bravo, P G; Santillán-Hernández, Y; Flores-Calderón, J; Mehta, R; Arellano-Valdés, C A; Carbajal-Rodríguez, L; Navarrete-Martínez, J I; Urbán-Reyes, M L; Valadez-Reyes, M T; Zárate-Mondragón, F; Consuelo-Sánchez, A

    Lysosomal acid lipase deficiency (LAL-D) causes progressive cholesteryl ester and triglyceride accumulation in the lysosomes of hepatocytes and monocyte-macrophage system cells, resulting in a systemic disease with various manifestations that may go unnoticed. It is indispensable to recognize the deficiency, which can present in patients at any age, so that specific treatment can be given. The aim of the present review was to offer a guide for physicians in understanding the fundamental diagnostic aspects of LAL-D, to successfully aid in its identification. The review was designed by a group of Mexican experts and is presented as an orienting algorithm for the pediatrician, internist, gastroenterologist, endocrinologist, geneticist, pathologist, radiologist, and other specialists that could come across this disease in their patients. An up-to-date review of the literature in relation to the clinical manifestations of LAL-D and its diagnosis was performed. The statements were formulated based on said review and were then voted upon. The structured quantitative method employed for reaching consensus was the nominal group technique. A practical algorithm of the diagnostic process in LAL-D patients was proposed, based on clinical and laboratory data indicative of the disease and in accordance with the consensus established for each recommendation. The algorithm provides a sequence of clinical actions from different studies for optimizing the diagnostic process of patients suspected of having LAL-D. Copyright © 2017 Asociación Mexicana de Gastroenterología. Publicado por Masson Doyma México S.A. All rights reserved.

  13. Fabrication of enzyme-immobilized halloysite nanotubes for affinity enrichment of lipase inhibitors from complex mixtures.

    PubMed

    Wang, Haibo; Zhao, Xiaoping; Wang, Shufang; Tao, Shan; Ai, Ni; Wang, Yi

    2015-05-01

    Lipase is the key enzyme for catalyzing triglyceride hydrolysis in vivo, and lipase inhibitors have been used in the management of obesity. We present the first report on the use of lipase-adsorbed halloysite nanotubes as an efficient medium for the selective enrichment of lipase inhibitors from natural products. A simple and rapid approach was proposed to fabricate lipase-adsorbed nanotubes through electrostatic interaction. Results showed that more than 85% lipase was adsorbed into nanotubes in 90 min, and approximately 80% of the catalytic activity was maintained compared with free lipase. The specificity and reproducibility of the proposed approach were validated by screening a known lipase inhibitor (i.e., orlistat) from a mixture that contains active and inactive compounds. Moreover, we applied this approach with high performance liquid chromatography-mass spectrometry technique to screen lipase inhibitors from the Magnoliae cortex extract, a medicinal plant used for treating obesity. Two novel biphenyl-type natural lipase inhibitors magnotriol A and magnaldehyde B were identified, and their IC50 values were determined as 213.03 and 96.96 μM, respectively. The ligand-enzyme interactions of magnaldehyde B were further investigated by molecular docking. Our findings proved that enzyme-adsorbed nanotube could be used as a feasible and selective affinity medium for the rapid screening of enzyme inhibitors from complex mixtures. Copyright © 2015 Elsevier B.V. All rights reserved.

  14. Production of Omega-3 Fatty Acid Ethyl Esters from Menhaden Oil Using Proteus vulgaris Lipase-Mediated One-Step Transesterification and Urea Complexation.

    PubMed

    Kim, Soo-Jin; Kim, Hyung Kwoun

    2016-05-01

    An organic solvent-stable lipase from Proteus vulgaris K80 was used to produce the omega-3 polyunsaturated fatty acid ethyl esters (ω-3 PUFA EEs). First, the lyophilized recombinant lipase K80 (LyoK80) was used to perform the transesterification reaction of menhaden oil and ethanol. LyoK80 produced the ω-3 PUFA EEs with a conversion yield of 82 % in the presence of 20 % water content via a three-step ethanol-feeding process; however, in a non-aqueous condition, LyoK80 produced only a slight amount of the ω-3 PUFA EEs. To enhance its reaction properties, the lipase K80 was immobilized on a hydrophobic bead to derive ImmK80; the biochemical properties and substrate specificity of ImmK80 are similar to those of LyoK80. ImmK80 was then used to produce ω-3 PUFA EEs in accordance with the same transesterification reaction. Unlike LyoK80, ImmK80 achieved a high ω-3 PUFA EE conversion yield of 86 % under a non-aqueous system via a one-step ethanol-feeding reaction. The ω-3 PUFA EEs were purified up to 92 % using a urea complexation method.

  15. Coconut oil induced production of a surfactant-compatible lipase from Aspergillus tamarii under submerged fermentation.

    PubMed

    Das, Arijit; Bhattacharya, Sourav; Shivakumar, Srividya; Shakya, Sujina; Sogane, Swathi Shankar

    2017-02-01

    Filamentous fungi are efficient producers of lipases. The present study focuses on identification of a potent lipolytic fungus and enhancement of lipase production through optimization of nutritional and cultural conditions under submerged fermentation. Molecular characterization of the fungus by 18S rDNA sequencing revealed its identity as Aspergillus tamarii with 98% homology. Maximum lipase production was noted in mineral salts medium supplemented with coconut oil (2.5%, v/v). A combination of ammonium chloride (2%, w/v) and tryptone (2%, w/v) facilitated maximum lipase production at pH 5 of the production medium. A carbon: nitrogen ratio of 1:4 led to significant (p < 0.00008) increase in the enzyme production in the presence of surfactant cetyltrimethylammonium bromide (0.5%, w/v). Maximum lipase activity (2,32,500 ± 192 U/ml/min) was recorded after 7 days of incubation at 25 °C on a rotary shaker at 120 rpm. A 9.8-fold increase in lipase activity was recorded after optimization of the process parameters. Addition of crude lipase enhanced the oil stain removal activity of a commercially available detergent by 2.2-fold. The current findings suggest the potentiality of this fungal lipase to be used in detergent formulation. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  16. Parathyroid hormone is not an inhibitor of lipoprotein lipase activity.

    PubMed

    Arnadottir, M; Nilsson-Ehle, P

    1994-01-01

    The reduced lipoprotein lipase (LPL) activities in uraemia are reflected by increased serum triglyceride concentrations and reduced HDL cholesterol concentrations. Both hyperparathyroidism and circulating inhibitor(s) of LPL have been associated with the disturbances of lipid metabolism in uraemia. The aim of the present study was to investigate if parathyroid hormone (PTH) had an inhibitory effect on LPL activity. Plasma post-heparin LPL activities, plasma LPL inhibitory activities, serum PTHintact and serum PTHC-terminal concentrations were analysed in 20 patients on haemodialysis and 20 healthy controls. The effects of purified, human PTHintact and a carboxyterminal fragment of PTH (PTH39-84) on LPL activities in post-heparin plasma from healthy individuals and on the enzyme activity of purified, bovine milk LPL, activated with apolipoprotein CII, were studied. Patients had significantly higher plasma LPL inhibitory activities than controls, but there was no correlation between plasma LPL inhibitory activities and serum PTH concentrations. Neither PTHintact nor PTH39-84 had a significant effect on LPL activities in vitro. Thus there was no evidence of a direct inhibition of LPL activity by PTH under the present in-vivo or in-vitro conditions.

  17. [Lipase and phospholipase C from Staphylococcus aureus of different origin. I. Determination and occurrence (author's transl)].

    PubMed

    Berete, Y J; Schaeg, W; Brückler, J; Blobel, H

    1980-11-01

    Lipase and phospholipase C from Staphylococcus aureus of different origin were demonstrated qualitatively by agar diffusion on tributyrin- and lecithin agar. On test media with either 0,3% Na-azide or 0,3% KCN lipase-activity was not inhibited, phospholipase C, on the other hand, completely blocked (Table 1, Fig. 2). In this manner a tentative differentiation was possible between lipase and phospholipase C. For the quantitative determination of lipase the hydrolysis of p-nitrophenyl palmitate proved to be most useful (Fig. 1). S. aureus-cultures of human origin produced more often and more actively lipase and phospholipase C than those from cattle (Table 2).

  18. Factors influencing production of lipase under metal supplementation by bacterial strain, Bacillus subtilis BDG-8.

    PubMed

    Dhevahi, B; Gurusamy, R

    2014-11-01

    Lipases are biocatalyst having wide applications in industries due to their versatile properties. In the present study, a lipolytic bacterial strain, Bacillus subtilis BDG-8 was isolated from an oil based industrial soil. The effect of selenium and nickel as a media supplement on enhancement of lipase production, was studied individually with the isolated strain by varying the concentration of selected metal. 60 μg l(-1) selenium enhanced lipase production to an enzyme activity measuring 7.8 U ml(-1) while 40 μgI(-1) nickel gave the maximum enzyme activity equivalent to 7.5 U ml(-1). However, nickel and selenium together at a range of concentration with an equal w/v ratio, at 60 μg l(-1) each, showed the maximum lipase activity of 8.5 U ml(-1). The effect of pH and temperature on lipase production showed maximum enzyme activity in the presence of each of the metals at pH 7 and 35°C among the other tested ranges. After optimisation of the parameters such as metal concentration, pH and temperature lipase production by Bacillus subtilis BDG-8 had increased several folds. This preliminary investigation may consequently lead as to various industrial applications such as treatment of wastewater contaminated with metal or oil with simultaneous lipase production.

  19. Immobilization of Lipase Inhibitor on the Biopolymers from Agaricus bisporus Cell Walls

    PubMed Central

    2017-01-01

    Summary One of the methods for curing obesity is the inclusion of some substances with the antilipase activity in the diet and thus reducing the uptake of fat components from food. The aim of this research is to provide a stabilized form of lipase inhibitor by immobilization of enzyme on the biopolymers from Agaricus bisporus cell walls. The phenolic compounds extracted from the rapeseed were considered as the lipase inhibitor. The activity of the inhibitor was considerably reduced in the gastric juice, as well as at temperatures above 37 °C and during its storage, which determined the suitability of the inhibitor for stabilization on the matrix. The effectiveness of the phenolic compound stabilization was investigated by means of immobilization on the biopolymers from Agaricus bisporus cell wall matrix. The biopolymers used were β-glucan, chitin, melanin and proteins. A number of samples, which differed both in the content of the inhibitor (from 1 to 16%) and in the ratio of biopolymers in the matrix composition, was obtained. The conditions of immobilization (temperature, duration of the process) were also varied. The expediency of obtaining the sample with the inhibitor content of 12% and matrix containing 47.9% of glucan, 18.8% of chitin, 18.8% of melanin and 11.1% of proteins was shown. The best immobilization was carried out at 20–25 °C for 30 min. Thermal analysis and infrared spectroscopy data confirmed that immobilization of the lipase inhibitor on the matrix was due to the hydrogen bonds. The immobilized inhibitor had higher pH stability and higher thermal stability than the original one. The remaining activity of the immobilized inhibitor was higher than the original one after incubation in the gastric acid and bile. The immobilized inhibitor was characterized by a low loss of activity after 12 months of storage. PMID:29540987

  20. Monoacylglycerol lipase inhibition by organophosphorus compounds leads to elevation of brain 2-arachidonoylglycerol and the associated hypomotility in mice

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Quistad, Gary B.; Klintenberg, Rebecka; Caboni, Pierluigi

    2006-02-15

    Three components of the cannabinoid system are sensitive to selected organophosphorus (OP) compounds: monoacylglycerol (MAG) lipase that hydrolyzes the major endogenous agonist 2-arachidonoylglycerol (2-AG); fatty acid amide hydrolase (FAAH) that cleaves the agonist anandamide present in smaller amounts; the CB1 receptor itself. This investigation considers which component of the cannabinoid system is the most likely contributor to OP-induced hypomotility in mice. Structure-activity studies by our laboratory and others rule against major involvement of a direct toxicant-CB1 receptor interaction for selected OPs. Attention was therefore focused on the OP sensitivities of MAG lipase and FAAH, assaying 19 structurally diverse OP chemicalsmore » (pesticides, their metabolites and designer compounds) for in vitro inhibition of both enzymes. Remarkably high potency and low selectivity is observed with three O-alkyl (C{sub 1}, C{sub 2}, C{sub 3}) alkylphosphonofluoridates (C{sub 8}, C{sub 12}) (IC50 0.60-3.0 nM), five S-alkyl (C{sub 5}, C{sub 7}, C{sub 9}) and alkyl (C{sub 1}, C{sub 12}) benzodioxaphosphorin oxides (IC50 0.15-5.7 nM) and one OP insecticide metabolite (chlorpyrifos oxon, IC50 34-40 nM). In ip-treated mice, the OPs at 1-30 mg/kg more potently inhibit brain FAAH than MAG lipase, but FAAH inhibition is not correlated with hypomotility. However, the alkylphosphonofluoridate-treated mice show dose-dependent increases in severity of hypomotility, inhibition of MAG lipase activity and elevation of 2-AG. Moderate to severe hypomotility is accompanied by 64 to 86% MAG lipase inhibition and about 6-fold elevation of brain 2-AG level. It therefore appears that OP-induced MAG lipase inhibition leads to elevated 2-AG and the associated hypomotility.« less

  1. High-level heterologous expression and properties of a novel lipase from Ralstonia sp. M1.

    PubMed

    Quyen, Dinh Thi; Giang Le, Thi Thu; Nguyen, Thi Thao; Oh, Tae-Kwang; Lee, Jung-Kee

    2005-01-01

    The mature lipase LipA and its 56aa-truncated chaperone DeltaLipBhis (with 6xhis-tag) from Ralstonia sp. M1 were over-expressed in Escherichia coli BL21 under the control of T7 promoter with a high level of 70 and 12mg protein per gram of wet cells, respectively. The simply purified lipase LipA was effectively refolded by Ni-NTA purified chaperone DeltaLipBhis in molar ratio 1:1 at 4 degrees C for 24 hours in H2O. The in vitro refolded lipase LipA had an optimal activity in the temperature range of 50-55 degrees C and was stable up to 45 degrees C with more than 84% activity retention. The maximal activity was observed at pH 10.75 for hydrolysis of olive oil and found to be stable over alkaline pH range 8.0-10.5 with more than 52% activity retention. The enzyme was found to be highly resistant to many organic solvents especially induced by ethanolamine (remaining activity 137-334%), but inhibited by 1-butanol and acetonitrile (40-86%). Metal ions Cu2+, Sn2+, Mn2+, Mg2+, and Ca2+ stimulated the lipase slightly with increase in activity by up to 22%, whereas Zn2+ significantly inhibited the enzyme with the residual activity of 30-65% and Fe3+ to a lesser degree (activity retention of 77-86%). Tween 80, Tween 60, and Tween 40 induced the activation of the lipase LipA (222-330%) and 0.2-1% (w/v) of Triton X-100, X-45, and SDS increased the lipase activity by up to 52%. However, 5% (w/v) of Triton X-100, X-45, and SDS inhibited strongly the activity by 31-89%. The inhibitors including DEPC, EDTA, PMSF, and 2-mercaptoethanol (0.1-10mM) inhibited moderately the lipase with remaining activity of 57-105%. The lipase LipA hydrolyzed a wide range of triglycerides, but preferentially short length acyl chains (C4 and C6). In contrast to the triglycerides, medium length acyl chains (C8 and C14) of p-nitrophenyl (p-NP) esters were preferential substrates of this lipase. The enzyme preferentially catalyzed the hydrolysis of cottonseed oil (317%), cornoil (227%), palm oil (222

  2. A stereospecific carboxyl esterase from Bacillus coagulans hosting nonlipase activity within a lipase-like fold.

    PubMed

    De Vitis, Valerio; Nakhnoukh, Cristina; Pinto, Andrea; Contente, Martina L; Barbiroli, Alberto; Milani, Mario; Bolognesi, Martino; Molinari, Francesco; Gourlay, Louise J; Romano, Diego

    2018-03-01

    Microbial carboxylesterases are important biocatalysts that selectively hydrolyze an extensive range of esters. Here, we report the biochemical and structural characterization of an atypical carboxylesterase from Bacillus coagulans (BCE), endowed with high enantioselectivity toward different 1,2-O-isopropylideneglycerol (IPG or solketal) esters. BCE efficiently catalyzes the production of enantiopure (S)-IPG, a chiral building block for the synthesis of β-blockers, glycerophospholipids, and prostaglandins; efficient hydrolysis was observed up to 65 °C. To gain insight into the mechanistic bases of such enantioselectivity, we solved the crystal structures of BCE in apo- and glycerol-bound forms at resolutions of 1.9 and 1.8 Å, respectively. In silico docking studies on the BCE structure confirmed that IPG esters with small acyl chains (≤ C6) were easily accommodated in the active site pocket, indicating that small conformational changes are necessary to accept longer substrates. Furthermore, docking studies suggested that enantioselectivity may be due to an improved stabilization of the tetrahedral reaction intermediate for the S-enantiomer. Contrary to the above functional data implying nonlipolytic functions, BCE displays a lipase-like 3D structure that hosts a "lid" domain capping the main entrance to the active site. In lipases the lid mediates catalysis through interfacial activation, a process that we did not observe for BCE. Overall, we present the functional-structural properties of an atypical carboxyl esterase that has nonlipase-like functions, yet possesses a lipase-like 3D fold. Our data provide original enzymatic information in view of BCE applications as an inexpensive, efficient biocatalyst for the production of enantiopure (S)-IPG. Coordinates and structure factors have been deposited in the Protein Data Bank (www.rcsb.org) under accession numbers 5O7G (apo-BCE) and 5OLU (glycerol-bound BCE). © 2017 Federation of European Biochemical

  3. Biodiesel production from Nannochloropsis gaditana lipids through transesterification catalyzed by Rhizopus oryzae lipase.

    PubMed

    Navarro López, Elvira; Robles Medina, Alfonso; González Moreno, Pedro Antonio; Esteban Cerdán, Luis; Martín Valverde, Lorena; Molina Grima, Emilio

    2016-03-01

    Biodiesel (fatty acid methyl esters, FAMEs) was produced from saponifiable lipids (SLs) extracted from wet Nannochloropsis gaditana biomass using methanolysis catalyzed by Rhizopus oryzae intracellular lipase. SLs were firstly extracted with ethanol to obtain 31 wt% pure SLs. But this low SL purity also gave a low biodiesel conversion (58%). This conversion increased up to 80% using SLs purified by crystallization in acetone (95 wt% purity). Polar lipids play an important role in decreasing the reaction velocity - using SLs extracted with hexane, which have lower polar lipid content (37.4% versus 49.0% using ethanol), we obtained higher reaction velocities and less FAME conversion decrease when the same lipase batch was reused. 83% of SLs were transformed to biodiesel using a 70 wt% lipase/SL ratio, 11:1 methanol/SL molar ratio, 10 mL t-butanol/g SLs after 72 h. The FAME conversion decreased to 71% after catalyzing three reactions with the same lipase batch. Copyright © 2015 Elsevier Ltd. All rights reserved.

  4. Hydrolysis of mixed monomolecular films of triglyceride/lecithin by pancreatic lipase.

    PubMed

    Pieroni, G; Verger, R

    1979-10-25

    The main purpose of this study was to describe the influence of lecithin upon lipolysis of mixed monomolecular films of trioctanoylglycerol/didodecanoylphosphatidycholine by pancreatic lipase in order to mimic some physiological situations. The quantity of enzyme adsorbed to the interface was simultaneously determined using 5-thio-2-nitro[14C]benzoyl lipase. Lipolytic activity was enhanced 3- to 4-fold in the presence of colipase, an effect which is attributed to increased enzyme turnover number. When a pure triglyceride film was progressively diluted with lecithin, the minimum specific activity of lipase exhibited a bell-shaped curve: a mixed film containing only 20% trioctanoylglycerol was hydrolyzed at the same rate as a monolayer of pure triglyceride.

  5. The combined use of whole Cuphea seeds containing medium chain fatty acids and an exogenous lipase in piglet nutrition.

    PubMed

    Dierick, N A; Decuypere, J A; Degeyter, I

    2003-02-01

    In search for an alternative for nutritional antimicrobials in piglet feeding, the effects of adding whole Cuphea seeds, as a natural source of medium chain fatty acids (MCFA), with known antimicrobial effects, and an exogenous lipase to a weaner diet were studied. The foregut flora, the gut morphology, some digestive parameters and the zootechnical performance of weaned piglets were investigated. Thirty newly weaned piglets, initial weight 7.0 +/- 0.4 kg, were divided according to litter, sex and weight in two groups (control diet; Cuphea + lipase diet). The Cuphea seeds (lanceolata and ignea) (50 g kg(-1)) were substituted for soybean oil (15 g kg(-1)), Alphacell (25 g kg(-1)) and soy protein isolate (10 g kg(-1)) in the control diet. Also 500 mg kg(-1) microbial lipase was added to the Cuphea diet. The piglets were weighted individually on days 0, 3. 7, 14 and 16. Feed intake was recorded per pen during days 0 to 3, 3 to 7, 7 to 14 and 14 to 16. On day 7 five piglets of each experimental group were euthanized for counting the gastric and small intestinal gut flora and for gut morphology at two sites of the small intestine (proximal, distal). The results indicate a trend towards improved performances parameters by feeding Cuphea + lipase. The enzymic released MCFA (1.7 g kg(-1) fresh gastric contents) tended to decrease the number of Coliforms in the proximal small intestine, but increased the number in the stomach and distal small intestine. With Culphea, the number of Streptococci was significantly lower in small intestine, but not in the stomach, while the number of Lactobacilli was significantly lower in the distal small intestine and tended to be lower in the stomach and proximal small intestine. No differences between the diets were noted for the total anaerobic microbial load in the stomach or in the gut. Feeding Cuphea + lipase resulted in a significantly greater villus height (distal small intestine) and a lesser crypt depth (proximal and distal small

  6. Activation of lipoprotein lipase by lipoprotein fractions of human serum.

    PubMed

    Bier, D M; Havel, R J

    1970-11-01

    Triglycerides in fat emulsions are hydrolyzed by lipoprotein lipase only when they are "activated" by serum lipoproteins. The contribution of different lipoprotein fractions to hydrolysis of triglycerides in soybean oil emulsion was assessed by determining the quantity of lipoprotein fraction required to give half-maximal hydrolysis. Most of the activator property of whole serum from normolipidemic, postabsorptive subjects was in high density lipoproteins. Low density lipoproteins and serum from which all lipoprotein classes were removed had little or no activity. Also, little activator was present in guinea pig serum or in very low density poor serum from an individual with lecithin:cholesterol acyltransferase deficiency, both of which are deficient in high density lipoproteins. Human very low density lipoproteins are potent activators and are much more active than predicted from their content of high density lipoprotein-protein. Per unit weight of protein, very low density lipoproteins had 13 times the activity of high density lipoproteins. These observations suggest that one or more of the major apoproteins of very low density lipoproteins, present as a minor constituent of high density lipoproteins, may be required for the activation process.

  7. Saturation mutagenesis in selected amino acids to shift Pseudomonas sp. acidic lipase Lip I.3 substrate specificity and activity.

    PubMed

    Panizza, Paola; Cesarini, Silvia; Diaz, Pilar; Rodríguez Giordano, Sonia

    2015-01-25

    Several Pseudomonas sp. CR611 Lip I.3 mutants with overall increased activity and a shift towards longer chain substrates were constructed. Substitution of residues Y29 and W310 by smaller amino acids provided increased activity on C18-substrates. Residues G152 and S154, modified to study their influence on interfacial activation, displayed a five and eleven fold increased activity.

  8. Solvent-Free Lipase-Catalyzed Synthesis of Diacylgycerols as Low-Calorie Food Ingredients.

    PubMed

    Vázquez, Luis; González, Noemí; Reglero, Guillermo; Torres, Carlos

    2016-01-01

    Problems derived from obesity and overweight have recently promoted the development of fat substitutes and other low-calorie foods. On the one hand, fats with short- and medium-chain fatty acids are a source of quick energy, easily hydrolyzable and hardly stored as fat. Furthermore, 1,3-diacylglycerols are not hydrolyzed to 2-monoacylglycerols in the gastrointestinal tract, reducing the formation of chylomicron and lowers the serum level of triacylglycerols by decreasing its resynthesis in the enterocyte. In this work, these two effects were combined to synthesize short- and medium-chain 1,3-diacylglycerols, leading to a product with great potential as for their low-calorie properties. Lipase-catalyzed transesterification reactions were performed between short- and medium-chain fatty acid ethyl esters and glycerol. Different variables were investigated, such as the type of biocatalyst, the molar ratio FAEE:glycerol, the adsorption of glycerol on silica gel, or the addition of lecithin. Best reaction conditions were evaluated considering the percentage of 1,3-DAG produced and the reaction rate. Except Novozym 435 (Candida antarctica), other lipases required the adsorption of glycerol on silica gel to form acylglycerols. Lipases that gave the best results with adsorption were Novozym 435 and Lipozyme RM IM (Rhizomucor miehei) with 52 and 60.7% DAG at 32 h, respectively. Because of its specificity for sn-1 and sn-3 positions, lipases leading to a higher proportion of 1,3-DAG vs. 1,2-DAG were Lipozyme RM IM (39.8 and 20.9%, respectively) and Lipase PLG (Alcaligenes sp.) (35.9 and 19.3%, respectively). By adding 1% (w/w) of lecithin to the reaction with Novozym 435 and raw glycerol, the reaction rate was considerably increased from 41.7 to 52.8% DAG at 24 h.

  9. Solvent-Free Lipase-Catalyzed Synthesis of Diacylgycerols as Low-Calorie Food Ingredients

    PubMed Central

    Vázquez, Luis; González, Noemí; Reglero, Guillermo; Torres, Carlos

    2016-01-01

    Problems derived from obesity and overweight have recently promoted the development of fat substitutes and other low-calorie foods. On the one hand, fats with short- and medium-chain fatty acids are a source of quick energy, easily hydrolyzable and hardly stored as fat. Furthermore, 1,3-diacylglycerols are not hydrolyzed to 2-monoacylglycerols in the gastrointestinal tract, reducing the formation of chylomicron and lowers the serum level of triacylglycerols by decreasing its resynthesis in the enterocyte. In this work, these two effects were combined to synthesize short- and medium-chain 1,3-diacylglycerols, leading to a product with great potential as for their low-calorie properties. Lipase-catalyzed transesterification reactions were performed between short- and medium-chain fatty acid ethyl esters and glycerol. Different variables were investigated, such as the type of biocatalyst, the molar ratio FAEE:glycerol, the adsorption of glycerol on silica gel, or the addition of lecithin. Best reaction conditions were evaluated considering the percentage of 1,3-DAG produced and the reaction rate. Except Novozym 435 (Candida antarctica), other lipases required the adsorption of glycerol on silica gel to form acylglycerols. Lipases that gave the best results with adsorption were Novozym 435 and Lipozyme RM IM (Rhizomucor miehei) with 52 and 60.7% DAG at 32 h, respectively. Because of its specificity for sn-1 and sn-3 positions, lipases leading to a higher proportion of 1,3-DAG vs. 1,2-DAG were Lipozyme RM IM (39.8 and 20.9%, respectively) and Lipase PLG (Alcaligenes sp.) (35.9 and 19.3%, respectively). By adding 1% (w/w) of lecithin to the reaction with Novozym 435 and raw glycerol, the reaction rate was considerably increased from 41.7 to 52.8% DAG at 24 h. PMID:26904539

  10. Lipase inhibition and antiobesity effect of Atractylodes lancea.

    PubMed

    Jiao, Ping; Tseng-Crank, Julie; Corneliusen, Brandon; Yimam, Mesfin; Hodges, Mandee; Hong, Mei; Maurseth, Catherine; Oh, Misun; Kim, Hyunjin; Chu, Min; Jia, Qi

    2014-05-01

    The ethanol extract of Atractylodes lancea rhizome displayed significant lipase inhibition with an IC50 value of 9.06 µg/mL in a human pancreatic lipase assay from high-throughput screening. Bioassay-guided isolation led to the identification of one new polyacetylene, syn-(5E,11E)-3-acetoxy-4-O-(3-methylbutanoyl)-1,5,11-tridecatriene-7,9-diyne-3,4-diol (7), along with six known compounds (1-6). The structure of compound 7 was determined based on the analysis of NMR and MS data. Among these seven lipase inhibitors, the major compound atractylodin (1) showed the highest lipase inhibitory activity (IC50 = 39.12 µM). The antiobesity effect of the ethanol extract of Atractylodes lancea rhizome was evaluated in a high-fat diet-induced obesity mice model at daily dosages of 250 mg/kg and 500 mg/kg body weight for 4 weeks, and treatment with this extract demonstrated a moderate efficacy at the 500 mg/kg dose level. Georg Thieme Verlag KG Stuttgart · New York.

  11. Lipases in Medicine: An Overview.

    PubMed

    Loli, Heni; Narwal, Sunil Kumar; Saun, Nitin Kumar; Gupta, Reena

    2015-01-01

    Lipases are part of the family of hydrolases that act on carboxylic ester bonds. They are involved in catalyzing the hydrolysis of triglycerides (TG) into chylomicrons and very low density lipoprotein (VLDL) particles. Uses of lipases are evolving rapidly and currently they are reported to show high potential in medicine. Intensive study and investigations have led researchers to explore lipases for their use in substitution therapy, where in enzyme deficiency during diseased conditions is compensated by their external administration. In our body, they are used to break down fats present in food so that they can be absorbed in the intestine and deficiency of lipases leads to malabsorption of fats and fat-soluble vitamins. Lipases help a person who has cystic fibrosis, Alzheimer's disease, atherosclerosis and act as a candidate target for cancer prevention and therapy. They act as diagnostic tool and their presence or increasing levels can indicate certain infection or disease. Obesity causes metabolic disease and is a serious health problem around the world. Thus inhibiting digestive lipase to reduce fat absorption has become the main pharmacological approach to the treatment of obesity in recent years.

  12. Molecular and enzymatic characterization of alkaline lipase from Bacillus amyloliquefaciens E1PA isolated from lipid-rich food waste.

    PubMed

    Saengsanga, Thanakorn; Siripornadulsil, Wilailak; Siripornadulsil, Surasak

    2016-01-01

    Bacillus amyloliquefaciens E1PA is a lipase-producing strain that was originally isolated from lipid-rich food waste, and the production of its lipase was found to be induced by vegetable oils. The E1PA lipase was successfully expressed and secreted in a heterologous Escherichia coli host and was ultimately purified. The conserved pentapeptide motif Ala-His-Ser-Met-Gly was observed at positions 108-112. The purified recombinant lipase was stable over a pH range of 4.0-11.0 at 40 °C and exhibited maximal activity at pH 10. The recombinant E1PA lipase hydrolyzed a wide range of acyl esters (C4-C18). However, the highest activity (3.5 units mg(-1)) was observed when the p-nitrophenyl ester of myristate (C14) was used as a substrate. Compared to the lipases produced by Bacillus spp., the E1PA lipase displayed a structural molecular mass excluding the leader sequence (19.22 kDa) and a pI (9.82) that were similar to those reported for B. amyloliquefaciens lipases and lipase subfamily I.4 but that were quite distinct from those of lipase subfamily I.5 (approximately 43 kDa, pI 6). These results suggested that Bacillus lipases are closely related. Although the recombinant E1PA lipase digested only certain oils, the wild-type E1PA lipase degraded a variety of oils, including blended and re-used cooking oils. The recombinant and wild-type forms of the E1PA lipase were able to digest heterogeneous lipid-rich food waste at similar levels; this result suggests that this lipase can function even when it solely consists of its structural enzyme component. The enzyme exhibited lipid hydrolysis ability as either an intracellular domain of the recombinant protein or an extracellular domain secreted by the E1PA strain. However, the recombinant lipase showed higher activity than the wild-type E1PA lipase, indicating that the recombinant protein from E. coli possessed effective lipase activity. Thus, the inducible alkaline E1PA lipase exhibited the ability to act on a broad spectrum

  13. Comparative study of thermostability and ester synthesis ability of free and immobilized lipases on cross linked silica gel.

    PubMed

    Kumari, Annapurna; Mahapatra, Paramita; Kumar, Garlapati Vijay; Banerjee, Rintu

    2008-06-01

    A novel support has been utilized for immobilization of lipase, which was prepared by amination of silica with ethanolamine followed by cross linking with glutaraldehyde. Lipases from Rhizopus oryzae 3562 and Enterobacter aerogenes were immobilized on activated silica gel, where they retained 60 and 50% of respective original activity. The thermal stability of the immobilized lipases was significantly improved in comparison to the free forms while the pH stability remained unchanged. E. aerogenes and R. oryzae 3562 lipases retained 75 and 97% of respective initial activity on incubation at 90 degrees C, whereas both the free forms became inactive at this temperature. The conversion yield of isoamyl acetate was found to be higher with the immobilized fungal (90 vs. 21%) and bacterial lipases (64 vs. 18%) than the respective free forms. Immobilized R. oryzae 3562 lipases retained 50% activity for isoamyl acetate synthesis up to ten cycles whereas it was eight cycles for E. aerogenes.

  14. Quantitative approach to track lipase producing Pseudomonas sp. S1 in nonsterilized solid state fermentation.

    PubMed

    Sahoo, R K; Subudhi, E; Kumar, M

    2014-06-01

    Proliferation of the inoculated Pseudomonas sp. S1 is quantitatively evaluated using ERIC-PCR during the production of lipase in nonsterile solid state fermentation an approach to reduce the cost of enzyme production. Under nonsterile solid state fermentation with olive oil cake, Pseudomonas sp. S1 produced 57·9 IU g(-1) of lipase. DNA fingerprints of unknown bacterial isolates obtained on Bushnell Haas agar (BHA) + tributyrin exactly matched with that of Pseudomonas sp. S1. Using PCR-based enumeration, population of Pseudomonas sp. S1 was proliferated from 7·6 × 10(4) CFU g(-1) after 24 h to 4·6 × 10(8) CFU g(-1) after 96 h, which tallied with the maximum lipase activity as compared to control. Under submerged fermentation (SmF), Pseudomonas sp. S1 produced maximum lipase (49 IU ml(-1) ) using olive oil as substrate, while lipase production was 9·754 IU ml(-1) when Pseudomonas sp. S1 was grown on tributyrin. Optimum pH and temperature of the crude lipase was 7·0 and 50°C. Crude enzyme activity was 71·2% stable at 50°C for 360 min. Pseudomonas sp. S1 lipase was also stable in methanol showing 91·6% activity in the presence of 15% methanol, whereas 75·5 and 51·1% of activity were retained in the presence of 20 and 30% methanol, respectively. Thus, lipase produced by Pseudomonas sp. S1 is suitable for the production of biodiesel as well as treatment of oily waste water. This study presents the first report on the production of thermophilic organic solvent tolerant lipase using agro-industry waste in nonsterile solid state fermentation. Positive correlation between survival of Pseudomonas sp. S1 and lipase production under nonsterile solid state fermentation was established, which may emphasize the need to combine molecular tools and solid state fermentation in future studies. Our study brings new insights into the lipase production in cost-effective manner, which is an industrially relevant approach. © 2014 The Society for Applied Microbiology.

  15. Thermo-kinetics of lipase-catalyzed synthesis of 6-O-glucosyldecanoate.

    PubMed

    Gumel, A M; Annuar, M S M; Heidelberg, T; Chisti, Y

    2011-10-01

    Lipase-catalyzed synthesis of 6-O-glucosyldecanoate from d-glucose and decanoic acid was performed in dimethyl sulfoxide (DMSO), a mixture of DMSO and tert-butanol and tert-butanol alone with a decreasing order of polarity. The highest conversion yield (> 65%) of decanoic acid was obtained in the blended solvent of intermediate polarity mainly because it could dissolve relatively large amounts of both the reactants. The reaction obeyed Michaelis-Menten type of kinetics. The affinity of the enzyme towards the limiting substrate (decanoic acid) was not affected by the polarity of the solvent, but increased significantly with temperature. The esterification reaction was endothermic with activation energy in the range of 60-67 kJ mol⁻¹. Based on the Gibbs energy values, in the solvent blend of DMSO and tert-butanol the position of the equilibrium was shifted more towards the products compared to the position in pure solvents. Monoester of glucose was the main product of the reaction. Copyright © 2011 Elsevier Ltd. All rights reserved.

  16. Synthesis of structured triacylglycerols containing caproic acid by lipase-catalyzed acidolysis: optimization by response surface methodology.

    PubMed

    Zhou, D; Xu, X; Mu, H; Høy, C E; Adler-Nissen, J

    2001-12-01

    Production in a batch reactor with a solvent-free system of structured triacylglycerols containing short-chain fatty acids by Lipozyme RM IM-catalyzed acidolysis between rapeseed oil and caproic acid was optimized using response surface methodology (RSM). Reaction time (t(r)), substrate ratio (S(r)), enzyme load (E(l), based on substrate), water content (W(c), based on enzyme), and reaction temperature (T(e)), the five most important parameters for the reaction, were chosen for the optimization. The range of each parameter was selected as follows: t(r) = 5-17 h; E(l) = 6-14 wt %; T(e) = 45-65 degrees C; S(r) = 2-6 mol/mol; and W(c) = 2-12 wt %. The biocatalyst was Lipozyme RM IM, in which Rhizomucor miehei lipase is immobilized on a resin. The incorporation of caproic acid into rapeseed oil was the main monitoring response. In addition, the contents of mono-incorporated structured triacylglycerols and di-incorporated structured triacylglycerols were also evaluated. The optimal reaction conditions for the incorporation of caproic acid and the content of di-incorporated structured triacylglycerols were as follows: t(r) = 17 h; S(r) = 5; E(l) = 14 wt %; W(c) = 10 wt %; T(e) = 65 degrees C. At these conditions, products with 55 mol % incorporation of caproic acid and 55 mol % di-incorporated structured triacylglycerols were obtained.

  17. Production and application of a thermostable lipase from Serratia marcescens in detergent formulation and biodiesel production.

    PubMed

    García-Silvera, Edgar Edurman; Martínez-Morales, Fernando; Bertrand, Brandt; Morales-Guzmán, Daniel; Rosas-Galván, Nashbly Sarela; León-Rodríguez, Renato; Trejo-Hernández, María R

    2018-03-01

    In this study, extracellular lipase was produced by Serratia marcescens wild type and three mutant strains. The maximum lipase activity (80 U/mL) was obtained with the SMRG4 mutant strain using soybean oil. Using a 2 2 factorial design, the lipase production increased 1.55-fold (124 U/mL) with 4% and 0.05% of soybean oil and Triton X-100, respectively. The optimum conditions for maximum lipase activity were 50 °C and pH 8. However, the enzyme was active in a broad range of pH (6-10) and temperatures (5-55 °C). This lipase was stable in organic solvents and in the presence of oxidizing agents. The enzyme also proved to be efficient for the removal of triacylglycerol from olive oil in cotton cloth. A Box-Behnken experimental design was used to evaluate the effects of the interactions between total lipase activity, buffer pH, and wash temperatures on oil removal. The model obtained suggested that all selected factors had a significant impact on oil removal, with optimum conditions of 550 U lipase, 45 °C, pH 9.5, with 79.45% removal. Biotransformation of waste frying oil using the enzyme and in presence of methanol resulted in the synthesis of methyl esters such as methyl oleate, methyl palmitate, and methyl stearate. © 2017 International Union of Biochemistry and Molecular Biology, Inc.

  18. Hydrolytic resolution of (R,S)-naproxen 2,2,2-trifluoroethyl thioester by Carica papaya lipase in water-saturated organic solvents.

    PubMed

    Ng, I-Son; Tsai, Shau-Wei

    2005-01-05

    For the first time, the Carica papaya lipase (CPL) stored in crude papain is explored as a potential enantioselective biocatalyst for obtaining chiral acids from their racemic thioesters. Hydrolytic resolution of (R,S)-naproxen 2,2,2-trifluoroethyl thioester in water-saturated organic solvents is employed as a model system for studying the effects of temperature and solvents on lipase activity and enantioselectivity. An optimal temperature of 60 degrees C, based on the initial rate of (S)-thioester and a high enantiomeric ratio (i.e., E-value defined as the ratio of initial rates for both substrates) of >100 at 45 degrees C in isooctane, is obtained. Kinetic analysis, considering product inhibition and enzyme deactivation, is also performed, showing agreement between the experimental and best-fit conversions for (S)-thioester. A comparison of the kinetic and thermodynamic behaviors of CPL and Candida rugosa lipase (CRL) in isooctane and cyclohexane indicates that both lipases are very similar in terms of thermodynamic parameters DeltaDeltaH and DeltaDeltaS, initial rate of (S)-substrate, and E-value when (R,S)-naproxen 2,2,2-trifluoroethyl thioester or ester is employed as substrate. (c) 2004 Wiley Periodicals, Inc.

  19. Inhibitors of Fatty Acid Amide Hydrolase and Monoacylglycerol Lipase: New Targets for Future Antidepressants.

    PubMed

    Ogawa, Shintaro; Kunugi, Hiroshi

    2015-01-01

    Cannabis and analogs of Δ<sup>9</sup>-tetrahydrocannabinol have been used for therapeutic purposes, but their therapeutic use remains limited because of various adverse effects. Endogenous cannabinoids have been discovered, and dysregulation of endocannabinoid signaling is implicated in the pathophysiology of major depressive disorder (MDD). Recently, endocannabinoid hydrolytic enzymes such as fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL) have become new therapeutic targets in the treatment of MDD. Several FAAH or MAGL inhibitors are reported to have no cannabimimetic side effects and, therefore, are new potential therapeutic options for patients with MDD who are resistant to first-line antidepressants (selective serotonin and serotonin-norepinephrine reuptake inhibitors). In this review, we focus on the possible relationships between MDD and the endocannabinoid system as well as the inhibitors' therapeutic potential. MAGL inhibitors may reduce inflammatory responses through activation of cannabinoid receptor type 2. In the hypothalamic-pituitary-adrenal axis, repeated FAAH inhibitor administration may be beneficial for reducing circulating glucocorticoid levels. Both FAAH and MAGL inhibitors may contribute to dopaminergic system regulation. Recently, several new inhibitors have been developed with strong potency and selectivity. FAAH inhibitor, MAGL inhibitor, or dual blocker use would be promising new treatments for MDD. Further pre-clinical studies and clinical trials using these inhibitors are warranted.

  20. Effect of lipolytic activity of Candida adriatica, Candida diddensiae and Yamadazyma terventina on the acidity of extra-virgin olive oil with a different polyphenol and water content.

    PubMed

    Ciafardini, G; Zullo, B A

    2015-05-01

    Previous microbiological research demonstrated the presence of a rich micro-flora composed mainly of yeasts in the suspended fraction of freshly produced olive oil. Some of the yeasts are considered harmful as they can damage the quality of the olive oil through the hydrolysis of the triacylglycerols. Present research has demonstrated that the lipolytic activity of some lipase-producer strains belonging to a yeast species called Candida adriatica, Candida diddensiae and Yamadazyma terventina can be modulated by the water and the polyphenol content of olive oil. Laboratory tests highlighted a substantial increase in free fatty acid in the inoculated olive oil characterized by high water content and low polyphenol concentration. The acidity of the olive oil samples containing 0.06% and 0.31% of water increased significantly by 33% in the lipase-producer yeast strains tested during a period of 2 weeks of incubation at 30 °C. All other yeasts showed strong lipolytic activity in the presence of 1.31% of water - the only exception to this was the C. adriatica 1985 strain. The phenolic compounds typical of olive oil represent another important factor able to condition the viability and the lipolytic activity of the lipase-producer yeasts. From the tests performed on the olive oil characterized by an increasing content of total polyphenols equal to 84, 150 and 510 mg per kg of oil, the percentage of the lipase-producer yeasts able to hydrolyse the triacylglycerols was respectively 100%, 67% and 11%. Copyright © 2014 Elsevier Ltd. All rights reserved.

  1. Secreted Fungal Effector Lipase Releases Free Fatty Acids to Inhibit Innate Immunity-Related Callose Formation during Wheat Head Infection[W][OPEN

    PubMed Central

    Blümke, Antje; Falter, Christian; Herrfurth, Cornelia; Sode, Björn; Bode, Rainer; Schäfer, Wilhelm; Feussner, Ivo; Voigt, Christian A.

    2014-01-01

    The deposition of the (1,3)-β-glucan cell wall polymer callose at sites of attempted penetration is a common plant defense response to intruding pathogens and part of the plant’s innate immunity. Infection of the Fusarium graminearum disruption mutant Δfgl1, which lacks the effector lipase FGL1, is restricted to inoculated wheat (Triticum aestivum) spikelets, whereas the wild-type strain colonized the whole wheat spike. Our studies here were aimed at analyzing the role of FGL1 in establishing full F. graminearum virulence. Confocal laser-scanning microscopy revealed that the Δfgl1 mutant strongly induced the deposition of spot-like callose patches in vascular bundles of directly inoculated spikelets, while these callose deposits were not observed in infections by the wild type. Elevated concentrations of the polyunsaturated free fatty acids (FFAs) linoleic and α-linolenic acid, which we detected in F. graminearum wild type-infected wheat spike tissue compared with Δfgl1-infected tissue, provided clear evidence for a suggested function of FGL1 in suppressing callose biosynthesis. These FFAs not only inhibited plant callose biosynthesis in vitro and in planta but also partially restored virulence to the Δfgl1 mutant when applied during infection of wheat spikelets. Additional FFA analysis confirmed that the purified effector lipase FGL1 was sufficient to release linoleic and α-linolenic acids from wheat spike tissue. We concluded that these two FFAs have a major function in the suppression of the innate immunity-related callose biosynthesis and, hence, the progress of F. graminearum wheat infection. PMID:24686113

  2. Carica papaya lipase: a naturally immobilized enzyme with interesting biochemical properties.

    PubMed

    Abdelkafi, Slim; Barouh, Nathalie; Fouquet, Benjamin; Fendri, Imen; Pina, Michel; Scheirlinckx, Frantz; Villeneuve, Pierre; Carrière, Frédéric

    2011-03-01

    Triacylglycerol (TAG) lipases have been thoroughly characterized in mammals and microorganisms, whereas very little is known about plant TAG lipases. The lipolytic activity occurring in all the laticies is known to be associated with sedimentable particles, and all attempts to solubilize the lipolytic activity of Carica papaya latex have been unsuccessful so far. However, some of the biochemical properties of the lipase from Carica papaya latex (CPL) were determined from the insoluble fraction of the latex. The activity was optimum at a temperature of 37°C and a pH of 9.0, and the specific activities of CPL were found to be 2,000 ± 185 and 256 ± 8 U/g when tributyrin and olive oil were used as substrates, respectively. CPL was found to be active in the absence of any detergent, whereas many lipases require detergent to prevent the occurrence of interfacial denaturation. CPL was inactive in the presence of micellar concentrations of Triton X-100, sodium dodecyl sulfate (SDS) and tetradecyl trimethylammonium bromide (TTAB), and still showed high levels of activity in the presence of sodium taurodeoxycholate (NaTDC) and the zwitterionic Chaps detergent. The effects of various proteases on the lipolytic activity of CPL were studied, and CPL was found to be resistant to treatment with various enzymes, except in the presence of trypsin. All these properties suggest that CPL may be a good candidate for various biotechnological applications.

  3. Exploring the specific features of interfacial enzymology based on lipase studies.

    PubMed

    Aloulou, Ahmed; Rodriguez, Jorge A; Fernandez, Sylvie; van Oosterhout, Dirk; Puccinelli, Delphine; Carrière, Frédéric

    2006-09-01

    Many enzymes are active at interfaces in the living world (such as in the signaling processes at the surface of cell membranes, digestion of dietary lipids, starch and cellulose degradation, etc.), but fundamental enzymology remains largely focused on the interactions between enzymes and soluble substrates. The biochemical and kinetic characterization of lipolytic enzymes has opened up new paths of research in the field of interfacial enzymology. Lipases are water-soluble enzymes hydrolyzing insoluble triglyceride substrates, and studies on these enzymes have led to the development of specific interfacial kinetic models. Structure-function studies on lipases have thrown light on the interfacial recognition sites present in the molecular structure of these enzymes, the conformational changes occurring in the presence of lipids and amphiphiles, and the stability of the enzymes present at interfaces. The pH-dependent activity, substrate specificity and inhibition of these enzymes can all result from both "classical" interactions between a substrate or inhibitor and the active site, as well as from the adsorption of the enzymes at the surface of aggregated substrate particles such as oil drops, lipid bilayers or monomolecular lipid films. The adsorption step can provide an alternative target for improving substrate specificity and developing specific enzyme inhibitors. Several data obtained with gastric lipase, classical pancreatic lipase, pancreatic lipase-related protein 2 and phosphatidylserine-specific phospholipase A1 were chosen here to illustrate these specific features of interfacial enzymology.

  4. Fucosyltransferase 2 (FUT2) non-secretor status and blood group B are associated with elevated serum lipase activity in asymptomatic subjects, and an increased risk for chronic pancreatitis: a genetic association study.

    PubMed

    Weiss, Frank Ulrich; Schurmann, Claudia; Guenther, Annett; Ernst, Florian; Teumer, Alexander; Mayerle, Julia; Simon, Peter; Völzke, Henry; Radke, Dörte; Greinacher, Andreas; Kuehn, Jens-Peter; Zenker, Martin; Völker, Uwe; Homuth, Georg; Lerch, Markus M

    2015-04-01

    Serum lipase activities above the threefold upper reference limit indicate acute pancreatitis. We investigated whether high lipase activity-within the reference range and in the absence of pancreatitis-are associated with genetic single nucleotide polymorphisms (SNP), and whether these identified SNPs are also associated with clinical pancreatitis. Genome-wide association studies (GWAS) on phenotypes 'serum lipase activity' and 'high serum lipase activity' were conducted including 3966 German volunteers from the population-based Study-of-Health-in-Pomerania (SHIP). Lead SNPs associated on a genome-wide significance level were replicated in two cohorts, 1444 blood donors and 1042 pancreatitis patients. Initial discovery GWAS detected SNPs within or near genes encoding the ABO blood group specifying transferases A/B (ABO), Fucosyltransferase-2 (FUT2), and Chymotrypsinogen-B2 (CTRB2), to be significantly associated with lipase activity levels in asymptomatic subjects. Replication analyses in blood donors confirmed the association of FUT-2 non-secretor status (OR=1.49; p=0.012) and ABO blood-type-B (OR=2.48; p=7.29×10(-8)) with high lipase activity levels. In pancreatitis patients, significant associations were found for FUT-2 non-secretor status (OR=1.53; p=8.56×10(-4)) and ABO-B (OR=1.69, p=1.0×10(-4)) with chronic pancreatitis, but not with acute pancreatitis. Conversely, carriers of blood group O were less frequently affected by chronic pancreatitis (OR=0.62; p=1.22×10(-05)) and less likely to have high lipase activity levels (OR=0.59; p=8.14×10(-05)). These are the first results indicating that ABO blood type-B as well as FUT2 non-secretor status are common population-wide risk factors for developing chronic pancreatitis. They also imply that, even within the reference range, elevated lipase activities may indicate subclinical pancreatic injury in asymptomatic subjects. Published by the BMJ Publishing Group Limited. For permission to use (where not already

  5. Screening and characterization of a novel thermostable lipase with detergent-additive potential from the metagenomic library of a mangrove soil.

    PubMed

    Tang, Leli; Xia, Yulin; Wu, Xiuling; Chen, Xiaoli; Zhang, Xueling; Li, He

    2017-08-20

    One clone (Lip906) exhibiting lipase activity was screened from a metagenomic library by using a medium containing tricaprylin. A novel lipase gene from the inserted fragment of Lip906 was obtained by sequencing. The phylogenetic analysis of Lip906 lipase exhibited 34% and 32% homologue to lipases from Streptomyces sp. MspMP-M5 and Rhodopirellula europaea. This gene was expressed in Escherichia coli (E. coli) BL21 (DE3), and the recombinant protein was purified and characterized. The best substrate of the recombinant Lip906 lipase was p-nitrophenyl myristate (C14). The lipase expressed maximum activity at 74°C and pH7.8, and it was found to be stable at pH values and temperatures ranging from 6.0-8.0 and 4-78°C, respectively. Furthermore, the lipase was found to be highly resistant to commercial detergent, DMSO, and EDTA, whereas its activity was stimulated in the presence of methanol and ethanol at low concentrations. The lipase showed enhanced activity in the presence of Hg 2+ , whereas the presence of the metal ions Fe 2+ , Ca 2+ , Co 2+ , and Mg 2+ inhibited the activity. These beneficial characteristics of Lip906 lipase provide some advantages for its potential application in industry. Copyright © 2017. Published by Elsevier B.V.

  6. Optimisation of flavour ester biosynthesis in an aqueous system of coconut cream and fusel oil catalysed by lipase.

    PubMed

    Sun, Jingcan; Yu, Bin; Curran, Philip; Liu, Shao-Quan

    2012-12-15

    Coconut cream and fusel oil, two low-cost natural substances, were used as starting materials for the biosynthesis of flavour-active octanoic acid esters (ethyl-, butyl-, isobutyl- and (iso)amyl octanoate) using lipase Palatase as the biocatalyst. The Taguchi design method was used for the first time to optimize the biosynthesis of esters by a lipase in an aqueous system of coconut cream and fusel oil. Temperature, time and enzyme amount were found to be statistically significant factors and the optimal conditions were determined to be as follows: temperature 30°C, fusel oil concentration 9% (v/w), reaction time 24h, pH 6.2 and enzyme amount 0.26 g. Under the optimised conditions, a yield of 14.25mg/g (based on cream weight) and signal-to-noise (S/N) ratio of 23.07 dB were obtained. The results indicate that the Taguchi design method was an efficient and systematic approach to the optimisation of lipase-catalysed biological processes. Copyright © 2012 Elsevier Ltd. All rights reserved.

  7. Characterization of Proteus vulgaris K80 lipase immobilized on amine-terminated magnetic microparticles.

    PubMed

    Natalia, Agnes; Kristiani, Lidya; Kim, Hyung Kwoun

    2014-10-01

    Proteus vulgaris K80 lipase was expressed in Escherichia coli BL21 (DE3) cells and immobilized on amine-terminated magnetic microparticles (Mag-MPs). The immobilization yield and activity retention were 84.15% and 7.87%, respectively. A homology model of lipase K80 was constructed using P. mirabilis lipase as the template. Many lysine residues were located on the protein surface, remote from active sites. The biochemical characteristics of immobilized lipase K80 were compared with the soluble free form of lipase K80. The optimum temperature of K80-Mag-MPs was 60°C, which was 20°C higher than that of the soluble form. K80-Mag-MPs also tended to be more stable than the soluble form at elevated temperatures and a broad range of pH. K80-Mag-MP maintained its stable form at up to 40°C and in a pH range of 5.0- 10.0, whereas soluble K80 maintained its activity up to 35°C and pH 6.0-10.0. K80-Mag-MPs had broader substrate specificity compared with that of soluble K80. K80-Mag-MPs showed about 80% residual relative activity after five recovery trials. These results indicate the potential benefit of K80-Mag-MPs as a biocatalyst in various industries.

  8. Immobilised lipases in the cosmetics industry.

    PubMed

    Ansorge-Schumacher, Marion B; Thum, Oliver

    2013-08-07

    Commercial products for personal care, generally perceived as cosmetics, have an important impact on everyday life worldwide. Accordingly, the market for both consumer products and specialty chemicals comprising their ingredients is considerable. Lipases have started to play a minor role as active ingredients in so-called 'functional cosmetics' as well as a major role as catalysts for the industrial production of various specialty esters, aroma compounds and active agents. Interestingly, both applications almost always require preparation by appropriate immobilisation techniques. In addition, for catalytic use special reactor concepts often have to be employed due to the mostly limited stability of these preparations. Nevertheless, these processes show distinct advantages based on process simplification, product quality and environmental footprint and are therefore apt to more and more replace traditional chemical processes. Here, for the first time a review on the various aspects of using immobilised lipases in the cosmetics industry is given.

  9. Transesterification of Waste Frying Oil and Soybean Oil by Combi-lipases Under Ultrasound-Assisted Reactions.

    PubMed

    Poppe, Jakeline Kathiele; Matte, Carla Roberta; Fernandez-Lafuente, Roberto; Rodrigues, Rafael C; Ayub, Marco Antônio Záchia

    2018-04-21

    This work describes the use of an ultrasound system for the enzymatic transesterification of oils using combi-lipases as biocatalyst. The reactions were carried out evaluating the individual use of waste oil and fresh soybean oil, and the immobilized lipases CALB, TLL, and RML were used as biocatalysts. It was performed in a mixture design of three factors to obtain the ideal mixture of lipases according to the composition of fatty acids present in each oil, and the main reaction variables were optimized. After 18 h of reaction, ultrasound provided a biodiesel yield of about 90% when using soybean oil and 70% using the waste oil. The results showed that ultrasound technology, in combination with the application of enzyme mixtures, known as combi-lipases, and the use of waste oil, could be a promising route to reduce the overall process costs of enzymatic production of biodiesel.

  10. 21 CFR 184.1415 - Animal lipase.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 3 2014-04-01 2014-04-01 false Animal lipase. 184.1415 Section 184.1415 Food and....1415 Animal lipase. (a) Animal lipase (CAS Reg. No. 9001-62-1) is an enzyme preparation obtained from edible forestomach tissue of calves, kids, or lambs, or from animal pancreatic tissue. The enzyme...

  11. Lipase Test: MedlinePlus Lab Test Information

    MedlinePlus

    ... page: https://medlineplus.gov/labtests/lipasetest.html Lipase Test To use the sharing features on this page, please enable JavaScript. What is a lipase test? Lipase is a type of protein made by ...

  12. Maturation of hepatic lipase. Formation of functional enzyme in the endoplasmic reticulum is the rate-limiting step in its secretion.

    PubMed

    Ben-Zeev, Osnat; Doolittle, Mark H

    2004-02-13

    Among three lipases in the lipase gene family, hepatic lipase (HL), lipoprotein lipase, and pancreatic lipase, HL exhibits the lowest intracellular specific activity (i.e. minimal amounts of catalytic activity accompanied by massive amounts of inactive lipase mass in the endoplasmic reticulum (ER)). In addition, HL has a distinctive sedimentation profile, where the inactive mass overlaps the region containing active dimeric HL and trails into progressively larger molecular forms. Eventually, at least half of the HL inactive mass in the ER reaches an active, dimeric conformation (t(1/2) = 2 h) and is rapidly secreted. The remaining inactive mass is degraded. HL maturation occurs in the ER and is strongly dependent on binding to calnexin in the early co-/post-translational stages. Later stages of HL maturation occur without calnexin assistance, although inactive HL at all stages appears to be associated in distinct complexes with other ER proteins. Thus, unlike other lipases in the gene family, HL maturation is the rate-limiting step in its secretion as a functional enzyme.

  13. GDSL lipases modulate immunity through lipid homeostasis in rice

    PubMed Central

    Lam, Sin Man; Tong, Xiaohong; Liu, Jiyun; Wang, Xin; Shui, Guanghou

    2017-01-01

    Lipids and lipid metabolites play important roles in plant-microbe interactions. Despite the extensive studies of lipases in lipid homeostasis and seed oil biosynthesis, the involvement of lipases in plant immunity remains largely unknown. In particular, GDSL esterases/lipases, characterized by the conserved GDSL motif, are a subfamily of lipolytic enzymes with broad substrate specificity. Here, we functionally identified two GDSL lipases, OsGLIP1 and OsGLIP2, in rice immune responses. Expression of OsGLIP1 and OsGLIP2 was suppressed by pathogen infection and salicylic acid (SA) treatment. OsGLIP1 was mainly expressed in leaf and leaf sheath, while OsGLIP2 showed high expression in elongating internodes. Biochemical assay demonstrated that OsGLIP1 and OsGLIP2 are functional lipases that could hydrolyze lipid substrates. Simultaneous down-regulation of OsGLIP1 and OsGLIP2 increased plant resistance to both bacterial and fungal pathogens, whereas disease resistance in OsGLIP1 and OsGLIP2 overexpression plants was significantly compromised, suggesting that both genes act as negative regulators of disease resistance. OsGLIP1 and OsGLIP2 proteins mainly localize to lipid droplets and the endoplasmic reticulum (ER) membrane. The proper cellular localization of OsGLIP proteins is indispensable for their functions in immunity. Comprehensive lipid profiling analysis indicated that the alteration of OsGLIP gene expression was associated with substantial changes of the levels of lipid species including monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG). We show that MGDG and DGDG feeding could attenuate disease resistance. Taken together, our study indicates that OsGLIP1 and OsGLIP2 negatively regulate rice defense by modulating lipid metabolism, thus providing new insights into the function of lipids in plant immunity. PMID:29131851

  14. 21 CFR 184.1415 - Animal lipase.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 3 2011-04-01 2011-04-01 false Animal lipase. 184.1415 Section 184.1415 Food and... Substances Affirmed as GRAS § 184.1415 Animal lipase. (a) Animal lipase (CAS Reg. No. 9001-62-1) is an enzyme preparation obtained from edible forestomach tissue of calves, kids, or lambs, or from animal pancreatic...

  15. 21 CFR 184.1415 - Animal lipase.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 3 2013-04-01 2013-04-01 false Animal lipase. 184.1415 Section 184.1415 Food and... Substances Affirmed as GRAS § 184.1415 Animal lipase. (a) Animal lipase (CAS Reg. No. 9001-62-1) is an enzyme preparation obtained from edible forestomach tissue of calves, kids, or lambs, or from animal pancreatic...

  16. 21 CFR 184.1415 - Animal lipase.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 3 2012-04-01 2012-04-01 false Animal lipase. 184.1415 Section 184.1415 Food and... Substances Affirmed as GRAS § 184.1415 Animal lipase. (a) Animal lipase (CAS Reg. No. 9001-62-1) is an enzyme preparation obtained from edible forestomach tissue of calves, kids, or lambs, or from animal pancreatic...

  17. 21 CFR 184.1415 - Animal lipase.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Animal lipase. 184.1415 Section 184.1415 Food and... Substances Affirmed as GRAS § 184.1415 Animal lipase. (a) Animal lipase (CAS Reg. No. 9001-62-1) is an enzyme preparation obtained from edible forestomach tissue of calves, kids, or lambs, or from animal pancreatic...

  18. Assembly of lipase and P450 fatty acid decarboxylase to constitute a novel biosynthetic pathway for production of 1-alkenes from renewable triacylglycerols and oils.

    PubMed

    Yan, Jinyong; Liu, Yi; Wang, Cong; Han, Bingnan; Li, Shengying

    2015-01-01

    Biogenic hydrocarbons (biohydrocarbons) are broadly accepted to be the ideal 'drop-in' biofuel alternative to petroleum-based fuels due to their highly similar chemical composition and physical characteristics. The biological production of aliphatic hydrocarbons is largely dependent on engineering of the complicated enzymatic network surrounding fatty acid biosynthesis. In this work, we developed a novel system for bioproduction of terminal fatty alkenes (1-alkenes) from renewable and low-cost triacylglycerols (TAGs) based on the lipase hydrolysis coupled to the P450 catalyzed decarboxylation. This artificial biosynthetic pathway was constituted using both cell-free systems including purified enzymes or cell-free extracts, and cell-based systems including mixed resting cells or growing cells. The issues of high cost of fatty acid feedstock and complicated biosynthesis network were addressed by replacement of the de novo biosynthesized fatty acids with the fed cheap TAGs. This recombinant tandem enzymatic pathway consisting of the Thermomyces lanuginosus lipase (Tll) and the P450 fatty acid decarboxylase OleTJE resulted in the production of 1-alkenes from purified TAGs or natural oils with 6.7 to 46.0% yields. Since this novel hydrocarbon-producing pathway only requires two catalytically efficient enzymatic steps, it may hold great potential for industrial application by fulfilling the large-scale and cost-effective conversion of renewable TAGs into biohydrocarbons. This work highlights the power of designing and implementing an artificial pathway for production of advanced biofuels.

  19. Inhibitors of pancreatic lipase: state of the art and clinical perspectives

    PubMed Central

    Lunagariya, Nitin A.; Patel, Neeraj K.; Jagtap, Sneha C.; Bhutani, Kamlesh K.

    2014-01-01

    Obesity is a disorder of lipid metabolism and continues to be a global problem, ranking fifth for deaths worldwide. It also leads to diabetes, cardiovascular disorders, musculoskeletal disorders and some types of cancer. Obesity is regarded as the output of a long-term imbalance between energy intake and energy expenditure. Digestion and absorption of dietary lipids by pancreatic lipase, a major source of excess calorie intake, can be targeted for development of anti-obesity agents. Being the major factor of concern, food materials and edible plants are most widely studied for the anti-obesity activity, so that they can be incorporated in the routine diet. In this review, an attempt was made to present a current scenario of the bioactive compounds from plant and microbial origin that have been investigated for their pancreatic lipase inhibition. Compounds belonging to various classes of natural products such as alkaloids, carotenoids, glycosides, polyphenols, polysaccharides, saponins and terpenoids are well studied while lipophilic compounds from microbial sources are the most active against the pancreatic lipase. Few studies on the synthetic analogues, structurally similar to the triglycerides have been described in the review. Despite of tremendous research on the finding of potential pancreatic lipase inhibitor, very few compounds have entered the clinical studies and no new molecule after orlistat has been marketed. Along with HTS based screening, detailed structure-activity relationship studies on semi-synthetic and synthetic derivatives might also provide a direction for the development of potential lead(s) or pharmacophore for pancreatic lipase inhibition in order to treat and/or prevent obesity and related disorders. PMID:26417311

  20. Biodiesel production with special emphasis on lipase-catalyzed transesterification.

    PubMed

    Bisen, Prakash S; Sanodiya, Bhagwan S; Thakur, Gulab S; Baghel, Rakesh K; Prasad, G B K S

    2010-08-01

    The production of biodiesel by transesterification employing acid or base catalyst has been industrially accepted for its high conversion and reaction rates. Downstream processing costs and environmental problems associated with biodiesel production and byproducts recovery have led to the search for alternative production methods. Recently, enzymatic transesterification involving lipases has attracted attention for biodiesel production as it produces high purity product and enables easy separation from the byproduct, glycerol. The use of immobilized lipases and immobilized whole cells may lower the overall cost, while presenting less downstream processing problems, to biodiesel production. The present review gives an overview on biodiesel production technology and analyzes the factors/methods of enzymatic approach reported in the literature and also suggests suitable method on the basis of evidence for industrial production of biodiesel.

  1. Effects of dietary energy and lipase levels on nutrient digestibility, digestive physiology and noxious gas emission in weaning pigs.

    PubMed

    Liu, Jingbo; Cao, S C; Liu, J; Pu, J; Chen, L; Zhang, H F

    2018-05-31

    This study was conducted to evaluate the effect of dietary energy and lipase supplementation on growth performance, nutrient digestibility, serum profiles, intestinal morphology, small intestinal digestive enzyme activities, biochemical index of intestinal development and noxious gas emission in weaning pigs. A total of 240 weaning pigs [(Yorkshire×Landrace)×Duroc)] with an average BW of 7.3 ± 0.12 kg were used in this 28-d experiment. Weaning pigs were randomly allocated to 4 dietary treatments in a 2 × 2 factorial arrangement with 2 levels of energy (NE = 2,470 kcal/kg for low energy diet and 2,545 kcal/kg for basal diet) and 2 levels of lipase (0 and 1.5 U/g of lipase) according to BW and sex. There were 6 replications (pens) per treatment and 10 pigs per pen (5 barrows and 5 gilts). Weaning pigs fed the low energy diet had lower (p<0.05) G:F throughout the experiment, apparent digestibility of DM, N, EE, and GE during d 0 to 14, ADG during d 15 to 28, lipase activity in duodenum and ileum and protein/DNA in jejunum (p<0.05), respectively. Lipase supplementation had no effect on growth performance but affected apparent nutrient digestibility (p<0.05) on d 14 and enhanced lipase activity in the duodenum and ileum and protease activity in duodenum and jejunum of pigs (p<0.05) fed the low energy diet. Lipase reduced serum low-density lipoprotein cholesterol (LDL-C) and triglyceride (TG), NH3 production (p<0.05) from the feces. The low energy diet decreased G:F throughout the experiment and nutrient digestibility during d 0 to 14 as well as lipase activity in duodenum and ileum. Lipase supplementation increased nutrient digestibility during d 0 to 14 and exerted beneficial effects on lipase activity in duodenum and ileum as well as protease activity in duodenum and jejunum, while reduced serum LDL-C, TG and fecal NH3.

  2. Predicting the impact of mutations on the specific activity of Bacillus thermocatenulatus lipase using a combined approach of docking and molecular dynamics.

    PubMed

    Yukselen, Onur; Timucin, Emel; Sezerman, Ugur

    2016-10-01

    Lipases are important biocatalysts owing to their ability to catalyze diverse reactions with exceptional substrate specificities. A combined docking and molecular dynamics (MD) approach was applied to study the chain-length selectivity of Bacillus thermocatenulatus lipase (BTL2) towards its natural substrates (triacylglycerols). A scoring function including electrostatic, van der Waals (vdW) and desolvation energies along with conformational entropy was developed to predict the impact of mutation. The native BTL2 and its 6 mutants (F17A, V175A, V175F, D176F, T178V and I320F) were experimentally analyzed to determine their specific activities towards tributyrin (C4) or tricaprylin (C8), which were used to test our approach. Our scoring methodology predicted the chain-length selectivity of BTL2 with 85.7% (6/7) accuracy with a positive correlation between the calculated scores and the experimental activity values (r = 0.82, p = 0.0004). Additionally, the impact of mutation on activity was predicted with 75% (9/12) accuracy. The described study represents a fast and reliable approach to accurately predict the effect of mutations on the activity and selectivity of lipases and also of other enzymes. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

  3. Lipoprotein lipase regulates hematopoietic stem progenitor cell maintenance through DHA supply.

    PubMed

    Liu, Chao; Han, Tianxu; Stachura, David L; Wang, Huawei; Vaisman, Boris L; Kim, Jungsu; Klemke, Richard L; Remaley, Alan T; Rana, Tariq M; Traver, David; Miller, Yury I

    2018-04-03

    Lipoprotein lipase (LPL) mediates hydrolysis of triglycerides (TGs) to supply free fatty acids (FFAs) to tissues. Here, we show that LPL activity is also required for hematopoietic stem progenitor cell (HSPC) maintenance. Knockout of Lpl or its obligatory cofactor Apoc2 results in significantly reduced HSPC expansion during definitive hematopoiesis in zebrafish. A human APOC2 mimetic peptide or the human very low-density lipoprotein, which carries APOC2, rescues the phenotype in apoc2 but not in lpl mutant zebrafish. Creating parabiotic apoc2 and lpl mutant zebrafish rescues the hematopoietic defect in both. Docosahexaenoic acid (DHA) is identified as an important factor in HSPC expansion. FFA-DHA, but not TG-DHA, rescues the HSPC defects in apoc2 and lpl mutant zebrafish. Reduced blood cell counts are also observed in Apoc2 mutant mice at the time of weaning. These results indicate that LPL-mediated release of the essential fatty acid DHA regulates HSPC expansion and definitive hematopoiesis.

  4. Lipoprotein lipase variants interact with polyunsaturated fatty acids for obesity traits in women: replication in two populations.

    PubMed

    Ma, Y; Tucker, K L; Smith, C E; Lee, Y C; Huang, T; Richardson, K; Parnell, L D; Lai, C Q; Young, K L; Justice, A E; Shao, Y; North, K E; Ordovás, J M

    2014-12-01

    Lipoprotein lipase (LPL) is a candidate gene for obesity based on its role in triglyceride hydrolysis and the partitioning of fatty acids towards storage or oxidation. Whether dietary fatty acids modify LPL associated obesity risk is unknown. We examined five single nucleotide polymorphisms (SNPs) (rs320, rs2083637, rs17411031, rs13702, rs2197089) for potential interaction with dietary fatty acids for obesity traits in 1171 participants (333 men and 838 women, aged 45-75 y) of the Boston Puerto Rican Health Study (BPRHS). In women, SNP rs320 interacted with dietary polyunsaturated fatty acids (PUFA) for body mass index (BMI) (P = 0.002) and waist circumference (WC) (P = 0.001) respectively. Higher intake of PUFA was associated with lower BMI and WC in homozygotes of the major allele (TT) (P = 0.01 and 0.005) but not in minor allele carriers (TG and GG). These interactions were replicated in an independent population, African American women of the Atherosclerosis Risk in Communities (ARIC) study (n = 1334). Dietary PUFA modulated the association of LPL rs320 with obesity traits in two independent populations. These interactions may be relevant to the dietary management of obesity, particularly in women. Copyright © 2014. Published by Elsevier B.V.

  5. Lipoprotein lipase variants interact with polyunsaturated fatty acids for obesity traits in women: Replication in two populations

    PubMed Central

    Ma, Y.; Tucker, K.L.; Smith, C.E.; Lee, Y.C.; Huang, T.; Richardson, K.; Parnell, L.D.; Lai, C.Q.; Young, K.L.; Justice, A.E.; Shao, Y.; North, K.E.; Ordovás, J.M.

    2015-01-01

    Background and aims Lipoprotein lipase (LPL) is a candidate gene for obesity based on its role in triglyceride hydrolysis and the partitioning of fatty acids towards storage or oxidation. Whether dietary fatty acids modify LPL associated obesity risk is unknown. Methods and results We examined five single nucleotide polymorphisms (SNPs) (rs320, rs2083637, rs17411031, rs13702, rs2197089) for potential interaction with dietary fatty acids for obesity traits in 1171 participants (333 men and 838 women, aged 45–75 y) of the Boston Puerto Rican Health Study (BPRHS). In women, SNP rs320 interacted with dietary polyunsaturated fatty acids (PUFA) for body mass index (BMI) (P = 0.002) and waist circumference (WC) (P = 0.001) respectively. Higher intake of PUFA was associated with lower BMI and WC in homozygotes of the major allele (TT) (P = 0.01 and 0.005) but not in minor allele carriers (TG and GG). These interactions were replicated in an independent population, African American women of the Atherosclerosis Risk in Communities (ARIC) study (n = 1334). Conclusion Dietary PUFA modulated the association of LPL rs320 with obesity traits in two independent populations. These interactions may be relevant to the dietary management of obesity, particularly in women. PMID:25156894

  6. Tetrahydrolipstatin Inhibition, Functional Analyses, and Three-dimensional Structure of a Lipase Essential for Mycobacterial Viability

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Crellin, Paul K.; Vivian, Julian P.; Scoble, Judith

    2010-09-17

    The highly complex and unique mycobacterial cell wall is critical to the survival of Mycobacteria in host cells. However, the biosynthetic pathways responsible for its synthesis are, in general, incompletely characterized. Rv3802c from Mycobacterium tuberculosis is a partially characterized phospholipase/thioesterase encoded within a genetic cluster dedicated to the synthesis of core structures of the mycobacterial cell wall, including mycolic acids and arabinogalactan. Enzymatic assays performed with purified recombinant proteins Rv3802c and its close homologs from Mycobacterium smegmatis (MSMEG{_}6394) and Corynebacterium glutamicum (NCgl2775) show that they all have significant lipase activities that are inhibited by tetrahydrolipstatin, an anti-obesity drug that coincidentlymore » inhibits mycobacterial cell wall biosynthesis. The crystal structure of MSMEG{_}6394, solved to 2.9 {angstrom} resolution, revealed an {alpha}/{beta} hydrolase fold and a catalytic triad typically present in esterases and lipases. Furthermore, we demonstrate direct evidence of gene essentiality in M. smegmatis and show the structural consequences of loss of MSMEG{_}6394 function on the cellular integrity of the organism. These findings, combined with the predicted essentiality of Rv3802c in M. tuberculosis, indicate that the Rv3802c family performs a fundamental and indispensable lipase-associated function in mycobacteria.« less

  7. Tetrahydrolipstatin inhibition, functional analyses, and three-dimensional structure of a lipase essential for mycobacterial viability.

    PubMed

    Crellin, Paul K; Vivian, Julian P; Scoble, Judith; Chow, Frances M; West, Nicholas P; Brammananth, Rajini; Proellocks, Nicholas I; Shahine, Adam; Le Nours, Jerome; Wilce, Matthew C J; Britton, Warwick J; Coppel, Ross L; Rossjohn, Jamie; Beddoe, Travis

    2010-09-24

    The highly complex and unique mycobacterial cell wall is critical to the survival of Mycobacteria in host cells. However, the biosynthetic pathways responsible for its synthesis are, in general, incompletely characterized. Rv3802c from Mycobacterium tuberculosis is a partially characterized phospholipase/thioesterase encoded within a genetic cluster dedicated to the synthesis of core structures of the mycobacterial cell wall, including mycolic acids and arabinogalactan. Enzymatic assays performed with purified recombinant proteins Rv3802c and its close homologs from Mycobacterium smegmatis (MSMEG_6394) and Corynebacterium glutamicum (NCgl2775) show that they all have significant lipase activities that are inhibited by tetrahydrolipstatin, an anti-obesity drug that coincidently inhibits mycobacterial cell wall biosynthesis. The crystal structure of MSMEG_6394, solved to 2.9 Å resolution, revealed an α/β hydrolase fold and a catalytic triad typically present in esterases and lipases. Furthermore, we demonstrate direct evidence of gene essentiality in M. smegmatis and show the structural consequences of loss of MSMEG_6394 function on the cellular integrity of the organism. These findings, combined with the predicted essentiality of Rv3802c in M. tuberculosis, indicate that the Rv3802c family performs a fundamental and indispensable lipase-associated function in mycobacteria.

  8. Facile fabrication of a stable and recyclable lipase@amine-functionalized ZIF-8 nanoparticles for esters hydrolysis and transesterification

    NASA Astrophysics Data System (ADS)

    Cheong, Ling-Zhi; Wei, Yayu; Wang, Hongbin; Wang, Zhiying; Su, Xiurong; Shen, Cai

    2017-08-01

    Zeolitic imidazolate frameworks (ZIF) represent one of the metal organic frameworks (MOF) with high potential for enzyme immobilization due to their exceptional chemical and thermal stability, negligible cytotoxicity, and easy synthesis under mild biocompatible conditions. Amine-functionalized ZIF-8 (An-ZIF-8) are capable of forming multipoint attachment via hydrogen bonding with lipase which will immobilize and further enhance stabilization of lipase. In addition, increased hydrophilicity of An-ZIF-8 will increase partitioning of An-ZIF-8 immobilized lipase at the aqueous/organic interface which enable lipase to expose its active site and retain its catalytic activity at its highest. Present study reports the use of ZIF-8 and An-ZIF-8 nanoparticles as carrier for Burkholderia cepacia lipase (BCL), compares the ester hydrolysis and transesterification activities of immobilized lipase with those of free lipase, and evaluates the reusability and recovery rate of the immobilized lipase. An-ZIF-8 nanoparticles (average 130.42 ± 0.55 nm) were facilely synthesized via mixing ZIF-8 nanoparticles with ammonia hydroxide solution. Despite having similar characteristics of high crystallinity and forming cuboid-like particles, An-ZIF-8 demonstrated significantly ( P < 0.05) lower Brunauer-Emmett-Teller (BET) surface area and higher thermal stability than ZIF-8. BCL were successfully immobilized on ZIF-8 (BCL@ZIF-8) and An-ZIF-8 (BCL@An-ZIF-8) nanoparticles with an average lipase loading rate of 8 mg/g MOF. The immobilized BCL demonstrated no significant differences in terms of esters hydrolysis and transesterification activities with those of free BCL. BCL@An-ZIF-8 demonstrated superior catalytic stability in comparison to BCL@ZIF-8 with retainment of more than 80% of its initial hydrolysis and transesterification activity for at least 10 repeated runs. In addition, more than 80% of the BCL@An-ZIF-8 can be easily recovered during each cycle of the reusability test through

  9. The Purification and Characterization of Lipases from Lasiodiplodia theobromae, and Their Immobilization and Use for Biodiesel Production from Coconut Oil.

    PubMed

    Venkatesagowda, Balaji; Ponugupaty, Ebenezer; Barbosa-Dekker, Aneli M; Dekker, Robert F H

    2017-12-18

    The coconut kernel-associated fungus, Lasiodiplodia theobromae VBE1, was grown on coconut cake with added coconut oil as lipase inducer under solid-state fermentation conditions. The extracellular-produced lipases were purified and resulted in two enzymes: lipase A (68,000 Da)-purified 25.41-fold, recovery of 47.1%-and lipase B (32,000 Da)-purified 18.47-fold, recovery of 8.2%. Both lipases showed optimal activity at pH 8.0 and 35 °C, were activated by Ca 2+ , exhibited highest specificity towards coconut oil and p-nitrophenyl palmitate, and were stable in iso-octane and hexane. Ethanol supported higher lipase activity than methanol, and n-butanol inactivated both lipases. Crude lipase immobilized by entrapment within 4% (w/v) calcium alginate beads was more stable than the crude-free lipase preparation within the range pH 2.5-10.0 and 20-80 °C. The immobilized lipase preparation was used to catalyze the transesterification/methanolysis of coconut oil to biodiesel (fatty acyl methyl esters (FAMEs)) and was quantified by gas chromatography. The principal FAMEs were laurate (46.1%), myristate (22.3%), palmitate (9.9%), and oleate (7.2%), with minor amounts of caprylate, caprate, and stearate also present. The FAME profile was comparatively similar to NaOH-mediated transesterified biodiesel from coconut oil, but distinctly different to petroleum-derived diesel. This study concluded that Lasiodiplodia theobromae VBE1 lipases have potential for biodiesel production from coconut oil.

  10. Efficient production of biodiesel from waste grease: one-pot esterification and transesterification with tandem lipases.

    PubMed

    Yan, Jinyong; Li, Aitao; Xu, Yi; Ngo, Thao P N; Phua, Szechao; Li, Zhi

    2012-11-01

    A novel concept and efficient method for producing biodiesel (FAME) from grease (15-40wt% free fatty acid, FFA) were developed by using tandem lipases for one-pot esterification of FFA and transesterification of triglyceride with methanol in a solvent-free system. Combining immobilized Candida antarctica lipase B (CALB) (Novozyme 435) favoring the esterification and immobilized Thermomyces lanuginosus lipase (TLL) (Lipozyme TLIM) preferring the transesterification at 2:8 (wt/wt) gave FAME in 80% yield, being better than that with Novozyme 435 or Lipozyme TLIM. Recombinant Escherichia coli (Calb/Tll) co-expressing CALB and TLL was engineered as a more efficient tandem-lipases system. Using wet or dry cells (4wt%) gave FAME in 87% or 95% yield, which is much better than that with E. coli cells expressing either CALB or TLL alone. Cells of E. coli (Calb/Tll) were recycled for five times and retained 75% productivity, thus being practical for producing biodiesel from grease. Copyright © 2012 Elsevier Ltd. All rights reserved.

  11. Production of a solvent, detergent, and thermotolerant lipase by a newly isolated Acinetobacter sp. in submerged and solid-state fermentations.

    PubMed

    Khoramnia, Anahita; Ebrahimpour, Afshin; Beh, Boon Kee; Lai, Oi Ming

    2011-01-01

    The lipase production ability of a newly isolated Acinetobacter sp. in submerged (SmF) and solid-state (SSF) fermentations was evaluated. The results demonstrated this strain as one of the rare bacterium, which is able to grow and produce lipase in SSF even more than SmF. Coconut oil cake as a cheap agroindustrial residue was employed as the solid substrate. The lipase production was optimized in both media using artificial neural network. Multilayer normal and full feed forward backpropagation networks were selected to build predictive models to optimize the culture parameters for lipase production in SmF and SSF systems, respectively. The produced models for both systems showed high predictive accuracy where the obtained conditions were close together. The produced enzyme was characterized as a thermotolerant lipase, although the organism was mesophile. The optimum temperature for the enzyme activity was 45°C where 63% of its activity remained at 70°C after 2 h. This lipase remained active after 24 h in a broad range of pH (6-11). The lipase demonstrated strong solvent and detergent tolerance potentials. Therefore, this inexpensive lipase production for such a potent and industrially valuable lipase is promising and of considerable commercial interest for biotechnological applications.

  12. Lipase-Catalyzed Kinetic Resolution of Novel Antifungal N-Substituted Benzimidazole Derivatives.

    PubMed

    Łukowska-Chojnacka, Edyta; Staniszewska, Monika; Bondaryk, Małgorzata; Maurin, Jan K; Bretner, Maria

    2016-04-01

    A series of new N-substituted benzimidazole derivatives was synthesized and their antifungal activity against Candida albicans was evaluated. The chemical step included synthesis of appropriate ketones containing benzimidazole ring, reduction of ketones to the racemic alcohols, and acetylation of alcohols to the esters. All benzimidazole derivatives were obtained with satisfactory yields and in relatively short times. All synthesized compounds exhibit significant antifungal activity against Candida albicans 900028 ATCC (% cell inhibition at 0.25 μg concentration > 98%). Additionally, racemic mixtures of alcohols were separated by lipase-catalyzed kinetic resolution. In the enzymatic step a transesterification reaction was applied and the influence of a lipase type and solvent on the enantioselectivity of the reaction was studied. The most selective enzymes were Novozyme SP 435 and lipase Amano AK from Pseudomonas fluorescens (E > 100). © 2016 Wiley Periodicals, Inc.

  13. Comparative genomic analysis of the Lipase3 gene family in five plant species reveals distinct evolutionary origins.

    PubMed

    Wang, Dan; Zhang, Lin; Hu, JunFeng; Gao, Dianshuai; Liu, Xin; Sha, Yan

    2018-04-01

    Lipases are physiologically important and ubiquitous enzymes that share a conserved domain and are classified into eight different families based on their amino acid sequences and fundamental biological properties. The Lipase3 family of lipases was reported to possess a canonical fold typical of α/β hydrolases and a typical catalytic triad, suggesting a distinct evolutionary origin for this family. Genes in the Lipase3 family do not have the same functions, but maintain the conserved Lipase3 domain. There have been extensive studies of Lipase3 structures and functions, but little is known about their evolutionary histories. In this study, all lipases within five plant species were identified, and their phylogenetic relationships and genetic properties were analyzed and used to group them into distinct evolutionary families. Each identified lipase family contained at least one dicot and monocot Lipase3 protein, indicating that the gene family was established before the split of dicots and monocots. Similar intron/exon numbers and predicted protein sequence lengths were found within individual groups. Twenty-four tandem Lipase3 gene duplications were identified, implying that the distinctive function of Lipase3 genes appears to be a consequence of translocation and neofunctionalization after gene duplication. The functional genes EDS1, PAD4, and SAG101 that are reportedly involved in pathogen response were all located in the same group. The nucleotide diversity (Dxy) and the ratio of nonsynonymous to synonymous nucleotide substitutions rates (Ka/Ks) of the three genes were significantly greater than the average across the genomes. We further observed evidence for selection maintaining diversity on three genes in the Toll-Interleukin-1 receptor type of nucleotide binding/leucine-rich repeat immune receptor (TIR-NBS LRR) immunity-response signaling pathway, indicating that they could be vulnerable to pathogen effectors.

  14. Production and partial characterization of lipases from a newly isolated Penicillium sp. using experimental design.

    PubMed

    Wolski, E; Rigo, E; Di Luccio, M; Oliveira, J V; de Oliveira, D; Treichel, H

    2009-07-01

    The objective of this work was to investigate the lipase production by a newly isolated Penicillium sp., using experimental design technique, in submerged fermentation using a medium based on peptone, yeast extract, NaCl and olive oil, as well as to characterize the crude enzymatic extracts obtained. Lipase activity values of 9.5 U ml(-1) in 96 h of fermentation was obtained at the maximized operational conditions of peptone, yeast extract, NaCl and olive oil concentrations (g l(-1)) of 20.0, 5.0, 5.0 and of 10.0 respectively. The partial characterization of crude enzymatic extract obtained by submerged fermentation showed optimum activity at pH range from 4.9 to 5.5 and temperature from 37 degrees C to 42 degrees C. The crude extract maintained its initial activity at freezing temperatures up to 100 days. A newly isolated strain of Penicillium sp. used in this work yielded good lipase activities compared to the literature. The growing interest in lipase production is related to the potential biotechnological applications that these enzymes present. New lipase producers are relevant to finding enzymes with different catalytic properties of commercial interest could be obtained, without using genetically modified organisms (GMO).

  15. Lipase production in solid-state fermentation monitoring biomass growth of aspergillus niger using digital image processing.

    PubMed

    Dutra, Júlio C V; da C Terzi, Selma; Bevilaqua, Juliana Vaz; Damaso, Mônica C T; Couri, Sônia; Langone, Marta A P; Senna, Lilian F

    2008-03-01

    The aim of this study was to monitor the biomass growth of Aspergillus niger in solid-state fermentation (SSF) for lipase production using digital image processing technique. The strain A. niger 11T53A14 was cultivated in SSF using wheat bran as support, which was enriched with 0.91% (m/v) of ammonium sulfate. The addition of several vegetable oils (castor, soybean, olive, corn, and palm oils) was investigated to enhance lipase production. The maximum lipase activity was obtained using 2% (m/m) castor oil. In these conditions, the growth was evaluated each 24 h for 5 days by the glycosamine content analysis and digital image processing. Lipase activity was also determined. The results indicated that the digital image process technique can be used to monitor biomass growth in a SSF process and to correlate biomass growth and enzyme activity. In addition, the immobilized esterification lipase activity was determined for the butyl oleate synthesis, with and without 50% v/v hexane, resulting in 650 and 120 U/g, respectively. The enzyme was also used for transesterification of soybean oil and ethanol with maximum yield of 2.4%, after 30 min of reaction.

  16. Lipase Production in Solid-State Fermentation Monitoring Biomass Growth of Aspergillus niger Using Digital Image Processing

    NASA Astrophysics Data System (ADS)

    Dutra, Julio C. V.; da Terzi, Selma C.; Bevilaqua, Juliana Vaz; Damaso, Mônica C. T.; Couri, Sônia; Langone, Marta A. P.; Senna, Lilian F.

    The aim of this study was to monitor the biomass growth of Aspergillus niger in solid-state fermentation (SSF) for lipase production using digital image processing technique. The strain A. niger 11T53A14 was cultivated in SSF using wheat bran as support, which was enriched with 0.91% (m/v) of ammonium sulfate. The addition of several vegetable oils (castor, soybean, olive, corn, and palm oils) was investigated to enhance lipase production. The maximum lipase activity was obtained using 2% (m/m) castor oil. In these conditions, the growth was evaluated each 24 h for 5 days by the glycosamine content analysis and digital image processing. Lipase activity was also determined. The results indicated that the digital image process technique can be used to monitor biomass growth in a SSF process and to correlate biomass growth and enzyme activity. In addition, the immobilized esterification lipase activity was determined for the butyl oleate synthesis, with and without 50% v/v hexane, resulting in 650 and 120 U/g, respectively. The enzyme was also used for transesterification of soybean oil and ethanol with maximum yield of 2.4%, after 30 min of reaction.

  17. Cloning, expression and characterization of a lipase gene from marine bacterium Pseudoalteromonas lipolytica SCSIO 04301

    NASA Astrophysics Data System (ADS)

    Su, Hongfei; Mai, Zhimao; Zhang, Si

    2016-12-01

    A lipase gene, lip1233, isolated from Pseudoalteromonas lipolytica SCSIO 04301, was cloned and expressed in E. coli. The enzyme comprised 810 amino acid residues with a deduced molecular weight of 80 kDa. Lip1233 was grouped into the lipase family X because it contained a highly conserved motif GHSLG. The recombinant enzyme was purified with Ni-NTA affinity chromatography. The optimal temperature and pH value of Lip1233 were 45°C and 8.0, respectively. It retained more than 70% of original activity after being incubated in pH ranging from 6.0 to 9.5 for 30 min. It was stable when the temperature was below 45°C, but was unstable when the temperature was above 55°C. Most metal ions tested had no significant effect on the activity of Lip1233. Lip1233 remained more than original activity in some organic solvents at the concentration of 30% (v/v). It retained more than 30% activity after incubated in pure organic solvents for 12 h, while in hexane the activity was nearly 100%. Additionally, Lip1233 exhibited typical halotolerant characteristic as it was active under 4M NaCl. Lip1233 powder could catalyze efficiently the synthesis of fructose esters in hexane at 40°C. These characteristics demonstrated that Lip1233 is applicable to elaborate food processing and organic synthesis.

  18. Synthesis of oleoylethanolamide using lipase.

    PubMed

    Wang, Xiaosan; Wang, Xingguo; Wang, Tong

    2012-01-11

    An effective process for the enzymatic synthesis of oleoylethanolamide is described in this study. The process included purification of a commercial oleic acid product and then optimization of the reaction between the purified oleic acid and ethanolamine in the presence of hexane and a lipase. Under the optimal amidation reaction conditions identified, oleoylethanolamide was obtained with 96.6% purity. The synthesis was also conducted on a large scale (50 mmol of each of the reactants), and oleoylethanolamide purity and yield after crystallization purification were 96.1 and 73.5%, respectively. Compared to the previous studies, the current method of preparing high-purity oleoylethanolamide is more effective and economically feasible. The scalability and ease for such synthesis make it possible to study the biological and nutritional functions of the cannabinoid-like oleoylethanolamide in animal or human subjects.

  19. Lipase-catalyzed acylation of microbial mannosylerythritol lipids (biosurfactants) and their characterization.

    PubMed

    Recke, Verena K; Beyrle, Catharina; Gerlitzki, Melanie; Hausmann, Rudolf; Syldatk, Christoph; Wray, Victor; Tokuda, Harukuni; Suzuki, Nobutaka; Lang, Siegmund

    2013-05-24

    Culturing Pseudozyma aphidis on glucose as main carbon source and soybean oil as co-substrate the mannosylerythritol lipids MEL-A and MEL-B were produced. Based on their excellent surface/interfacial active behavior they possess a high potential among all known biosurfactants. The components of a microbial MEL mixture were purified by medium pressure liquid chromatography (MPLC) and were used as substrates for in vitro enzymatic modifications. Lipase-catalyzed acylations of MEL-A and MEL-B with uncommon fatty acids from other microbial glycolipids-3-hydroxydecanoic acid from rhamnolipids and 17-hydroxyoctadecanoic acid from classical sophorolipids-yielded functionalized products at the C-1 position of the erythritol. The novel products were purified by MPLC and their structures elucidated by (1)H and (13)C nuclear magnetic resonance spectroscopy and mass spectrometry. In physicochemical characterization experiments two of the three new glycoconjugates lowered the surface tension of water from 72 mN m(-1) to 27-38 mN m(-1). Moreover the novel compounds inhibited the growth of gram-positive bacteria and showed a potential for anti-tumor-promoting activity. Copyright © 2013 Elsevier Ltd. All rights reserved.

  20. Role of Methoxypolyethylene Glycol on the Hydration, Activity, Conformation and Dynamic Properties of a Lipase in a Dry Film

    PubMed Central

    Secundo, Francesco; Barletta, Gabriel; Mazzola, Giorgio

    2009-01-01

    A combined approach based on the use of ATR-FT/IR and steady-state fluorescence spectroscopy allowed to shed light on the effects of the additive methoxypolyethylene glycol (MePEG) on the hydration, conformation and dynamic properties of lipase from Burkholderia cepacia dehydrated to form a film. Spectroscopic data show that the additive has little effect on the structure of the protein; however, H/D exchange kinetic and fluorescence anisotropy suggest a more flexible enzyme molecule when in the presence of MePEG. By infrared spectroscopy, we estimated that, after conditioning the films at water activity of 1, the water content in the lipase dehydrated with MePEG is 5.4-and 4.7-fold higher than in the absence of the additive and the additive alone, respectively. Additionally, our infrared data suggest that MePEG acts by hindering intermolecular protein–protein interactions and contributing to increase the accessibility and flexibility of the lipase in the dehydrated solid film. These factors also explain the enhancement of the enzyme catalytic activity (i.e., up to 3.7-fold in neat organic solvent) when in the presence of MePEG. The method and results presented might better address the use of additives for the preparation of enzymes employed in non-aqueous media or of proteins used in a dry form in different fields of biotechnology. PMID:18727030

  1. A Sensitive Microplate Assay for Lipase Activity Measurement Using Olive Oil Emulsion Substrate: Modification of the Copper Soap Colorimetric Method.

    PubMed

    Mustafa, Ahmad; Karmali, Amin; Abdelmoez, Wael

    2016-01-01

    The present work involves a sensitive high-throughput microtiter plate based colorimetric assay for estimating lipase activity using cupric acetate pyridine reagent (CAPR). In the first approach, three factors two levels factorial design methodology was used to evaluate the interactive effect of different parameters on the sensitivity of the assay method. The optimization study revealed that the optimum CAPR concentration was 7.5% w/v, the optimum solvent was heptane and the optimum CAPR pH was 6. In the second approach, the optimized colorimetric microplate assay was used to measure lipase activity based on enzymatic hydrolysis of olive oil emulsion substrate at 37°C and 150 rpm. The emulsion substrates were formulated by using olive oil, triton X-100 (10% v/v in pH 8) and sodium phosphate buffer of pH 8 in ratio of 1:1:1 in the case of Candida sp. lipase. While in the case of immobilized lipozyme RMIM, The emulsion substrates were formulated by using olive oil, triton X-100 (1% v/v in pH 8) and sodium phosphate buffer of pH 8 in ratio of 2:1:1. Absorbance was measured at 655 nm. The stability of this assay (in terms of colored heptane phase absorbance readings) retained more than 92.5% after 24 h at 4°C compared to the absorbance readings measured at zero time. In comparison with other lipase assay methods, beside the developed sensitivity, the reproducibility and the lower limit of detection (LOD) of the proposed method, it permits analyzing of 96 samples at one time in a 96-well microplate. Furthermore, it consumes small quantities of chemicals and unit operations.

  2. Thermostable, alkaline and detergent-tolerant lipase from a newly isolated thermophilic Bacillus stearothermophilus.

    PubMed

    Ben Bacha, Abir; Moubayed, Nadine M S; Abid, Islam

    2015-04-01

    Lipases are the enzymes of choice for laundry detergent industries, owing to their triglyceride removing ability from the soiled fabric, which eventually reduces the usage of phosphate-based chemical cleansers in the detergent formulation. In this study, a novel thermo-alkaline lipase-producing strain identified as Bacillus stearothermophilus was isolated from the soil samples of olive oil mill. Enhanced lipase production was observed at 55 degrees C, pH 11 and after 48 h of incubation. Among the substrates tested, xylose (a carbon source), peptone (a nitrogen source) and olive oil at a concentration of 1% were suitable substrates for enhancing lipase production. MgSO4 and Tween-80 were suitable substrates for maximizing lipase production. The enzyme was purified to homogeneity by a single CM-Sephadex column chromatography and revealed molecular mass of 67 kDa. The enzyme (BL1) was active over a wide range of pH from 9.0 to 13.0, with an optimum at pH 11.0, exhibited maximal activity at 55 degreesC and retained more than 70% of its activity after incubation at 70 degrees C or pH 13 for 0.5 h or 24 h, respectively. The enzyme hydrolyzed both short and long-chain triacylglycerols at comparable rates. BL1 was studied in a preliminary evaluation for use in detergent formulation solutions. This novel lipase showed extreme stability towards non-ionic and anionic surfactants after pre-incubation for 1 h at 40 degrees C, and good stability towards oxidizing agents. Additionally, the enzyme showed excellent stability and compatibility with various commercial detergents, suggesting its potential as an additive in detergent formulations.

  3. Kinetic properties of dromedary pancreatic lipase: a comparative study on emulsified and monomolecular substrate.

    PubMed

    Jemel, Ikram; Fendri, Ahmed; Gargouri, Youssef; Bezzine, Sofiane

    2009-05-01

    Using the classical emulsified system and the monomolecular film technique, we compared several interfacial properties of dromedary pancreatic lipase (DrPL) with those of a mammal (human) and an avian (turkey) model. Like turkey pancreatic lipase (TPL) and unlike human pancreatic lipase (HPL), in the absence of colipase and bile salts, using tributyrin emulsion or monomolecular films of dicaprin at low surface pressure, DrPL hydrolyses pure tributyrin emulsion, as well as dicaprin films maintained at low surface pressures. DrPL was also able to hydrolyse triolein emulsion in the absence of any additive and despite the accumulation of long-chain free fatty acids at the interface. The difference of behaviours between the two mammal pancreatic lipases (DrPL and HPL) can be explained by the penetration capacity of each enzyme. DrPL presents a critical surface pressure value (21 m Nm(-1)) that is more important than this of HPL. Subsequently, the dromedary pancreatic lipase interacts efficiently with interfaces and it is not denaturated at high interfacial energy. A kinetic study on the surface pressure dependency, stereospecificity and regioselectivity of DrPL was performed using optically pure stereoisomers of either three dicaprin isomers containing a single hydrolysable decanoyl ester bond that were spread as monomolecular films at the air/water interface. Interestingly, in comparison with all the previously studied mammal pancreatic lipases, DrPL presents the highest preference for adjacent ester groups of dicaprin isomers (1,2-sn-dicaprin and 2,3-sn-dicaprin) at high surface pressure. Furthermore, DrPL forms a pancreatic lipase subgroup in which the stereopreference switches from sn-3 position to the sn-1 position when increasing the surface pressure.

  4. Extracellular lipase of an entomopathogenic fungus effecting larvae of a scale insect.

    PubMed

    Ali, Shaukat; Ren, Shunxiang; Huang, Zhen

    2014-11-01

    Lipases play an important role in the infection process of entomopathogenic fungi by hydrolyzing the ester bonds of lipoproteins, fats and waxes present on the insect surface and in the body. Here we report the purification and characterization of an extracellular lipase from Isaria fumosorosea. The enzyme was purified (138.46-fold) in three steps using (NH4 )2 SO4 precipitation followed by DEAE-cellulose and Sephadex G-100 column chromatography. The molecular weight of purified enzyme was determined to be 31 KDa by SDS-PAGE. The optimum temperature and pH for enzyme activity were 35 °C and 7.0, respectively, using p-nitrophenylpalmitate as the substrate. Lipolytic activity was enhanced in the presence of Ca(+2) , Mg(+2) , Na(+) , and NH4 (+) salts, while Zn(+2) , Fe(+2) , and Cu(+2) inhibited enzyme activity. The enzyme displayed broad substrate specificity with the highest activity observed for coconut oil and p-nitrophenyl carprate. Topical co-application of purified lipase with fungal conidial suspensions decreased the median survival time (ST50 ) of Dysmicoccus neobrevipes nymphs as compared to the fungus alone. Our results indicate that an extracellular lipase produced by I. fumosorosea can be exploited for development of enzyme-based insect management. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  5. Characterization of pancreatic lipase-related protein 2 isolated from human pancreatic juice.

    PubMed

    De Caro, Josiane; Sias, Barbara; Grandval, Philippe; Ferrato, Francine; Halimi, Hubert; Carrière, Frédéric; De Caro, Alain

    2004-09-01

    Human pancreatic lipase-related protein 2 (HPLRP2) was identified for the first time in pancreatic juice using specific anti-peptide antibodies and purified to homogeneity. Antibodies were raised in the rabbit using a synthetic peptide from the HPLRP2 protein sequence deduced from cDNA. Western blotting analysis showed that these antibodies did not react with classical human pancreatic lipase (HPL) or human pancreatic lipase-related protein 1 (HPLRP1) but cross-reacted with native rat PLRP2 (RPLRP2), as well as with recombinant rat and guinea-pig PLRP2 (GPLRP2). Immunoaffinity chromatography was performed on immobilized anti-recombinant HPLRP2 polyclonal antibodies to purify native HPLRP2 after conventional chromatographic steps including gel filtration and chromatrography on an anion-exchanger. The substrate specificity of HPLRP2 was investigated using various triglycerides, phospholipids and galactolipids as substrates. The lipase activity on triglycerides was inhibited by bile salts and weakly restored by colipase. The phospholipase activity of HPLRP2 on phospholipid micelles was very low. A significant level of galactolipase activity was measured using monogalactosyldiglyceride monomolecular films. These data suggest that the main physiological function of HPLRP2 is the hydrolysis of galactolipids, which are the main lipids present in vegetable food.

  6. Isolation of a novel alkaline-stable lipase from a metagenomic library and its specific application for milkfat flavor production.

    PubMed

    Peng, Qing; Wang, Xu; Shang, Meng; Huang, Jinjin; Guan, Guohua; Li, Ying; Shi, Bo

    2014-01-04

    Lipolytic enzymes are commonly used to produce desired flavors in lipolyzed milkfat (LMF) manufacturing processes. However, the choice of enzyme is critical because it determines the final profile of fatty acids released and the consequent flavor of the product. We previously constructed a metagenomic library from marine sediments, to explore the novel enzymes which have unique properties useful in flavor-enhancing LMF. A novel lipase Est_p6 was isolated from a metagenomic library and was expressed highly in E.coli. Bioinformatic analysis indicated that Est_p6 belongs to lipolytic enzyme family IV, the molecular weight of purified Est_p6 was estimated at 36 kDa by SDS-PAGE. The hydrolytic activity of the enzyme was stable under alkaline condition and the optimal temperature was 50°C. It had a high specific activity (2500 U/mg) toward pNP butyrate (pNP-C4), with K(m) and V(max) values of 1.148 mM and 3497 μmol∙min⁻¹∙mg⁻¹, respectively. The enzyme activity was enhanced by DTT and was not significantly inhibited by PMSF, EDTA or SDS. This enzyme also showed high hydrolysis specificity for myristate (C14) and palmitate (C16). It seems that Est_p6 has safety for commercial LMF flavor production and food manufacturing processes. The ocean is a vast and largely unexplored resource for enzymes. According the outstanding alkaline-stability of Est_p6 and it produced myristic acid and palmitic acid more efficiently than other free fatty acids in lipolyzed milkfat. This novel lipase may be used to impart a distinctive and desirable flavor and odor in milkfat flavor production.

  7. The Lp_3561 and Lp_3562 Enzymes Support a Functional Divergence Process in the Lipase/Esterase Toolkit from Lactobacillus plantarum

    PubMed Central

    Esteban-Torres, María; Reverón, Inés; Santamaría, Laura; Mancheño, José M.; de las Rivas, Blanca; Muñoz, Rosario

    2016-01-01

    Lactobacillus plantarum species is a good source of esterases since both lipolytic and esterase activities have been described for strains of this species. No fundamental biochemical difference exists among esterases and lipases since both share a common catalytic mechanism. L. plantarum WCFS1 possesses a protein, Lp_3561, which is 44% identical to a previously described lipase, Lp_3562. In contrast to Lp_3562, Lp_3561 was unable to degrade esters possessing a chain length higher than C4 and the triglyceride tributyrin. As in other L. plantarum esterases, the electrostatic potential surface around the active site in Lp_3561 is predicted to be basic, whereas it is essentially neutral in the Lp_3562 lipase. The fact that the genes encoding both proteins were located contiguously in the L. plantarum WCFS1 genome, suggests that they originated by tandem duplication, and therefore are paralogs as new functions have arisen during evolution. The presence of the contiguous lp_3561 and lp_3562 genes was studied among L. plantarum strains. They are located in a 8,903 bp DNA fragment that encodes proteins involved in the catabolism of sialic acid and are predicted to increase bacterial adaptability under certain growth conditions. PMID:27486450

  8. High-level expression and characterization of a chimeric lipase from Rhizopus oryzae for biodiesel production

    PubMed Central

    2013-01-01

    Background Production of biodiesel from non-edible oils is receiving increasing attention. Tung oil, called “China wood oil” is one kind of promising non-edible biodiesel oil in China. To our knowledge, tung oil has not been used to produce biodiesel by enzymatic method. The enzymatic production of biodiesel has been investigated extensively by using Rhizopus oryzae lipase as catalyst. However, the high cost of R. oryzae lipase remains a barrier for its industrial applications. Through different heterologous expression strategies and fermentation techniques, the highest expression level of the lipase from R. oryzae reached 1334 U/mL in Pichia pastoris, which is still not optimistic for industry applications. Results The prosequence of lipases from Rhizopus sp. is very important for the folding and secretion of an active lipase. A chimeric lipase from R. oryzae was constructed by replacing the prosequence with that from the R. chinensis lipase and expressed in P. pastoris. The maximum activity of the chimera reached 4050 U/mL, which was 11 fold higher than that of the parent. The properties of the chimera were studied. The immobilized chimera was used successfully for biodiesel production from tung oil, which achieved higher FAME yield compared with the free chimeric lipase, non-chimeric lipase and mature lipase. By response surface methodology, three variables, water content, methanol to tung oil molar ratio and enzyme dosage were proved to be crucial parameters for biosynthesis of FAME and the FAME yield reached 91.9±2.5% at the optimized conditions by adding 5.66 wt.% of the initial water based on oil weight, 3.88 of methanol to tung oil molar ratio and 13.24 wt.% of enzyme concentration based on oil weight at 40°C. Conclusions This is the first report on improving the expression level of the lipase from R. oryzae by replacing prosequences. The immobilized chimera was used successfully for biodiesel production from tung oil. Using tung oil as non-edible raw

  9. High-level expression and characterization of a chimeric lipase from Rhizopus oryzae for biodiesel production.

    PubMed

    Yu, Xiao-Wei; Sha, Chong; Guo, Yong-Liang; Xiao, Rong; Xu, Yan

    2013-02-21

    Production of biodiesel from non-edible oils is receiving increasing attention. Tung oil, called "China wood oil" is one kind of promising non-edible biodiesel oil in China. To our knowledge, tung oil has not been used to produce biodiesel by enzymatic method. The enzymatic production of biodiesel has been investigated extensively by using Rhizopus oryzae lipase as catalyst. However, the high cost of R. oryzae lipase remains a barrier for its industrial applications. Through different heterologous expression strategies and fermentation techniques, the highest expression level of the lipase from R. oryzae reached 1334 U/mL in Pichia pastoris, which is still not optimistic for industry applications. The prosequence of lipases from Rhizopus sp. is very important for the folding and secretion of an active lipase. A chimeric lipase from R. oryzae was constructed by replacing the prosequence with that from the R. chinensis lipase and expressed in P. pastoris. The maximum activity of the chimera reached 4050 U/mL, which was 11 fold higher than that of the parent. The properties of the chimera were studied. The immobilized chimera was used successfully for biodiesel production from tung oil, which achieved higher FAME yield compared with the free chimeric lipase, non-chimeric lipase and mature lipase. By response surface methodology, three variables, water content, methanol to tung oil molar ratio and enzyme dosage were proved to be crucial parameters for biosynthesis of FAME and the FAME yield reached 91.9±2.5% at the optimized conditions by adding 5.66 wt.% of the initial water based on oil weight, 3.88 of methanol to tung oil molar ratio and 13.24 wt.% of enzyme concentration based on oil weight at 40°C. This is the first report on improving the expression level of the lipase from R. oryzae by replacing prosequences. The immobilized chimera was used successfully for biodiesel production from tung oil. Using tung oil as non-edible raw material and a chimeric lipase

  10. Recent Advances in Lipase-Mediated Preparation of Pharmaceuticals and Their Intermediates

    PubMed Central

    Carvalho, Ana Caroline Lustosa de Melo; Fonseca, Thiago de Sousa; de Mattos, Marcos Carlos; de Oliveira, Maria da Conceição Ferreira; de Lemos, Telma Leda Gomes; Molinari, Francesco; Romano, Diego; Serra, Immacolata

    2015-01-01

    Biocatalysis offers an alternative approach to conventional chemical processes for the production of single-isomer chiral drugs. Lipases are one of the most used enzymes in the synthesis of enantiomerically pure intermediates. The use of this type of enzyme is mainly due to the characteristics of their regio-, chemo- and enantioselectivity in the resolution process of racemates, without the use of cofactors. Moreover, this class of enzymes has generally excellent stability in the presence of organic solvents, facilitating the solubility of the organic substrate to be modified. Further improvements and new applications have been achieved in the syntheses of biologically active compounds catalyzed by lipases. This review critically reports and discusses examples from recent literature (2007 to mid-2015), concerning the synthesis of enantiomerically pure active pharmaceutical ingredients (APIs) and their intermediates in which the key step involves the action of a lipase. PMID:26690428

  11. Childhood/adult-onset lysosomal acid lipase deficiency: A serious metabolic and vascular phenotype beyond liver disease-four new pediatric cases.

    PubMed

    Poinsot, Pierre; Collardeau Frachon, Sophie; Restier, Lioara; Sérusclat, André; Di Filippo, Mathilde; Charrière, Sybil; Moulin, Philippe; Lachaux, Alain; Peretti, Noel

    The childhood/adult-onset lysosomal acid lipase deficiency (LALD; late-onset LALD) is a rare genetic disease. Children present severe fatty liver disease with early cirrhosis. Before enzyme replacement therapy, statins were the standard treatment to improve the severe dyslipidemia. However, late-onset LALD should be considered as a systemic metabolic disease: chronic hyper-low-density lipoprotein and hypo-high-density lipoprotein cholesterolemia induces early atherosclerosis in addition to the liver morbidity. To assess 4 new pediatric cases of late-onset LALD with an evaluation of hepatic, metabolic, and vascular evolution under statin. Four patients were retrospectively described. Anthropometric data (weight, height, and body mass index) and laboratory data (LIPA mutations, acid lipase residual activity, liver and lipid profile, and homeostatic model assessment index) were collected. Liver histology was assessed by the noninvasive tests FibroScan and FibroTest and confirmed by liver biopsy. Vascular impact was followed up by carotid intima-media thickness (cIMT) assessment. The 4 cases of late-onset LALD came from 2 families, each with a boy (aged 8.6 and 11 years at diagnosis) and a girl (aged 10.6 and 13 years at diagnosis). Treatment with statins was performed for 8 and 5 years, respectively, from diagnosis. Statins decreased the low-density lipoprotein cholesterol mean value of 40%. All children showed significant liver fibrosis (F3 [n = 3]; F2 [n = 1]). cIMT showed the following for all children: abnormal measures without improvement and atherosclerotic plaques. One child developed a deleterious metabolic phenotype with obesity and insulin resistance (homeostatic model assessment = 3.08) associated with higher mean hepatic transaminases (149 vs 98, 88, and 61 IU/L) and increased mean cIMT values (raising from 0.47 to 0.5 mm vs 0.43 and 0.43 mm). Late-onset LALD is a rare metabolic disease with a larger impact than liver disease. Our work shows the

  12. Update on lysosomal acid lipase deficiency: Diagnosis, treatment and patient management.

    PubMed

    Camarena, Carmen; Aldamiz-Echevarria, Luis J; Polo, Begoña; Barba Romero, Miguel A; García, Inmaculada; Cebolla, Jorge J; Ros, Emilio

    2017-05-10

    Lysosomal acid lipase deficiency (LALD) is an ultra-rare disease caused by a congenital disorder of the lipid metabolism, characterized by the deposition of cholesterol esters and triglycerides in the organism. In patients with no enzyme function, the disease develops during the perinatal period and is invariably associated with death during the first year of life. In all other cases, the phenotype is heterogeneous, although most patients develop chronic liver diseases and may also develop an early cardiovascular disease. Treatment for LALD has classically included the use of supportive measures that do not prevent the progression of the disease. In 2015, regulatory agencies approved the use of a human recombinant LAL for the treatment of LALD. This long-term enzyme replacement therapy has been associated with significant improvements in the hepatic and lipid profiles of patients with LALD, increasing survival rates in infants with a rapidly progressive disease. Both the severity of LALD and the availability of a specific treatment highlight the need to identify these patients in clinical settings, although its low prevalence and the existing clinical overlap with other more frequent pathologies limit its diagnosis. In this paper we set out practical recommendations to identify and monitor patients with LALD, including a diagnostic algorithm, along with an updated treatment. Copyright © 2017 Elsevier España, S.L.U. All rights reserved.

  13. Effect of ethyleneoxide groups of anionic surfactants on lipase activity.

    PubMed

    Magalhães, Solange S; Alves, Luís; Sebastião, Marco; Medronho, Bruno; Almeida, Zaida L; Faria, Tiago Q; Brito, Rui M M; Moreno, Maria J; Antunes, Filipe E

    2016-09-01

    The use of enzymes in laundry and dish detergent products is growing. Such tendency implies dedicated studies to understand surfactant-enzyme interactions. The interactions between surfactants and enzymes and their impact on the catalytic efficiency represent a central problem and were here evaluated using circular dichroism, dynamic light scattering, and enzyme activity determinations. This work focuses on this key issue by evaluating the role of the ethyleneoxide (EO) groups of anionic surfactants on the structure and activity of a commercial lipase, and by focusing on the protein/surfactant interactions at a molecular level. The conformational changes and enzymatic activity of the protein were evaluated in the presence of sodium dodecyl sulfate (SDS also denoted as SLE 0 S) and of sodium lauryl ether sulfate with two EO units (SLE 2 S). The results strongly suggest that the presence of EO units in the surfactant polar headgroup determines the stability and the activity of the enzyme. While SDS promotes enzyme denaturation and consequent loss of activity, SLE 2 S preserves the enzyme structure and activity. The data further highlights that the electrostatic interactions among the protein groups are changed by the presence of the adsorbed anionic surfactants being such absorption mainly driven by hydrophobic interactions. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1276-1282, 2016. © 2016 American Institute of Chemical Engineers.

  14. Purification and characterization of halophilic lipase of Chromohalobacter sp. from ancient salt well.

    PubMed

    Ai, Li; Huang, Yaping; Wang, Chuan

    2018-06-04

    A halophilic lipase (LipS2) was produced by Chromohalobacter canadensis strain which was isolated from ancient salt well of Zigong, China. LipS2 was purified to homogeneity and showed a single band with molecular mass of 58 kDa by SDS-PAGE. LipS2 preferred middle-to-long acyl chain esters with C14 triglycerides as optimum substrate. It was noteworthy that LipS2 displayed efficient hydrolysis activity to some vegetable oils which were composed of polyunsaturated fatty acid. LipS2 showed high activity in range of 2.5-3.5 M NaCl, no activity without salt. Optimum temperature and pH were 55 °C and pH 8.5, respectively. Notably, the thermostability and pH stability of LipS2, varying with salt concentration, reached optimum in the presence of 3.0 M NaCl. LipS2 was stimulated by Ca 2+ and Mg 2+ , inhibited by Zn 2+ , Cu 2+ , Mn 2+ , Fe 2+ , and Hg 2+ . Moreover, LipS2 displayed significant tolerance to organic solvents including methanol, ethanol, ethyl acetate and acetone, especially, LipS2 activity was enhanced markedly by the hexane and benzene. Non-ionic surfactants increased LipS2 activity, while ionic surfactants decreased activity. This was the first report on halophilic lipase of Chromohalobacter from ancient salt well. The results suggested that LipS2 may have considerable potential for biotechnological applications. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  15. Engineering Yarrowia lipolytica to Simultaneously Produce Lipase and Single Cell Protein from Agro-industrial Wastes for Feed.

    PubMed

    Yan, Jinyong; Han, Bingnan; Gui, Xiaohua; Wang, Guilong; Xu, Li; Yan, Yunjun; Madzak, Catherine; Pan, Dujie; Wang, Yaofeng; Zha, Genhan; Jiao, Liangcheng

    2018-01-15

    Lipases are scarcely exploited as feed enzymes in hydrolysis of lipids for increasing energy supply and improving nutrient use efficiency. In this work, we performed homologous overexpression, in vitro characterization and in vivo assessment of a lipase from the yeast Yarrowia lipolytica for feed purpose. Simultaneously, a large amount of yeast cell biomass was produced, for use as single cell protein, a potential protein-rich feed resource. Three kinds of low cost agro-industrial wastes were tested as substrates for simultaneous production of lipase and single cell protein (SCP) as feed additives: sugarcane molasses, waste cooking oil and crude glycerol from biodiesel production. Sugarcane molasses appeared as the most effective cheap medium, allowing production of 16420 U/ml of lipase and 151.2 g/L of single cell protein at 10 liter fermentation scale. In vitro characterization by mimicking a gastro-intestinal environment and determination of essential amino acids of the SCP, and in vivo oral feeding test on fish all revealed that lipase, SCP and their combination were excellent feed additives. Such simultaneous production of this lipase and SCP could address two main concerns of feed industry, poor utilization of lipid and shortage of protein resource at the same time.

  16. Identification and characterization of lipases from Malassezia restricta, a causative agent of dandruff.

    PubMed

    Sommer, Bettina; Overy, David P; Kerr, Russell G

    2015-11-01

    Dandruff, a skin disorder affecting 50% of the world population, is linked with proliferation of lipophilic yeasts of the genus Malassezia (particularly Malassezia globosa and M. restricta). Most Malassezia species show a unique lipid dependency and require external lipids for growth. Genome mining of the incomplete M. restricta genome led to the identification of eight lipase sequences. Sequences representing the class 3 and LIP lipase families were used to clone the lipases MrLip1, MrLip2 and MrLip3, recombinantly expressed in Pichia pastoris, and tested for their activity using mono-, di- and triacylglycerol substrates. Hydrolysis by the M. restricta lipase MrLip1 and MrLip2 (family class 3) was limited to the mono- and diacylglycerol, while MrLip3 (family LIP) hydrolyzed all three substrates. This result confirms that Malassezia family LIP lipases are responsible for the hydrolysis of triacylglycerols, the main component of human sebum. Furthermore, the information regarding lipases from M. restricta presented here might aid in the search for anti-dandruff agents. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  17. Adsorption and activity of Thermomyces lanuginosus lipase on hydrophobic and hydrophilic surfaces measured with dual polarization interferometry (DPI) and confocal microscopy.

    PubMed

    Sonesson, Andreas W; Callisen, Thomas H; Brismar, Hjalmar; Elofsson, Ulla M

    2008-02-15

    The adsorption and activity of Thermomyces lanuginosus lipase (TLL) was measured with dual polarization interferometry (DPI) and confocal microscopy at a hydrophilic and hydrophobic surface. In the adsorption isotherms, it was evident that TLL both had higher affinity for the hydrophobic surface and adsorbed to a higher adsorbed amount (1.90 mg/m(2)) compared to the hydrophilic surface (1.40-1.50mg/m(2)). The thickness of the adsorbed layer was constant (approximately 3.5 nm) on both surfaces at an adsorbed amount >1.0mg/m(2), but decreased on the hydrophilic surface at lower surface coverage, which might be explained by partially unfolding of the TLL structure. However, a linear dependence of the refractive index of the adsorbed layer on adsorbed amount of TLL on C18 surfaces indicated that the structure of TLL was similar at low and high surface coverage. The activity of adsorbed TLL was measured towards carboxyfluorescein diacetate (CFDA) in solution, which upon lipase activity formed a fluorescent product. The surface fluorescence intensity increase was measured in a confocal microscope as a function of time after lipase adsorption. It was evident that TLL was more active on the hydrophilic surface, which suggested that a larger fraction of adsorbed TLL molecules were oriented with the active site facing the solution compared to the hydrophobic surface. Moreover, most of the activity remained when the TLL surface coverage decreased. Earlier reports on TLL surface mobility on the same surfaces have found that the lateral diffusion was highest on hydrophilic surfaces and at low surface coverage of TLL. Hence, a high lateral mobility might lead to a longer exposure time of the active site towards solution, thereby increasing the activity against a water-soluble substrate.

  18. Palmitic acid follows a different metabolic pathway than oleic acid in human skeletal muscle cells; lower lipolysis rate despite an increased level of adipose triglyceride lipase.

    PubMed

    Bakke, Siril S; Moro, Cedric; Nikolić, Nataša; Hessvik, Nina P; Badin, Pierre-Marie; Lauvhaug, Line; Fredriksson, Katarina; Hesselink, Matthijs K C; Boekschoten, Mark V; Kersten, Sander; Gaster, Michael; Thoresen, G Hege; Rustan, Arild C

    2012-10-01

    Development of insulin resistance is positively associated with dietary saturated fatty acids and negatively associated with monounsaturated fatty acids. To clarify aspects of this difference we have compared the metabolism of oleic (OA, monounsaturated) and palmitic acids (PA, saturated) in human myotubes. Human myotubes were treated with 100μM OA or PA and the metabolism of [(14)C]-labeled fatty acid was studied. We observed that PA had a lower lipolysis rate than OA, despite a more than two-fold higher protein level of adipose triglyceride lipase after 24h incubation with PA. PA was less incorporated into triacylglycerol and more incorporated into phospholipids after 24h. Supporting this, incubation with compounds modifying lipolysis and reesterification pathways suggested a less influenced PA than OA metabolism. In addition, PA showed a lower accumulation than OA, though PA was oxidized to a relatively higher extent than OA. Gene set enrichment analysis revealed that 24h of PA treatment upregulated lipogenesis and fatty acid β-oxidation and downregulated oxidative phosphorylation compared to OA. The differences in lipid accumulation and lipolysis between OA and PA were eliminated in combination with eicosapentaenoic acid (polyunsaturated fatty acid). In conclusion, this study reveals that the two most abundant fatty acids in our diet are partitioned toward different metabolic pathways in muscle cells, and this may be relevant to understand the link between dietary fat and skeletal muscle insulin resistance. Copyright © 2012 Elsevier B.V. All rights reserved.

  19. New insights into the pH-dependent interfacial adsorption of dog gastric lipase using the monolayer technique.

    PubMed

    Bénarouche, Anaïs; Point, Vanessa; Parsiegla, Goetz; Carrière, Frédéric; Cavalier, Jean-François

    2013-11-01

    The access to kinetic parameters of lipolytic enzyme adsorption onto lipids is essential for a better understanding of interfacial enzymology and lipase-lipid interactions. The interfacial adsorption of dog gastric lipase (DGL) was monitored as a function of pH and surface pressure (Π), independently from the catalytic activity, using non-hydrolysable 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC) monomolecular films. The acid-stable DGL, which initiates fat digestion in the stomach, was then selected because its adsorption kinetics onto hydrophobic solid surfaces were already studied. This gastric lipase was therefore used as a model enzyme to validate both experimental and theoretical approaches. Results show that the adsorption process of DGL at the lipid/water interface depends on a pH-dependent adsorption equilibrium coefficient which is optimum at pH 5.0 (K(Ads) = 1.7 ± 0.05 × 10(8)M(-1)). KAds values further allowed an indirect estimation of the molar fraction (ΦE*(%), mol%) as well as the molecular area (AE*) of DGL adsorbed onto DLPC monolayer. Based on these data, a model for DGL adsorption onto DLPC monolayer at pH 5.0 is proposed for a surface pressure range of 15-25 mNm(-1). Copyright © 2013 Elsevier B.V. All rights reserved.

  20. Optimized preparation and characterization of CLEA-lipase from cocoa pod husk.

    PubMed

    Khanahmadi, Soofia; Yusof, Faridah; Amid, Azura; Mahmod, Safa Senan; Mahat, Mohd Khairizal

    2015-05-20

    Cross-linked enzyme aggregate (CLEA) is easily prepared from crude enzyme and has many advantages to the environment and it is considered as an economic method in the context of industrial biocatalysis compared to free enzyme. In this work, a highly active and stable CLEA-lipase from cocoa pod husk (CPH) which is a by-product after removal of cocoa beans, were assayed for their hydrolytic activity and characterized under the optimum condition successfully. Face centered central composite design (FCCCD) under response surface methodology (RSM) was used to get the optimal conditions of the three significant factors (concentration of ammonium sulfate, concentration of glutaraldehyde and concentration of additive) to achieve higher enzyme activity of CLEA. From 20 runs, the highest activity recorded was around 9.407U (83% recovered activity) under the condition of using 20% saturated ammonium sulfate, 60mM glutaraldehyde as cross-linker and 0.17mM bovine serum albumin as feeder. Moreover, the optimal reaction temperature and pH value in enzymatic reaction for both crude enzyme and immobilized were found to be 45°C at pH 8 and 60°C at pH 8.2, respectively. A systematic study of the stability of CLEA and crude enzyme was taken with regards to temperature (25-60°C) and pH (5-10) value and in both factors, CLEA-lipase showed more stability than free lipase. The Km value of CLEA was higher compared to free enzyme (0.55mM vs. 0.08mM). The CLEA retained more than 60% of the initial activity after six cycles of reuse compared to free enzyme. The high stability and recyclability of CLEA-lipase from CPH make it efficient for different industrial applications. Copyright © 2014 Elsevier B.V. All rights reserved.

  1. [Nonesterified fatty acids and the titrable acidity of breast milk. Consequences for collection conditions in milk bans].

    PubMed

    Luzeau, R; Barrois, V; Odièvre, M

    1983-01-01

    The study of breast-milk samples, fresh or after storage, shows that the titrable acidity (expressed in degrees Dornic) is directly correlated with their nonesterified fatty acid concentration. Those fresh samples which contain a high activity of lipoprotein lipase can develop in situ lipolysis. The resulting elevated titrable acidity may lead to consider these samples as unsuitable for infant nutrition. These results suggest that collection and storage of breast-milk have to be reassessed in order to avoid in situ lipolysis.

  2. Novel Strategy of Using Methyl Esters as Slow Release Methanol Source during Lipase Expression by mut+ Pichia pastoris X33

    PubMed Central

    Kumari, Arti; Gupta, Rani

    2014-01-01

    One of the major issues with heterologous production of proteins in Pichia pastoris X33 under AOX1 promoter is repeated methanol induction. To obviate repeated methanol induction, methyl esters were used as a slow release source of methanol in lipase expressing mut+ recombinant. Experimental design was based on the strategy that in presence of lipase, methyl esters can be hydrolysed to release their products as methanol and fatty acid. Hence, upon break down of methyl esters by lipase, first methanol will be used as a carbon source and inducer. Then P. pastoris can switch over to fatty acid as a carbon source for multiplication and biomass maintenance till further induction by methyl esters. We validated this strategy using recombinant P. pastoris expressing Lip A, Lip C from Trichosporon asahii and Lip11 from Yarrowia lipolytica. We found that the optimum lipase yield under repeated methanol induction after 120 h was 32866 U/L, 28271 U/L and 21978 U/L for Lip C, Lip A and Lip 11 respectively. In addition, we found that a single dose of methyl ester supported higher production than repeated methanol induction. Among various methyl esters tested, methyl oleate (0.5%) caused 1.2 fold higher yield for LipA and LipC and 1.4 fold for Lip11 after 120 h of induction. Sequential utilization of methanol and oleic acid by P. pastoris was observed and was supported by differential peroxisome proliferation studies by transmission electron microscopy. Our study identifies a novel strategy of using methyl esters as slow release methanol source during lipase expression. PMID:25170843

  3. Myeloid-Derived Suppressor Cells Are Involved in Lysosomal Acid Lipase Deficiency-Induced Endothelial Cell Dysfunctions

    PubMed Central

    Zhao, Ting; Ding, Xinchun; Du, Hong; Yan, Cong

    2014-01-01

    The underlying mechanisms that lysosomal acid lipase (LAL) deficiency causes infiltration of myeloid-derived suppressor cells (MDSCs) in multiple organs and subsequent inflammation remain incompletely understood. Endothelial cells (ECs), lining the inner layer of blood vessels, constitute barriers regulating leukocytes transmigration to the site of inflammation. Therefore, we hypothesized that ECs are dysfunctional in LAL-deficient (lal−/−) mice. We found that Ly6G+ cells transmigrated more efficiently across lal−/− ECs than wild-type (lal+/+) ECs, which was associated with increased level of platelet endothelial cell adhesion molecule-1 (PECAM-1) and monocyte chemoattractant protein-1 (MCP-1) in lal−/− ECs. In addition, lal−/−ECs showed enhanced migration and proliferation, decreased apoptosis, but impaired tube formation and angiogenesis. lal−/− ECs also suppressed T cell proliferation in vitro. Interestingly, lal−/− Ly6G+ cells promoted in vivo angiogenesis (including a tumor model), EC tube formation and proliferation. Finally, the mammalian target of rapamycin (mTOR) pathway was activated in lal−/− ECs, and inhibition of mTOR reversed EC dysfunctions, including decreasing Ly6G+ cell transmigration, delaying migration, and relieving suppression of T cell proliferation, which was mediated by decreasing production of reactive oxygen species (ROS). Our results indicate that LAL regulates EC functions through interaction with MDSCs and modulation of the mTOR pathway, which may provide a mechanistic basis for targeting MDSCs or mTOR to rejuvenate EC functions in LAL-deficiency related diseases. PMID:25000979

  4. Purification and Characterization of a Cold-Adapted Lipase from Oceanobacillus Strain PT-11

    PubMed Central

    Jiewei, Tian; Zuchao, Lei; Peng, Qiu; Lei, Wang; Yongqiang, Tian

    2014-01-01

    We isolated a moderately halophilic lipase-producing bacterium from the saline soil. Based on the morphological, physiological, chemotaxonomic and phylogenetic analysis, the isolate PT-11 was postulated to be a novel species identified as Oceanobacillus rekensis PT-11. The lipase was purified 2.50-fold by Q-Sepharose FF and SP-Sepharose FF chromatography and its molecular mass was estimated to be 23.5 kDa by SDS-PAGE. It was highly active over the broad temperature ranging from 10 to 35°C and showed up to 80% of the maximum activity at 10°C indicating the lipase to be a typical cold-adapted enzyme. The enzyme activity was slightly enhanced by Na+, Li+ and K+. Incubation with detergents, such as Tween-20 and Tween-80, slightly inhibited the enzyme activity; while Triton X-100decreased the enzyme activity. The enzyme was fairly stable in the presence of long-chain alcohols but was highly denatured in hydrophilic solvents such as acetone or short-chain alcohols (C1–C3). PMID:24984141

  5. Lipoprotein lipase activity in surgical patients: influence of trauma and infection.

    PubMed

    Robin, A P; Askanazi, J; Greenwood, M R; Carpentier, Y A; Gump, F E; Kinney, J M

    1981-08-01

    Hypertriglyceridemia commonly accompanies clinical sepsis and may be caused by increased hepatic production or decreased clearance of triglyceride from the bloodstream. In contrast, enhanced lipid clearing capacity is usually seen after uncomplicated trauma. The purpose of the study was to determine the role of lipoprotein lipase (LPL) in effecting the above changes. Enzyme activity was assayed in skeletal muscle and adipose tissue biopsy samples from 11 normal subjects and from 17 injured and 11 infected surgical patients. Normal subjects after 4 days of 5% dextrose infusion (D5) showed a significant decrease in adipose tissue LPL activity but no change in skeletal muscle activity. Trauma patients after several days of D5 had higher activity in adipose tissue and higher plasma insulin levels than diet-matched control subjects but showed no change in skeletal muscle activity. Infected patients with high plasma triglyceride levels had significantly decreased LPL activity in both tissues. A linear relationship was found between insulin concentration and adipose tissue LPL activity in normal subjects. We conclude that: (1) low tissue LPL activity in sepsis may result in diminished lipid clearance and contribute to hypertriglyceridemia, (2) after trauma, changes in tissue LPL activity as well as other factors such as altered hemodynamics play a role in determining in vivo lipid clearance, and (3) adipose tissue LPL activity is related to the plasma insulin concentration in normal subjects.

  6. Candida antarctica Lipase B Immobilized onto Chitin Conjugated with POSS® Compounds: Useful Tool for Rapeseed Oil Conversion

    PubMed Central

    Zdarta, Jakub; Wysokowski, Marcin; Norman, Małgorzata; Kołodziejczak-Radzimska, Agnieszka; Moszyński, Dariusz; Maciejewski, Hieronim; Ehrlich, Hermann; Jesionowski, Teofil

    2016-01-01

    A new method is proposed for the production of a novel chitin-polyhedral oligomeric silsesquioxanes (POSS) enzyme support. Analysis by such techniques as X-ray photoelectron spectroscopy (XPS) and Raman spectroscopy confirmed the effective functionalization of the chitin surface. The resulting hybrid carriers were used in the process of immobilization of the lipase type b from Candida antarctica (CALB). Fourier transform infrared spectroscopy (FTIR) confirmed the effective immobilization of the enzyme. The tests of the catalytic activity showed that the resulting support-biocatalyst systems remain hydrolytically active (retention of the hydrolytic activity up to 87% for the chitin + Methacryl POSS® cage mixture (MPOSS) + CALB after 24 h of the immobilization), as well as represents good thermal and operational stability, and retain over 80% of its activity in a wide range of temperatures (30–60 °C) and pH (6–9). Chitin-POSS-lipase systems were used in the transesterification processes of rapeseed oil at various reaction conditions. Produced systems allowed the total conversion of the oil to fatty acid methyl esters (FAME) and glycerol after 24 h of the process at pH 10 and a temperature 40 °C, while the Methacryl POSS® cage mixture (MPOSS) was used as a chitin-modifying agent. PMID:27657054

  7. A newly thermoactive and detergent-stable lipase from annular sea bream (Diplodus annularis): Biochemical properties.

    PubMed

    Smichi, Nabil; Miled, Nabil; Gargouri, Youssef; Fendri, Ahmed

    2017-01-01

    A lipolytic activity was located in the annular seabream pyloric caeca, from which a digestive lipase (AsDL) was purified. Pure AsDL has an apparent molecular mass of 50 kDa. The purified lipase is thermoactive as it displays its maximal activity on short- and long-chain triacylglycerols at a temperature of 50 °C. The enzyme is alkaline resistant as it retains 90% of its maximal activity when incubated during 1 H at pH 10. No colipase was detected in the annular seabream pyloric caeca. Similar results were reported for the sardine and the gray mullet digestive systems. This is in line with the idea that colipase might have evolved in mammal animals simultaneously with the appearance of an exocrine pancreas. AsDL is a serine enzyme, like all known lipases from different origins. Interestingly, the pure lipase was found to be insensitive to Triton X-100, a synthetic detergent, addition even at a concentration as high as 12 mM. The purified enzyme has potential applications in detergent and food industry because of its thermal activity and alkaline nature. © 2015 International Union of Biochemistry and Molecular Biology, Inc.

  8. New ether-functionalized ionic liquids for lipase-catalyzed synthesis of biodiesel.

    PubMed

    Zhao, Hua; Song, Zhiyan; Olubajo, Olarongbe; Cowins, Janet V

    2010-09-01

    Ionic liquids (ILs) are being explored as solvents for the enzymatic methanolysis of triglycerides. However, most available ILs (especially hydrophobic ones) have poor capability in dissolving lipids, while hydrophilic ILs tend to cause enzyme inactivation. Recently, we synthesized a new type of ether-functionalized ionic liquids (ILs) carrying anions of acetate or formate; they are capable of dissolving a variety of substrates and are also lipase-compatible (Green Chem., 2008, 10, 696-705). In the present study, we carried out the lipase-catalyzed transesterifications of Miglyol oil 812 and soybean oil in these novel ILs. These ILs are capable of dissolving oils at the reaction temperature (50 degrees C); meanwhile, lipases maintained high catalytic activities in these media even in high concentrations of methanol (up to 50% v/v). High conversions of Miglyol oil were observed in mixtures of IL and methanol (70/30, v/v) when the reaction was catalyzed by a variety of lipases and different enzyme preparations (free and immobilized), especially with the use of two alkylammonium ILs 2 and 3. The preliminary study on the transesterification of soybean oil in IL/methanol mixtures further confirms the potential of using oil-dissolving and lipase-stabilizing ILs in the efficient production of biodiesels.

  9. Ultrasound-assisted chemoenzymatic epoxidation of soybean oil by using lipase as biocatalyst.

    PubMed

    Bhalerao, Machhindra S; Kulkarni, Vaishali M; Patwardhan, Anand V

    2018-01-01

    The present work reports the use of ultrasonic irradiation for enhancing lipase catalyzed epoxidation of soybean oil. Higher degree of unsaturated fatty acids, present in the soybean oil was converted to epoxidized soybean oil by using an immobilized lipase, Candida antarctica (Novozym 435). The effects of various parameters on the relative percentage conversion of the double bond to oxirane oxygen were investigated and the optimum conditions were established. The parameters studied were temperature, hydrogen peroxide to ethylenic unsaturation mole ratio, stirring speed, solvent ratio, catalyst loading, ultrasound frequency, ultrasound input power and duty cycle. The main objective of this work was to intensify chemoenzymatic epoxidation of the soybean oil by using ultrasound, to reduce the time required for epoxidation. Epoxidation of the soybean oil was achieved under mild reaction conditions by indirect ultrasonic irradiations (using ultrasonic bath). The relative percentage conversion to oxirane oxygen of 91.22% was achieved within 5h. The lipase was remarkably stable under optimized reaction conditions, later was recovered and reused six times to produce epoxidized soybean oil (ESO). Copyright © 2017 Elsevier B.V. All rights reserved.

  10. Preparation of Chiral Triacylglycerols, sn-POO and sn-OOP, via Lipase-mediated Acidolysis Reaction.

    PubMed

    Yamamoto, Yukihiro; Yoshida, Hiroki; Nagai, Toshiharu; Hara, Setsuko

    2018-02-01

    It is well known that lipases are useful tools for preparing various structured triacylglycerols (TAGs). However, the lipase-mediated preparation of chiral TAGs has never been reported. This study aimed to prepare chiral TAGs (viz., 1-palmitoyl-2,3-dioleoyl-sn-glycerol (sn-POO) or 1,2-dioleoyl-3-palmitoyl-sn-glycerol (sn-OOP)) via lipase mediated acidolysis, using triolein (TO) and palmitic acid (P) as substrates. Three commercially available lipases (viz., Lipozyme RM-IM ® , Lipozyme TL-IM ® , and Lipase OF ® ) were used. Lipozyme RM-IM ® resulted in an increase 1P-2O (sn-POO + sn-OOP + 1,3-dioleoyl-2-palmitoyl-sn-glycerol) content with reaction time, which plateaued at 2~24 h (max. yield 47.1% at 4 h). The highest sn-POO/sn-OOP ratio of ca. 9 was obtained at 0.25 h, and the rate got close to 1 with reaction time (sn-POO/sn-OOP = 1.3 at 24 h). Lipozyme TL-IM ® resulted in a lower 1P-2O synthesis rate than Lipozyme RM-IM ® , where its highest sn-POO/sn-OOP ratio of ca. 2 was obtained at 0.25 h and did not vary much further with reaction time. In the case of Lipase OF ® , its reaction rate for 1P-2O synthesis was lower than that of the other two lipases, and the highest sn-POO/sn-OOP ratio of ca. 1.4 was obtained at 0.5 h, reaching closer to 1 with a longer reaction time. Reaction solvents (viz., hexane, acetone, and benzene) also affected the 1P-2O preparation, where the highest 1P-2O content was obtained with the solvent-free system. Furthermore, the solvent-free system showed a higher reaction rate for 1P-2O synthesis than did the hexane system, with no effect on chiral specificity of the lipase for the TAG molecules. These results suggested that among three types of commercial lipase, Lipozyme RM-IM ® is the most useful for the preparation of chiral TAGs by acidolysis reaction.

  11. Autodisplay for the co-expression of lipase and foldase on the surface of E. coli: washing with designer bugs

    PubMed Central

    2014-01-01

    Background Lipases including the lipase from Burkholderia cepacia are in a main focus in biotechnology research since many years because of their manifold possibilities for application in industrial processes. The application of Burkholderia cepacia lipase for these processes appears complicated because of the need for support by a chaperone, the lipase specific foldase. Purification and reconstitution protocols therefore interfere with an economic implementation of such enzymes in industry. Autodisplay is a convenient method to express a variety of passenger proteins on the surface of E. coli. This method makes subsequent purification steps to obtain the protein of interest unnecessary. If enzymes are used as passengers, the corresponding cells can simply be applied as whole cell biocatalysts. Furthermore, enzymes surface displayed in this manner often acquire stabilization by anchoring within the outer membrane of E. coli. Results The lipase and its chaperone foldase from B. cepacia were co-expressed on the surface of E. coli via autodisplay. The whole cell biocatalyst obtained thereby exhibited an enzymatic activity of 2.73 mU mL-1 towards the substrate p-nitrophenyl palmitate when applied in an OD578 =1. Outer membrane fractions prepared from the same culture volume showed a lipase activity of 4.01 mU mL-1. The lipase-whole cell biocatalyst as well as outer membrane preparations thereof were used in a standardized laundry test, usually adopted to determine the power of washing agents. In this test, the lipase whole cell biocatalyst and the membrane preparation derived thereof exhibited the same lipolytic activity as the purified lipase from B. cepacia and a lipase preparation which is already applied in commercial washing agents. Conclusions Co-expression of both the lipase and its chaperone foldase on the surface of E. coli yields a lipid degrading whole cell biocatalyst. Therefore the chaperone supported folding process, absolutely required for the lipolytic

  12. Conformation Analysis of T1 Lipase on Alcohols Solvent using Molecular Dynamics Simulation

    NASA Astrophysics Data System (ADS)

    Putri, A. M.; Sumaryada, T.; Wahyudi, S. T.

    2017-07-01

    Biodiesel usually is produced commercially via a transesterification reaction of vegetable oil with alcohol and alkali catalyst. The alkali catalyst has some drawbacks, such as the soap formation during the reaction. T1 Lipase enzyme had been known as a thermostable biocatalyst which is able to produce biodiesel through a cleaner process. In this paper the performance of T1 lipase enzyme as catalyst for transesterification reaction in pure ethanol, methanol, and water solvents were studied using a Molecular Dynamics (MD) Simulation at temperature of 300 K for 10 nanoseconds. The results have shown that in general the conformation of T1 lipase enzyme in methanol is more dynamics as shown by the value of root mean square deviation (RMSD), root mean squared fluctuation (RMSF), and radius of gyration. The highest solvent accessible surface area (SASA) total was also found in methanol due to the contribution of non-polar amino acid in the interior of the protein. Analysis of MD simulation has also revealed that the enzyme structure tend to be more rigid in ethanol environment. The analysis of electrostatic interactions have shown that Glu359-Arg270 salt-bridge pair might hold the key of thermostability of T1 lipase enzyme as shown by its strong and stable binding in all three solvents.

  13. Towards single crystalline, highly monodisperse and catalytically active gold nanoparticles capped with probiotic Lactobacillus plantarum derived lipase

    NASA Astrophysics Data System (ADS)

    Khan, Imran; Nagarjuna, Ravikiran; Ray Dutta, Jayati; Ganesan, Ramakrishnan

    2018-03-01

    Owing to the eco-friendly nature of biomolecules, there lies a huge interest in exploring them as capping agents for nanoparticles to achieve stability and biocompatibility. Lipase extracted from the probiotic Lactobacillus plantarum is utilized for the first time to study its efficacy in capping gold nanoparticles (GNPs) in the room temperature synthesis using HAuCl4. The synthesized lipase-capped GNPs are characterized using UV-visible spectroscopy, FT-IR, HR-TEM, DLS and zeta potential measurements. Importantly, selected area electron diffraction (SAED) studies with HR-TEM have revealed the effect of lipase capping in tuning the polycrystallinity of the GNPs. The lipase-capped GNPs are explored for their catalytic efficiency towards an environmentally and industrially important conversion of 4-nitrophenol to 4-aminophenol. Exploiting the amine functional groups in the protein, the recoverability and reusability of the GNPs have been demonstrated through immobilization over amine-functionalized Fe3O4 nanoparticles.

  14. Beauveria bassiana Lipase A expressed in Komagataella (Pichia) pastoris with potential for biodiesel catalysis.

    PubMed

    Vici, Ana C; da Cruz, Andrezza F; Facchini, Fernanda D A; de Carvalho, Caio C; Pereira, Marita G; Fonseca-Maldonado, Raquel; Ward, Richard J; Pessela, Benevides C; Fernandez-Lorente, Gloria; Torres, Fernando A G; Jorge, João A; Polizeli, Maria L T M

    2015-01-01

    Lipases (EC 3.1.1.3) comprise a biotechnologically important group of enzymes because they are able to catalyze both hydrolysis and synthesis reactions, depending on the amount of water in the system. One of the most interesting applications of lipase is in the biofuel industry for biodiesel production by oil and ethanol (or methanol) transesterification. Entomopathogenic fungi, which are potential source of lipases, are still poorly explored in biotechnological processes. The present work reports the heterologous expression and biochemical characterization of a novel Beauveria bassiana lipase with potential for biodiesel production. The His-tagged B. bassiana lipase A (BbLA) was produced in Komagataella pastoris in buffered methanol medium (BMM) induced with 1% methanol at 30°C. Purified BbLA was activated with 0.05% Triton X-100 and presented optimum activity at pH 6.0 and 50°C. N-glycosylation of the recombinant BbLA accounts for 31.5% of its molecular weight. Circular dichroism and molecular modeling confirmed a structure composed of α-helix and β-sheet, similar to α/β hydrolases. Immobilized BbLA was able to promote transesterification reactions in fish oil, demonstrating potential for biodiesel production. BbLA was successfully produced in K. pastoris and shows potential use for biodiesel production by the ethanolysis reaction.

  15. Symbiotic effects of a lipase-secreting bacterium, Burkholderia arboris SL1B1, and a glycerol-assimilating yeast, Candida cylindracea SL1B2, on triacylglycerol degradation.

    PubMed

    Matsuoka, Hiroshi; Miura, Atsuto; Hori, Katsutoshi

    2009-04-01

    Although microbial degradation of oils and fats has been developed for application in wastewater treatment, microbial degraders are not always effective in the field, for example, in grease-traps installed for the treatment of wastewater from restaurants and food industries. Wastewater in grease-traps is usually in a pH range of 5.5 to 6.5 due to hydrolysis of triacylglycerol (TAG). Because many microorganisms commercialized for use in grease-traps cannot grow at pH 6.0, we screened oil-degrading microorganisms from the environment by growing in a medium at pH 6.0 containing canola oil as the sole carbon source. We succeeded in isolating the bacterial strain Burkholderia arboris SL1B1, which secretes lipase and assimilates fatty acids, and the yeast strain Candida cylindracea SL1B2, which assimilates glycerol. The former cannot utilize glycerol as a carbon source while the latter shows only faint lipase activity that cannot support its active growth on TAG. Canola oil was degraded rapidly by a pure culture of SL1B1 at pH 6.0. However, the degradation was markedly enhanced by a mixed culture of SL1B1 and SL1B2, although lipase activity during cultivation was similar between the pure and mixed cultures. This suggests that the reversible reaction proceeds in the direction of hydrolysis of TAG due to consumption of the reaction product, glycerol, by the symbiotic yeast strain. The optimum pH and temperature of lipase secreted by B. arboris SL1B1 were 8.0 and 60 degrees C, respectively. This lipase showed highly thermal stability; the residual activity after incubation at 70 degrees C for 2 h did not decline.

  16. Isolation of a novel alkaline-stable lipase from a metagenomic library and its specific application for milkfat flavor production

    PubMed Central

    2014-01-01

    Background Lipolytic enzymes are commonly used to produce desired flavors in lipolyzed milkfat (LMF) manufacturing processes. However, the choice of enzyme is critical because it determines the final profile of fatty acids released and the consequent flavor of the product. We previously constructed a metagenomic library from marine sediments, to explore the novel enzymes which have unique properties useful in flavor-enhancing LMF. Results A novel lipase Est_p6 was isolated from a metagenomic library and was expressed highly in E.coli. Bioinformatic analysis indicated that Est_p6 belongs to lipolytic enzyme family IV, the molecular weight of purified Est_p6 was estimated at 36 kDa by SDS-PAGE. The hydrolytic activity of the enzyme was stable under alkaline condition and the optimal temperature was 50°C. It had a high specific activity (2500 U/mg) toward pNP butyrate (pNP-C4), with Km and Vmax values of 1.148 mM and 3497 μmol∙min-1∙mg-1, respectively. The enzyme activity was enhanced by DTT and was not significantly inhibited by PMSF, EDTA or SDS. This enzyme also showed high hydrolysis specificity for myristate (C14) and palmitate (C16). It seems that Est_p6 has safety for commercial LMF flavor production and food manufacturing processes. Conclusions The ocean is a vast and largely unexplored resource for enzymes. According the outstanding alkaline-stability of Est_p6 and it produced myristic acid and palmitic acid more efficiently than other free fatty acids in lipolyzed milkfat. This novel lipase may be used to impart a distinctive and desirable flavor and odor in milkfat flavor production. PMID:24387764

  17. Modeling of lipase catalyzed ring-opening polymerization of epsilon-caprolactone.

    PubMed

    Sivalingam, G; Madras, Giridhar

    2004-01-01

    Enzymatic ring-opening polymerization of epsilon-caprolactone by various lipases was investigated in toluene at various temperatures. The determination of molecular weight and structural identification was carried out with gel permeation chromatography and proton NMR, respectively. Among the various lipases employed, an immobilized lipase from Candida antartica B (Novozym 435) showed the highest catalytic activity. The polymerization of epsilon-caprolactone by Novozym 435 showed an optimal temperature of 65 degrees C and an optimum toluene content of 50/50 v/v of toluene and epsilon-caprolactone. As lipases can degrade polyesters, a maximum in the molecular weight with time was obtained due to the competition of ring opening polymerization and degradation by specific chain end scission. The optimum temperature, toluene content, and the variation of molecular weight with time are consistent with earlier observations. A comprehensive model based on continuous distribution kinetics was developed to model these phenomena. The model accounts for simultaneous polymerization, degradation and enzyme deactivation and provides a technique to determine the rate coefficients for these processes. The dependence of these rate coefficients with temperature and monomer concentration is also discussed.

  18. Immobilization of Pseudomonas fluorescens lipase onto magnetic nanoparticles for resolution of 2-octanol.

    PubMed

    Xun, Er-na; Lv, Xiao-li; Kang, Wei; Wang, Jia-xin; Zhang, Hong; Wang, Lei; Wang, Zhi

    2012-10-01

    The lipase from Pseudomonas fluorescens (Lipase AK, AKL) was immobilized onto the magnetic Fe(3)O(4) nanoparticles via hydrophobic interaction. Enzyme loading and immobilization yield were determined as 21.4±0.5 mg/g and 49.2±1.8 %, respectively. The immobilized AKL was successfully used for resolution of 2-octanol with vinyl acetate used as acyl donor. Effects of organic solvent, water activity, substrate ratio, and temperature were investigated. Under the optimum conditions, the preferred isomer for AKL is the (R)-2-octanol and the highest enantioselectivity (E=71.5±2.2) was obtained with a higher enzyme activity (0.197±0.01 μmol/mg/min). The results also showed that the immobilized lipase could be easily separated from reaction media by the magnetic steel and remained 89 % of its initial activity as well as the nearly unchanged enantioselectivity after five consecutive cycles, indicating a high stability in practical operation.

  19. In silico identification of potent pancreatic triacylglycerol lipase inhibitors from traditional Chinese medicine.

    PubMed

    Chen, Kuan-Yu; Chang, Su-Sen; Chen, Calvin Yu-Chian

    2012-01-01

    Pancreatic triacylglycerol lipase (PNLIP) are primary lipases that are critical for triacylglyceride digestion in human. Since reduced metabolism of triacylglyceride might be a plausible concept for weight loss, we screened for potential PNLIP inhibitors from traditional Chinese medicine (TCM) with the aim to identify weight loss candidate compounds. TCM candidates Aurantiamide, Cnidiadin, and 2-hexadecenoic acid exhibited higher Dock Scores than the commercial drug Orlistat, and were also predicted to have inhibitory characteristics against PNLIP using constructed MLR (R(2) = 0.8664) and SVM (R(2) = 0.9030) models. Molecular dynamics indicated that the TCM-PNLIP complexes formed were stable. We identified that the PNLIP binding site has several residues that can serve as anchors, and a hydrophobic corridor that provides additional stability to the complex. Aurantiamide, Cnidiadin, and 2-hexadecenoic acid all have features that correspond to these binding site features, indicating their potential as candidates for PNLIP inhibitors. The information presented in this study may provide helpful insights to designing novel weight-control drugs.

  20. Rapid screening natural-origin lipase inhibitors from hypolipidemic decoctions by ultrafiltration combined with liquid chromatography-mass spectrometry.

    PubMed

    Xiao, Shun; Yu, Runru; Ai, Ni; Fan, Xiaohui

    2015-02-01

    Lipase inhibitors generate hypolipidemic effect that is helpful to control or treat some obesity diseases by inactivating catalytic activity of human pancreatic lipase, a key enzyme involved in triglyceride hydrolysis in vivo. Many traditional Chinese medicine (TCM) formulae have been effectively used to treat obesity and other fat related diseases for centuries and modern biological experiments demonstrate therapeutic effect of these formulae can be linked to their lipid-lowering capability in blood. These observations suggest that these hypolipidemic decoctions (HDs) could be a promising resource of natural-origin lipase inhibitors. This work described a rapid approach for screening lipase inhibitors from four widely used HDs, including Wu-Ling-San (WLS), Ze-Xie decoction (ZX), Xiao-Xian-Xiong decoction (XXX) and Xiao-Chai-Hu decoction (XCH), by ultrafiltration combing with high performance liquid chromatography-mass spectrometry (HPLC-MS). Our results showed sixteen natural-origin lipase inhibitors were discovered and identified by high resolution and multistage mass spectrometry. Inhibitory activities of two compounds were confirmed by a functional assay of lipase, which validated the reliability of our approach. Molecular docking simulation was then performed to investigate potential mechanism of action for these compounds. Together we present an efficient method for rapid screening lipase inhibitors from complex natural products, which can be easily accommodated to other important enzymatic system with therapeutic values. Copyright © 2014 Elsevier B.V. All rights reserved.

  1. Utilization of fish meal and fish oil for production of Cryptococcus sp. MTCC 5455 lipase and hydrolysis of polyurethane thereof.

    PubMed

    Thirunavukarasu, K; Purushothaman, S; Gowthaman, M K; Nakajima-Kambe, T; Rose, C; Kamini, N R

    2015-09-01

    Fish meal has been used as an additional nitrogen source and fish oil as inducer for the growth and production of lipase from Cryptococcus sp. MTCC 5455. A response surface design illustrated that the optimum factors influencing lipase production were fish meal, 1.5 %, w/v, Na2HPO4, 0.2 %, w/v, yeast extract, 0.25 %, w/v and sardine oil, 2.0 %, w/v with an activity of 71.23 U/mL at 96 h and 25 °C, which was 48.39 % higher than the conventional one-factor-at-a-time method. The crude concentrated enzyme hydrolyzed polyurethane (PUR) efficiently and hydrolysis was 94 % at 30 °C and 96 h. The products, diethylene glycol and adipic acid were quantified by HPLC and scanning electron microscopic studies of the degraded polymer showed significant increase in size of the holes from 24 to 72 h of incubation. Hydrolysis of PUR within 96 h makes the lipase novel for disposal of PUR and provides an innovative solution to the problems created by plastic wastes.

  2. Cellulosic fraction of rice bran fibre alters the conformation and inhibits the activity of porcine pancreatic lipase

    USDA-ARS?s Scientific Manuscript database

    The anti-lipase properties of insoluble dietary fiber obtained from rice bran treated with H2SO4 followed by 1.25% KOH were investigated and compared. Porcine pancreatic lipase (PL) adsorbed with higher velocity and saturated at a higher level on the rice bran fibers prepared with higher concentrat...

  3. Fatty acids bind tightly to the N-terminal domain of angiopoietin-like protein 4 and modulate its interaction with lipoprotein lipase.

    PubMed

    Robal, Terje; Larsson, Mikael; Martin, Miina; Olivecrona, Gunilla; Lookene, Aivar

    2012-08-24

    Angiopoietin-like protein 4 (Angptl4), a potent regulator of plasma triglyceride metabolism, binds to lipoprotein lipase (LPL) through its N-terminal coiled-coil domain (ccd-Angptl4) inducing dissociation of the dimeric enzyme to inactive monomers. In this study, we demonstrate that fatty acids reduce the inactivation of LPL by Angptl4. This was the case both with ccd-Angptl4 and full-length Angptl4, and the effect was seen in human plasma or in the presence of albumin. The effect decreased in the sequence oleic acid > palmitic acid > myristic acid > linoleic acid > linolenic acid. Surface plasmon resonance, isothermal titration calorimetry, fluorescence, and chromatography measurements revealed that fatty acids bind with high affinity to ccd-Angptl4. The interactions were characterized by fast association and slow dissociation rates, indicating formation of stable complexes. The highest affinity for ccd-Angptl4 was detected for oleic acid with a subnanomolar equilibrium dissociation constant (K(d)). The K(d) values for palmitic and myristic acid were in the nanomolar range. Linoleic and linolenic acid bound with much lower affinity. On binding of fatty acids, ccd-Angptl4 underwent conformational changes resulting in a decreased helical content, weakened structural stability, dissociation of oligomers, and altered fluorescence properties of the Trp-38 residue that is located close to the putative LPL-binding region. Based on these results, we propose that fatty acids play an important role in modulating the effects of Angptl4.

  4. Development of an indirect method for measuring porcine pancreatic lipase in human duodenal fluid.

    PubMed

    Tuvignon, N; Abousalham, A; Tocques, F; De Caro, J; De Caro, A; Laugier, R; Carrière, F

    2008-12-15

    Patients with exocrine pancreatic insufficiency are usually treated with porcine pancreatic enzymes but the bioavailability of these enzymes in the gut remains a matter of discussion. In order to determine the duodenal availability of porcine pancreatic lipase (PPL) present in pancreatic extracts (PE) taken orally, we developed a method for quantifying PPL in samples containing both PPL and human pancreatic lipase (HPL). Total pancreatic lipase activity measurements using the pH-stat technique and tributyrin as substrate were combined with an HPL-specific ELISA. Based on the known specific activity of the purified HPL, its activity was deduced from the ELISA measurements, and the PPL activity was obtained by subtracting the HPL activity from the total pancreatic lipase activity. This assay was established and validated using various samples containing pure PPL and recombinant HPL or PE, mixed or not with human duodenal juice. Samples collected in vivo from patients treated with PE were also tested. It was found that PPL did not affect the HPL ELISA, and the indirect PPL assay gave a measurement accuracy of 6.6% with the samples containing pure PPL and 10% with those containing PE. This assay was also used successfully to discriminate between PPL and the endogenous HPL present in the duodenal contents of patients with severe pancreatic insufficiency treated with PE. This method might provide a useful means of assessing the availability of PEs at their site of action, in the absence of a PPL-specific ELISA.

  5. Optimized Production of Biodiesel from Waste Cooking Oil by Lipase Immobilized on Magnetic Nanoparticles

    PubMed Central

    Yu, Chi-Yang; Huang, Liang-Yu; Kuan, I-Ching; Lee, Shiow-Ling

    2013-01-01

    Biodiesel, a non-toxic and biodegradable fuel, has recently become a major source of renewable alternative fuels. Utilization of lipase as a biocatalyst to produce biodiesel has advantages over common alkaline catalysts such as mild reaction conditions, easy product separation, and use of waste cooking oil as raw material. In this study, Pseudomonas cepacia lipase immobilized onto magnetic nanoparticles (MNP) was used for biodiesel production from waste cooking oil. The optimal dosage of lipase-bound MNP was 40% (w/w of oil) and there was little difference between stepwise addition of methanol at 12 h- and 24 h-intervals. Reaction temperature, substrate molar ratio (methanol/oil), and water content (w/w of oil) were optimized using response surface methodology (RSM). The optimal reaction conditions were 44.2 °C, substrate molar ratio of 5.2, and water content of 12.5%. The predicted and experimental molar conversions of fatty acid methyl esters (FAME) were 80% and 79%, respectively. PMID:24336109

  6. Enzymatic synthesis of ethyl esters from waste oil using mixtures of lipases in a plug-flow packed-bed continuous reactor.

    PubMed

    Kathiele Poppe, Jakeline; Matte, Carla Roberta; Olave de Freitas, Vitória; Fernandez-Lafuente, Roberto; Rodrigues, Rafael C; Záchia Ayub, Marco Antônio

    2018-04-30

    This work describes the continuous synthesis of ethyl esters via enzymatic catalysis on a packed-bed continuous reactor, using mixtures of immobilized lipases (combi-lipases) of Candida antarctica (CALB), Thermomyces lanuginosus (TLL), and Rhizomucor miehei (RML). The influence of the addition of glass beads to the reactor bed, evaluation of the use of different solvents, and flow rate on reaction conditions were studied. All experiments were conducted using the best combination of lipases according to the fatty acid composition of the waste oil (combi-lipase composition: 40% of TLL, 35% of CALB, and 25% of RML), and soybean oil (combi-lipase composition: 22.5% of TLL, 50% of CALB, and 27.5% of RML). The best general reaction conditions were found to be using tert-butanol as solvent, and the flow rate of 0.08 mL min -1 . The combi-lipase reactors operating at steady state for over 30 days (720 h), kept conversion yields of approximately 50%, with average productivity of 1.94 g ethyl esters g substrate -1 h -1 , regardless of the type of oil in use. This article is protected by copyright. All rights reserved. © 2018 American Institute of Chemical Engineers.

  7. Critical evaluation of a specific ELISA and two enzymatic assays of pancreatic lipases in human sera.

    PubMed

    Grandval, Philippe; De Caro, Alain; De Caro, Josiane; Sias, Barbara; Carrière, Frédéric; Verger, Robert; Laugier, René

    2004-01-01

    Human pancreatic lipases (HPL) include the classical HPL, and two related proteins known as pancreatic lipase-related proteins 1 and 2 (HPLRP1 and 2). The aim of this study was to develop an ELISA for specifically quantifying the classical-HPL level in sera of patients with and without pancreatic disorders. The specific activity of various human (including classical-HPL) and microbial lipases was measured using Lipa Vitros and potentiometric (pH-stat) assays. A double sandwich ELISA was also set up, using an anti-classical-HPL polyclonal antibody and a biotinylated monoclonal antibody (mAb 146-40) specific to the classical-HPL. Sera (n = 53) were collected from patients with and without pancreatic disorders. The lipase concentration was deduced from the measured lipolytic activity and compared with the corresponding classical-HPL concentration, measured with the ELISA. Both the purified HPLRP2 and 3 lipases of microbial origin were found to have a significant and unexpected lipolytic activity under the standard Lipa Vitros assay, whereas the ELISA test developed in the present study was found to be specific for the classical-HPL, due to the absence of cross-reactivity between mAb 146-40, HPLRP1 and HPLRP2. The efficiency of the ELISA was assessed in terms of its reproducibility and accuracy. The lower detection limit of classical-HPL was found to be 0.03 microg/l. A good correlation was found to exist between the lipase concentrations obtained in the ELISA, pH-stat and Lipa Vitros tests, in both the control and pathological groups. This is the first time a specific method of measuring classical-HPL in human serum has been proposed. Using this ELISA, we established with the 53 sera selected in the present study, that the Lipa Vitros assay as well as the pH-stat assay were mostly detecting classical pancreatic lipase. However, it is possible that other lipases such as HPLRP2 or lipases of microbial origin, present in some pathological sera, may well interfere with

  8. Clinical Effect and Safety Profile of Recombinant Human Lysosomal Acid Lipase in Patients with Cholesteryl Ester Storage Disease

    PubMed Central

    Balwani, Manisha; Breen, Catherine; Enns, Gregory M; Deegan, Patrick B; Honzík, Tomas; Jones, Simon; Kane, John P; Malinova, Vera; Sharma, Reena; Stock, Eveline O; Valayannopoulos, Vassili; Wraith, J Edmond; Burg, Jennifer; Eckert, Stephen; Schneider, Eugene; Quinn, Anthony G

    2013-01-01

    Background & Aims Cholesteryl Ester Storage Disease, an inherited deficiency of lysosomal acid lipase, is an underappreciated cause of progressive liver disease with no approved therapy. Presenting features include dyslipidemia, elevated transaminases, and hepatomegaly. Methods To assess the clinical effects and safety of the recombinant human lysosomal acid lipase, sebelipase alfa, 9 patients received 4 once-weekly infusions (0.35, 1, or 3 mg·kg−1) in LAL-CL01 which is the first human study of this investigational agent. Patients completing LAL-CL01 were eligible to enroll in the extension study (LAL-CL04) in which they again received 4 once-weekly infusions of sebelipase alfa (0.35, 1, or 3 mg·kg−1) before transitioning to long term every other week infusions (1 or 3 mg·kg−1). Results Sebelipase alfa was well-tolerated with mostly mild adverse events unrelated sebelipase alfa. No anti-drug antibodies were detected. Transaminases decreased in patients in LAL-CL01 and increased between studies. In 7 patients receiving ongoing sebelipase alfa treatment in LAL-CL04, mean±SD decreases for alanine transaminase and aspartate aminotransferase at week 12 compared to the baseline values in LAL-CL01 were 46±21U/L (-52%) and 21±14U/L (-36%), respectively (p<0.05). Through week 12 of LAL-CL04, these 7 patients also showed mean decreases from baseline in total cholesterol of 44±41mg/dL (-22%; p=0.047), low density lipoprotein-cholesterol of 29±31mg/dL (-27%; p=0.078), and triglycerides of 50±38mg/dL (-28%, p=0.016) and increases in high density lipoprotein-cholesterol of 5mg/dL (15%; p=0.016). Conclusions These data establish that sebelipase alfa, an investigational enzyme replacement, in patients with Cholesteryl Ester Storage Disease is well tolerated, rapidly decreases serum transaminases and that these improvements are sustained with long term dosing and are accompanied by improvements in serum lipid profile. PMID:23348766

  9. Microbial lipase mediated by health beneficial modification of cholesterol and flavors in food products: A review.

    PubMed

    Sharma, Ranjana; Sharma, Nivedita

    2017-06-14

    The tremendous need of lipase in varied applications in biotechnological increases its economical value in food and allied industries. Lipase has an impressive number of applications viz. enhancements of flavor in food products (Cheese, butter, alcoholic beverages, milk chocolate and diet control food stuffs), detergent industry in removing oil, grease stain, organic chemical processing, textile industry, oleochemical industry, cosmetic industry and also as therapeutic agents in pharmaceutical industries. This communication extends the frontier of lipase catalyzed benefits to human body by lowering serum cholesterol and enhancement of flavor in different food products. Among all, multiple innovations going on in the field of lipase applications are widening its scope in food industries consistently. Therefore in the present work an effort has been made to explore the utilization of lipase in the field of food product enhancement. Supplementation of food products with lipase results in modification of its physical, chemical and biochemical properties by enhancing its therapeutic activity. Lipases are the most important enzymes used in food industries. They are utilized as industrial catalysts for lipid hydrolysis. Because of lipases hydrolysis nature it is widely exploited to catalyze lipids or fats in different food products and enhancement of food flavors. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  10. Whole genome sequencing analysis of the cutaneous pathogenic yeast Malassezia restricta and identification of the major lipase expressed on the scalp of patients with dandruff.

    PubMed

    Park, Minji; Cho, Yong-Joon; Lee, Yang Won; Jung, Won Hee

    2017-03-01

    Malassezia species are opportunistic pathogenic fungi that are frequently associated with seborrhoeic dermatitis, including dandruff. Most Malassezia species are lipid dependent, a property that is compensated by breaking down host sebum into fatty acids by lipases. In this study, we aimed to sequence and analyse the whole genome of Malassezia restricta KCTC 27527, a clinical isolate from a Korean patient with severe dandruff, to search for lipase orthologues and identify the lipase that is the most frequently expressed on the scalp of patients with dandruff. The genome of M. restricta KCTC 27527 was sequenced using the Illumina MiSeq and PacBio platforms. Lipase orthologues were identified by comparison with known lipase genes in the genomes of Malassezia globosa and Malassezia sympodialis. The expression of the identified lipase genes was directly evaluated in swab samples from the scalps of 56 patients with dandruff. We found that, among the identified lipase-encoding genes, the gene encoding lipase homolog MRES_03670, named LIP5 in this study, was the most frequently expressed lipase in the swab samples. Our study provides an overview of the genome of a clinical isolate of M. restricta and fundamental information for elucidating the role of lipases during fungus-host interaction. © 2016 Blackwell Verlag GmbH.

  11. New Strategy for the Immobilization of Lipases on Glyoxyl-Agarose Supports: Production of Robust Biocatalysts for Natural Oil Transformation.

    PubMed

    Godoy, César A

    2017-10-12

    Immobilization on Glyoxyl-agarose support (Gx) is one of the best strategies to stabilize enzymes. However, the strategy is difficult to apply at neutral pH when most enzymes are stable and, even when possible, produces labile derivatives. This work contributes to overcoming this hurdle through a strategy that combines solid-phase amination, presence of key additives, and derivative basification. To this end, aminated industrial lipases from Candida artarctica (CAL), Thermomyces lunuginosus (TLL), and the recombinant Geobacillus thermocatenulatus (BTL2) were immobilized on Gx for the first time at neutral pH using anthranilic acid (AA) or DTT as additives (immobilization yields >70%; recovered activities 37.5-76.7%). The spectroscopic evidence suggests nucleophilic catalysis and/or adsorption as the initial lipase immobilization events. Subsequent basification drastically increases the stability of BTL2-glyoxyl derivatives under harsh conditions (t 1/2 , from 2.1-54.5 h at 70 °C; from 10.2 h-140 h in 80% dioxane). The novel BTL2-derivatives were active and selective in fish oil hydrolysis (1.0-1.8 μmol of polyunsaturated fatty acids (PUFAs) min -1 ·g -1 ) whereas the selected TLL-derivative was as active and stable in biodiesel production (fatty ethyl esters, EE) as the commercial Novozyme ® -435 after ten reaction cycles (~70% EE). Therefore, the potential of the proposed strategy in producing suitable biocatalysts for industrial processes was demonstrated.

  12. New Strategy for the Immobilization of Lipases on Glyoxyl–Agarose Supports: Production of Robust Biocatalysts for Natural Oil Transformation

    PubMed Central

    2017-01-01

    Immobilization on Glyoxyl–agarose support (Gx) is one of the best strategies to stabilize enzymes. However, the strategy is difficult to apply at neutral pH when most enzymes are stable and, even when possible, produces labile derivatives. This work contributes to overcoming this hurdle through a strategy that combines solid-phase amination, presence of key additives, and derivative basification. To this end, aminated industrial lipases from Candida artarctica (CAL), Thermomyces lunuginosus (TLL), and the recombinant Geobacillus thermocatenulatus (BTL2) were immobilized on Gx for the first time at neutral pH using anthranilic acid (AA) or DTT as additives (immobilization yields >70%; recovered activities 37.5–76.7%). The spectroscopic evidence suggests nucleophilic catalysis and/or adsorption as the initial lipase immobilization events. Subsequent basification drastically increases the stability of BTL2–glyoxyl derivatives under harsh conditions (t1/2, from 2.1–54.5 h at 70 °C; from 10.2 h–140 h in 80% dioxane). The novel BTL2-derivatives were active and selective in fish oil hydrolysis (1.0–1.8 μmol of polyunsaturated fatty acids (PUFAs) min−1·g−1) whereas the selected TLL-derivative was as active and stable in biodiesel production (fatty ethyl esters, EE) as the commercial Novozyme®-435 after ten reaction cycles (~70% EE). Therefore, the potential of the proposed strategy in producing suitable biocatalysts for industrial processes was demonstrated. PMID:29023423

  13. PNPLA3 has retinyl-palmitate lipase activity in human hepatic stellate cells

    PubMed Central

    Pirazzi, Carlo; Valenti, Luca; Motta, Benedetta Maria; Pingitore, Piero; Hedfalk, Kristina; Mancina, Rosellina Margherita; Burza, Maria Antonella; Indiveri, Cesare; Ferro, Yvelise; Montalcini, Tiziana; Maglio, Cristina; Dongiovanni, Paola; Fargion, Silvia; Rametta, Raffaela; Pujia, Arturo; Andersson, Linda; Ghosal, Saswati; Levin, Malin; Wiklund, Olov; Iacovino, Michelina; Borén, Jan; Romeo, Stefano

    2014-01-01

    Retinoids are micronutrients that are stored as retinyl esters in the retina and hepatic stellate cells (HSCs). HSCs are key players in fibrogenesis in chronic liver diseases. The enzyme responsible for hydrolysis and release of retinyl esters from HSCs is unknown and the relationship between retinoid metabolism and liver disease remains unclear. We hypothesize that the patatin-like phospholipase domain-containing 3 (PNPLA3) protein is involved in retinol metabolism in HSCs. We tested our hypothesis both in primary human HSCs and in a human cohort of subjects with non-alcoholic fatty liver disease (N = 146). Here we show that PNPLA3 is highly expressed in human HSCs. Its expression is regulated by retinol availability and insulin, and increased PNPLA3 expression results in reduced lipid droplet content. PNPLA3 promotes extracellular release of retinol from HSCs in response to insulin. We also show that purified wild-type PNPLA3 hydrolyzes retinyl palmitate into retinol and palmitic acid. Conversely, this enzymatic activity is markedly reduced with purified PNPLA3 148M, a common mutation robustly associated with liver fibrosis and hepatocellular carcinoma development. We also find the PNPLA3 I148M genotype to be an independent (P = 0.009 in a multivariate analysis) determinant of circulating retinol-binding protein 4, a reliable proxy for retinol levels in humans. This study identifies PNPLA3 as a lipase responsible for retinyl-palmitate hydrolysis in HSCs in humans. Importantly, this indicates a potential novel link between HSCs, retinoid metabolism and PNPLA3 in determining the susceptibility to chronic liver disease. PMID:24670599

  14. S5 Lipase: an organic solvent tolerant enzyme.

    PubMed

    Rahman, Raja Noor Zaliha Abdul; Baharum, Syarul Nataqain; Salleh, Abu Bakar; Basri, Mahiran

    2006-12-01

    In this study, an organic solvent tolerant bacterial strain was isolated. This strain was identified as Pseudomonas sp. strain S5, and was shown to degrade BTEX (Benzene, Toluene, Ethyl-Benzene, and Xylene). Strain S5 generates an organic solvent-tolerant lipase in the late logarithmic phase of growth. Maximum lipase production was exhibited when peptone was utilized as the sole nitrogen source. Addition of any of the selected carbon sources to the medium resulted in a significant reduction of enzyme production. Lower lipase generation was noted when an inorganic nitrogen source was used as the sole nitrogen source. This bacterium hydrolyzed all tested triglycerides and the highest levels of production were observed when olive oil was used as a natural triglyceride. Basal medium containing Tween 60 enhanced lipase production to the most significant degree. The absence of magnesium ions (Mg2+) in the basal medium was also shown to stimulate lipase production. Meanwhile, an alkaline earth metal ion, Na+, was found to stimulate the production of S5 lipase.

  15. Clean synthesis of biolubricant range esters using novel liquid lipase enzyme in solvent free medium.

    PubMed

    Trivedi, Jayati; Aila, Mounika; Sharma, Chandra Dutt; Gupta, Piyush; Kaul, Savita

    2015-01-01

    In view of the rising global problems of environment pollution and degradation, the present process provides a 'green solution' to the synthesis of higher esters of lubricant range, more specifically in the range C12-C36, using different combinations of acids and alcohols, in a single step reaction. The esters produced are biodegradable in nature and have a plethora of uses, such as in additives, as lubricating oils and other hydraulic fluids. The enzymatic esterification was performed using liquid (non-immobilized or free) lipase enzyme, without any additional organic solvent. Soluble lipase proves to be superior to immobilized enzymes as it is more cost effective and provides a faster process for the production of higher esters of lubricant range. An interesting finding was, that the lipase enzyme showed higher conversion rates with increasing carbon number of straight chain alcohols and acids. Reactions were carried out for the optimization of initial water concentration, temperature, pH of the substrate mixture and the chain length of the substrates. Under optimized conditions, the method was suitable to achieve ~ 99% conversion. Thus, the process provides an environment friendly, enzymatic alternative to the chemical route which is currently used in the industrial synthesis of lubricant components.

  16. Engineering of baker's yeasts, E. coli and Bacillus hosts for the production of Bacillus subtilis Lipase A.

    PubMed

    Sánchez, Marta; Prim, Núria; Rández-Gil, Francisca; Pastor, F I Javier; Diaz, Pilar

    2002-05-05

    Lipases are versatile biocatalists showing multiple applications in a wide range of biotechnological processes. The gene lipA coding for Lipase A from Bacillus subtilis was isolated by PCR amplification, cloned and expressed in Escherichia coli, Saccharomyces cerevisiae and Bacillus subtilis strains, using pBR322, YEplac112 and pUB110-derived vectors, respectively. Lipase activity analysis of the recombinant strains showed that the gene can be properly expressed in all hosts assayed, this being the first time a lipase from bacterial origin can be expressed in baker's S. cerevisiae strains. An important increase of lipase production was obtained in heterologous hosts with respect to that of parental strains, indicating that the described systems can represent a useful tool to enhance productivity of the enzyme for biotechnological applications, including the use of the lipase in bread making, or as a technological additive. Copyright 2002 Wiley Periodicals, Inc.

  17. Solvent Flux Method (SFM): A Case Study of Water Access to Candida antarctica Lipase B.

    PubMed

    Benson, Sven P; Pleiss, Jürgen

    2014-11-11

    The solvent flux method (SFM) was developed to comprehensively characterize the influx of solvent molecules from the solvent environment into the active site of a protein in the framework of molecular dynamics simulations. This was achieved by introducing a solvent concentration gradient as well as partially reorienting and rescaling the velocity vector of all solvent molecules contained within a spherical volume enclosing the protein, thus inducing an accelerated solvent influx toward the active site. In addition to the detection of solvent access pathway within the protein structure, it is hereby possible to identify potential amino acid positions relevant to solvent-related enzyme engineering with high statistical significance. The method is particularly aimed at improving the reverse hydrolysis reaction rates in nonaqueous media. Candida antarctica lipase B (CALB) binds to a triglyceride-water interface with its substrate entrance channel oriented toward the hydrophobic substrate interface. The lipase-triglyceride-water system served as a model system for SFM to evaluate the influx of water molecules to the active site. As a proof of principle for SFM, a previously known water access pathway in CALB was identified as the primary water channel. In addition, a secondary water channel and two pathways for water access which contribute to water leakage between the protein and the triglyceride-water interface were identified.

  18. Molecular characterization of human ABHD2 as TAG lipase and ester hydrolase

    PubMed Central

    M., Naresh Kumar; V.B.S.C., Thunuguntla; G.K., Veeramachaneni; B., Chandra Sekhar; Guntupalli, Swapna; J.S., Bondili

    2016-01-01

    Alterations in lipid metabolism have been progressively documented as a characteristic property of cancer cells. Though, human ABHD2 gene was found to be highly expressed in breast and lung cancers, its biochemical functionality is yet uncharacterized. In the present study we report, human ABHD2 as triacylglycerol (TAG) lipase along with ester hydrolysing capacity. Sequence analysis of ABHD2 revealed the presence of conserved motifs G205XS207XG209 and H120XXXXD125. Phylogenetic analysis showed homology to known lipases, Drosophila melanogaster CG3488. To evaluate the biochemical role, recombinant ABHD2 was expressed in Saccharomyces cerevisiae using pYES2/CT vector and His-tag purified protein showed TAG lipase activity. Ester hydrolase activity was confirmed with pNP acetate, butyrate and palmitate substrates respectively. Further, the ABHD2 homology model was built and the modelled protein was analysed based on the RMSD and root mean square fluctuation (RMSF) of the 100 ns simulation trajectory. Docking the acetate, butyrate and palmitate ligands with the model confirmed covalent binding of ligands with the Ser207 of the GXSXG motif. The model was validated with a mutant ABHD2 developed with alanine in place of Ser207 and the docking studies revealed loss of interaction between selected ligands and the mutant protein active site. Based on the above results, human ABHD2 was identified as a novel TAG lipase and ester hydrolase. PMID:27247428

  19. Molecular characterization of human ABHD2 as TAG lipase and ester hydrolase.

    PubMed

    M, Naresh Kumar; V B S C, Thunuguntla; G K, Veeramachaneni; B, Chandra Sekhar; Guntupalli, Swapna; J S, Bondili

    2016-08-01

    Alterations in lipid metabolism have been progressively documented as a characteristic property of cancer cells. Though, human ABHD2 gene was found to be highly expressed in breast and lung cancers, its biochemical functionality is yet uncharacterized. In the present study we report, human ABHD2 as triacylglycerol (TAG) lipase along with ester hydrolysing capacity. Sequence analysis of ABHD2 revealed the presence of conserved motifs G(205)XS(207)XG(209) and H(120)XXXXD(125) Phylogenetic analysis showed homology to known lipases, Drosophila melanogaster CG3488. To evaluate the biochemical role, recombinant ABHD2 was expressed in Saccharomyces cerevisiae using pYES2/CT vector and His-tag purified protein showed TAG lipase activity. Ester hydrolase activity was confirmed with pNP acetate, butyrate and palmitate substrates respectively. Further, the ABHD2 homology model was built and the modelled protein was analysed based on the RMSD and root mean square fluctuation (RMSF) of the 100 ns simulation trajectory. Docking the acetate, butyrate and palmitate ligands with the model confirmed covalent binding of ligands with the Ser(207) of the GXSXG motif. The model was validated with a mutant ABHD2 developed with alanine in place of Ser(207) and the docking studies revealed loss of interaction between selected ligands and the mutant protein active site. Based on the above results, human ABHD2 was identified as a novel TAG lipase and ester hydrolase. © 2016 The Author(s).

  20. Beauveria bassiana Lipase A expressed in Komagataella (Pichia) pastoris with potential for biodiesel catalysis

    PubMed Central

    Vici, Ana C.; da Cruz, Andrezza F.; Facchini, Fernanda D. A.; de Carvalho, Caio C.; Pereira, Marita G.; Fonseca-Maldonado, Raquel; Ward, Richard J.; Pessela, Benevides C.; Fernandez-Lorente, Gloria; Torres, Fernando A. G.; Jorge, João A.; Polizeli, Maria L. T. M.

    2015-01-01

    Lipases (EC 3.1.1.3) comprise a biotechnologically important group of enzymes because they are able to catalyze both hydrolysis and synthesis reactions, depending on the amount of water in the system. One of the most interesting applications of lipase is in the biofuel industry for biodiesel production by oil and ethanol (or methanol) transesterification. Entomopathogenic fungi, which are potential source of lipases, are still poorly explored in biotechnological processes. The present work reports the heterologous expression and biochemical characterization of a novel Beauveria bassiana lipase with potential for biodiesel production. The His-tagged B. bassiana lipase A (BbLA) was produced in Komagataella pastoris in buffered methanol medium (BMM) induced with 1% methanol at 30°C. Purified BbLA was activated with 0.05% Triton X-100 and presented optimum activity at pH 6.0 and 50°C. N-glycosylation of the recombinant BbLA accounts for 31.5% of its molecular weight. Circular dichroism and molecular modeling confirmed a structure composed of α-helix and β-sheet, similar to α/β hydrolases. Immobilized BbLA was able to promote transesterification reactions in fish oil, demonstrating potential for biodiesel production. BbLA was successfully produced in K. pastoris and shows potential use for biodiesel production by the ethanolysis reaction. PMID:26500628