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Sample records for acid lipase activity

  1. Acid Lipase Disease

    MedlinePlus

    ... Awards Enhancing Diversity Find People About NINDS NINDS Acid Lipase Disease Information Page Synonym(s): Cholesterol Ester Storage ... Trials Related NINDS Publications and Information What is Acid Lipase Disease ? Acid lipase disease or deficiency occurs ...

  2. Lysosomal acid lipase deficiency in rats: Lipid analyses and lipase activities in liver and spleen

    SciTech Connect

    Kuriyama, M.; Yoshida, H.; Suzuki, M.; Fujiyama, J.; Igata, A. )

    1990-09-01

    We report the biological characterization of an animal model of a genetic lipid storage disease analogous to human Wolman's disease. Affected rats accumulated cholesteryl esters (13.3-fold), free cholesterol (2.8-fold), and triglycerides (5.4-fold) in the liver, as well as cholesteryl esters (2.5-fold) and free cholesterol (1.33-fold) in the spleen. Triglycerides did not accumulate, and the levels actually decreased in the spleen. Analysis of the fatty acid composition of the cholesteryl esters and triglycerides showed high percentages of linoleic acid (18:2) and arachidonic acid (20:4) in both organs, especially in the liver. No accumulation of phospholipids, neutral glycosphingolipids, or gangliosides was found in the affected rats. Acid lipase activity for (14C)triolein, (14C)cholesteryl oleate, and 4-methyl-umbelliferyl oleate was deficient in both the liver and spleen of affected rats. Lipase activity at neutral pH was normal in both liver and spleen. Heterozygous rats showed intermediate utilization of these substrates in both organs at levels between those for affected rats and those for normal controls, although they did not accumulate any lipids. These data suggest that these rats represent an animal counterpart of Wolman's disease in humans.

  3. Fatty acid alcohol ester-synthesizing activity of lipoprotein lipase.

    PubMed

    Tsujita, T; Sumiyoshi, M; Okuda, H

    1999-12-01

    The fatty acid alcohol ester-synthesizing activity of lipoprotein lipase (LPL) was characterized using bovine milk LPL. Synthesizing activities were determined in an aqueous medium using oleic acid or trioleylglycerol as the acyl donor and equimolar amounts of long-chain alcohols as the acyl acceptor. When oleic acid and hexadecanol emulsified with gum arabic were incubated with LPL, palmityl oleate was synthesized, in a time- and dose-dependent manner. Apo-very low density lipoprotein (apoVLDL) stimulated LPL-catalyzed palmityl oleate synthesis. The apparent equilibrium ratio of fatty acid alcohol ester/oleic acid was estimated using a high concentration of LPL and a long (20 h) incubation period. The equilibrium ratio was affected by the incubation pH and the alcohol chain length. When the incubation pH was below pH 7.0 and long chain fatty acyl alcohols were used as substrates, the fatty acid alcohol ester/free fatty acid equilibrium ratio favored ester formation, with an apparent equilibrium ratio of fatty acid alcohol ester/fatty acid of about 0.9/0.1. The equilibrium ratio decreased sharply at alkaline pH (above pH 8.0). The ratio also decreased when fatty alcohols with acyl chains shorter than dodecanol were used. When a trioleoylglycerol/fatty acyl alcohol emulsion was incubated with LPL, fatty acid alcohol esters were synthesized in a dose- and time-dependent fashion. Fatty acid alcohol esters were easily synthesized from trioleoylglycerol when fatty alcohols with acyl chains longer than dodecanol were used, but synthesis was decreased with fatty alcohols with acyl chain lengths shorter than decanol, and little synthesizing activity was detected with shorter-chain fatty alcohols such as butanol or ethanol. PMID:10578059

  4. Improvement of catalytic activity of lipase in the presence of calix[4]arene valeric acid or hydrazine derivative.

    PubMed

    Akoz, Enise; Sayin, Serkan; Kaplan, Selcuk; Yilmaz, Mustafa

    2015-03-01

    Sol-gel encapsulation is a simple but powerful method to enhance the enantioselectivity of lipase-catalyzed transformations in an isooctane/aqueous buffer solution. Candida rugosa lipase was encapsulated according to a sol-gel procedure in the presence and absence of calix[4]arene hydrazine or carboxylic acid derivatives with Fe3O4 magnetic nanoparticles as an additive. The activity of the encapsulated lipases was evaluated for the enantioselective hydrolysis of racemic Naproxen methyl ester and the hydrolysis of p-Nitrophenylpalmitate. The results indicate that the encapsulated lipase without calix[4]arene derivative has lower conversion and enantioselectivity compared to the encapsulated lipase with calix[4]arene derivative. It was found that the calix[4]arene hydrazine and carboxylic acid-based encapsulated lipases have excellent activity and enantioselectivity (E >300) compared to encapsulated lipase without the calix[4]arene derivatives. PMID:25326059

  5. Synthesis of monoacylglycerol containing pinolenic acid via stepwise esterification using a cold active lipase.

    PubMed

    Pyo, Young-Gil; Hong, Seung In; Kim, Yangha; Kim, Byung Hee; Kim, In-Hwan

    2012-01-01

    High purity monoacylglycerol (MAG) containing pinolenic acid was synthesized via stepwise esterification of glycerol and fatty acids from pine nut oil using a cold active lipase from Penicillium camembertii as a biocatalyst. Effects of temperature, molar ratio, water content, enzyme loading, and vacuum on the synthesis of MAG by lipase-catalyzed esterification of glycerol and fatty acid from pine nut oil were investigated. Diacylglycerol (DAG) as well as MAG increased significantly when temperature was increased from 20 to 40 °C. At a molar ratio of 1:1, MAG content decreased because of the significant increase in DAG content. Water has a profound influence on both MAG and DAG content through the entire course of reaction. The reaction rate increased significantly as enzyme loading increased up to 600 units. Vacuum was an effective method to reduce DAG content. The optimum temperature, molar ratio, water content, enzyme loading, vacuum, and reaction time were 20 °C, 1:5 (fatty acid to glycerol), 2%, 600 units, 5 torr, and 24 h, respectively. MAG content further increased via lipase-catalyzed second step esterification at subzero temperature. P. camembertii lipase exhibited esterification activity up to -30 °C. PMID:22753389

  6. Fatty acid-releasing activities in Sinorhizobium meliloti include unusual diacylglycerol lipase.

    PubMed

    Sahonero-Canavesi, Diana X; Sohlenkamp, Christian; Sandoval-Calderón, Mario; Lamsa, Anne; Pogliano, Kit; López-Lara, Isabel M; Geiger, Otto

    2015-09-01

    Phospholipids are well known for their membrane-forming properties and thereby delimit any cell from the exterior world. In addition, membrane phospholipids can act as precursors for signals and other biomolecules during their turnover. Little is known about phospholipid signalling, turnover and remodelling in bacteria. Recently, we showed that a FadD-deficient mutant of Sinorhizobium meliloti, unable to convert free fatty acids to their coenzyme A derivatives, accumulates free fatty acids during the stationary phase of growth. Enzymatic activities responsible for the generation of these free fatty acids were unknown in rhizobia. Searching the genome of S. meliloti, we identified a potential lysophospholipase (SMc04041) and two predicted patatin-like phospholipases A (SMc00930, SMc01003). Although SMc00930 as well as SMc01003 contribute to the release of free fatty acids in S. meliloti, neither one can use phospholipids as substrates. Here we show that SMc01003 converts diacylglycerol to monoacylglycerol and a fatty acid, and that monoacylglycerol can be further degraded by SMc01003 to another fatty acid and glycerol. A SMc01003-deficient mutant of S. meliloti transiently accumulates diacylglycerol, suggesting that SMc01003 also acts as diacylglycerol lipase (DglA) in its native background. Expression of the DglA lipase in Escherichia coli causes lysis of cells in stationary phase of growth. PMID:25711932

  7. Wax ester-synthesizing activity of lipases.

    PubMed

    Tsujita, T; Sumiyoshi, M; Okuda, H

    1999-11-01

    The synthesis/hydrolysis of wax esters was studied in an aqueous solution using purified rat pancreatic lipase, porcine pancreatic carboxylester lipase, and Pseudomonas fluorescens lipase. The equilibrium between wax ester synthesis and hydrolysis favored ester formation at neutral pH. The synthesizing activities were measured using free fatty acid or triacylglycerol as the acyl donor and an equimolar amount of long-chain alcohol as the acyl acceptor. When oleic acid and hexadecanol emulsified with gum arabic were incubated with these lipases, wax ester was synthesized, in a dose- and time-dependent manner, and the apparent equilibrium ratio of palmityl oleate/free oleic acid was about 0.9/0.1. These lipases catalyzed the hydrolysis of palmityl oleate emulsified with gum arabic, and the apparent equilibrium ratio of palmityl oleate/free oleic acid was also about 0.9/0.1. The apparent equilibrium ratio of wax ester/free fatty acid catalyzed by lipase depended on incubation pH and fatty alcohol chain length. When equimolar amounts of trioleoylglycerol and fatty acyl alcohol were incubated with pancreatic lipase, carboxylester lipase, or P. fluorescens lipase, wax esters were synthesized dose-dependently. These results suggest that lipases can catalyze the synthesis of wax esters from free fatty acids or through degradation of triacylglycerol in an aqueous medium. PMID:10606038

  8. Low Serum Lysosomal Acid Lipase Activity Correlates with Advanced Liver Disease

    PubMed Central

    Shteyer, Eyal; Villenchik, Rivka; Mahamid, Mahmud; Nator, Nidaa; Safadi, Rifaat

    2016-01-01

    Fatty liver has become the most common liver disorder and is recognized as a major health burden in the Western world. The causes for disease progression are not fully elucidated but lysosomal impairment is suggested. Here we evaluate a possible role for lysosomal acid lipase (LAL) activity in liver disease. To study LAL levels in patients with microvesicular, idiopathic cirrhosis and nonalcoholic fatty liver disease (NAFLD). Medical records of patients with microvesicular steatosis, cryptogenic cirrhosis and NAFLD, diagnosed on the basis of liver biopsies, were included in the study. Measured serum LAL activity was correlated to clinical, laboratory, imaging and pathological data. No patient exhibited LAL activity compatible with genetic LAL deficiency. However, serum LAL activity inversely predicted liver disease severity. A LAL level of 0.5 was the most sensitive for detecting both histologic and noninvasive markers for disease severity, including lower white blood cell count and calcium, and elevated γ-glutamyltransferase, creatinine, glucose, glycated hemoglobin, uric acid and coagulation function. Serum LAL activity <0.5 indicates severe liver injury in patients with fatty liver and cirrhosis. Further studies should define the direct role of LAL in liver disease severity and consider the possibility of replacement therapy. PMID:26927097

  9. Low Serum Lysosomal Acid Lipase Activity Correlates with Advanced Liver Disease.

    PubMed

    Shteyer, Eyal; Villenchik, Rivka; Mahamid, Mahmud; Nator, Nidaa; Safadi, Rifaat

    2016-01-01

    Fatty liver has become the most common liver disorder and is recognized as a major health burden in the Western world. The causes for disease progression are not fully elucidated but lysosomal impairment is suggested. Here we evaluate a possible role for lysosomal acid lipase (LAL) activity in liver disease. To study LAL levels in patients with microvesicular, idiopathic cirrhosis and nonalcoholic fatty liver disease (NAFLD). Medical records of patients with microvesicular steatosis, cryptogenic cirrhosis and NAFLD, diagnosed on the basis of liver biopsies, were included in the study. Measured serum LAL activity was correlated to clinical, laboratory, imaging and pathological data. No patient exhibited LAL activity compatible with genetic LAL deficiency. However, serum LAL activity inversely predicted liver disease severity. A LAL level of 0.5 was the most sensitive for detecting both histologic and noninvasive markers for disease severity, including lower white blood cell count and calcium, and elevated γ-glutamyltransferase, creatinine, glucose, glycated hemoglobin, uric acid and coagulation function. Serum LAL activity <0.5 indicates severe liver injury in patients with fatty liver and cirrhosis. Further studies should define the direct role of LAL in liver disease severity and consider the possibility of replacement therapy. PMID:26927097

  10. Fatty acid ethyl ester-synthesizing activity of lipoprotein lipase from rat postheparin plasma.

    PubMed

    Tsujita, T; Okuda, H

    1994-02-25

    Lipoprotein lipase (LPL) was obtained from rat postheparin plasma by chromatographies on heparin-Sepharose and hydroxyapatite. The enzyme was associated with fatty acid ethyl ester synthase (FAEE synthase) as judged by their co-elution profiles and identical profiles of inhibition by diisopropyl fluorophosphate. Only one polypeptide of molecular weight 57,000 in purified LPL fraction was labeled by affinity labeling with [3H]-diisopropyl fluorophosphate. The FAEE synthase activity of LPL was not affected by addition of apolipoprotein C-II. Digestion of the enzyme with trypsin resulted in almost complete loss of the triolein-hydrolyzing activity without change in FAEE synthase activity. The tributyrin-hydrolyzing activity of LPL was also not affected by addition of apolipoprotein C-II or trypsin digestion. On addition at progressively higher concentrations, bovine serum albumin increased FAEE synthesis to a maximum at 2 mg/ml and at higher concentrations inhibited its activity. On incubation of purified LPL with chylomicrons in an ethanol/water mixture, FAEE was formed in the presence of a high concentration of bovine serum albumin. The specific activity of FAEE synthesis from chylomicrons was about 65 times that from oleic acid. Triolein/gum arabic emulsion was used for identification of reaction products. We propose the following mechanism of FAEE formation from chylomicrons by LPL. The enzyme attacks chylomicrons forming an acyl-enzyme intermediate, and during the deacylation process, ethanol binds to fatty acids as an acceptor. These results suggest that LPL contributes to nonoxidative ethanol metabolism (FAEE formation) through degradation of triglyceride-rich lipoproteins such as chylomicrons. PMID:8119932

  11. Immobilized MAS1 lipase showed high esterification activity in the production of triacylglycerols with n-3 polyunsaturated fatty acids.

    PubMed

    Wang, Xiumei; Li, Daoming; Qu, Man; Durrani, Rabia; Yang, Bo; Wang, Yonghua

    2017-02-01

    Immobilization of lipase MAS1 from marine Streptomyces sp. strain W007 and its application in catalyzing esterification of n-3 polyunsaturated fatty acids (PUFA) with glycerol were investigated. The resin XAD1180 was selected as a suitable support for the immobilization of lipase MAS1, and its absorption ability was 75mg/g (lipase/resin ratio) with initial buffer pH value of 8.0. The thermal stability of immobilized MAS1 was improved significantly compared with that of the free lipase. Immobilized MAS1 had no regiospecificity in the hydrolysis of triolein. The highest esterification degree (99.31%) and TAG content (92.26%) by immobilized MAS1-catalyzed esterification were achieved under the optimized conditions, which were significantly better than those (82.16% and 47.26%, respectively) by Novozym 435. More than 92% n-3 PUFA was incorporated into TAG that had similar fatty acids composition to the substrate (n-3 PUFA). The immobilized MAS1 exhibited 50% of its initial activity after being used for five cycles. PMID:27596418

  12. Small-angle X-ray scattering analysis of stearic acid modified lipase.

    PubMed

    Maruyama, T; Nakajima, M; Ichikawa, S; Sano, Y; Nabetani, H; Furusaki, S; Seki, M

    2001-04-01

    Stearic acid modified lipase (from Rhizopus japonicus) exhibited remarkable interesterification activity in n-hexane, but crude native lipase did not. The structure of the fatty acid modified lipase had not been analyzed until now. We analyzed the modified lipase by small-angle X-ray scattering (SAXS) measurements in order to clarify the structure. SAXS measurements showed that the modified lipase consisted of a lipid lamellar structure and implied that the lipase was incorporated into the lamellar structure of stearic acid. The long spacings in the lamellar structures of the modified lipase and stearic acid were measured. PMID:11388447

  13. Improvement of catalytic activity of Candida rugosa lipase in the presence of calix[4]arene bearing iminodicarboxylic/phosphonic acid complexes modified iron oxide nanoparticles.

    PubMed

    Ozyilmaz, Elif; Bayrakci, Mevlut; Yilmaz, Mustafa

    2016-04-01

    In the present study, iron oxide magnetite nanoparticles, prepared through a co-precipitation method, were coated with phosphonic acid or iminodicarboxylic acid derivatives of calix[4]arene to modulate their surfaces with different acidic groups. Candida rugosa lipase was then directly immobilized onto the modified nanoparticles through sol-gel encapsulation. The catalytic activities and enantioselectivities of the two encapsulated lipases in the hydrolysis reaction of (R/S)-naproxen methyl ester and (R/S)-2-phenoxypropionic acid methyl ester were assessed. The results showed that the activity and enantioselectivity of the lipase were improved when the lipase was encapsulated in the presence of calixarene-based additives; the encapsulated lipase with the phosphonic acid derivative of calix[4]arene had an excellent rate of enantioselectivity against the (R/S)-naproxen methyl and (R/S)-2-phenoxypropionic acid methyl esters, with E=350 and 246, respectively, compared to the free enzyme. The encapsulated lipases (Fe-Calix-N(COOH)) and (Fe-Calix-P) showed good loading ability and little loss of enzyme activity, and the stability of the catalyst was very good; they only lost 6-11% of the enzyme's activity after five batches. PMID:26698535

  14. Lysosomal Acid Lipase Activity Is Reduced Both in Cryptogenic Cirrhosis and in Cirrhosis of Known Etiology

    PubMed Central

    Vespasiani-Gentilucci, Umberto; Gallo, Paolo; Piemonte, Fiorella; Riva, Elisabetta; Porcari, Aldostefano; Vorini, Ferruccio; Tozzi, Giulia; Piccioni, Livia; Galati, Giovanni; De Vincentis, Antonio; Carotti, Simone; Morini, Sergio; D’Amico, Jessica; Angeletti, Silvia; Pedone, Claudio; Picardi, Antonio

    2016-01-01

    Lysosomal acid lipase deficiency (LAL-d) is a rare autosomal recessive disease in which LAL activity is almost absent, with consequent massive microvesicular steatosis evolving to cirrhosis and liver failure. We aimed to determine LAL-activity, and to investigate the most common single nucleotide polymorphism (SNP) affecting the LIPA gene and responsible for 50–70% of LAL-d cases (rs116928232 c.894G>A), in patients with cryptogenic cirrhosis. Sixty-three patients with cryptogenic cirrhosis, 88 cirrhotics of known etiology, and 97 healthy subjects were enrolled. LAL-activity was determined in dried-blood-spot (DBS). The c.894G>A mutation was analyzed by pyrosequencing method in SNP mode. LAL-activity was severely reduced in patients with cryptogenic cirrhosis with respect to healthy subjects [0.62 (0.44–0.86) Vs 0.96 (0.75–1.25) nmol/spot/h, p<0.001)], but it was also reduced in known-etiology cirrhotics [0.54 (0.42–0.79) nmol/spot/h, p<0.001 Vs healthy subjects; p = 0.5 Vs cryptogenic cirrhotics]. Fourteen percent of cryptogenic cirrhotics and 20% of known-etiology cirrhotics showed a LAL-activity in the range of heterozygous carriers of LIPA gene mutations (0.15–0.40 nmol/spot/h). However, none of the subjects with reduced LAL-activity carried the c.894G>A SNP except for one patient with HCV cirrhosis. By multivariate analysis, LAL-activity was not associated with age, sex, liver enzymes, liver function or lipid parameters, while it was independently associated with white blood cell (β = 0.2; p<0.01) and platelet (β = 0.4; p<0.001) counts and with the condition of cirrhosis (β = -0.2; p = 0.04). Conclusion Liver cirrhosis is characterized by a severe acquired reduction of LAL-activity, the precise causes and consequences of which need to be further addressed. DBS-determined lysosomal enzyme activities seem to be affected by white blood cell and platelet counts, and the specificity of these tests can be reduced when applied to determined populations

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

    SciTech Connect

    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 was 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.

  16. Design and synthesis of boronic acid inhibitors of endothelial lipase.

    PubMed

    O'Connell, Daniel P; LeBlanc, Daniel F; Cromley, Debra; Billheimer, Jeffrey; Rader, Daniel J; Bachovchin, William W

    2012-02-01

    Endothelial lipase (EL) and lipoprotein lipase (LPL) are homologous lipases that act on plasma lipoproteins. EL is predominantly a phospholipase and appears to be a key regulator of plasma HDL-C. LPL is mainly a triglyceride lipase regulating (V)LDL levels. The existing biological data indicate that inhibitors selective for EL over LPL should have anti-atherogenic activity, mainly through increasing plasma HDL-C levels. We report here the synthesis of alkyl, aryl, or acyl-substituted phenylboronic acids that inhibit EL. Many of the inhibitors evaluated proved to be nearly equally potent against both EL and LPL, but several exhibited moderate to good selectivity for EL. PMID:22225633

  17. Screening for lipase activity in the oil palm.

    PubMed

    Sambanthamurthi, R; Rajanaidu, N; Hasnah Parman, S

    2000-12-01

    The oil palm mesocarp contains an endogenous lipase which is strongly activated at low temperature. Lipase activity is thus very conveniently assayed by prior exposure of the fruits to low temperature. More than 100 oil palm samples from the germplasm collection of the Palm Oil Research Institute of Malaysia (now known as the Malaysian Palm Oil Board) were screened for non-esterified fatty acid activity using both the low-temperature activation assay and a radioactivity assay. The results showed good correlation between assay procedures. The different samples had a very wide range of lipase activity. Elaeis oleifera samples had significantly lower lipase activity compared with E. guineensis (var. tenera) samples. Even within E. guineensis (var. tenera), there was a wide range of activity. The results confirmed that lipase activity is genotype-dependent. Selection for lipase genotypes is thus possible and this will have obvious commercial value. PMID:11171201

  18. Study of Molecular Conformation and Activity-Related Properties of Lipase Immobilized onto Core-Shell Structured Polyacrylic Acid-Coated Magnetic Silica Nanocomposite Particles.

    PubMed

    Esmaeilnejad-Ahranjani, Parvaneh; Kazemeini, Mohammad; Singh, Gurvinder; Arpanaei, Ayyoob

    2016-04-01

    A facile approach for the preparation of core-shell structured poly(acrylic acid) (PAA)-coated Fe3O4 cluster@SiO2 nanocomposite particles as the support materials for the lipase immobilization is reported. Low- or high-molecular-weight (1800 and 100 000, respectively) PAA molecules were covalently attached onto the surface of amine-functionalized magnetic silica nanoacomposite particles. The successful preparation of particles were verified by scanning transmission electron microscopy (STEM), X-ray diffraction (XRD), vibrating sample magnetometer (VSM), thermogravimetric analysis (TGA), zeta potential measurement, and Fourier-transform infrared (FTIR) techniques. Once lipase is covalently immobilized onto the particles with an average diameter of 210 ± 50 nm, resulting from high binding sites concentrations on the low- and high-molecular-weight PAA-coated particles, high lipase immobilization efficiencies (86.2% and 89.9%, respectively), and loading capacities (786 and 816 mg g(-1), respectively) are obtained. Results from circular dichroism (CD) analysis and catalytic activity tests reveal an increase in the β-sheet content of lipase molecules upon immobilization, along with an enhancement in their activities and stabilities. The lipases immobilized onto the low- and high-molecular-weight PAA-coated particles show maximum activities at 55 and 50 °C, respectively, which are ∼28% and ∼15% higher than that of the free lipase at its own optimum temperature (40 °C), respectively. The immobilized lipases exhibit excellent performance at broader temperature and pH ranges and high thermal and storage stabilities, as well as superior reusability. These prepared magnetic nanocomposite particles can be offered as suitable support materials for efficient immobilization of enzymes and improvement of the immobilized enzymes properties. PMID:26986897

  19. Cold active microbial lipases: some hot issues and recent developments.

    PubMed

    Joseph, Babu; Ramteke, Pramod W; Thomas, George

    2008-01-01

    Lipases are glycerol ester hydrolases that catalyze the hydrolysis of triglycerides to free fatty acids and glycerol. Lipases catalyze esterification, interesterification, acidolysis, alcoholysis and aminolysis in addition to the hydrolytic activity on triglycerides. The temperature stability of lipases has regarded as the most important characteristic for use in industry. Psychrophilic lipases have lately attracted attention because of their increasing use in the organic synthesis of chiral intermediates due to their low optimum temperature and high activity at very low temperatures, which are favorable properties for the production of relatively frail compounds. In addition, these enzymes have an advantage under low water conditions due to their inherent greater flexibility, wherein the activity of mesophilic and thermophilic enzymes are severely impaired by an excess of rigidity. Cold-adapted microorganisms are potential source of cold-active lipases and they have been isolated from cold regions and studied. Compared to other lipases, relatively smaller numbers of cold active bacterial lipases were well studied. Lipases isolated from different sources have a wide range of properties depending on their sources with respect to positional specificity, fatty acid specificity, thermostability, pH optimum, etc. Use of industrial enzymes allows the technologist to develop processes that closely approach the gentle, efficient processes in nature. Some of these processes using cold active lipase from C. antarctica have been patented by pharmaceutical, chemical and food industries. Cold active lipases cover a broad spectrum of biotechnological applications like additives in detergents, additives in food industries, environmental bioremediations, biotransformation, molecular biology applications and heterologous gene expression in psychrophilic hosts to prevent formation of inclusion bodies. Cold active enzymes from psychrotrophic microorganisms showing high catalytic

  20. Cardiac lipoprotein lipase activity in the hypertrophied heart may be regulated by fatty acid flux

    PubMed Central

    Hauton, David; Caldwell, Germaine M.

    2012-01-01

    Cardiac hypertrophy is characterised by an imbalance between lipid uptake and fatty acid β-oxidation leading to an accumulation of lipids, particularly triacylglycerol (TAG). It is unclear whether uptake mechanisms such as lipoprotein lipase (LPL) can be attenuated to diminish this uptake. Rats were cold acclimated to induce cardiac hypertrophy and increase cardiac LPL. Lipid uptake and metabolism were altered by feeding a ‘Western-style’ high fat diet (WSD) or feeding oxfenicine (2 g/L) in the drinking water. Diastolic stiffness (increased volume change/unit pressure change) was induced in hypertrophied hearts for rats fed WSD (P < 0.05) or WSD + oxfenicine (P < 0.01), although absolute performance of cardiac muscle, estimated from stress–strain calculations was unchanged. Cold acclimation increased cardiac endothelial LPL (P < 0.05) but this was diminished following oxfenicine. Following WSD LPL was further decreased below WSD-fed control hearts (P < 0.05) with no further decrease by oxfenicine supplementation. A negative correlation was noted between plasma TAG and endothelial LPL (correlation coefficient = − 0.654; P < 0.001) but not cardiac TAG concentration. Transcript levels of angiopoietin-like protein-4 (ANGPTL4) were increased 6-fold by WSD (P < 0.05) and increased 15-fold following WSD + oxfenicine (P < 0.001). For CA-hearts fed WSD or WSD + oxfenicine ANGPTL4 mRNA levels were preserved at chow-fed levels. VLDLR protein levels were increased 10-fold (P < 0.01) by CA. ANGPTL4 protein levels were increased 2-fold (P < 0.05) by WSD, but restored following oxfenicine. For CA-hearts WSD increased ANGPTL4 protein levels 3-fold (P < 0.01) with WSD + oxfenicine increasing ANGPTL4 protein 4-fold (P < 0.01). These data suggest that endothelial LPL levels in the heart are altered to maintain FA flux and may exploit ANGPTL4. PMID:22226882

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  3. Identification of a novel boronic acid as a potent, selective, and orally active hormone sensitive lipase inhibitor.

    PubMed

    Ogiyama, Tomoko; Yamaguchi, Mitsuhiro; Kurikawa, Nobuya; Honzumi, Shoko; Yamamoto, Yuka; Sugiyama, Daisuke; Inoue, Shinichi

    2016-08-15

    Hormone sensitive lipase (HSL) is an attractive therapeutic target of dyslipidemia. We designed and synthesized several compounds as reversible HSL inhibitors with a focus on hydrophobic interactions, which was thought to be effective upon the HSL inhibitory activity. In these efforts, we identified boronated compound 12 showing a potent HSL inhibitory activity with an IC50 value of 7nM and a high selectivity against cholinesterases. Furthermore, compound 12 is the first boron containing HSL inhibitor that has shown an antilipolytic effect in rats after oral administration at 3mg/kg. PMID:27338659

  4. Acid lipase from Candida viswanathii: production, biochemical properties, and potential application.

    PubMed

    de Almeida, Alex Fernando; Tauk-Tornisielo, Sâmia Maria; 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. 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 (YL/S = 1.381 g/g), lipase yield (YL/S = 6.892 U/g), and biomass productivity (PX = 0.282 g/h). Addition of soybean lecithin increased lipase production in 1.45-fold, presenting lipase yield (YL/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

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  7. Inhibitory activity of chlorogenic acids in decaffeinated green coffee beans against porcine pancreas lipase and effect of a decaffeinated green coffee bean extract on an emulsion of olive oil.

    PubMed

    Narita, Yusaku; Iwai, Kazuya; Fukunaga, Taiji; Nakagiri, Osamu

    2012-01-01

    A decaffeinated green coffee bean extract (DGCBE) inhibited porcine pancreas lipase (PPL) activity with an IC50 value of 1.98 mg/mL. Six different chlorogenic acids in DGCBE contributed to this PPL inhibition, accounting for 91.8% of the inhibitory activity. DGCBE increased the droplet size and decreased the specific surface area of an olive oil emulsion. PMID:23221697

  8. In vitro effect of insulin and adrenaline on lung triglyceride-lipase activity in rats.

    PubMed

    Hadjiivanova, N; Koumanov, K; Georgiev, G

    1976-01-01

    The in vitro effect of insulin and adrenaline on the activity of lung triglyceride-lipases (alkaline and acid) has been investigated. Insulin inhibited strongly both triglyceride-lipases. Only caffein almost eliminated the inhibitory action of insulin, while adrenaline and dibutyryl cyclic adenosine monophosphate did not exhibit such an effect. It was assumed that the inhibition of lung triglyceride-lipases by insulin was effected through the activation of phosphodiesterases. On the other hand since adrenaline markedly activated lung triglyceride-lipases, this action was assumed to be carried out via the activation of lung adenylate cyclase and the increase of cyclic adenosine monophosphate. PMID:189868

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  10. Novel extremely acidic lipases produced from Bacillus species using oil substrates.

    PubMed

    Saranya, P; Kumari, H Sukanya; Jothieswari, M; Rao, B Prasad; Sekaran, G

    2014-01-01

    The extremely acidophilic microorganisms Bacillus pumilus and Bacillus subtilis were isolated from soil collected from the commercial edible oil and fish oil extraction industry. Optimization of conditions for acidic lipase production from B. pumilus and B. subtilis using palm oil and fish oil, respectively, was carried out using response surface methodology. The extremely acidic lipases, thermo-tolerant acidic lipase (TAL) and acidic lipase (AL), were produced by B. pumilus and B. subtilis, respectively. The optimum conditions for B. pumilus obtaining the maximum activity (1,100 U/mL) of TAL were fermentation time, 96 h; pH, 1; temperature, 50 °C; concentration of palm oil, 50 g/L. After purification, a 7.1-fold purity of lipase with specific activity of 5,173 U/mg protein was obtained. The molecular weight of the TAL was 55 kDa. The AL from B. subtilis activity was 214 U/mL at a fermentation time of 72 h; pH, 1; temperature, 35 °C; concentration of fish oil, 30 g/L; maltose concentration, 10 g/L. After purification, an 11.4-fold purity of lipase with specific activity of 2,189 U/mg protein was obtained. The molecular weight of the extremely acidic lipase was 22 kDa. The functional groups of lipases were determined by Fourier transform-infrared (FT-IR) spectroscopy. PMID:24185617

  11. A Bioactivity-Based Method for Screening, Identification of Lipase Inhibitors, and Clarifying the Effects of Processing Time on Lipase Inhibitory Activity of Polygonum Multiflorum.

    PubMed

    Chang, Yan-Xu; Ge, Ai-Hua; Jiang, Yan; Teye Azietaku, John; Li, Jin; Gao, Xiu-Mei

    2016-01-01

    Traditional Chinese medicine (TCM) has been used for the treatment of many complex diseases. However, the bioactive components are always undefined. In this study, a bioactivity-based method was developed and validated to screen lipase inhibitors and evaluate the effects of processing on the lipase inhibitory activity of TCM by ultrahigh performance liquid chromatography coupled with quadrupole-time-of-flight mass spectrometry and fraction collector (UHPLC/Q-TOF-MS-FC). The results showed that both Polygonum multiflorum and processed P. multiflorum extracts had inhibitory effect against lipase with IC50 values of 38.84 μg/mL and 190.6 μg/mL, respectively. Stilbenes, phenolic acid, flavonoids, and anthraquinones were considered to be the potential lipase inhibitors. Eleven potential lipase inhibitors were simultaneously determined by UHPLC. Principal component analysis (PCA) was employed in exploring the effects of processing time on lipase inhibitory activity of P. multiflorum. Compared with conventional methods, a bioactivity-based method could quantitatively analyze lipase inhibitory activity of individual constituent and provide the total lipase inhibitory activity of the samples. The results demonstrated that the activity integrated UHPLC/Q-TOF-MS-FC method was an effective and powerful tool for screening and identifying lipase inhibitors from traditional Chinese medicines. PMID:26925151

  12. A Bioactivity-Based Method for Screening, Identification of Lipase Inhibitors, and Clarifying the Effects of Processing Time on Lipase Inhibitory Activity of Polygonum Multiflorum

    PubMed Central

    Chang, Yan-xu; Ge, Ai-hua; Jiang, Yan; Teye Azietaku, John; Li, Jin; Gao, Xiu-mei

    2016-01-01

    Traditional Chinese medicine (TCM) has been used for the treatment of many complex diseases. However, the bioactive components are always undefined. In this study, a bioactivity-based method was developed and validated to screen lipase inhibitors and evaluate the effects of processing on the lipase inhibitory activity of TCM by ultrahigh performance liquid chromatography coupled with quadrupole-time-of-flight mass spectrometry and fraction collector (UHPLC/Q-TOF-MS-FC). The results showed that both Polygonum multiflorum and processed P. multiflorum extracts had inhibitory effect against lipase with IC50 values of 38.84 μg/mL and 190.6 μg/mL, respectively. Stilbenes, phenolic acid, flavonoids, and anthraquinones were considered to be the potential lipase inhibitors. Eleven potential lipase inhibitors were simultaneously determined by UHPLC. Principal component analysis (PCA) was employed in exploring the effects of processing time on lipase inhibitory activity of P. multiflorum. Compared with conventional methods, a bioactivity-based method could quantitatively analyze lipase inhibitory activity of individual constituent and provide the total lipase inhibitory activity of the samples. The results demonstrated that the activity integrated UHPLC/Q-TOF-MS-FC method was an effective and powerful tool for screening and identifying lipase inhibitors from traditional Chinese medicines. PMID:26925151

  13. Assaying lipase activity from oil palm fruit (Elaeis guineensis Jacq.) mesocarp.

    PubMed

    Ngando Ebongue, G F; Dhouib, R; Carrière, F; Amvam Zollo, P-H; Arondel, V

    2006-10-01

    The mesocarp of mature oil palm fruit undergoes intensive triglycerides hydrolysis upon abscission and bruising. This generates such a high amount of free fatty acids that the oil might become unfit for human consumption without appropriate refining. The lipase (EC 3.1.1.3) involved in the breakdown of the oil is not stable after homogenization of the tissue in aqueous buffers. In this study, we have devised a solvent-based procedure that allowed us to obtain fractions with stable lipase activity. Using these fractions, we have determined the optimal conditions for assaying mesocarp lipase activity. The activity was highest at a temperature of 35 degrees C and a pH of 9. The lipase was found to be strictly calcium dependent. The specific activity of the lipase measured in optimal conditions was found to be 33 mumol fatty acids released min(-1) mg(-1) protein using olive oil as substrate. The mesocarp contains about 190 U of lipase g(-1) fresh weight. This activity was found to be inhibited by the lipase inhibitor tetrahydrolipstatin (THL), suggesting that the lipase is a serine hydrolase. PMID:17064925

  14. 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). PMID:26724947

  15. Surfactant-Modified lipase for the catalysis of the interesterification of triglycerides and fatty acids.

    PubMed

    Basheer, S; Mogi, K; Nakajima, M

    1995-02-01

    The lipase-catalyzed intresterification of triglycerides and fatty acids in n-hexane was studied. Initially, lipase Saiken was modified with a surfactant of sorbitan esters so that its dispersibility in hydrophobic organic media was improved. The surfactant-modified lipase formed in the modification process carried out in a buffer solution has 1,3-positional specificity and predominantly catalyzed the interesterification reaction in a microaqueous n-hexane system. The modification technique converted inactive lipases to very active biocatalysts for the interesterification of triglycerides and fatty acids. The pH and the weight ratio of surfactant to enzyme used during the lipase modification process have shown significant effects in determining the recoveries of the protein and enzyme activity from the buffer solution, the protein content of the modified lipase complex after being freeze dried, and the interesterification activity of the complex. The water content in the reaction solution has strongly influenced the enzyme activity as well as the distribution of the products. (c) 1995 John Wiley & Sons, Inc. PMID:18623137

  16. The acidic domain of the endothelial membrane protein GPIHBP1 stabilizes lipoprotein lipase activity by preventing unfolding of its catalytic domain.

    PubMed

    Mysling, Simon; Kristensen, Kristian Kølby; Larsson, Mikael; Beigneux, Anne P; Gårdsvoll, Henrik; Fong, Loren G; Bensadouen, André; Jørgensen, Thomas Jd; Young, Stephen G; Ploug, Michael

    2016-01-01

    GPIHBP1 is a glycolipid-anchored membrane protein of capillary endothelial cells that binds lipoprotein lipase (LPL) within the interstitial space and shuttles it to the capillary lumen. The LPL•GPIHBP1 complex is responsible for margination of triglyceride-rich lipoproteins along capillaries and their lipolytic processing. The current work conceptualizes a model for the GPIHBP1•LPL interaction based on biophysical measurements with hydrogen-deuterium exchange/mass spectrometry, surface plasmon resonance, and zero-length cross-linking. According to this model, GPIHBP1 comprises two functionally distinct domains: (1) an intrinsically disordered acidic N-terminal domain; and (2) a folded C-terminal domain that tethers GPIHBP1 to the cell membrane by glycosylphosphatidylinositol. We demonstrate that these domains serve different roles in regulating the kinetics of LPL binding. Importantly, the acidic domain stabilizes LPL catalytic activity by mitigating the global unfolding of LPL's catalytic domain. This study provides a conceptual framework for understanding intravascular lipolysis and GPIHBP1 and LPL mutations causing familial chylomicronemia. PMID:26725083

  17. The acidic domain of the endothelial membrane protein GPIHBP1 stabilizes lipoprotein lipase activity by preventing unfolding of its catalytic domain

    PubMed Central

    Mysling, Simon; Kristensen, Kristian Kølby; Larsson, Mikael; Beigneux, Anne P; Gårdsvoll, Henrik; Fong, Loren G; Bensadouen, André; Jørgensen, Thomas JD; Young, Stephen G; Ploug, Michael

    2016-01-01

    GPIHBP1 is a glycolipid-anchored membrane protein of capillary endothelial cells that binds lipoprotein lipase (LPL) within the interstitial space and shuttles it to the capillary lumen. The LPL•GPIHBP1 complex is responsible for margination of triglyceride-rich lipoproteins along capillaries and their lipolytic processing. The current work conceptualizes a model for the GPIHBP1•LPL interaction based on biophysical measurements with hydrogen-deuterium exchange/mass spectrometry, surface plasmon resonance, and zero-length cross-linking. According to this model, GPIHBP1 comprises two functionally distinct domains: (1) an intrinsically disordered acidic N-terminal domain; and (2) a folded C-terminal domain that tethers GPIHBP1 to the cell membrane by glycosylphosphatidylinositol. We demonstrate that these domains serve different roles in regulating the kinetics of LPL binding. Importantly, the acidic domain stabilizes LPL catalytic activity by mitigating the global unfolding of LPL's catalytic domain. This study provides a conceptual framework for understanding intravascular lipolysis and GPIHBP1 and LPL mutations causing familial chylomicronemia. DOI: http://dx.doi.org/10.7554/eLife.12095.001 PMID:26725083

  18. Production of Lipase and Oxygenated Fatty Acids from Vegetable Oils

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Vegetable oils such as soybean oil and corn oil are cheap raw materials. Various value-added oxygenated fatty acids have been produced from unsaturated fatty acids such as oleic and linoleic acid by biotransformation. Lipase from the non-pathogenic yeast Candida cylindracea is another important va...

  19. Geotrichum candidum NRRL Y-553 lipase: purification, characterization and fatty acid specificity.

    PubMed

    Baillargeon, M W; McCarthy, S G

    1991-10-01

    Lipases from Geotrichum candidum NRRL Y-553 are of interest because of their unique specificity for cis-9-unsaturated fatty acids relative to both stearic and palmitic acids. The lipases were partially purified by chromatography on Octyl Sepharose, AG MP-1 macroporous anion exchanger, and chromatofocusing resin. The preparation was found to contain multiple, glycosylated lipases varying slightly in pI (pI 4.88, 4.78, 4.65, 4.57 and 4.52) as judged by both activity and silver staining. The molecular mass determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis was 64 kilodaltons for the main species, with minor species of 60 and 57 kilodaltons present as well. The specificity of the crude lipases for hydrolysis of 4-methylumbelliferyl esters of oleic vs. palmitic acid was 20-to-1. The specificity of the purified, partially separated lipases was similar to that of the crude preparation. Thus the lipases could be used even in crude form for the hydrolysis and restructuring of triacylglycerols on a large scale. PMID:1795605

  20. Lipase-catalyzed fractionation of conjugated linoleic acid isomers.

    PubMed

    Haas, M J; Kramer, J K; McNeill, G; Scott, K; Foglia, T A; Sehat, N; Fritsche, J; Mossoba, M M; Yurawecz, M P

    1999-09-01

    The abilities of lipases produced by the fungus Geotrichum candidum to selectively fractionate mixtures of conjugated linoleic acid (CLA) isomers during esterification of mixed CLA free fatty acids and during hydrolysis of mixed CLA methyl esters were examined. The enzymes were highly selective for cis-9,trans-11-18:2. A commercial CLA methyl ester preparation, containing at least 12 species representing four positional CLA isomers, was incubated in aqueous solution with either a commercial G. candidum lipase preparation (Amano GC-4) or lipase produced from a cloned high-selectivity G. candidum lipase B gene. In both instances selective hydrolysis of the cis-9,trans-11-18:2 methyl ester occurred, with negligible hydrolysis of other CLA isomers. The content of cis-9, trans-11-18:2 in the resulting free fatty acid fraction was between 94 (lipase B reaction) and 77% (GC-4 reaction). The commercial CLA mixture contained only trace amounts of trans-9,cis-11-18:2, and there was no evidence that this isomer was hydrolyzed by the enzyme. Analogous results were obtained with these enzymes in the esterification in organic solvent of a commercial preparation of CLA free fatty acids containing at least 12 CLA isomers. In this case, G. candidum lipase B generated a methyl ester fraction that contained >98% cis-9,trans-11-18:2. Geotrichum candidum lipases B and GC-4 also demonstrated high selectivity in the esterification of CLA with ethanol, generating ethyl ester fractions containing 96 and 80%, respectively, of the cis-9,trans-11 isomer. In a second set of experiments, CLA synthesized from pure linoleic acid, composed essentially of two isomers, cis-9,trans-11 and trans-10,cis-12, was utilized. This was subjected to esterification with octanol in an aqueous reaction system using Amano GC-4 lipase as catalyst. The resulting ester fraction contained up to 97% of the cis-9,trans-11 isomer. After adjustment of the reaction conditions, a concentration of 85% trans-10,cis-12

  1. Nanofibrous poly(acrylonitrile-co-maleic acid) membranes functionalized with gelatin and chitosan for lipase immobilization.

    PubMed

    Ye, Peng; Xu, Zhi-Kang; Wu, Jian; Innocent, Christophe; Seta, Patrick

    2006-08-01

    Nanofibrous membranes with an average diameter of 100 and 180 nm were fabricated from poly(acrylonitrile-co-maleic acid) (PANCMA) by the electrospinning process. These nanofibrous membranes contain reactive groups which can be used to covalently immobilize biomacromolecules. Two natural macromolecules, chitosan and gelatin, were tethered on these nanofibrous membranes to fabricate dual-layer biomimetic supports for enzyme immobilization in the presence of 1-ethyl-3-(dimethyl-aminopropyl) carbodiimide hydrochloride (EDC)/N-hydroxyl succinimide (NHS). Lipase from Candida rugosa was then immobilized on these dual-layer biomimetic supports using glutaraldehyde (GA), and on the nascent PANCMA fibrous membrane using EDC/NHS as coupling agent, respectively. The properties of the immobilized lipases were assayed. It was found that there is an increase of the activity retention of the immobilized lipase on the chitosan-modified nanofibrous membrane (45.6+/-1.8%) and on the gelatin-modified one (49.7+/-1.8%), compared to that on the nascent one (37.6+/-1.8%). The kinetic parameters of the free and immobilized lipases, K(m) and V(max), were also assayed. In comparison with the immobilized lipase on the nascent nanofibrous membrane, there is an increase of the V(max) value for the immobilized lipases on the chitosan- and gelatin-modified nanofibrous membranes. Results also indicate that the pH and thermal stabilities of lipases increase upon immobilization. The residual activities of the immobilized lipases are 55% on the chitosan-modified nanofibrous membrane and 60% on the gelatin-modified one, after 10 uses. PMID:16584770

  2. The effects of chemically modifying serum apolipoproteins on their ability to activate lipoprotein lipase.

    PubMed Central

    Dodds, P F; Lopez-Johnston, A; Welch, V A; Gurr, M I

    1987-01-01

    Lipoprotein lipase activity was measured in an acetone-dried-powder preparation from rat epididymal adipose tissue using pig serum or pig serum lipoprotein, which had been chemically modified, as activator. Modification of acidic amino acids of lipoproteins with NN-dimethyl-1,3-diamine resulted in a complete loss of ability to activate lipoprotein lipase. Modification of 34% of lipoprotein arginine groups with cyclohexanedione resulted in the loss of 75% of the activation of lipoprotein lipase; approx. 42% of the original activity was recovered after reversal of the modification. This effect was dependent on the cyclohexanedione concentration. Modification of 48% of lipoprotein lysine groups with malonaldehyde decreased the maximum activation by 20%, but three times as much lipoprotein was required to achieve this. Non-enzymic glycosylation of lipoprotein with glucose, under a variety of conditions resulting in up to 28 nmol of glucose/mg of protein, had no effect upon the ability to activate lipoprotein lipase. In contrast non-enzymic sialylation resulted in a time-dependent loss of up to 60% of ability to activate lipoprotein lipase. Reductive methylation and acetoacetylation of serum did not affect the ability to activate lipoprotein lipase. The results are compared to the effects of similar modifications to low density lipoproteins on receptor-mediated endocytosis. PMID:3593262

  3. [The characterization of microbial lipases. 1. The determination of lipase activity].

    PubMed

    Bariszlovich, M; Meusel, D; Tülsner, M

    1990-01-01

    In the selection of an appropriate method for activity determination of lipases existing technical equipment, kind of enzymes, number of samples investigated (e.g. in routine analysis), and expected sensitivity range have to be taken into account. Titrimetric methods and above all copper salt methods with their high detection sensitivity are the most suitable procedures for activity determination of lipases used in laboratories and institutions without equipment for radiochemical analysis. PMID:2233988

  4. Lipase

    MedlinePlus

    ... Lipase is used for indigestion, heartburn, allergy to gluten in wheat products (celiac disease), Crohn's disease, and ... that is associated with cystic fibrosis.Allergy to gluten in wheat products (celiac disease). Crohn's disease. Indigestion. ...

  5. Lysosomal Acid Lipase Hydrolyzes Retinyl Ester and Affects Retinoid Turnover.

    PubMed

    Grumet, Lukas; Eichmann, Thomas O; Taschler, Ulrike; Zierler, Kathrin A; Leopold, Christina; Moustafa, Tarek; Radovic, Branislav; Romauch, Matthias; Yan, Cong; Du, Hong; Haemmerle, Guenter; Zechner, Rudolf; Fickert, Peter; Kratky, Dagmar; Zimmermann, Robert; Lass, Achim

    2016-08-19

    Lysosomal acid lipase (LAL) is essential for the clearance of endocytosed cholesteryl ester and triglyceride-rich chylomicron remnants. Humans and mice with defective or absent LAL activity accumulate large amounts of cholesteryl esters and triglycerides in multiple tissues. Although chylomicrons also contain retinyl esters (REs), a role of LAL in the clearance of endocytosed REs has not been reported. In this study, we found that murine LAL exhibits RE hydrolase activity. Pharmacological inhibition of LAL in the human hepatocyte cell line HepG2, incubated with chylomicrons, led to increased accumulation of REs in endosomal/lysosomal fractions. Furthermore, pharmacological inhibition or genetic ablation of LAL in murine liver largely reduced in vitro acid RE hydrolase activity. Interestingly, LAL-deficient mice exhibited increased RE content in the duodenum and jejunum but decreased RE content in the liver. Furthermore, LAL-deficient mice challenged with RE gavage exhibited largely reduced post-prandial circulating RE content, indicating that LAL is required for efficient nutritional vitamin A availability. In summary, our results indicate that LAL is the major acid RE hydrolase and required for functional retinoid homeostasis. PMID:27354281

  6. Detection and determination of lipase (acylglycerol hydrolase) activity from various sources.

    PubMed

    Jensen, R G

    1983-09-01

    Methods for the detection and determination of lipases (acylglycerol hydrolases) and preparation of assays are reviewed including substrates, conditions and screening. Some newer methods for the determination of lipase activity are discussed. Several of these are: (a) titrimetry, (b) colorimetry of Cu soaps of free fatty acids (FFA), (c) colorimetry of chromophores in the acyl chain of FFA or in glycerol, (d) radioassay, (e) gas liquid chromatography, (f) enzymatic treatment of FFA and measurement of the resulting products, and (g) direct immunological determination of the lipase. Examples and sensitivities are given and advantages and disadvantages are described. PMID:6633171

  7. Fatty Acid Signaling: The New Function of Intracellular Lipases

    PubMed Central

    Papackova, Zuzana; Cahova, Monika

    2015-01-01

    Until recently, intracellular triacylglycerols (TAG) stored in the form of cytoplasmic lipid droplets have been considered to be only passive “energy conserves”. Nevertheless, degradation of TAG gives rise to a pleiotropic spectrum of bioactive intermediates, which may function as potent co-factors of transcription factors or enzymes and contribute to the regulation of numerous cellular processes. From this point of view, the process of lipolysis not only provides energy-rich equivalents but also acquires a new regulatory function. In this review, we will concentrate on the role that fatty acids liberated from intracellular TAG stores play as signaling molecules. The first part provides an overview of the transcription factors, which are regulated by fatty acids derived from intracellular stores. The second part is devoted to the role of fatty acid signaling in different organs/tissues. The specific contribution of free fatty acids released by particular lipases, hormone-sensitive lipase, adipose triacylglycerol lipase and lysosomal lipase will also be discussed. PMID:25674855

  8. Surfactant-activated lipase hybrid nanoflowers with enhanced enzymatic performance.

    PubMed

    Cui, Jiandong; Zhao, Yamin; Liu, Ronglin; Zhong, Cheng; Jia, Shiru

    2016-01-01

    Increasing numbers of materials have been extensively used as platforms for enzyme immobilization to improve catalytic performance. However, activity of the most of the enzymes was declined after immobilization. Here, we develop a surfactant-activated lipase-inorganic flowerlike hybrid nanomaterials with rational design based on interfacial activation and self-assembly. The resulting surfactant-activated lipase-inorganic hybird nanoflower (activated hNF-lipase) exhibited 460% and 200% higher activity than native lipase and conventional lipase-inorganic hybird nanoflower (hNF-lipase). Furthermore, the activated hNF-lipase displayed good reusability due to its monodispersity and mechanical properties, and had excellent long-time stability. The superior catalytic performances were attributed to both the conformational modulation of surfactants and hierarchical structure of nanoflowers, which not only anchored lipases in an active form, but also decreased the enzyme-support negative interaction and mass-transfer limitations. This new biocatalytic system is promising to find widespread use in applications related to biomedicine, biosensor, and biodiesel. PMID:27297609

  9. Surfactant-activated lipase hybrid nanoflowers with enhanced enzymatic performance

    PubMed Central

    Cui, Jiandong; Zhao, Yamin; Liu, Ronglin; Zhong, Cheng; Jia, Shiru

    2016-01-01

    Increasing numbers of materials have been extensively used as platforms for enzyme immobilization to improve catalytic performance. However, activity of the most of the enzymes was declined after immobilization. Here, we develop a surfactant-activated lipase-inorganic flowerlike hybrid nanomaterials with rational design based on interfacial activation and self-assembly. The resulting surfactant-activated lipase-inorganic hybird nanoflower (activated hNF-lipase) exhibited 460% and 200% higher activity than native lipase and conventional lipase-inorganic hybird nanoflower (hNF-lipase). Furthermore, the activated hNF-lipase displayed good reusability due to its monodispersity and mechanical properties, and had excellent long-time stability. The superior catalytic performances were attributed to both the conformational modulation of surfactants and hierarchical structure of nanoflowers, which not only anchored lipases in an active form, but also decreased the enzyme-support negative interaction and mass-transfer limitations. This new biocatalytic system is promising to find widespread use in applications related to biomedicine, biosensor, and biodiesel. PMID:27297609

  10. Catalyzed ester synthesis using Candida rugosa lipase entrapped by poly(N-isopropylacrylamide-co-itaconic acid) hydrogel.

    PubMed

    Milašinović, Nikola; Jakovetić, Sonja; Knežević-Jugović, Zorica; Milosavljević, Nedeljko; Lučić, Marija; Filipović, Jovanka; Kalagasidis Krušić, Melina

    2014-01-01

    This study reports the synthesis of polymeric matrices based on N-isopropylacrylamide and itaconic acid and its application for immobilization of lipase from Candida rugosa. The lipase was immobilized by entrapment method. Free and immobilized lipase activities, pH and temperature optima, and storage stability were investigated. The optimum temperature for free and entrapped lipase was found to be 40 and 45 °C, while the optimum pH was observed at pH 7 and 8, respectively. Both hydrolytic activity in an aqueous medium and esterolytic activity in an organic medium have been evaluated. Maximum reaction rate (V max) and Michaelis-Menten constants (K m ) were also determined for immobilized lipase. Storage stability of lipase was increased as a result of immobilization process. Furthermore, the operational stability and reusability of the immobilized lipase in esterification reaction have been studied, and it was observed that after 10 cycles, the residual activity for entrapped lipase was as high as 50%, implying that the developed hydrogel and immobilized system could provide a promising solution for the flavor ester synthesis at the industrial scale. PMID:24701136

  11. Catalyzed Ester Synthesis Using Candida rugosa Lipase Entrapped by Poly(N-isopropylacrylamide-co-itaconic Acid) Hydrogel

    PubMed Central

    Milašinović, Nikola; Jakovetić, Sonja; Knežević-Jugović, Zorica; Milosavljević, Nedeljko; Lučić, Marija; Filipović, Jovanka; Kalagasidis Krušić, Melina

    2014-01-01

    This study reports the synthesis of polymeric matrices based on N-isopropylacrylamide and itaconic acid and its application for immobilization of lipase from Candida rugosa. The lipase was immobilized by entrapment method. Free and immobilized lipase activities, pH and temperature optima, and storage stability were investigated. The optimum temperature for free and entrapped lipase was found to be 40 and 45°C, while the optimum pH was observed at pH 7 and 8, respectively. Both hydrolytic activity in an aqueous medium and esterolytic activity in an organic medium have been evaluated. Maximum reaction rate (Vmax) and Michaelis-Menten constants (Km) were also determined for immobilized lipase. Storage stability of lipase was increased as a result of immobilization process. Furthermore, the operational stability and reusability of the immobilized lipase in esterification reaction have been studied, and it was observed that after 10 cycles, the residual activity for entrapped lipase was as high as 50%, implying that the developed hydrogel and immobilized system could provide a promising solution for the flavor ester synthesis at the industrial scale. PMID:24701136

  12. 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

  13. ATGL is a major hepatic lipase that regulates TAG turnover and fatty acid signaling and partitioning

    PubMed Central

    Ong, Kuok Teong; Mashek, Mara T.; Bu, So Young; Greenberg, Andrew S.; Mashek, Douglas G.

    2010-01-01

    Despite advances into our understanding of how nutrient oversupply and triacylglycerol (TAG) anabolism contribute to hepatic steatosis, little is known about the lipases responsible for regulating hepatic TAG turnover. Recent studies have identified adipose triglyceride lipase (ATGL) as a major lipase in adipose tissue although its role in the liver is largely unknown. Thus, we tested the contribution of ATGL to hepatic lipid metabolism and signaling. Adenoviral-mediated knockdown of hepatic ATGL resulted in steatosis in mice and decreased hydrolysis of TAG in primary hepatocyte cultures and in vitro assays. In addition to altering TAG hydrolysis, ATGL is shown to play a significant role in partitioning hydrolyzed fatty acids between metabolic pathways. Whereas ATGL gain- and loss-of-function did not alter hepatic TAG secretion, fatty acid oxidation was increased by ATGL overexpression and decreased by ATGL knockdown. The effects on fatty acid oxidation coincided with decreased expression of PPAR-α and its target genes in mice with suppressed hepatic ATGL expression. However, PPAR-α agonism was unable to normalize the effects of ATGL knockdown on PPAR-α target gene expression suggesting that ATGL influences PPAR-α activity independent of ligand-induced activation. Taken together, these data show that ATGL is a major hepatic TAG lipase that plays an integral role in fatty acid partitioning and signaling to control energy metabolism. PMID:20967758

  14. Biocatalytic potential of lipase from Staphylococcus sp. MS1 for transesterification of jatropha oil into fatty acid methyl esters.

    PubMed

    Sharma, Monika; Singh, Shelley Sardul; Maan, Pratibha; Sharma, Rohit

    2014-11-01

    An extracellular lipase producing isolate Staphylococcus sp. MS1 was optimized for lipase production and its biocatalytic potential was assessed. Medium with tributyrin (0.25 %) and without any exogenous inorganic nitrogen source was found to be optimum for lipase production from Staphylococcus sp. MS1. The optimum pH and temperature for lipase production were found to be pH 7 and 37 °C respectively, showing lipase activity of 37.91 U. It showed good lipase production at pH 6-8. The lipase was found to be stable in organic solvents like hexane and petroleum ether, showing 98 and 88 % residual activity respectively. The biotransformation using the concentrated enzyme in petroleum ether resulted in the synthesis of fatty acid methyl esters like methyl oleate, methyl palmitate and methyl stearate. Thus, the lipase under study has got the potential to bring about transesterification of oils into methyl esters which can be exploited for various biotechnological applications. PMID:25115850

  15. Porcine pancreatic lipase related protein 2 has high triglyceride lipase activity in the absence of colipase.

    PubMed

    Xiao, Xunjun; Ross, Leah E; Sevilla, Wednesday A; Wang, Yan; Lowe, Mark E

    2013-09-01

    Efficient dietary fat digestion is essential for newborns who consume more dietary fat per body weight than at any other time of life. In many mammalian newborns, pancreatic lipase related protein 2 (PLRP2) is the predominant duodenal lipase. Pigs may be an exception since PLRP2 expression has been documented in the intestine but not in the pancreas. Because of the differences in tissue-specific expression, we hypothesized that the kinetic properties of porcine PLRP2 would differ from those of other mammals. To characterize its properties, recombinant porcine PLRP2 was expressed in HEK293T cells and purified to homogeneity. Porcine PLRP2 had activity against tributyrin, trioctanoin and triolein. The activity was not inhibited by bile salts and colipase, which is required for the activity of pancreatic triglyceride lipase (PTL), minimally stimulated PLRP2 activity. Similar to PLRP2 from other species, PLRP2 from pigs had activity against galactolipids and phospholipids. Importantly, porcine PLRP2 hydrolyzed a variety of dietary substrates including pasteurized human mother's milk and infant formula and its activity was comparable to that of PTL. In conclusion, porcine PLRP2 has broad substrate specificity and has high triglyceride lipase activity even in the absence of colipase. The data suggest that porcine PLRP2 would be a suitable lipase for inclusion in recombinant preparations for pancreatic enzyme replacement therapy. PMID:23770034

  16. Chitosan-tethered poly(acrylonitrile-co-maleic acid) hollow fiber membrane for lipase immobilization.

    PubMed

    Ye, Peng; Xu, Zhi-Kang; Che, Ai-Fu; Wu, Jian; Seta, Patrick

    2005-11-01

    A protocol was used to prepare a dual-layer biomimetic membrane as support for enzyme immobilization by tethering chitosan on the surface of poly(acrylonitrile-co-maleic acid) (PANCMA) ultrafiltration hollow fiber membrane in the presence of 1-ethyl-3-(dimethylaminopropyl) carbodiimide hydrochloride (EDC)/N-hydroxylsuccin-imide (NHS). The chemical change of the chitosan-modified PANCMA membrane surface was confirmed with Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy. Lipase from Candida rugosa was immobilized on this dual-layer biomimetic membrane using glutaraldehyde (GA), and on the nascent PANCMA membrane using EDC/NHS as coupling agent. The properties of the immobilized enzymes were assayed and compared with those of the free one. It was found that both the activity retention of the immobilized lipase and the amount of bound protein on the dual-layer biomimetic membrane (44.5% and 66.5 mg/m2) were higher than those on the nascent PANCMA membrane (33.9% and 53.7 mg/m2). The kinetic parameters of the free and immobilized lipases, Km and Vmax, were also assayed. The Km values were similar for the immobilized lipases, while the Vmax value of the immobilized lipase on the dual-layer biomimetic membrane was higher than that on the nascent PANCMA membrane. Results indicated that the pH and thermal stabilities of lipase increased upon immobilization. The residual activity of the immobilized lipase after 10 uses was 53% on the dual-layer biomimetic membrane and 62% on the nascent PANCMA membrane. PMID:15919112

  17. 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. PMID:23851449

  18. Endothelial lipase modulates pressure overload-induced heart failure through alternative pathway for fatty acid uptake.

    PubMed

    Nakajima, Hideto; Ishida, Tatsuro; Satomi-Kobayashi, Seimi; Mori, Kenta; Hara, Tetsuya; Sasaki, Naoto; Yasuda, Tomoyuki; Toh, Ryuji; Tanaka, Hidekazu; Kawai, Hiroya; Hirata, Ken-ichi

    2013-05-01

    Lipoprotein lipase has been considered as the only enzyme capable of generating lipid-derived fatty acids for cardiac energy. Endothelial lipase is another member of the triglyceride lipase family and hydrolyzes high-density lipoproteins. Although endothelial lipase is expressed in the heart, its function remains unclear. We assessed the role of endothelial lipase in the genesis of heart failure. Pressure overload-induced cardiac hypertrophy was generated in endothelial lipase(-/-) and wild-type mice by ascending aortic banding. Endothelial lipase expression in cardiac tissues was markedly elevated in the early phase of cardiac hypertrophy in wild-type mice, whereas lipoprotein lipase expression was significantly reduced. Endothelial lipase(-/-) mice showed more severe systolic dysfunction with left-ventricular dilatation compared with wild-type mice in response to pressure overload. The expression of mitochondrial fatty acid oxidation-related genes, such as carnitine palmitoyltransferase-1 and medium-chain acyl coenzyme A dehydrogenase, was significantly lower in the heart of endothelial lipase(-/-) mice than in wild-type mice. Also, endothelial lipase(-/-) mice had lower myocardial adenosine triphosphate levels than wild-type mice after aortic banding. In cultured cardiomyocytes, endothelial lipase was upregulated by inflammatory stimuli, whereas lipoprotein lipase was downregulated. Endothelial lipase-overexpression in cardiomyocytes resulted in an upregulation of fatty acid oxidation-related enzymes and intracellular adenosine triphosphate accumulation in the presence of high-density lipoprotein. Endothelial lipase may act as an alternative candidate to provide fatty acids to the heart and regulate cardiac function. This effect seemed relevant particularly in the diseased heart, where lipoprotein lipase action is downregulated. PMID:23460280

  19. A gene encoding a new cold-active lipase from an Antarctic isolate of Penicillium expansum.

    PubMed

    Mohammed, Suja; Te'o, Junior; Nevalainen, Helena

    2013-08-01

    Cold-active lipases are of significant interest as biocatalysts in industrial processes. We have identified a lipase that displayed activity towards long carbon-chain-p-nitrophenyl substrates (C12-C18) at 25 °C from the culture supernatant of an Antarctic Penicillium expansum strain assigned P. expansum SM3. Zymography revealed a protein band of around 30 kDa with activity towards olive oil. DNA fragments of a lipase gene designated as lipPE were isolated from the genomic DNA of P. expansum SM3 by genomic walking PCR. Subsequently, the complete genomic lipPE gene was amplified using gene-specific primers designed from the 5'- and 3'-regions. Reverse transcription PCR was used to amplify the lipPE cDNA. The deduced amino acid sequence consisted of 285 residues that included a predicted signal peptide. Three peptides identified by LC/MS/MS analysis of the proteins in the culture supernatant of P. expansum were also present in the deduced amino acid sequence of the lipPE gene suggesting that this gene encoded the lipase identified by initial zymogram activity analysis. Full analysis of the nucleotide and the deduced amino acid sequences indicated that the lipPE gene encodes a novel P. expansum lipase. The lipPE gene was expressed in E. coli for further characterization of the enzyme with a view of assessing its suitability for industrial applications. PMID:23779196

  20. The production and characterization of a new active lipase from Acremonium alcalophilum using a plant bioreactor

    PubMed Central

    2013-01-01

    Background Microorganisms are the most proficient decomposers in nature, using secreted enzymes in the hydrolysis of lignocellulose. As such, they present the most abundant source for discovery of new enzymes. Acremonium alcalophilum is the only known cellulolytic fungus that thrives in alkaline conditions and can be cultured readily in the laboratory. Its optimal conditions for growth are 30°C and pH 9.0-9.2. The genome sequence of Acremonium alcalophilum has revealed a large number of genes encoding biomass-degrading enzymes. Among these enzymes, lipases are interesting because of several industrial applications including biofuels, detergent, food processing and textile industries. Results We identified a lipA gene in the genome sequence of Acremonium alcalophilum, encoding a protein with a predicted lipase domain with weak sequence identity to characterized enzymes. Unusually, the predicted lipase displays ≈ 30% amino acid sequence identity to both feruloyl esterase and lipase of Aspergillus niger. LipA, when transiently produced in Nicotiana benthamiana, accumulated to over 9% of total soluble protein. Plant-produced recombinant LipA is active towards p-nitrophenol esters of various carbon chain lengths with peak activity on medium-chain fatty acid (C8). The enzyme is also highly active on xylose tetra-acetate and oat spelt xylan. These results suggests that LipA is a novel lipolytic enzyme that possesses both lipase and acetylxylan esterase activity. We determined that LipA is a glycoprotein with pH and temperature optima at 8.0 and 40°C, respectively. Conclusion Besides being the first heterologous expression and characterization of a gene coding for a lipase from A. alcalophilum, this report shows that LipA is very versatile exhibiting both acetylxylan esterase and lipase activities potentially useful for diverse industry sectors, and that tobacco is a suitable bioreactor for producing fungal proteins. PMID:23915965

  1. Kinetic modeling, production and characterization of an acidic lipase produced by Enterococcus durans NCIM5427 from fish waste.

    PubMed

    Ramakrishnan, Vrinda; Goveas, Louella Concepta; Halami, Prakash M; Narayan, Bhaskar

    2015-03-01

    Enterococcus durans NCIM5427 (ED-27), capable of producing an intracellular acid stable lipase, was isolated from fish processing waste. Its growth and subsequent lipase production was optimized by Box Behneken design (optimized conditions: 5 % v/v fish waste oil (FWO), 0.10 mg/ml fish waste protein hydrolysates (FWPH) at 48 h of fermentation time). Under optimized conditions, ED-27 showed a 3.0 fold increase (207.6 U/ml to 612.53 U/ml) in lipase production, as compared to un-optimized conditions. Cell growth and lipase production was modeled using Logistic and Luedeking-Piret model, respectively; and lipase production by ED-27 was found to be growth-associated. Lipase produced by ED-27 showed stability at low pH ranges from 2 to 5 with its optimal activity at 30 °C , pH 4.6; showed metal ion dependent activity wherein its catalytic activity was activated by barium, sodium, lithium and potassium (10 mM); reduced by calcium and magnesium (10 mM). However, iron and mercury (5 mM) completely inactivated the enzyme. In addition, modifying agents like SDS, DTT, β-ME (1%v/v) increased activity of lipase of ED-27; while, PMSF, DEPC and ascorbic acid resulted in a marked decrease. ED-27 had maximum cell growth of 9.90309 log CFU/ml under optimized conditions as compared to 13 log CFU/ml in MRS. The lipase produced has potential application in poultry and slaughterhouse waste management. PMID:25745201

  2. Immobilization of Yarrowia lipolytica lipase Ylip2 for the biocatalytic synthesis of phytosterol ester in a water activity controlled reactor.

    PubMed

    Cui, Caixia; Guan, Nan; Xing, Chen; Chen, Biqiang; Tan, Tianwei

    2016-10-01

    In this work, phytosterol ester was synthesized using Yarrowia lipolytica lipase Ylip2 that had been immobilized on inorganic support in a solvent-free system and reacted in a computer-aided water activity controlled bioreactor. The immobilization of Ylip2 on celite led to a remarkable increase in the phytosterol conversion compared to that of free lipase. An investigation of the reaction conditions were oleic acid as the fatty acid variety, 10,000U/g substrate, and a temperature of 50°C for phytosterol ester synthesis. Controlling of the water activity at a set point was accomplished by the introduction of dry air through the reaction medium at a digital feedback controlled flow rate. For the esterification of phytosterol ester, a low (15%) water activity resulted in a considerable improvement in phytosterol conversion (91.1%) as well as a decreased reaction time (78h). Furthermore, Ylip2 lipase immobilized on celite retained 90% esterification activity for the synthesis of phytosterol oleate after reused 8 cycles, while free lipase was only viable for 5 batches with 90% esterification activity remained. Finally, the phytosterol oleate space time yield increased from 1.65g/L/h with free lipase to 2.53g/L/h with immobilized lipase. These results illustrate that the immobilized Yarrowia lipolytica lipase Ylip2 in a water activity controlled reactor has great potential for the application in phytosterol esters synthesis. PMID:27416561

  3. Lipase Activity among Bacteria Isolated from Amazonian Soils.

    PubMed

    Willerding, André Luis; de Oliveira, Luiz Antonio; Moreira, Francisco Wesen; Germano, Mariana Gomes; Chagas, Aloísio Freitas

    2011-01-01

    The objective of this study was to select lipase-producing bacteria collected from different counties of the Amazon region. Of the 440 bacteria strains, 181 were selected for the lipase assay in qualitative tests at Petri dishes, being 75 (41%) lipase positive. The enzymatic index was determined during fifteen days at different temperatures (30°, 35°, 40°, and 45°C). The highest lipase activity was observed within 72 hours at 30°C. Twelve bacteria strains presented an index equal to or greater than the standard used like reference, demonstrating the potential of microbial resource. After the bioassay in Petri dishes, the selected bacteria strains were analyzed in quantitative tests on p-nitrophenyl palmitate (p-NPP). A group of the strains was selected for other phases of study with the use in oleaginous substrates of the Amazonian flora, aiming for the application in processes like oil biotransformation. PMID:22007294

  4. Lipase Activity among Bacteria Isolated from Amazonian Soils

    PubMed Central

    Willerding, André Luis; de Oliveira, Luiz Antonio; Moreira, Francisco Wesen; Germano, Mariana Gomes; Chagas, Aloísio Freitas

    2011-01-01

    The objective of this study was to select lipase-producing bacteria collected from different counties of the Amazon region. Of the 440 bacteria strains, 181 were selected for the lipase assay in qualitative tests at Petri dishes, being 75 (41%) lipase positive. The enzymatic index was determined during fifteen days at different temperatures (30°, 35°, 40°, and 45°C). The highest lipase activity was observed within 72 hours at 30°C. Twelve bacteria strains presented an index equal to or greater than the standard used like reference, demonstrating the potential of microbial resource. After the bioassay in Petri dishes, the selected bacteria strains were analyzed in quantitative tests on p-nitrophenyl palmitate (p-NPP). A group of the strains was selected for other phases of study with the use in oleaginous substrates of the Amazonian flora, aiming for the application in processes like oil biotransformation. PMID:22007294

  5. [Lipase-catalyzed production of biodiesel from high acid value waste oil with ultrasonic assistant].

    PubMed

    Wang, Jian-Xun; Huang, Qing-De; Huang, Feng-Hong; Wang, Jiang-Wei; Huang, Qin-Jie

    2007-11-01

    Biodiesel fuel produced with the enzyme-catalyzed esterification and transesterification of high acid value waste oil through ultrasonic assistant was explored. Propyl oleate, biodiesel, converted from high acid value waste oil and 1-proponal catalyzed with immobilized lipases from Candida antarctica and Aspergillus oryzae in conditions of ultrasonic assistant. Commercial immobilized lipase Novozym 435 from C. antarctica was used as biocatalyst catalyzing high acid value waste oil and 1-proponal esterification and transesterification to propyl oleate under the ultrasonic assistant conditions and different conditions such as lipases amounts, initiatory molar ratio of propanol to oil, frequency of ultrasonic and power of ultrasonic were investigated and optimized. It is revealed that the enzymatic activity of Novozym435 is enhanced and, in particular, enzyme-catalyzed transesterification activity is enhanced obviously under the ultrasonic assistant conditions. Low frequency and mild energy ultrasonic is a key factor for enhancing enzymatic activity, emulsifying oil-propanol system and accelerating the speed of produce diffusing in the system. Under the optimal ultrasonic assistant reaction conditions, such as Novozym435 amounts 8% by oil quantity, initiatory molar ratio of propanol to oil 3:1, frequency of ultrasonic 28 KHz, power of ultrasonic 100 W and temperature of water batch 40-45 degrees C, the conversion ratio to propyl oleate reached to 94.86% in 50 mins in comparison with the highest conversion ratio to propyl oleate 84.43% under the conventional mechanical agitation conditions. Furthermore, it is demonstrated that various short chain linear and branched alcohols (C1-C5) show high conversion ratio to fatty acid alkyl esters (biodiesel) under the optimal ultrasonic assistant reaction conditions. On the other hand, ultrasonic energy is propitious to reduce the adsorption of product propyl oleate, by-product glycerol and other emplastics in system on the

  6. Intercellular transport of lysosomal acid lipase mediates lipoprotein cholesteryl ester metabolism in a human vascular endothelial cell-fibroblast coculture system.

    PubMed Central

    Sando, G N; Ma, G P; Lindsley, K A; Wei, Y P

    1990-01-01

    We present results from studies of human cell culture models to support the premise that the extracellular transport of lysosomal acid lipase has a function in lipoprotein cholesteryl ester metabolism in vascular tissue. Vascular endothelial cells secreted a higher fraction of cellular acid lipase than did smooth muscle cells and fibroblasts. Acid lipase and lysosomal beta-hexosaminidase were secreted at approximately the same rate from the apical and basolateral surface of an endothelial cell monolayer. Stimulation of secretion with NH4Cl did not affect the polarity. We tested for the ability of secreted endothelial lipase to interact with connective tissue cells and influence lipoprotein cholesterol metabolism in a coculture system in which endothelial cells on a micropore filter were suspended above a monolayer of acid lipase-deficient (Wolman disease) fibroblasts. After 5-7 d, acid lipase activity in the fibroblasts reached 10%-20% of the level in normal cells; cholesteryl esters that had accumulated from growth in serum were cleared. Addition of mannose 6-phosphate to the coculture medium blocked acid lipase uptake and cholesterol clearance, indicating that lipase released from endothelial cells was packaged into fibroblast lysosomes by a phosphomannosyl receptor-mediated pathway. Supplementation of the coculture medium with serum was not required for lipase uptake and cholesteryl ester hydrolysis by the fibroblasts, but was necessary for cholesterol clearance. Results from our coculture model suggest that acid lipase may be transported from intact endothelium to cells in the lumen or the wall of a blood vessel. We postulate that delivery of acid hydrolases and lipoproteins to a common endocytic compartment may occur and have an impact on cellular lipoprotein processing. PMID:2150334

  7. Lipoproteini lipase-derived fatty acids: physiology and dysfunction.

    PubMed

    Lee, Jee; Goldberg, Ira J

    2007-12-01

    Under normal circumstances, most energy substrate used for heart contraction derives from fatty acids in the form of nonesterified fatty acids bound to albumin or fatty acids derived from lipolysis of lipoprotein-bound triglyceride by lipoprotein lipase (LpL). By creating LpL knockout mice (hLpL0), we learned that loss of cardiac LpL leads to myocardial dysfunction; therefore, neither nonesterified fatty acids nor increased glucose metabolism can replace LpL actions. hLpL0 mice do not survive abdominal aortic constriction and they develop more heart failure with hypertension. Conversely, we created a mouse overexpressing cardiomyocyte-anchored LpL. This transgene produced cardiac lipotoxicity and dilated cardiomyopathy. Methods to alter this phenotype and the causes of other models of lipotoxicity are currently being studied and will provide further insight into the physiology of lipid metabolism in the heart. PMID:18367009

  8. Synthesis of ascorbyl oleate by transesterification of olive oil with ascorbic acid in polar organic media catalyzed by immobilized lipases.

    PubMed

    Moreno-Perez, Sonia; Filice, Marco; Guisan, Jose M; Fernandez-Lorente, Gloria

    2013-09-01

    The reaction of transesterification between oils (e.g., olive oil) and ascorbic acid in polar anhydrous media (e.g., tert-amyl alcohol) catalyzed by immobilized lipases for the preparation of natural liposoluble antioxidants (e.g., ascorbyl oleate) was studied. Three commercial lipases were tested: Candida antarctica B lipase (CALB), Thermomyces lanuginosus lipase (TLL) and Rhizomucor miehei lipase (RML). Each lipase was immobilized by three different protocols: hydrophobic adsorption, anionic exchange and multipoint covalent attachment. The highest synthetic yields were obtained with CALB adsorbed on hydrophobic supports (e.g., the commercial derivative Novozym 435). The rates and yields of the synthesis of ascorbyl oleate were higher when using the solvent dried with molecular sieves, at high temperatures (e.g. 45°C) and with a small excess of oil (2 mol of oil per mol of ascorbic acid). The coating of CALB derivatives with polyethyleneimine (PEI) improved its catalytic behavior and allowed the achievement of yields of up to 80% of ascorbyl oleate in less than 24h. CALB adsorbed on a hydrophobic support and coated with PEI was 2-fold more stable than a non-coated derivative and one hundred-fold more stable than the best TLL derivative. The best CALB derivative exhibited a half-life of 3 days at 75°C in fully anhydrous media, and this derivative maintained full activity after 28 days at 45°C in dried tert-amyl alcohol. PMID:23891831

  9. Production of extracellular acidic lipase by Rhizopus arrhizus as a function of culture conditions.

    PubMed

    Kumar, K K; Deshpande, B S; Ambedkar, S S

    1993-01-01

    Thermophilic strain of Rhizopus arrhizus accumulates an acidic lipase in culture fluid when grown in a medium containing ground nut oil, milk powder and inorganic salts. Addition of 2.0% ground nut oil yielded the highest productivity of enzyme. Soyabean meal and arabinose were found to be the best nitrogen and carbon sources for enzyme production respectively. Addition of metal ions such as MnCl2, SnCl2 and CaCl2 increased the enzyme productivity by 4 fold. The enzyme productivity in the fermenter was much higher (310 U/ml) than in shake-flask (180 U/ml). Crude lipase preparation showed pH and temperature activity optima at 3.5 and 45 degrees C respectively. The enzyme is thermostable and highly active in hydrolysing triglycerides and failed to hydrolyse-methyl esters of caprylate and palmitate. PMID:8181953

  10. Lipases and whole cell biotransformations of 2-hydroxy-2-(ethoxyphenylphosphinyl)acetic acid and its ester.

    PubMed

    Majewska, Paulina; Serafin, Monika; Klimek-Ochab, Magdalena; Brzezińska-Rodak, Małgorzata; Żymańczyk-Duda, Ewa

    2016-06-01

    A wide spectrum of commercially available lipases and microbial whole cells catalysts were tested for biotransformations of 2-hydroxy-2-(ethoxyphenylphosphinyl)acetic acid 1 and its butyryl ester. The best results were achieved for biocatalytic hydrolysis of ester: 2-butyryloxy-2-(ethoxyphenylphosphinyl)acetic acid 2 performed by lipase from Candida cylindracea, what gave optically active products with 85% enantiomeric excess, 50% conversion degree and enantioselectivity 32.9 for one pair of enantiomers. Also enzymatic systems of Penicillium minioluteum and Fusarium oxysporum were able to hydrolyze tested compound with high enantiomeric excess (68-93% ee), enantioselectivity (44 for one pair of enantiomers) and conversion degree about 50-55%. Enzymatic acylation of hydroxyphosphinate was successful in case when porcine pancreas lipase was used. After 4days of biotransformation the conversion reaches 45% but the enantiomeric enrichment of the isomers mixture do not exceed 43%. Obtained chiral compounds are valuable derivatizing agents for spectroscopic (NMR) evaluation of enantiomeric excess for particular compounds (e.g. amino acids). PMID:26989983

  11. A simple strategy to monitor lipase activity using liquid crystal-based sensors.

    PubMed

    Hu, Qiong-Zheng; Jang, Chang-Hyun

    2012-09-15

    In this study, we developed a simple label-free technique for monitoring the enzymatic activity of lipase using liquid crystal (LC)-based sensors. The optical response of LCs changed from a bright to dark appearance when an aqueous solution of lipase was in contact with a nematic LC, 4-cyano-4'-pentylbiphenyl (5CB), that was doped with glyceryl trioleate, which is a glyceride that can be enzymatically hydrolyzed by lipase. Since the oleic acid released from the enzymatic reaction could spontaneously form a self-assembled monolayer at the aqueous/LC interface due to its amphiphilic property, the orientation of the LCs transited from a planar to homeotropic state, which induced a change in the optical response of the LCs. We did not observe a bright-to-dark shift in the optical appearance of LCs when pure 5CB was immersed into the lipase solution. Moreover, we further confirmed the specificity of the enzymatic reaction by transferring an aqueous buffer solution not containing an analyte, or with bovine serum albumin (BSA) or trypsin onto the interface of aqueous solutions and the glyceryl trioleate-doped 5CB, which did not produce any distinctive contrast in the optical appearance. These results suggest the feasibility of measuring the enzymatic activity of lipase using the LC-based sensing technique. Furthermore, our strategy could also be used for the preparation of a self-assembled monolayer of carboxylates at the aqueous/LC interface. PMID:22967518

  12. Investigating the influence of the interface in thiol-functionalized silver-gold nanoshells over lipase activity.

    PubMed

    Kisukuri, Camila M; Macedo, Alexandra; Oliveira, Caio C S; Camargo, Pedro H C; Andrade, Leandro H

    2013-12-23

    We employed thiol-funcionalized AgAu nanoshells (AgAu NSs) as supports for the covalent attachment of lipases (BCL, Burkholderia cepacia lipase; PPL, pancreatic porcine lipase). Specifically, we were interested in investigating the effect of the nature/size of the spacer in AgAu NSs-functionalized organic thiols over the covalent attachment of lipases. The catalytic performance of AgAu-lipase systems was measured in the kinetic resolution of (R,S)-1-(phenyl)ethanol via a transesterification reaction. In comparison to free BCL, the lipase attached to AgAu NSs using a small spacer such as cysteamine or mercaptoacetic acid, with the largest spacer mercaptoundecanoic acid, had the fastest conversion rate. The recycling potential for BCL was investigated. After three reaction cycles, the enzyme activity was kept at around 90% of the initial value. The results described herein show that the size of the spacer plays an important role in optimizing lipase activities in metallic nanoshells as solid supports. PMID:24313296

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

    SciTech Connect

    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 functionally 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.

  14. Branched Fatty Acid Esters of Hydroxy Fatty Acids Are Preferred Substrates of the MODY8 Protein Carboxyl Ester Lipase.

    PubMed

    Kolar, Matthew J; Kamat, Siddhesh S; Parsons, William H; Homan, Edwin A; Maher, Tim; Peroni, Odile D; Syed, Ismail; Fjeld, Karianne; Molven, Anders; Kahn, Barbara B; Cravatt, Benjamin F; Saghatelian, Alan

    2016-08-23

    A recently discovered class of endogenous mammalian lipids, branched fatty acid esters of hydroxy fatty acids (FAHFAs), possesses anti-diabetic and anti-inflammatory activities. Here, we identified and validated carboxyl ester lipase (CEL), a pancreatic enzyme hydrolyzing cholesteryl esters and other dietary lipids, as a FAHFA hydrolase. Variants of CEL have been linked to maturity-onset diabetes of the young, type 8 (MODY8), and to chronic pancreatitis. We tested the FAHFA hydrolysis activity of the CEL MODY8 variant and found a modest increase in activity as compared with that of the normal enzyme. Together, the data suggest that CEL might break down dietary FAHFAs. PMID:27509211

  15. Esterification of polyglycerol with polycondensed ricinoleic acid catalysed by immobilised Rhizopus oryzae lipase.

    PubMed

    Ortega, S; Máximo, M F; Montiel, M C; Murcia, M D; Arnold, G; Bastida, J

    2013-09-01

    The enzymatic method for synthesising polyglycerol polyricinoleate (PGPR), a food additive named E-476, was successfully carried out in the presence of immobilised Rhizopus oryzae lipase in a solvent-free medium. The great advantage of using the commercial preparation of R. oryzae lipase is that it is ten times cheaper than the commercial preparation of R. arrhizus lipase, the biocatalyst used in previous studies. The reaction, which is really a reversal of hydrolysis, takes place in the presence of a very limited amount of aqueous phase. Immobilisation of the lipase by physical adsorption onto an anion exchange resin provided good results in terms of activity, enzyme stability and reuse of the immobilised derivative. It has been established that the adsorption of R. oryzae lipase on Lewatit MonoPlus MP 64 follows a pseudo-second order kinetics, which means that immobilisation is a process of chemisorption, while the equilibrium adsorption follows a Langmuir isotherm. The use of this immobilised derivative as catalyst for obtaining PGPR under a controlled atmosphere in a vacuum reactor, with a dry nitrogen flow intake, allowed the synthesis of a product with an acid value lower than 6 mg KOH/g, which complies with the value established by the European Commission Directive. This product also fulfils the European specifications regarding the hydroxyl value and refractive index given for this food additive, one of whose benefits, as proved in our experiments, is that it causes a drastic decrease in the viscosity (by 50 %) and yield stress (by 82 %) of chocolate, comparable to the impact of customary synthesised PGPR. PMID:23263570

  16. Activities of amylase, proteinase, and lipase enzymes from Lactococcus chungangensis and its application in dairy products.

    PubMed

    Konkit, Maytiya; Kim, Wonyong

    2016-07-01

    Several enzymes are involved in the process of converting milk to lactic acid and coagulated milk to curd and, therefore, are important in dairy fermented products. Amylase, proteinase, and lipase are enzymes that play an important role in degrading milk into monomeric molecules such as oligosaccharides, amino acids, and fatty acids, which are the main molecules responsible for flavors in cheese. In the current study, we determined the amylase, proteinase, and lipase activities of Lactococcus chungangensis CAU 28(T), a bacterial strain of nondairy origin, and compared them with those of the reference strain, Lactococcus lactis ssp. lactis KCTC 3769(T), which is commonly used in the dairy industry. Lactococcus chungangensis CAU 28(T) and L. lactis ssp. lactis KCTC 3769(T) were both found to have amylase, proteinase, and lipase activities in broth culture, cream cheese, and yogurt. Notably, the proteinase and lipase activities of L. chungangensis CAU 28(T) were higher than those of L. lactis ssp. lactis KCTC 3769(T), with proteinase activity of 10.50 U/mL in tryptic soy broth and 8.64 U/mL in cream cheese, and lipase activity of 100 U/mL of tryptic soy broth, and 100 U/mL of cream cheese. In contrast, the amylase activity was low, with 5.28 U/mL in tryptic soy broth and 8.86 U/mL in cream cheese. These enzyme activities in L. chungangensis CAU 28(T) suggest that this strain has potential to be used for manufacturing dairy fermented products, even though the strain is of nondairy origin. PMID:27108177

  17. 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

  18. Infant case of lysosomal acid lipase deficiency: Wolman's disease

    PubMed Central

    Sadhukhan, Meghmala; Saha, Amit; Vara, Roshni; Bhaduri, Bim

    2014-01-01

    Lysosomal acid lipase (LAL) deficiency is a rare autosomal recessive disorder which causes two distinct clinical phenotypes: Wolman's disease and cholesterol ester storage disease. LAL hydrolyses LDL-derived triglycerides and cholesterol esters to glycerol or cholesterol and free fatty acids. Its deficiency leads to accumulation of intracellular triglycerides and/or cholesterol esters. In early onset LAL deficiency, clinical manifestations start in the first few weeks of life with persistent vomiting, failure to thrive, hepatosplenomegaly, liver dysfunction and hepatic failure. Adrenal calcification is a striking feature but is present in only about 50% of cases. We report a case of an infant presenting with vomiting, diarrhoea, hepatosplenomegaly and poor weight gain that was subsequently diagnosed as Wolman's disease. He was entered into a clinical trial for LAL replacement therapy. This case reinforces that early onset LAL deficiency should be considered in a baby presenting with failure to thrive, gastrointestinal symptoms and hepatosplenomegaly. PMID:24832708

  19. Enhancing the Acyltransferase Activity of Candida antarctica Lipase A by Rational Design.

    PubMed

    Müller, Janett; Sowa, Miriam A; Fredrich, Birte; Brundiek, Henrike; Bornscheuer, Uwe T

    2015-08-17

    A few lipases, such as Candida antarctica lipase A (CAL-A), are known to possess acyltransferase activity. This enables the enzyme to synthesize fatty acid esters from natural oils and alcohols even in the presence of bulk water. Unfortunately, fatty acids are still formed in these reactions as undesired side-products. To reduce the amount of fatty acids, several CAL-A variants were rationally designed based on its crystal structure. These variants were expressed in Escherichia coli and Pichia pastoris, purified, and their acyltransferase/hydrolase activities were investigated by various biocatalytic approaches. Among the investigated variants, mutant Asp122Leu showed a significant decrease in the hydrolytic activity, thus reducing the side-product yield during acylation. As desired, this variant retained wild-type process-relevant features like pH profile and thermostability. PMID:26058745

  20. Antioxidant property and [Formula: see text]-glucosidase, [Formula: see text]-amylase and lipase inhibiting activities of Flacourtia inermis fruits: characterization of malic acid as an inhibitor of the enzymes.

    PubMed

    Alakolanga, A G A W; Kumar, N Savitri; Jayasinghe, Lalith; Fujimoto, Yoshinori

    2015-12-01

    Flacourtia inermis Roxb. (Flacourtiaceae), is a moderate sized tree cultivated in Sri Lanka for its fruits known as Lovi. The current study was undertaken to study the biological activity of extracts of the fruits in an attempt to increase the value of the under exploited fruit crops. Fruits of F. inermis were found to be rich in phenolics and anthocyanins. Polyphenol content of the fruits was determined to be 1.28 g gallic acid equivalents per 100 g of fresh fruit and anthocyanin content was estimated as 108 mg cyanidin-3-glucoside equivalents per 100 g of fresh fruits. The EtOAc extract showed moderate antioxidant activity in the DPPH radical scavenging assay with IC50 value of 66.2 ppm. The EtOAc and MeOH extracts of the fruits also exhibited inhibitory activities toward α-glucosidase, α-amylase and lipase enzymes with IC50values ranging from 549 to 710 ppm, 1021 to 1949 ppm and 1290 to 2096 ppm, respectively. The active principle for the enzyme inhibition was isolated through activity-guided fractionation and was characterized as (S)-malic acid. The results of this study indicate that F. inermis fruits have the potential to be used in health foods and in nutritional supplements. PMID:26604419

  1. [Structure and Activity of Fungal Lipases in Bile Salt Solutions].

    PubMed

    Bogdanova, L R; Bakirova, D R; Valiullina, Yu A; Idiyatullin, B Z; Faizullin, D A; Zueva, O S; Zuev, Yu F

    2016-01-01

    The changes in structure and catalytic properties of fungal lipases (Candida rugosa, Rhizomucor miehei, Mucor javanicus) were investigated in micellar solutions of bile salts that differ in hydrophilic-lypophilic balance and reaction medium properties. The methods of circular dichroism and tryptophan fluorescence were applied to estimate the changes in peptide structure within complexes with bile salt micelles. Bile salts do not exert a significant influence on the structure of the enzymes under study: in Rh. miehei and M. javanicus lipases the alpha helix content slightly decreased, the influence of bile salts on the C. rugosa structure was not revealed. Despite negligible structural modifications in the enzymes, in bile salt solutions a considerable change in their catalytic properties was observed: an abrupt decrease in catalytic effectiveness. Substrate-bile salts micelles complex formation was demonstrated by the NMR self-diffusion method. The model of a regulation of fungal lipase activity was proposed. PMID:27192825

  2. 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. PMID:25466121

  3. Regulation of hepatic lipase activity by sphingomyelin in plasma lipoproteins.

    PubMed

    Yang, Peng; Subbaiah, Papasani V

    2015-10-01

    Hepatic lipase (HL) is an important enzyme in the clearance of triacylglycerol (TAG) from the circulation, and has been proposed to have pro-atherogenic as well as anti-atherogenic properties. It hydrolyzes both phospholipids and TAG of lipoproteins, and its activity is negatively correlated with HDL levels. Although it is known that HL acts preferentially on HDL lipids, the basis for this specificity is not known, since it does not require any specific apoprotein for activity. In this study, we tested the hypothesis that sphingomyelin (SM), whose concentration is much higher in VLDL and LDL compared to HDL, is an inhibitor of HL, and that this could explain the lipoprotein specificity of the enzyme. The results presented show that the depletion of SM from normal lipoproteins activated the HL roughly in proportion to their SM content. SM depletion stimulated the hydrolysis of both phosphatidylcholine (PC) and TAG, although the PC hydrolysis was stimulated more. In the native lipoproteins, HL showed specificity for PC species containing polyunsaturated fatty acids at sn-2 position, and produced more unsaturated lyso PC species. The enzyme also showed preferential hydrolysis of certain TAG species over others. SM depletion affected the specificity of the enzyme towards PC and TAG species modestly. These results show that SM is a physiological inhibitor of HL activity in lipoproteins and that the specificity of the enzyme towards HDL is at least partly due to its low SM content. PMID:26193433

  4. Hormone-sensitive lipase in differentiated 3T3-L1 cells and its activation by cyclic AMP-dependent protein kinase.

    PubMed Central

    Kawamura, M; Jensen, D F; Wancewicz, E V; Joy, L L; Khoo, J C; Steinberg, D

    1981-01-01

    Differentiation of 3T3-L1 fibroblasts to adipocyte-like cells was accompanied by a 19-fold increase in neutral triglyceride lipase activity, a 12-fold increase in diglyceride lipase activity, a 10-fold increase in monoglyceride lipase activity, and a 280-fold increase in cholesterol esterase activity. In contrast, acid acylhydrolase activities did not increase during differentiation. The rate of glycerol release from unstimulated intact cells increased by more than 1 order of magnitude upon differentiation. Isoproterenol (1 microM) and 1-methyl-3-isobutylxanthine (0.1 mM) further stimulated this rate of glycerol release 3-fold. The neutral triglyceride lipase activity in cell-free preparations of differentiated cells was activated 105% by cyclic AMP-dependent protein kinase. Neutral cholesterol esterase, diglyceride lipase, and monoglyceride lipase were also activated (117%, 10%, and 37+, respectively) by cyclic AMP-dependent protein kinase. In contrast, protein kinase had no effect on any of the four lysosomal acid acylhydrolase activities. Thus, hormone-sensitive lipase, the most characteristic and functionally important enzyme of adipose tissue, has been characterized in differentiated 3T3-L1 cells. The 3T3-L1 cell should be a valuable model system in which to study regulation of hormone-sensitive lipase, particularly its long-term regulation. PMID:6262767

  5. Identification and Characterization of Lipase Activity and Immunogenicity of LipL from Mycobacterium tuberculosis

    PubMed Central

    Cao, Jun; Dang, Guanghui; Li, Huafang; Li, Tiantian; Yue, Zhiguo; Li, Na; Liu, Yajun; Liu, Siguo; Chen, Liping

    2015-01-01

    Lipids and lipid-metabolizing esterases/lipases are highly important for the mycobacterial life cycle and, possibly, for mycobacterial virulence. In this study, we expressed 10 members of the Lip family of Mycobacterium tuberculosis. Among the 10 proteins, LipL displayed a significantly high enzymatic activity for the hydrolysis of long-chain lipids. The optimal temperature for the lipase activity of LipL was demonstrated to be 37°C, and the optimal pH was 8.0. The lipase active center was not the conserved motif G-x-S-x-G, but rather the S-x-x-K and GGG motifs, and the key catalytic amino acid residues were identified as G50, S88, and K91, as demonstrated through site-directed mutagenesis experiments. A three-dimensional modeling structure of LipL was constructed, which showed that the GGG motif was located in the surface of a pocket structure. Furthermore, the subcellular localization of LipL was demonstrated to be on the mycobacterial surface by Western blot analysis. Our results revealed that the LipL protein could induce a strong humoral immune response in humans and activate a CD8+ T cell-mediated response in mice. Overall, our study identified and characterized a novel lipase denoted LipL from M. tuberculosis, and demonstrated that LipL functions as an immunogen that activates both humoral and cell-mediated responses. PMID:26398213

  6. The activities of lipases and carnitine palmitoyl-transferase in muscles from vertebrates and invertebrates

    PubMed Central

    Crabtree, B.; Newsholme, E. A.

    1972-01-01

    1. The activities of tri-, di- and mono-glyceride lipase and carnitine palmitoyltransferase were measured in homogenates of a variety of muscles. These activities were used to estimate the rate of utilization of glycerides and fatty acids by muscle. In muscles whose estimated rates of fat utilization can be compared with rates calculated for the intact muscle from such information as O2 uptake, there is reasonable agreement between the estimated and calculated rates. 2. In all muscles investigated the maximum rates of hydrolysis of glycerides increase in the order triglyceride, diglyceride, monoglyceride. The activity of diglyceride lipase is highest in the flight muscles of insects such as the locust, waterbug and some moths and is lowest in the flight muscles of flies, bees and the wasp. These results are consistent with the utilization of diglyceride as a fuel for some insect flight muscles. 3. In many muscles from both vertebrates and invertebrates the activity of glycerol kinase is similar to that of lipase. It is concluded that in these muscles the metabolic role of glycerol kinase is the removal of glycerol produced during lipolysis. However, in some insect flight muscles the activity of glycerol kinase is much greater than that of lipase, which suggests a different role for glycerol kinase in these muscles. PMID:4664927

  7. Antioxidant and lipase inhibitory activities and essential oil composition of pomegranate peel extracts.

    PubMed

    Hadrich, Fatma; Cher, Slim; Gargouri, Youssef Talel; Adel, Sayari

    2014-01-01

    The chemical composition of essential oil, antioxidant and pancreatic lipase inhibitory activities of various solvent extracts obtained from pomegranate peelTunisian cultivar was evaluated. Gas chromatography/mass spectrometry was used to determine the composition of the PP essential oil. Nine-teen components were identified and the main compounds were the camphor (60.32%) and the benzaldehyde (20.98%). The phenolic and flavonoids content varied from 0 to 290.10 mg Gallic acid equivalent and from 5.2 to 20.43 mg catechin equivalent/g dried extract. The antioxidant activity of various solvent extracts from pomegranate peel was also investigated using various in vitro assays as the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical method, β-carotene bleaching and reducing power assays.Methanol and ethanol extracts showed the most potent antioxidant activity in all assays tested followed by water and acetone extracts. The inhibitory effect of the pomegranate peelextracts on porcine pancreatic lipase was evaluated and the results showed that ethanol and methanol extracts markedly reduced lipase activity. Generally, the highestlipase activity inhibitory (100%) was observed at a concentration of 1 mg/ml after 30 min of incubation. LC-MS analysis of ethanol extract showed the presence of four components which are cholorogenic acid, mannogalloylhexoside, gallic acid and ellagic acid. Our findings demonstrate that the ethanol extract from pomegranate peel might be a good candidate for furtherinvestigations of new bioactive substances. PMID:24770478

  8. Immobilization of active lipase B from Candida antarctica on the surface of polyhydroxyalkanoate inclusions.

    PubMed

    Jahns, Anika C; Rehm, Bernd H A

    2015-04-01

    Polyhydroxyalkanoate (PHA) beads, recombinantly produced in Escherichia coli, were functionalized to display lipase B from Candida antarctica as translational protein fusion. The respective beads were characterized in respect to protein content, functionality, long term storage capacity and re-usability. The direct fusion of the PHA synthase, PhaC, to lipase B yielded active PHA lipase beads capable of hydrolyzing glycerol tributyrate. Lipase B beads showed stable activity over several weeks and re-usability without loss of function. PMID:25407130

  9. 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 %. PMID:17898456

  10. Isolation and characterization of some moderately halophilic bacteria with lipase activity.

    PubMed

    Ghasemi, Y; Rasoul-Amini, S; Kazemi, A; Zarrinic, G; Morowvat, M H; Kargar, M

    2011-01-01

    Lipases are an important class of enzymes which catalyze the hydrolysis of long chain triglycerides and constitute the most prominent group ofbiocatalysts for biotechnological applications. There are a number of lipases, produced by some halophilic microorganisms. In this study, some lipase producing bacteria from Maharlu salt lake located in south of Iran were isolated. All isolates were screened for true lipase activity on plates containing olive oil. The lipase activity was measured using titrimetric methods. Among thirty three isolates, thirteen strains demonstrating orange zone around colonies under UV light, were selected for identification using the molecular methods and some morphological characteristics. The bacterium Bacillus vallismortis BCCS 007 with 3.41 +/- 0.14 U/mL lipase activity was selected as the highest lipase producing isolate. This is the first report of isolation and molecular identification of lipase producing bacteria from Maharlu lake. PMID:22073547

  11. 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. PMID:19560339

  12. 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. PMID:20580809

  13. Inhibition of endothelial lipase activity by sphingomyelin in the lipoproteins.

    PubMed

    Yang, Peng; Belikova, Natalia A; Billheimer, Jeff; Rader, Daniel J; Hill, John S; Subbaiah, Papasani V

    2014-10-01

    Endothelial lipase (EL) is a major determinant of plasma HDL concentration, its activity being inversely proportional to HDL levels. Although it is known that it preferentially acts on HDL compared to LDL and VLDL, the basis for this specificity is not known. Here we tested the hypothesis that sphingomyelin, a major phospholipid in lipoproteins is a physiological inhibitor of EL, and that the preference of the enzyme for HDL may be due to low sphingomyelin/phosphatidylcholine (PtdCho) ratio in HDL, compared to other lipoproteins. Using recombinant human EL, we showed that sphingomyelin inhibits the hydrolysis of PtdCho in the liposomes in a concentration-dependent manner. While the enzyme showed lower hydrolysis of LDL PtdCho, compared to HDL PtdCho, this difference disappeared after the degradation of lipoprotein sphingomyelin by bacterial sphingomyelinase. Analysis of molecular species of PtdCho hydrolyzed by EL in the lipoproteins showed that the enzyme preferentially hydrolyzed PtdCho containing polyunsaturated fatty acids (PUFA) such as 22:6, 20:5, 20:4 at the sn-2 position, generating the corresponding PUFA-lyso PtdCho. This specificity for PUFA-PtdCho species was not observed after depletion of sphingomyelin by sphingomyelinase. These results show that sphingomyelin not only plays a role in regulating EL activity, but also influences its specificity towards PtdCho species. PMID:25167836

  14. 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. PMID:24018659

  15. Enhancement of activity and selectivity of Candida rugosa lipase and Candida antarctica lipase A by bioimprinting and/or immobilization for application in the selective ethanolysis of fish oil.

    PubMed

    Kahveci, Derya; Xu, Xuebing

    2011-10-01

    Candida rugosa lipase (CRL) and Candida antarctica lipase A (CALA) with improved activity and selectivity were prepared for use in organic solvent media. CRL bioimprinted with fatty acids exhibited eightfold enhanced transesterification activity in hexane. Combination of bioimprinting and coating with lecithin or with immobilization did not improve the activity further. CALA was immobilized with and without bioimprinting, none of which improved the activity. All modified lipases were tested for selective ethanolysis of fish oil to concentrate omega-3 polyunsaturated fatty acids (PUFA). None of the preparations, except the immobilized ones catalysed ethanolysis. Immobilized CRL-catalyzed ethanolysis giving 27% (v/v) ethyl esters (EE) in 48 h, of which 43 mol% was oleic acid but no PUFA was detected in the EE fraction. Fatty acid selectivity of CALA was significantly improved by immobilization combined with bioimprinting, resulting in 5.5-fold lower omega-3 PUFA in EE. PMID:21695486

  16. 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%. PMID:23574345

  17. Optimization of biomass production of a mutant of Yarrowia lipolytica with an increased lipase activity using raw glycerol.

    PubMed

    Galvagno, Miguel A; Iannone, Leopoldo J; Bianchi, Jorgelina; Kronberg, Florencia; Rost, Enrique; Carstens, Maria R; Cerrutti, Patricia

    2011-01-01

    The yeast Yarrowia lipolytica accumulates oils and is able to produce extracellular lipases when growing in different carbon sources including glycerol, the principal by-product of the biodiesel industry. In this study, biomass production of a novel mutant strain of Y. lipolytica was statistically optimized by Response Surface Methodology in media containing biodiesel-derived glycerol as main carbon source. This strain exhibited distinctive morphological and fatty acid profile characteristics, and showed an increased extracellular lipase activity. An organic source of nitrogen and the addition of 1.0 g/l olive oil were necessary for significant lipase production. Plackett-Burman and Central Composite Statistical Designs were employed for screening and optimization of fermentation in shaken flasks cultures, and the maximum values obtained were 16.1 g/l for biomass and 12.2 Units/ml for lipase, respectively. Optimized batch bioprocess was thereafter scaled in aerated bioreactors and the values reached for lipase specific activity after 95 % of the glycerol had been consumed, were three-fold higher than those obtained in shaken flasks cultures. A sustainable bioprocess to obtain biomass and extracellular lipase activity was attained by maximizing the use of the by-products of biodiesel industry. PMID:22430997

  18. Uml2 is a novel CalB-type lipase of Ustilago maydis with phospholipase A activity.

    PubMed

    Buerth, Christoph; Kovacic, Filip; Stock, Janpeter; Terfrüchte, Marius; Wilhelm, Susanne; Jaeger, Karl-Erich; Feldbrügge, Michael; Schipper, Kerstin; Ernst, Joachim F; Tielker, Denis

    2014-06-01

    CalB of Pseudozyma aphidis (formerly named Candida antarctica) is one of the most widely applied enzymes in industrial biocatalysis. Here, we describe a protein with 66 % sequence identity to CalB, designated Ustilago maydis lipase 2 (Uml2), which was identified as the product of gene um01422 of the corn smut fungus U. maydis. Sequence analysis of Uml2 revealed the presence of a typical lipase catalytic triad, Ser-His-Asp with Ser125 located in a Thr-Xaa-Ser-Xaa-Gly pentapeptide. Deletion of the uml2 gene in U. maydis diminished the ability of cells to hydrolyse fatty acids from tributyrin or Tween 20/80 substrates, thus demonstrating that Uml2 functions as a lipase that may contribute to nutrition of this fungal pathogen. Uml2 was heterologously produced in Pichia pastoris and recombinant N-glycosylated Uml2 protein was purified from the culture medium. Purified Uml2 released short- and long-chain fatty acids from p-nitrophenyl esters and Tween 20/80 substrates. Furthermore, phosphatidylcholine substrates containing long-chain saturated or unsaturated fatty acids were effectively hydrolysed. Both esterase and phospholipase A activity of Uml2 depended on the Ser125 catalytic residue. These results indicate that Uml2, in contrast to CalB, exhibits not only esterase and lipase activity but also phospholipase A activity. Thus, by genome mining, we identified a novel CalB-like lipase with different substrate specificities. PMID:24469105

  19. 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

  20. Colorimetric assay for heterogeneous-catalyzed lipase activity: enzyme-regulated gold nanoparticle aggregation.

    PubMed

    Zhang, Wei; Tang, Yan; Liu, Jia; Jiang, Ling; Huang, Wei; Huo, Feng-Wei; Tian, Danbi

    2015-01-14

    Lipase is a neglected enzyme in the field of gold nanoparticle-based enzyme assays. This paper reports a novel colorimetric probe to rapidly visualize lipase activities by using Tween 20 functioned GNPs (Tween 20-GNPs) as a reporter. The present strategy hence could overcome the limitations caused by the heterogeneous interface in lipase assay. Catalytic hydrolytic cleavage of the ester bond in Tween 20-GNPs by lipase will trigger the rapid aggregation of GNPs at a high salt solution. The color change from red to purple could be used to sense the activity of lipase. The detection limit (3σ) is as low as 2.8 × 10-2 mg/mL. A preliminary enzyme activity screening was carried out for seven commercially purchased lipase samples. It also has been successfully applied to detecting lipase in fermentation broth of Bacillus subtilis without any pretreatment. PMID:25516269

  1. A convenient test for lipase activity in aqueous-based solutions.

    PubMed

    Guo, Jin; Chen, Cheng-Peng; Wang, Shu-Gen; Huang, Xiao-Jun

    2015-04-01

    We proposed a convenient and accurate method for the measurement of lipase activity in a uniform aqueous-based substrate solution. In this work, lipase from Candida rugosa was used as the model lipase to test its catalytic ability toward p-nitrophenyl palmitate (p-NPP), which was suspended in a mixture of p-NPP ethanol solution and buffer. An ultraviolet-visible spectrophotometer was used to efficiently measure the liberated p-nitrophenol without extraction or centrifugation. Several factors that affected lipase activity were investigated, such as the ratio of p-NPP ethanol solution to buffer, the concentrations of p-NPP and lipase, as well as the temperature, reaction time, pH and agitation rate. Additionally, enzyme catalytic parameters such as Km, Vm and "activation energy" were also assessed. We determined the optimal conditions for lipase in this homogeneous system and demonstrated lipase's catalytic performance in this condition followed Michealis-Menten kinetics. PMID:25765304

  2. The differential hepatic uptake of chylomicron remnants of different fatty acid composition is not mediated by hepatic lipase.

    PubMed

    Lambert, M S; Avella, M A; Berhane, Y; Shervill, E; Botham, K M

    2001-05-01

    The hypothesis that hepatic lipase mediates the differential hepatic uptake of chylomicron remnants of different fatty acid composition, demonstrated in previous work from our laboratory, was tested by investigating the effect of antibodies to the enzyme on the uptake of remnants enriched with saturated or n-3 polyunsaturated fatty acids by the perfused rat liver. After perfusion of rat livers with polyclonal antibodies to rat hepatic lipase raised in rabbits or with rabbit non-immune serum for 15 min, [3H]oleate-labelled chylomicron remnants, derived from chylomicrons of rats given a bolus of either palm (rich in saturated fatty acids) oil or fish (rich in n-3 polyunsaturated fatty acids) oil, were added. The disappearance of radioactivity from the perfusate during 120 min and its recovery in the liver at the end of the experiments were then measured. Although the rabbit anti-rat hepatic lipase antiserum was shown to inhibit hepatic lipase activity by up to 90%, and to bind extensively to hepatic sinusoidal surfaces when added to the perfusate, radioactivity from remnants of chylomicrons from rats given a bolus of fish oil as compared with palm oil disappeared from the perfusate and appeared in the liver more rapidly in the presence both the antiserum and the non-immune serum, and the differences between the uptake of the two types of remnants were similar. We conclude, therefore, that differential interaction with hepatic lipase is not responsible for the differences in the rate of removal of chylomicron remnants of different fatty acid composition from the blood. PMID:11348572

  3. Identification of lipase encoding genes from Antarctic seawater bacteria using degenerate primers: expression of a cold-active lipase with high specific activity.

    PubMed

    Parra, Loreto P; Espina, Giannina; Devia, Javier; Salazar, Oriana; Andrews, Barbara; Asenjo, Juan A

    2015-01-01

    Cold-active enzymes are valuable catalysts showing high activity at low and moderate temperatures and low thermostability. Among cold-active enzymes, lipases offer a great potential in detergent, cosmetic, biofuel and food or feed industries. In this paper we describe the identification of novel lipase coding genes and the expression of a lipase with high activity at low temperatures. The genomic DNA from Antarctic seawater bacteria showing lipolytic activity at 4°C was used to amplify five DNA fragments that partially encode novel lipases using specifically designed COnsensus-DEgenerate Hybrid Oligonucleotide Primers (CODEHOP). All the fragments were found to have a high identity with an α/β-hydrolase domain-containing protein identified by the sequencing of the complete genome of Shewanella frigidimarina NCIMB 400. The complete sequence of one of the lipase-coding gene fragments, lipE13, was obtained by genome walking. Considering that the other fragments had a high identity to the putative lipase from S. frigidimarina NCIMB 400, the complete lipase genes were amplified using oligonucleotide primers designed based on the 5' and 3' regions of the coding sequence of the related protein. This strategy allowed the amplification of 3 lipase-encoding genes of which one was expressed in the periplasm using the Escherichia coli BL21(DE3)/pET-22b(+) expression system. The recombinant protein was obtained with activity toward p-nitrophenyl caproate showing a high specific activity between 15 and 25°C. PMID:25435506

  4. The Inhibition of Lipase and Glucosidase Activities by Acacia Polyphenol

    PubMed Central

    Ikarashi, Nobutomo; Takeda, Rumi; Ito, Kiyomi; Ochiai, Wataru; Sugiyama, Kiyoshi

    2011-01-01

    Acacia polyphenol (AP) extracted from the bark of the black wattle tree (Acacia mearnsii) is rich in unique catechin-like flavan-3-ols, such as robinetinidol and fisetinidol. In an in vitro study, we measured the inhibitory activity of AP on lipase and glucosidase. In addition, we evaluated the effects of AP on absorption of orally administered olive oil, glucose, maltose, sucrose and starch solution in mice. We found that AP concentration-dependently inhibited the activity of lipase, maltase and sucrase with an IC50 of 0.95, 0.22 and 0.60 mg ml−1, respectively. In ICR mice, olive oil was administered orally immediately after oral administration of AP solution, and plasma triglyceride concentration was measured. We found that AP significantly inhibited the rise in plasma triglyceride concentration after olive oil loading. AP also significantly inhibited the rise in plasma glucose concentration after maltose and sucrose loading, and this effect was more potent against maltose. AP also inhibited the rise in plasma glucose concentration after glucose loading and slightly inhibited it after starch loading. Our results suggest that AP inhibits lipase and glucosidase activities, which leads to a reduction in the intestinal absorption of lipids and carbohydrates. PMID:21660093

  5. Structure of product-bound SMG1 lipase: active site gating implications.

    PubMed

    Guo, Shaohua; Xu, Jinxin; Pavlidis, Ioannis V; Lan, Dongming; Bornscheuer, Uwe T; Liu, Jinsong; Wang, Yonghua

    2015-12-01

    Monoacylglycerol and diacylglycerol lipases are industrially interesting enzymes, due to the health benefits that arise from the consumption of diglycerides compared to the traditional triglyceride oils. Most lipases possess an α-helix (lid) directly over the catalytic pocket which regulates the activity of the enzyme. Generally, lipases exist in active and inactive conformations, depending on the positioning of this lid subdomain. However, lipase SMG1, a monoacylglycerol and diacylglycerol specific lipase, has an atypical activation mechanism. In the present study we were able to prove by crystallography, in silico analysis and activity tests that only two positions, residues 102 and 278, are responsible for a gating mechanism that regulates the active and inactive states of the lipase, and that no significant structural changes take place during activation except for oxyanion hole formation. The elucidation of the gating effect provided data enabling the rational design of improved lipases with 6-fold increase in the hydrolytic activity toward diacylglycerols, just by providing additional substrate stabilization with a single mutation (F278N or F278T). Due to the conservation of F278 among the monoacylglycerol and diacylglycerol lipases in the Rhizomucor miehei lipase-like family, the gating mechanism described herein might represent a general mechanism applicable to other monoacylglycerol and diacylglycerol lipases as well. Database: Structural data are available in the Protein Data Bank under the accession numbers 4ZRE (F278D mutant) and 4ZRD (F278N mutant). PMID:26365206

  6. 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

  7. [Lysosomal acid lipase deficiency. Overview of Czech patients].

    PubMed

    Elleder, M; Poupĕtová, H; Ledvinová, J; Hyánek, J; Zeman, J; Sýkora, J; Stozický, F; Chlumská, A; Lohse, P

    1999-11-29

    Lysosomal lipase deficiency is a hereditary autosomal recessive enzymopathy leading to lysosomal storage of triacylglycerols (TAG) and cholesterol esters (CE). In particular cells with a permanently high receptor-mediated LDL endocytosis are affected (liver, kidneys). There are two basic phenotypes. The fatal infantile phenotype (Wolman's disease) with generalized storage of both types of apolar lipids. This form was diagnosed in this country only once. The opposite is the protracted, oligosymptomatic form encountered in all age groups. It is characterized by the storage of CE (which gave this entity the name of cholesteryl storage disease--CESD). Its main sign is affection of the liver (hepatomegaly, hepatopathy), which in some instances may lead to organ failure, directly or after cirrhotic transformation. Furthermore there is permanent hypercholesterolaemia (high LDL cholesterol) due to increased VLDL synthesis by hepatocytes, low HDL cholesterol and variably raised TAG. This constellation of blood lipids is a risk factor for the development of atherosclerosis. In the course of 25 years in the Czech Republic 13 cases of CESD were diagnosed in 11 families. Ten of these cases were characterized by clinically manifest hepatopathy with hepatomegaly, detected incidentally during medical examinations (at the age of 2-14 years). In three adult patients with permanent hypercholesterolaemia the storage process was subclinical and the diagnosis was established quite incidentally by examination of non-specific secondary and tertiary manifestations of the disease. The diagnosis was established in all cases of CESD at the tissue level (liver biopsy), at the biochemical (acid lipase deficiency) and molecular genetic level (mutation in enzyme locus). In all instances mutation of G934A was found leading to reduction and loss of the eighth exon. This mutation was present in five patients in a homozygous state. Six mutations were heterozygous. In one instance for technical

  8. Synthesis of a novel biologically active amide ester of 7,10-dihydroxy-8(E)-octadecanoic acid (DOD) using lipase

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Hydroxy fatty acids (HFA) are known to have industrial potential because of their special properties such as high viscosity and reactivity. Among the hydroxy fatty acids, 7,10-dihydroxy-8(E)-octadecenoic acid (DOD) was successfully produced from oleic acid and lipid containing oleic acid by a bacter...

  9. QSAR study and the hydrolysis activity prediction of three alkaline lipases from different lipase-producing microorganisms

    PubMed Central

    2012-01-01

    The hydrolysis activities of three alkaline lipases, L-A1, L-A2 and L-A3 secreted by different lipase-producing microorganisms isolated from the Bay of Bohai, P. R. China were characterized with 16 kinds of esters. It was found that all the lipases have the ability to catalyze the hydrolysis of the glycerides, methyl esters, ethyl esters, especially for triglycerides, which shows that they have broad substrate spectra, and this property is very important for them to be used in detergent industry. Three QSAR models were built for L-A1, L-A2 and L-A3 respectively with GFA using Discovery studio 2.1. The models equations 1, 2 and 3 can explain 95.80%, 97.45% and 97.09% of the variances (R2adj) respectively while they could predict 95.44%, 89.61% and 93.41% of the variances (R2cv) respectively. With these models the hydrolysis activities of these lipases to mixed esters were predicted and the result showed that the predicted values are in good agreement with the measured values, which indicates that this method can be used as a simple tool to predict the lipase activities for single or mixed esters. PMID:23016923

  10. QSAR study and the hydrolysis activity prediction of three alkaline lipases from different lipase-producing microorganisms.

    PubMed

    Wang, Haikuan; Wang, Xiaojie; Li, Xiaolu; Zhang, Yehong; Dai, Yujie; Guo, Changlu; Zheng, Heng

    2012-01-01

    The hydrolysis activities of three alkaline lipases, L-A1, L-A2 and L-A3 secreted by different lipase-producing microorganisms isolated from the Bay of Bohai, P. R. China were characterized with 16 kinds of esters. It was found that all the lipases have the ability to catalyze the hydrolysis of the glycerides, methyl esters, ethyl esters, especially for triglycerides, which shows that they have broad substrate spectra, and this property is very important for them to be used in detergent industry. Three QSAR models were built for L-A1, L-A2 and L-A3 respectively with GFA using Discovery studio 2.1. The models equations 1, 2 and 3 can explain 95.80%, 97.45% and 97.09% of the variances (R(2)(adj)) respectively while they could predict 95.44%, 89.61% and 93.41% of the variances (R(2)(cv)) respectively. With these models the hydrolysis activities of these lipases to mixed esters were predicted and the result showed that the predicted values are in good agreement with the measured values, which indicates that this method can be used as a simple tool to predict the lipase activities for single or mixed esters. PMID:23016923

  11. [Prediction of lipases types by different scale pseudo-amino acid composition].

    PubMed

    Zhang, Guangya; Li, Hongchun; Gao, Jiaqiang; Fang, Baishan

    2008-11-01

    Lipases are widely used enzymes in biotechnology. Although they catalyze the same reaction, their sequences vary. Therefore, it is highly desired to develop a fast and reliable method to identify the types of lipases according to their sequences, or even just to confirm whether they are lipases or not. By proposing two scales based pseudo amino acid composition approaches to extract the features of the sequences, a powerful predictor based on k-nearest neighbor was introduced to address the problems. The overall success rates thus obtained by the 10-fold cross-validation test were shown as below: for predicting lipases and nonlipase, the success rates were 92.8%, 91.4% and 91.3%, respectively. For lipase types, the success rates were 92.3%, 90.3% and 89.7%, respectively. Among them, the Z scales based pseudo amino acid composition was the best, T scales was the second. They outperformed significantly than 6 other frequently used sequence feature extraction methods. The high success rates yielded for such a stringent dataset indicate predicting the types of lipases is feasible and the different scales pseudo amino acid composition might be a useful tool for extracting the features of protein sequences, or at lease can play a complementary role to many of the other existing approaches. PMID:19256347

  12. Influence of fatty acid on lipase-catalyzed synthesis of ascorbyl esters and their free radical scavenging capacity.

    PubMed

    Stojanović, Marija; Carević, Milica; Mihailović, Mladen; Veličković, Dušan; Dimitrijević, Aleksandra; Milosavić, Nenad; Bezbradica, Dejan

    2015-01-01

    Fatty acid (FA) ascorbyl esters are recently emerging food, cosmetic, and pharmaceutical additives, which can be prepared in an eco-friendly way by using lipases as catalysts. Because they are amphiphilic molecules, which possess high free radical scavenging capacity, they can be applied as liposoluble antioxidants as well as emulsifiers and biosurfactants. In this study, the influence of a wide range of acyl donors on ester yield in lipase-catalyzed synthesis and ester antioxidant activity was examined. Among saturated acyl donors, higher yields and antioxidant activities of esters were achieved when short-chain FAs were used. Oleic acid gave the highest yield overall and its ester exhibited a high antioxidant activity. Optimization of experimental factors showed that the highest conversion (60.5%) in acetone was achieved with 5 g L(-1) of lipase, 50 mM of vitamin C, 10-fold molar excess of oleic acid, and 0.7 mL L(-1) of initial water. Obtained results showed that even short- and medium-chain ascorbyl esters could be synthesized with high yields and retained (or even exceeded) free radical scavenging capacity of l-ascorbic acid, indicating prospects of broadening their application in emulsions and liposomes. PMID:25224149

  13. Comparative fatty acid selectivity of lipases in esterification reactions with glycerol and diol analogues in organic media.

    PubMed

    Lee, C H; Parkin, K L

    2000-01-01

    Reaction selectivity of Pseudomonas cepacia, Rhizomucor miehei, and Candida antarctica B lipases was assessed in multicompetitive esterification reaction mixtures containing an homologous series of n-chain even carbon number fatty acid (FA; C4-C18) substrates and a single alcohol cosubstrate (glycerol, 1,2-propanediol (1,2-PD), or 1, 3-propanediol (1,3-PD)) in tert-butyl methyl ether at water activity of 0.69 or 0.90 and a reaction temperature of 35 degrees C. For P. cepacia lipase, the ordinal patterns of FA selectivities observed were, with glycerol, C8 > C10, C6, C16 > other FA; with 1,2-PD and 1, 3-PD, C16 > C8 > C14 > other FA. For R. miehei lipase, the ordinal patterns of FA selectivities observed were, with glycerol, C8 > C12 > C10, C14 > other FA; with 1,2-PD and 1,3-PD, C8 > C12 > other FA. For C. antarctica B lipase, the ordinal patterns of FA selectivities observed were, with glycerol, C8 > C10, C6, C12 > other FA; with 1, 2-PD, C8 > C10, C6 > other FA; and with 1,3-PD, C8 > C10 > C6 > other FA. The differences in selectivity among FA ranged up to 16-fold, depending upon the lipase and alcohol cosubstrate used. These findings represent intrinsic and substrate-modulated features of FA selectivities that are of particular relevance to the use of lipases for acylglycerol synthesis reactions. PMID:10835238

  14. Pancreas-specific lipase concentrations and amylase and lipase activities in the peritoneal fluid of dogs with suspected pancreatitis.

    PubMed

    Chartier, Marie A; Hill, Steve L; Sunico, Sarena; Suchodolski, Jan S; Robertson, Jane E; Steiner, Joerg M

    2014-09-01

    Diagnosing acute pancreatitis in the dog can be challenging. The aim of this study was to determine the concentrations of pancreas-specific lipase immunoreactivity (cPLI), and the activities of amylase and lipase, in the peritoneal fluid from a population of dogs diagnosed with acute pancreatitis based on clinical signs, ultrasonographic findings and serum cPLI concentrations. In a prospective study, cPLI concentrations, and amylase and lipase activities, were measured in the peritoneal fluid of 14 dogs with pancreatitis and 19 dogs with non-pancreatic disease. The sensitivity and specificity of peritoneal fluid cPLI concentration (cut-off value 500 µg/L) were 100.0% (95% confidence interval, CI, 80.7-100.0%) and 94.7% (95% CI 76.7-99.7%), respectively. The sensitivity and specificity of peritoneal fluid amylase (cut-off value 1050 U/L) and lipase activities (cut-off value 500 U/L) were 71.4% (95% CI 44.5-90.2%) and 84.2% (95% CI 62.8-95.8%) for amylase activity, and 92.9% (95% CI 69.5-99.6%) and 94.7% (95% CI 76.7-99.7%) for lipase activity, respectively. In conclusion, peritoneal fluid cPLI concentration was highly sensitive as a complementary diagnostic tool in a group of dogs with suspected acute pancreatitis. Peritoneal fluid lipase activity was not as sensitive as cPLI concentration, but may also support a diagnosis of acute pancreatitis in dogs. PMID:25106805

  15. Production, characterization, and application of an organic solvent-tolerant lipase present in active inclusion bodies.

    PubMed

    Li, Suxia; Lin, Kang; Pang, Huaiyu; Wu, Yixin; Xu, Jianhe

    2013-01-01

    An organic solvent-tolerant lipase from Serratia marcescens ECU1010 (rSML) was overproduced in Escherichia coli in an insoluble form. High concentrations of both biomass (50 g cell wet weight/L culture broth) and inclusion bodies (10.5 g/L) were obtained by applying a high-cell-density cultivation procedure. Activity assays indicated that the enzymatic activity of rSML reached 600 U/L. After treatment with isopropyl ether for 12 h, the maximum lipase activity reached 6,000 U/L. Scanning electron microscopy and Fourier transform infrared microspectroscopy revealed the activation mechanism of rSML in the presence of organic solvents. rSML was stable in broad ranges of temperatures and pH values, as well as in a series of organic solvents. Besides, rSML showed the best enantioselectivity for the kinetic resolution of (±)-trans-3-(4-methoxyphenyl)glycidic acid methyl ester. These features render the S. marcescens ECU1010 lipase attractive for biotechnological applications in the field of organic synthesis and pharmaceutical industry. PMID:23269633

  16. Lipase-catalyzed ethanolysis of milk fat with a focus on short-chain fatty acid selectivity.

    PubMed

    Lubary, Marta; ter Horst, Joop H; Hofland, Gerard W; Jansens, Peter J

    2009-01-14

    Mixtures of fatty acid ethyl esters were produced by lipase-catalyzed ethanolysis of milk fat triglycerides. Three commercial immobilized lipases (Lipozyme TL, Lipozyme RM, and Novozym 435) were tested in different reaction conditions with the aim of maximizing the conversion of the short-chain fatty acid fraction of milk fat to flavor ethyl esters. The influence of the reactants molar ratio was investigated, as well as three different reaction media, that is, hexane, CO(2)-expanded liquid (GXL), and the solvent-free mixture. Novozym 435 showed the highest activity in all conditions. This lipase also exhibited selectivity for short-chain fatty acids, which, at short reaction times, resulted in a product mixture richer in short-chain fatty acids than the original milk fat. The highest selectivities were obtained in hexane and in CO(2)-expanded liquid fat, at low ethanol to fat ratios. Using dense CO(2) as the reaction cosolvent is attractive because it results in the largest short-chain fatty acid enrichment in the product mixture, while leaving no residues in the product. PMID:19072544

  17. Screening of microbial lipases and evalutaion of their potential to produce glycerides with high gamma linolenic acid concentration.

    PubMed

    Fregolente, Patricia B L; Fregolente, Leonardo V; Maciel, Maria R W; Carvalho, Patricia O

    2009-10-01

    Gamma linolenic acid (GLA, 18:3, cis- 6,9,12- octadecatrienoic acid), an important compound in n- 6 eicosanoid family biosynthesis, occurs in the lipids of a few plant and microbial sources. This study focused on the screening of microbial strains with suitable lipase activity for enrichment of GLA by selective hydrolysis of the borage oil (21.6 % of GLA/total fatty acids). Firstly, 352 microrganisms were tested for their lipolytic capacity using screening techniques on agar plates containing borage oil, strains were then selected and screened for their activity (U/mg) using both submerged fermentation (SmF) and solid state fermentation (SSF). The rate of hydrolysis and the selective preference of these hydrolytic enzymes towards fatty acids, with a special focus on enrichment of GLA were studied and compared with those obtained by two commercially-available lipases. Only one of the lipases tested during this study displayed selectivity, discriminating the GLA during the hydrolysis reaction. Using the enzymatic extract from Geotrichum candidum as a biocatalyst of the reaction, it was possible to obtain a percentage of 41.7% of GLA in acylglycerols fraction when the borage oil was treated in a fixed-bed reactor for 24 hours at 30ºC. PMID:24031421

  18. Investigation of the Reuse of Immobilized Lipases in Biodiesel Synthesis: Influence of Different Solvents in Lipase Activity.

    PubMed

    Aguieiras, Erika C G; Ribeiro, Douglas S; Couteiro, Pedro P; Bastos, Caenam M B; de Queiroz, Danielle S; Parreira, Juliana M; Langone, Marta A P

    2016-06-01

    Biodiesel production catalyzed by immobilized lipases offers the possibility of easy reuse of the catalyst, which is very important to minimize costs and to make this process economically feasible. In this study, the reuse of three commercial immobilized lipases (Novozym 435, Lipozyme RM IM, and Lipozyme TL IM) was investigated in ethanolysis of soybean oil. The effect of the use of solvents (ethanol, butanol, and hexane) to wash the immobilized lipases before the enzyme reuse was evaluated, as well as the lipase reuse without solvent washing. The washing with butanol and ethanol led to the lowest decrease in ester yield after the first batch and allowed the highest glycerol removal (>85 %) from biocatalysts. The biocatalysts were incubated at 50 °C for 2 h in these three solvents. Esterification activities of the enzyme preparations, scanning electron microscopy (SEM) analyses of the beads, and protein content in organic phase were evaluated before and after incubation in the solvent. SEM analysis showed a significant change in beads morphology of Novozym 435 after contact with hexane. For Lipozyme TL IM lipase, this effect was visualized with ethanol. PMID:26883757

  19. A novel oriented immobilized lipase on magnetic nanoparticles in reverse micelles system and its application in the enrichment of polyunsaturated fatty acids.

    PubMed

    Liu, Tao; Zhao, Yuandi; Wang, Xiaofeng; Li, Xiang; Yan, Yunjun

    2013-03-01

    A novel oriented immobilized lipase was derived from Yarrowia lipolytica lipase LIP2 covalently immobilized on functionalized Fe3O4 magnetic nanoparticles (MNPs) in reverse micelles system (RMS). The activity recovery reached 382% compared with 29% in aqueous phase, and further ran up to 1425% under optimum conditions. (3-Aminopropyl) triethoxysilane (APTES) coated Fe3O4 nanoparticles were characterized by Fourier transform infrared (FT-IR) and X-ray diffraction (XRD). A significant alteration in the secondary structure of the lipase in RMS with a 15.5% increase of α-helix content and a 12.5% decrease of β-sheet content was detected by circular dichroism (CD). The immobilized lipase was employed to enrich polyunsaturated fatty acids in fish oil, a 90% increase of DHA content was obtained after 12h, and after 20 cycles of successive usage, it still remained over 80% of relative hydrolysis degree, which shows a good recyclability. PMID:23395761

  20. 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. PMID:25529980

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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) (...

  2. Potential of essential fatty acid deficiency with extremely low fat diet in lipoprotein lipase deficiency during pregnancy: A case report

    PubMed Central

    Tsai, Elaine C; Brown, Judy A; Veldee, Megan Y; Anderson, Gregory J; Chait, Alan; Brunzell, John D

    2004-01-01

    Background Pregnancy in patients with lipoprotein lipase deficiency is associated with high risk of maternal pancreatitis and fetal death. A very low fat diet (< 10% of calories) is the primary treatment modality for the prevention of acute pancreatitis, a rare but potentially serious complication of severe hypertriglyceridemia. Since pregnancy can exacerbate hypertriglyceridemia in the genetic absence of lipoprotein lipase, a further reduction of dietary fat intake to < 1–2% of total caloric intake may be required during the pregnancy, along with the administration of a fibrate. It is uncertain if essential fatty acid deficiency will develop in the mother and fetus with this extremely low fat diet, or whether fibrates will cross the placenta and concentrate in the fetus. Case presentation A 23 year-old gravida 1 woman with primary lipoprotein lipase deficiency was seen at 7 weeks of gestation in the Lipid Clinic for management of severe hypertriglyceridemia that had worsened with pregnancy. While on her habitual fat intake of 10% of total calories, her pregnancy resulted in an exacerbation of the hypertriglyceridemia, which prompted further restriction of fat intake to < 2% of total calories, as well as administration of gemfibrozil at a lower than average dose. The level of gemfibrozil, as the active metabolite, in the venous and arterial fetal cord blood was within the expected therapeutic range for adults. The clinical signs and a biomarker of essential fatty acid deficiency, namely the ratio of 20:3 [n-9] to 20:4 [n-6] fatty acids, were closely monitored throughout her pregnancy. Despite her extremely low fat diet, the levels of essential fatty acids measured in the mother and in the fetal blood immediately postpartum were normal. Normal essential fatty acid levels may have been achieved by the topical application of sunflower oil. Conclusions An extremely low fat diet in combination with topical sunflower oil and gemfibrozil administration was safely

  3. Interesterification activity of Rhizopus delemar lipase in phospholipid microemulsions.

    PubMed

    Komatsu, Takayoshi; Nagayama, Kazuhito; Imai, Masanao

    2004-11-15

    The interesterification of olive oil with palmitic acid catalyzed by Rhizopus delemar lipase was investigated in phospholipid microemulsion systems. Soybean lecithin was used as the amphiphilic component. The maximal reaction rate was obtained at a buffer pH of 5.5-6.0. The reaction rate was also dependent on the W(L) (= [H2O]/[lecithin]) value and attained a maximum at W(L)=5. The reaction rate reached a maximum at a palmitic acid concentration of 350 mM. The molar fraction of the interesterified product 1,3-dipalmitoyl-3-oleoyl glycerol (POP) in the olive oil was enhanced from 2.8 to 65.6 mol% after 24 h of the reaction. PMID:15542321

  4. Plant lipases: biocatalyst aqueous environment in relation to optimal catalytic activity in lipase-catalyzed synthesis reactions.

    PubMed

    Caro, Yanis; Pina, Michel; Turon, Fabrice; Guilbert, Stephane; Mougeot, Estelle; Fetsch, David V; Attwool, Philip; Graille, Jean

    2002-03-20

    Adsorption and desorption isotherms of two commercial enzyme preparations of papain and bromelain were determined with a Dynamic Vapor System. The Guggenheim-Anderson-deBoer (GAB) modeling of the obtained sorption isotherms allowed the definition of different levels of hydration of those samples. Afterward, these enzyme preparations were used as biocatalysts in water and solvent-free esterification and alcoholysis reactions. The evolution of the obtained fatty acid ester level as a function of the initial hydration level of the biocatalyst, i.e., thermodynamic water activity (a(w)) and water content, was studied. The results show an important correlation between the initial hydration level of the biocatalyst and its catalytic activity during the lipase-catalyzed synthesis reactions. Thus, the Carica papaya lipase (crude papain preparation) catalytic activity is highly dependent on the biocatalyst hydration state. The optimized synthesis reaction yield is obtained when the a(w) value of the enzyme preparation is stabilized at 0.22, which corresponds to 2% water content. This optimal level of hydration occurs on the linear part of the biocatalyst's sorption isotherm, where the water molecules can form a mono- or multiple layer with the protein network. The synthesis reaction yield decreases when the a(w) of the preparation is higher than 0.22, because the excess water molecules modify the system equilibrium leading to the reverse and competitive reaction, i.e., hydrolysis. These results show also that an optimal storage condition for the highly hydrophilic crude papain preparation is a relative humidity strictly lower than 70% to avoid an irreversible structural transition leading to a useless biocatalyst. Concerning the bromelain preparation, no effect of the hydration level on the catalytic activity during esterification reactions was observed. This biocatalyst has too weak a catalytic activity which makes it difficult to observe any differences. Furthermore, the

  5. An activity-based probe for high-throughput measurements of triacylglycerol lipases.

    PubMed

    Tam, John; Henault, Martin; Li, Lianhai; Wang, Zhaoyin; Partridge, Anthony W; Melnyk, Roman A

    2011-07-15

    Modulating the activity of lipases involved in the metabolism of plasma lipoproteins is an attractive approach for developing lipid raising/lowering therapies to treat cardiovascular disease. Identifying small molecule inhibitors for these membrane-active enzymes, however, is complicated by difficulties associated with measuring lipase activity and inhibition at the water-membrane interface; substrate and compound dynamics at the particle interface have the potential to confound data interpretation. Here, we describe a novel ELISA-based lipase activity assay that employs as "bait" a biotinylated active-site probe that irreversibly binds to the catalytic active-site serine of members of the triacylglycerol lipase family (hepatic lipase, lipoprotein lipase, and endothelial lipase) in solution with high affinity. Detection of "captured" (probe-enzyme) complexes on streptavidin-coated plates using labeled secondary antibodies to specific primary antibodies offers several advantages over conventional assays, including the ability to eliminate enzyme-particle and compound-particle effects; specifically measure lipase activity in complex mixtures in vitro; preferentially identify active-site-directed inhibitors; and distinguish between reversible and irreversible inhibitors through a simple assay modification. Using EL as an exemplar, we demonstrate the versatility of this assay both for high-throughput screening and for compound mechanism-of-action studies. PMID:21397586

  6. Production of high-oleic acid tallow fractions using lipase-catalyzed directed interesterification, using both batch and continuous processing.

    PubMed

    MacKenzie; Stevenson

    2000-08-01

    Immobilized lipases were used to catalyze batch-directed interesterification of tallow, resulting in oleins containing significantly higher levels of unsaturated fatty acids than obtained by fractionation without lipase. After 14 days, a reaction catalyzed by 2% Novozym 435 yielded 57% olein unsaturation, compared with 45% in a no-enzyme control. Free fatty acid levels increased to 2-3% during reactions. Incubation of the enzyme in multiple batches of melted fat caused a gradual loss of interesterification activity, apparently due to progressive dehydration. The activity could be restored by addition of water to the reaction medium. Immobilized lipase was also used to catalyze directed interesterification in a continuous flow reactor. Melted tallow was circulated through a packed bed enzyme reactor and a separate crystallization vessel. The temperatures of the two parts of the apparatus were controlled separately to allow crystallization to occur separately from interesterification. Operation of the reactor with conventionally dry, prefractionated tallow allowed the formation of an olein consisting of up to 60% unsaturated fatty acids. The greatest changes in olein fatty acid composition were achieved when the fractionation temperature was kept constant at a value that promoted selective crystallization of trisaturated triglycerides that were continuously produced by enzymic interesterification. The enzyme could be reused without apparent loss of activity, and its activity was apparently enhanced by preincubation in melted tallow for up to several days. Control of both the water activity of the enzyme and tallow feedstock and of the absorption of atmospheric water vapor were required to maintain enzyme activity, during multiple reuse and minimize free fatty acid formation. This method may form the basis for a process to produce highly mono-unsaturated tallow fractions for use in food applications (e.g. frying) where a "healthy" low saturated fat product is required

  7. Release of endothelial cell lipoprotein lipase by plasma lipoproteins and free fatty acids

    SciTech Connect

    Saxena, U.; Witte, L.D.; Goldberg, I.J.

    1989-03-15

    Lipoprotein lipase (LPL) bound to the lumenal surface of vascular endothelial cells is responsible for the hydrolysis of triglycerides in plasma lipoproteins. Studies were performed to investigate whether human plasma lipoproteins and/or free fatty acids would release LPL which was bound to endothelial cells. Purified bovine milk LPL was incubated with cultured porcine aortic endothelial cells resulting in the association of enzyme activity with the cells. When the cells were then incubated with media containing chylomicrons or very low density lipoproteins (VLDL), a concentration-dependent decrease in the cell-associated LPL enzymatic activity was observed. In contrast, incubation with media containing low density lipoproteins or high density lipoproteins produced a much smaller decrease in the cell-associated enzymatic activity. The addition of increasing molar ratios of oleic acid:bovine serum albumin to the media also reduced enzyme activity associated with the endothelial cells. To determine whether the decrease in LPL activity was due to release of the enzyme from the cells or inactivation of the enzyme, studies were performed utilizing radioiodinated bovine LPL. Radiolabeled LPL protein was released from endothelial cells by chylomicrons, VLDL, and by free fatty acids (i.e. oleic acid bound to bovine serum albumin). The release of radiolabeled LPL by VLDL correlated with the generation of free fatty acids from the hydrolysis of VLDL triglyceride by LPL bound to the cells. Inhibition of LPL enzymatic activity by use of a specific monoclonal antibody, reduced the extent of release of /sup 125/I-LPL from the endothelial cells by the added VLDL. These results demonstrated that LPL enzymatic activity and protein were removed from endothelial cells by triglyceride-rich lipoproteins (chylomicrons and VLDL) and oleic acid.

  8. Cloning, Expression, and Characterization of a Cold-Active and Organic Solvent-Tolerant Lipase from Aeromicrobium sp. SCSIO 25071.

    PubMed

    Su, Hongfei; Mai, Zhimao; Yang, Jian; Xiao, Yunzhu; Tian, Xinpeng; Zhang, Si

    2016-06-28

    The gene encoding lipase (Lip98) from Aeromicrobium sp. SCSIO 25071 was cloned and functionally expressed in Escherichia coli. Lip98 amino acid sequence shares the highest (49%) identity to Rhodococcus jostii RHA1 lipase and contains a novel motif (GHSEG), which is different from other clusters in the lipase superfamily. The recombinant lipase was purified to homogeneity with Ni-NTA affinity chromatography. Lip98 showed an apparent molecular mass of 30 kDa on SDS gel. The optimal temperature and pH value for enzymatic activity were recorded at 30°C and 7.5, respectively. Lip98 exhibited high activity at low temperatures with 35% maximum activity at 0°C and good stability at temperatures below 35°C. Its calculated activation energy was 4.12 kcal/mol at the low temperature range of 15-30°C. Its activity was slightly affected by some metal ions such as K(+), Ca(2+), and Na(+). The activity of Lip98 was increased by various organic solvents such as DMSO, ethanol, acetone, and hexane with the concentration of 30% (v/v) and retained more than 30% residual activity in neat organic solvent. The unique characteristics of Lip98 imply that it is a promising candidate for industrial application as a nonaqueous biocatalyst and food additive. PMID:26975765

  9. Effect of plant oils upon lipase and citric acid production in Yarrowia lipolytica yeast.

    PubMed

    Darvishi, Farshad; Nahvi, Iraj; Zarkesh-Esfahani, Hamid; Momenbeik, Fariborz

    2009-01-01

    The nonconventional yeast Yarrowia lipolytica degrades very efficiently hydrophobic substrates to produce organic acids, single-cell oil, lipases, and so forth. The aim of this study was to investigate the biochemical behavior and simultaneous production of valuable metabolites such as lipase, citric acid (CA), and single-cell protein (SCP) by Yarrowia lipolytica DSM 3286 grown on various plant oils as sole carbon source. Among tested plant oils, olive oil proved to be the best medium for lipase and CA production. The Y. lipolytica DSM 3286 produced 34.6 +/- 0.1 U/mL of lipase and also CA and SCP as by-product on olive oil medium supplemented with yeast extract. Urea, as organic nitrogen, was the best nitrogen source for CA production. The results of this study suggest that the two biotechnologically valuable products, lipase and CA, could be produced simultaneously by this strain using renewable low-cost substrates such as plant oils in one procedure. PMID:19826636

  10. Effect of Plant Oils upon Lipase and Citric Acid Production in Yarrowia lipolytica Yeast

    PubMed Central

    Darvishi, Farshad; Nahvi, Iraj; Zarkesh-Esfahani, Hamid; Momenbeik, Fariborz

    2009-01-01

    The nonconventional yeast Yarrowia lipolytica degrades very efficiently hydrophobic substrates to produce organic acids, single-cell oil, lipases, and so forth. The aim of this study was to investigate the biochemical behavior and simultaneous production of valuable metabolites such as lipase, citric acid (CA), and single-cell protein (SCP) by Yarrowia lipolytica DSM 3286 grown on various plant oils as sole carbon source. Among tested plant oils, olive oil proved to be the best medium for lipase and CA production. The Y. lipolytica DSM 3286 produced 34.6 ± 0.1 U/mL of lipase and also CA and SCP as by-product on olive oil medium supplemented with yeast extract. Urea, as organic nitrogen, was the best nitrogen source for CA production. The results of this study suggest that the two biotechnologically valuable products, lipase and CA, could be produced simultaneously by this strain using renewable low-cost substrates such as plant oils in one procedure. PMID:19826636

  11. The Influence of Different Physicochemical Parameters of the Solvent On The Activity and Selectivity of Lipase.

    PubMed

    Gao, Xiu-Gong; Cao, Shu-Gui; Zhang, Ke-Chang

    1997-01-01

    The asymmetric esterification of octanoic acid with racemic 2-octanol catalyzed by a lipase from Pseudomonas sp. was investigated in several typical solvents. It was found that the catalytic activity and the enantioselectivity of the enzyme were governed by different physicochemical parameters of the solvent employed. While the former depended on the hydrophobicity (lgP) of the solvent, by contrast the latter was a function of the dielectric constant and the dipole moment. A mechanistic model for the binding site of the enzyme was postulated to rationalize this phenomenon based on the results of the kinetic studies of the reaction in some representative solvents. PMID:12215797

  12. Isolation and biochemical characterization of Bacillus pumilus lipases from the Antarctic.

    PubMed

    Arifin, Arild Ranlym; Kim, Soon-Ja; Yim, Joung Han; Suwanto, Antonius; Kim, Hyung Kwoun

    2013-05-01

    Lipase-producing bacterial strains were isolated from Antarctic soil samples using the tricaprylin agar plate method. Seven strains with relatively strong lipase activities were selected. All of them turned out to be Bacillus pumilus strains by the 16S rRNA gene sequence analysis. Their corresponding lipase genes were cloned, sequenced, and compared. Finally, three different Bacillus pumilus lipases (BPL1, BPL2, and BPL3) were chosen. Their amino acid sequence identities were in the range of 92-98% with the previous Bacillus pumilus lipases. Their optimum temperatures and pHs were measured to be 40 degrees C and pH 9. Lipase BPL1 and lipase BPL2 were stable up to 30 degrees C, whereas lipase BPL3 was stable up to 20 degrees C. Lipase BPL2 was stable within a pH range of 6-10, whereas lipase BPL1 and lipase BPL3 were stable within a pH range of 5-11, showing strong alkaline tolerance. All these lipases exhibited high hydrolytic activity toward pnitrophenyl caprylate (C8). In addition, lipase BPL1 showed high hydrolytic activity toward tributyrin, whereas lipase BPL2 and lipase BPL3 hydrolyzed tricaprylin and castor oil preferentially. These results demonstrated that the three Antarctic Bacillus lipases were alkaliphilic and had a substrate preference toward short- and mediumchain triglycerides. These Antarctic Bacillus lipases might be used in detergent and food industries. PMID:23648856

  13. 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)

  14. Mechanism for release of arachidonic acid during guinea pig platelet aggregation: a role for the diacylglycerol lipase inhibitor RHC 80267

    SciTech Connect

    Amin, D.

    1986-01-01

    The mechanism of the release of arachidonic acid from phospholipids after the stimulation of guinea pig platelets with collagen, thrombin and platelet activating factor (PAF) was studied. RHC 80267, a diacylglycerol lipase inhibitor, and indomethacin, a cyclooxygenase inhibitor, were used. Various in vitro assays for enzymes involved in arachidonic acid release and metabolism were conducted. Platelet aggregation and simultaneous release of ADP from platelets were monitored using a Chrono-log Lumiaggregometer. Platelets were labeled with (/sup 14/C)arachidonic acid to facilitate sensitive determination of small changes in platelet phospholipids during platelet aggregation. In the present investigation it is shown that collagen, thrombin and PAF increased phospholipase C activity. It was also discovered that cyclooxygenase products were responsible for further stimulation (a positive feed-back) of phospholipase C activity, while diacylglycerol provided a negative feed-back control over receptor-stimulated phospholipase C activity and inhibited ADP release. The guinea pig platelet is an ideal model to study phospholipase C-diacylglycerol lipase pathway for the release of arachidonic acid from platelet phospholipids because it does not have any phospholipase A/sub 2/ activity. It was observed that cyclooxygenase products were responsible for collagen-induced guinea pig platelet aggregation. Indomethacin completely inhibited collagen-induced platelet aggregation, was less effective against thrombin, and had no effect on PAF-induced platelet aggregation. On the other hand, RHC 80267 was a powerful inhibitor of aggregation and ADP release induced by all three of these potent aggregating agents.

  15. 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. PMID:7870347

  16. Oscillatory changes in muscle lipoprotein lipase activity of fed and starved rats.

    PubMed

    Kotlar, T J; Borensztajn, J

    1977-10-01

    Lipoprotein lipase activity was measured at short time intervals in cardiac and skeletal muscles of normal and streptozotocin-treated diabetic rats fed ad libitum or deprived of food. In normal animals fed ad libitum, lipoprotein lipase activities of heart, diaphragm, soleus, and fast-twitch red fibers of the quadriceps muscle showed rhythmic oscillations that appeared to coincide with the nocturnal feeding habits of the animals. During the day (7 A.M. to 7 P.M.), when food consumption by the rats was greatly reduced, lipoprotein lipase activity in all muscles increased, followed by a decline to basal levels during the night. Similar oscillatory changes in lipoprotein lipase activity were observed in the muscles of diabetic rats fed ad libitum. In normal rats deprived of food, however, the oscillatory changes in muscle lipoprotein lipase activity were not abolished and persisted for at least 48 h. In diabetic rats starved during a 48-h period, the oscillatory changes in muscle lipoprotein lipase activity were markedly altered. In all animals, muscle lipoprotein lipase activities were not correlated to plasma glucagon levels. PMID:143895

  17. Enhanced catalysis and enantioselective resolution of racemic naproxen methyl ester by lipase encapsulated within iron oxide nanoparticles coated with calix[8]arene valeric acid complexes.

    PubMed

    Sayin, Serkan; Akoz, Enise; Yilmaz, Mustafa

    2014-09-14

    In this study, two types of nanoparticles have been used as additives for the encapsulation of Candida rugosa lipase via the sol-gel method. In one case, the nanoparticles were covalently linked with a new synthesized calix[8]arene octa valeric acid derivative (C[8]-C4-COOH) to produce new calix[8]arene-adorned magnetite nanoparticles (NP-C[8]-C4-COOH), and then NP-C[8]-C4-COOH was used as an additive in the sol-gel encapsulation process. In the other case, iron oxide nanoparticles were directly added into the sol-gel encapsulation process in order to interact electrostatically with both C[8]-C4-COOH and Candida rugosa lipase. The catalytic activities and enantioselectivities of two novel encapsulated lipases (Enc-NP-C[8]-C4-COOH and Enc-C[8]-C4-COOH@Fe3O4) in the hydrolysis reaction of racemic naproxen methyl ester were evaluated. The results showed that the activity and enantioselectivity of the lipase were improved when the lipase was encapsulated in the presence of calixarene-based additives. Indeed, the encapsulated lipases have an excellent rate of enantioselectivity, with E = 371 and 265, respectively, as compared to the free enzyme (E = 137). The lipases encapsulated with C[8]-C4-COOH and iron oxide nanoparticles (Enc-C[8]-C4-COOH@Fe3O4) retained more than 86% of their initial activities after 5 repeated uses and 92% with NP-C[8]-C4-COOH. PMID:25012138

  18. An interactive study of influential parameters for shikimic acid production using statistical approach, scale up and its inhibitory action on different lipases.

    PubMed

    Rawat, Garima; Tripathi, Priyanka; Yadav, Sweta; Saxena, R K

    2013-09-01

    Shikimic acid is the promising candidate as a building block for the industrial synthesis of drug Tamiflu used for the treatment of Swine flu. The fermentative production process using microbes present an excellent and even more sustainable alternative to the traditional plants based extraction methods. In the present study, the fermentative production of shikimic acid by Citrobacter freundii GR-21 (KC466031) was optimized by process engineering using a statistical modeling approach and a maximum amount of 16.78 g L(-1) was achieved. The process was also scaled up to 14L bioreactor to validate the production of shikimic acid. Further, the potential of anti-enzymatic nature of purified shikimic acid was evaluated for different lipases wherein, shikimic acid inhibited the hydrolysis of triglycerides by 55-60%. Shikimic acid also profoundly inhibited pancreatic lipase activity by 66%, thus providing another valuable therapeutic aspect for treating diet induced obesity in humans. PMID:23871288

  19. 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. PMID:26672452

  20. Purification and properties of a phospholipase A2/lipase preferring phosphatidic acid, bis(monoacylglycerol) phosphate, and monoacylglycerol from rat testis.

    PubMed

    Ito, Masafumi; Tchoua, Urbain; Okamoto, Mitsuhiro; Tojo, Hiromasa

    2002-11-15

    Phospholipase A(2) (PLA(2)) was purified to homogeneity from the supernatant fraction of rat testis homogenate. The purified 63-kDa enzyme did not require Ca(2+) ions for activity and exhibited both phosphatidic acid-preferring PLA(2) and monoacylglycerol lipase activities with a modest specificity toward unsaturated acyl chains. Anionic detergents enhanced these activities. Serine-modifying irreversible inhibitors, (p-amidinophenyl) methanesulfonyl fluoride and methylarachidonyl fluorophosphonate, inhibited both activities to a similar extent, indicating a single active site is involved in PLA(2) and lipase activities. The sequence of NH(2)-terminal 12 amino acids of purified enzyme was identical to that of a carboxylesterase from rat liver. The optimal pH for PLA(2) activity (around 5.5) differed from that for lipase activity (around 8.0). At pH 5.5 the enzyme also hydrolyzed bis(monoacylglycerol) phosphate, or lysobisphosphatidic acid (LBPA), that has been hitherto known as a secretory PLA(2)-resistant phospholipid and a late endosome marker. LBPA-enriched fractions were prepared from liver lysosome fractions of chloroquine-treated rats, treated with excess of pancreatic PLA(2), and then used for assaying LBPA-hydrolyzing activity. LBPA and the reaction products were identified by microbore normal phase high performance liquid chromatography/electrospray ionization ion-trap mass spectrometry. These enzymatic properties suggest that the enzyme can metabolize phosphatidic and lysobisphosphatidic acids in cellular acidic compartments. PMID:12223468

  1. 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

  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. Immobilized lipase from Schizophyllum commune ISTL04 for the production of fatty acids methyl esters from cyanobacterial oil.

    PubMed

    Singh, Jyoti; Singh, Manoj Kumar; Kumar, Madan; Thakur, Indu Shekhar

    2015-01-01

    Novel lipase from model mushroom Schizophyllum commune strain ISTL04 produced by solid state fermentation of Leucaena leucocephala seeds, was immobilized onto Celite for enzymatic FAMEs production from cyanobacterial endolith Leptolyngbya ISTCY101. The isolate showed vigorous growth and produced remarkable lipase activity of 146.5 U g(-1) dry solid substrate, without any external lipase inducer. Single-factor experiments were carried out to study the effects of various reaction parameters on the FAMEs yield. The best conditions for enzymatic transesterification as revealed by the results were: 1:3 oil to methanol molar ratio, added at 3h intervals, 12% water content, 1581.5 U g(-1) immobilized lipase, temperature 45 °C, and time 24h. Under these conditions, the maximum FAMEs yield reached 94%. The immobilized lipase was able to produce >90% of the relative FAMEs yield after four repeated transesterification cycles. This immobilized lipase exhibited potential for application in biodiesel industry. PMID:25670399

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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. Effect of temperature on Candida antartica lipase B activity in the kinetic resolution of acebutolol

    NASA Astrophysics Data System (ADS)

    Rajin, Mariani; Kamaruddin, A. H.

    2016-06-01

    Thermodynamic studies of free Candida antartica lipase B in kinetic resolution of acebutolol have been carried out to characterize the temperature effects towards enzyme stability and activity. A decreased in reaction rate was observed in temperature above 40oC. Thermodynamic studies on lipase deactivation exhibited a first-order kinetic pattern. The activation and deactivation energies were 39.63 kJ/mol and 54.90 kJ/mol, respectively. The enthalpy and entropy of the lipase deactivation were found to be 52.12 kJ/mol and -0.18 kJ/mol, respectively.

  6. Biochemical properties of a new cold-active mono- and diacylglycerol lipase from marine member Janibacter sp. strain HTCC2649.

    PubMed

    Yuan, Dongjuan; Lan, Dongming; Xin, Ruipu; Yang, Bo; Wang, Yonghua

    2014-01-01

    Mono- and di-acylglycerol lipase has been applied to industrial usage in oil modification for its special substrate selectivity. Until now, the reported mono- and di-acylglycerol lipases from microorganism are limited, and there is no report on the mono- and di-acylglycerol lipase from bacteria. A predicted lipase (named MAJ1) from marine Janibacter sp. strain HTCC2649 was purified and biochemical characterized. MAJ1 was clustered in the family I.7 of esterase/lipase. The optimum activity of the purified MAJ1 occurred at pH 7.0 and 30 °C. The enzyme retained 50% of the optimum activity at 5 °C, indicating that MAJ1 is a cold-active lipase. The enzyme activity was stable in the presence of various metal ions, and inhibited in EDTA. MAJ1 was resistant to detergents. MAJ1 preferentially hydrolyzed mono- and di-acylglycerols, but did not show activity to triacylglycerols of camellia oil substrates. Further, MAJ1 is low homologous to that of the reported fungal diacylglycerol lipases, including Malassezia globosa lipase 1 (SMG1), Penicillium camembertii lipase U-150 (PCL), and Aspergillus oryzae lipase (AOL). Thus, we identified a novel cold-active bacterial lipase with a sn-1/3 preference towards mono- and di-acylglycerides for the first time. Moreover, it has the potential, in oil modification, for special substrate selectivity. PMID:24927145

  7. Activity of Lipase and Chitinase Immobilized on Superparamagnetic Particles in a Rotational Magnetic Field

    PubMed Central

    Mizuki, Toru; Sawai, Miyuki; Nagaoka, Yutaka; Morimoto, Hisao; Maekawa, Toru

    2013-01-01

    We immobilize hydrolases such as lipase and chitinase on superparamagnetic particles, which are subjected to a rotational magnetic field, and measure the activities of the enzymes. We find that the activities of lipase and chitinase increase in the rotational magnetic field compared to those in the absence of a magnetic field and reach maximum at certain frequencies. The present methodology may well be utilized for the design and development of efficient micro reactors and micro total analysis systems (μ-TASs). PMID:23799111

  8. 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

  9. Interfacial activation of Candida antarctica lipase B: combined evidence from experiment and simulation

    PubMed Central

    Zisis, Themistoklis; Freddolino, Peter L.; Turunen, Petri; van Teeseling, Muriel C. F.

    2015-01-01

    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. PMID:26346632

  10. A thiol-activated lipase from Trichosporon asahii MSR 54: detergent compatibility and presoak formulation for oil removal from soiled cloth at ambient temperature.

    PubMed

    Kumar, S Suresh; Kumar, Lalit; Sahai, Vikram; Gupta, Rani

    2009-03-01

    An alkaline lipase from Trichosporon asahii MSR 54 was used to develop presoak formulation for removing oil stains at ambient temperature. The lipase was produced in a reactor followed by concentration by ultrafiltration and then it was dried with starch. The biochemical characteristics of enzyme showed that it was an alkaline lipase having pH activity in the range of pH 8.0-10.0 and temperature in the range of 25-50 degrees C. The present lipase was active >80% at 25 degrees C. The lipase was cystein activated with fourfold enhancement in presence of 5 mM cystein and likewise the activity was also stimulated in presence of papain hydrolysate which served as source of cystein. The presoak formulation consisted of two components A and B, component A was enzyme additive and B was a mixture of carbonate/bicarbonate source of alkali and papain hydrolysate as source of cystein. The results indicated that the presoaking in enzyme formulation followed by detergent washing was a better strategy for stain removal than direct washing with detergent in presence of lipase. Further, it was observed that 0.25% presoak component B in presence of 100 U enzyme component A (0.1 g) was the best formulation in removing maximum stain from mustard oil/triolein soiled clothes as indicated by increase in reflectance which was found equal to that of control cloth. The lipase action in presoaked formulation was clearly indicated by quantitated fatty acid release and also the TLC results of wash water, where oil hydrolytic products were visible only in presence of enzyme in the treatment. The wash performance carried at 25 degrees C indicated that washing at 25 degrees C was at par with that at 40 degrees C as indicated by similar reflectance of the washed cloth piece though qualitative fatty acid release was higher at 40 degrees C. PMID:19165516

  11. Role of lipase-generated free fatty acids in converting mesenteric lymph from a noncytotoxic to a cytotoxic fluid.

    PubMed

    Qin, Xiaofa; Dong, Wei; Sharpe, Susan M; Sheth, Sharvil U; Palange, David C; Rider, Therese; Jandacek, Ronald; Tso, Patrick; Deitch, Edwin A

    2012-10-15

    Recent studies have shown that mesenteric lymph plays a very important role in the development of multiple-organ dysfunction syndrome under critical conditions. Great efforts have been made to identify the biologically active molecules in the lymph. We used a trauma-hemorrhagic shock (T/HS) model and the superior mesenteric artery occlusion (SMAO) model, representing a global and a localized intestinal ischemia-reperfusion insult, respectively, to investigate the role of free fatty acids (FFAs) in the cytotoxicity of mesenteric lymph in rats. Lymph was collected before, during, and after (post) shock or SMAO. The post-T/HS and SMAO lymph, but not the sham lymph, manifested cytotoxicity for human umbilical vein endothelial cells (HUVECs). HUVEC cytotoxicity was associated with increased FFAs, especially the FFA-to-protein ratio. Addition of albumin, especially delipidated albumin, reduced this cytotoxicity. Lipase treatment of trauma-sham shock (T/SS) lymph converted it from a noncytotoxic to a cytotoxic fluid, and its toxicity correlated with the FFA-to-protein ratio in a fashion similar to that of the T/HS lymph, further suggesting that FFAs were the key components leading to HUVEC cytotoxicity. Analysis of lymph by gas chromatography revealed that the main FFAs in the post-T/HS or lipase-treated T/SS lymph were palmitic, stearic, oleic, and linoleic acids. When added to the cell culture at levels comparable to those in T/HS lymph, all these FFAs were cytotoxic, with linoleic acid being the most potent. In conclusion, this study suggests that lipase-generated FFAs are the key components resulting in the cytotoxicity of T/HS and SMAO mesenteric lymph. PMID:22899820

  12. Cholesterol efflux from THP-1 macrophages is impaired by the fatty acid component from lipoprotein hydrolysis by lipoprotein lipase

    SciTech Connect

    Yang, Yanbo; Thyagarajan, Narmadaa; Coady, Breanne M.; Brown, Robert J.

    2014-09-05

    Highlights: • Lipoprotein hydrolysis products were produced by lipoprotein lipase. • Hydrolysis products lowers expression of macrophage cholesterol transporters. • Hydrolysis products reduces expression of select nuclear receptors. • Fatty acid products lowers cholesterol transporters and select nuclear receptors. • Fatty acid products reduces cholesterol efflux from macrophages. - Abstract: 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.

  13. Novel LIPA mutations in Mexican siblings with lysosomal acid lipase deficiency

    PubMed Central

    Santillán-Hernández, Yuritzi; Almanza-Miranda, Enory; Xin, Winnie W; Goss, Kendrick; Vera-Loaiza, Aurea; Gorráez-de la Mora, María T; Piña-Aguilar, Raul E

    2015-01-01

    Lysosomal acid lipase (LAL) deficiency is an under-recognized lysosomal disease caused by deficient enzymatic activity of LAL. In this report we describe two affected female Mexican siblings with early hepatic complications. At two months of age, the first sibling presented with alternating episodes of diarrhea and constipation, and later with hepatomegaly, elevated transaminases, high levels of total and low-density lipoprotein cholesterol, and low levels of high-density lipoprotein. Portal hypertension and grade 2 esophageal varices were detected at four years of age. The second sibling presented with hepatomegaly, elevated transaminases and mildly elevated low-density lipoprotein and low high-density lipoprotein at six months of age. LAL activity was deficient in both patients. Sequencing of LIPA revealed two previously unreported heterozygous mutations in exon 4: c.253C>A and c.294C>G. These cases highlight the clinical continuum between the so-called Wolman disease and cholesteryl ester storage disease, and underscore that LAL deficiency represents a single disease with a degree of clinical heterogeneity. PMID:25624737

  14. 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. PMID:24792990

  15. Increase of Oleic Acid Content in Phosphatidylcholine through Lipase-catalyzed Interesterification: Optimization by Response Surface Methodology.

    PubMed

    Yang, Guolong; Yang, Lihui

    2015-01-01

    In order to obtain phosphatidylcholine (PC) with higher amount of oleic acid, the interesterification between soybean PC and Camellia oleifera oil (COO) rich in oleic acid catalyzed by lipase was studied in hexane. For this aim three commercially available immobilized lipases (Novozym 435, Lipozyme TLIM and Lipozyme RMIM) were assayed and Novozym 435 was finally selected for further optimization. The effects of the factors, such as PC concentration, substrate ratio, water amount, lipase dosage and temperature, on the oleic acid content in PC and PC recovery during the interesterification were investigated. The conditions of the interesterification were optimized using response surface methodology. The optimum conditions were as follows: lipase dosage 13 % (based on the mass of PC and COO), reaction temperature 55°C, water amount 5% (based on the mass of PC), reaction time 8 h, PC concentration 0.3g/mL (PC/hexane), PC-to-COO ratio 1:3 (acyl groups in PC/acyl groups in COO, mol/mol). Under these conditions, oleic acid content and PC recovery were 40.8 ± 0.5% and 69.0 ± 2.8%, respectively. Analysis of variance (ANOVA) showed that the regression models were adequate for predicting the interesterifiction. The orders of reaction variables affecting on oleic acid content and PC recovery were water amount > reaction time > lipase dosage > reaction temperature, and water amount > reaction temperature > lipase dosage > reaction time, respectively. PMID:25891113

  16. Lipase-catalyzed acidolysis of menhaden oil with conjugated linoleic acid: effect of water content.

    PubMed

    Torres, Carlos F; Hill, Charles G

    2002-06-01

    The effect of the water content on the lipase-catalyzed (Candida rugosa) interesterification (acidolysis) of menhaden oil with conjugated linoleic acid was studied for amounts of added water ranging from 0-4% (w/w). The rate of the acidolysis reaction increased with increasing water content, but the corresponding percentage of n-3 fatty acids liberated also increased. The implications of water content for minimization of the release of n-3 fatty acid residues while maximizing incorporation of CLA are discussed. PMID:12115120

  17. Biophysical characterization of mutants of Bacillus subtilis lipase evolved for thermostability: Factors contributing to increased activity retention

    PubMed Central

    Augustyniak, Wojciech; Brzezinska, Agnieszka A; Pijning, Tjaard; Wienk, Hans; Boelens, Rolf; Dijkstra, Bauke W; Reetz, Manfred T

    2012-01-01

    Previously, Lipase A from Bacillus subtilis was subjected to in vitro directed evolution using iterative saturation mutagenesis, with randomization sites chosen on the basis of the highest B-factors available from the crystal structure of the wild-type (WT) enzyme. This provided mutants that, unlike WT enzyme, retained a large part of their activity after heating above 65°C and cooling down. Here, we subjected the three best mutants along with the WT enzyme to biophysical and biochemical characterization. Combining thermal inactivation profiles, circular dichroism, X-ray structure analyses and NMR experiments revealed that mutations of surface amino acid residues counteract the tendency of Lipase A to undergo precipitation under thermal stress. Reduced precipitation of the unfolding intermediates rather than increased conformational stability of the evolved mutants seems to be responsible for the activity retention. PMID:22267088

  18. Production and regulation of lipase activity from Penicillium restrictum in submerged and solid-state fermentations.

    PubMed

    de Azeredo, Luciana A I; Gomes, Patrícia M; Sant'Anna, Geraldo L; Castilho, Leda R; Freire, Denise M G

    2007-05-01

    Different carbon (C) sources, mainly carbohydrates and lipids, have been screened for their capacity to support growth and lipase production by Penicillium restrictum in submerged fermentation (SmF) and in solid-state fermentation (SSF). Completely different physiological behaviors were observed after the addition of easily (oleic acid and glucose) and complex (olive oil and starch) assimilable C sources to the liquid and solid media. Maximal lipolytic activities (12.1 U/mL and 17.4 U/g) by P. restrictum were obtained with olive oil in SmF and in SSF, respectively. Biomass levels in SmF (12.2-14.1 mg/mL) and SSF (7.0-8.0 mg/g) did not varied greatly with the distinct C sources used. High lipase production (12.3 U/g) using glucose was only attained in SSF, perhaps due to the ability of this fermentation process to minimize catabolite repression. PMID:17457647

  19. Lipoprotein lipase activity is required for cardiac lipid droplet production.

    PubMed

    Trent, Chad M; Yu, Shuiqing; Hu, Yunying; Skoller, Nathan; Huggins, Lesley A; Homma, Shunichi; Goldberg, Ira J

    2014-04-01

    The rodent heart accumulates TGs and lipid droplets during fasting. The sources of heart lipids could be either FFAs liberated from adipose tissue or FAs from lipoprotein-associated TGs via the action of lipoprotein lipase (LpL). Because circulating levels of FFAs increase during fasting, it has been assumed that albumin transported FFAs are the source of lipids within heart lipid droplets. We studied mice with three genetic mutations: peroxisomal proliferator-activated receptor α deficiency, cluster of differentiation 36 (CD36) deficiency, and heart-specific LpL deletion. All three genetically altered groups of mice had defective accumulation of lipid droplet TGs. Moreover, hearts from mice treated with poloxamer 407, an inhibitor of lipoprotein TG lipolysis, also failed to accumulate TGs, despite increased uptake of FFAs. TG storage did not impair maximal cardiac function as measured by stress echocardiography. Thus, LpL hydrolysis of circulating lipoproteins is required for the accumulation of lipids in the heart of fasting mice. PMID:24493834

  20. Lipoprotein lipase activity and chylomicron clearance in rats fed a high fat diet

    SciTech Connect

    Brown, C.M.; Layman, D.K.

    1988-11-01

    The relationships of tissue and plasma lipoprotein lipase (LPL) activities to tissue uptake and plasma clearance of UC-labeled chylomicron-triglyceride ( UC-CM-TG) were studied in female rats fed isoenergetic and isonitrogenous control (12% kJ from fat) or high fat diets (72% kJ from fat) for 8 wk. Animals fed the high-fat diet had higher levels of fasting plasma triglycerides and lower LPL activities in heart, renal adipose tissue and post-heparin plasma. Changes in LPL activities of skeletal muscles varied among muscles with higher values in the soleus and plantaris (32-61%) and no differences in the gastrocnemius. The lower LPL activity in renal adipose tissue was associated with lower uptake of fatty acids from UC-CM-TG by adipose. Fatty-acid uptake from labeled TG was not associated with tissue LPL activity in other tissues. Clearance of UC-CM-TG from plasma and the half-lives of UC-CM-TG were similar in both dietary groups. These data indicate that tissue and plasma LPL activities are not a direct index of uptake of fatty acids by tissues or clearance of chylomicron triglycerides.

  1. Differential induction, purification and characterization of cold active lipase from Yarrowia lipolytica NCIM 3639.

    PubMed

    Yadav, K N Sathish; Adsul, M G; Bastawde, K B; Jadhav, D D; Thulasiram, H V; Gokhale, D V

    2011-11-01

    The production, purification and characterization of cold active lipases by Yarrowia lipolytica NCIM 3639 is described. The study presents a new finding of production of cell bound and extracellular lipase activities depending upon the substrate used for growth. The strain produced cell bound and extracellular lipase activity when grown on olive oil and Tween 80, respectively. The organism grew profusely at 20 °C and at initial pH of 5.5, producing maximum extracellular lipase. The purified lipase has a molecular mass of 400 kDa having 20 subunits forming a multimeric native protein. Further the enzyme displayed an optimum pH of 5.0 and optimum temperature of 25 °C. Peptide mass finger printing reveled that some peptides showed homologues sequence (42%) to Yarrowia lipolytica LIP8p. The studies on hydrolysis of racemic lavandulyl acetate revealed that extracellular and cell bound lipases show preference over the opposite antipodes of irregular monoterpene, lavandulyl acetate. PMID:21967715

  2. 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 Δ9-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. PMID:26630956

  3. Inhibitors of Fatty Acid Amide Hydrolase and Monoacylglycerol Lipase: New Targets for Future Antidepressants

    PubMed Central

    Ogawa, Shintaro; Kunugi, Hiroshi

    2015-01-01

    Cannabis and analogs of Δ9-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. PMID:26630956

  4. Lipases, industrial uses

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Lipases are enzymes that catalyze the hydrolysis of triglycerides to glycerol and fatty acids. Microbial lipases are relatively stable and are capable of catalyzing a variety of reactions; they are potentially of importance for diverse industrial applications. Lipases can be divided generally into...

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

    PubMed

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

    2015-04-14

    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. PMID:25766647

  6. Cross-linked enzyme aggregates (CLEAs) of Pencilluim notatum lipase enzyme with improved activity, stability and reusability characteristics.

    PubMed

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

    2016-10-01

    Cross-linked enzyme aggregates (CLEAs) are considered as an effective tool for the immobilization of enzyme. In this study, Pencillium notatum lipase (PNL) was immobilized as carrier free cross-linked enzyme aggregates using glutaraldehyde (GLA) and Ethylene glycol-bis [succinic acid N-hydroxysuccinimide] (EG-NHS) as cross-linking agents. The optimal conditions for the synthesis of an efficient lipase CLEAs such as precipitant type, the nature and amount of cross-linking reagent, and cross-linking time were optimized. The recovered activities of CLEAs were considerably dependent on the concentration of GLA; however, the activity recovery was not severely affected by EG-NHS as a mild cross-linker. The EG-NHS aggregates displayed superior hydrolytic (52.08±2.52%) and esterification (64.42%) activities as compared to GLA aggregates which showed 23.8±1.86 and 34.54% of hydrolytic and esterification activity, respectively. Morphological analysis by fluorescence and scanning electron microscope revealed that EG-NHS aggregates were smaller in size with larger surface area compared to GLA aggregates. The pH optima of both types of CLEAs were displaced to slightly alkaline region and higher temperature as compared to native enzyme. Highest enzyme activity of CLEAs was achieved at the pH of 9.0 and 42°C temperature. Moreover, a significant improvement in the thermal resistance was also recorded after immobilization. After ten reusability cycles in aqueous medium, GLA and EG-NHS cross-linked lipase CLEAs preserved 63.62% and 70.9% of their original activities, respectively. The results suggest that this novel CLEA-lipase is potentially usable in many industrial applications. PMID:27365121

  7. Physiological factors affecting production of extracellular lipase (LipA) in Acinetobacter calcoaceticus BD413: fatty acid repression of lipA expression and degradation of LipA.

    PubMed Central

    Kok, R G; Nudel, C B; Gonzalez, R H; Nugteren-Roodzant, I M; Hellingwerf, K J

    1996-01-01

    The extracellular lipase (LipA) produced by Acinetobacter calcoaceticus BD413 is required for growth of the organism on triolein, since mutant strains that lack an active lipase fail to grow with triolein as the sole carbon source. Surprisingly, extracellular lipase activity and expression of the structural lipase gene (lipA), the latter measured through lacZ as a transcriptional reporter, are extremely low in triolein cultures of LipA+ strains. The explanation for this interesting paradox lies in the effect of fatty acids on the expression of lipA. We found that long-chain fatty acids, especially, strongly repress the expression of lipA, thereby negatively influencing the production of lipase. We propose the involvement of a fatty acyl-responsive DNA-binding protein in regulation of expression of the A. calcoaceticus lipBA operon. The potential biological significance of the observed physiological competition between expression and repression of lipA in the triolein medium is discussed. Activity of the extracellular lipase is also negatively affected by proteolytic degradation, as shown in in vitro stability experiments and by Western blotting (immunoblotting) of concentrated supernatants of stationary-phase cultures. In fact, the relatively high levels of extracellular lipase produced in the early stationary phase in media which contain hexadecane are due only to enhanced stability of the extracellular enzyme under those conditions. The rapid extracellular degradation of LipA of A. calcoaceticus BD413 by an endogenous protease is remarkable and suggests that proteolytic degradation of the enzyme is another important factor in regulating the level of active extracellular lipase. PMID:8830702

  8. Lipase immobilization on differently functionalized vinyl-based amphiphilic polymers: influence of phase segregation on the enzyme hydrolytic activity.

    PubMed

    Bellusci, Mariangela; Francolini, Iolanda; Martinelli, Andrea; D'Ilario, Lucio; Piozzi, Antonella

    2012-03-12

    Microbial lipase from Candida rugosa was immobilized by physical adsorption onto an ethylene-vinyl alcohol polymer (EVAL) functionalized with acyl chlorides. To evaluate the influence of the reagent chain-length on the amount and activity of immobilized lipase, three differently long aliphatic fatty acids were employed (C8, C12, C18), obtaining EVAL functionalization degrees ranging from 5% to 65%. The enzyme-polymer affinity increased with both the length of the alkyl chain and the matrix hydrophobicity. In particular, the esterified polymers showed a tendency to give segregated hydrophilic and hydrophobic domains. It was observed the formation of an enzyme multilayer at both low and high protein concentrations. Desorption experiments showed that Candida rugosa lipase may be adsorbed in a closed form on the polymer hydrophilic domains and in an open, active structure on the hydrophobic ones. The best results were found for the EVAL-C18 13% matrix that showed hyperactivation with both the soluble and unsoluble substrate after enzyme desorption. In addition, this supported biocatalyst retained its activity for repetitive cycles. PMID:22295868

  9. The effect of ultrasonic pre-treatment on the catalytic activity of lipases in aqueous and non-aqueous media

    PubMed Central

    Shah, Shweta; Gupta, Munishwar Nath

    2008-01-01

    Background Ultrasound has been used to accelerate the rates of numerous chemical reactions, however its effects on enzymatic reactions have been less extensively studied. While known to result in the acceleration of enzyme-catalysed reactions, ultrasonication has also been shown to induce enzyme inactivation. In this study we investigated the effects of ultrasonic pretreatment on lipases in both aqueous and non-aqueous media. Results Our results show that the ultrasonic pre-irradiation of lipases (from Burkholderia cepacia and Pseudomonas fluorescens) in aqueous buffer and organic solvents enhanced enzymic activities. In addition, we report the enhancement of hydrolytic (esterase) and transesterification activities. On using pre-irradiated enzyme, we found that the conversion rate for the transesterification of ethyl butyrate to butyl butyrate, increased from 66% to 82%. Similarly, a 79% conversion of Jatropha oil to biodiesel was observed upon employing pre-irradiated enzyme, in contrast to a 34% conversion with untreated enzyme. CD spectra showed that while the enzyme's secondary structure remained largely unaffected, the microenvironments of aromatic amino acids were altered, with perturbation of the tertiary structure having also occurred. SEM analysis demonstrated significant morphological changes in the enzyme preparation as a result of ultrasonication. Conclusion In contrast to the effects of ultrasonic irradiation on other enzymes, for the lipases focused upon in this study, we report an enhancement of biocatalytic activity, which is thought to originate from morphological changes on the macro and molecular levels. PMID:18234100

  10. Influence of surface hydrophobicity on immobilized lipase activity

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Candida antarctica lipase B (CALB) has considerable commercial synthetic utility, particularly in the nonaqueous preparation of chiral pharmaceuticals, polymers, and various bio-based materials. CALB is typically used in an immobilized form, such as Novozym 435, in which the enzyme is non-covalentl...

  11. Effect of oil and oil products on lipase activity in gray forest soil

    NASA Astrophysics Data System (ADS)

    Kireeva, N. A.; Tarasenko, E. M.; Shamaeva, A. A.; Novoselova, E. I.

    2006-08-01

    The effect of different rates of oil and oil products on the lipase activity in gray forest soil was studied under field and laboratory conditions. It was found that hydrocarbons activate the lipolytic activity of the soil. Along with the activation of lipolysis, an increase in the number of hydrocarbon-oxidizing microorganisms and a decrease in the content of oil products were observed.

  12. Synergistic effects of amine and protein modified epoxy-support on immobilized lipase activity.

    PubMed

    Cui, Caixia; Tao, Yifeng; Ge, Chunling; Zhen, Yueju; Chen, Biqiang; Tan, Tianwei

    2015-09-01

    We have developed an improved and effective method to immobilize Yarrowia lipolytica lipase Lip2 (YLIP2) on an epoxy poly-(glycidylmethacrylate-triallyisocyanurate-ethyleneglycoldimethacrylate) (PGMA-TAIC-EGDMA) support structure with or without amine or/and protein modifications. Our results show that there is an increase in the activity of the immobilized lipase on n-butylamine (BA) modified support (420U/g support) and the biocompatible gelatin modified support (600U/g support) when compared to the support without modification (240U/g support). To further study the influences of BA and gelatin modification on the activity of the immobilized lipase, gelatin and BA were concurrently used to decorate the support structure. Lipase immobilized on 2% BA/gelatin (1:1) modified support obtained the highest activity (1180U/g support), which was five-fold higher than that on a native support structure. These results suggest that the activity of a support-immobilized lipase depends on the support surface properties and a moderate support surface micro-environment was crucial for elevated activity. Collectively, these data show that a combined gelatin and BA modification regulates the support surface more suitable for immobilizing YLIP2. PMID:26073154

  13. 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

    SciTech Connect

    Anderson, R.A.; Rao, N.; Byrum, R.S.; Rothschild, C.B.; Bowden, D.W.; Hayworth, R.; Pettenati, M. )

    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 regulation 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.

  14. 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

    2013-01-01

    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. PMID:24287996

  15. Spectroscopic studies on the inhibitory effects of ionic liquids on lipase activity.

    PubMed

    Fan, Yunchang; Dong, Xing; Li, Xiaojing; Zhong, Yingying; Kong, Jichuan; Hua, Shaofeng; Miao, Juan; Li, Yan

    2016-04-15

    The effects of ionic liquids (ILs) on the lipase activity were studied by UV-Vis spectroscopy and the IL-lipase interaction mechanism at the molecular level was investigated by fluorescence technique. Experimental results indicated that the lipase activity was inhibited by ILs and the degree of inhibition highly depended on the chemical structures of ILs. The inhibitory ability of the Cl(-)- and Br(-)-based ILs increased with increasing the alkyl chain length in the IL cation. Thermodynamic parameters, enthalpy change (ΔH) and entropy change (ΔS) were obtained by analyzing the fluorescence behavior of lipase with the addition of ILs. Both ΔH and ΔS were positive suggesting hydrophobicity was the major driven force for the Cl(-)- and Br(-)-based ILs. For the BF4(-)-, CF3SO3(-)-, ClO4(-)- and N(CN)2(-)-based ILs, hydrogen bonding was the main driven force. For a more comprehensive understanding of the effects of ILs on lipase activity, the roles of hydrophobicity and hydrogen bonding must be considered simultaneously. A regression-based equation was developed to describe the relationship of the inhibitory ability of ILs and their hydrophobicity and hydrogen bonding ability. PMID:26836454

  16. Spectroscopic studies on the inhibitory effects of ionic liquids on lipase activity

    NASA Astrophysics Data System (ADS)

    Fan, Yunchang; Dong, Xing; Li, Xiaojing; Zhong, Yingying; Kong, Jichuan; Hua, Shaofeng; Miao, Juan; Li, Yan

    2016-04-01

    The effects of ionic liquids (ILs) on the lipase activity were studied by UV-Vis spectroscopy and the IL-lipase interaction mechanism at the molecular level was investigated by fluorescence technique. Experimental results indicated that the lipase activity was inhibited by ILs and the degree of inhibition highly depended on the chemical structures of ILs. The inhibitory ability of the Cl-- and Br--based ILs increased with increasing the alkyl chain length in the IL cation. Thermodynamic parameters, enthalpy change (ΔH) and entropy change (ΔS) were obtained by analyzing the fluorescence behavior of lipase with the addition of ILs. Both ΔH and ΔS were positive suggesting hydrophobicity was the major driven force for the Cl-- and Br--based ILs. For the BF4--, CF3SO3--, ClO4-- and N(CN)2--based ILs, hydrogen bonding was the main driven force. For a more comprehensive understanding of the effects of ILs on lipase activity, the roles of hydrophobicity and hydrogen bonding must be considered simultaneously. A regression-based equation was developed to describe the relationship of the inhibitory ability of ILs and their hydrophobicity and hydrogen bonding ability.

  17. Toluene promotes lid 2 interfacial activation of cold active solvent tolerant lipase from Pseudomonas fluorescens strain AMS8.

    PubMed

    Yaacob, Norhayati; Mohamad Ali, Mohd Shukuri; Salleh, Abu Bakar; Rahman, Raja Noor Zaliha Raja Abdul; Leow, Adam Thean Chor

    2016-07-01

    The utilization of cold active lipases in organic solvents proves an excellent approach for chiral synthesis and modification of fats and oil due to the inherent flexibility of lipases under low water conditions. In order to verify whether this lipase can function as a valuable synthetic catalyst, the mechanism concerning activation of the lid and interacting solvent residues in the presence of organic solvent must be well understood. A new alkaline cold-adapted lipase, AMS8, from Pseudomonas fluorescens was studied for its structural adaptation and flexibility prior to its exposure to non-polar, polar aprotic and protic solvents. Solvents such as ethanol, toluene, DMSO and 2-propanol showed to have good interactions with active sites. Asparagine (Asn) and tyrosine (Tyr) were key residues attracted to solvents because they could form hydrogen bonds. Unlike in other solvents, Phe-18, Tyr-236 and Tyr-318 were predicted to have aromatic-aromatic side-chain interactions with toluene. Non-polar solvent also was found to possess highest energy binding compared to polar solvents. Due to this circumstance, the interaction of toluene and AMS8 lipase was primarily based on hydrophobicity and molecular recognition. The molecular dynamic simulation showed that lid 2 (residues 148-167) was very flexible in toluene and Ca(2+). As a result, lid 2 moves away from the catalytic areas, leaving an opening for better substrate accessibility which promotes protein activation. Only a single lid (lid 2) showed the movement following interactions with toluene, although AMS8 lipase displayed double lids. The secondary conformation of AMS8 lipase that was affected by toluene observed a reduction of helical strands and increased coil structure. Overall, this work shows that cold active lipase, AMS8 exhibits distinguish interfacial activation and stability in the presence of polar and non-polar solvents. PMID:27474867

  18. The effects of an inhibitor of diglyceride lipase on collagen-induced platelet activation.

    PubMed

    Jackson, Elke C G; Ortar, Giorgio; McNicol, Archie

    2013-12-01

    Human platelet activation by collagen occurs in a dose-dependent manner. High concentrations of collagen bind to a pair of receptors, the α2β1 integrin and glycoprotein (GP)VI/Fc-receptor γ-chain (FcRγ), which stimulate a cascade of events including Syk, LAT, Btk, Gads, and phospholipase Cγ2, leading to calcium release and protein kinase C (PKC) activation. Calcium and PKC are responsible for a range of platelet responses including exocytosis and aggregation, as well as the cytosolic phospholipase A2 (cPLA2)-mediated release of arachidonic acid, which is converted to thromboxane (Tx)A2. In contrast, low concentrations of collagen are acutely aspirin-sensitive, and calcium release and aggregation are TxA2-dependent. Under these conditions, cPLA2 is not involved and it has been suggested that phospholipase C generates 1,2-diacylglycerol (DG) from which arachidonic acid is liberated by diglyceride lipase (DGL). Here a novel DGL blocker (OMDM-188) inhibited collagen-, but not arachidonic acid-induced aggregation and TxA2 synthesis. Furthermore, OMDM-188 inhibited collagen-induced arachidonic acid release. Finally OMDM-188 inhibited collagen-induced p38(MAPK) phosphorylation, but not extracellular signal-regulated kinase (ERK) phosphorylation, with no effect on the phosphorylation of either enzyme in response to arachidonic acid. Taken together, these data suggest a role for a pathway involving phospholipase C liberating DG from membrane phospholipids in response to minimally activating concentrations of collagen. The DG serves as a substrate for DGL, potentially under the regulations of p38(MAPK), to release arachidonic acid, which is subsequently converted to TxA2, which mediates the final platelet response. PMID:24042163

  19. Purification of stearidonic acid (18:4(n-3)) and hexadecatetraenoic acid (16:4(n-3)) from algal fatty acid with lipase and medium pressure liquid chromatography.

    PubMed

    Ishihara, K; Murata, M; Kaneniwa, M; Saito, H; Komatsu, W; Shinohara, K

    2000-11-01

    Stearidonic acid (18:4(n-3)) and hexadecatetraenoic acid (16:4(n-3)) are included in some edible marine algae such as Undaria pinnatifida and Ulva pertusa with relatively high compositions (up to 40%) of total fatty acids. In order to prepare 16:4(n-3) and 18:4(n-3) enriched fatty acid concentrates, we screened for a suitable lipase which concentrates these acids by the removal of other fatty acids in the selective esterification reaction reported by Shimada et al. (Shimada et al. (1997), J. Am. Oil Chem. Soc., 74, 1465-1470). In combination with the lipase reaction and reversed-phase medium pressure liquid chromatography, we purified 18:4(n-3) and 16:4(n-3) to more than 95% purity. PMID:11193415

  20. A highly selective, reversible inhibitor identified by comparative chemoproteomics modulates diacylglycerol lipase activity in neurons

    PubMed Central

    Baggelaar, Marc P.; Chameau, Pascal J. P.; Kantae, Vasudev; Hummel, Jessica; Hsu, Ku-Lung; Janssen, Freek; van der Wel, Tom; Soethoudt, Marjolein; Deng, Hui; den Dulk, Hans; Allarà, Marco; Florea, Bogdan I.; Di Marzo, Vincenzo; Wadman, Wytse J.; Kruse, Chris G.; Overkleeft, Herman S.; Hankemeier, Thomas; Werkman, Taco R.; Cravatt, Benjamin F.; van der Stelt, Mario

    2016-01-01

    Diacylglycerol lipase (DAGL)-α and -β are enzymes responsible for the biosynthesis of the endocannabinoid 2-arachidonoylglycerol (2-AG). Selective and reversible inhibitors are required to study the function of DAGLs in neuronal cells in an acute and temporal fashion, but they are currently lacking. Here, we describe the identification of a highly selective DAGL inhibitor using structure-guided and a chemoproteomics strategy to characterize the selectivity of the inhibitor in complex proteomes. Key to the success of this approach is the use of comparative and competitive activity-based proteome profiling (ABPP), in which broad-spectrum and tailor-made activity-based probes are combined to report on the inhibition of a protein family in its native environment. Competitive ABPP with broad-spectrum fluorophosphonate-based probes and specific β-lactone-based probes led to the discovery of α-ketoheterocycle LEI105 as a potent, highly selective and reversible dual DAGL-α/DAGL-β inhibitor. LEI105 did not affect other enzymes involved in endocannabinoid metabolism including abhydrolase domain-containing protein 6, abhydrolase domain-containing protein 12, monoacylglycerol lipase and fatty acid amide hydrolase and did not display affinity for the cannabinoid CB1 receptor. Targeted lipidomics revealed that LEI105 concentration-dependently reduced 2-AG levels, but not anandamide levels, in Neuro2A cells. We show that cannabinoid CB1-receptor-mediated short-term synaptic plasticity in a mouse hippocampal slice model can be reduced by LEI105. Thus, we have developed a highly selective DAGL inhibitor and provide new pharmacological evidence to support the hypothesis that ‘on demand biosynthesis’ of 2-AG is responsible for retrograde signaling. PMID:26083464

  1. Optimization of environmental conditions for production of a novel cold-active lipase from Pichia lynferdii Y-7723

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Lipases with abnormal functionality 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 lipase(CAL)s have gained special attention in various industrial fields...

  2. Analysis of Comparative Sequence and Genomic Data to Verify Phylogenetic Relationship and Explore a New Subfamily of Bacterial Lipases.

    PubMed

    Masomian, Malihe; Rahman, Raja Noor Zaliha Raja Abd; Salleh, Abu Bakar; Basri, Mahiran

    2016-01-01

    Thermostable and organic solvent-tolerant enzymes have significant potential in a wide range of synthetic reactions in industry due to their inherent stability at high temperatures and their ability to endure harsh organic solvents. In this study, a novel gene encoding a true lipase was isolated by construction of a genomic DNA library of thermophilic Aneurinibacillus thermoaerophilus strain HZ into Escherichia coli plasmid vector. Sequence analysis revealed that HZ lipase had 62% identity to putative lipase from Bacillus pseudomycoides. The closely characterized lipases to the HZ lipase gene are from thermostable Bacillus and Geobacillus lipases belonging to the subfamily I.5 with ≤ 57% identity. The amino acid sequence analysis of HZ lipase determined a conserved pentapeptide containing the active serine, GHSMG and a Ca(2+)-binding motif, GCYGSD in the enzyme. Protein structure modeling showed that HZ lipase consisted of an α/β hydrolase fold and a lid domain. Protein sequence alignment, conserved regions analysis, clustal distance matrix and amino acid composition illustrated differences between HZ lipase and other thermostable lipases. Phylogenetic analysis revealed that this lipase represented a new subfamily of family I of bacterial true lipases, classified as family I.9. The HZ lipase was expressed under promoter Plac using IPTG and was characterized. The recombinant enzyme showed optimal activity at 65 °C and retained ≥ 97% activity after incubation at 50 °C for 1h. The HZ lipase was stable in various polar and non-polar organic solvents. PMID:26934700

  3. Analysis of Comparative Sequence and Genomic Data to Verify Phylogenetic Relationship and Explore a New Subfamily of Bacterial Lipases

    PubMed Central

    Salleh, Abu Bakar; Basri, Mahiran

    2016-01-01

    Thermostable and organic solvent-tolerant enzymes have significant potential in a wide range of synthetic reactions in industry due to their inherent stability at high temperatures and their ability to endure harsh organic solvents. In this study, a novel gene encoding a true lipase was isolated by construction of a genomic DNA library of thermophilic Aneurinibacillus thermoaerophilus strain HZ into Escherichia coli plasmid vector. Sequence analysis revealed that HZ lipase had 62% identity to putative lipase from Bacillus pseudomycoides. The closely characterized lipases to the HZ lipase gene are from thermostable Bacillus and Geobacillus lipases belonging to the subfamily I.5 with ≤ 57% identity. The amino acid sequence analysis of HZ lipase determined a conserved pentapeptide containing the active serine, GHSMG and a Ca2+-binding motif, GCYGSD in the enzyme. Protein structure modeling showed that HZ lipase consisted of an α/β hydrolase fold and a lid domain. Protein sequence alignment, conserved regions analysis, clustal distance matrix and amino acid composition illustrated differences between HZ lipase and other thermostable lipases. Phylogenetic analysis revealed that this lipase represented a new subfamily of family I of bacterial true lipases, classified as family I.9. The HZ lipase was expressed under promoter Plac using IPTG and was characterized. The recombinant enzyme showed optimal activity at 65°C and retained ≥ 97% activity after incubation at 50°C for 1h. The HZ lipase was stable in various polar and non-polar organic solvents. PMID:26934700

  4. Structured lipids via lipase-catalyzed incorporation of eicosapentaenoic acid into borage (Borago officinalis L.) and evening primrose (Oenothera biennis L.) oils.

    PubMed

    Senanayake, S P J Namal; Shahidi, Fereidoon

    2002-01-30

    Enzymatic acidolysis of borage oil (BO) or evening primrose oil (EPO) with eicosapentaenoic acid (20:5n-3; EPA) was studied. Of the six lipases that were tested in the initial screening, nonspecific lipase PS-30 from Pseudomonas sp. resulted in the highest incorporation of EPA into both oils. This enzyme was further studied for the influence of enzyme load, temperature, time, type of organic solvent, and mole ratio of substrates. The products from the acidolysis reaction were analyzed by gas chromatography (GC). The highest incorporation of EPA in both oils occurred at 45-55 degrees C and at 150-250 enzyme activity units. One unit of lipase activity was defined as nanomoles of fatty acids (oleic acid equivalents) produced per minute per gram of enzyme. Time course studies indicated that EPA incorporation was increased up to 26.8 and 25.2% (after 24 h) in BO and EPO, respectively. Among the solvents examined, n-hexane served best for the acidolysis of EPA with both oils. The effect of the mole ratio of oil to EPA was studied from 1:1 to 1:3. As the mole ratio of EPA increased, the incorporation increased from 25.2-26.8 to 37.4-39.9% (after 24 h). The highest EPA incorporations of 39.9 and 37.4% in BO and EPO, respectively, occurred at the stoichiometric mole ratio of 1:3 for oil to EPA. PMID:11804516

  5. 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

    2014-09-01

    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. PMID:25130461

  6. 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. PMID:17629478

  7. 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. PMID:26343557

  8. Unlocking the mystery behind the activation phenomenon of T1 lipase: A molecular dynamics simulations approach

    PubMed Central

    Rahman, Mohd Zulhilmi Abdul; Salleh, Abu Bakar; Rahman, Raja Noor Zaliha Raja Abdul; Rahman, Mohd Basyaruddin Abdul; Basri, Mahiran; Leow, Thean Chor

    2012-01-01

    The activation of lipases has been postulated to proceed by interfacial activation, temperature switch activation, or aqueous activation. Recently, based on molecular dynamics (MD) simulation experiments, the T1 lipase activation mechanism was proposed to involve aqueous activation in addition to a double-flap mechanism. Because the open conformation structure is still unavailable, it is difficult to validate the proposed theory unambiguously to understand the behavior of the enzyme. In this study, we try to validate the previous reports and uncover the mystery behind the activation process using structural analysis and MD simulations. To investigate the effects of temperature and environmental conditions on the activation process, MD simulations in different solvent environments (water and water-octane interface) and temperatures (20, 50, 70, 80, and 100°C) were performed. Based on the structural analysis of the lipases in the same family of T1 lipase (I.5 lipase family), we proposed that the lid domain comprises α6 and α7 helices connected by a loop, thus forming a helix-loop-helix motif involved in interfacial activation. Throughout the MD simulations experiments, lid displacements were only observed in the water-octane interface, not in the aqueous environment with respect to the temperature effect, suggesting that the activation process is governed by interfacial activation coupled with temperature switch activation. Examining the activation process in detail revealed that the large structural rearrangement of the lid domain was caused by the interaction between the hydrophobic residues of the lid with octane, a nonpolar solvent, and this conformation was found to be thermodynamically favorable. PMID:22692819

  9. Adipose triglyceride lipase regulates eicosanoid production in activated human mast cells.

    PubMed

    Dichlberger, Andrea; Schlager, Stefanie; Maaninka, Katariina; Schneider, Wolfgang J; Kovanen, Petri T

    2014-12-01

    Human mast cells (MCs) contain TG-rich cytoplasmic lipid droplets (LDs) with high arachidonic acid (AA) content. Here, we investigated the functional role of adipose TG lipase (ATGL) in TG hydrolysis and the ensuing release of AA as substrate for eicosanoid generation by activated human primary MCs in culture. Silencing of ATGL in MCs by siRNAs induced the accumulation of neutral lipids in LDs. IgE-dependent activation of MCs triggered the secretion of the two major eicosanoids, prostaglandin D2 (PGD2) and leukotriene C4 (LTC4). The immediate release of PGD2 from the activated MCs was solely dependent on cyclooxygenase (COX) 1, while during the delayed phase of lipid mediator production, the inducible COX-2 also contributed to its release. Importantly, when ATGL-silenced MCs were activated, the secretion of both PGD2 and LTC4 was significantly reduced. Interestingly, the inhibitory effect on the release of LTC4 was even more pronounced in ATGL-silenced MCs than in cytosolic phospholipase A2-silenced MCs. These data show that ATGL hydrolyzes AA-containing TGs present in human MC LDs and define ATGL as a novel regulator of the substrate availability of AA for eicosanoid generation upon MC activation. PMID:25114172

  10. Relationship between skeletal muscle lipoprotein lipase activity and 24-hour macronutrient oxidation.

    PubMed Central

    Ferraro, R T; Eckel, R H; Larson, D E; Fontvieille, A M; Rising, R; Jensen, D R; Ravussin, E

    1993-01-01

    A low ratio of whole-body 24-h fat/carbohydrate (CHO) oxidation has been shown to be a predictor of subsequent body weight gain. We tested the hypothesis that the variability of this ratio may be related to differences in skeletal muscle metabolism. Since lipoprotein lipase (LPL) plays a pivotal role in partitioning lipoprotein-borne triglycerides to adipose (storage) and skeletal muscle (mostly oxidation), we postulated that a low ratio of fat/CHO oxidation was associated with a low skeletal muscle LPL (SMLPL) activity. As an index of substrate oxidation, 24-h RQ was measured under sedentary and eucaloric conditions in 16 healthy nondiabetic Pima males. During a 6-h euglycemic, hyperinsulinemic clamp, muscle biopsies were obtained at baseline, 3, and 6 h. Heparin-elutable SMLPL activity was 2.92 +/- 0.56 nmol free fatty acids/g.min (mean +/- SD) at baseline, was unchanged (2.91 +/- 0.51) at the third hour, and increased significantly (P < 0.05) to 3.13 +/- 0.57 at the sixth hour of the clamp. The mean (of baseline and 3-h) SMLPL activity correlated inversely with 24-h RQ (r = 0.57, P < 0.03) but not with body size, body composition, or insulin-mediated glucose uptake. Since SMLPL activity is related to the ratio of whole body fat/CHO oxidation rate, a decreased muscle LPL activity may, therefore, predispose to obesity. PMID:8326010

  11. Adipose triglyceride lipase regulates eicosanoid production in activated human mast cells

    PubMed Central

    Dichlberger, Andrea; Schlager, Stefanie; Maaninka, Katariina; Schneider, Wolfgang J.; Kovanen, Petri T.

    2014-01-01

    Human mast cells (MCs) contain TG-rich cytoplasmic lipid droplets (LDs) with high arachidonic acid (AA) content. Here, we investigated the functional role of adipose TG lipase (ATGL) in TG hydrolysis and the ensuing release of AA as substrate for eicosanoid generation by activated human primary MCs in culture. Silencing of ATGL in MCs by siRNAs induced the accumulation of neutral lipids in LDs. IgE-dependent activation of MCs triggered the secretion of the two major eicosanoids, prostaglandin D2 (PGD2) and leukotriene C4 (LTC4). The immediate release of PGD2 from the activated MCs was solely dependent on cyclooxygenase (COX) 1, while during the delayed phase of lipid mediator production, the inducible COX-2 also contributed to its release. Importantly, when ATGL-silenced MCs were activated, the secretion of both PGD2 and LTC4 was significantly reduced. Interestingly, the inhibitory effect on the release of LTC4 was even more pronounced in ATGL-silenced MCs than in cytosolic phospholipase A2-silenced MCs. These data show that ATGL hydrolyzes AA-containing TGs present in human MC LDs and define ATGL as a novel regulator of the substrate availability of AA for eicosanoid generation upon MC activation. PMID:25114172

  12. 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. PMID:25225262

  13. 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

  14. Improved Performance of Lipase Immobilized on Tannic Acid-Templated Mesoporous Silica Nanoparticles.

    PubMed

    Jiang, Yanjun; Sun, Wenya; Zhou, Liya; Ma, Li; He, Ying; Gao, Jing

    2016-08-01

    Mesoporous silica nanoparticles were synthesized by using tannic acid as a pore-forming agent, which is an environmentally friendly, cheap, and non-surfactant template. SEM and TEM images indicated that the tannic acid-templated mesoporous silica nanoparticles (TA-MSNs) are monodisperse spherical-like particles with an average diameter of 195 ± 16 nm. The Brunauer-Emmett-Teller (BET) results showed that the TA-MSNs had a relatively high surface area (447 m(2)/g) and large pore volume (0.91 cm(3)/g), and the mean pore size was ca. 10.1 nm. Burkholderia cepacia lipase was immobilized on the TA-MSNs by physical adsorption for the first time, and the properties of immobilized lipase (BCL@TA-MSNs) were investigated. The BCL@TA-MSNs exhibited satisfactory thermal stability; strong tolerance to organic solvents such as methanol, ethanol, isooctane, n-hexane, and tetrahydrofuran; and high operational reusability when BCL@TA-MSNs were applied in esterification and transesterification reactions. After recycling 15 times in the transesterification reaction for biodiesel production, over 85 % of biodiesel yield can be maintained. With these desired characteristics, the TA-MSNs may provide excellent candidates for enzyme immobilization. PMID:27011329

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

    PubMed

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

    2016-02-14

    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. PMID:26790821

  16. 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. PMID:25737230

  17. 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

  18. Modification of pancreatic lipase properties by directed molecular evolution.

    PubMed

    Colin, Damien Yann; Deprez-Beauclair, Paule; Silva, Noella; Infantes, Lourdes; Kerfelec, Brigitte

    2010-05-01

    Cystic fibrosis is associated with pancreatic insufficiency and acidic intraluminal conditions that limit the action of pancreatic enzyme replacement therapy, especially that of lipase. Directed evolution combined with rational design was used in the aim of improving the performances of the human pancreatic lipase at acidic pH. We set up a method for screening thousands of lipase variants for activity at low pH. A single round of random mutagenesis yielded one lipase variant with an activity at acidic pH enhanced by approximately 50% on medium- and long-chain triglycerides. Sequence analysis revealed two substitutions (E179G/N406S) located in specific regions, the hydrophobic groove accommodating the sn-1 chain of the triglyceride (E179G) and the surface loop that is likely to mediate lipase/colipase interaction in the presence of lipids (N406S). Interestingly, these two substitutions shifted the chain-length specificity of lipase toward medium- and long-chain triglycerides. Combination of those two mutations with a promising one at the entrance of the catalytic cavity (K80E) negatively affected the lipase activity at neutral pH but not that at acidic pH. Our results provide a basis for the design of improved lipase at acidic pH and identify for the first time key residues associated with chain-length specificity. PMID:20150178

  19. Substitution of Val72 residue alters the enantioselectivity and activity of Penicillium expansum lipase.

    PubMed

    Tang, Lianghua; Su, Min; Zhu, Ling; Chi, Liying; Zhang, Junling; Zhou, Qiong

    2013-01-01

    Error-prone PCR was used to create more active or enantioselective variants of Penicillium expansum lipase (PEL). A variant with a valine to glycine substitution at residue 72 in the lid structure exhibited higher activity and enantioselectivity than those of wild-type PEL. Site-directed saturation mutagenesis was used to explore the sequence-function relationship and the substitution of Val72 of P. expansum lipase changed both catalytic activity and enantioselectivity greatly. The variant V72A, displayed a highest enantioselectivity enhanced to about twofold for the resolution of (R, S)-naproxen (E value increased from 104 to 200.7 for wild-type PEL and V72A variant, respectively). In comparison to PEL, the variant V72A showed a remarkable increase in specific activity towards p-nitrophenyl palmitate (11- and 4-fold increase at 25 and 35 °C, respectively) whereas it had a decreased thermostability. The results suggest that the enantioselective variant V72A could be used for the production of pharmaceutical drugs such as enantiomerically pure (S)-naproxen and the residue Val 72 of P. expansum lipase plays a significant role in the enantioselectivity and activity of this enantioselective lipase. PMID:22972595

  20. Functional characterization of a novel aspartic acid rich lipase, TALipC, from Trichosporon asahii MSR54: solvent-dependent enantio inversion during esterification of 1-phenylethanol.

    PubMed

    Kumari, Arti; Gupta, Rani

    2015-01-01

    A novel lipase gene TAlipC was isolated from Trichosporon asahii MSR54 and functionally expressed in Pichia pastoris. The protein was His-tagged and purified to homogeneity by affinity chromatography. Sequence comparison revealed a high homology with lipases from Cryptococcus sp. It had a GX type oxyanion hole and a GHSLG-type conserved penta-peptide similar to those in the lipases from Yarrowia lipolytica. The enzyme had optimal activity at pH 8 and 50 °C. It was specific for long chain fatty acid groups of p-nitrophenol esters and triacylglycerols, showing regio- and enantio-selectivity. It was activated by Mg(2+) ions (20 mM) and had a predicted Mg-binding domain at the aspartic acid-rich C-terminal. Solvent-based enantio- inversion was the key feature of the enzyme where it showed (S)-selectivity in 1,4-dioxane and 2-propanol and (R)-selectivity in hexane during chiral separation of (±)1-phenylethanol by esterification. PMID:25214220

  1. A solid-state bioprocess for selecting lipase-producing filamentous fungi.

    PubMed

    Colla, Luciane Maria; Rezzadori, Kátia; Câmara, Stela Kochenborger; Debon, Janaina; Tibolla, Márcia; Bertolin, Telma Elita; Costa, Jorge Alberto Vieira

    2009-01-01

    A solid-state bioprocess with wheat bran and rice husk as substrate was used to isolate filamentous fungi with lipase activity from dairy effluent and soil contaminated with diesel oil. The lipase activity was measured in units, with one unit (U) being defined as the amount of enzyme required to liberate 1 micromol of fatty acids per minute per gram of bran substrate (1 U = 1 micromol min(-1) g(-1)). We obtained 24 isolates of filamentous fungi with lipase activity, 17 from the dairy effluent and 7 from the diesel oil-contaminated soil. The best lipase producers were the dairy effluent isolate Aspergillus E-6, with a maximum lipase activity of 49.81 U, and Aspergillus isolate O-4 recovered from the diesel oil-contaminated soil, with a maximum lipase activity of 45.49 U. Both isolates produced their maximum lipase activity eight days after the start of the bioprocess. PMID:19323278

  2. PLTP activity in premenopausal women. Relationship with lipoprotein lipase, HDL, LDL, body fat, and insulin resistance.

    PubMed

    Murdoch, S J; Carr, M C; Hokanson, J E; Brunzell, J D; Albers, J J

    2000-02-01

    Plasma phospholipid transfer protein (PLTP) is thought to play a major role in the facilitated transfer of phospholipids between lipoproteins and in the modulation of high density lipoprotein (HDL) particle size and composition. However, little has been reported concerning the relationships of PLTP with plasma lipoprotein parameters, lipolytic enzymes, body fat distribution, insulin, and glucose in normolipidemic individuals, particularly females. In the present study, 50 normolipidemic healthy premenopausal females were investigated. The relationships between the plasma PLTP activity and selected variables were assessed. PLTP activity was significantly and positively correlated with low density lipoprotein (LDL) cholesterol (r(s) = 0.53), apoB (r(s) = 0.44), glucose (r(s) = 0.40), HDL cholesterol (r(s) = 0.38), HDL(3) cholesterol (r(s) = 0.37), lipoprotein lipase activity (r(s) = 0.36), insulin (r(s) = 0.33), subcutaneous abdominal fat (r(s) = 0.36), intra-abdominal fat (r(s) = 0.29), and body mass index (r(s) = 0.29). HDL(2) cholesterol, triglyceride, and hepatic lipase were not significantly related to PLTP activity. As HDL(2) can be decreased by hepatic lipase and hepatic lipase is increased in obesity with increasing intra-abdominal fat, the participants were divided into sub-groups of non-obese (n = 35) and obese (n = 15) individuals and the correlation of PLTP with HDL(2) cholesterol was re-examined. In the non-obese subjects, HDL(2) cholesterol was found to be significantly and positively related to PLTP activity (r(s) = 0.44). Adjustment of the HDL(2) values for the effect of hepatic lipase activity resulted in a significant positive correlation between PLTP and HDL(2) (r(s) = 0.41), indicating that the strength of the relationship between PLTP activity and HDL(2) can be reduced by the opposing effect of hepatic lipase on HDL(2) concentrations. We conclude that PLTP-facilitated lipid transfer activity is related to HDL and LDL metabolism, as well as

  3. Organic Solvent Tolerant Lipases and Applications

    PubMed Central

    Kanwar, Shamsher S.

    2014-01-01

    Lipases are a group of enzymes naturally endowed with the property of performing reactions in aqueous as well as organic solvents. The esterification reactions using lipase(s) could be performed in water-restricted organic media as organic solvent(s) not only improve(s) the solubility of substrate and reactant in reaction mixture but also permit(s) the reaction in the reverse direction, and often it is easy to recover the product in organic phase in two-phase equilibrium systems. The use of organic solvent tolerant lipase in organic media has exhibited many advantages: increased activity and stability, regiospecificity and stereoselectivity, higher solubility of substrate, ease of products recovery, and ability to shift the reaction equilibrium toward synthetic direction. Therefore the search for organic solvent tolerant enzymes has been an extensive area of research. A variety of fatty acid esters are now being produced commercially using immobilized lipase in nonaqueous solvents. This review describes the organic tolerance and industrial application of lipases. The main emphasis is to study the nature of organic solvent tolerant lipases. Also, the potential industrial applications that make lipases the biocatalysts of choice for the present and future have been presented. PMID:24672342

  4. Lipases in lipophilization reactions.

    PubMed

    Villeneuve, Pierre

    2007-01-01

    Lipases are used in various sectors, as pharmaceutical, food or detergency industry. Their advantage versus classical chemical catalysts is that they exhibit a better selectivity and operate in milder reaction conditions. Theses enzymes can also be used in lipophilization reactions corresponding to the grafting of a lipophilic moiety to a hydrophilic one such as sugar, amino acids and proteins, or phenolic compounds. The major difficulty to overcome in such enzyme-catalyzed reaction resides in the fact that the two involved substrates greatly differ in term of polarity and solvent affinity. Therefore, several key parameters are to be considered in order to achieve the reaction in satisfactory kinetics and yields. The present review discusses the nature of such parameters (eg solvent nature, water activity, chemical modification of substrates) and illustrates their effect with examples of lipase-catalyzed lipophilization reactions of various sugar, amino acids or phenolic derivatives. PMID:17681737

  5. Effects of different dietary lipid contents on growth and lipase activity of Eriocheir sinensis larvae

    NASA Astrophysics Data System (ADS)

    Zheng, Debin; Pan, Luqing; Fang, Bo

    2011-03-01

    The effects of different dietary lipid content on the growth and lipase activity of Eriocheir sinensis larvae were studied in the paper. The results showed that the survival, metamorphic rate and weight gain of E. sinensis larvae at different stages of growth all varied significantly with lipid content ( P<0.05). Further, the survival and metamorphosis rates were the highest during the larval phases Z3 to Z4, and the weight gain was the highest during the larval phases Z5 to M. During the first 20 h after metamorphosis of every larval stage, the lipase activity increased over time at Z1, Z2, Z3 and M and declined at Z4 and Z5, and was influenced significantly by lipid content ( P<0.05). In addition, lipase activity at each larval stage began to respond to dietary lipid contents 4 h after the larvae were fed, and tended to be stable after 12 h. The diets with higher lipase activity and lower lipid content were selected to give the suitable recipe of lipid requirements at each larval stage. It was concluded that the suitable lipid requirements at Z1, Z2, Z3, Z4, Z5 and M were 6%, 4%, 6%, 8%, 8% and 10%, respectively.

  6. EFFECT OF WATER POLLUTANTS AND OTHER CHEMICALS UPON THE ACTIVITY OF LIPASE 'IN VITRO'

    EPA Science Inventory

    Lipase preparations were treated in vitro with 100 chemicals of various classes, many of which are environmental pollutants, to determine their effect upon enzyme activity. The greatest inhibition was caused by mercuric ion and certain heavy metal cations; almost as inhibiting we...

  7. Predicting lipase types by improved Chou's pseudo-amino acid composition.

    PubMed

    Zhang, Guang-Ya; Li, Hong-Chun; Gao, Jia-Qiang; Fang, Bai-Shan

    2008-01-01

    By proposing a improved Chou's pseudo amino acid composition approach to extract the features of the sequences, a powerful predictor based on k-nearest neighbor was introduced to identify the types of lipases according to their sequences. To avoid redundancy and bias, demonstrations were performed on a dataset where none of the proteins has > or =25% sequence identity to any other. The overall success rate thus obtained by the 10-fold cross-validation test was over 90%, indicating that the improved Chou's pseudo amino acid composition might be a useful tool for extracting the features of protein sequences, or at lease can play a complementary role to many of the other existing approaches. PMID:19075826

  8. Activation of Immobilized Lipase in Non-Aqueous Systems by Hydrophobic Poly-DL-Tryptophan Tethers

    PubMed Central

    Schilke, Karl F.; Kelly, Christine

    2014-01-01

    Many industrially important reactions use immobilized enzymes in non-aqueous, organic systems, particularly for the production of chiral compounds such as pharmaceutical precursors. The addition of a spacer molecule (“tether”) between a supporting surface and enzyme often substantially improves the activity and stability of enzymes in aqueous solution. Most “long” linkers (e.g. polyethylene oxide derivatives) are relatively hydrophilic, improving the solubility of the linker-enzyme conjugate in polar environments, but this provides little benefit in non-polar environments such as organic solvents. We present a novel method for the covalent immobilization of enzymes on solid surfaces using a long, hydrophobic polytryptophan tether. Candida antarctica lipase B (CALB) was covalently immobilized on non-porous, functionalized 1-μm silica microspheres, with and without an intervening hydrophobic poly-DL-tryptophan tether (n ≈ 78). The polytryptophan-tethered enzyme exhibited 35 times greater esterification of n-propanol with lauric acid in the organic phase and five times the hydrolytic activity against pnitrophenol palmitate, compared to the activity of the same enzyme immobilized without tethers. In addition, the hydrophobic tethers caused the silica microspheres to disperse more readily in the organic phase, while the surface-immobilized control treatment was less lipophilic and quickly settled out of the organic phase when the suspensions were not vigorously mixed. PMID:18393315

  9. Stability and activity of lipase in subcritical 1,1,1,2-tetrafluoroethane (R134a).

    PubMed

    Yu, Gang; Xue, Yong; Xu, Wei; Zhang, Jing; Xue, Chang Hu

    2007-12-01

    The stability and activity of commercial immobilized lipase from Candida antarctica (Novozym 435) in subcritical 1,1,1,2-tetrafluoroethane (R134a) was investigated. The esterification of oleic acid with glycerol was studied as a model reaction in subcritical R134a and in solvent-free conditions. The results indicated that subcritical R134a treatment led to significant increase of activity of Novozym 435, and a maximum residual activity of 300% was measured at 4 MPa, 30 degrees C after 7 h incubation. No deactivation of Novozym 435 treated with subcritical R134a under different operation factors (pressure 2-8 MPa, temperature 30-60 degrees C, incubation time 1-12 h, water content 1:1, 1:2, 1:5 enzyme/water, depressurization rate 4 MPa/1 min, 4 MPa/30 min, 4 MPa/90 min) was observed. While the initial reaction rate was high in subcritical R134a, higher conversion was obtained in solvent-free conditions. Though the apparent conversion of the reaction is lower in subcritical R134a, it is more practicable, especially at low enzyme concentrations desired at commercial scales. PMID:17909872

  10. Simple amino acid tags improve both expression and secretion of Candida antarctica lipase B in recombinant Escherichia coli.

    PubMed

    Kim, Sun-Ki; Park, Yong-Cheol; Lee, Hyung Ho; Jeon, Seung Taeg; Min, Won-Ki; Seo, Jin-Ho

    2015-02-01

    Escherichia coli is the best-established microbial host strain for production of proteins and chemicals, but has a weakness for not secreting high amounts of active heterologous proteins to the extracellular culture medium, of which origins belong to whether prokaryotes or eukaryotes. In this study, Candida antarctica lipase B (CalB), a popular eukaryotic enzyme which catalyzes a number of biochemical reactions and barely secreted extracellularly, was expressed functionally at a gram scale in culture medium by using a simple amino acid-tag system of E. coli. New fusion tag systems consisting of a pelB signal sequence and various anion amino acid tags facilitated both intracellular expression and extracellular secretion of CalB. Among them, the N-terminal five aspartate tag changed the quaternary structure of the dimeric CalB and allowed production of 1.9 g/L active CalB with 65 U/mL activity in culture medium, which exhibited the same enzymatic properties as the commercial CalB. This PelB-anion amino acid tag-based expression system for CalB can be extended to production of other industrial proteins hardly expressed and exported from E. coli, thereby increasing target protein concentrations and minimizing purification steps. PMID:25182473

  11. 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. PMID:26340506

  12. Development and validation of a lipase nasogastric tube position test

    PubMed Central

    Anderson, Oliver; Carr, Reuben; Harbinson, Merrilee; Hanna, George Bushra

    2016-01-01

    Background Nasogastric tube position should be checked every day by either aspirate pH or chest radiography to prevent fatal misplaced feeding into the lungs. Many patients do not have acidic gastric aspirates and require daily chest radiographs. We developed and validated a lipase test that was compatible with non-acidic gastric aspirates. Methods We conducted evaluations of diagnostic test accuracy at a teaching hospital in development and validation stages. Development: We collected gastric and lung aspirates from 34 consecutive patients. We measured pH and human gastric lipase activity in the laboratory. These data helped us develop the lipase test. Ingenza Ltd (Roslin, Scotland) created tributyrin-coated pH test paper, which human gastric lipase converted into butyric acid, thus correcting false negatives. Validation: We tested nasogastric feeding tube aspirates from 36 consecutive patients with pH and lipase tests, using chest radiography or trial by use as the reference standard. Results Development: We demonstrated human gastric lipase activity in the non-acidic stomach aspirates. Validation: The accuracy of the lipase test (sensitivity 97.2%, specificity 100%) was significantly better than pH (sensitivity 65.7%, specificity 100%, p<0.05). Conclusions When nasogastric tube stomach aspirates were not acidic and pH was falsely negative, the lipase test showed a true positive and was significantly more accurate. PMID:26966548

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

    PubMed

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

    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

  14. 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. PMID:26078966

  15. 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

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

    PubMed Central

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

    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

  17. Antibiofilm Properties of Interfacially Active Lipase Immobilized Porous Polycaprolactam Prepared by LB Technique

    PubMed Central

    Prabhawathi, Veluchamy; Boobalan, Thulasinathan; Sivakumar, Ponnurengam Malliappan; Doble, Mukesh

    2014-01-01

    Porous biomaterial is the preferred implant due to the interconnectivity of the pores. Chances of infection due to biofilm are also high in these biomaterials because of the presence of pores. Although biofilm in implants contributes to 80% of human infections [1], there are no commercially available natural therapeutics against it. In the current study, glutaraldehyde cross linked lipase was transferred onto a activated porous polycaprolactam surface using Langmuir-Blodgett deposition technique, and its thermostability, slimicidal, antibacterial, biocompatibility and surface properties were studied. There was a 20% increase in the activity of the covalently crosslinked lipase when compared to its free form. This immobilized surface was thermostable and retained activity and stability until 100°C. There was a 2 and 7 times reduction in carbohydrate and 9 and 5 times reduction in biofilm protein of Staphylococcus aureus and Escherichia coli respectively on lipase immobilized polycaprolactam (LIP) when compared to uncoated polycaprolactam (UP). The number of live bacterial colonies on LIP was four times less than on UP. Lipase acted on the cell wall of the bacteria leading to its death, which was confirmed from AFM, fluorescence microscopic images and amount of lactate dehydrogenase released. LIP allowed proliferation of more than 90% of 3T3 cells indicating that it was biocompatible. The fact that LIP exhibits antimicrobial property at the air-water interface to hydrophobic as well as hydrophilic bacteria along with lack of cytotoxicity makes it an ideal biomaterial for biofilm prevention in implants. PMID:24798482

  18. Activation and deformation of immobilized lipase on self-assembled monolayers with tailored wettability.

    PubMed

    Chen, Peng-Cheng; Huang, Xiao-Jun; Xu, Zhi-Kang

    2015-05-28

    In this work, lipase from Candida rugosa (CRL) was immobilized on self-assembled monolayers (SAMs) with various wettabilities ranging from highly hydrophilic to highly hydrophobic by adsorption in order to clearly elucidate the interfacial activation character of lipases. The SAMs were made of 11-hydroxyundecane-1-thiol and 1-dodecanethiol. The adsorption behavior was monitored in situ by quartz crystal microbalance with dissipation (QCM-D), and the enzyme binding constants indicated a stronger affinity between CRL and more hydrophobic surfaces. Atomic force microscopy (AFM) and X-ray photoelectron spectroscopy (XPS) were used to characterize the morphologies of the adsorbed lipases. Amide I band attenuated total reflection/Fourier transformed infrared (ART/FTIR) spectroscopy showed an increasing fraction of intermolecular β-sheet content on surfaces with higher hydrophilicities. Moreover, liquid chromatography (LC) verified that the activity of CRL adsorbed on a hydrophobic surface was higher than that of CRL adsorbed on a hydrophilic surface. This work related the enzyme activity to the substrate properties, adsorption behavior, distribution, and morphology of lipases, helping to achieve the external control of both the immobilization process and enzyme utilization. PMID:25929434

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

    SciTech Connect

    Dousset, N.; Negre, A.; Salvayre, R.; Rogalle, P.; Dang, Q.Q.; Douste-Blazy, L.

    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.

  20. Contractions Activate Hormone-Sensitive Lipase in Rat Muscle by Protein Kinase C and Mitogen-Activated Protein Kinase

    PubMed Central

    Donsmark, Morten; Langfort, Jozef; Holm, Cecilia; Ploug, Thorkil; Galbo, Henrik

    2003-01-01

    Intramuscular triacylglycerol is an important energy store and is also related to insulin resistance. The mobilization of fatty acids from this pool is probably regulated by hormone-sensitive lipase (HSL), which has recently been shown to exist in muscle and to be activated by both adrenaline and contractions. Adrenaline acts via cAMP-dependent protein kinase (PKA). The signalling mediating the effect of contractions is unknown and was explored in this study. Incubated soleus muscles from 70 g male rats were electrically stimulated to perform repeated tetanic contractions for 5 min. The contraction-induced activation of HSL was abolished by the protein kinase C (PKC) inhibitors bisindolylmaleimide I and calphostin C and reduced 50 % by the mitogen-activated protein kinase kinase (MEK) inhibitor U0126, which also completely blocked extracellular signal-regulated kinase (ERK) 1 and 2 phosphorylation. None of the inhibitors reduced adrenaline-induced HSL activation in soleus muscle. Both phorbol-12-myristate-13-acetate (PMA), which activates PKC and, in turn, ERK, and caffeine, which increases intracellular Ca2+ without eliciting contraction, increased HSL activity. Activated ERK increased HSL activity in supernatant from basal but not from electrically stimulated muscle. In conclusion, in muscle, PKC can stimulate HSL through ERK. Contractions and adrenaline enhance muscle HSL activity by different signalling mechanisms. The effect of contractions is mediated by PKC, at least partly via the ERK pathway. PMID:12794177

  1. Enzymatic Synthesis of Furfuryl Alcohol Ester with Oleic Acid by Candida antarctica Lipase B and Its Kinetic Study

    NASA Astrophysics Data System (ADS)

    Sengupta, Avery; Dey, Tanmoy; Ghosh, Mahua; Ghosh, Jaydip; Ghosh, Santinath

    2012-08-01

    This study investigated the successful enzymatic production of furfuryl oleate and its detailed kinetic study by Michaelis-Menten model. Esterification of oleic acid and furfuryl alcohol by Candida antarctica lipase B (Novozym 435 preparation) in a solvent free system was studied in the present work at 1:1 molar ratio of furfuryl alcohol and oleic acid. About 99 % conversion (on the basis of oleic acid) has been achieved within 6 h at 5 % enzyme concentration. Ping-pong bi-bi mechanism (inhibition phenomenon taken into account) was applied to describe the ratios as a complex kinetic model. The kinetic parameters were determined using MATLAB language programme. The two initial rate constants KA and KB respectively were found out by different progress curves plotted with the help of MATLAB language programme. It was concluded from the results that furfuryl alcohol considerably inhibited the enzymatic reaction while oleic acid had negligible inhibitory effect. It was clearly seen that the initial rate was increased with the increase in the furfuryl alcohol concentration until 2 M/L after which there was a drop in the initial rate depicting the inhibitory effect of furfuryl alcohol. Surprisingly, it has been observed that addition of 0.1 mol of product activated the esterification reaction. Finally, the model was found to be statistically fitting well with the experimental data.

  2. Adaptational properties and applications of cold-active lipases from psychrophilic bacteria.

    PubMed

    Maiangwa, Jonathan; Ali, Mohd Shukuri Mohamad; Salleh, Abu Bakar; Rahman, Raja Noor Zaliha Raja Abd; Shariff, Fairolniza Mohd; Leow, Thean Chor

    2015-03-01

    Psychrophilic microorganisms are cold-adapted with distinct properties from other thermal classes thriving in cold conditions in large areas of the earth's cold environment. Maintenance of functional membranes, evolving cold-adapted enzymes and synthesizing a range of structural features are basic adaptive strategies of psychrophiles. Among the cold-evolved enzymes are the cold-active lipases, a group of microbial lipases with inherent stability-activity-flexibility property that have engaged the interest of researchers over the years. Current knowledge regarding these cold-evolved enzymes in psychrophilic bacteria proves a display of high catalytic efficiency with low thermal stability, which is a differentiating feature with that of their mesophilic and thermophilic counterparts. Improvement strategies of their adaptive structural features have significantly benefited the enzyme industry. Based on their homogeneity and purity, molecular characterizations of these enzymes have been successful and their properties make them unique biocatalysts for various industrial and biotechnological applications. Although, strong association of lipopolysaccharides from Antarctic microorganisms with lipid hydrolases pose a challenge in their purification, heterologous expression of the cold-adapted lipases with affinity tags simplifies purification with higher yield. The review discusses these cold-evolved lipases from bacteria and their peculiar properties, in addition to their potential biotechnological and industrial applications. PMID:25472009

  3. 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

  4. SorLA regulates the activity of lipoprotein lipase by intracellular trafficking.

    PubMed

    Klinger, Stine C; Glerup, Simon; Raarup, Merete K; Mari, Muriel C; Nyegaard, Mette; Koster, Gerbrand; Prabakaran, Thaneas; Nilsson, Stefan K; Kjaergaard, Maj M; Bakke, Oddmund; Nykjær, Anders; Olivecrona, Gunilla; Petersen, Claus Munck; Nielsen, Morten S

    2011-04-01

    Many different tissues and cell types exhibit regulated secretion of lipoprotein lipase (LPL). However, the sorting of LPL in the trans Golgi network has not, hitherto, been understood in detail. Here, we characterize the role of SorLA (officially known as SorLA-1 or sortilin-related receptor) in the intracellular trafficking of LPL. We found that LPL bound to SorLA under neutral and acidic conditions, and in cells this binding mainly occurred in vesicular structures. SorLA expression changed the subcellular distribution of LPL so it became more concentrated in endosomes. From the endosomes, LPL was further routed to the lysosomes, which resulted in a degradation of newly synthesized LPL. Consequently, an 80% reduction of LPL activity was observed in cells that expressed SorLA. By analogy, SorLA regulated the vesicle-like localization of LPL in primary neuronal cells. Thus, LPL binds to SorLA in the biosynthetic pathway and is subsequently transported to endosomes. As a result of this SorLA mediated-transport, newly synthesized LPL can be routed into specialized vesicles and eventually sent to degradation, and its activity thereby regulated. PMID:21385844

  5. Gastric lipase: localization of the enzyme in the stomach

    SciTech Connect

    DeNigris, S.J.; Hamosh, M.; Hamosh, P.; Kasbekar, D.K.

    1986-03-05

    Isolated gastric glands prepared from human and rabbit stomach secrete lipase in response to secretagogues. They have investigated the localization of this enzyme in three species (rabbit, baboon, guinea pig). Gastric mucosa was sampled from the cardia (C), fundus-smooth (FS), fundus-ruggae (FR) and the antral area (A). Lipase activity was measured in mucosal homogenates using /sup 3/H-triolein as substrate and is expressed in units (U) = nmols free fatty acid released/min/mg wet weight. The localization of lipase is compared with that of pepsin (measured by hydrolysis of 2% hemoglobin at pH 1.8 and expressed in I.U.). Lipase is localized in a well defined area in the rabbit and is diffusely distributed in both guinea pig and baboon. The distribution of lipase and pepsin containing cells differs in all three species. The cellular origin of gastric lipase remains to be determined.

  6. Biocatalytic methanolysis activities of cross-linked protein-coated microcrystalline lipase toward esterification/transesterification of relevant palm products.

    PubMed

    Raita, Marisa; Laosiripojana, Navadol; Champreda, Verawat

    2015-03-01

    Biocatalysis by immobilized lipase is an efficient alternative process for conversion of crude vegetable oil with high free fatty acid content to biodiesel, which is the limit of the conventional alkaline-catalyzed reaction. In this study, influences of solid-state organic and inorganic buffer core matrices with different pKa on catalytic performance of cross-linked protein coated microcrystalline biocatalysts prepared from Thermomyces lanuginosus lipase (CL-PCMC-LIP) toward esterification of palmitic acid (PA), transesterification of refined palm oil (RPO), and co-ester/transesterification of crude palm oil (CPO) to fatty acid methyl ester (FAME) was studied. Glycine, CAPSO (3-(cyclohexylamino)-2-hydroxy-1-propanesulfonic acid), and TAPS ([(2-hydroxy-1,1-bis(hydroxymethyl)ethyl)amino]-1-propanesulfonic acid) were shown to be potent core matrices for these reactions. The optimal reaction contained 4:1 [methanol]/[fatty acid] molar equivalence ratio with 20% (w/w) CL-PCMC-LIP on glycine in the presence of tert-butanol as a co-solvent. Deactivation effect of glycerol on the biocatalyst reactive surface was shown by FTIR, which could be alleviated by increasing co-solvent content. The maximal FAME yields from PA, RPO, and CPO reached 97.6, 94.9, and 95.5%, respectively on a molar basis under the optimum conditions after incubation at 50°C for 6h. The biocatalyst retained >80% activity after recycling in five consecutive batches. The work demonstrates the potential of CL-PCMC-LIP on one-step conversion of inexpensive crude fatty acid-rich feedstock to biodiesel. PMID:25659629

  7. Altered Skeletal Muscle Lipase Expression and Activity Contribute to Insulin Resistance in Humans

    PubMed Central

    Badin, Pierre-Marie; Louche, Katie; Mairal, Aline; Liebisch, Gerhard; Schmitz, Gerd; Rustan, Arild C.; Smith, Steven R.; Langin, Dominique; Moro, Cedric

    2011-01-01

    OBJECTIVE Insulin resistance is associated with elevated content of skeletal muscle lipids, including triacylglycerols (TAGs) and diacylglycerols (DAGs). DAGs are by-products of lipolysis consecutive to TAG hydrolysis by adipose triglyceride lipase (ATGL) and are subsequently hydrolyzed by hormone-sensitive lipase (HSL). We hypothesized that an imbalance of ATGL relative to HSL (expression or activity) may contribute to DAG accumulation and insulin resistance. RESEARCH DESIGN AND METHODS We first measured lipase expression in vastus lateralis biopsies of young lean (n = 9), young obese (n = 9), and obese-matched type 2 diabetic (n = 8) subjects. We next investigated in vitro in human primary myotubes the impact of altered lipase expression/activity on lipid content and insulin signaling. RESULTS Muscle ATGL protein was negatively associated with whole-body insulin sensitivity in our population (r = −0.55, P = 0.005), whereas muscle HSL protein was reduced in obese subjects. We next showed that adenovirus-mediated ATGL overexpression in human primary myotubes induced DAG and ceramide accumulation. ATGL overexpression reduced insulin-stimulated glycogen synthesis (−30%, P < 0.05) and disrupted insulin signaling at Ser1101 of the insulin receptor substrate-1 and downstream Akt activation at Ser473. These defects were fully rescued by nonselective protein kinase C inhibition or concomitant HSL overexpression to restore a proper lipolytic balance. We show that selective HSL inhibition induces DAG accumulation and insulin resistance. CONCLUSIONS Altogether, the data indicate that altered ATGL and HSL expression in skeletal muscle could promote DAG accumulation and disrupt insulin signaling and action. Targeting skeletal muscle lipases may constitute an interesting strategy to improve insulin sensitivity in obesity and type 2 diabetes. PMID:21498783

  8. Glycerol Ester Hydrolase Activity of Lactic Acid Bacteria

    PubMed Central

    Oterholm, Anders; Ordal, Z. John; Witter, Lloyd D.

    1968-01-01

    Seventeen strains of lactic acid bacteria were assayed for their glycerol ester hydrolase activity by using an improved agar-well technique, and eight strains by determining the activity in cell-free extracts using a pH-stat procedure. All cultures tested showed activity and hydrolyzed tributyrin more actively than they did tricaproin. The cell extract studies demonstrated that the cells contained intracellular esterases and lipases. The culture supernatant fluid was without activity. The lipase and the esterase differed in their relative activity to each other in the different extracts and in the ease by which they could be freed from the cellular debris. It is suggested that the lipase of these organisms is an endoenzyme and the esterase an ectoenzyme. PMID:5649866

  9. Inhibitory activities of pancreatic lipase and phosphodiesterase from Korean medicinal plant extracts.

    PubMed

    Lee, Yun Mi; Kim, Young Sook; Lee, Youngseop; Kim, Junghyun; Sun, Hang; Kim, Joo Hwan; Kim, Jin Sook

    2012-05-01

    To find new pancreatic lipase (triacylglycerol acylhydrolase, EC 3.1.1.3) inhibitors from natural products, 61 medicinal plants from Korea were screened for their antilipase activity for prevention of obesity. Dried and powdered plants were extracted three times with EtOH and extracts were obtained by removal of the solvent in vacuo. Lipase activity was determined by measuring the hydrolysis of p-nitrophenyl butyrate to p-nitrophenol. Also, the inhibitory effect was measured on phosphodiesterase (PDE), another therapeutic target for obesity. Of the extracts tested, Sorbus commixta (stem, leaf) and Viscum album (whole plant) exhibited antilipase activity (with IC(50) values of 29.6 µg/mL and 33.3 µg/mL, respectively) and only anti-PDE activity (IC(50) values of 20.08 µg/mL and 35.15 µg/mL, respectively). PMID:22069182

  10. Ionic liquid activated Bacillus subtilis lipase A variants through cooperative surface substitutions.

    PubMed

    Zhao, Jing; Jia, Ning; Jaeger, Karl-Erich; Bocola, Marco; Schwaneberg, Ulrich

    2015-10-01

    The interest in performing enzyme-catalyzed reactions in amphiphilic systems, e.g., imidazolium-based ionic liquids (ILs) or surfactants, has been increased over the past decades. Directed protein evolution has been successful in tailoring enzymes for desired properties. Herein, nine IL-resistant Bacillus subtilis lipase A variants, particularly an IL-activated variant M1 (M134N/N138S/L140S), were identified by directed evolution. For instance, variant M2 (M134R/L140S) showed almost doubled specific activity (16.9 vs. 9.4 U/mg) and resistance (233% vs. 111%) at 9 vol% 1-butyl-3-methylimidazolium trifluoromethanesulfonate ([C4 mim][TfO]) compared with wild-type. The specific activities and resistance of purified individual single and double variants have been studied in five different IL-aqueous mixtures. The re-activation of lipase variant M1 (not wild-type) at high IL concentration was attributed to the cooperative effect of three surface substitutions (M134N, N138S, L140S) near the substrate-binding cleft. The presence of IL/substrate clusters under assay conditions was likely related to the re-activation effect. This study provides first example of IL-activated lipase variant generated by protein engineering, and helps to better understand the protein-IL interaction. PMID:25899108

  11. Metal ion activated lipase from halotolerant Bacillus sp. VITL8 displays broader operational range.

    PubMed

    Balaji, Lavanya; Jayaraman, Gurunathan

    2014-06-01

    Lipase producing halo tolerant Bacillus sp. VITL8 was isolated from oil contaminated areas of Vellore. The identity of the organism was established by 16S rDNA sequence, in addition to the morphological and biochemical characterization. The purified enzyme (22kDa, 8680U/mg) exhibited optimal activity at pH 7.0 and 40°C and retained more than 50% of its activity in the NaCl concentration range of 0-3.0M, pH 6.0-10.0 and 10-60°C. Secondary structure analysis, using circular dichroism, revealed that the enzyme is composed of 38% α-helix and 29% β-turns. The lipase activity significantly increased in the presence of (1mM) Mn(2+) (139%), Ca(2+) (134%) and Mg(2+) (130%). Organic solvents such as butanol and acetonitrile (25%, v/v) enhanced the activity whereas DMSO (25% v/v) retained the activity. The Km of enzyme-p-Nitrophenyl palmitate complex was determined to be 191μM with a Vmax of 68μM/mg/min. Though halotolerant Bacillus sp. has been explored for hydrocarbon degradation, to our knowledge this is the first report on the lipase activity of the isolate. The characteristics of the enzyme presented in this report, imply broader operational range of the enzyme and therefore could be suitable for many of the industrial chemical processes. PMID:24704541

  12. Homologous yeast lipases/acyltransferases exhibit remarkable cold-active properties.

    PubMed

    Neang, Pisey M; Subileau, Maeva; Perrier, Véronique; Dubreucq, Eric

    2014-11-01

    Lipases/acyltransferases catalyse acyltransfer to various nucleophiles preferentially to hydrolysis even in aqueous media with high thermodynamic activity of water (a w >0.9). Characterization of hydrolysis and acyltransfer activities in a large range of temperature (5 to 80 °C) of secreted recombinant homologous lipases of the Pseudozyma antarctica lipase A superfamily (CaLA) expressed in Pichia pastoris, enlighten the exceptional cold-activity of two remarkable lipases/acyltransferases: CpLIP2 from Candida parapsilosis and CtroL4 from Candida tropicalis. The activation energy of the reactions catalysed by CpLIP2 and CtroL4 was 18-23 kJ mol(-1) for hydrolysis and less than 15 kJ mol(-1) for transesterification between 5 and 35 °C, while it was respectively 43 and 47 kJ mol(-1) with the thermostable CaLA. A remarkable consequence is the high rate of the reactions catalysed by CpLIP2 and CtroL4 at very low temperatures, with CpLIP2 displaying at 5 °C 65 % of its alcoholysis activity and 45 % of its hydrolysis activity at 30 °C. These results suggest that, within the CaLA superfamily and its homologous subgroups, common structural determinants might allow both acyltransfer and cold-active properties. Such biocatalysts are of great interest for the efficient synthesis or functionalization of temperature-sensitive lipid derivatives, or more generally to lessen the environmental impact of biocatalytic processes. PMID:24770385

  13. Fatty acid specificity of hormone-sensitive lipase. Implication in the selective hydrolysis of triacylglycerols.

    PubMed

    Raclot, T; Holm, C; Langin, D

    2001-12-01

    The selective mobilization of fatty acids from white fat cells depends on their molecular structure, in particular the degree of unsaturation. The present study was designed to examine if the release of fatty acids by hormone-sensitive lipase (HSL) in vitro i) is influenced by the amount of unsaturation, ii) depends on the temperature, and iii) could explain the selective pattern of fatty acid mobilization and notably the preferential mobilization of certain highly unsaturated fatty acids. Recombinant rat and human HSL were incubated with a lipid emulsion. The hydrolysis of 35 individual fatty acids, ranging in chain length from 12 to 24 carbon atoms and in unsaturation from 0 to 6 double bonds was measured. Fatty acid composition of in vitro released NEFA was compared with that of fat cell triacylglycerols (TAG), the ratio % NEFA/% TAG being defined as the relative hydrolysis. The relative hydrolysis of individual fatty acids differed widely, ranging from 0.44 (24:1n-9) to 1.49 (18:1n-7) with rat HSL, and from 0.38 (24:1n-9) to 1.67 (18:1n-7) with human HSL. No major difference was observed between rat and human HSL. The relative release was dependent on the number of double bonds according to chain length. The amount of fatty acid released by recombinant rat HSL was decreased but remained robust at 4 degrees C compared with 37 degrees C, and the relative hydrolysis of some individual fatty acids was affected. The relative hydrolysis of fatty acids moderately, weakly, and highly mobilized by adipose tissue in vivo was similar and close to unity in vitro. We conclude that i) the release of fatty acids by HSL is only slightly affected by their degree of unsaturation, ii) the ability of HSL to efficiently and selectively release fatty acids at low temperature could reflect a cold adaptability for poikilotherms or hibernators when endogenous lipids are needed, and iii) the selectivity of fatty acid hydrolysis by HSL does not fully account for the selective pattern of

  14. Comparing the effect of immobilization methods on the activity of lipase biocatalysts in ester hydrolysis.

    PubMed

    Costa, L; Brissos, V; Lemos, F; Ribeiro, F Ramôa; Cabral, J M S

    2008-06-01

    The activity of various lipases was compared, in both free and immobilized forms, using the kinetics of the hydrolysis reaction of p-nitrophenyl butyrate, which was followed with in situ UV/Vis diode array spectrophotometry. Several enzymes were used to catalyze the reaction, namely Candida antarctica lipase B and Fusarium solani pisi cutinase wildtype and three single-mutation variants. The enzymes were tested in three different forms: free, immobilized as cross-linked aggregates and supported on zeolite NaY. A simple kinetic model was used to allow a quantitative comparison of the behavior of the different catalysts. It was concluded that although immobilization reduces the activity of the enzyme, the zeolite offers a much higher specific activity when compared to the cross-linked aggregates, thus supplying a heterogeneous catalyst with promising catalytic properties. PMID:17940805

  15. Anti-obesity effect of Dioscorea nipponica Makino with lipase-inhibitory activity in rodents.

    PubMed

    Kwon, Chong-Suk; Sohn, Ho Yong; Kim, Sung Hee; Kim, Ji Hyun; Son, Kun Ho; Lee, Jeong Soon; Lim, Jin Kyu; Kim, Jong-Sang

    2003-07-01

    In the process of screening for pancreatic lipase inhibitors, which could be used as an anti-obesity measure, the methanol extract of Dioscorea nipponica Makino powder (DP) appeared to have potent inhibitory activity against porcine pancreatic lipase with an IC50 value of 5-10 microg/ml, where the enzyme activity was assayed by using 4-methylumbelliferyl oleate as a substrate. Further purification of active components present in the herb generated dioscin that belongs to the saponin family. Dioscin and its aglycone, diosgenin, both suppressed the time-dependent increase of blood triacylglycerol level when orally injected with corn oil to mice, suggesting their inhibitory potential against fat absorption. Sprague-Dawley rats fed on a high-fat diet containing 5% Dioscorea nipponica Makino and 40% beef tallow gained significantly less body weight and adipose tissue than control animals fed on a high-fat diet alone during an 8-week experimental period (P<0.05). PMID:12913286

  16. Enhanced esterification activity through interfacial activation and cross-linked immobilization mechanism of Rhizopus oryzae lipase in a nonaqueous medium.

    PubMed

    Kartal, Funda

    2016-07-01

    Interfacial activation via surfactant (Tween 80, Triton X-100) treatment was conducted to improve the esterification activity of Rhizopus oryzae lipase that had undergone immobilization through cross-linked enzyme aggregates (CLEA®) technique. Surfactant pretreated immobilized enzymes exhibited better esterification activity compared to free and non-pretreated immobilized enzyme (Control CLEAs) since higher conversion rates were obtained within shorter times. The superiority of surfactant pretreated CLEAs, especially Tween 80 pretreated CLEAs (T 80 PT CLEAs), were clearly pronounced when longer alcohols were used as substrates. Conversion values exceeded 90% for octyl octanoate, oleyl octanoate and oleyl oleate synthesis with T 80 PT CLEAs whereas Control CLEAs and free enzyme showed no activity. Maximum conversions were achieved in the case equal molars of the substrates or in the case excess of the alcohol to acid in cyclohexane. In solvent free medium containing equal molars of substrates the conversion rates were 85% and 87% with T 80 PT CLEAs respectively for octyl octanoate and oleyl oleate within 2 hours. T 80 PT CLEAs showed 59% of its original activity after 7 consecutive usage for oleyl oleate synthesis. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:899-904, 2016. PMID:27111483

  17. 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

  18. 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. PMID:26954083

  19. Potential pancreatic lipase inhibitory activity of an endophytic Penicillium species.

    PubMed

    Gupta, Mahiti; Saxena, Sanjai; Goyal, Dinesh

    2015-02-01

    Pancreatic lipase (PL) is considered as one of the safest target for diet-induced anti-obesity drug development. Orlistat is the only PL inhibitor approved for anti-obesity treatment till date. In the process of exploration of new PL inhibitors, we have screened culture filtrates of 70 endophytic fungi of medicinal plants using qualitative as well as quantitative in-vitro PL assays. The qualitative assays indicated potential PL inhibition in only three isolates, namely #57 TBBALM, #33 TBBALM and #1 CSSTOT. Only ethyl acetate extracts of the culture filtrates of these isolates exhibited the PL inhibition. #57 TBBLAM ethyl acetate extract of culture filtrate exhibited potential PL inhibition with an IC50 of 3.69 µg/ml which was comparable to the positive control, i.e. Orlistat exhibiting IC50 value of 2.73 µg/ml. Further molecular phylogenetic tools and morphological studies were used to identify the isolate #57 TBBALM as Penicillium species. PMID:24417211

  20. 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

  1. Beta-adrenergic blockade and lipoprotein lipase activity in rat tissues after acute exercise.

    PubMed

    Paulin, A; Lalonde, J; Deshaies, Y

    1991-10-01

    The present experiments were aimed at evaluating the acute effects of exercise on lipoprotein lipase (LPL) activity in untrained rats. The activity of LPL was measured in postheparin plasma (PHP) before and at various times after a 1-h run on a treadmill (22 m/min, O degrees grade). LPL in PHP was 50% below pre-exercise levels immediately and 3 h after the run but was increased 65% over resting levels 24 h postexercise. To further characterize the very early fall in LPL activity in response to exercise and to assess the possible involvement therein of the beta-adrenergic pathway, LPL in heart, vastus lateralis muscle (VLM), and white (WAT) and brown (BAT) adipose tissues was determined at rest and immediately after exercise in rats that were treated or not with nadolol (25 mg.kg-1.day-1 for 30 days). Immediately after 1 h of exercise, there was a reduction in total enzyme activity in WAT (40% below resting levels), BAT (-58%), VLM (-53%), and heart (-30%). Exercise reduced serum triacylglycerol levels (-64%) and doubled those of nonesterified fatty acids. beta-Adrenergic blockade did not affect any of these variables. Both exercise and nadolol lowered serum cholesterol levels by approximately 20%, but the effects were not additive. These results show that the global intravascular pool of LPL undergoes divergent, time-dependent alterations in response to a single bout of moderate exercise. The acute downregulation of postheparin plasma LPL immediately after exercise reflected a fall in the total enzyme pool of all tissues studied.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1681747

  2. Influence of Gender, Obesity, and Muscle Lipase Activity on Intramyocellular Lipids in Sedentary Individuals

    PubMed Central

    Moro, Cedric; Galgani, Jose E.; Luu, LanChi; Pasarica, Magdalena; Mairal, Aline; Bajpeyi, Sudip; Schmitz, Gerd; Langin, Dominique; Liebisch, Gerhard; Smith, Steven R.

    2009-01-01

    Context: Obesity and type 2 diabetes are associated with elevated intramyocellular lipids (IMCLs) and insulin resistance. Objective: We tested the hypothesis that skeletal muscle lipases activity could influence IMCL content (including diacylglycerol and ceramides). Design and Patients: The present study included 48 subjects with a wide range of age (19–68 yr) and body mass index (20–45 kg/m2) who underwent skeletal muscle biopsy, dual-energy x-ray absorptiometry and a hyperinsulinemic euglycemic clamp. Main Outcome Measures: Insulin sensitivity by hyperinsulinemic clamp, and intramyocellular triacylglycerol (IMTG), diacylglycerol (DAG), and ceramides content, and triacylglycerol and diacylglycerol hydrolase activities were measured in biopsies of vastus lateralis. IMCL was measured by 1H-magnetic resonance spectroscopy in a subgroup of 25 subjects. Multivariate regression analyses were performed to identify the main predictors of IMCL. Results: Body fat was the main predictor of IMTG independently of the method and the type of muscle; IMTG concentration was higher in females vs. males and obese vs. nonobese subjects. Muscle DAG and ceramides concentrations were elevated in obese and type 2 diabetic subjects and were not related to body fat and fasting free fatty acids, whereas a direct association with the ratio of diacylglycerol hydrolase to triacylglycerol hydrolase activity (an index of incomplete triacylglycerol hydrolysis) was observed, which explained 54 and 38% of the variance in DAG and ceramides (P < 0.001), respectively. DAG content was the main determinant of insulin resistance. Conclusions: These data suggest that intramyocellular DAG is an independent predictor of insulin resistance in humans and that its levels correlate with lipolytic enzymes activity in skeletal muscle but not with markers of adiposity. PMID:19531593

  3. Endothelial lipase is a major determinant of HDL level

    SciTech Connect

    Ishida, Tatsuro; Choi, Sungshin; Kundu, Ramendra K.; Hirata, Ken-Ichi; Rubin, Edward M.; Cooper, Allen D.; Quertermous, Thomas

    2003-01-30

    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.

  4. Inhibitory Activities of Cudrania tricuspidata Leaves on Pancreatic Lipase In Vitro and Lipolysis In Vivo

    PubMed Central

    Kim, Young Sook; Lee, Youngseop; Kim, Junghyun; Sohn, Eunjin; Kim, Chan Sik; Lee, Yun Mi; Jo, Kyuhyung; Shin, Sodam; Song, Yoojin; Kim, Joo Hwan; Kim, Jin Sook

    2012-01-01

    To identify effective herb to treat obesity, we screened 115 herbal extracts for inhibition of porcine pancreatic lipase (triacylg-ycerol acylhydrolase, EC 3.1.1.3) activity in vitro. Of the extracts tested, Cudrania tricuspidata leaves exhibited the most pronounced inhibitory effect on lipase activity with an IC50 value of 9.91 μg/mL. Antilipid absorption effects of C. tricuspidata leaves were examined in rats after oral administration of lipid emulsions containing 50 or 250 mg  C. tricuspidata/kg body weight. Plasma triacylglycerol levels 2 h after the oral administration of emulsions containing C. tricuspidata were significantly reduced compared to the untreated group (P < 0.05). These results suggest that C. tricuspidata leaves may be useful for the treatment of obesity. PMID:23365603

  5. Activity of Candida rugosa lipase immobilized on gamma-Fe2O3 magnetic nanoparticles.

    PubMed

    Dyal, Ansil; Loos, Katja; Noto, Mayumi; Chang, Seung W; Spagnoli, Chiara; Shafi, Kurikka V P M; Ulman, Abraham; Cowman, Mary; Gross, Richard A

    2003-02-19

    We report the stability and enzymatic activity of Candida rugosa Lipase (E.C.3.1.1.3) immobilized on gamma-Fe2O3 magnetic nanoparticles. The immobilization strategies were either reacting the enzyme amine group with a nanoparticle surface acetyl, or amine groups. In the former, the enzyme was attached through a C=N bond, while in the latter it was connected using glutaraldehyde. AFM images show an average particle size of 20 +/- 10 nm after deconvolution. The enzymatic activity of the immobilized lipase was determined by following the ester cleavage of p-nitrophenol butyrate. The covalently immobilized enzyme was stabile and reactive over 30 days. PMID:12580578

  6. Immobilization of Lipases on Alkyl Silane Modified Magnetic Nanoparticles: Effect of Alkyl Chain Length on Enzyme Activity

    PubMed Central

    Wang, Jiqian; Meng, Gang; Tao, Kai; Feng, Min; Zhao, Xiubo; Li, Zhen; Xu, Hai; Xia, Daohong; Lu, Jian R.

    2012-01-01

    Background Biocatalytic processes often require a full recycling of biocatalysts to optimize economic benefits and minimize waste disposal. Immobilization of biocatalysts onto particulate carriers has been widely explored as an option to meet these requirements. However, surface properties often affect the amount of biocatalysts immobilized, their bioactivity and stability, hampering their wide applications. The aim of this work is to explore how immobilization of lipases onto magnetite nanoparticles affects their biocatalytic performance under carefully controlled surface modification. Methodology/Principal Findings Magnetite nanoparticles, prepared through a co-precipitation method, were coated with alkyl silanes of different alkyl chain lengths to modulate their surface hydrophobicity. Candida rugosa lipase was then directly immobilized onto the modified nanoparticles through hydrophobic interaction. Enzyme activity was assessed by catalytic hydrolysis of p-nitrophenyl acetate. The activity of immobilized lipases was found to increase with increasing chain length of the alkyl silane. Furthermore, the catalytic activities of lipases immobilized on trimethoxyl octadecyl silane (C18) modified Fe3O4 were a factor of 2 or more than the values reported from other surface immobilized systems. After 7 recycles, the activities of the lipases immobilized on C18 modified nanoparticles retained 65%, indicating significant enhancement of stability as well through hydrophobic interaction. Lipase immobilized magnetic nanoparticles facilitated easy separation and recycling with high activity retaining. Conclusions/Significance The activity of immobilized lipases increased with increasing alkyl chain length of the alkyl trimethoxy silanes used in the surface modification of magnetite nanoparticles. Lipase stability was also improved through hydrophobic interaction. Alkyl silane modified magnetite nanoparticles are thus highly attractive carriers for enzyme immobilization

  7. Differential modulation of cytosolic lipases activities in liver and adipose tissue by high-carbohydrate diets.

    PubMed

    Rodrigues, Angélica Heringer; Moreira, Carolina Campos Lima; Mario, Érica Guilhen; de Souza Cordeiro, Letícia Maria; Avelar, Gleide Fernandes; Botion, Leida Maria; Chaves, Valéria Ernestânia

    2016-08-01

    Several studies have demonstrated that a high-fructose (FRUC) diet induces metabolic and haemodynamic abnormalities, known as the metabolic syndrome, which are characterised by obesity, glucose intolerance, insulin resistance, dyslipidaemia and hypertension. In this study, the effect of a FRUC diet (60 % fructose) for 8 weeks on the metabolism of lipids in liver and epididymal adipose tissue from Wistar rats was compared with the AIN-93M diet and the effects of the AIN-93M diet were compared with a chow diet. The FRUC diet induced marked increases in both hepatocyte lipid droplet volume and postprandial serum levels of triacylglycerol (TAG), but reduced the postprandial serum levels of insulin. The AIN-93M diet induced marked increases in the hepatocyte lipid droplet volume and the serum levels of insulin, without affecting the serum levels of TAG. We found that isocaloric substitution of cornstarch, dextrinised cornstarch and sucrose (AIN-93M diet) for fructose did not affect the hepatic VLDL-TAG secretion and adipose tissue glucose uptake, lipolysis and cytosolic lipases activities in rats. However, the high-fructose diet induced a severe steatosis in liver accompanied by a decrease in cytosolic lipases activities. In adipose tissue, the FRUC diet induced a decrease in the lipoprotein lipase activity, and an increase in lipogenesis. FRUC and AIN-93M diets induced changes in lipid homeostasis in liver and adipose tissue by distinct biochemical mechanisms. PMID:26874528

  8. Lipase-catalyzed process in an anhydrous medium with enzyme reutilization to produce biodiesel with low acid value.

    PubMed

    Azócar, Laura; Ciudad, Gustavo; Heipieper, Hermann J; Muñoz, Robinson; Navia, Rodrigo

    2011-12-01

    One major problem in the lipase-catalyzed production of biodiesel or fatty acid methyl esters (FAME) is the high acidity of the product, mainly caused by water presence, which produces parallel hydrolysis and esterification reactions instead of transesterification to FAME. Therefore, the use of reaction medium in absence of water (anhydrous medium) was investigated in a lipase-catalyzed process to improve FAME yield and final product quality. FAME production catalyzed by Novozym 435 was carried out using waste frying oil (WFO) as raw material, methanol as acyl acceptor, and 3Å molecular sieves to extract the water. The anhydrous conditions allowed the esterification of free fatty acids (FFA) from feedstock at the initial reaction time. However, after the initial esterification process, water absence avoided the consecutives reactions of hydrolysis and esterification, producing FAME mainly by transesterification. Using this anhydrous medium, a decreasing in both the acid value and the diglycerides content in the product were observed, simultaneously improving FAME yield. Enzyme reuse in the anhydrous medium was also studied. The use of the moderate polar solvent tert-butanol as a co-solvent led to a stable catalysis using Novozym 435 even after 17 successive cycles of FAME production under anhydrous conditions. These results indicate that a lipase-catalyzed process in an anhydrous medium coupled with enzyme reuse would be suitable for biodiesel production, promoting the use of oils of different origin as raw materials. PMID:21889401

  9. 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. PMID:27317984

  10. CHARACTERIZATION OF LEPTOSPIRAL LIPASE

    PubMed Central

    Patel, Virendra; Goldberg, Herbert S.; Blenden, Donald

    1964-01-01

    Patel, Virendra (University of Missouri, Columbia), Herbert S. Goldberg, and Donald Blenden. Characterization of leptospiral lipase. J. Bacteriol. 88:877–884. 1964.—A technique for leptospiral lipase extraction which yielded a highly active, stable, and concentrated lipase preparation was developed. The chief characteristics of leptospiral lipase were determined and are summarized below. Leptospiral lipase was soluble in water and stable in both the dry state and in aqueous solution. Tributyrin was found to be the substrate upon which the enzyme was most active. With this substrate, leptospiral lipase was found to display optimal activity at pH 7 and at 30 C. The Michaelis constant of leptospiral lipase with tributyrin substrate was determined to be 4.76 × 10-2m. The enzyme was not inhibited by low concentrations of mercury, iron, cobalt, or copper or by —SH blocking agents. Bile and calcium chloride in low concentrations were able to increase lipase activity at alkaline pH. The isoelectric point of leptospiral lipase was determined to be in the range of pH 5.2 to 5.4. PMID:14219049

  11. Chain length affects pancreatic lipase activity and the extent and pH-time profile of triglyceride lipolysis.

    PubMed

    Benito-Gallo, Paloma; Franceschetto, Alessandro; Wong, Jonathan C M; Marlow, Maria; Zann, Vanessa; Scholes, Peter; Gershkovich, Pavel

    2015-06-01

    Triglycerides (TG) are one of the most common excipients used in oral lipid-based formulations. The chain length of the TG plays an important role in the oral bioavailability of the co-administered drug. Fatty acid (FA) chain-length specificity of porcine pancreatic lipase was studied by means of an in vitro lipolysis model under bio-relevant conditions at pH 6.80. In order to determine the total extent of lipolysis, back-titration experiments at pH 11.50 were performed. Results suggest that there is a specific chain length range (C2-C8) for which pancreatic lipase shows higher activity. This specificity could result from a combination of physicochemical properties of TGs, 2-monoglycerides (2-MGs) and FAs, namely the droplet size of the TGs, the solubility of 2-MGs within mixed micelles, and the relative stability of the FAs as leaving groups in the hydrolysis reaction. During experimentation, it was evident that an optimisation of lipolysis conditions was needed for tighter control over pH levels so as to better mimic in vivo conditions. 1M NaOH, 3.5 mL/min maximum dosing rate, and 3 μL/min minimum dosing rate were the optimised set of conditions that allowed better pH control, as well as the differentiation of the lipolysis of different lipid loads. PMID:25936853

  12. Liquid crystal based sensors monitoring lipase activity: a new rapid and sensitive method for cytotoxicity assays.

    PubMed

    Hussain, Zakir; Zafiu, Christian; Küpcü, Seta; Pivetta, Lucineia; Hollfelder, Nadine; Masutani, Akira; Kilickiran, Pinar; Sinner, Eva-Kathrin

    2014-06-15

    In this work we present liquid crystal (LC) based sensor devices to monitor cell viability. The sensing layer is composed by the LC and a planar monolayer of phospholipids. In the presence of minute traces of phospholipases, which hydrolyze enzymatically phospholipids, the LC-lipid interface is disintegrated. This event causes a change in orientation of the LC, which was followed in a polarized microscope. The lipase activity can be used to measure the cell viability, since members of this enzyme family are released by cells, as they undergo necrosis. The described sensor was used to monitor the presence of the lipases released from three different cell lines, which were either exposed to highly cytotoxic model compounds (sodium azide and paracetamol) or subjected to freeze-thaw cycles to induce cell death by a non-chemical based inducer for apoptosis, such as temperature. Finally, the comparison of lipase activity detected by a state-of-the-art fluorescence assay to the LC based system resulted in the superiority of the LC system concerning incubation time and sensitivity. PMID:24508543

  13. Improvement in extraction and catalytic activity of Mucor javanicus lipase by modification of AOT reverse micelle.

    PubMed

    Talukder, Mahabubur Rahman; Susanto, Diana; Feng, Gao; Wu, Jinchuan; Choi, Won Jae; Chow, Yvonne

    2007-11-01

    Reverse micelles are formed in apolar solvents by spontaneous aggregation of surfactants. Surfactant sodium bis (2-ethylhexyl) sulfosuccinate (AOT) is most often used for the reverse micellar extraction of enzymes. However, the inactivation of enzyme due to strong interaction with AOT molecules is a severe problem. To overcome this problem, the AOT/water/isooctane reverse micellar system was modified by adding short chain polyethylene glycol 400 (PEG 400). The modified AOT reverse micellar system was used to extract Mucor javanicus lipase from the aqueous phase to the reverse micellar phase. The extraction efficiency (E) increased with the increase in PEG 400 addition and the maximum E in PEG 400 modified system was twofold higher than that in the PEG 400-free system. Upon addition of PEG 400, the water activity (a(w)) of aqueous phase decreased, whereas a(w) of reverse micellar phase increased. The circular dichroism spectroscopy analysis revealed that PEG 400 changes the secondary and tertiary structure of lipase. The maximum specific activity of lipase extracted in PEG 400-modified reverse micellar system was threefold higher than that in the PEG-free system. PMID:17639532

  14. Purification, characterization and preliminary X-ray diffraction analysis of a cold-active lipase (CpsLip) from the psychrophilic bacterium Colwellia psychrerythraea 34H.

    PubMed

    Do, Hackwon; Lee, Jun Hyuck; Kwon, Mi Hyun; Song, Hye Eun; An, Jun Yop; Eom, Soo Hyun; Lee, Sung Gu; Kim, Hak Jun

    2013-08-01

    The putative lipase CpsLip from the psychrophilic bacterium Colwellia psychrerythraea 34H encodes a 34,538 Da, 308-amino-acid protein. In this study, CpsLip (UniProtKB code Q486T5) was expressed as an N-terminal hexahistidine fusion protein in Escherichia coli and purified by affinity and size-exclusion chromatography. The expression and purification of CpsLip enabled characterization of the lipase enzymatic properties of the protein. The optimal activity temperature and pH of the recombinant protein were 298 K and pH 7, respectively. CpsLip maintained over 80% activity in the low-temperature range (278-288 K), thereby suggesting that CpsLip is a cold-active lipase. Substrate-specificity analysis demonstrated that CpsLip exhibits maximum activity towards the C12 acyl group. In addition, sequence-alignment results revealed that CpsLip has a highly conserved catalytic triad in the active site consisting of residues Ser111, Asp135 and His283. Moreover, purified CpsLip was successfully crystallized using the hanging-drop vapour-diffusion method and a complete diffraction data set was collected to 4.0 Å resolution using synchrotron radiation on the BL-5A beamline of the Photon Factory. PMID:23908044

  15. Stepwise esterification of phytosterols with conjugated linoleic acid catalyzed by Candida rugosa lipase in solvent-free medium.

    PubMed

    Torres, Carlos F; Torrelo, Guzman; Vazquez, Luis; Señorans, F Javier; Reglero, Guillermo

    2008-12-01

    We conducted a near quantitative esterification of phytosterols from soybean oil deodorizer distillate with conjugated linoleic acid. We used a 1:1 molar ratio of sterols to conjugated linoleic acid. For that matter, stepwise addition of sterols was investigated. Total sterols were divided into several portions and added sequentially to the reaction mixture. Using this methodology, purities of up to 80% steryl esters were obtained that consumed more than 90% of the total conjugated linoleic acid. In addition, the effects of temperature, amount, and stability of lipase were also evaluated. PMID:19134551

  16. Enantiomeric perylene-glycerolipids as fluorogenic substrates for a dual wavelength assay of lipase activity and stereoselectivity.

    PubMed

    Zandonella, G; Haalck, L; Spener, F; Faber, K; Paltauf, F; Hermetter, A

    1996-01-01

    A new type of fluorogenic alkyldiacyl glycerols was synthesized and used as fluorogenic substrates for the analysis of lipase activities and stereoselectivities. These compounds contain perylene as a fluorophore and the trinitrophenylamino (TNP) residue as a quencher. Both substituents are covalently bound to the omega-ends of the sn-2 and sn-1 (3) acyl chains, respectively. Upon glycerolipid hydrolysis, the residues are separated from each other thus allowing determination of lipase activity by the continuous increase in fluorescence intensity which is caused by dequenching. Using enantiomeric pairs of these compounds, we were able to analyze lipase stereoselectivity depending on the reaction medium. Mixtures of enantiomeric fluorogenic alkyldiacyl glycerols, selectively labelled with pyrene or perylene as fluorophores, can be used for a dual-wavelength "stereoassay" of lipases. Since absorption and emission maxima of both labels are clearly separated, hydrolysis of the respective enantiomeric substrates can be determined simultaneously, and the difference in the rates of hydrolysis can be taken as a parameter for the stereopreference of a lipase. Hydrolysis rates measured with perylene-substituted lipids are generally lower than those obtained with the pyrene analogs. Thus, with a mixture of perylene and pyrene-substituted lipids, we observe a higher apparent stereoselectivity of lipases since we measure a combination of stereo- and substrate selectivity. In the presence of albumin, all microbial lipases tested so far exhibit stereopreference for the sn-1 glycerol position. In our assay, the apparent stereoselectivities are highest if in the presence of albumin, the sn-1 position carries pyrene and the sn-3 position is substituted with perylene. The lipase stereoselectivity assay described here requires the simultaneous measurement of the fluorescence intensities at two different wavelengths in a single cuvette and can thus be carried out using existing and cheap

  17. Cloning and characterization of a new cold-active lipase from a deep-sea sediment metagenome.

    PubMed

    Jeon, Jeong Ho; Kim, Jun-Tae; Kim, Yun Jae; Kim, Hyung-Kwoun; Lee, Hyun Sook; Kang, Sung Gyun; Kim, Sang-Jin; Lee, Jung-Hyun

    2009-01-01

    To search for new cold-active lipases, a metagenomic library was constructed using cold-sea sediment samples at Edison Seamount and was screened for lipolytic activities by plating on a tricaprylin medium. Subsequently, a fosmid clone was selected, and the whole sequence of 36 kb insert of the fosmid clone was determined by shotgun sequencing. The sequence analysis revealed the presence of 25 open reading frames (ORF), and ORF20 (EML1) showed similarities to lipases. Phylogenetic analysis of EML1 suggested that the protein belonged to a new family of esterase/lipase together with LipG. The EML1 gene was expressed in Escherichia coli, and purified by metal-chelating chromatography. The optimum activity of the purified EML1 (rEML1) occurred at pH 8.0 and 25 degrees C, respectively, and rEML1 displayed more than 50% activity at 5 degrees C. The activation energy for the hydrolysis of olive oil was determined to be 3.28 kcal/mol, indicating that EML1 is a cold-active lipase. rEML1 preferentially hydrolyzed triacylglycerols acyl-group chains with long chain lengths of > or = 8 carbon atoms and displayed hydrolyzing activities toward various natural oil substrates. rEML1 was resistant to various detergents such as Triton X-100 and Tween 80. This study represents an example which developed a new cold-active lipase from a deep-sea sediment metagenome. PMID:18773201

  18. Structure-activity studies in the development of a hydrazone based inhibitor of adipose-triglyceride lipase (ATGL).

    PubMed

    Mayer, Nicole; Schweiger, Martina; Melcher, Michaela-Christina; Fledelius, Christian; Zechner, Rudolf; Zimmermann, Robert; Breinbauer, Rolf

    2015-06-15

    Adipose triglyceride lipase (ATGL) catalyzes the degradation of cellular triacylglycerol stores and strongly determines the concentration of circulating fatty acids (FAs). High serum FA levels are causally linked to the development of insulin resistance and impaired glucose tolerance, which eventually progresses to overt type 2 diabetes. ATGL-specific inhibitors could be used to lower circulating FAs, which can counteract the development of insulin resistance. In this article, we report about structure-activity relationship (SAR) studies of small molecule inhibitors of ATGL based on a hydrazone chemotype. The SAR indicated that the binding pocket of ATGL requests rather linear compounds without bulky substituents. The best inhibitor showed an IC50=10μM in an assay with COS7-cell lysate overexpressing murine ATGL. PMID:25778769

  19. Glucose ingestion blunts hormone-sensitive lipase activity in contracting human skeletal muscle.

    PubMed

    Watt, Matthew J; Krustrup, Peter; Secher, Niels H; Saltin, Bengt; Pedersen, Bente K; Febbraio, Mark A

    2004-01-01

    To examine the effect of attenuated epinephrine and elevated insulin on intramuscular hormone sensitivity lipase activity (HSLa) during exercise, seven men performed 120 min of semirecumbent cycling (60% peak pulmonary oxygen uptake) on two occasions while ingesting either 250 ml of a 6.4% carbohydrate (GLU) or sweet placebo (CON) beverage at the onset of, and at 15 min intervals throughout, exercise. Muscle biopsies obtained before and immediately after exercise were analyzed for HSLa. Blood samples were simultaneously obtained from a brachial artery and a femoral vein before and during exercise, and leg blood flow was measured by thermodilution in the femoral vein. Net leg glycerol and lactate release and net leg glucose and free fatty acid (FFA) uptake were calculated from these measures. Insulin and epinephrine were also measured in arterial blood before and throughout exercise. During GLU, insulin was elevated (120 min: CON, 11.4 +/- 2.4, GLU, 35.3 +/- 6.9 pM, P < 0.05) and epinephrine suppressed (120 min: CON, 6.1 +/- 2.5, GLU, 2.1 +/- 0.9 nM; P < 0.05) compared with CON. Carbohydrate feeding also resulted in suppressed (P < 0.05) HSLa relative to CON (120 min: CON, 1.71 +/- 0.18, GLU, 1.27 +/- 0.16 mmol.min-1.kg dry mass-1). There were no differences in leg lactate or glycerol release when trials were compared, but leg FFA uptake was lower (120 min: CON, 0.29 +/- 0.06, GLU, 0.82 +/- 0.09 mmol/min) and leg glucose uptake higher (120 min: CON, 3.16 +/- 0.59, GLU, 1.37 +/- 0.37 mmol/min) in GLU compared with CON. These results demonstrate that circulating insulin and epinephrine play a role in HSLa in contracting skeletal muscle. PMID:14506077

  20. Lysosomal acid lipase deficiency: diagnosis and treatment of Wolman and Cholesteryl Ester Storage Diseases.

    PubMed

    Porto, Anthony F

    2014-09-01

    Lysosomal acid lipase (LAL) is responsible for the hydrolysis of cholesterol esters and triglycerides. LAL is coded by the LIPA gene on chromosome 10q23.31. Its deficiency leads to two autosomal recessive disorders, Wolman disease (WD) and Cholesteryl Ester Storage Disease (CESD). WD has an estimated incidence of 1 in 500,000 live births and is the result of a complete loss of LAL and presents in infancy with vomiting, diarrhea, poor weight gain and hepatomegaly subsequently leading to death. CESD is the result of partial loss of LAL and its presentation is more variable. Patients may be asymptomatic or present with nonspecific gastrointestinal symptoms, hepatomegaly, elevated transaminases and dystipidemia which may be confused with the diagnosis of Non-alcoholic Fatty Liver Disease. CESD is currently underdiagnosed and has an estimated prevalence as high as I in 40,000 individuals. Radiologic findings in WD is calcification of the adrenal glands. Hepatomegaly is noted on CT scan in both WD and CESD. MRI may demonstrate accumulation of cholesterol esters and may be useful to study effects of potential medical therapies. The diagnosis of WD and CESD is based on LIPA gene sequencing and the measurement of LAL levels in peripheral blood leukocytes. Treatment of LAL deficiency is currently limited to control of cholesterol levels and to prevent premature atherosclerosis. Use of enzyme replacement therapy with recombinant human LAL in short-term studies has shown to be safe and effective. PMID:25345094

  1. Simultaneous production of fatty acid methyl esters and diglycerides by four recombinant Candida rugosa lipase's isozymes.

    PubMed

    Chang, Shu-Wei; Huang, Myron; Hsieh, Yu-Hsun; Luo, Ying-Ting; Wu, Tsung-Ta; Tsai, Chia-Wen; Chen, Chin-Shuh; Shaw, Jei-Fu

    2014-07-15

    In this study, the catalytic efficiency of four recombinant CRL (Candida rugosa lipase) isozymes (LIP1-LIP4) towards the production of fatty acid methyl ester (FAME) was compared and evaluated as an alternative green method for industrial applications. The results indicated that the recombinant C. rugosa LIP1 enzyme exhibited the highest catalytic efficiency for FAME production compared to the recombinant C. rugosa LIP2-LIP4 enzymes. The optimal conditions were as follows: pH 7.0, methanol/soybean oil molar ratio: 3/1, enzyme amount: 2U (1.6 μL), reaction temperature: 20°C, 22 h of reaction time, and 3 times of methanol addition (1 mol/6h), and resulted in 61.5 ± 1.5 wt.% of FAME conversion. The reaction product contained also 10 wt.% of DAG with a ratio of 1,3-DAG to 1,2-DAG of approximately 4:6, and can be potentially used in industrial applications as a food emulsifier. PMID:24594166

  2. Rapid progression and mortality of lysosomal acid lipase deficiency presenting in infants

    PubMed Central

    Jones, Simon A.; Valayannopoulos, Vassili; Schneider, Eugene; Eckert, Stephen; Banikazemi, Maryam; Bialer, Martin; Cederbaum, Stephen; Chan, Alicia; Dhawan, Anil; Di Rocco, Maja; Domm, Jennifer; Enns, Gregory M.; Finegold, David; Gargus, J. Jay; Guardamagna, Ornella; Hendriksz, Christian; Mahmoud, Iman G.; Raiman, Julian; Selim, Laila A.; Whitley, Chester B.; Zaki, Osama; Quinn, Anthony G.

    2016-01-01

    Purpose: The purpose of this study was to enhance understanding of lysosomal acid lipase deficiency (LALD) in infancy. Genet Med 18 5, 452–458. Methods: Investigators reviewed medical records of infants with LALD and summarized data for the overall population and for patients with and without early growth failure (GF). Kaplan–Meier survival analyses were conducted for the overall population and for treated and untreated patients. Genet Med 18 5, 452–458. Results: Records for 35 patients, 26 with early GF, were analyzed. Prominent symptom manifestations included vomiting, diarrhea, and steatorrhea. Median age at death was 3.7 months; estimated probability of survival past age 12 months was 0.114 (95% confidence interval (CI): 0.009-0.220). Among patients with early GF, median age at death was 3.5 months; estimated probability of survival past age 12 months was 0.038 (95% CI: 0.000-0.112). Treated patients (hematopoietic stem cell transplant (HSCT), n = 9; HSCT and liver transplant, n = 1) in the overall population and the early GF subset survived longer than untreated patients, but survival was still poor (median age at death, 8.6 months). Genet Med 18 5, 452–458. Conclusions: These data confirm and expand earlier insights on the progression and course of LALD presenting in infancy. Despite variations in the nature, onset, and severity of clinical manifestations, and treatment attempts, clinical outcome was poor. Genet Med 18 5, 452–458. PMID:26312827

  3. Conversion of sunflower oil to biodiesel by alcoholysis using immobilized lipase.

    PubMed

    Sagiroglu, Ayten

    2008-01-01

    Transesterification reaction was performed using sunflower oil and short-chain alcohol by immobilized lipases in organic solvents. The fatty acid ester, which is the product of this reaction, can be used as a diesel fuel that does not produce sulfur oxide and minimize the soot particulate. Immobilized porcine pancreatic lipase (PPL) and Candida rugosa lipase (CRL) showed the satisfactory activity in these reactions. Immobilization of lipases was carried out using inorganic absorbance Celit 545 particle as a carrier. Organic solvent like hexane in reactions was required when methanol and ethanol were used as alcoholic substrate. The reaction could be performed in absence of solvent when 1-propanol and 1-butanol were used as short-chain alcohol. The activities of immobilized lipases were highly increased in comparison with free lipases because its activity sites became more effective. Immobilized enzyme could be repeatedly used without difficult method of separation and the decrease in its activity was not largely observed. PMID:18437590

  4. 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

    2016-03-01

    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. PMID:26853559

  5. Metabolic engineering of Lactococcus lactis influence of the overproduction of lipase enzyme.

    PubMed

    Raftari, Mohammad; Ghafourian, Sobhan; Bakar, Fatimah Abu

    2013-11-01

    The dairy industry uses lipase extensively for hydrolysis of milk fat. Lipase is used in the modification of the fatty acid chain length, to enhance the flavours of various chesses. Therefore finding the unlimited source of lipase is a concern of dairy industry. Due to the importance of lipase, this study was an attempt to express the lipase from Burkholderia cepacia in Lactococcus lactis. To achieve this, a gene associated with lipase transport was amplified and subcloned in inducible pNZ8148 vector, and subsequently transformed into Lc. lactis NZ9000. The enzyme assay as well as SDS-PAGE and western blotting were carried out to analysis the recombinant lipase expression. Nucleotide sequencing of the DNA insert from the clone revealed that the lipase activity corresponded to an open reading frame consisting of 1092 bp coding for a 37·5-kDa size protein. Blue colour colonies on nile blue sulphate agar and sharp band on 37·5-kD size on SDS-PAGE and western blotting results confirm the successful expression of lipase by Lc. lactis. The protein assay also showed high expression, approximately 152·2 μg/ml.h, of lipase by recombinant Lc. lactis. The results indicate that Lc. lactis has high potential to overproduce the recombinant lipase which can be used commercially for industrially purposes. PMID:24063299

  6. Nutrient regulation of post-heparin lipoprotein lipase activity in obese subjects.

    PubMed

    Ranganath, L R; Beety, J M; Wright, J; Morgan, L M

    2001-01-01

    This study examines the immediate effect of ingestion of oral carbohydrate and fat on lipoprotein lipase (LPL) activity post-heparin in six lean and six obese age-matched women. Subjects were given, on two separate occasions, 340 kcal carbohydrate or an equicaloric amount of fat, both in 300 ml of water. Post-heparin LPL activity (10,000 U) was measured on each occasion 120 minutes after ingestion of the meal. Following oral carbohydrate postprandial plasma insulin levels were significantly higher in obese subjects than in lean (p < 0.01). Impaired glucose tolerance was seen in the obese group. GIP secretion was similar in lean and obese subjects both during oral fat and carbohydrate ingestion. GLP-1 secretion post-carbohydrate was lower in obese subjects. Total LPL activity unadjusted for body weight was similar in the two groups after carbohydrate administration but was significantly lower when adjusted per kg body weight. Total LPL activity was lower in the lean group at 130 minutes after fat administration (p < 0.02). Fasting serum triglycerides were higher in the obese group and were inversely related to the post-carbohydrate LPL activity (r = - 0.65, p < 0.02). Intraluminal lipoprotein lipase activity is not increased in established obesity. Fat and carbohydrate nutrients may affect LPL activity differently in lean and obese subjects. PMID:11280717

  7. Contribution of novel ATGL missense mutations to the clinical phenotype of NLSD-M: a strikingly low amount of lipase activity may preserve cardiac function

    PubMed Central

    Tavian, Daniela; Missaglia, Sara; Redaelli, Chiara; Pennisi, Elena M.; Invernici, Gloria; Wessalowski, Ruediger; Maiwald, Robert; Arca, Marcello; Coleman, Rosalind A.

    2012-01-01

    The lack of adipose triglyceride lipase (ATGL), a patatin-like phospholipase domain-containing enzyme that hydrolyzes fatty acids from triacylglycerol (TAG) stored in multiple tissues, causes the autosomal recessive disorder neutral lipid storage disease with myopathy (NLSD-M). In two families of Lebanese and Italian origin presenting with NLSD-M, we identified two new missense mutations in highly conserved regions of ATGL (p.Arg221Pro and p.Asn172Lys) and a novel nonsense mutation (p.Trp8X). The Lebanese patients harbor homozygous p.Arg221Pro, whereas the Italian patients are heterozygotes for p.Asn172Lys and the p.Trp8X mutation. The p.Trp8X mutation results in a complete absence of ATGL protein, while the p.Arg221Pro and p.Asn172Lys mutations result in proteins with minimal lipolytic activity. Although these mutations did not affect putative catalytic residues or the lipid droplet (LD)-binding domain of ATGL, cytosolic LDs accumulated in cultured skin fibroblasts from the patients. The missense mutations might destabilize a random coil (p.Asn172Lys) or a helix (p.Arg221Pro) structure within or proximal to the patatin domain of the lipase, thereby interfering with the enzyme activity, while leaving intact the residues required to localize the protein to LDs. Overexpressing wild-type ATGL in one patient's fibroblasts corrected the metabolic defect and effectively reduced the number and area of cellular LDs. Despite the poor lipase activity in vitro, the Lebanese siblings have a mild myopathy and not clinically evident myocardial dysfunction. The patients of Italian origin show a late-onset and slowly progressive skeletal myopathy. These findings suggest that a small amount of correctly localized lipase activity preserves cardiac function in NLSD-M. PMID:22990388

  8. Synthesis of some glucose-fatty acid esters by lipase from Candida antarctica and their emulsion functions.

    PubMed

    Ren, Kangzi; Lamsal, Buddhi P

    2017-01-01

    The synthesis of glucose esters with palmitic acid, lauric acid and hexanoic acid using lipase enzyme was studied and their emulsion functionality in oil-in-water system were compared. Reactions at 3:1M ratio of fatty acids-to-glucose had the highest conversion percentages (over 90% for each of the fatty acid). Initial conversion rate increased as substrate solubility increased. Ester bond formation was confirmed by nuclear magnetic resonance technique that the chemical shifts of glucose H-6 and α-carbon protons of fatty acids in the ester molecules shifted to the higher fields. Contact angle of water on esters' pelleted surface increased as the hydrophobicity increased. Glucose esters' and commercial sucrose esters' functionality as emulsifiers were compared. Glucose esters delayed, but did not prevent coalescence, because the oil droplets diameter doubled during 7days. Sucrose esters prevented coalescence during 7days since the droplets diameter did not have significant change. PMID:27507510

  9. Expression of lipases and lipid receptors in sperm storage tubules and possible role of fatty acids in sperm survival in the hen oviduct.

    PubMed

    Huang, A; Isobe, N; Obitsu, T; Yoshimura, Y

    2016-04-15

    The aim of this study was to determine the role of fatty acids for sperm survival in the sperm storage tubules (SSTs) of the hen oviduct. The mucosa tissues of uterovaginal junction (UVJ) of White Leghorn laying hens with or without artificial insemination using semen from Barred Plymouth Rock roosters were collected. The lipid density in the epithelium of UVJ and SST was analyzed by Sudan black B staining. The expressions of genes encoding lipid receptors and lipases were assayed by polymerase chain reaction in UVJ mucosa and SST cells isolated by laser microdissection. Fatty acid composition was analyzed by gas chromatography, and sperm were cultured with or without the identified predominant fatty acids for 24 hours to examine their effect on sperm viability. The lipid droplets were localized in the epithelium of UVJ mucosa and SSTs. The expression of genes encoding very low-density lipoprotein receptor(VLDLR), low-density lipoprotein receptor (LDLR), and fatty acid translocase (FAT/CD36) were found in SST cells. Expression of genes encoding endothelial lipase (EL), lipase H (LIPH), adipose triglyceride lipase (ATGL), and lipoprotein lipase (LPL) were found in UVJ. In contrast, only ATGL was found in SST cells, and its expression was significantly upregulated after artificial insemination. In UVJ mucosal tissues, five fatty acids, namely myristic acid (C14), palmitic acid (C16), stearic acid (C18), oleic acid (C18:1n9), and linoleic acid (C18:2n6), were identified as predominant fatty acids. The viability of sperm cultured with 1 mM oleic acid or linoleic acid was significantly higher than the sperm in the control culture without fatty acids. These results suggest that lipids in the SST cells may be degraded by ATGL, and fatty acids including oleic acid and linoleic acid may be released into the SST lumen to support sperm survival. PMID:26777559

  10. PPARalpha and PPARgamma activators direct a distinct tissue-specific transcriptional response via a PPRE in the lipoprotein lipase gene.

    PubMed Central

    Schoonjans, K; Peinado-Onsurbe, J; Lefebvre, A M; Heyman, R A; Briggs, M; Deeb, S; Staels, B; Auwerx, J

    1996-01-01

    Increased activity of lipoprotein lipase (LPL) may explain the hypotriglyceridemic effects of fibrates, thiazolidinediones and fatty acids, which are known activators (and/or ligands) of the various peroxisome proliferator-activated receptors (PPARs). Treatment with compounds which activate preferentially PPARalpha, such as fenofibrate, induced LPL expression exclusively in rat liver. In contrast, the antidiabetic thiazolidinedione BRL 49653, a high affinity ligand for PPARgamma, had no effect on liver, but induced LPL expression in rat adipose tissue. In the hepatocyte cell line AML-12, fenofibric acid, but not BRL 49653, induced LPL mRNA, whereas in 3T3-L1 preadipocytes, the PPARgamma ligand induced LPL mRNA levels much quicker and to a higher extent than fenofibric acid. In both the in vivo and in vitro studies, inducibility by either PPARalpha or gamma activators, correlated with the tissue distribution of the respective PPARs: an adipocyte-restricted expression of PPARgamma, whereas PPARalpha was expressed predominantly in liver. A sequence element was identified in the human LPL promoter that mediates the functional responsiveness to fibrates and thiazolidinediones. Methylation interference and gel retardation assays demonstrated that a PPARalpha or gamma and the 9-cis retinoic acid receptor (RXR) heterodimers bind to this sequence -169 TGCCCTTTCCCCC -157. These data provide evidence that transcriptional activation of the LPL gene by fibrates and thiazolidinediones is mediated by PPAR-RXR heterodimers and contributes significantly to their hypotriglyceridemic effects in vivo. Whereas thiazolidinediones predominantly affect adipocyte LPL production through activation of PPARgamma, fibrates exert their effects mainly in the liver via activation of PPARalpha. Images PMID:8895578

  11. Study of Antiobesity Effect through Inhibition of Pancreatic Lipase Activity of Diospyros kaki Fruit and Citrus unshiu Peel

    PubMed Central

    Kim, Gyo-Nam; Shin, Mi-Rae; Shin, Sung Ho; Lee, Ah Reum; Lee, Joo Young; Seo, Bu-Il; Kim, Min Yeong; Kim, Tae Hoon; Noh, Jeong Sook; Rhee, Man Hee

    2016-01-01

    Pancreatic lipase is the enzyme responsible for digestion and absorption of triglycerides, being its inhibition one of the widest studied methods used to determine the potential activity of natural products to inhibit dietary fat absorption. Decrease of energy intake from dietary fat through inhibition of this enzyme may be an excellent strategy to prevent and treat obesity. The inhibitory activity on pancreatic lipase enzyme of Diospyros kaki fruit and Citrus unshiu peel mixture extract (PCM) was evaluated in vitro and its antiobesity effects were studied based on the serum lipid parameters analysis from high-fat diet- (HFD-) fed mice in vivo. PCM was orally administered at a dose of 50 and 200 mg/kg body weight for 6 weeks. In addition, the activity of pancreatic lipase was assessed using orlistat (positive control). PCM exhibited inhibitory effect on lipase activity with IC50 value of 507.01 μg/mL. Moreover, serum triacylglycerol, total cholesterol levels, and visceral fat weight were significantly reduced compared to HFD control mice in PCM 200 mg/kg-treated mice (p < 0.05). These results suggest that PCM administration may be a novel potential antiobesity agent for reduction of fat absorption via inhibition of pancreatic lipase. PMID:27529064

  12. Study of Antiobesity Effect through Inhibition of Pancreatic Lipase Activity of Diospyros kaki Fruit and Citrus unshiu Peel.

    PubMed

    Kim, Gyo-Nam; Shin, Mi-Rae; Shin, Sung Ho; Lee, Ah Reum; Lee, Joo Young; Seo, Bu-Il; Kim, Min Yeong; Kim, Tae Hoon; Noh, Jeong Sook; Rhee, Man Hee; Roh, Seong-Soo

    2016-01-01

    Pancreatic lipase is the enzyme responsible for digestion and absorption of triglycerides, being its inhibition one of the widest studied methods used to determine the potential activity of natural products to inhibit dietary fat absorption. Decrease of energy intake from dietary fat through inhibition of this enzyme may be an excellent strategy to prevent and treat obesity. The inhibitory activity on pancreatic lipase enzyme of Diospyros kaki fruit and Citrus unshiu peel mixture extract (PCM) was evaluated in vitro and its antiobesity effects were studied based on the serum lipid parameters analysis from high-fat diet- (HFD-) fed mice in vivo. PCM was orally administered at a dose of 50 and 200 mg/kg body weight for 6 weeks. In addition, the activity of pancreatic lipase was assessed using orlistat (positive control). PCM exhibited inhibitory effect on lipase activity with IC50 value of 507.01 μg/mL. Moreover, serum triacylglycerol, total cholesterol levels, and visceral fat weight were significantly reduced compared to HFD control mice in PCM 200 mg/kg-treated mice (p < 0.05). These results suggest that PCM administration may be a novel potential antiobesity agent for reduction of fat absorption via inhibition of pancreatic lipase. PMID:27529064

  13. Isolation of lipase producing thermophilic bacteria: optimization of production and reaction conditions for lipase from Geobacillus sp.

    PubMed

    Mehta, Akshita; Kumar, Rakesh; Gupta, Reena

    2012-12-01

    Lipases catalyze the hydrolysis and the synthesis of esters formed from glycerol and long chain fatty acids. Lipases occur widely in nature, but only microbial lipases are commercially significant. In the present study, thirty-two bacterial strains, isolated from soil sample of a hot spring were screened for lipase production. The strain TS-4, which gave maximum activity, was identified as Geobacillus sp. at MTCC, IMTECH, Chandigarh. The isolated lipase producing bacteria were grown on minimal salt medium containing olive oil. Maximal quantities of lipase were produced when 30 h old inoculum was used at 10% (v/v) in production medium and incubated in shaking conditions (150 rpm) for 72 h. The optimal temperature and pH for the bacterial growth and lipase production were found to be 60°C and 9.5, respectively. Maximal enzyme production resulted when mustard oil was used as carbon source and yeast extract as sole nitrogen source at a concentration of 1% (v/v) and 0.15% (w/v), respectively. The different optimized reaction parameters were temperature 65°C, pH 8.5, incubation time 10 min and substrate p-nitrophenyl palmitate. The Km and Vmax values of enzyme were found to be 14 mM and 17.86 μmol ml-1min-1, respectively, with p-nitrophenyl palmitate as substrate. All metal ions studied (1 mM) increased the lipase activity. PMID:23195552

  14. Sequence of the lid affects activity and specificity of Candida rugosa lipase isoenzymes.

    PubMed

    Brocca, Stefania; Secundo, Francesco; Ossola, Mattia; Alberghina, Lilia; Carrea, Giacomo; Lotti, Marina

    2003-10-01

    The fungus Candida rugosa produces multiple lipase isoenzymes (CRLs) with distinct differences in substrate specificity, in particular with regard to selectivity toward the fatty acyl chain length. Moreover, isoform CRL3 displays high activity towards cholesterol esters. Lipase isoenzymes share over 80% sequence identity but diverge in the sequence of the lid, a mobile loop that modulates access to the active site. In the active enzyme conformation, the open lid participates in the substrate-binding site and contributes to substrate recognition. To address the role of the lid in CRL activity and specificity, we substituted the lid sequences from isoenzymes CRL3 and CRL4 in recombinant rCRL1, thus obtaining enzymes differing only in this stretch of residues. Swapping the CRL3 lid was sufficient to confer to CRL1 cholesterol esterase activity. On the other hand, a specific shift in the chain-length specificity was not observed. Chimeric proteins displayed different sensitivity to detergents in the reaction medium. PMID:14500889

  15. Sequence of the lid affects activity and specificity of Candida rugosa lipase isoenzymes

    PubMed Central

    Brocca, Stefania; Secundo, Francesco; Ossola, Mattia; Alberghina, Lilia; Carrea, Giacomo; Lotti, Marina

    2003-01-01

    The fungus Candida rugosa produces multiple lipase isoenzymes (CRLs) with distinct differences in substrate specificity, in particular with regard to selectivity toward the fatty acyl chain length. Moreover, isoform CRL3 displays high activity towards cholesterol esters. Lipase isoenzymes share over 80% sequence identity but diverge in the sequence of the lid, a mobile loop that modulates access to the active site. In the active enzyme conformation, the open lid participates in the substrate-binding site and contributes to substrate recognition. To address the role of the lid in CRL activity and specificity, we substituted the lid sequences from isoenzymes CRL3 and CRL4 in recombinant rCRL1, thus obtaining enzymes differing only in this stretch of residues. Swapping the CRL3 lid was sufficient to confer to CRL1 cholesterol esterase activity. On the other hand, a specific shift in the chain-length specificity was not observed. Chimeric proteins displayed different sensitivity to detergents in the reaction medium. PMID:14500889

  16. Immobilization of lipase on epoxy activated (1-->3)-alpha-D-glucan isolated from Penicillium chrysongenum.

    PubMed

    Wang, Tianqi; Li, Hanxiang; Nie, Kaili; Tan, Tianwei

    2006-12-01

    A water-insoluble linear (1-->3)-alpha-D-glucan was isolated from Penicillium mycelia. Three kinds of epoxy-activated microspheres of this glucan were prepared as supports for Candida sp. lipase (EC3.1.1.3) immobilization. The highest immobilization yield was 36.4%. The specific activity was 26.85 U/mg, and only 4.1% of activity was lost in comparison with the free enzyme used for immobilization. The higher thermal stability, storage stability, and reusability of the immobilized lipase make it a potential candidate for wide application. PMID:17151459

  17. Easy preparation of enantiomerically enriched heteroaromatic alcohols through lipase-catalyzed acylation with succinic anhydride under unconventional activation.

    PubMed

    Melais, Nedjma; Boukachabia, Mourad; Aribi-Zouioueche, Louisa; Riant, Olivier

    2015-08-01

    This study reports the lipase-catalyzed resolution of heteroaromatic secondary alcohols by succinic anhydride under different activation conditions by convenient procedure with succinic anhydride. The effects of succinic anhydride and the nature of the heteroatom are discussed in standard conditions in the kinetic resolution with lipases. The results recorded under microwave activation and ultrasonication is compared. (R)-4-chromanol was obtained in optically pure form (ee > 99%) with a high selectivity E > 200 by Pseudomonas cepacia lipase (PCL) in diethyl ether, using microwave radiation and under ultrasonication. The reaction time is reduced compared to the conventional heating with a better control of the selectivity of the lipase PCL. A significant effect of the nature of the heteroatoms on the reactivity and selectivity of the lipase with succinic anhydride has been disclosed, regardless the conditions of activation. This method proved to be clean, fast, interesting alternative, and facilitates the use of a cyclic anhydride, by microwave or ultrasound especially with secondary alcohols. The process is a valuable prerequisite for the preparative scale production of enantiomerically heteroaromatic alcohols in sustainable chemistry. PMID:25957778

  18. Regulation of fatty acid sup 18 O exchange catalyzed by pancreatic carboxylester lipase. 1. Mechanism and kinetic properties

    SciTech Connect

    Muderhwa, J.M.; Schmid, P.C.; Brockman, H.L. )

    1992-01-14

    The exchange of {sup 18}O between H{sub 2}O and long-chain free fatty acids is catalyzed by pancreatic carboxylester lipase. For palmitic, oleic, and arachidonic acid in aqueous suspension and for 13,16-cis,cis-docosadienoic acid (DA) in monomolecular films, carboxyl oxygens were completely exchanged with water oxygens of the bulk aqueous phase. With enzyme at either substrate or catalytic concentrations in the argon-buffer interface, the exchange of DA oxygens obeyed a random sequential mechanism, i.e., {sup 18}O, {sup 18}O-DA {rightleftharpoons} {sup 18}O, {sup 16}O-DA {rightleftharpoons} {sup 16}O, {sup 16}O-DA. This indicates that the dissociation of the enzyme{center dot}DA complex is much faster than the rate-limiting step in the overall exchange reaction. Kinetic analysis of {sup 18}O exchange showed a first-order dependence on surface enzyme and DA concentrations, i.e., the reaction was limited by the acylation rate. The values of k{sub cat}/K{sub m}, 0.118 cm{sup 2} pmol{sup {minus}1} s{sup {minus}1}, for the exchange reaction was comparable to that for methyl oleate hydrolysis and 5-fold higher than that for cholesteryl oleate hydrolysis in monolayers. Thus, fatty acids are good substrates' for carboxylester lipase. With substrate levels of carboxylester lipase in the interfacial phase, the acylation rate constant k{sub cat}/K{sub m} was 200-fold lower than that obtained with catalytic levels of enzyme. This suggests a possible restriction of substrate diffusion in the protein-covered substrate monolayer.

  19. Production of L2 lipase by Bacillus sp. strain L2: nutritional and physical factors.

    PubMed

    Shariff, Fairolniza Mohd; Leow, Thean Chor; Mukred, A D; Salleh, Abu Bakar; Basri, Mahiran; Rahman, Raja Noor Zaliha Raja Abd

    2007-10-01

    A thermophilic bacterium, Bacillus sp. strain L2 was isolated from a hot spring in Perak, Malaysia. An extracellular lipase activity was detected through plate and broth assays at 70 degrees C after 28 h of incubation. The L2 lipase production was growth dependent as revealed by a number of factors affecting the secretion of extracelullar lipase. As for nutritional factors, casamino acids, trehalose, Ca(2+) and Tween 60 were found to be more effective for lipase production. The optimum physical condition for L2 lipase production was obtained at 70 degrees C after 28 h of cultivation time, at pH 7.0, 150 rpm of agitation rate and 1% of starting inoculum size. The activity staining of crude L2 lipase revealed a clearing zone at 39 kDa. PMID:17910105

  20. Purification and characterization of an extracellular lipase from Geotrichum marinum.

    PubMed

    Huang, Youliang; Locy, Robert; Weete, John D

    2004-03-01

    An extracellular lipase (EC 3.1.1.3) from Geotrichum marinum was purified 76-fold with 46% recovery using Octyl Sepharose 4 Fast Flow and Bio-Gel A 1.5 m chromatography. The purified enzyme showed a prominent band on SDS-PAGE and a single band on native PAGE based on the activity staining. The molecular mass of the lipase was estimated to be 62 kDa using SDS-PAGE and Bio-Gel A chromatography, indicating that the lipase likely functions as a monomer. The pl of the lipase was determined to be 4.54. The apparent V(max) and Km were 1000 micromol/min/mg protein and 11.5 mM, respectively, using olive oil emulsified with taurocholic acid as substrate. The lipase demonstrated a pH optimum at pH 8.0 and a temperature optimum at 40 degrees C. At 6 mM, Na+, K+, Ca2+, and Mg2+ stimulated activity, but Na+ and K+ at 500 mM and Fe2+ and Mn2+ at 6 mM reduced lipase activity. The anionic surfactant, taurocholic acid, and the zwitterionic surfactant, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, enhanced the activity at 0.1 mM. Other anionic surfactants such as SDS and sodium dioctyl sulfosuccinate, the cationic surfactants methylbenzethonium bromide and cetyltriethylammonium bromide, and the nonionic surfactants Tween-20 and Triton X-100 inhibited the lipase activity to different extents. The lipase was found to have a preference for TG containing cis double bonds in their FA side chains, and the reaction rate increased with an increasing number of double bonds in the side chain. The lipase had a preference for ester bonds at the sn-1 and sn-3 positions over the ester bond at the sn-2 position. PMID:15233404

  1. Interesterification of phosphatidylcholine with lipases in organic media.

    PubMed

    Svensson, I; Adlercreutz, P; Mattiasson, B

    1990-06-01

    Lipases were investigated with respect to their ability to catalyse the incorporation of fatty acids into phosphatidylcholine (PC) by interesterification reactions. The enzymes were dried onto solid support materials and the conversions were carried out in water-saturated toluene. Three lipases (two fungal and one plant enzyme) had the desired activity; immobilized lipase from Mucor miehei (Lipozyme) was the most active enzyme. The Lipozyme-catalysed interesterification was selective for the sn-1 position of PC and during 48 h of reaction around 50% of the fatty acids in this position were replaced with heptadecanoic acid, a fatty acid which was practically absent in the original phospholipid. Due to adsorption on the support material and the competing hydrolysis reaction the total amount of PC in the reaction solution decreased to about 40% of the original amount. Higher interesterification rates were obtained with free fatty acids as acyl donors than with fatty acid esters. PMID:1366637

  2. 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. PMID:26672465

  3. Improving palm oil quality through identification and mapping of the lipase gene causing oil deterioration.

    PubMed

    Morcillo, F; Cros, D; Billotte, N; Ngando-Ebongue, G-F; Domonhédo, H; Pizot, M; Cuéllar, T; Espéout, S; Dhouib, R; Bourgis, F; Claverol, S; Tranbarger, T J; Nouy, B; Arondel, V

    2013-01-01

    The oil palm fruit mesocarp contains high lipase activity that increases free fatty acids and necessitates post-harvest inactivation by heat treatment of fruit bunches. Even before heat treatment the mesocarp lipase activity causes consequential oil losses and requires costly measures to limit free fatty acids quantities. Here we demonstrate that elite low-lipase lines yield oil with substantially less free fatty acids than standard genotypes, allowing more flexibility for post-harvest fruit processing and extended ripening for increased yields. We identify the lipase and its gene cosegregates with the low-/high-lipase trait, providing breeders a marker to rapidly identify potent elite genitors and introgress the trait into major cultivars. Overall, economic gains brought by wide adoption of this material could represent up to one billion dollars per year. Expected benefits concern all planters but are likely to be highest for African smallholders who would be more able to produce oil that meets international quality standards. PMID:23857501

  4. Biological Activities of a Mixture of Biosurfactant from Bacillus subtilis and Alkaline Lipase from Fusarium oxysporum

    PubMed Central

    Pereira de Quadros, Cedenir; Cristina Teixeira Duarte, Marta; Maria Pastore, Gláucia

    2011-01-01

    In this study, we investigate the antimicrobial effects of a mixture of a biosurfactant from Bacillus subtilis and an alkaline lipase from Fusarium oxysporum (AL/BS mix) on several types of microorganisms, as well as their abilities to remove Listeria innocua ATCC 33093 biofilm from stainless steel coupons. The AL/BS mix had a surface tension of around 30 mN.m-1, indicating that the presence of alkaline lipase did not interfere in the surface activity properties of the tensoactive component. The antimicrobial activity of the AL/BS mix was determined by minimum inhibitory concentration (MIC) micro-assays. Among all the tested organisms, the presence of the mixture only affected the growth of B. subtilis CCT 2576, B. cereus ATCC 10876 and L. innocua. The most sensitive microorganism was B. cereus (MIC 0.013 mg.mL-1). In addition, the effect of the sanitizer against L. innocua attached to stainless steel coupons was determined by plate count after vortexing. The results showed that the presence of the AL/BS mix improved the removal of adhered cells relative to treatment done without the sanitizer, reducing the count of viable cells by 1.72 log CFU.cm-2. However, there was no significant difference between the sanitizers tested and an SDS detergent standard (p<0.05). PMID:24031642

  5. Influence of environmental factors on lipase production by Lactobacillus plantarum.

    PubMed

    Lopes, M de F; Cunha, A E; Clemente, J J; Carrondo, M J; Crespo, M T

    1999-02-01

    A strain of Lactobacillus plantarum, DSMZ 12028 (Deutsch Sammlung von Mikroorganismen und Zellkulturen), isolated from a Portuguese dry fermented sausage, "chouriço", was found to produce true lipase, producing free fatty acids from triolein (olive oil). This enzymatic activity was found in whole cells, but was negligible in comparison to lipolytic activity in culture supernatant. Therefore, only extracellular activity was studied. The effect of pH, temperature and glucose concentration on extracellular lipase production was studied in continuously stirred tank reactors, the first time this technology has been used to study the production of this enzyme in lactobacilli. Maximum lipase production was achieved at a pH of 5.5 and 30 degrees C and was kept at a significant level over a wide range of dilution rates (0.05-0.4 h-1); the production of lipase was still significant for low pH values, temperature and glucose concentration, conditions that are close to the ones present during chouriço ripening. The effect of glucose concentration was also studied in a batch system. The control of lipase production was found to be related both to glucose concentration in the medium and to the growth rate/dilution rate. Glucose concentration was found to be important for fast lipase production, although it did not influence the maximum lipase activity reached in a batch culture. PMID:10091332

  6. Lipase immobilized on hydrophobic porous polymer supports prepared by concentrated emulsion polymerization and their activity in the hydrolysis of triacylglycerides.

    PubMed

    Ruckenstein, E; Wang, X

    1993-09-20

    Microporous polymer supports for the immobilization of lipase have been prepared by the polymerization of a concentrated emulsion precursor. The concentrated emulsion consists of a mixture of styrene and divinyl-benzene containing a suitable surfactant and an initiator as the continuous phase and water as the dispersed phase. The volume fraction of the latter phase was greater than 0.74, which is the volume fraction of the dispersed phase for the most compact arrangement of spheres of equal radius. The lipase from Candida rugosa has been immobilized on the internal surface of the hydrophobic microporous poly(styrene-divinyl benzene) supports and used as biocatalysts for the hydrolysis of triacylglycerides. The effects of the amount of surfactant, of the molar ratio of divinylbenzene/styrene in the continuous phase, and of the aquaphilicity of the supports on the adsorption, activity, and stability of the immobilized lipase have been investigated. The microporous poly(styrene-divinylbenzene) adsorbents constitute excellent supports for lipase because both the amount adsorbed is large and the rate of enzymatic reaction per molecule of lipase is higher for the immobilized enzyme than for the free one. PMID:18613129

  7. Optimization of cold-active lipase production from psychrophilic bacterium Moritella sp. 2-5-10-1 by statistical experimental methods.

    PubMed

    Wang, Quanfu; Zhang, Chunyu; Hou, Yanhua; Lin, Xuezheng; Shen, Jihong; Guan, Xiangyu

    2013-01-01

    Statistical experimental designs were applied to optimize cold-active lipase production by the psychrophilic bacterium Moritella sp. 2-5-10-1. First, a Plackett-Burmen design (PBD) was used to evaluate the significant effects of various fermentation parameters. The results indicated that soybean meal, temperature, and Tween-80 had significant influences on lipase production. The levels of these variables were optimized subsequently using central composite design (CCD). A quadratic regression model of cold-active lipase production was built, and verification experiments confirmed its validity. On subsequent scale-up in a 10-L bioreactor using optimized conditions, cold-active lipase production (30.56 U/mL) was obtained. The results clearly indicated that the model was adequate even on a large scale. To our knowledge, this is the first report of statistical optimization of cold-active lipase production by a psychrophilic bacterium. PMID:23291744

  8. Expression of human hormone-sensitive lipase in white adipose tissue of transgenic mice increases lipase activity but does not enhance in vitro lipolysis.

    PubMed

    Lucas, Stéphanie; Tavernier, Geneviève; Tiraby, Claire; Mairal, Aline; Langin, Dominique

    2003-01-01

    Hormone-sensitive lipase (HSL) catalyzes the hydrolysis of acylglycerols and cholesteryl esters (CEs). The enzyme is highly expressed in adipose tissues (ATs), where it is thought to play an important role in fat mobilization. The purpose of the present work was to study the effect of a physiological increase of HSL expression in vivo. Transgenic mice were produced with a 21 kb human genomic fragment encompassing the exons encoding the adipocyte form of HSL. hHSL mRNA was expressed at 3-fold higher levels than murine HSL mRNA in white adipocytes. Transgene expression was also observed in brown adipose tissue (BAT) and skeletal muscle. The human protein was detected in ATs of transgenic (Tg) mice. The hydrolytic activities against triacylglycerol (TG), diacylglycerol (DG) analog, and CE were increased in transgenic mouse AT. However, cAMP-inducible adipocyte lipolysis was lower in transgenic animals. In the B6CBA genetic background, transgenic mice up to 14 weeks of age showed lower body weight and fat mass. The phenotype was not observed in older animals and in mice fed a high-fat diet (HFD). In the OF1 genetic background, there was no difference in fat mass of mice fed ad libitum. However, transgenic mice became leaner than their wild-type (WT) littermates after a 4 day calorie restriction. The data show that overexpression of HSL, despite increased lipase activity, does not lead to enhanced lipolysis. PMID:12518034

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

    PubMed

    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 96h. 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 46.3kDa 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 1h, and the optimum enzyme-substrate reaction time was 30min. 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 Zn(2+), followed by Mg(2+) and Fe(2+). Lipase activity was not significantly affected in the presence of ethylenediaminetetraacetic acid, sodium lauryl sulfate and Triton X-100. Phenylmethylsulfonyl fluoride (1mM) 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

  10. Purification and characterization of an organic solvent-tolerant lipase from Pseudomonas aeruginosa CS-2.

    PubMed

    Peng, Ren; Lin, Jinping; Wei, Dongzhi

    2010-10-01

    An extracellular lipase secreted by Pseudomonas aeruginosa CS-2 was purified to homogeneity about 25.5-fold with an overall yield of 45.5%. The molecular mass of the lipase was estimated to be 33.9 kDa by SDS-PAGE and 36 kDa by gel filtration. The optimum temperature and pH were 50 degrees C and 8.0. The lipase was found to be stable at pH 4-10 and below 50 degrees C. Its hydrolytic activity was highest against p-nitrophenyl palmitate (p-NPP) among p-nitrophenyl esters of fatty acids with various chain lengths. The lipase was activated in the presence of Ca(2+), while it was inactivated by other metal ions more or less. EDTA significantly reduced the lipase activity, indicating the lipase was a metalloenzyme. Gum Arabic and polyvinyl alcohol 124 enhanced lipase activity but Tween-20, Tween-80, and hexadecyltrimethyl ammonium bromide strongly inhibited the lipase. It exhibited stability in some organic solvents. The lipase was activated in the presence of acetonitrile. Conversely, it was drastically inactivated by methanol and ethanol. PMID:19936633

  11. Physical inactivity amplifies the sensitivity of skeletal muscle to the lipid-induced downregulation of lipoprotein lipase activity.

    PubMed

    Zderic, Theodore W; Hamilton, Marc T

    2006-01-01

    Physical inactivity is a risk factor for lipoprotein disorders and the metabolic syndrome. Physical inactivity has a powerful effect on suppressing lipoprotein lipase (LPL) activity in skeletal muscle, the rate-limiting enzyme for hydrolysis of triglyceride (TG)-rich lipoproteins. We tested the ability of several compounds to prevent the decrease in LPL. The present study minimized standing and ordinary light nonexercise movements in rats to compare the effects of inactivity and nonexercise activity thermogenesis (NEAT) on LPL activity. The key new insight was that the typically quick decrease in LPL activity of oxidative muscle caused by physical inactivity was prevented by nicotinic acid (NA), whereas inhibitors of TNF-alpha, inducible nitric oxide synthase, and NF-kappaB had no such effect. NA was administered at a dose known to acutely impede the appearance of plasma TG from the liver and free fatty acids from adipose tissue, and it was effective at intentionally lowering plasma lipid concentrations to the same level in active and inactive groups. As measured from heparin-releasable LPL activity, LPL in the microvasculature of the most oxidative muscles was approximately 90% lower in the inactive group compared with controls, and this suppression was completely blocked by NA. In contrast to inactivity, NA did not raise muscle LPL in ambulatory controls, whereas a large exogenous fat delivery did decrease LPL activity. In vitro control studies revealed that NA did not have a direct effect on skeletal muscle LPL activity. In conclusion, physical inactivity amplifies the ability of plasma lipids to suppress muscle LPL activity. The light ambulatory contractions responsible for NEAT are sufficient for mitigating these deleterious effects. PMID:16195388

  12. Interesterification of butter fat by partially purified extracellular lipases from Pseudomonas putida, Aspergillus niger and Rhizopus oryzae.

    PubMed

    Pabai, F; Kermasha, S; Morin, A

    1995-11-01

    Three extracellular lipases were produced by batch fermentation of Pseudomonas putida ATCC 795, Aspergillus niger CBS 131.52 and Rhizopus oryzae ATCC 34612 during the late phase of growth, at 72, 96 and 96 h, respectively. The lipases were partially purified by (NH4)2SO4 fractionation. The lipase of P. putida was optimal at pH 8.0 whereas those from A. niger and R. oryzae were optimal at pH 7.5. The A. niger lipase had the lowest V max value (0.51×10(-3) U/min) and R. oryzae the highest (1.86×10(-3) U/min). The K m values for P. putida, A. niger and R. oryzae lipases were 1.18, 0.97, and 0.98 mg/ml, respectively. Native PAGE of the partially-purified lipase extracts showed two to four major bands. The interesterification of butter fat by A. niger lipase decreased the water activity as well as the hydrolytic activity. The A. niger lipase had the highest interesterification yield value (26%) and the R. oryzae lipase the lowest (4%). In addition, A. niger lipase exhibited the highest decrease (17%) in long-chain hypercholesterolemic fatty acids (C12:0, C14:0 and C16:0) at the sn-2-position; the P. putida lipase demonstrated the least favourable changes in specificity at the same position. PMID:24415019

  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. Differential Expression of Extracellular Lipase and Protease Activities of Mycelial and Yeast Forms in Malassezia furfur.

    PubMed

    Juntachai, Weerapong; Kajiwara, Susumu

    2015-10-01

    Malassezia furfur is a dimorphic yeast that is part of the human skin microflora. This fungus is a pathogen of a certain skin diseases, such as pityriasis versicolor, and in rare cases causes systemic infection in neonates. However, the role of dimorphism in the pathogenicity remains unclear. A modified induction medium (IM) was successfully able to induce mycelial growth of M. furfur under both solid and liquid condition. Filamentous elements with branching hyphae were observed when cultured in the IM. Furthermore, addition of bovine fetus serum into the liquid IM did not promote hyphal formation; on the contrary, it retrograded hyphae to the yeast form. Plate-washing assay showed that M. furfur hyphae did not possess the ability of invasive growth. Secretory proteins from both yeast and hyphal forms were isolated, and lipase and protease activities were analyzed. Intriguingly, the hyphal form showed higher activities than those of the yeast form, particularly the protease activity. PMID:26173769

  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. A continuous fluorescence displacement assay for the measurement of phospholipase A2 and other lipases that release long-chain fatty acids.

    PubMed Central

    Wilton, D C

    1990-01-01

    1. A new continuous fluorescence assay for phospholipase A2 is described which involves the displacement of the highly fluorescent fatty-acid probe 11-(dansylamino)undecanoic acid from rat liver fatty-acid-binding protein by long-chain fatty acids released as a result of phospholipase A2-catalysed hydrolysis of phospholipids. The initial rate of decrease in fluorescence is linearly related to enzyme activity. 2. The assay will detect enzyme activity down to about 10 pmol/min per ml and gives a linear response up to about 10 nmol/min per ml. 3. The assay will work with all phospholipids that have been tested including phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol and phosphatidylglycerol. Substrates carrying a net negative charge showed the highest rates of hydrolysis. 4. The assay will work, in principle, with an enzyme catalysing the release of long-chain fatty acids from a fatty-acylated substrate. This has been confirmed with pancreatic lipase and cholesterol esterase. PMID:2317197

  17. Apolipoproteins C-I and C-III Inhibit Lipoprotein Lipase Activity by Displacement of the Enzyme from Lipid Droplets*

    PubMed Central

    Larsson, Mikael; Vorrsjö, Evelina; Talmud, Philippa; Lookene, Aivar; Olivecrona, Gunilla

    2013-01-01

    Apolipoproteins (apo) C-I and C-III are known to inhibit lipoprotein lipase (LPL) activity, but the molecular mechanisms for this remain obscure. We present evidence that either apoC-I or apoC-III, when bound to triglyceride-rich lipoproteins, prevent binding of LPL to the lipid/water interface. This results in decreased lipolytic activity of the enzyme. Site-directed mutagenesis revealed that hydrophobic amino acid residues centrally located in the apoC-III molecule are critical for attachment to lipid emulsion particles and consequently inhibition of LPL activity. Triglyceride-rich lipoproteins stabilize LPL and protect the enzyme from inactivating factors such as angiopoietin-like protein 4 (angptl4). The addition of either apoC-I or apoC-III to triglyceride-rich particles severely diminished their protective effect on LPL and rendered the enzyme more susceptible to inactivation by angptl4. These observations were seen using chylomicrons as well as the synthetic lipid emulsion Intralipid. In the presence of the LPL activator protein apoC-II, more of apoC-I or apoC-III was needed for displacement of LPL from the lipid/water interface. In conclusion, we show that apoC-I and apoC-III inhibit lipolysis by displacing LPL from lipid emulsion particles. We also propose a role for these apolipoproteins in the irreversible inactivation of LPL by factors such as angptl4. PMID:24121499

  18. Apolipoproteins C-I and C-III inhibit lipoprotein lipase activity by displacement of the enzyme from lipid droplets.

    PubMed

    Larsson, Mikael; Vorrsjö, Evelina; Talmud, Philippa; Lookene, Aivar; Olivecrona, Gunilla

    2013-11-22

    Apolipoproteins (apo) C-I and C-III are known to inhibit lipoprotein lipase (LPL) activity, but the molecular mechanisms for this remain obscure. We present evidence that either apoC-I or apoC-III, when bound to triglyceride-rich lipoproteins, prevent binding of LPL to the lipid/water interface. This results in decreased lipolytic activity of the enzyme. Site-directed mutagenesis revealed that hydrophobic amino acid residues centrally located in the apoC-III molecule are critical for attachment to lipid emulsion particles and consequently inhibition of LPL activity. Triglyceride-rich lipoproteins stabilize LPL and protect the enzyme from inactivating factors such as angiopoietin-like protein 4 (angptl4). The addition of either apoC-I or apoC-III to triglyceride-rich particles severely diminished their protective effect on LPL and rendered the enzyme more susceptible to inactivation by angptl4. These observations were seen using chylomicrons as well as the synthetic lipid emulsion Intralipid. In the presence of the LPL activator protein apoC-II, more of apoC-I or apoC-III was needed for displacement of LPL from the lipid/water interface. In conclusion, we show that apoC-I and apoC-III inhibit lipolysis by displacing LPL from lipid emulsion particles. We also propose a role for these apolipoproteins in the irreversible inactivation of LPL by factors such as angptl4. PMID:24121499

  19. Maternal High-Fat Feeding Increases Placental Lipoprotein Lipase Activity by Reducing SIRT1 Expression in Mice.

    PubMed

    Qiao, Liping; Guo, Zhuyu; Bosco, Chris; Guidotti, Stefano; Wang, Yunfeng; Wang, Mingyong; Parast, Mana; Schaack, Jerome; Hay, William W; Moore, Thomas R; Shao, Jianhua

    2015-09-01

    This study investigated how maternal overnutrition and obesity regulate expression and activation of proteins that facilitate lipid transport in the placenta. To create a maternal overnutrition and obesity model, primiparous C57BL/6 mice were fed a high-fat (HF) diet throughout gestation. Fetuses from HF-fed dams had significantly increased serum levels of free fatty acid and body fat. Despite no significant difference in placental weight, lipoprotein lipase (LPL) protein levels and activity were remarkably elevated in placentas from HF-fed dams. Increased triglyceride content and mRNA levels of CD36, VLDLr, FABP3, FABPpm, and GPAT2 and -3 were also found in placentas from HF-fed dams. Although both peroxisome proliferator-activated receptor-γ (PPARγ) and CCAAT/enhancer binding protein-α protein levels were significantly increased in placentas of the HF group, only PPARγ exhibited a stimulative effect on LPL expression in cultured JEG-3 human trophoblasts. Maternal HF feeding remarkably decreased SIRT1 expression in placentas. Through use of an SIRT1 activator and inhibitor and cultured trophoblasts, an inhibitory effect of SIRT1 on LPL expression was demonstrated. We also found that SIRT1 suppresses PPARγ expression in trophoblasts. Most importantly, inhibition of PPARγ abolished the SIRT1-mediated regulatory effect on LPL expression. Together, these results indicate that maternal overnutrition induces LPL expression in trophoblasts by reducing the inhibitory effect of SIRT1 on PPARγ. PMID:25948680

  20. Lipase maturation factor 1: a lipase chaperone involved in lipid metabolism.

    PubMed

    Péterfy, Miklós

    2012-05-01

    Mutations in lipase maturation factor 1 (LMF1) are associated with severe hypertriglyceridemia in mice and human subjects. The underlying cause is impaired lipid clearance due to lipase deficiency. LMF1 is a chaperone of the endoplasmic reticulum (ER) and it is critically required for the post-translational activation of three vascular lipases: lipoprotein lipase (LPL), hepatic lipase (HL) and endothelial lipase (EL). As LMF1 is only required for the maturation of homodimeric, but not monomeric, lipases, it is likely involved in the assembly of inactive lipase subunits into active enzymes and/or the stabilization of active dimers. Herein, we provide an overview of current understanding of LMF1 function and propose that it may play a regulatory role in lipase activation and lipid metabolism. Further studies will be required to test this hypothesis and elucidate the full spectrum of phenotypes in combined lipase deficiency. This article is part of a Special Issue entitled Triglyceride Metabolism and Disease. PMID:22063272

  1. Open and closed states of Candida antarctica lipase B: protonation and the mechanism of interfacial activation1

    PubMed Central

    Stauch, Benjamin; Fisher, Stuart J.; Cianci, Michele

    2015-01-01

    Lipases (EC 3.1.1.3) are ubiquitous hydrolases for the carboxyl ester bond of water-insoluble substrates, such as triacylglycerols, phospholipids, and other insoluble substrates, acting in aqueous as well as in low-water media, thus being of considerable physiological significance with high interest also for their industrial applications. The hydrolysis reaction follows a two-step mechanism, or “interfacial activation,” with adsorption of the enzyme to a heterogeneous interface and subsequent enhancement of the lipolytic activity. Among lipases, Candida antarctica lipase B (CALB) has never shown any significant interfacial activation, and a closed conformation of CALB has never been reported, leading to the conclusion that its behavior was due to the absence of a lid regulating the access to the active site. The lid open and closed conformations and their protonation states are observed in the crystal structure of CALB at 0.91 Å resolution. Having the open and closed states at atomic resolution allows relating protonation to the conformation, indicating the role of Asp145 and Lys290 in the conformation alteration. The findings explain the lack of interfacial activation of CALB and offer new elements to elucidate this mechanism, with the consequent implications for the catalytic properties and classification of lipases. PMID:26447231

  2. A novel live cell assay to measure diacylglycerol lipase α activity.

    PubMed

    Singh, Praveen K; Markwick, Rachel; Howell, Fiona V; Williams, Gareth; Doherty, Patrick

    2016-06-01

    Diacylglycerol lipase α (DAGLα) hydrolyses DAG to generate the principal endocannabinoid (eCB) 2-arachidonoylglycerol (2-AG) in the central nervous system. DAGLα dependent cannabinoid (CB) signalling has been implicated in numerous processes including axonal growth and guidance, adult neurogenesis and retrograde signalling at the synapse. Recent studies have implicated DAGLα as an emerging drug target for several conditions including pain and obesity. Activity assays are critical to the drug discovery process; however, measurement of diacylglycerol lipase (DAGL) activity using its native substrate generally involves low-throughput MS techniques. Some relatively high-throughput membrane based assays utilizing surrogate substrates have been reported, but these do not take into account the rate-limiting effects often associated with the ability of a drug to cross the cell membrane. In the present study, we report the development of a live cell assay to measure DAGLα activity. Two previously reported DAGLα surrogate substrates, p-nitrophenyl butyrate (PNPB) and 6,8-difluoro-4-methylumbelliferyl octanoate (DiFMUO), were evaluated for their ability to detect DAGLα activity in live cell assays using a human cell line stably expressing the human DAGLα transgene. Following optimization, the small molecule chromogenic substrate PNPB proved to be superior by providing lower background activity along with a larger signal window between transfected and parental cells when compared with the fluorogenic substrate DiFMUO. The assay was further validated using established DAGL inhibitors. In summary, the live cell DAGLα assay reported here offers an economical and convenient format to screen for novel inhibitors as part of drug discovery programmes and compliments previously reported high-throughput membrane based DAGL assays. PMID:27013337

  3. A novel live cell assay to measure diacylglycerol lipase α activity

    PubMed Central

    Singh, Praveen K.; Markwick, Rachel; Howell, Fiona V.; Williams, Gareth; Doherty, Patrick

    2016-01-01

    Diacylglycerol lipase α (DAGLα) hydrolyses DAG to generate the principal endocannabinoid (eCB) 2-arachidonoylglycerol (2-AG) in the central nervous system. DAGLα dependent cannabinoid (CB) signalling has been implicated in numerous processes including axonal growth and guidance, adult neurogenesis and retrograde signalling at the synapse. Recent studies have implicated DAGLα as an emerging drug target for several conditions including pain and obesity. Activity assays are critical to the drug discovery process; however, measurement of diacylglycerol lipase (DAGL) activity using its native substrate generally involves low-throughput MS techniques. Some relatively high-throughput membrane based assays utilizing surrogate substrates have been reported, but these do not take into account the rate-limiting effects often associated with the ability of a drug to cross the cell membrane. In the present study, we report the development of a live cell assay to measure DAGLα activity. Two previously reported DAGLα surrogate substrates, p-nitrophenyl butyrate (PNPB) and 6,8-difluoro-4-methylumbelliferyl octanoate (DiFMUO), were evaluated for their ability to detect DAGLα activity in live cell assays using a human cell line stably expressing the human DAGLα transgene. Following optimization, the small molecule chromogenic substrate PNPB proved to be superior by providing lower background activity along with a larger signal window between transfected and parental cells when compared with the fluorogenic substrate DiFMUO. The assay was further validated using established DAGL inhibitors. In summary, the live cell DAGLα assay reported here offers an economical and convenient format to screen for novel inhibitors as part of drug discovery programmes and compliments previously reported high-throughput membrane based DAGL assays. PMID:27013337

  4. Epistatic interaction between two nonstructural loci on chromosomes 7 and 3 influences hepatic lipase activity in BSB mice.

    PubMed

    Yi, Nengjun; Chiu, Sally; Allison, David B; Fisler, Janis S; Warden, Craig H

    2004-11-01

    BSB mice exhibit a wide range of obesity despite being produced by a backcross of lean C57BL/6J (B) x lean Mus spretus (SPRET/Pt) F1 animals x B. Previous linkage studies identified a quantitative trait locus (QTL) on mouse chromosome 7 with coincident peaks for hepatic lipase activity, obesity, and plasma cholesterol. However, these mice were not analyzed for gene x gene epistasis. Hepatic lipase activity is correlated with obesity and plasma cholesterol levels. In this study, we identified QTLs for plasma hepatic lipase activity with three statistical mapping methods: maximum likelihood interval mapping, Bayesian nonepistatic mapping, and Bayesian epistatic mapping. Bayesian epistatic mapping detected not only the QTL on chromosome 7 but also an additional QTL on chromosome 3, which has a weak main effect but a strong interaction with chromosome 7. SPRET/Pt alleles of the QTL on each chromosome promote hepatic lipase activity. The proportion of phenotypic variance explained by the epistatic effect is higher than that explained by the main effect of the QTL on chromosome 7. PMID:15314098

  5. Crystal structure of the catalytic domain of human bile salt activated lipase.

    PubMed Central

    Terzyan, S.; Wang, C. S.; Downs, D.; Hunter, B.; Zhang, X. C.

    2000-01-01

    Bile-salt activated lipase (BAL) is a pancreatic enzyme that digests a variety of lipids in the small intestine. A distinct property of BAL is its dependency on bile salts in hydrolyzing substrates of long acyl chains or bulky alcoholic motifs. A crystal structure of the catalytic domain of human BAL (residues 1-538) with two surface mutations (N186D and A298D), which were introduced in attempting to facilitate crystallization, has been determined at 2.3 A resolution. The crystal form belongs to space group P2(1)2(1)2(1) with one monomer per asymmetric unit, and the protein shows an alpha/beta hydrolase fold. In the absence of bound bile salt molecules, the protein possesses a preformed catalytic triad and a functional oxyanion hole. Several surface loops around the active site are mobile, including two loops potentially involved in substrate binding (residues 115-125 and 270-285). PMID:11045623

  6. Measurement of the phospholipase activity of endothelial lipase in mouse plasma.

    PubMed

    Basu, Debapriya; Lei, Xia; Josekutty, Joby; Hussain, M Mahmood; Jin, Weijun

    2013-01-01

    Endothelial lipase (EL) is a major negative regulator of plasma HDL levels in mice, rabbits, and most probably, humans. Although this regulatory function is critically dependent on EL's hydrolysis of HDL phospholipids, as yet there is no phospholipase assay specific for EL in plasma. We developed such an assay for the mouse enzyme using a commercially available phospholipid-like fluorescent substrate in combination with an EL neutralizing antibody. The specificity of the assay was established using EL knockout mice and its utility demonstrated by detection of an increase in plasma EL phospholipase activity following exposure of wild-type mice to lipopolysaccharide. The assay revealed that murine pre-heparin plasma does not contain measurable EL activity, indicating that the hydrolysis of HDL phospholipids by EL in vivo likely occurs on the cell surface. PMID:23103358

  7. 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. PMID:18373071

  8. Comparative gene identification-58/α/β hydrolase domain 5: more than just an adipose triglyceride lipase activator?

    PubMed Central

    Zierler, Kathrin A.; Zechner, Rudolf; Haemmerle, Guenter

    2014-01-01

    Purpose of review Comparative gene identification-58 (CGI-58) is a lipid droplet-associated protein that controls intracellular triglyceride levels by its ability to activate adipose triglyceride lipase (ATGL). Additionally, CGI-58 was described to exhibit lysophosphatidic acid acyl transferase (LPAAT) activity. This review focuses on the significance of CGI-58 in energy metabolism in adipose and nonadipose tissue. Recent findings Recent studies with transgenic and CGI-58-deficient mouse strains underscored the importance of CGI-58 as a regulator of intracellular energy homeostasis by modulating ATGL-driven triglyceride hydrolysis. In accordance with this function, mice and humans that lack CGI-58 accumulate triglyceride in multiple tissues. Additionally, CGI-58-deficient mice develop an ATGL-independent severe skin barrier defect and die soon after birth. Although the premature death prevented a phenotypical characterization of adult global CGI-58 knockout mice, the characterization of mice with tissue-specific CGI-58 deficiency revealed new insights into its role in neutral lipid and energy metabolism. Concerning the ATGL-independent function of CGI-58, a recently identified LPAAT activity for CGI-58 was shown to be involved in the generation of signaling molecules regulating inflammatory processes and insulin action. Summary Although the function of CGI-58 in the catabolism of cellular triglyceride depots via ATGL is well established, further studies are required to consolidate the function of CGI-58 as LPAAT and to clarify the involvement of CGI-58 in the metabolism of skin lipids. PMID:24565921

  9. Isolation and characterization of a metagenome-derived and cold-active lipase with high stereospecificity for (R)-ibuprofen esters.

    PubMed

    Elend, C; Schmeisser, C; Hoebenreich, H; Steele, H L; Streit, W R

    2007-07-15

    We report on the isolation and biochemical characterization of a novel, cold-active and metagenome-derived lipase with a high stereo-selectivity for pharmaceutically important substrates. The respective gene was isolated from a cosmid library derived from oil contaminated soil and designated lipCE. The deduced aa sequence indicates that the protein belongs to the lipase family l.3, with high similarity to Pseudomonas fluorescens lipases containing a C-terminal secretion signal for ABC dependent transport together with possible motifs for Ca(2+)-binding sites. The overexpressed protein revealed a molecular weight of 53.2kDa and was purified by refolding from inclusion bodies after expression in Escherichia coli. The optimum temperature of LipCE was determined to be 30 degrees C. However, the enzyme still displayed 28% residual activity at 0 degrees C and 16% at -5 degrees C. Calcium ions strongly increased activity and thermal stability of the protein. Further detailed biochemical characterization of the recombinant enzyme showed an optimum pH of 7 and that it retained activity in the presence of a range of metal ions and solvents. A detailed analysis of the enzyme's substrate spectrum with more than 34 different substrates indicated that the enzyme was able to hydrolyze a wide variety of substrates including the conversion of long chain fatty acid substrates with maximum activity for pNP-caprate (C(10)). Furthermore LipCE was able to hydrolyze stereo-selectively ibuprofen-pNP ester with a high preference for the (R) enantiomer of >91% ee and it demonstrated selectivity for esters of primary alcohols, whereas esters of secondary or tertiary alcohols were nearly not converted. PMID:17601620

  10. Stimuli-sensitive hydrogel based on N-isopropylacrylamide and itaconic acid for entrapment and controlled release of Candida rugosa lipase under mild conditions.

    PubMed

    Milašinović, Nikola; Knežević-Jugović, Zorica; Milosavljević, Nedeljko; Lučić Škorić, Marija; Filipović, Jovanka; Kalagasidis Krušić, Melina

    2014-01-01

    Stimuli responsive pH- and temperature-sensitive hydrogel drug delivery systems, as those based on N-isopropylacrylamide (NiPAAm) and itaconic acid (IA), have been attracting much of the attention of the scientific community nowadays, especially in the field of drug release. By adjusting comonomer composition, the matrix is enabled to protect the incorporated protein in the highly acidic environment of upper gastrointestinal tract and deliver it in the neutral or slightly basic region of the lower intestine. The protein/poly(NiPAAm-co-IA) hydrogels were synthetized by free radical crosslinking copolymerization and were characterized concerning their swelling capability, mechanical properties, and morphology. The pore structure and sizes up to 1.90 nm allowed good entrapment of lipase molecules. Model protein, lipase from Candida rugosa, was entrapped within hydrogels upon mild conditions that provided its protection from harmful environmental influences. The efficiency of the lipase entrapment reached 96.7%, and was dependent on the initial concentration of lipase solution. The swelling of the obtained hydrogels in simulated pH and temperature of gastrointestinal tract, the lipase entrapment efficiency, and its release profiles from hydrogels were investigated as well. PMID:24982870

  11. Stimuli-Sensitive Hydrogel Based on N-Isopropylacrylamide and Itaconic Acid for Entrapment and Controlled Release of Candida rugosa Lipase under Mild Conditions

    PubMed Central

    Milašinović, Nikola; Knežević-Jugović, Zorica; Milosavljević, Nedeljko; Lučić Škorić, Marija; Filipović, Jovanka; Kalagasidis Krušić, Melina

    2014-01-01

    Stimuli responsive pH- and temperature-sensitive hydrogel drug delivery systems, as those based on N-isopropylacrylamide (NiPAAm) and itaconic acid (IA), have been attracting much of the attention of the scientific community nowadays, especially in the field of drug release. By adjusting comonomer composition, the matrix is enabled to protect the incorporated protein in the highly acidic environment of upper gastrointestinal tract and deliver it in the neutral or slightly basic region of the lower intestine. The protein/poly(NiPAAm-co-IA) hydrogels were synthetized by free radical crosslinking copolymerization and were characterized concerning their swelling capability, mechanical properties, and morphology. The pore structure and sizes up to 1.90 nm allowed good entrapment of lipase molecules. Model protein, lipase from Candida rugosa, was entrapped within hydrogels upon mild conditions that provided its protection from harmful environmental influences. The efficiency of the lipase entrapment reached 96.7%, and was dependent on the initial concentration of lipase solution. The swelling of the obtained hydrogels in simulated pH and temperature of gastrointestinal tract, the lipase entrapment efficiency, and its release profiles from hydrogels were investigated as well. PMID:24982870

  12. 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. PMID:2586234

  13. Inhibition of key enzymes linked to obesity by preparations from Mediterranean dietary plants: effects on α-amylase and pancreatic lipase activities.

    PubMed

    2013-12-01

    One of the most important strategy in the treatment of obesity includes the development of nutrient digestion and absorption inhibitors. Inhibition of digestive enzymes is one of the most widely studied mechanisms used to determine the potential efficacy of natural products as hypolipidemic and hypoglycaemic agents. In vitro studies here reported were performed to evaluate the inhibitory activity of five species(as hydroalcoholic extracts) of edible plants from Calabria region (Italy) on amylase and lipase by monitoring the hydrolysis of p-NPC and the hydrolysis of glycoside bonds indigestible carbohydrate foods. The formulation obtained from Clematis vitalba L. exhibited the strongest inhibitory effect on pancreatic lipase (IC50=0.99 mg/ml) and on α-amylase(IC50=31.52 μg/ml). In order to explore metabolome production HPTLC analysis of the extracts was performed, revealing the predominance of (±)-catechin, caffeic acid and chlorogenic acid in C. vital ba formulation at concentration of 23.18±3.14,13.63±0.65 and 18.88±0.76 mg/g, respectively. GC/MS analysis was used to identify fatty acids and terpene composition. PMID:24122547

  14. Lipase-catalyzed synthesis of structured phenolic lipids in solvent-free system using flaxseed oil and selected phenolic acids as substrates.

    PubMed

    Sorour, Noha; Karboune, Salwa; Saint-Louis, Richard; Kermasha, Selim

    2012-04-15

    Structured phenolic lipids (PLs) were obtained by lipase-catalyzed transesterification of flaxseed oil, in a solvent-free system (SFS), with selected phenolic acids, including hydroxylated and/or methoxylated derivatives of cinnamic, phenyl acetic and benzoic acids. A bioconversion yield of 65% was obtained for the transesterification of flaxseed oil with 3,4-dihydroxyphenyl acetic acid (DHPA). However, the effect of the chemical structure of phenolic acids on the transesterification of flaxseed oil in SFS was of less magnitude as compared to that in organic solvent system (OSS). Using DHPA, the APCI-MS analysis confirmed the synthesis of monolinolenyl, dilinolenyl, linoleyl linolenyl and oleyl linolenyl dihydroxyphenyl acetates as phenolic lipids. A significant increase in the enzymatic activity from 200 to 270 nmol of PLs/g solid enzyme/min was obtained upon the addition of the non-ionic surfactant Span 65. However, upon the addition of the anionic surfactant, sodium bis-2-ethylhexyl sulfosuccinate (AOT), and the cationic one, hexadecyltrimethylammonium bromide (CTAB), the enzymatic activity was decreased slightly from 200 to 192 and 190 nmol of PLs/g solid enzyme/min, respectively. The results also showed that the increase in DHPA concentration from 20 to 60 mM resulted in a significant increase in the volumetric productivity (P(V)) from 1.61 to 4.74 mg PLs per mL reaction mixture per day. PMID:22329891

  15. Microbial lipases: production and applications.

    PubMed

    Ghosh, P K; Saxena, R K; Gupta, R; Yadav, R P; Davidson, S

    1996-01-01

    Lipases occupy a prominent place among biocatalysts and have a wide spectrum of biotechnological applications. Lipases are unique as they hydrolyse fats into fatty acids and glycerol at the water-lipid interface and can reverse the reaction in non-aqueous media. The stability of these enzymes in organic solvents have pushed them into the frontier areas of organic synthesis leading to the designing of novel drugs, surfactants, bioactive compounds and oleochemicals. In addition, lipase-catalysed trans-esterification and inter-esterification reactions have been exploited in the fat industry. Looking into the wide scenario of lipase applications, commercialization of lipase production is a prime area of interest for microbiologists, process engineers and biochemists. Research carried out in this field has revealed that microbes, especially fungi and bacteria, are the tools of choice for commercial production. Recently, the structure determination of a few microbial lipases has widened our knowledge about the unique mechanism of catalysis of this enzyme. PMID:8828407

  16. Enhancement of the organic solvent-stability of the LST-03 lipase by directed evolution.

    PubMed

    Kawata, Takuya; Ogino, Hiroyasu

    2009-01-01

    LST-03 lipase from an organic solvent-tolerant Pseudomonas aeruginosa LST-03 has high stability and activity in the presence of various organic solvents. In this research, enhancement of organic solvent-stability of LST-03 lipase was attempted by directed evolution. The structural gene of the LST-03 lipase was amplified by the error prone-PCR method. Organic solvent-stability of the mutated lipases was assayed by formation of a clear zone of agar which contained dimethyl sulfoxide (DMSO) and tri-n-butyrin and which overlaid a plate medium. And the organic solvent-stability was also confirmed by measuring the half-life of activity in the presence of DMSO. Four mutated enzymes were selected on the basis of their high organic solvent-stability in the presence of DMSO. The organic solvent-stabilities of mutated LST-03 lipase in the presence of various organic solvents were measured and their mutated amino acid residues were identified. The half-lives of the LST-03-R65 lipase in the presence of cyclohexane and n-decane were about 9 to 11-fold longer than those of the wild-type lipase, respectively. Some substituted amino acid residues of mutated LST-03 lipases have been located at the surface of the enzyme molecules, while some other amino acid residues have been changed from neutral to basic residues. PMID:19731302

  17. 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

  18. Assessment of two immobilized lipases activity and stability to low temperatures in organic solvents under ultrasound-assisted irradiation.

    PubMed

    Batistella, Luciane; Ustra, Mara K; Richetti, Aline; Pergher, Sibele B C; Treichel, Helen; Oliveira, J V; Lerin, Lindomar; de Oliveira, Débora

    2012-03-01

    Both stability and catalytic activity of two commercial immobilized lipases were investigated in the presence of different organic solvents in ultrasound-assisted system. In a general way, for Novozym 435, the use of ethanol as solvent led to a loss of activity of 35% after 10 h of contact. The use of iso-octane conducted to a gradual increase in lipase activity in relation to the contact time, reaching a maximum value of relative activity of 126%. For Lipozyme RM IM, after 5 h of exposure, the enzyme presented no residual activity when ethanol was used as solvent. The solvents tert-butanol and iso-octane showed an enhancement of about 20 and 17% in the enzyme activity in 6 h of exposure, respectively. Novozym 435 and Lipozyme IM presented high stability to storage after treatment under ultrasound-assisted system using n-hexane and tert-butanol as solvents. PMID:21779888

  19. Lipase cocktail for efficient conversion of oils containing phospholipids to biodiesel.

    PubMed

    Amoah, Jerome; Ho, Shih-Hsin; Hama, Shinji; Yoshida, Ayumi; Nakanishi, Akihito; Hasunuma, Tomohisa; Ogino, Chiaki; Kondo, Akihiko

    2016-07-01

    The presence of phospholipid has been a challenge in liquid enzymatic biodiesel production. Among six lipases that were screened, lipase AY had the highest hydrolysis activity and a competitive transesterification activity. However, it yielded only 21.1% FAME from oil containing phospholipids. By replacing portions of these lipases with a more robust bioFAME lipase, CalT, the combination of lipase AY-CalT gave the highest FAME yield with the least amounts of free fatty acids and partial glycerides. A higher methanol addition rate reduced FAME yields for lipase DF-CalT and A10D-CalT combinations while that of lipase AY-CalT combination improved. Optimizing the methanol addition rate for lipase AY-CalT resulted in a FAME yield of 88.1% at 2h and more than 95% at 6h. This effective use of lipases could be applied for the rapid and economic conversion of unrefined oils to biodiesel. PMID:27019125

  20. Thiadiazole Carbamates: Potent Inhibitors of Lysosomal Acid Lipase and Potential Niemann-Pick Type C Disease Therapeuticsa

    PubMed Central

    Rosenbaum, Anton I.; Cosner, Casey C.; Mariani, Christopher J.; Maxfield, Frederick R.; Wiest, Olaf; Helquist, Paul

    2010-01-01

    Niemann-Pick type C (NPC) disease is a lysosomal storage disorder characterized at the cellular level by abnormal accumulation of cholesterol and other lipids in lysosomal storage organelles. Lysosomal acid lipase (LAL) has been recently identified as a potential therapeutic target for NPC. LAL can be specifically inhibited by a variety of 3,4-disubstituted thiadiazole carbamates. An efficient synthesis of the C(3) oxygenated/C(4) aminated analogues has been developed that furnishes the products in high yields and high degrees of purity. Common intermediates can also be used for the synthesis of the C(3) carbon substituted derivatives. Herein we tested various thiadiazole carbamates, amides, esters, and ketones for inhibition of LAL. In addition, we tested a diverse selection of commercially available non-thiadiazole carbamates. Our studies show that, among the compounds examined herein, only thiadiazole carbamates are effective inhibitors of LAL. We present a mechanism for LAL inhibition by these compounds whereby LAL transiently carbamoylates the enzyme similarly to previously described inhibition of acetylcholinesterase by rivastigmine and other carbamates as well as acylation of various lipases by orlistat. PMID:20557099

  1. Maté tea inhibits in vitro pancreatic lipase activity and has hypolipidemic effect on high-fat diet-induced obese mice.

    PubMed

    Martins, Fernanda; Noso, Tatiana M; Porto, Viviane B; Curiel, Alline; Gambero, Alessandra; Bastos, Deborah H M; Ribeiro, Marcelo L; Carvalho, Patrícia de O

    2010-01-01

    The inhibitory effects of maté tea (MT), a beverage produced with leaves from Ilex paraguariensis, in vitro lipase activity and on obesity in obese mice models were examined. For the in vitro experiment, porcine and human pancreatic lipase (PL) activities were determined by measuring the rate of release of oleic acid from hydrolysis of olive oil emulsified with taurocholate, phospholipids, gum arabic, or polyvinyl alcohol. For the in vivo experiments, animals were fed with a standard diet (SD, n = 10) or high-fat diet (HFD, n = 30) for 16 weeks. After the first 8 weeks on the HFD, the animals were treated with 1 and 2 g/kg of body weight of MT. The time course of the body weight and obesity-related biochemical parameters were evaluated. The results showed that MT inhibited both porcine and human PL (half-maximal inhibitory concentration = 1.5 mg MT/ml) and induced a strong inhibition of the porcine lipase activity in the hydrolysis of substrate emulsified with taurocholate + phosphatidylcholine (PC) (83 +/- 3.8%) or PC alone (62 +/- 4.3%). MT suppressed the increases in body weight (P < 0.05) and decreased the serum triglycerides and low-density lipoprotein (LDL)-cholesterol concentrations at both doses (from 190.3 +/- 5.7 to 135.0 +/- 8.9 mg/dl, from 189.1 +/- 7.3 to 129.3 +/- 17.6 mg/dl; P < 0.05, respectively) after they had been increased by the HFD. The liver lipid content was also decreased by the diet containing MT (from 132.6 +/- 3.9 to 95.6 +/- 6.1 mg/g of tissue; P < 0.05). These results suggest that MT could be a potentially therapeutic alternative in the treatment of obesity caused by a HFD. PMID:19543216

  2. 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. PMID:26887328

  3. Covalent binding of hyper-activated Rhizomucor miehei lipase (RML) on hetero-functionalized siliceous supports.

    PubMed

    Garmroodi, Maryam; Mohammadi, Mehdi; Ramazani, Ali; Ashjari, Maryam; Mohammadi, Javad; Sabour, Behrouz; Yousefi, Maryam

    2016-05-01

    Physical adsorption onto hydrophobic supports has proven to be an effective way to improve the activity of lipases. Covalent binding, on the other hand, enhances the active lifetime of the immobilized biocatalysts. To combine the benefits of adsorption and covalent binding, immobilization of RML on new hetero-functional supports are reported. For this, chemical modification of silica and silica mesoporous nanoparticles was performed by the simultaneous use of two coupling linkers; Octyltriethoxysilane (OTES) for hydrophobic interaction and glycidoxypropyltrimethoxylsilane (GPTMS) for covalent linkage of RML. Altering the GPTMS/OTES ratio makes possible to have different amount of octyl and epoxy groups on the supports. The results showed that immobilization of RML on octyl-functionalized supports produces specific activity almost 1.5-2 folds greater than the specific activity of the free enzyme. The observed hyper-activation decreased with increasing epoxy groups on the supports confirming the enhancement of covalent nature of the attachment. Leaching experiment was also confirmed positive effect of the presence of epoxy groups on the supports. Regarding the specific activity of the immobilized preparations and desorption percentages of RML from each support, the most suitable carrier obtains from the functionalization of the supports in presence of GPTMS and OTES in the ratio of 1:1. PMID:26812114

  4. Relationship between lipoprotein lipase activity and plasma sex steroid level in obese women.

    PubMed Central

    Iverius, P H; Brunzell, J D

    1988-01-01

    In obese women (n = 16) at their weight, fasting adipose tissue lipoprotein lipase (LPL) activity, obtained by elution with serum and heparin at 4 degrees and 37 degrees C, was inversely correlated to plasma estradiol levels (r = -0.724; P = 0.002) and (r = -0.641; P = 0.010), respectively. Furthermore, fasting postheparin plasma LPL activity during a heparin infusion, showed an even stronger inverse correlation to plasma estradiol when measured at 60 min (r = -0.815; P less than 0.001). None of the above parameters was correlated to the body mass index. Postprandial LPL activity in postheparin plasma, measured 10 min after a heparin injection, showed a strong positive correlation with plasma free testosterone (r = 0.780; P = 0.001). Neither of these parameters was correlated with the body mass index. The origin of this LPL activity is presently unknown but could conceivably represent a pool of LPL from skeletal muscle. Since it has been shown convincingly that estrogen decreases adipose tissue LPL activity in the rat, the present studies strongly suggest that estradiol is a major negative regulator of fasting adipose tissue LPL activity in women. PMID:3417867

  5. Enzymatic interesterification of triglyceride with surfactant-coated lipase in organic media.

    PubMed

    Goto, M; Goto, M; Kamiya, N; Nakashio, F

    1995-01-01

    Several surfactant-coated enzymes have been prepared by coating lipases of various origins with a nonionic surfactant, glutamic acid dioleylester ribitol (2C(18)Delta(9)GE). Enzymatic interesterification of tripalmitin with oleic acid using the surfactant-coated lipase was carried out in organic media. The surfactant-coated lipases could effectively catalyze the interesterification of glycerides better than did the powder lipases. A suitable organic solvent was an aliphatic hydrocarbon such as isooctane. The enzymatic activity for the interesterification strongly depended on the origin of the lipase. The surfactant-coated lipase prepared by Mucor javanicus showed the highest enzymatic activity for the interesterification of glycerides, although its powder lipase did not show enzymatic activity. Selective interesterification of glycerides could be performed by adjusting the concentration ratio of oleic acid to tripalmitin in isooctane. Di-substituted glyceride could be selectively produced when the concentration ratio of carboxylic acid to glycerides was 7. (c) 1995 John Wiley & Sons, Inc. PMID:18623048

  6. Lipases in autolysed cultures of filamentous fungi.

    PubMed

    García-Lepe, R; Nuero, O M; Reyes, F; Santamaría, F

    1997-08-01

    Fifty-one fungi from different genera and strains were checked in plate to determine lipase activity in protein precipitates from their autolysed cultures. Each of them was then analysed at 3.5, 6.5 and 9.2 pH units and, as a consequence, basic lipases with high activity at 9.2 pH were found after 1 h of incubation. Only 25% of the studied fungi showed this lipase activity, among them the best producers were fungi from genus Fusarium (47% of fungi had lipase activity). In addition to lipase activity, Fusaria showed a low hydrolytic activity on cutin and suberin. The genus Aspergillus produced lipase and cutinase activity to a similar extent. Aspergillus nidulans 2544 also showed suberinase activity in a considerable amount. Penicillium species had very low activities. Other species and strains from genus Trichoderma, order Mucorales and class Basidiomycetes, did not show lipase activity in their degradative processes. PMID:9281862

  7. Polymer-like polyphenols of black tea and their lipase and amylase inhibitory activities.

    PubMed

    Kusano, Rie; Andou, Hisashi; Fujieda, Miho; Tanaka, Takashi; Matsuo, Yosuke; Kouno, Isao

    2008-03-01

    Lipase and amylase inhibitory activities of black tea were examined. After solvent partitioning of a black tea extract with the ethyl acetate and n-butanol, the two soluble fractions showed comparable inhibitory activities. Activity in the ethyl acetate fraction was mainly attributable to polyphenols with low-molecular weights, such as theaflavin gallates. On the other hand, the active substance in the n-butanol layer was ascertained to be a polymer-like substance. 1H- and 13C-NMR spectra showed signals arising from the flavan A-ring and galloyl groups, although signals due to flavan B-rings were not detected, suggesting that the polymer-like substances were generated by oxidative condensation of flavan B-rings, a result which was previously deduced from our results of in vitro catechin oxidation experiments. Enzymatic oxidation of epicatechin 3-O-gallate produced a similar polymer-like substance and suggested that condensation between a B-ring and galloyl groups was involved in the polymerization reaction. PMID:18310934

  8. Optimization of pancreatic lipase inhibitory and antioxidant activities of Ilex paraguariensis by using response surface methodology.

    PubMed

    Oh, Kyung-Eon; Shin, Hyeji; Jeon, Young Ho; Jo, Yang Hee; Lee, Mi Kyeong; Lee, Ken S; Park, Byoungduck; Lee, Ki Yong

    2016-07-01

    Response surface methodology (RSM) using a Box-Behnken design was used to optimize the extraction conditions for obtaining pancreatic lipase inhibitory and antioxidant principles from Ilex paraguariensis leaves. Three influencing factors: extraction time (min), the liquid-solid ratio, and ethanol concentration (%, v/v) were investigated in the ultrasonic extraction process. Optimization of the extraction conditions to obtain a product with minimum PL activity, maximum antioxidant activity, and maximum yield was performed using RSM by focusing on the three target influencing factors. The optimum conditions were established as the ethanol concentration (54.8 %), liquid-solid ratio (35.4), and extraction time (70.0 min). Under these conditions, the 2,2-diphenyl-1-picrylhydrazyl scavenging activity, PL activity, extraction yield were 59.3 ± 3.5, 35.3 ± 3.0, and 34.4 ± 0.4 %, respectively, similar to the theoretical predicted values of 59.7, 35.2, and 34.3 %, respectively. PMID:27277165

  9. Screening and characterization of a thermostable lipase from marine Streptomyces sp. strain W007.

    PubMed

    Yuan, Dongjuan; Lan, Dongming; Xin, Ruipu; Yang, Bo; Wang, Yonghua

    2016-02-01

    A screening method along with the combination of genome sequence of microorganism, pairwise alignment, and lipase classification was used to search the thermostable lipase. Then, a potential thermostable lipase (named MAS1) from marine Streptomyces sp. strain W007 was expressed in Pichia pastoris X-33, and the biochemical properties were characterized. Lipase MAS1 belongs to the subfamily I.7, and it has 38% identity to the well-characterized Bacillus subtilis thermostable lipases in the subfamily I.4. The purified enzyme was estimated to be 29 kDa. The enzyme showed optimal temperature at 40 °C, and retained more than 80% of initial activity after 1 H incubation at 60 °C, suggesting that MAS1 was a thermostable lipase. MAS1 was an alkaline enzyme with optimal pH value at 7.0 and had stable activity for 12 H of incubation at pH 6.0-9.0. It was stable and retained about 90% of initial activity in the presence of Cu(2+) , Ca(2+) , Ni(2+) , and Mg(2+) , whereas 89.05% of the initial activity was retained when ethylene diamine tetraacetic acid was added. MAS1 showed the tolerance to organic solvents, but was inhibited by various surfactants. MAS1 was verified to be a triglyceride lipase and could hydrolyze triacylglycerol and diacylglycerol. The result represents a good example for researchers to discover thermostable lipase for industrial application. PMID:25639796

  10. Comparative study of free and immobilized lipase from Bacillus aerius and its application in synthesis of ethyl ferulate.

    PubMed

    Saun, Nitin Kumar; Narwal, Sunil Kumar; Dogra, Priyanka; Chauhan, Ghanshyam Singh; Gupta, Reena

    2014-01-01

    In the present study, a purified lipase from Bacillus aerius immobilized on celite matrix was used for synthesis of ethyl ferulate. The celite-bound lipase exposed to glutaraldehyde showed 90.02% binding efficiency. It took two hours to bind maximally onto the support. The pH and temperature optima of the immobilized lipase were same as those of free enzyme i.e 9.5 and 55°C. Among different substrates both free and immobilized lipase showed maximum affinity towards p-nitrophenyl palmitate (p-NPP). The lipase activity was found to be stimulated in the presence of Mg(2+) in case of free enzyme while Zn(2+) and Fe(3+) showed stimulatory effect on immobilized lipase whereas salt ions as well as chelating agents inhibited activity of both free and immobilized lipase. Maximum enzyme activity was observed in n-hexane as organic solvent followed by n-heptane for both free and immobilized lipase, however CCl4, acetone and benzene inhibited the enzyme activity. Moreover, all the selected detergents (SDS, Triton X-100, Tween 80 and Tween 20) had an inhibitory effect on both free and immobilized enzyme activity. The celite bound lipase (1.5%) efficiently performed maximum esterification (2.51 moles/l) of ethanol and ferulic acid (100 mM each, at a molar ratio of 1:3) when incubated at 55°C for 48 h resulting in the formation of ester ethyl ferulate. PMID:25099909

  11. Lipase production by Aspergillus niger under various growth conditions using solid state fermentation.

    PubMed

    Olama, Z A; el-Sabaeny, A H

    1993-12-01

    Ricinus seed litters were chosen as a cheap carbon source for lipase production by A. niger under solid state fermentation (SSF). Maximum lipase production was achieved upon using an enriched (potassium citrate and casein) waste at pH 7.8 and 30 degrees C for 8 days incubation. Nitrogen sources as NH4Cl, NH4NO3, (NH4)2SO4, urea and amino acids repressed the lipolytic activity. The chloride salts of Ba2+, Co2+, Cu2+, Fe3+, Hg2+, K+, Mg2+, Mn2+, Na+ and Sn2+ inhibited, while Zn+2 did not affect lipase production. Compounds containing hydrolyzable ester group, such as Tween(s), were found to inhibit lipase activity. When the effect of different additives such as EDTA, gum acacia, span(s), mineral and vitamins, were studied, it was found that they all exhibit decreased lipase production by the tested fungus. PMID:8172691

  12. Extracellular lipase production by a sapwood-staining fungus, Ophiostoma piceae.

    PubMed

    Gao, Y; Breuil, C

    1995-11-01

    The extracellular lipase production of a sapwood-staining fungus, Ophiostoma piceae, grown in liquid media, was optimally active at pH 5.5 and 37°C. Although glucose, fructose, sucrose, starch and dextrin, as carbon sources for growth gave similar mycelial yields, which were higher than those obtained with arabinose, galactose or raffinose, the cells growing on those carbohydrates produced little extracellular lipase. However, both high biomass and lipase activity were obtained when plant oils (olive, soybean, corn, sunflower seed, sesame, cotton seed or peanut) were used as carbon sources. Among the nitrogen sources examined, Casamino acids gave the best growth, whereas (NH4)2SO4 gave the best lipase production. The highest lipase productivity seen was obtained in a medium with olive oil as carbon source and a combination of (NH4)2SO4and peptone as nitrogen source. PMID:24415011

  13. Studies of the in vitro cytotoxic, antioxidant, lipase inhibitory and antimicrobial activities of selected Thai medicinal plants

    PubMed Central

    2012-01-01

    Background Traditional folk medicinal plants have recently become popular and are widely used for primary health care. Since Thailand has a great diversity of indigenous (medicinal) plant species, this research investigated 52 traditionally used species of Thai medicinal plants for their in vitro cytotoxic, antioxidant, lipase inhibitory and antimicrobial activities. Methods The 55 dried samples, derived from the medicinally used parts of the 52 plant species were sequentially extracted by hexane, dichloromethane, ethanol and water. These 220 extracts were then screened for in vitro (i) cytotoxicity against four cell lines, derived from human lung (A549), breast (MDA-MB-231), cervical (KB3-1) and colon (SW480) cancers, using the MTT cytotoxicity assay; (ii) antioxidant activity, analyzed by measuring the scavenging activity of DPPH radicals; (iii) lipase inhibitory activity, determined from the hydrolytic reaction of p-nitrophenyllaurate with pancreatic lipase; and (iv) antimicrobial activity against three Gram-positive and two Gram-negative bacteria species plus one strain of yeast using the disc-diffusion method and determination of the minimum inhibitory concentration by the broth micro-dilution assay. Results The crude dichloromethane and/or ethanol extracts from four plant species showed an effective in vitro cytotoxic activity against the human cancer cell lines that was broadly similar to that of the specific chemotherapy drugs (etoposide, doxorubicin, vinblastine and oxaliplatin). In particular, this is the first report of the strong in vitro cytotoxic activity of Bauhinia strychnifolia vines. The tested tissue parts of only six plant species (Allium sativum, Cocoloba uvifera, Dolichandrone spathacea, Lumnitzera littorea, Sonneratia alba and Sonneratia caseolaris) showed promising potential antioxidant activity, whereas lipase inhibitory activity was only found in the ethanol extract from Coscinum fenestratum and this was weak at 17-fold lower than Orlistat

  14. Clearing-factor lipase in adipose tissue. A medium in which the enzyme activity of tissue from starved rats increases in vitro

    PubMed Central

    Salaman, M. R.; Robinson, D. S.

    1966-01-01

    1. When epididymal fat bodies from starved rats are incubated for 3·5hr. at 37° in a defined medium in vitro the total clearing-factor lipase activity rises to approximately twice its initial value. 2. During the incubation period part of the tissue clearing-factor lipase activity appears in the medium. 3. Heparin, glucose, insulin, and HCO3− and K+ ions are shown to be important medium constituents. PMID:5964961

  15. Effects of Porous Polystyrene Resin Parameters on Candida antarctica Lipase B Adsorption, Distribution, and Polyester Synthesis Activity

    SciTech Connect

    Chen,B.; Miller, M.; Gross, R.

    2007-01-01

    Polystyrene resins with varied particle sizes (35 to 350-600 {mu}m) and pore diameters (300-1000 {angstrom}) were employed to study the effects of immobilization resin particle size and pore diameter on Candida antarctica Lipase B (CALB) loading, distribution within resins, fraction of active sites, and catalytic properties for polyester synthesis. CALB adsorbed rapidly (saturation time {<=}4 min) for particle sizes 120 {mu}m (pore size = 300 {angstrom}). Infrared microspectroscopy showed that CALB forms protein loading fronts regardless of resin particle size at similar enzyme loadings ({approx}8%). From the IR images, the fractions of total surface area available to the enzyme are 21, 33, 35, 37, and 88% for particle sizes 350-600, 120, 75, 35 {mu}m (pore size 300 {angstrom}), and 35 {mu}m (pore size 1000 {angstrom}), respectively. Titration with methyl p-nitrophenyl n-hexylphosphate (MNPHP) showed that the fraction of active CALB molecules adsorbed onto resins was {approx}60%. The fraction of active CALB molecules was invariable as a function of resin particle and pore size. At {approx}8% (w/w) CALB loading, by increasing the immobilization support pore diameter from 300 to 1000 {angstrom}, the turnover frequency (TOF) of {var_epsilon}-caprolactone ({var_epsilon}-CL) to polyester increased from 12.4 to 28.2 s{sup -1}. However, the {var_epsilon}-CL conversion rate was not influenced by changes in resin particle size. Similar trends were observed for condensation polymerizations between 1,8-octanediol and adipic acid. The results herein are compared to those obtained with a similar series of methyl methacrylate resins, where variations in particle size largely affected CALB distribution within resins and catalyst activity for polyester synthesis.

  16. Candida antarctica Lipase B Chemically Immobilized on Epoxy-Activate Micro- and Nanobeads: Catalysts for Polyester Synthesis

    SciTech Connect

    Chen,B.; Hu, J.; Miller, E.; Xie, W.; Cai, M.; Gross, R.

    2008-01-01

    Candida antarctica Lipase B (CALB) was covalently immobilized onto epoxy-activated macroporous poly(methyl methacrylate) Amberzyme beads (235 {mu}m particle size, 220 Angstroms pore size) and nanoparticles (nanoPSG, diameter 68 nm) with a poly(glycidyl methacrylate) outer region. Amberzyme beads allowed CALB loading up to 0.16 g of enzyme per gram of support. IR microspectroscopy generated images of Amberzyme-CALB beads showed CALB is localized within a 50 {mu}m thick loading front. IR microspectroscopy images, recorded prior to and after treatment of Amberzyme-CALB with DMSO/aqueous Triton X-100, are similar, confirming that CALB is largely chemically linked to Amberzyme. The activity of CALB immobilized on Amberzyme, Lewatit (i.e., Novozym 435 catalyst), and nanoPSG was assessed for lactone ring-opening and step-condensation polymerizations. For example, the percent conversion of -caprolactone using the same amount of enzyme catalyzed by Amberzym-CALB, Novozym 435, and nanoPSG-CALB for 20 min was 7.0, 16, and 65%, respectively. Differences in CALB reactivity were discussed based on resin physical parameters and availability of active sites determined by active site titrations. Regardless of the matrix used and chemical versus physical immobilization, -CL ring-opening polymerizations occur by a chain growth mechanism without chain termination. To test Amberzyme-CALB stability, the catalyst was reused over three reaction cycles for -CL ring-opening polymerization (70 C, 70 min reactions) and glycerol/1, 8-octanediol/adipic acid polycondensation reactions (90 C, 64 h). Amberzyme-CALB was found to have far better stability for reuse relative to Novozym 435 for the polycondensation reaction.

  17. Candida antarctica lipase B chemically immobilized on epoxy-activated micro- and nanobeads: catalysts for polyester synthesis.

    PubMed

    Chen, Bo; Hu, Jun; Miller, Elizabeth M; Xie, Wenchun; Cai, Minmin; Gross, Richard A

    2008-02-01

    Candida antarctica Lipase B (CALB) was covalently immobilized onto epoxy-activated macroporous poly(methyl methacrylate) Amberzyme beads (235 microm particle size, 220 A pore size) and nanoparticles (nanoPSG, diameter 68 nm) with a poly(glycidyl methacrylate) outer region. Amberzyme beads allowed CALB loading up to 0.16 g of enzyme per gram of support. IR microspectroscopy generated images of Amberzyme-CALB beads showed CALB is localized within a 50 microm thick loading front. IR microspectroscopy images, recorded prior to and after treatment of Amberzyme-CALB with DMSO/aqueous Triton X-100, are similar, confirming that CALB is largely chemically linked to Amberzyme. The activity of CALB immobilized on Amberzyme, Lewatit (i.e., Novozym 435 catalyst), and nanoPSG was assessed for lactone ring-opening and step-condensation polymerizations. For example, the percent conversion of -caprolactone using the same amount of enzyme catalyzed by Amberzym-CALB, Novozym 435, and nanoPSG-CALB for 20 min was 7.0, 16, and 65%, respectively. Differences in CALB reactivity were discussed based on resin physical parameters and availability of active sites determined by active site titrations. Regardless of the matrix used and chemical versus physical immobilization, -CL ring-opening polymerizations occur by a chain growth mechanism without chain termination. To test Amberzyme-CALB stability, the catalyst was reused over three reaction cycles for -CL ring-opening polymerization (70 degrees C, 70 min reactions) and glycerol/1,8-octanediol/adipic acid polycondensation reactions (90 degrees C, 64 h). Amberzyme-CALB was found to have far better stability for reuse relative to Novozym 435 for the polycondensation reaction. PMID:18197630

  18. Secretion and apparent activation of human hepatic lipase requires proper oligosaccharide processing in the endoplasmic reticulum.

    PubMed Central

    Verhoeven, A J; Neve, B P; Jansen, H

    1999-01-01

    Human hepatic lipase (HL) is a glycoprotein with four N-linked oligosaccharide side chains. The importance of glycosylation for the secretion of catalytically active HL was studied in HepG2 cells by using inhibitors of intracellular trafficking, N-glycosylation and oligosaccharide processing. Secretion of HL was inhibited by carbonyl cyanide m-chlorophenylhydrazone (CCCP), monensin, brefeldin A (BFA), tunicamycin, castanospermine and N-methyldeoxynojirimycin, but not by 1-deoxymannojirimycin. Secretion of alpha1-antitrypsin, an unrelated N-glycoprotein, was also inhibited by monensin, BFA and tunicamycin, but not by CCCP, castanospermine or N-methyldeoxynojirimycin. Intracellular HL activity decreased with CCCP, tunicamycin, castanospermine and N-methyldeoxynojirimycin, but increased with monensin and BFA. In the absence of protein synthesis de novo, HL activity secreted into the medium was 7.8+/-2.1-fold higher (mean+/-S.D., n=7) than the simultaneous fall in intracellular HL activity. In cells pretreated with monensin or BFA, this factor decreased to 1.3+/-0.5, indicating that the apparent increase in HL activity had already occurred within these cells. After chromatography on Sepharose-heparin, the specific triacylglycerol hydrolase activity of secreted HL was only 1.7+/-0. 3-fold higher than that of intracellular HL, indicating that the secretion-coupled increase in HL activity is only partly explained by true activation. We conclude that oligosaccharide processing by glucosidases in the endoplasmic reticulum is necessary for the transport of newly synthesized human HL, but not alpha1-antitrypsin, to the Golgi, where the catalytic activity of HL is unmasked. PMID:9854035

  19. Lipase-catalyzed interesterification of soybean oil with an omega-3 polyunsaturated fatty acid concentrate prepared from sardine oil.

    PubMed

    Akimoto, Masamichi; Izawa, Maki; Hoshino, Kazumi; Abe, Ken-Ichi; Takahashi, Hiromi

    2003-02-01

    To reduce the content of linoleoyl moiety in soybean oil, soybean oil that contains 53.0% linoleoyl moiety as molar acyl moiety composition was interesterified with an omega-3 polyunsaturated fatty acid (PUFA) concentrate (24.0 mol% eicosapentaenoic acid [EPA], 40.4 mol% docosahexaenoic acid [DHA]) prepared from sardine oil, using an immobilized sn-1,3-specific lipase from Rhizomucor miehei (Lipozyme IM). The reaction was carried out in a batch reactor at 37 degrees C under the following conditions: 500 micromol of soybean oil, molar ratio of omega-3 PUFA concentrate to soybean oil = 1.0-6.0,5.0 mL of heptane, and 30 batch interesterification units of enzyme. After the reaction time of 72 h, modified soybean oil, which contains 34.9% linoleoyl, 10.1% eicosapentaenoyl, and 14.2% docosahexaenoyl moieties, was produced at the molar reactant ratio of 6.0. In this oil, the total omega-3 acyl moiety composition reached 34.1%; the molar ratio of omega-3 to omega-6 acyl moieties was enhanced by five times compared with soybean oil. Compared with palmitic acid, DHA was kinetically six times less reactive, although the EPA was by 16% more reactive. PMID:12603099

  20. 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. PMID:26706798

  1. In vitro screening of medicinal plants used in Mexico as antidiabetics with glucosidase and lipase inhibitory activities.

    PubMed

    Ramírez, Guillermo; Zavala, Miguel; Pérez, Julia; Zamilpa, Alejandro

    2012-01-01

    This work shows the inhibitory effect on glucosidase and lipase enzymes of 23 medicinal plants described as traditional treatments for diabetes in several Mexican sources. Hydroalcoholic extracts of selected plants were evaluated at 1 mg/mL for glucosidase and 0.25 mg/mL for lipase inhibitory activities, respectively. Camellia sinensis, acarbose, and orlistat were used as positive controls. Dose-response curves were done with the most active species. Sixty percent of all tested extracts inhibited more than 25% of α-glucosidase activity. C. sinensis displayed an inhibition of 85% (IC(50) = 299 μg/mL), while Ludwigia octovalvis and Iostephane heterophylla showed the highest inhibition (82.7 %, IC(50) = 202 μg/mL and 60.6%, CI(50) = 509 μg/mL, resp.). With respect to lipase activity, L. octovalvis and Tecoma stans were the most inhibiting treatments (31.4%, IC(50) = 288 μg/mL; 27.2%, IC(50) = 320 μg/mL), while C. sinensis displayed 45% inhibition (IC(50) = 310 μg/mL). These results indicate that a high proportion of plants used in Mexico as treatment for diabetes displays significant inhibition of these digestive enzymes. PMID:23082084

  2. In Vitro Screening of Medicinal Plants Used in Mexico as Antidiabetics with Glucosidase and Lipase Inhibitory Activities

    PubMed Central

    Ramírez, Guillermo; Zavala, Miguel; Pérez, Julia; Zamilpa, Alejandro

    2012-01-01

    This work shows the inhibitory effect on glucosidase and lipase enzymes of 23 medicinal plants described as traditional treatments for diabetes in several Mexican sources. Hydroalcoholic extracts of selected plants were evaluated at 1 mg/mL for glucosidase and 0.25 mg/mL for lipase inhibitory activities, respectively. Camellia sinensis, acarbose, and orlistat were used as positive controls. Dose-response curves were done with the most active species. Sixty percent of all tested extracts inhibited more than 25% of α-glucosidase activity. C. sinensis displayed an inhibition of 85% (IC50 = 299 μg/mL), while Ludwigia octovalvis and Iostephane heterophylla showed the highest inhibition (82.7 %, IC50 = 202 μg/mL and 60.6%, CI50 = 509 μg/mL, resp.). With respect to lipase activity, L. octovalvis and Tecoma stans were the most inhibiting treatments (31.4%, IC50 = 288 μg/mL; 27.2%, IC50 = 320 μg/mL), while C. sinensis displayed 45% inhibition (IC50 = 310 μg/mL). These results indicate that a high proportion of plants used in Mexico as treatment for diabetes displays significant inhibition of these digestive enzymes. PMID:23082084

  3. Comparative analyses of lipoprotein lipase, hepatic lipase, and endothelial lipase, and their binding properties with known inhibitors.

    PubMed

    Wang, Ziyun; Li, Shen; Sun, Lidan; Fan, Jianglin; Liu, Zhenming

    2013-01-01

    The triglyceride lipase gene subfamily plays a central role in lipid and lipoprotein metabolism. There are three members of this subfamily: lipoprotein lipase, hepatic lipase, and endothelial lipase. Although these lipases are implicated in the pathophysiology of hyperlipidemia and atherosclerosis, their structures have not been fully solved. In the current study, we established homology models of these three lipases, and carried out analysis of their activity sites. In addition, we investigated the kinetic characteristics for the catalytic residues using a molecular dynamics simulation strategy. To elucidate the molecular interactions and determine potential key residues involved in the binding to lipase inhibitors, we analyzed the binding pockets and binding poses of known inhibitors of the three lipases. We identified the spatial consensus catalytic triad "Ser-Asp-His", a characteristic motif in all three lipases. Furthermore, we found that the spatial characteristics of the binding pockets of the lipase molecules play a key role in ligand recognition, binding poses, and affinities. To the best of our knowledge, this is the first report that systematically builds homology models of all the triglyceride lipase gene subfamily members. Our data provide novel insights into the molecular structures of lipases and their structure-function relationship, and thus provides groundwork for functional probe design towards lipase-based therapeutic inhibitors for the treatment of hyperlipidemia and atherosclerosis. PMID:23991054

  4. Continuous lipase-catalyzed esterification of soybean fatty acids under ultrasound irradiation.

    PubMed

    Trentin, Claudia M; Scherer, Robison P; Dalla Rosa, C; Treichel, H; Oliveira, D; Oliveira, J Vladimir

    2014-05-01

    This work investigates the continuous production of alkyl esters from soybean fatty acid (FA) charges using immobilized Novozym 435 as catalyst. The experiments were performed in a packed-bed bioreactor evaluating the effects of FA charge to alcohol (methanol and ethanol) molar ratio, from 1:1 to 1:6, substrate flow rate in the range of 0.5-2.5 mL/min and output irradiation power up to 154 W, at fixed temperature of 65 °C, on the reaction conversion. Results showed that almost complete conversions to fatty acids ethyl esters were achieved at mild ultrasonic power (61.6 W), FA to ethanol molar ratio of 1:6, operating temperature (65 °C) and remained nearly constant for long-term reactions without negligible enzyme activity losses. PMID:24078183

  5. New Atglistatin closely related analogues: Synthesis and structure-activity relationship towards adipose triglyceride lipase inhibition.

    PubMed

    Roy, Pierre-Philippe; D'Souza, Kenneth; Cuperlovic-Culf, Miroslava; Kienesberger, Petra C; Touaibia, Mohamed

    2016-08-01

    Adipose Triglyceride Lipase (ATGL) performs the first and rate-limiting step in lipolysis by hydrolyzing triacylglycerols stored in lipid droplets to diacylglycerols. By mediating lipolysis in adipose and non-adipose tissues, ATGL is a major regulator of overall energy metabolism and plasma lipid levels. Since chronically high levels of plasma lipids are linked to metabolic disorders including insulin resistance and type 2 diabetes, ATGL is an interesting therapeutic target. In the present study, fourteen closely related analogues of Atglistatin (1), a newly discovered ATGL inhibitor, were synthesized, and their ATGL inhibitory activity was evaluated. The effect of these analogues on lipolysis in 3T3-L1 adipocytes clearly shows that inhibition of the enzyme by Atglistatin (1) is due to the presence of the carbamate and N,N-dimethyl moieties on the biaryl central core at meta and para position, respectively. Mono carbamate-substituted analogue C2, in which the carbamate group was in the meta position as in Atglistatin (1), showed slight inhibition. Low dipole moment of Atglistatin (1) compared to the synthesized analogues possibly explains the lower inhibitory activities. PMID:27155760

  6. Lignin binding to pancreatic lipase and its influence on enzymatic activity.

    PubMed

    Zhang, Juan; Xiao, Lin; Yang, Yucai; Wang, Zhaoxia; Li, Genxi

    2014-04-15

    In this paper, we find that the effect of lignin on pancreatic lipase (PL) is dependent on reaction medium and substrate used. Experimental results reveal that lignin can gradually bind to PL to form a PL-lignin complex, resulting in an increased activity of the enzyme. The binding process is spontaneous and the PL-lignin complex formation is an endothermic reaction induced by hydrophobic and electrostatic interaction. There is a non-radiation energy transfer from PL to lignin during the binding process, and the binding of lignin to PL conforms to a secondary exponential decay function. Moreover, the α-helix content of the enzyme will be changed and the rigidity of its side chain will be enhanced due to the formation of lignin-PL complex. This study has not only provided the activation effect of lignin on PL, but also given an insight into the interaction between lignin and the enzyme, which would benefit the application of lignin in the pharmacy and food industry, as well as other fields. PMID:24295682

  7. Improving adsorption and activation of the lipase immobilized in amino-functionalized ordered mesoporous SBA-15

    NASA Astrophysics Data System (ADS)

    Xu, Yun-qiang; Zhou, Guo-wei; Wu, Cui-cui; Li, Tian-duo; Song, Hong-bin

    2011-05-01

    Ordered mesoporous SBA-15 was prepared by hydrothermal process and was functionalized with(3-aminopropyl) triethoxysilane (APTES) by post-synthesis-grafting method. The materials were characterized by scanning electron microscopy (SEM), transmission electron microscopy (TEM), energy-dispersive spectrometer (EDS), small-angle X-ray powder diffraction (SAXRD), N 2 adsorption-desorption and Fourier transform infrared spectroscopy (FT-IR). The results indicated that SBA-15 had a 2-dimensional hexagonal p6 mm mesoscopic structure and the mesoscopic structure was remained after the functionalization procedure. The activities of porcine pancreatic lipase (PPL) immobilized in SBA-15 by physical adsorption and in APTES functionalized SBA-15 by chemical adsorption were studied by hydrolysis of triacetin. Chemically adsorbed PPL showed higher loading amount and catalytic activity comparing with physically adsorbed PPL. The stability of immobilized PPL against thermal and pH of reaction medium was significantly improved. Recycling experiments showed that chemically adsorbed PPL exhibited better reusability than physically adsorbed PPL.

  8. Altering the interfacial activation mechanism of a lipase by solid-phase selective chemical modification.

    PubMed

    López-Gallego, Fernando; Abian, Olga; Guisán, Jose Manuel

    2012-09-01

    This study presents a combined protein immobilization, directed mutagenesis, and site-selective chemical modification approach, which was used to create a hyperactivated semisynthetic variant of BTL2. Various alkane chains were tethered at three different positions in order to mimic the lipase interfacial activation exogenously triggered by detergents. Optimum results were obtained when a dodecane chain was introduced at position 320 by solid-phase site-selective chemical modification. The resulting semisynthetic variant showed a 2.5-fold higher activity than the wild-type nonmodified variant in aqueous conditions. Remarkably, this is the maximum hyperactivation ever observed for BTL2 in the presence of detergents such as Triton X-100. We present evidence to suggest that the endogenous dodecane chain hyperactivates the enzyme in a similar fashion as an exogenous detergent molecule. In this way, we also observe a faster irreversible enzyme inhibition and an altered detergent sensitivity profile promoted by the site-selective chemical modification. These findings are also supported by fluorescence studies, which reveal that the structural conformation changes of the semisynthetic variant are different to those of the wild type, an effect that is more pronounced in the presence of detergent. Finally, the optimal immobilized semisynthetic variant was successfully applied to the selective synthesis of oxiran-2-yl butyrate. Significantly, this biocatalyst is 12-fold more efficient than the immobilized wild-type enzyme, producing the S-enantiomer with higher enantiospecificity (ee = 92%). PMID:22876885

  9. Understanding the thermostability and activity of Bacillus subtilis lipase mutants: insights from molecular dynamics simulations.

    PubMed

    Singh, Bipin; Bulusu, Gopalakrishnan; Mitra, Abhijit

    2015-01-15

    Improving the thermostability of industrial enzymes is an important protein engineering challenge. Point mutations, induced to increase thermostability, affect the structure and dynamics of the target protein in several ways and thus can also affect its activity. There appears to be no general rules for improving the thermostabilty of enzymes without adversely affecting their enzymatic activity. We report MD simulations, of wild type Bacillus subtilis lipase (WT) and its six progressively thermostable mutants (2M, 3M, 4M, 6M, 9M, and 12M), performed at different temperatures, to address this issue. Less thermostable mutants (LTMs), 2M to 6M, show WT-like dynamics at all simulation temperatures. However, the two more thermostable mutants (MTMs) show the required flexibility at appropriate temperature ranges and maintain conformational stability at high temperature. They show a deep and rugged free-energy landscape, confining them within a near-native conformational space by conserving noncovalent interactions, and thus protecting them from possible aggregation. In contrast, the LTMs having marginally higher thermostabilities than WT show greater probabilities of accessing non-native conformations, which, due to aggregation, have reduced possibilities of reverting to their respective native states under refolding conditions. Our analysis indicates the possibility of nonadditive effects of point mutations on the conformational stability of LTMs. PMID:25495458

  10. Computer-aided control of water activity for lipase-catalyzed esterification in solvent-free systems.

    PubMed

    Won, K; Lee, S B

    2001-01-01

    A computer system for on-line monitoring and control of the water activity (a(w)) in solvent-free media has been developed. The performance of this system was investigated by carrying out the lipase-catalyzed esterification of n-capric acid with n-decyl alcohol. A humidity sensor measured the relative humidity in the reactor headspace, which was then transmitted electrically to a digital computer that was used as a feedback controller. The water activity control was achieved by sparging either humidified air or dried air through the reaction medium at a flow rate determined by the digital feedback controller. The use of humid air and dry air for a(w) control made it possible to induce a larger a(w) gradient and thereby higher water transfer rate. As a result, the water activity quickly reached the desired a(w) values. We tested whether water activity in the reaction medium can be monitored by measuring relative humidity in the headspace. When the water activity in the liquid phase was determined from measurements of water content in the medium and compared to that measured directly with the humidity sensor, the a(w) in the reaction medium did not differ significantly from that in the headspace. This indicates that there is a near-equilibrium between the liquid medium and the exit air stream. Water activity was also successfully maintained close to the set point despite the massive production of water during the esterification process. Thus, the control system developed in this study is particularly useful for systems where large amounts of water are produced and where conventional methods make it difficult to control water activity as a result of a low water transfer rate. The effects that computer control of the water activity had on the reaction rate and yield were also examined. The reaction yield was significantly improved with water activity control. The conversions obtained at 28 h without and those with water activity control were 70% and 96%, respectively

  11. Immobilization of lipases on hydrophobilized zirconia nanoparticles: highly enantioselective and reusable biocatalysts.

    PubMed

    Chen, Yi Zhao; Yang, Cai Ting; Ching, Chi Bun; Xu, Rong

    2008-08-19

    Our study has demonstrated for the first time that zirconia nanoparticles modified by a simple carboxylic surfactant of a very long alkyl chain can significantly enhance the activity of the immobilized lipases for asymmetric synthesis in organic media. Zirconia nanoparticles of ca. 20 nm diameter were grafted with carboxylic surfactant modifiers from Tween 85 and erucic acid. The surface of nanoparticles was successfully changed from hydrophilic to hydrophobic. Lipases from Candida rugosa and Pseudomonas cepacia were immobilized on the modified zirconia nanoparticles by adsorption in aqueous solution. The immobilized lipases were used for the resolution of ( R, S)-ibuprofen and ( R, S)-1-phenylethanol through esterification and acylation, respectively, in isooctane organic solvent. When immobilized on erucic acid-modified zirconia, both lipases gave significantly higher activity and enantioselectivity compared with those from their corresponding crude lipase powders. The nanohybrid biocatalysts are stable and can be reused for eight cycles without loss in activity and selectivity. The interaction between the hydrophobic surface of zirconia support and lipases probably induces the conformational rearrangement of lipases into an active, stable form. PMID:18656972

  12. Use of stable emulsion to improve stability, activity, and enantioselectivity of lipase immobilized in a membrane reactor.

    PubMed

    Giorno, L; Li, N; Drioli, E

    2003-12-20

    The enantiocatalytic performance of immobilized lipase in an emulsion membrane reactor using stable emulsion prepared by membrane emulsification technology was studied. The production of optical pure (S)-naproxen from racemic naproxen methyl ester was used as a model reaction system. The O/W emulsion, containing the substrate in the organic phase, was fed to the enzyme membrane reactor from shell-to-lumen. The enzyme was immobilized in the sponge layer (shell side) of capillary polyamide membrane with 50 kDa cut-off. The aqueous phase was able to permeate through the membrane while the microemulsion was retained by the thin selective layer. Therefore, the substrate was kept in the enzyme-loaded membrane while the water-soluble product was continuously removed from the reaction site. The results show that lipase maintained stable activity during the entire operation time (more than 250 h), showing an enantiomeric excess (96 +/- 2%) comparable to the free enzyme (98 +/- 1%) and much higher compared to similar lipase-loaded membrane reactors used in two-separate phase systems (90%). The results demonstrate that immobilized enzymes can achieve high stability as well as high catalytic activity and enantioselectivity. PMID:14595780

  13. Lipase test

    MedlinePlus

    ... for disease of the pancreas, most often acute pancreatitis . Lipase appears in the blood when the pancreas ... Forsmark CE. Pancreatitis. In: Goldman L, Schafer AI, eds. Goldman's Cecil Medicine . 25th ed. Philadelphia, PA: Elsevier Saunders; 2016:chap 144. ...

  14. Lipase test

    MedlinePlus

    ... the bowel (bowel obstruction) Celiac disease Duodenal ulcer Cancer of the pancreas Infection or swelling of the pancreas This test may also be done for familial lipoprotein lipase deficiency . Risks ... Update Date 2/4/2015 Updated ...

  15. 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. PMID:27581492

  16. Effect of the lipoprotein lipase activator NO-1886 on adriamycin-induced nephrotic syndrome in rats.

    PubMed

    Nakayama, K; Hara, T; Kusunoki, M; Tsutsumi, K; Minami, A; Okada, K; Sakamoto, S; Ohnaka, M; Miyata, T; Nakamura, T; Aoki, T; Fukatsu, A; Nakaya, Y; Kakumu, S

    2000-05-01

    Hyperlipidemia associated with nephrotic syndrome may play a role in the deterioration of renal function. Tsutsumi et al have previously reported that the novel compound NO-1886 increases lipoprotein lipase (LPL) activity, resulting in a reduction of plasma triglycerides and an elevation of high-density lipoprotein (HDL) cholesterol in normal rats. The aim of this study was to ascertain whether NO-1886 suppresses the renal injury by treatment of the hyperlipidemia in an Adriamycin (Kyowa Hakko Kogyo, Tokyo, Japan) induced nephrosis rat model fed a high-protein diet that induced renal dysfunction and tubulointerstitial injury. Administration of Adriamycin caused hyperlipidemia, proteinuria, and edema with ascites in rats in 4 weeks. Furthermore, a combination of Adriamycin and a high-protein diet increased plasma creatinine and blood urea nitrogen (BUN) and decreased plasma albumin. Histologically, in Adriamycin-treated rats, marked interstitial cellular infiltration, tubular lumen dilation, and tubular cast formation in the kidney were observed. NO-1886 decreased plasma triglyceride and increased HDL cholesterol in Adriamycin-induced nephrotic rats. NO-1886 treatment reduced plasma creatinine and BUN levels and increased plasma albumin in Adriamycin-treated rats; it also ameliorated the ascites and proteinuria. Histologically, NO-1886-treated rats showed a quantitatively significant preservation of tubulointerstitial lesions. These data suggest that NO-1886 may have a protective effect against Adriamycin-induced nephrosis with tubulointerstitial nephritis in rats by a modification of the plasma lipid disorder. PMID:10831167

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

    SciTech Connect

    Bian, Chuanbing; Yuan, Cai; Chen, Liqing; Meehan, Edward J.; Jiang, Longguang; Huang, Zixiang; Lin, Lin; Huang, Mingdong

    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 from 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 published

  18. Linking nutritional regulation of Angptl4, Gpihbp1, and Lmf1 to lipoprotein lipase activity in rodent adipose tissue

    PubMed Central

    2012-01-01

    Background Lipoprotein lipase (LPL) hydrolyzes triglycerides in lipoproteins and makes fatty acids available for tissue metabolism. The activity of the enzyme is modulated in a tissue specific manner by interaction with other proteins. We have studied how feeding/fasting and some related perturbations affect the expression, in rat adipose tissue, of three such proteins, LMF1, an ER protein necessary for folding of LPL into its active dimeric form, the endogenous LPL inhibitor ANGPTL4, and GPIHBP1, that transfers LPL across the endothelium. Results The system underwent moderate circadian oscillations, for LPL in phase with food intake, for ANGPTL4 and GPIHBP1 in the opposite direction. Studies with cycloheximide showed that whereas LPL protein turns over rapidly, ANGPTL4 protein turns over more slowly. Studies with the transcription blocker Actinomycin D showed that transcripts for ANGPTL4 and GPIHBP1, but not LMF1 or LPL, turn over rapidly. When food was withdrawn the expression of ANGPTL4 and GPIHBP1 increased rapidly, and LPL activity decreased. On re-feeding and after injection of insulin the expression of ANGPTL4 and GPIHBP1 decreased rapidly, and LPL activity increased. In ANGPTL4−/− mice adipose tissue LPL activity did not show these responses. In old, obese rats that showed signs of insulin resistance, the responses of ANGPTL4 and GPIHBP1 mRNA and of LPL activity were severely blunted (at 26 weeks of age) or almost abolished (at 52 weeks of age). Conclusions This study demonstrates directly that ANGPTL4 is necessary for rapid modulation of LPL activity in adipose tissue. ANGPTL4 message levels responded very rapidly to changes in the nutritional state. LPL activity always changed in the opposite direction. This did not happen in Angptl4−/− mice. GPIHBP1 message levels also changed rapidly and in the same direction as ANGPTL4, i.e. increased on fasting when LPL activity decreased. This was unexpected because GPIHBP1 is known to stabilize LPL. The

  19. Changes with starvation in the rat of the lipoprotein lipase activity and hydrolysis of triacylglycerols from triacylglycerol-rich lipoproteins in adipose tissue preparations.

    PubMed Central

    Lasunción, M A; Herrera, E

    1983-01-01

    Lipoprotein lipase activity was higher in fat-pad pieces than in isolated adipocytes from the same fed rats, whereas hydrolysis of triacylglycerols from triacylglycerol-rich lipoproteins was similar in the two preparations when incubated either in basal conditions or in the presence of heparin. In both preparations there was a similar release of lipoprotein lipase activity into the medium during basal incubation, enhanced by the presence of heparin. In fat-pad pieces, but not in isolated adipocytes, incubation with heparin produced a decrease in the lipoprotein lipase activity measured in the tissue preparation. In fat-pad pieces from 24 h-starved rats, lipoprotein lipase activity was the same as in isolated adipocytes from the same animals and incubation with heparin did not affect the appearance of lipoprotein lipase in the medium or the utilization of triacylglycerols from triacylglycerol-rich lipoproteins. These results support the following conclusions. (1) The effectiveness of lipoprotein lipase in adipose tissue preparations in vitro depends more on its availability to the substrate than on its total activity. (2) Heparin acts on adipose tissue preparations from fed animals both by enhancing the release of pre-existing extracellular enzyme (which is absent in isolated adipocytes) and by enhancing the transfer outside the cells of the intracellular (and mainly undetectable) enzyme that is activated in the secretion process. (3) In adipose tissue from starved animals there is not only a decrease in the active extracellular form of lipoprotein lipase activity but also a reduction in the intracellular (and mainly undetectable) pool of the enzyme. PMID:6870799

  20. Synthesis of esters by immobilized-lipase-catalyzed condensation reaction of sugars and fatty acids in water-miscible organic solvent.

    PubMed

    Adachi, Shuji; Kobayashi, Takashi

    2005-02-01

    A lipase-catalyzed condensation reaction in an organic solvent is a promising means of synthesizing esters. Reaction equilibrium constant, which is usually defined on the basis of reactant concentration, is an important parameter for estimating equilibrium yield. It is shown that the constant is markedly, affected by some factors, such as the hydration of a sugar substrate and the interaction of a reactant with a solvent. To reasonably design the reaction system or determine the reaction conditions, attention should be paid to these factors. From the viewpoint of kinetics, substrate selectivity for carboxylic acids also numerically correlates to the electrical and steric properties of these acids. Reactor systems for continuously producing esters through an immobilized-lipase-catalyzed condensation reaction are developed. PMID:16233762

  1. Lipoprotein ApoC-II activation of lipoprotein lipase. Modulation by apolipoprotein A-IV.

    PubMed

    Goldberg, I J; Scheraldi, C A; Yacoub, L K; Saxena, U; Bisgaier, C L

    1990-03-15

    Lipoprotein lipase (LPL)-mediated hydrolysis of triglycerides (TG) contained in chylomicrons requires the presence of a cofactor, apolipoprotein (apo) C-II. The physiological mechanism by which chylomicrons gain apoC-II necessary for LPL activation in whole plasma is not known. Using a gum arabic stabilized TG emulsion, activation of LPL by lipoprotein apoC-II was studied. Hydrolysis of TG by LPL was greater in the presence of serum than with addition of either high density lipoproteins (HDL) or very low density lipoproteins (VLDL). LPL activation by either VLDL or HDL increased with addition of the lipoprotein-free fraction of plasma. A similar increase in LPL activity by addition of the lipoprotein-free fraction together with HDL or VLDL was observed when another TG emulsion (Intralipid) or TG-rich lipoproteins from an apoC-II deficient subject were used as a substrate. Human apoA-IV, apoA-I, apoE, and cholesteryl ester transfer protein were assessed for their ability to increase LPL activity in the presence of VLDL. At and below physiological concentrations, only apoA-IV increased LPL activity. One hundred percent of LPL activity measured in the presence of serum was achieved using VLDL plus apoA-IV. In the absence of an apoC-II source, apoA-IV had no effect on LPL activity. Removal of greater than 80% of the apoA-IV from the nonlipoprotein-containing fraction of plasma by incubation with Intralipid markedly reduced its ability to activate LPL in the presence of VLDL or HDL. Gel filtration chromatography demonstrated that incubation of the nonlipoprotein-containing fraction of plasma with HDL and the TG emulsion caused increased transfer of apoC-II to the emulsion and association of apoA-IV with HDL. Our studies demonstrate that apoA-IV increases LPL activation in the presence of lipoproteins. We hypothesize that apoA-IV is required for efficient release of apoC-II from either HDL or VLDL, which then allows for LPL-mediated hydrolysis of TG in nascent

  2. Esterification of oleic acid with methanol by immobilized lipase on wrinkled silica nanoparticles with highly ordered, radially oriented mesochannels.

    PubMed

    Pang, Jinli; Zhou, Guowei; Liu, Ruirui; Li, Tianduo

    2016-02-01

    Mesoporous silica nanoparticles with a wrinkled structure (wrinkled silica nanoparticles, WSNs) having highly ordered, radially oriented mesochannels were synthesized by a solvothermal method. The method used a mixture of cyclohexane, ethanol, and water as solvent, tetraethoxysilane (TEOS) as source of inorganic silica, ammonium hydroxide as hydrolysis additive, cetyltrimethylammonium bromide (CTAB) as surfactant, and polyvinylpyrrolidone (PVP) as stabilizing agent of particle growth. Particle size (240nm to 540nm), specific surface areas (490m(2)g(-1) to 634m(2)g(-1)), surface morphology (radial wrinkled structures), and pore structure (radially oriented mesochannels) of WSN samples were varied using different molar ratios of CTAB to PVP. Using synthesized WSN samples with radially oriented mesochannels as support, we prepared immobilized Candida rugosa lipase (CRL) as a new biocatalyst for biodiesel production through the esterification of oleic acid with methanol. These results suggest that WSNs with highly ordered, radially oriented mesochannels have promising applications in biocatalysis, with the highest oleic acid conversion rate of about 86.4% under the optimum conditions. PMID:26652346

  3. Ezetimibe markedly attenuates hepatic cholesterol accumulation and improves liver function in the lysosomal acid lipase-deficient mouse, a model for cholesteryl ester storage disease.

    PubMed

    Chuang, Jen-Chieh; Lopez, Adam M; Posey, Kenneth S; Turley, Stephen D

    2014-01-17

    Lysosomal acid lipase (LAL) plays a critical role in the intracellular handling of lipids by hydrolyzing cholesteryl esters (CE) and triacylglycerols (TAG) contained in newly internalized lipoproteins. In humans, mutations in the LAL gene result in cholesteryl ester storage disease (CESD), or in Wolman disease (WD) when the mutations cause complete loss of LAL activity. A rat model for WD and a mouse model for CESD have been described. In these studies we used LAL-deficient mice to investigate how modulating the amount of intestinally-derived cholesterol reaching the liver might impact its mass, cholesterol content, and function in this model. The main experiment tested if ezetimibe, a potent cholesterol absorption inhibitor, had any effect on CE accumulation in mice lacking LAL. In male Lal(-/-) mice given ezetimibe in their diet (20 mg/day/kg bw) for 4 weeks starting at 21 days of age, both liver mass and hepatic cholesterol concentration (mg/g) were reduced to the extent that whole-liver cholesterol content (mg/organ) in the treated mice (74.3±3.4) was only 56% of that in those not given ezetimibe (133.5±6.7). There was also a marked improvement in plasma alanine aminotransferase (ALT) activity. Thus, minimizing cholesterol absorption has a favorable impact on the liver in CESD. PMID:24370824

  4. Production of butyl acetate ester by lipase from novel strain of Rhizopus oryzae.

    PubMed

    Ben Salah, Riadh; Ghamghui, Hanen; Miled, Nabil; Mejdoub, Hafedh; Gargouri, Youssef

    2007-04-01

    A new lipase preparation from Rhizopus oryzae was used to catalyze the esterification reaction between acetic acid and butanol to produce butyl acetate ester (pineapple flavor). This flavor compound can be used in food, cosmetic and pharmaceutical industries. Only 3% of butyl acetate was obtained when free lipase was used in the synthesis containing only the substrates. In contrast, the conversion yield reached 25% when immobilized lipase was used under the same conditions. The synthesis of butyl acetate catalyzed by immobilized lipase in nonconventional media was optimized. A maximum conversion yield of 60% in a solvent-free system was obtained under the following conditions: amount of immobilized lipase, 500 IU; amount of initially added water, 45%; acetic acid/butanol molar ratio, 1:1; and in incubation temperature, 37 degrees C. Immobilized lipase could be repeatedly used for three cycles without a decrease in synthesis activity. The production of butyl acetate esters by immobilized R. oryzae lipase was also studied in the presence of organic solvents. Compared with a solvent-free system, the synthesis activity was improved in the presence of heptane and hexane with conversion yields of 80% and 76%, respectively. However, solvent-free systems tend to purify more easily the products without any toxicity and inflammability problems. PMID:17502279

  5. Evaluation of immobilized lipases on poly-hydroxybutyrate beads to catalyze biodiesel synthesis.

    PubMed

    Mendes, Adriano A; Oliveira, Pedro C; Vélez, Ana M; Giordano, Roberto C; Giordano, Raquel de L C; de Castro, Heizir F

    2012-04-01

    Five microbial lipase preparations from several sources were immobilized by hydrophobic adsorption on small or large poly-hydroxybutyrate (PHB) beads and the effect of the support particle size on the biocatalyst activity was assessed in the hydrolysis of olive oil, esterification of butyric acid with butanol and transesterification of babassu oil (Orbignya sp.) with ethanol. The catalytic activity of the immobilized lipases in both olive oil hydrolysis and biodiesel synthesis was influenced by the particle size of PHB and lipase source. In the esterification reaction such influence was not observed. Geobacillus thermocatenulatus lipase (BTL2) was considered to be inadequate to catalyze biodiesel synthesis, but displayed high esterification activity. Butyl butyrate synthesis catalyzed by BTL2 immobilized on small PHB beads gave the highest yield (≈90 mmol L(-1)). In biodiesel synthesis, the catalytic activity of the immobilized lipases was significantly increased in comparison to the free lipases. Full conversion of babassu oil into ethyl esters was achieved at 72 h in the presence of Pseudozyma antarctica type B (CALB), Thermomyces lanuginosus lipase (Lipex(®) 100 L) immobilized on either small or large PHB beads and Pseudomonas fluorescens (PFL) immobilized on large PHB beads. The latter preparation presented the highest productivity (40.9 mg of ethyl esters mg(-1) immobilized protein h(-1)). PMID:22285987

  6. 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

  7. Screening, gene sequencing and characterising of lipase for methanolysis of crude palm oil.

    PubMed

    Ratnaningsih, Enny; Handayani, Dewi; Khairunnisa, Fatiha; Ihsanawati; Kurniasih, Sari Dewi; Mangindaan, Bill; Rismayani, Sinta; Kasipah, Cica; Nurachman, Zeily

    2013-05-01

    Staphylococcus sp. WL1 lipase (LipFWS) was investigated for methanolysis of crude palm oil (CPO) at moderate temperatures. Experiments were conducted in the following order: searching for the suitable bacterium for producing lipase from activated sludge, sequencing lipase gene, identifying lipase activity, then synthesising CPO biodiesel using the enzyme. From bacterial screening, one isolated specimen which consistently showed the highest extracellular lipase activity was identified as Staphylococcus sp. WL1 possessing lipFWS (lipase gene of 2,244 bp). The LipFWS deduced was a protein of 747 amino acid residues containing an α/β hydrolase core domain with predicted triad catalytic residues to be Ser474, His704 and Asp665. Optimal conditions for the LipFWS activity were found to be at 55 °C and pH 7.0 (in phosphate buffer but not in Tris buffer). The lipase had a K(M) of 0.75 mM and a V(max) of 0.33 mMmin(-1) on p-nitrophenyl palmitate substrate. The lyophilised crude LipFWS performed as good as the commonly used catalyst potassium hydroxide for methanolysis of CPO. ESI-IT-MS spectra indicated that the CPO was converted into biodiesel, suggesting that free LipFWS is a worthy alternative for CPO biodiesel synthesis. PMID:23463327

  8. Influence of self-assembled monolayer surface chemistry on Candida antarctica lipase B adsorption and specific activity

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Immobilization of Candida antarctica B lipase was examined on gold surfaces modified with either methyl- or hydroxyl-terminated self-assembled alkylthiol monolayers (SAMs), representing hydrophobic and hydrophilic surfaces, respectively. Lipase adsorption was monitored gravimetrically using a quart...

  9. Effects of conjugated linoleic acid supplementation and exercise on post-heparin lipoprotein lipase, butyrylcholinesterase, blood lipid profile and glucose metabolism in young men.

    PubMed

    Bulut, Suleyman; Bodur, Ebru; Colak, Ridvan; Turnagol, Husrev

    2013-03-25

    This study was designed to investigate the effects of conjugated linoleic acid (CLA) supplementation and endurance exercise training-induced changes on post-heparin lipoprotein lipase (PH-LPL) and butyrylcholinesterase (BChE) activities along with leptin, insulin and lipid levels in plasma by a randomized double blind experiment. Eighteen sedentary male volunteers were randomly divided into CLA and Placebo (PLC) supplementation groups. Both groups underwent daily supplementation of either 3g CLA or 3g placebo for 30 days, respectively, and performed exercise on a bicycle ergometer 3 times per week for 30-40 min at 50% VO2 peak workload. For plasma glucose, insulin and leptin levels and BChE activity fasting blood was used. For PH-LPL measurements, blood was collected 15 min after 50 IU/kg iv heparin injection. In all groups, there is a statistically significant decrease in BChE (p = 0.03, p = 0.02) and leptin (p = 0.002), insulin and HOMA-IR levels (p = 0.02). Exercise with or without CLA supplementation decreased insulin levels and increased insulin sensitivity. PH-LPL activity was increased significantly in both groups, displaying increased fatty acid mobilization. We conclude that though CLA supplementation and exercise can affect these parameters, CLA is not more effective than exercise alone. Hence, a prolonged supplementation regime may be more effective. Taken together in our small study group, our findings display that BChE is a potential marker for synthetic function of liver, fat metabolism, an obesity marker, a function long overlooked. PMID:23073171

  10. Selectivity of Candida antarctica B lipase toward fatty acid and (Iso)propanol substrates in esterification reactions in organic media.

    PubMed

    Arsan, J; Parkin, K L

    2000-08-01

    Fatty acid (FA) selectivity of immobilized Candida antarctica B lipase was assessed as influenced by various cosubstrate systems for ester synthesis. Reaction mixtures contained a homologous series of even-chain n-acyl donor (C(4)(-)(16)) substrates (FA or their methyl esters, FAME) and a single alcohol cosubstrate (propanol, 2-propanol, or their acetate derivatives) in hexane. Multiple FA optima were often observed, with preferences for C(6) (or C(4)) followed by C(14) and sometimes C(10). The degree of selectivity among acyl donors was modest (up to 1.28-2.60, based on ratios of selectivity constants) and was dependent on the choice of cosubstrate system. Acyl group selectivity ranged up to 1.31-1.36 for [FA + alcohol], 1. 48-2.60 for [FAME + alcohol], 1.30-1.72 for [FA + alcohol acetate], and 1.28-1.88 [FAME + alcohol acetate] reaction systems. General shifts in selectivity were observed between short-chain (C(4)(-)(8)) and long-chain (C(10)(-)(16)) FA as groups with propanol cosubstrate, whereas shifts in reaction selectivity were observed toward specific FA(s) for 2-propanol cosubstrate. Selectivity among a series of alcohol cosubstrates ranged up to 13-fold in esterification reactions with C(6) FA. PMID:10956180

  11. Mycelium-bound lipase from a locally isolated strain of Geotrichum candidum.

    PubMed

    Loo, Joo Ling; Khoramnia, Anahita; Lai, Oi Ming; Long, Kamariah; Ghazali, Hasanah Mohd

    2014-01-01

    Mycelium-bound lipase (MBL), from a locally isolated Geotrichum candidum strain, was produced and characterized as a natural immobilized lipase. A time course study of its lipolytic activity in 1 L liquid broth revealed the maximum MBL activity at 4 h for mycelium cells harvested after 54 h. The yield and specific activity of MBL were 3.87 g/L dry weight and 508.33 U/g protein, respectively, while less than 0.2 U/mL lipase activity was detected in the culture supernatant. Prolonged incubation caused release of the bound lipase into the growth medium. The growth pattern of G. candidum, and production and properties of MBL were not affected by the scale. The stability of mycelia harboring lipase (MBL), harvested and lyophilized after 54 h, studied at 4 °C depicted a loss of 4.3% and 30% in MBL activity after 1 and 8 months, while the activity of free lipase was totally lost after 14 days of storage. The MBL from G. candidum displayed high substrate selectivity for unsaturated fatty acids containing a cis-9 double bond, even in crude form. This unique specificity of MBL could be a direct, simple and inexpensive way in the fats and oil industry for the selective hydrolysis or transesterification of cis-9 fatty acid residues in natural triacylglycerols. PMID:24959682

  12. [Lipoprotein lipase and diabetic cardiomyopathy].

    PubMed

    Liu, Xiang-Yu; Yin, Wei-Dong; Tang, Chao-Ke

    2014-02-01

    Lipoprotein lipase (LPL) hydrolyzes plasma triglyceride-rich lipoproteins into free fatty acids (FFA) to provide energy for cardiac tissue. During diabetes, cardiac energy supply is insufficient due to defected utilization of glucose. As a compensation of cardiac energy supply, FFAs are released through the hydrolysis of very low density lipoprotein (VLDL) and chylomicrons (CM) due to activation of LPL activity. In diabetic patients, activated LPL activity and elevated FFAs result in the intracellular accumulation of reactive oxygen species and lipids in myocardium and potentially induce the diabetic cardiomyopathy (DCM). The present review summarizes the regulatory mechanisms of myocardial LPL and the pathogenesis of DCM induced by LPL and provides novel therapeutic targets and pathways for DCM. PMID:24873138

  13. Characterization of fatty amides produced by lipase-catalyzed amidation of multihydroxylated fatty acids

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Novel multi-hydroxylated primary fatty amides produced by direct amidation of 7,10-dihydroxy-8(E)-octadecenoic acid (DOD) and 7,10,12-trihydroxy-8(E)-octadecenoic acid (TOD) were characterized by GC-MS and NMR. The amidation reactions were catalyzed by immobilized Pseudozyma (Candida) antarctica li...

  14. Lipoprotein particle distribution and skeletal muscle lipoprotein lipase activity after acute exercise

    PubMed Central

    2012-01-01

    Background Many of the metabolic effects of exercise are due to the most recent exercise session. With recent advances in nuclear magnetic resonance spectroscopy (NMRS), it is possible to gain insight about which lipoprotein particles are responsible for mediating exercise effects. Methods Using a randomized cross-over design, very low density lipoprotein (VLDL) responses were evaluated in eight men on the morning after i) an inactive control trial (CON), ii) exercising vigorously on the prior evening for 100 min followed by fasting overnight to maintain an energy and carbohydrate deficit (EX-DEF), and iii) after the same exercise session followed by carbohydrate intake to restore muscle glycogen and carbohydrate balance (EX-BAL). Results The intermediate, low and high density lipoprotein particle concentrations did not differ between trials. Fasting triglyceride (TG) determined biochemically, and mean VLDL size were lower in EX-DEF but not in EX-BAL compared to CON, primarily due to a reduction in VLDL-TG in the 70–120 nm (large) particle range. In contrast, VLDL-TG was lower in both EX-DEF and EX-BAL compared to CON in the 43–55 nm (medium) particle range. VLDL-TG in smaller particles (29–43 nm) was unaffected by exercise. Because the majority of VLDL particles were in this smallest size range and resistant to change, total VLDL particle concentration was not different between any of these conditions. Skeletal muscle lipoprotein lipase (LPL) activity was also not different across these 3 trials. However, in CON only, the inter-individual differences in LPL activity were inversely correlated with fasting TG, VLDL-TG, total, large and small VLDL particle concentration and VLDL size, indicating a regulatory role for LPL in the non-exercised state. Conclusions These findings reveal a high level of differential regulation between different sized triglyceride-rich lipoproteins following exercise and feeding, in the absence of changes in LPL activity. PMID

  15. Cloning, expression and characterization of a lipase gene from the Candida antarctica ZJB09193 and its application in biosynthesis of vitamin A esters.

    PubMed

    Liu, Zhi-Qiang; Zheng, Xiao-Bo; Zhang, Su-Ping; Zheng, Yu-Guo

    2012-09-01

    Lipase is one of the most important industrial enzymes, which has been widely used in the preparation of food additives, cosmetics and pharmaceuticals industries. In order to obtain a large amount of lipase, the lipase gene from Candida antarctica ZJB09193 was cloned, and expressed in Pichia pastoris with the vector pPICZαA. Under the optimal conditions, the yield of recombinant lipase in the culture broth reached 3.0 g/L. After purification, the properties of recombinant lipase were studied: the optimum pH and temperature were pH 8.0 and 52°C, Ca(2+) activated the activity of lipase, and the apparent K(m) and V(max) values for p-nitrophenyl acetate were 0.34 mM and 7.36 μmol min(-1) mg(-1), respectively. Furthermore, the recombinant lipase was immobilized on pretreated textile for biosynthesis of vitamin A esters. In a system of n-hexane, 0.3 g immobilized recombinant lipase was used in the presence of 0.06 g vitamin A acetate and 0.55 mmol fatty acid (nine different fatty acids were tested). The yield of all vitamin A esters exceeded 78% in 7h at 30°C except using lactic acid and hexanoic acid as substrates. After optimization, the yield of vitamin A palmitate reached 87%. This study has the potential to be developed into industrial application. PMID:22281522

  16. Lipase inactivation in wheat germ by gamma irradiation

    NASA Astrophysics Data System (ADS)

    Jha, Pankaj Kumar; Kudachikar, V. B.; Kumar, Sourav

    2013-05-01

    An attempt was made to improve the shelf life of wheat germ by optimizing processing conditions involving γ-irradiation. Studies were carried out to investigate the effect of γ-irradiation (0-30 kGy doses) on the chemical composition of wheat germ with respect to variation in moisture, total ash, crude fat, free fatty acid, protein and lipase activity. The results demonstrate that shelf stability of wheat germ was achieved by inactivation of lipase at doses of γ-irradiation greater than 12 kGy.

  17. Lipoprotein Lipase, Tissue Expression and Effects on Genes Related to Fatty Acid Synthesis in Goat Mammary Epithelial Cells

    PubMed Central

    Zhao, Wang-Sheng; Hu, Shi-Liang; Yu, Kang; Wang, Hui; Wang, Wei; Loor, Juan; Luo, Jun

    2014-01-01

    Lipoprotein lipase (LPL) serves as a central factor in hydrolysis of triacylglycerol and uptake of free fatty acids from the plasma. However, there are limited data concerning the action of LPL on the regulation of milk fat synthesis in goat mammary gland. In this investigation, we describe the cloning and sequencing of the LPL gene from Xinong Saanen dairy goat mammary gland, along with a study of its phylogenetic relationships. Sequence analysis showed that goat LPL shares similarities with other species including sheep, bovine, human and mouse. LPL mRNA expression in various tissues determined by RT-qPCR revealed the highest expression in white adipose tissue, with lower expression in heart, lung, spleen, rumen, small intestine, mammary gland, and kidney. Expression was almost undetectable in liver and muscle. The expression profiles of LPL gene in mammary gland at early, peak, mid, late lactation, and the dry period were also measured. Compared with the dry period, LPL mRNA expression was markedly greater at early lactation. However, compared with early lactation, the expression was lower at peak lactation and mid lactation. Despite those differences, LPL mRNA expression was still greater at peak, mid, and late lactation compared with the dry period. Using goat mammary epithelial cells (GMEC), the in vitro knockdown of LPL via shRNA or with Orlistat resulted in a similar degree of down-regulation of LPL (respectively). Furthermore, knockdown of LPL was associated with reduced mRNA expression of SREBF1, FASN, LIPE and PPARG but greater expression of FFAR3. There was no effect on ACACA expression. Orlistat decreased expression of LIPE, FASN, ACACA, and PPARG, and increased FFAR3 and SREBF1 expression. The pattern of LPL expression was similar to the changes in milk fat percentage in lactating goats. Taken together, results suggest that LPL may play a crucial role in fatty acid synthesis. PMID:25501331

  18. Phenolic profiles of 20 Canadian lentil cultivars and their contribution to antioxidant activity and inhibitory effects on α-glucosidase and pancreatic lipase.

    PubMed

    Zhang, Bing; Deng, Zeyuan; Ramdath, D Dan; Tang, Yao; Chen, Peter X; Liu, Ronghua; Liu, Qiang; Tsao, Rong

    2015-04-01

    Phenolic extracts from 20 Canadian lentil cultivars (Lens culinaris) were evaluated for total phenolic contents and composition, antioxidant activities (DPPH, FRAP, ORAC), and inhibitory properties against α-glucosidase and pancreatic lipase. Twenty one phenolic compounds were identified in the present study, with the majority being flavonoids, including kaempeferol glycosides, catechin/epicatechin glucosides and procyanidins. These phenolic compounds not only contributed significantly to the antioxidant activities, but they were also good inhibitors of α-glucosidase and lipase, two enzymes, respectively, associated with glucose and lipid digestion in the human intestine, thus contributing significantly to the control of blood glucose levels and obesity. More interestingly, it was the flavonols, not the flavanols, which showed the inhibitory activities against α-glucosidase and pancreatic lipase. Our result provides supporting information for developing lentil cultivars and functional foods with improved health benefits and suggests a potential role of lentil consumption in managing weight and control of blood glucose. PMID:25442631

  19. 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

  20. Synthesis of hepatic lipase in liver and extrahepatic tissues

    SciTech Connect

    Doolittle, M.H.; Wong, H.; Davis, R.C.; Schotz, M.C.

    1987-11-01

    Immunoprecipitations of hepatic lipase from pulse-labeled rat liver have demonstrated that hepatic lipase is synthesized in two distinct molecular weight forms, HL-I (Mr = 51,000) and HL-II (Mr = 53,000). Both forms are immunologically related to purified hepatic lipase, but not to lipoprotein lipase. HL-I and HL-II are also kinetically related and represent different stages of intracellular processing. Glycosidase experiments suggest that HL-I is the high mannose microsomal form of the mature, sialylated HL-II enzyme. Hepatic lipase activity was detected in liver and adrenal gland but was absent in brain, heart, kidney, testes, small intestine, lung, and spleen. The adrenal and liver lipase activities were inhibited in a similar dose-dependent manner by hepatic lipase antiserum. Immunoblot analysis of partially purified adrenal lipase showed an immunoreactive band co-migrating with HL-II at 53,000 daltons which was absent in a control blot treated with preimmune serum. Adrenal lipase and authentic hepatic lipase yielded similar peptide maps, confirming the presence of the lipase in adrenal gland. However, incorporation of L-(/sup 35/S)methionine into immunoprecipitable hepatic lipase was not detected in this tissue. In addition, Northern blot analysis showed the presence of hepatic lipase mRNA in liver but not adrenal gland. The presence of hepatic lipase in adrenal gland in the absence of detectable synthesis or messenger suggests that hepatic lipase originates in liver and is transported to this extrahepatic site.

  1. Effects of Macroporous Resin Size on Candida antarctica Lipase B Adsorption, Fraction of Active Molecules, and Catalytic Activity for Polyester Synthesis

    SciTech Connect

    Chen,B.; Miller, E.; Miller, L.; Maikner, J.; Gross, R.

    2007-01-01

    Methyl methacrylate resins with identical average pore diameter (250 {angstrom}) and surface area (500 m{sup 2}/g) but with varied particle size (35 to 560-710 {mu}m) were employed to study how immobilization resin particle size influences Candida antarctica Lipase B (CALB) loading, fraction of active sites, and catalytic properties for polyester synthesis. CALB adsorbed more rapidly on smaller beads. Saturation occurred in less than 30 s and 48 h for beads with diameters 35 and 560-710 {mu}m, respectively. Linearization of adsorption isotherm data by the Scatchard analysis showed for the 35 {mu}m resin that: (1) CALB loading at saturation was well below that required to form a monolayer and fully cover the support surface and (2) CALB has a high affinity for this resin surface. Infrared microspectroscopy showed that CALB forms protein loading fronts for resins with particle sizes 560-710 and 120 {mu}m. In contrast, CALB appears evenly distributed throughout 35 {mu}m resins. By titration with p-nitrophenyl n-hexyl phosphate (MNPHP), the fraction of active CALB molecules adsorbed onto resins was <50% which was not influenced by particle size. The fraction of active CALB molecules on the 35 {mu}m support increased from 30 to 43% as enzyme loading was increased from 0.9 to 5.7% (w/w) leading to increased activity for {epsilon}-caprolactone ({epsilon}-CL) ring-opening polymerization. At about 5% w/w CALB loading, by decreasing the immobilization support diameter from 560-710 to 120, 75, and 35 {mu}m, conversion of {epsilon}-CL % to polyester increased (20 to 36, 42, and 61%, respectively, at 80 min). Similar trends were observed for condensation polymerizations between 1,8-octanediol and adipic acid.

  2. Lipase in the Lipid Bodies of Corn Scutella during Seedling Growth 1

    PubMed Central

    Lin, Yon-Hui; Wimer, Larry T.; Huang, Anthony H. C.

    1983-01-01

    In the scutella of corn (Zea mays), lipase activity is absent in ungerminated seeds and increases during seedling growth. At the peak stage of lipolysis, about 50% of the lipase activity is recovered in the lipid body fraction after flotation centrifugation. The lipase is tightly bound to the lipid bodies, and resists solubilization by repeated washing with buffers or NaCl solutions. Isolated lipid bodies undergo autolysis of internal triacylglycerols, resulting in the release of fatty acids. After the triacylglycerols in isolated lipid bodies have been extracted with diethyl ether, the lipase is recovered in the membrane fraction. The lipase has an optimal activity at pH 7.5 in the autolysis of lipid bodies, or on trilinolein or N-methylindoxylmyristate. Of the various acylglycerols examined, the enzyme is active only on acylglycerols of linoleic and oleic acids which are the major fatty acid constituents of corn oil. The activity is not greatly affected by NaCl, CaCl2, or pretreatment of the enzyme with p-chloromercuribenzoate or mersalyl, and detergents abolish the activity. The enzyme hydrolyzes trilinolein completely to fatty acids; during the course of reaction, there is little accumulation of di- or mono-linolein. PMID:16663239

  3. 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. PMID:26842598

  4. Lipase catalyzed synthesis of neutral glycerides rich in micronutrients from rice bran oil fatty acid distillate.

    PubMed

    Nandi, Sumit; Gangopadhyay, Sarbani; Ghosh, Santinath

    2008-01-01

    Neutral glycerides with micronutrients like sterols, tocopherols and squalene may be prepared from cheap raw material like rice bran oil fatty acid distillate (RBO FAD). RBO FAD is an important byproduct of vegetable oil refining industries in the physical refining process. Glycerides like triacylglycerols (TAG), diacylglycerols (DAG) and monoacylglycerols (MAG) containing significant amounts of unsaponifiable matter like sterols, tocopherols and hydrocarbons (mainly squalene) may certainly be considered as novel functional food ingredients. Fatty acids present in RBO FAD were esterified with glycerol of varying amount (1:0.33, 1:0.5, 1:1 and 1:1.5 of FAD : glycerol ratio) for 8 h using non-specific enzyme NS 40013 (Candida antartica). After esterification the product mixture containing mono, di- and triglycerides was purified by molecular distillation to remove excess free fatty acids and also other volatile undesirable components. The purified product containing sterols, tocopherols and squalene can be utilized in various food formulations. PMID:18838832

  5. Cross-linked enzyme aggregates (CLEAs) of selected lipases: a procedure for the proper calculation of their recovered activity

    PubMed Central

    2013-01-01

    In the last few years, synthesis of carrier-free immobilized biocatalysts by cross-linking of enzyme aggregates has appeared as a promising technique. Cross-linked enzyme aggregates (CLEAs) present several interesting advantages over carrier-bound immobilized enzymes, such as highly concentrated enzymatic activity, high stability of the produced superstructure, important production costs savings by the absence of a support, and the fact that no previous purification of the enzyme is needed. However, the published literature evidences that a) much specific non-systematic exploratory work is being done and, b) recovered activity calculations in CLEAs still need to be optimized. In this context, this contribution presents results of an optimized procedure for the calculation of the activity retained by CLEAs, based on the comparison of their specific activity relative to their free enzyme counterparts. The protocol implies determination of precipitable protein content in commercial enzyme preparations through precipitation with ammonium sulphate and a protein co-feeder. The identification of linear ranges of activity versus concentration/amount of protein in the test reaction is also required for proper specific activity determinations. By use of mass balances that involve the protein initially added to the synthesis medium, and the protein remaining in the supernatant and washing solutions (these last derived from activity measurements), the precipitable protein present in CLEAs is obtained, and their specific activity can be calculated. In the current contribution the described protocol was applied to CLEAs of Thermomyces lanuginosa lipase, which showed a recovered specific activity of 11.1% relative to native lipase. The approach described is simple and can easily be extended to other CLEAs and also to carrier-bound immobilized enzymes for accurate determination of their retained activity. PMID:23663379

  6. Factors affecting the resolution of dl-menthol by immobilized lipase-catalyzed esterification in organic solvent.

    PubMed

    Wang, Dong-Lin; Nag, Ahindra; Lee, Guan-Chun; Shaw, Jei-Fu

    2002-01-16

    Among 10 lipases tested, Candida rugosa lipase exhibited the best ability to catalyze the resolution of dl-menthol in organic solvent. The lipase was immobilized on different carriers, and the experiment was carried out with different acyl donors. The high yield and optical purity of the product were achieved in cyclohexane with valeric acid as acyl donor using C. rugosa lipase immobilized on DEAE-Sephadex A-25. The conversion of dl-menthol depended on the water content of immobilized lipase and on the pH of the aqueous solution from which lipase was immobilized. The operational stability of the DEAE-Sephadex A-25 immobilized lipase in catalysis of the esterification reaction showed that >85% activity remained after 34 days of repeated use. The resolution of racemic menthol in organic medium catalyzed by immobilized C. rugosa lipase-catalyzed esterification is very convenient, and it represents a significant improvement in the use of enzyme for the preparative production of optically active menthol. This process is readily applicable to large-scale preparation. PMID:11782192

  7. Involvement of Glyoxysomal Lipase in the Hydrolysis of Storage Triacylglycerols in the Cotyledons of Soybean Seedlings 1

    PubMed Central

    Lin, Yon-Hui; Moreau, Robert A.; Huang, Anthony H. C.

    1982-01-01

    The total cotyledon extract of soybean (Glycine max [L.] Merr. var. Coker 136) seedlings underwent lipolysis as measured by the release of fatty acids. The highest lipolytic activity occurred at pH 9. This lipolytic activity was absent in the dry seeds and increased after germination concomitant with the decrease in total lipids. Using spherosomes (lipid bodies) isolated from the cotyledons during the peak stage of lipolysis (5-7 days) as substrates, about 40% of the lipase activity was found in the glyoxysomes after organelle breakage had been accounted for; the remaining activity was distributed among other subcellular fractions but none was found in the spherosomal fraction. The glyoxysomal lipase had maximal activity at pH 9, and catalyzed the hydrolysis of tri-, di-, and monoacylglycerols of linoleic acid, the most abundant fatty acid in soybean. The spherosomes contained a neutral lipase that could hydrolyze monolinolein and N-methylindoxylmyristate, but not trilinolein. This spherosomal lipase activity dropped off rapidly during early seedling growth, preceding lipolysis. Spherosomes isolated from either dry or germinated seeds did not possess lipolytic activity, and spherosomes from germinated seeds but not from dry seeds could serve as substrates for the glyoxysomal lipase. It is concluded that the glyoxysomal lipase is the enzyme catalyzing the initial hydrolysis of storage triacylglycerols. PMID:16662427

  8. Aspergillus niger lipase: Heterologous expression in Pichia pastoris, molecular modeling prediction and the importance of the hinge domains at both sides of the lid domain to interfacial activation.

    PubMed

    Shu, Zhengyu; Duan, Mojie; Yang, Jiangke; Xu, Li; Yan, Yunjun

    2009-01-01

    Aspergillus niger lipase (ANL) is an important biocatalyst in the food processing industry. However, there is no report of its detailed three-dimensional structure because of difficulties in crystallization. In this article, based on experimental data and bioinformational analysis results, the structural features of ANL were simulated. Firstly, two recombinant ANLs expressed in Pichia pastoris were purified to homogeneity and their corresponding secondary structure compositions were determined by circular dichroism spectra. Secondly, the primary structure, the secondary structure and the three-dimensional structure of ANL were modeled by comparison with homologous lipases with known three-dimensional structures using the BioEdit software, lipase engineering database (http://www.led.uni-stuttgart.de/), PSIPRED server and SwissModel server. The predicted molecular structure of ANL presented typical features of the alpha/beta hydrolase fold including positioning of the putative catalytic triad residues and the GXSXG signature motif. Comparison of the predicted three-dimensional structure of ANL with the X-ray three-dimensional structure of A. niger feruloyl esterase showed that the functional difference of interfacial activation between lipase and esterase was concerned with the difference in position of the lid. Our three-dimensional model of ANL helps to modify lipase structure by protein engineering, which will further expand the scope of application of ANL. PMID:19248178

  9. Lung Epithelial Cell-Specific Expression of Human Lysosomal Acid Lipase Ameliorates Lung Inflammation and Tumor Metastasis in Lipa(-/-) Mice.

    PubMed

    Zhao, Ting; Ding, Xinchun; Du, Hong; Yan, Cong

    2016-08-01

    Lysosomal acid lipase (LAL), a key enzyme in the metabolic pathway of neutral lipids, has a close connection with inflammation and tumor progression. One major manifestation in LAL-deficient (Lipa(-/-)) mice is an increase of tumor growth and metastasis associated with expansion of myeloid-derived suppressor cells. In the lung, LAL is highly expressed in alveolar type II epithelial cells. To assess how LAL in lung epithelial cells plays a role in this inflammation-related pathogenic process, lung alveolar type II epithelial cell-specific expression of human LAL (hLAL) in Lipa(-/-) mice was established by crossbreeding of CCSP-driven rtTA transgene and (TetO)7-CMV-hLAL transgene into Lipa(-/-) mice (CCSP-Tg/KO). hLAL expression in lung epithelial cells not only reduced tumor-promoting myeloid-derived suppressor cells in the lung, but also down-regulated the synthesis and secretion of tumor-promoting cytokines and chemokines into the bronchoalveolar lavage fluid of Lipa(-/-) mice. hLAL expression reduced the immunosuppressive functions of bronchoalveolar lavage fluid cells, inhibited bone marrow cell transendothelial migration, and inhibited endothelial cell proliferation and migration in Lipa(-/-) mice. As a result, hLAL expression in CCSP-Tg/KO mice corrected pulmonary damage, and inhibited tumor cell proliferation and migration in vitro, and tumor metastasis to the lung in vivo. These results support a concept that LAL is a critical metabolic enzyme in lung epithelial cells that regulates lung homeostasis, immune response, and tumor metastasis. PMID:27461363

  10. Isolation and Characterization of a Staphylococcal Lipase

    PubMed Central

    Troller, J. A.; Bozeman, M. A.

    1970-01-01

    A number of coagulase-negative staphylococci isolated from human skin were found to produce lipase. Lipolytic activity appeared in the growth medium during the stationary phase of growth but did not appear as a result of autolysis of the cells. Maximal lipase synthesis was obtained when the medium was adjusted to pH 7.5 before inoculation. The purified enzyme hydrolyzed tributyrin and tridecanoin most actively, and a relatively high rate of hydrolysis of triolein was also noted. The optimal activity of the purified lipase was at pH 7.5. The characteristics of the concentrated crude enzyme and purified lipase were compared. PMID:5485729

  11. Incorporation of omega-3 polyunsaturated fatty acids into soybean lecithin: effect of amines and divalent cations on transesterification by lipases.

    PubMed

    Marsaoui, Nabil; Laplante, Serge; Raies, Aly; Naghmouchi, Karim

    2013-12-01

    The transesterification of soybean lecithin with methyl esters of EPA and DHA in an organic solvent (hexane) using various commercially available lipases was studied. Lipases produced by Candida antarctica, Pseudomonas fluorescens, Burkholderia cepacia, Mucor miehei, Thermomyces lanuginosus and Rhizomucor miehei were compared, in the absence or presence of histidine, arginine, urea, Ca²⁺, Mg²⁺, or a combination of urea and divalent cations (additives at 5 % of the total lipid mass). Transesterification using the R. miehei enzyme reached 11.32 and 12.30 % in the presence of Ca²⁺ or Mg²⁺ respectively, and 8.58 and 9.31 % when urea was also added. These were the greatest degrees of transesterification obtained. The results suggest the potential use of this immobilized lipase as a catalyst for interesterification reactions in organic solvent systems with low water content. PMID:23749246

  12. Marine Fungal and Bacterial Isolates for Lipase Production: A Comparative Study.

    PubMed

    Patnala, H S; Kabilan, U; Gopalakrishnan, L; Rao, R M D; Kumar, D S

    2016-01-01

    Lipases, belonging to the class of enzymes called hydrolases, can catalyze triglycerides to fatty acids and glycerol. They are produced by microbes of plant and animal origin, and also by marine organisms. As marine microorganisms thrive in extreme conditions, lipases isolated from their origin possess characteristics of extremozymes, retain its activity in extreme conditions and can catalyze few chemical reactions which are impossible otherwise relative to the lipase produced from terrestrial microorganisms. Lipases are useful in many industries like detergent, food, leather, pharmaceutical, diary, etc. Few commercial enzymes have been developed and the use of them in certain industries like dairy, soaps are proved to be beneficial. There are few research papers reporting the production of lipase from marine bacteria and fungi. Lipase production involves two types of fermentation processes-solid-state fermentation (SSF) and submerged fermentation (SmF). Although SmF process is used conventionally, SSF process produces lipase in higher amounts. The production is also influenced by the composition of the medium, physiochemical parameters like temperature, pH, carbon, and nitrogen sources. PMID:27452166

  13. Identification of a triacylglycerol lipase in the diatom Phaeodactylum tricornutum.

    PubMed

    Barka, Frederik; Angstenberger, Max; Ahrendt, Tilman; Lorenzen, Wolfram; Bode, Helge B; Büchel, Claudia

    2016-03-01

    Diatoms accumulate triacylglycerols (TAGs) as storage lipids, but the knowledge about the molecular mechanisms of lipid metabolism is still sparse. Starting from a partial sequence for a putative TAG-lipase of the diatom Phaeodactylum tricornutum retrieved from the data bases, we have identified the full length coding sequence, tgl1. The gene encodes an 813 amino acid sequence that shows distinct motifs for so called "true" TAG-lipases [EC 3.1.1.3] that have been functionally characterized in model organisms like Arabidopsis thaliana and Saccharomyces cerevisiae. These lipases mediate the first initial step of TAG breakdown from storage lipids. To test whether Tgl1 can act as a TAG-lipase, a His-tagged version was overexpressed in Escherichia coli and the protein indeed showed esterase activity. To identify the TAG degrading function of Tgl1 in P. tricornutum, knock-down mutant strains were created using an antisense RNA approach. In the mutant cell lines the relative tgl1-mRNA-level was reduced up to 20% of that of the wild type, accompanied by a strong increase of TAG in the lipid extracts. In spite of the TAG accumulation, the polar lipid species pattern appeared to be unchanged, confirming the TAG-lipase function of Tgl1. PMID:26747649

  14. 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

  15. Synergistic interactions of apigenin, naringin, quercetin and emodin on inhibition of 3T3-L1 preadipocyte differentiation and pancreas lipase activity.

    PubMed

    Guo, XiaoXuan; Liu, Jia; Cai, ShengBao; Wang, Ou; Ji, BaoPing

    2016-01-01

    The interactions of four natural compounds including apigenin, naringin, emodin and quercetin were investigated on inhibiting 3T3-L1 preadipocyte differentiation and pancreas lipase activity. Oil Red O staining was conducted to visualise and quantify lipid accumulation. The difference between experimental and calculated results was utilised for determining the interaction types. Interestingly, emodin synergistically interacted with the other three compounds, and the combination of emodin and apigenin exhibited the strongest synergistic effect in both differentiation and pancreas lipase assays. Results implied that the combination of apigenin and emodin may be regarded as a promising complementary therapy for management of overweight or obesity. PMID:26314502

  16. Organization of the human lipoprotein lipase gene and evolution of the lipase gene family

    SciTech Connect

    Kirchgessner, T.G.; Heinzmann, C.; Svenson, K.; Ameis, D.; Lusis, A.J. ); Chuat, J.C.; Etienne, J.; Guilhot, S.; Pilon, C.; D'Auriol, L.; Galibert, F. ); Schotz, M.C. Wadsworth Medical Center, Los Angeles, CA )

    1989-12-01

    The human lipoprotein lipase gene was cloned and characterized. It is composed of 10 exons spanning {approx} 30 kilobase. The first exon encodes the 5{prime}-untranslated region, the signal peptide plus the first two amino acids of the mature protein. The next eight exons encode the remaining 446 amino acids, and the tenth exon encodes the long 3{prime}-untranslated region of 1948 nucleotides. The lipoprotein lipase transcription start site and the sequence of the 5{prime}-flanking region were also determined. The authors compared the organization of genes for lipoprotein lipase, hepatic lipase, pancreatic lipase, and Drosophila yolk protein 1, which are members of a family of related genes. A model for the evolution of the lipase gene family is presented that involves multiple rounds of gene duplication plus exon-shuffling and intron-loss events.

  17. Adsorption of lipase on polypropylene powder.

    PubMed

    Gitlesen, T; Bauer, M; Adlercreutz, P

    1997-04-01

    Adsorption of different lipases by EP-100 polypropylene powder from crude and pure lipase preparations was studied. Langmuir isotherms described the adsorption equilibria well both for protein and lipase activity adsorption. Adsorption isotherms for five different proteins all gave a similar saturation level of 220 mg protein per g carrier. Twelve commercial lipase preparations were tested for selectivity in the adsorption of lipase. For all preparations the selectivity factor was larger than one. In a crude lipase preparation from Pseudomonas fluorescence, the specific activity in solution decreased by two orders of magnitude after adsorption. The adsorption was not significantly influenced by pH changes in the adsorption buffer, indicating that hydrophobic and not electrostatic interactions are the dominating adsorption forces. Adsorption of a crude lipase from Candida rugosa (Sigma) was fast and equilibrium was reached in 30 and 100 min for protein and lipase activity adsorption respectively. Desorption in aqueous solution was negligible. Investigations with seven different lipases showed no correlation between the specific lipolytic activity of dissolved enzyme in aqueous solution and the specific activity of adsorbed enzyme in an esterification reaction in organic solvent. PMID:9106498

  18. 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

  19. 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. PMID:18571389

  20. Elucidation of a key position for acyltransfer activity in Candida parapsilosis lipase/acyltransferase (CpLIP2) and in Pseudozyma antarctica lipase A (CAL-A) by rational design.

    PubMed

    Jan, Anne-Hélène; Subileau, Maeva; Deyrieux, Charlotte; Perrier, Véronique; Dubreucq, Éric

    2016-02-01

    Performing transesterifications in aqueous media is becoming a priority challenge in lipid biotechnology in order to develop more eco-friendly and efficient biocatalytic processes in systems containing both polar and apolar substrates. In this context, our group has explored for several years the high potential of the lipase/acyltransferase CpLIP2 from Candida parapsilosis and of several of its homologs, that catalyze efficiently acyltransfer reactions in lipid/water media with high water activity (aw>0.9). The discovery of a new member of this group, CduLAc from Candida dubliniensis, with a higher acyltransferase activity than CpLIP2, has provided a new insight on structure-function relationships in this group. Indeed, the comparison of sequences and 3D models, especially of CpLIP2 and CduLAc, with those of the phylogenetically related lipase A from Pseudozyma antarctica (CAL-A), allowed elucidating a key structural determinant of the acyltransferase activity: serine S369 in CpLIP2 and its equivalents E370 in CAL-A and A366 in CduLAc. Mutants obtained by rational design at this key position showed significant changes in acyltransfer activity. Whereas mutation S369E resulted in an increase in the hydrolytic activity of CpLIP2, S369A increased alcoholysis. More strikingly, the single E370A mutation in CAL-A drastically increased the acyltransferase activity of this enzyme, giving it the character of a lipase/acyltransferase. Indeed, this single mutation lowered the methanol concentration for which the initial rates of alcoholysis and hydrolysis are equal from 2M in CAL-A down to 0.3M in its mutant, while the exceptional stability of the parental enzyme toward alcohol and temperature was conserved. PMID:26602447

  1. The activities of lipoprotein lipase and of enzymes involved in triacylglycerol synthesis in rat adipose tissue. Effects of starvation, dietary modification and of corticotropin injection.

    PubMed Central

    Lawson, N; Pollard, A D; Jennings, R J; Gurr, M I; Brindley, D N

    1981-01-01

    1. The effects of dietary modification, including starvation, and of corticotropin injection on the activities of acyl-CoA synthetase, glycerol phosphate acyltransferase, dihydroxyacetone phosphate acyltransferase, phosphatidate phosphohydrolase, diacylglycerol acyltransferase and lipoprotein lipase were measured in adipose tissue. 2. Lipoprotein lipase activities in heart were increased and those in adipose tissue were decreased when rats were fed on diets enriched with corn oil or beef tallow rather than with sucrose or starch. The lipoprotein lipase activity was lower in the adipose tissue of rats fed on the sucrose rather than on the starch diet. 3. Rats fed on the beef tallow diet had slightly higher activities of the total glycerol phosphate acyltransferase in adipose tissue than did rats fed on the sucrose or starch diet. The diacylglycerol acyltransferase and the mitochondrial glycerol phosphate acyltransferase activities were higher for the rats fed on the tallow diet than for those fed on the corn-oil diet. 4. Starvation significantly decreased the activities of lipoprotein lipase (after 24 and 48 h), acyl-CoA synthetase (after 24 h) and of the mitochondrial glycerol phosphate acyltransferase and the N-ethylmaleimide-insensitive dihydroxyacetone phosphate acyltransferase (after 48 h) in adipose tissue. The activities of the microsomal glycerol phosphate acyltransferase, diacylglycerol acyltransferase and the soluble phosphatidate phosphohydrolase were not significantly changed after 24 or 48 h of starvation. 5. The activities of lipoprotein lipase and phosphatidate phosphohydrolase in adipose tissue were decreased 15 min after corticotropin was injected into rats during November to December. No statistically significant differences were found when these experiments were performed during March to September. These differences may be related to the seasonal variation in acute lipolytic responses. 6. These results are discussed in relation to the control of

  2. Simultaneous Inhibition of Fatty Acid Amide Hydrolase and Monoacylglycerol Lipase Shares Discriminative Stimulus Effects with Δ9-Tetrahydrocannabinol in Mice

    PubMed Central

    Hruba, Lenka; Seillier, Alexandre; Zaki, Armia; Cravatt, Benjamin F.; Lichtman, Aron H.; Giuffrida, Andrea

    2015-01-01

    Monoacylglycerol lipase (MAGL) and fatty acid amide hydrolase (FAAH) inhibitors exert preclinical effects indicative of therapeutic potential (i.e., analgesia). However, the extent to which MAGL and FAAH inhibitors produce unwanted effects remains unclear. Here, FAAH and MAGL inhibition was examined separately and together in a Δ9-tetrahydrocannabinol (Δ9-THC; 5.6 mg/kg i.p.) discrimination assay predictive of subjective effects associated with cannabis use, and the relative contribution of N-arachidonoyl ethanolamine (AEA) and 2-arachidonoylglycerol (2-AG) in the prefrontal cortex, hippocampus, and caudate putamen to those effects was examined. Δ9-THC dose-dependently increased Δ9-THC appropriate responses (ED50 value = 2.8 mg/kg), whereas the FAAH inhibitors PF-3845 [N-3-pyridinyl-4-[[3-[[5-(trifluoromethyl)-2-pyridinyl]oxy]phenyl]methyl]-1-piperidinecarboxamide] and URB597 [(3′-​(aminocarbonyl)[1,​1′-​biphenyl]-​3-​yl)-​cyclohexylcarbamate] or a MAGL inhibitor JZL184 [4-​nitrophenyl-​4-​(dibenzo[d][1,​3]dioxol-​5-​yl(hydroxy)methyl)piperidine-​1-​carboxylate] alone did not substitute for the Δ9-THC discriminative stimulus. The nonselective FAAH/MAGL inhibitors SA-57 [4-[2-(4-chlorophenyl)ethyl]-1-piperidinecarboxylic acid 2-(methylamino)-2-oxoethyl ester] and JZL195 [4-​nitrophenyl 4-​(3-​phenoxybenzyl)piperazine-​1-​carboxylate] fully substituted for Δ9-THC with ED50 values equal to 2.4 and 17 mg/kg, respectively. Full substitution for Δ9-THC was also produced by a combination of JZL184 and PF-3845, but not by a combination of JZL184 and URB597 (i.e., 52% maximum). Cannabinoid receptor type 1 antagonist rimonabant attenuated the discriminative stimulus effects of Δ9-THC, SA-57, JZL195, and the combined effects of JZL184 and PF-3845. Full substitution for the Δ9-THC discriminative stimulus occurred only when both 2-AG and AEA were significantly elevated, and the patterns of increased endocannabinoid content were

  3. 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

  4. Chemical modification for improving activity and stability of lipase B from Candida antarctica with imidazolium-functional ionic liquids.

    PubMed

    Jia, Ru; Hu, Yi; Liu, Luo; Jiang, Ling; Huang, He

    2013-11-01

    Various imidazolium-functional ionic liquids (ILs) composed of different cations and anions were grafted onto Candida antarctica lipase B (CALB) through lysine coupling, and 4-6 of the 9 primary amino groups of the lysine residues were modified. The catalytic activity and stability were investigated in a p-nitrophenyl palmitate hydrolysis reaction. After modification, CALB was activated and achieved a high catalytic efficiency in the aqueous phase (e.g., [HOOCMMIm][Cl] modification showed a 1.5-fold increase in the catalytic efficiency). The thermostability and organic solvent tolerance were significantly increased with the ILs modification (e.g., [HOOCBMIm][Cl] owned kosmotropic cation and chaotropic anion modification showed a 7-fold thermostability increase at 70 °C, 1-fold increase in 50% aqueous dimethylformamide and 5-fold increase in 50% aqueous methanol). Conformation changes were confirmed by fluorescence spectroscopy, circular dichroism spectroscopy and attenuated total reflection Fourier transform infrared spectroscopy. PMID:24057321

  5. New eutectic ionic liquids for lipase activation and enzymatic preparation of biodiesel†

    PubMed Central

    Zhao, Hua; Baker, Gary A.; Holmes, Shaletha

    2012-01-01

    The enzymatic preparation of biodiesel has been hampered by the lack of suitable solvents with desirable properties such as high lipase compatibility, low cost, low viscosity, high biodegradability, and ease of product separation. Recent interest in using ionic liquids (ILs) as advanced reaction media has led to fast reaction rates and high yields in the enzymatic synthesis of biodiesel. However, conventional (i.e., cation–anion paired) ILs based on imidazolium and other quaternary ammonium salts remain too expensive for wide application at industrial scales. In this study, we report on newly-synthesized eutectic ILs derived from choline acetate or choline chloride coupled with biocompatible hydrogen-bond donors, such as glycerol. These eutectic solvents have favorable properties including low viscosity, high biodegradability, and excellent compatibility with Novozym® 435, a commercial immobilized Candida antarctica lipase B. Furthermore, in a model biodiesel synthesis system, we demonstrate high reaction rates for the enzymatic transesterification of Miglyol® oil 812 with methanol, catalyzed by Novozym® 435 in choline acetate/glycerol (1 : 1.5 molar ratio). The high conversion (97%) of the triglyceride obtained within 3 h, under optimal conditions, suggests that these novel eutectic solvents warrant further exploration as potential media in the enzymatic production of biodiesel. PMID:21283901

  6. 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. PMID:27374548

  7. Gene cloning and catalytic characterization of cold-adapted lipase of Photobacterium sp. MA1-3 isolated from blood clam.

    PubMed

    Kim, Young Ok; Khosasih, Vivia; Nam, Bo-Hye; Lee, Sang-Jun; Suwanto, Antonius; Kim, Hyung Kwoun

    2012-12-01

    A lipase-producing Photobacterium strain (MA1-3) was isolated from the intestine of a blood clam caught at Namhae, Korea. The lipase gene was cloned by shotgun cloning and encoded 340 amino acids with a molecular mass of 38,015 Da. It had a very low sequence identity with other bacterial lipases, with the exception of that of Photobacterium lipolyticum M37 (83.2%). The MA1-3 lipase was produced in soluble form when Escherichia coli cells harboring the gene were cultured at 18°C. Its optimum temperature and pH were 45°C and pH 8.5, respectively. Its activation energy was calculated to be 2.69 kcal/mol, suggesting it to be a cold-adapted lipase. Its optimum temperature, temperature stability, and substrate specificity were quite different from those of M37 lipase, despite the considerable sequence similarities. Meanwhile, MA1-3 lipase performed a transesterification reaction using olive oil and various alcohols including methanol, ethanol, 1-propanol, and 1-butanol. In the presence of t-butanol as a co-solvent, this lipase produced biodiesel using methanol and plant or waste oils. The highest biodiesel conversion yield (73%) was achieved using waste soybean oil and methanol at a molar ratio of 1:5 after 12 h using 5 units of lipase. PMID:22841866

  8. General characterization of noncommercial microbial lipases in hydrolytic and synthetic reactions.

    PubMed

    Otero, C; Berrendero, M A; Cardenas, F; Alvarez, E; Elson, S W

    2005-03-01

    Fourteen noncommercial preparations of microbial lipases were investigated with respect to their catalytic activity for hydrolysis and synthesis of ester bonds. Six of the lipases were derived from microorganisms that have not previously been described as lipase producers, and another four were characterized for the first time. The synthetic reactions were carried out in two solvents of different polarities (n-heptane and acetone) using a series of fatty acids and primary and secondary alcohols with different chain lengths. Under the culture conditions employed, Pseudomonas cepacia produced more active enzyme than the other microorganisms. The lipase preparations produced using Ovadendron sulphureo-ochraceum, Monascus mucoroides, Monascus sp., Fusarium oxysporum, Penicillium chrysogenum, Rhodotorula araucariae, Pseudomonas cepacia, Streptomyces halstedii, and Streptomyces sp.were the most efficient catalysts for hydrolysis at lipid-water interfaces. Enzyme preparations from P. cepacia, Streptomyces sp., S. halstedii, and R. araucariae were good biocatalysts for esterification in the polar medium (acetone). When the lipase preparations with the greatest activity for hydrolytic reactions were excluded, regression analysis of the data for the hydrolytic and synthetic activities of the remaining lipase preparations yielded high multiple correlation coefficients for these reactions in both n-heptane and acetone (R = 0.82 and 0.91, respectively). PMID:15767695

  9. Prolonged Monoacylglycerol Lipase Blockade Causes Equivalent Cannabinoid Receptor Type 1 Receptor–Mediated Adaptations in Fatty Acid Amide Hydrolase Wild-Type and Knockout Mice

    PubMed Central

    Kinsey, Steven G.; Ignatowska-Jankowska, Bogna; Ramesh, Divya; Abdullah, Rehab A.; Tao, Qing; Booker, Lamont; Long, Jonathan Z.; Selley, Dana E.; Cravatt, Benjamin F.; Lichtman, Aron H.

    2014-01-01

    Complementary genetic and pharmacological approaches to inhibit monoacylglycerol lipase (MAGL) and fatty acid amide hydrolase (FAAH), the primary hydrolytic enzymes of the respective endogenous cannabinoids 2-arachidonoylglycerol (2-AG) and N-arachidonoylethanolamine, enable the exploration of potential therapeutic applications and physiologic roles of these enzymes. Complete and simultaneous inhibition of both FAAH and MAGL produces greatly enhanced cannabimimetic responses, including increased antinociception, and other cannabimimetic effects, far beyond those seen with inhibition of either enzyme alone. While cannabinoid receptor type 1 (CB1) function is maintained following chronic FAAH inactivation, prolonged excessive elevation of brain 2-AG levels, via MAGL inhibition, elicits both behavioral and molecular signs of cannabinoid tolerance and dependence. Here, we evaluated the consequences of a high dose of the MAGL inhibitor JZL184 [4-nitrophenyl 4-(dibenzo[d][1,3]dioxol-5-yl(hydroxy)methyl)piperidine-1-carboxylate; 40 mg/kg] given acutely or for 6 days in FAAH(−/−) and (+/+) mice. While acute administration of JZL184 to FAAH(−/−) mice enhanced the magnitude of a subset of cannabimimetic responses, repeated JZL184 treatment led to tolerance to its antinociceptive effects, cross-tolerance to the pharmacological effects of Δ9-tetrahydrocannabinol, decreases in CB1 receptor agonist–stimulated guanosine 5′-O-(3-[35S]thio)triphosphate binding, and dependence as indicated by rimonabant-precipitated withdrawal behaviors, regardless of genotype. Together, these data suggest that simultaneous elevation of both endocannabinoids elicits enhanced cannabimimetic activity but MAGL inhibition drives CB1 receptor functional tolerance and cannabinoid dependence. PMID:24849924

  10. TCDD (2,3,7,8-tetrachlorodibenzo-P-dioxin) reduces lipoprotein lipase activity in the adipose tissue of the guinea pig

    SciTech Connect

    Brewster, D.W.; Matsumura, F.

    1984-07-31

    TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin) adminstered to young male guinea pigs at a dose of 1 ..mu..g/kg (single intraperitoneal injection) caused a large reduction in adipose tissue lipoprotein lipase (LPL) activity. This effect occurred rapidly; a 70% decrease was noticed after 24 hour and 80% of LPL activity was lost by 48 hours when the serum triglyceride levels increased to 175% of control levels. LPL is known to play an important role in controlling the amount of free fatty acids supplied to adipose tissues. Administration of a large dose of glucose to fasted guinea pigs, which have shown a similar weight loss, but less LPL loss than TCDD-treated animals, had the effect of elevating their adipose LPL levels back to a near normal level, whereas the same treatment caused no significant increase in the LPL levels of TCDD-treated animals. Evidence indicates that the TCDD-caused decline in LPL activity is irreversible. As a consequence, the affected guinea pigs are incapable of responding to changes in nutritional status. 36 references, 2 figures, 1 table.

  11. 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. PMID:25748441

  12. 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

  13. Identification and characterization of a new true lipase isolated through metagenomic approach

    PubMed Central

    2011-01-01

    Background Metagenomics, the application of molecular genomics to consortia of non-cultivated microbes, has the potential to have a substantial impact on the search for novel industrial enzymes such as esterases (carboxyl ester hydrolases, EC 3.1.1.1) and lipases (triacylglycerol lipases, EC 3.1.1.3). In the current work, a novel lipase gene was identified from a fosmid metagenomic library constructed with the "prokaryotic-enriched" DNA from a fat-contaminated soil collected from a wastewater treatment plant. Results In preliminary screening on agar containing 1% tributyrin, 2661 of the approximately 500,000 clones in the metagenomic library showed activity. Of these, 127 showed activity on agar containing 1% tricaprylin, while 32 were shown to be true lipase producers through screening on agar containing 1% triolein. The clone with the largest halo was further characterized. Its lipase gene showed 72% identity to a putative lipase of Yersinia enterocolitica subsp. palearctica Y11. The lipase, named LipC12, belongs to family I.1 of bacterial lipases, has a chaperone-independent folding, does not possess disulfide bridges and is calcium ion dependent. It is stable from pH 6 to 11 and has activity from pH 4.5 to 10, with higher activities at alkaline pH values. LipC12 is stable up to 3.7 M NaCl and from 20 to 50°C, with maximum activity at 30°C over a 1 h incubation. The pure enzyme has specific activities of 1722 U/mg and 1767 U/mg against olive oil and pig fat, respectively. Moreover, it is highly stable in organic solvents at 15% and 30% (v/v). Conclusions The combination of the use of a fat-contaminated soil, enrichment of prokaryotic DNA and a three-step screening strategy led to a high number of lipase-producing clones in the metagenomic library. The most notable properties of the new lipase that was isolated and characterized were a high specific activity against long chain triacylglycerols, activity and stability over a wide range of pH values, good thermal

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

    PubMed Central

    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. PMID:26240816

  15. Elevated hepatic lipase activity and low levels of high density lipoprotein in a normotriglyceridemic, nonobese Turkish population.

    PubMed

    Bersot, T P; Vega, G L; Grundy, S M; Palaoglu, K E; Atagündüz, P; Ozbayrakçi, S; Gökdemir, O; Mahley, R W

    1999-03-01

    Low levels of high density lipoprotein cholesterol (HDL-C) are associated with increased risk of coronary heart disease and, in the United States, are often associated with hypertriglyceridemia and obesity. In Turkey, low HDL-C levels are highly prevalent, 53% of men and 26% of women having HDL-C levels <35 mg/dl, in the absence of hypertriglyceridemia and obesity. In this study to investigate the cause of low HDL-C levels in Turks, various factors affecting HDL metabolism were assessed in normotriglyceridemic Turkish men and women living in Istanbul and in non-Turkish men and women living in San Francisco. Turkish men and women had significantly lower HDL-C levels than the San Francisco men and women, as well as markedly lower apolipoprotein A-I levels (25 and 39 mg/dl lower, respectively). In both Turkish and non-Turkish subjects, the mean body mass index was <27 kg/m2, the mean triglyceride level was <120 mg/dl, and the mean total cholesterol was 170-180 mg/dl. The mean hepatic triglyceride lipase activity was 21% and 31% higher in Turkish men and women, respectively, than in non-Turkish men and women, and remained higher even after subjects with a body mass index >50th percentile for men and women in the United States were excluded from the analysis. As no dietary or behavioral factors have been identified in the Turkish population that account for increased hepatic triglyceride lipase activity, the elevation most likely has a genetic basis. high density lipoprotein in a normotriglyceridemic, nonobese Turkish population. PMID:10064731

  16. 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. PMID:25449652

  17. 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

  18. Stimulation of Lipase Production During Bacterial Growth on Alkanes

    PubMed Central

    Breuil, Colette; Shindler, D. B.; Sijher, J. S.; Kushner, D. J.

    1978-01-01

    Acinetobacter lwoffi strain O16, a facultative psychrophile, can grow on crude oil, hexadecane, octadecane, and most alkanes when tested at 20 but not at 30°C. Growth occurred on a few alkanes at 30°C but after a longer lag than at 20°C. Cells grown on alkanes as sole carbon sources had high levels of cell-bound lipase. In contrast, previous work has shown that those grown on complex medium produced cell-free lipase and those grown on defined medium without alkanes produced little or no lipase. Low concentrations of the detergent Triton X-100 caused the liberation of most of the lipase activity of alkane-grown cells and increased total lipase activity. When ethanol and hexadecane were both present in a mineral medium, diauxic growth occurred; until the ethanol was completely used up, hexadecane was not utilized, and the lipase activity was very low. When growth on hexadecane began, lipase activity increased, reaching a level 50- to 100-fold higher than that of cells growing on ethanol. A similar pattern of lipase formation and hexadecane utilization was observed with Pseudomonas aeruginosa. Whenever A. lwoffi and other bacteria degraded alkanes they exhibited substantial lipase activity. Not all bacteria that produced lipase, however, could attack alkanes. Bacteria that could not produce lipase did not attack alkanes. The results suggest that a correlation may exist between lipase formation and alkane utilization. PMID:627533

  19. Screening, purification and characterization of a novel cold-active and organic solvent-tolerant lipase from Stenotrophomonas maltophilia CGMCC 4254.

    PubMed

    Li, Mu; Yang, Li-Rong; Xu, Gang; Wu, Jian-Ping

    2013-11-01

    An extracellular organic solvent-tolerant and cold-active lipase producing bacterium was isolated from oil-contaminated soil samples, and identified taxonomically as Stenotrophomonas maltophilia. The lipase from S. maltophilia CGMCC 4254 (SML) was purified 60.5-fold to homogeneity with 38.9 U/mg specific activity. Partially purified SML displayed remarkable stability in 50% and 100% (v/v) hydrophobic organic solvents after incubation for 7 days. The enzyme also retained more than 50% of its residual activity in several pure hydrophilic organic solvents after incubation for 7 days. SML showed 57% maximum activity at 5°C, and had optimal activity at 35°C. These unique properties of SML make it promising as a biocatalyst for industrial processes. PMID:24050922

  20. Enhancing the thermostability of a cold-active lipase from Penicillium cyclopium by in silico design of a disulfide bridge.

    PubMed

    Tan, Zhongbiao; Li, Jianfang; Wu, Minchen; Wang, Junqing

    2014-08-01

    Cysteine mutants of a cold-active lipase (PcLipI) from Penicillium cyclopium were designed by the software Disulfide by Design Ver. 1.20 in an effort to improve enzyme thermostability by addition of a disulfide bridge. Those mutants predicted by molecular dynamics simulation to have better thermostability than the wild type were first expressed in Escherichia coli BL21(DE3) and then, for further investigation, in Pichia pastoris GS115. By replacing Val248 and Thr251 with cysteines to create a disulfide bridge, the recombinant lipases reE-PcLipV248C-T251C (expressed in E. coli) and reP-PcLipV248C-T251C (expressed in P. pastoris) were obtained. Both had enhanced thermostability with half-lives at 35 °C about 4.5- and 12.8-fold longer than that of the parent PcLipI expressed in E. coli and P. pastoris, respectively. The temperature optima of reE-PcLipV248C-T251C and reP-PcLipV248C-T251C were 35 and 30 °C, which were each 5 °C higher than those of the parent PcLipI expressed in E. coli and P. pastoris. The K ms of reE-PcLipV248C-T251C and reP-PcLipV248C-T251C toward tributyrin were 53.2 and 39.5 mM, while their V maxs were 1,460 and 3,800 U/mg, respectively. PcLipV248C-T251C had better thermostability and catalytic efficiency than the other mutants and the parent PcLipI. PMID:24867629

  1. Relationship between lipoprotein lipase and peroxisome proliferator-activated receptor-alpha expression in rat liver during development.

    PubMed

    Panadero, M; Bocos, C; Herrera, E

    2006-09-01

    The present study was addressed to determine whether the high expression of peroxisome proliferator-activated receptor-alpha (PPAR-alpha) in rat liver during the perinatal stage plays a role in the induction of liver lipoprotein lipase (LPL) expression and activity. Parallel increases in liver mRNA PPAR-alpha and LPL activity were found in newborn rats, and after a slight decline, values remained elevated until weaning. Anticipated weaning for 3 days caused a decline in those two variables as well as in the mRNA LPL level, and a similar change was also found in liver triacylglycerol concentration. Force-feeding with Intralipid in 10-day-old rats or animals kept fasted for 5 h showed high mRNA-PPARalpha and -LPL levels as well as LPL activity with low plasma insulin and high FFA levels, whereas glucose and a combination of glucose and Intralipid produced low mRNA-PPARalpha and -LPL levels as well as LPL activity. Under these latter conditions, plasma insulin and FFA levels were high in those rats receiving the combination of glucose and Intralipid, whereas plasma FFA levels were low in those force-fed with glucose. It is proposed that the hormonal and nutritional induction of liver PPAR-alpha expression around birth and its maintained elevated level throughout suckling is responsible for the induction of liver LPL-expression and activity during suckling. PMID:17451160

  2. 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. PMID:24197522

  3. Activation of hormone-sensitive lipase requires two steps, protein phosphorylation and binding to the PAT-1 domain of lipid droplet coat proteins.

    PubMed

    Wang, Hong; Hu, Liping; Dalen, Knut; Dorward, Heidi; Marcinkiewicz, Amy; Russell, Deanna; Gong, Dawei; Londos, Constantine; Yamaguchi, Tomohiro; Holm, Cecilia; Rizzo, Mark A; Brasaemle, Dawn; Sztalryd, Carole

    2009-11-13

    Lipolysis is an important metabolic pathway controlling energy homeostasis through degradation of triglycerides stored in lipid droplets and release of fatty acids. Lipid droplets of mammalian cells are coated with one or more members of the PAT protein family, which serve important functions in regulating lipolysis. In this study, we investigate the mechanisms by which PAT family members, perilipin A, adipose differentiation-related protein (ADFP), and LSDP5, control lipolysis catalyzed by hormone-sensitive lipase (HSL), a major lipase in adipocytes and several non-adipose cells. We applied fluorescence microscopic tools to analyze proteins in situ in cultured Chinese hamster ovary cells using fluorescence recovery after photobleaching and anisotropy Forster resonance energy transfer. Fluorescence recovery after photobleaching data show that ADFP and LSDP5 exchange between lipid droplet and cytoplasmic pools, whereas perilipin A does not. Differences in protein mobility do not correlate with PAT protein-mediated control of lipolysis catalyzed by HSL or endogenous lipases. Forster resonance energy transfer and co-immunoprecipitation experiments reveal that each of the three PAT proteins bind HSL through interaction of the lipase with amino acids within the highly conserved amino-terminal PAT-1 domain. ADFP and LSDP5 bind HSL under basal conditions, whereas phosphorylation of serine residues within three amino-terminal protein kinase A consensus sequences of perilipin A is required for HSL binding and maximal lipolysis. Finally, protein kinase A-mediated phosphorylation of HSL increases lipolysis in cells expressing ADFP or LSDP5; in contrast, phosphorylation of perilipin A exerts the major control over HSL-mediated lipolysis when perilipin is the main lipid droplet protein. PMID:19717842

  4. A lipasin/Angptl8 monoclonal antibody lowers mouse serum triglycerides involving increased postprandial activity of the cardiac lipoprotein lipase

    PubMed Central

    Fu, Zhiyao; Abou-Samra, Abdul B.; Zhang, Ren

    2015-01-01

    Lipasin/Angptl8 is a feeding-induced hepatokine that regulates triglyceride (TAG) metabolism; its therapeutical potential, mechanism of action, and relation to the lipoprotein lipase (LPL), however, remain elusive. We generated five monoclonal lipasin antibodies, among which one lowered the serum TAG level when injected into mice, and the epitope was determined to be EIQVEE. Lipasin-deficient mice exhibited elevated postprandial activity of LPL in the heart and skeletal muscle, but not in white adipose tissue (WAT), suggesting that lipasin suppresses the activity of LPL specifically in cardiac and skeletal muscles. Consistently, mice injected with the effective antibody or with lipasin deficiency had increased postprandial cardiac LPL activity and lower TAG levels only in the fed state. These results suggest that lipasin acts, at least in part, in an endocrine manner. We propose the following model: feeding induces lipasin, activating the lipasin-Angptl3 pathway, which inhibits LPL in cardiac and skeletal muscles to direct circulating TAG to WAT for storage; conversely, fasting induces Angptl4, which inhibits LPL in WAT to direct circulating TAG to cardiac and skeletal muscles for oxidation. This model suggests a general mechanism by which TAG trafficking is coordinated by lipasin, Angptl3 and Angptl4 at different nutritional statuses. PMID:26687026

  5. 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

  6. [Gene cloning, expression and characterization of two cold-adapted lipases from Penicillium sp. XMZ-9].

    PubMed

    Zheng, Xiaomei; Wu, Ningfeng; Fan, Yunliu

    2012-04-01

    Cold-adapted lipases are attractive biocatalysts that can be used at low temperatures as additives in food products, laundry detergents, and the organic synthesis of chiral intermediates. Cold-adapted lipases are normally found in microorganisms that survive at low temperatures. A fungi strain XMZ-9 exhibiting lipolytic activity was isolated from the soil of glaciers in Xinjiang by the screening plates using 1% tributyrin as the substrate and Victoria blue as an indicator. Based on morphological characteristics and phylogenetic comparisons of its 18S rDNA, the strain was identified as Penicillium sp. The partial nucleotide sequences of these two lipase related genes, LipA and LipB, were obtained by touchdown PCR using the degenerate primers designed according to the conservative domains of lipase. The full-length sequences of two genes were obtained by genome walking. The gene lipA contained 1 014 nucleotides, without any intron, comprising one open reading frame encoding a polypeptide of 337 amino acids. The gene lipB comprised two introns (61 bp and 49 bp) and a coding region sequence of 1 122 bp encoding a polypeptide of 373 amino acids, cDNA sequences of both lipA and lipB were cloned and expressed in Escherichia coli BL21 (DE3). The recombinant LipA was mostly expressed as inclusion bodies, and recovered lipase activity at low temperature after in vitro refolded by dilution. Differently, the recombinant LipB was expressed in the soluble form and then purified by Ni-NTA affinity chromatography Column. It showed high lipase activity at low temperature. These results indicated that they were cold-adapted enzymes. This study paves the way for the further research of these cold-adapted lipases for application in the industry. PMID:22803398

  7. Preparation Fe3O4@chitosan magnetic particles for covalent immobilization of lipase from Thermomyces lanuginosus.

    PubMed

    Wang, Xiang-Yu; Jiang, Xiao-Ping; Li, Yue; Zeng, Sha; Zhang, Ye-Wang

    2015-04-01

    Magnetic Fe3O4@chitosan nanoparticles were prepared by a simple in situ co-precipitation method and characterized by transmission electron microscope (TEM) and Fourier transform infrared spectroscopy (FTIR). The prepared Fe3O4@chitosan nanoparticles were used for covalent immobilization of lipase from Thermomyces lanuginosus by chemical conjugation after electrostatic entrapment (CCEE). The optimal immobilization conditions were obtained as follows: enzyme/support 19.8 mg/g, pH 5.0, time 4h and temperature 30 °C. Under these conditions, a high immobilization efficiency of 75% and a protein loading of 16.8 mg/g-support were obtained. Broad pH tolerance and high thermostability could be achieved by immobilization. The immobilized lipase retained 70% initial activity after ten cycles. Kinetic parameters Vmax and Km of free and immobilized lipase were determined as 5.72 mM/min, 2.26 mM/min and 21.25 mM, 28.73 mM, respectively. Ascorbyl palmitate synthesis with immobilized lipase was carried out in tert-butanol at 50 °C, and the conversion of ascorbic acid was obtained higher than 50%. These results showed that the immobilization of lipase onto magnetic chitosan nanoparticles by the method of CCEE is an efficient and simple way for preparation of stable lipase. PMID:25603148

  8. 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. PMID:24534493

  9. Effect of di(2-ethylhexyl) phthalate (DEHP) on lipolysis and lipoprotein lipase activities in adipose tissue of rats.

    PubMed

    Martinelli, Marcela I; Mocchiutti, Norberto O; Bernal, Claudio A

    2010-09-01

    The di(2-ethylhexyl) phthalate (DEHP) is an ubiquitous environmental chemical with detrimental health effects. The present work was designed to asses some potential mechanisms by which DEHP causes, among others, a reduced body fat retention. Since this effect could be related to an alteration of adipocyte triacylglycerol (TG) metabolism, we evaluated the effects of dietary DEHP in adipose tissues upon (1) the number and size of fat cells; (2) the basal and stimulated lipolysis and (3) the lipoprotein lipase (LPL) activity. Groups of male Wistar rats were fed for 21 days a control diet alone (control group) or the same control diet supplemented with 2% (w/w) of DEHP (DEHP group). The LPL activity of DEHP-fed rats was increased in lumbar and epididymal adipose tissues. These rats had significantly reduced weight in epididymal and lumbar tissues, together with reduced size of epididymal adipocytes. These alterations do not seem to be associated with higher lipid mobility because neither basal lipolysis nor 'in vitro' stimulated lipolysis by noradrenaline (NA) showed to be modified by DEHP. Based on these results, we concluded that the adipose tissue size reduction induced by DEHP intake is not due to changes in lipolysis nor to a decreased LPL activity. More research is needed to achieve a comprehensive understanding of the potential mechanisms by which DEHP causes, among others, a reduced body fat retention. PMID:20144957

  10. Immobilization of Burkholderia sp. lipase on a ferric silica nanocomposite for biodiesel production.

    PubMed

    Tran, Dang-Thuan; Chen, Ching-Lung; Chang, Jo-Shu

    2012-04-15

    In this work, lipase produced from an isolated strain Burkholderia sp. C20 was immobilized on magnetic nanoparticles to catalyze biodiesel synthesis. Core-shell nanoparticles were synthesized by coating Fe(3)O(4) core with silica shell. The nanoparticles treated with dimethyl octadecyl [3-(trimethoxysilyl) propyl] ammonium chloride were used as immobilization supporters. The Burkholderia lipase was then bound to the synthesized nanoparticles for immobilization. The protein binding efficiency on alkyl-functionalized Fe(3)O(4)-SiO(2) was estimated as 97%, while the efficiency was only 76% on non-modified Fe(3)O(4)-SiO(2). Maximum adsorption capacity of lipase on alkyl-functionalized Fe(3)O(4)-SiO(2) was estimated as 29.45 mg g(-1) based on Langmuir isotherm. The hydrolytic kinetics (using olive oil as substrate) of the lipase immobilized on alkyl-grafted Fe(3)O(4)-SiO(2) followed Michaelis-Menten model with a maximum reaction rate and a Michaelis constant of 6251 Ug(-1) and 3.65 mM, respectively. Physical and chemical properties of the nanoparticles and the immobilized lipase were characterized by Brunauer-Emmett-Teller (BET) analysis, scanning electron microscope (SEM), and Fourier transform infrared spectroscopy (FT-IR). Moreover, the immobilized lipase was used to catalyze the transesterification of olive oil with methanol to produce fatty acid methyl esters (FAMEs), attaining a FAMEs conversion of over 90% within 30 h in batch operation when 11 wt% immobilized lipase was employed. The immobilized lipase could be used for ten cycles without significant loss in its transesterification activity. PMID:22306108

  11. Lipid biomarkers and bacterial lipase activities as indicators of organic matter and bacterial dynamics in contrasted regimes at the DYFAMED site, NW Mediterranean

    NASA Astrophysics Data System (ADS)

    Bourguet, Nicolas; Goutx, Madeleine; Ghiglione, Jean-François; Pujo-Pay, Mireille; Mével, Geneviève; Momzikoff, André; Mousseau, Laure; Guigue, Catherine; Garcia, Nicole; Raimbault, Patrick; Pete, Romain; Oriol, Louise; Lefèvre, Dominique

    2009-08-01

    This study investigated the relationships between dissolved organic matter (DOM) composition and bacterial dynamics on short time scale during spring mesotrophic (March 2003) and summer oligotrophic (June 2003) regimes, in a 0-500 m depth water column with almost no advection, at the DYFAMED site, NW Mediterranean. DOM was characterized by analyzing dissolved organic carbon (DOC), colored dissolved organic matter (CDOM) and lipid class biotracers. Bacterial dynamic was assessed through the measurement of in situ bacterial lipase activity, abundance, production and bacterial community structure. We made the assumption that by coupling the ambient concentration of hydrolysable acyl-lipids with the measurement of their in situ bacterial hydrolysis rates (i.e. the free fatty acids release rate) would provide new insights about bacterial response to change in environmental conditions. The seasonal transition from spring to summer was accompanied by a significant accumulation of excess DOC (+5 μM) (ANOVA, p<0.05, n=8) in the upper layer (0-50 m). In this layer, the free fatty acids release rate to the bacterial carbon demand (BCD) ratio increased from 0.6±0.3 in March to 1.3±1.0 in June (ANOVA, p<0.05, n=8) showing that more uncoupling between the hydrolysis of the acyl-lipids and the BCD occurred during the evolution of the season, and that free fatty acids contributed to the excess DOC. The increase of lipolysis index and CDOM absorbance (from 0.24±0.17 to 0.39±0.13 and from 0.076±0.039 to 0.144±0.068; ANOVA, p<0.05, n=8, respectively), and the higher contribution of triglycerides, wax esters and phospholipids (from <5% to 12-31%) to the lipid pool reflected the change in the DOM quality. In addition to a strong increase of bacterial lipase activity per cell (51.4±29.4-418.3±290.6 Ag C cell -1 h -1), a significant percentage of ribotypes (39%) was different between spring and summer in the deep chlorophyll maximum (DCM) layer in particular, suggesting a shift

  12. 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

  13. 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. PMID:25242958

  14. Acylation of Antioxidant of Bamboo Leaves with Fatty Acids by Lipase and the Acylated Derivatives' Efficiency in the Inhibition of Acrylamide Formation in Fried Potato Crisps.

    PubMed

    Ma, Xiang; Wang, Erpei; Lu, Yuyun; Wang, Yong; Ou, Shiyi; Yan, Rian

    2015-01-01

    This study selectively acylated the primary hydroxyl groups on flavonoids in antioxidant of bamboo leaves (AOB) using lauric acid with Candida antarctica lipase B in tert-amyl-alcohol. The separation and isolation of acylated derivatives were performed using silica gel column chromatography with a mixture of dichloromethane/diethyl ether/methanol as eluents. Both thin layer chromatography and high-performance liquid chromatography analyses confirmed the high efficiency of the isolation process with the purified orientin-6″-laurate, isoorientin-6″-laurate, vitexin-6″-laurate, and isovitexin-6″-laurate that were obtained. The addition of AOB and acylated AOB reduced acrylamide formation in fried potato crisps. Results showed that 0.05% AOB and 0.05% and 0.1% acylated AOB groups significantly (p < 0.05) reduced the content of acrylamide in potato crisps by 30.7%, 44.5%, and 46.9%, respectively. PMID:26098744

  15. Acylation of Antioxidant of Bamboo Leaves with Fatty Acids by Lipase and the Acylated Derivatives’ Efficiency in the Inhibition of Acrylamide Formation in Fried Potato Crisps

    PubMed Central

    Ma, Xiang; Wang, Erpei; Lu, Yuyun; Wang, Yong; Ou, Shiyi; Yan, Rian

    2015-01-01

    This study selectively acylated the primary hydroxyl groups on flavonoids in antioxidant of bamboo leaves (AOB) using lauric acid with Candida antarctica lipase B in tert-amyl-alcohol. The separation and isolation of acylated derivatives were performed using silica gel column chromatography with a mixture of dichloromethane/diethyl ether/methanol as eluents. Both thin layer chromatography and high-performance liquid chromatography analyses confirmed the high efficiency of the isolation process with the purified orientin-6″-laurate, isoorientin-6″-laurate, vitexin-6″-laurate, and isovitexin-6″-laurate that were obtained. The addition of AOB and acylated AOB reduced acrylamide formation in fried potato crisps. Results showed that 0.05% AOB and 0.05% and 0.1% acylated AOB groups significantly (p < 0.05) reduced the content of acrylamide in potato crisps by 30.7%, 44.5%, and 46.9%, respectively. PMID:26098744

  16. The Collaborative Study on the Enzymatic Analysis of Positional Distribution of Short- and Medium-chain Fatty Acids in Milk Fat Using Immobilized Candida antarctica Lipase B.

    PubMed

    Yoshinaga, Kazuaki; Sato, Shinichi; Sasaki, Ryo; Asada, Mihoko; Hori, Ryuji; Imagi, Jun; Miyazaki, Yosuke; Nagai, Toshiharu; Saito, Katsuyoshi; Sano, Takashi; Sasaki, Akiko; Sato, Chiemi; Tsukahara, Yuki; Yamashita, Atsushi; Watanabe, Shimpei; Watanabe, Yomi

    2016-01-01

    The positional distributions of fatty acids (FAs) in milk fat containing short- and medium-chain FAs were analyzed by sn-1(3)-selective transesterification of triacylglycerols (TAGs) with ethanol using immobilized Candida antarctica lipase B (CALB), in a collaborative study conducted by 10 laboratories. The mean C4:0, C6:0, and C8:0 FA contents, when analyzed as propyl esters (PEs) using gas chromatography (GC) with a DB-23 capillary column, were found to be 3.0, 2.0, and, 1.3 area%, respectively. Their reproducibility standard deviations were 0.33, 0.18, and 0.19, respectively. The mean C4:0, C6:0, and C8:0 contents at the sn-2 position were 0.3, 0.4, and 1.0 area%, respectively. Their reproducibility standard deviations were 0.17, 0.11, and 0.19, respectively. The reproducibility standard deviations of C4:0, C6:0, and C8:0 FAs at the sn-2 position were either the same as or smaller than those for milk fat, although the FA contents at the sn-2 position were smaller than those in the milk fat. Therefore, it was concluded that the CALB method for estimating the regiospecific distribution is applicable to TAGs containing short- and medium-chain FAs. When estimating the short-chain (SC) FA contents in fats and oils by GC, it is better to analyze SCFAs as PEs or butyl esters, and not as methyl esters, in order to prevent loss of SCFAs during the experimental procedure because of their volatility and water solubility. This study also revealed that the stationary phase of the GC capillary column affected the flame ionization detector (FID) response of SCFAs. The theoretical FID correction factor (MWFA / active carbon number / atomic weight of carbon) fitted well with the actual FID responses of C4:0-C12:0 FAs when they were analyzed as PEs using a DB-23 column; however, this was not the case when the GC analysis was performed using wax-type columns. PMID:26972465

  17. The selectivity of some fungal lipases.

    PubMed

    Adamczak, M; Bednarski, W

    2003-01-01

    Selectivity is one of the most important lipase properties which depends on a wide range of factors. In order to choose the right enzyme for a special purpose, it is necessary to check its selectivity. Fatty acid selectivity of lipases determined for natural substrales was different from that determined for p-nitrophenyl esters and those determined for each substrate. Enantoiselectivity of lipase from Mucor circinelloides (MCL) determined for 2 was over 100 (E > > 100). In this case, inversion of enantiopreferences was observed; the conversion was 10% and (R)-alcohol was preferentially produced PMID:24757816

  18. Burkholderia cepacia lipase is a promising biocatalyst for biofuel production.

    PubMed

    Sasso, Francesco; Natalello, Antonino; Castoldi, Simone; Lotti, Marina; Santambrogio, Carlo; Grandori, Rita

    2016-07-01

    Lipases resistant to inhibition and denaturation by methanol are valuable tools for biotechnological applications, in particular for biofuel production. Microbial lipases have attracted a great deal of interest because of their stability at high concentrations of organic solvents. Burkholderia cepacia lipase (BCL) is tested here for robustness towards methanol in terms of conformational stability and catalytic activity in transesterification assays. This lipase turns out to be even more tolerant than the homologous and better characterized enzyme from Burkholderia glumae. BCL unfolding transition, as monitored by far-UV circular dichroism (CD) and intrinsic fluorescence, displays a Tm above 60°C in the presence of 50% methanol. The protein unfolds at low pH, and the organic solvent affects the nature of the denatured state under acidic conditions. The protein performs well in transesterification assays upon prolonged incubations at high methanol concentrations. BCL is highly tolerant to methanol and displays particularly high conformational stability under conditions employed for transesterification reactions. These features depict BCL as a promising enzyme for biofuel industry. PMID:27067648

  19. Solvent-free lipase-catalyzed preparation of diacylglycerols.

    PubMed

    Weber, Nikolaus; Mukherjee, Kumar D

    2004-08-25

    Various methods have been applied for the enzymatic preparation of diacylglycerols that are used as dietary oils for weight reduction in obesity and related disorders. Interesterification of rapeseed oil triacylglycerols with commercial preparations of monoacylglycerols, such as Monomuls 90-O18, Mulgaprime 90, and Nutrisoft 55, catalyzed by immobilized lipase from Rhizomucor miehei (Lipozyme RM IM) in vacuo at 60 degrees C led to extensive (from 60 to 75%) formation of diacylglycerols. Esterification of rapeseed oil fatty acids with Nutrisoft, catalyzed by Lipozyme RM in vacuo at 60 degrees C, also led to extensive (from 60 to 70%) formation of diacylglycerols. Esterification of rapeseed oil fatty acids with glycerol in vacuo at 60 degrees C, catalyzed by Lipozyme RM and lipases from Thermomyces lanuginosus (Lipozyme TL IM) and Candida antarctica (lipase B, Novozym 435), also provided diacylglycerols, however, to a lower extent (40-45%). Glycerolysis of rapeseed oil triacylglycerols with glycerol in vacuo at 60 degrees C, catalyzed by Lipozyme TL and Novozym 435, led to diacylglycerols to the extent of acids with Monomuls resulted in minor reduction of its activity. The products of esterification of rapeseed oil fatty acids with Monomuls and glycerol yielded upon short-path vacuum distillation residues (diacylglycerol oils) containing 66-70% diacylglycerols. PMID:15315368

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  1. Nanonets Derived from Turnip Mosaic Virus as Scaffolds for Increased Enzymatic Activity of Immobilized Candida antarctica Lipase B

    PubMed Central

    Cuenca, Sol; Mansilla, Carmen; Aguado, Marta; Yuste-Calvo, Carmen; Sánchez, Flora; Sánchez-Montero, Jose M.; Ponz, Fernando

    2016-01-01

    Elongated flexuous plant viral nanoparticles (VNPs) represent an interesting platform for developing different applications in nanobiotechnology. In the case of potyviruses, the virion external surface is made up of helically arrayed domains of the viral structural coat protein (CP), repeated over 2000 times, in which the N- and C-terminal domains of each CP are projected toward the exterior of the external virion surface. These characteristics provide a chemical environment rich in functional groups susceptible to chemical conjugations. We have conjugated Candida antarctica lipase B (CALB) onto amino groups of the external surface of the potyvirus turnip mosaic virus (TuMV) using glutaraldehyde as a conjugating agent. Using this approach, TuMV virions were transformed into scaffolds for CALB nanoimmobilization. Analysis of the resulting structures revealed the formation of TuMV nanonets onto which large CALB aggregates were deposited. The functional enzymatic characterization of the CALB-bearing TuMV nanonets showed that CALB continued to be active in the nanoimmobilized form, even gaining an increased relative specific activity, as compared to the non-immobilized form. These novel virus-based nanostructures may provide a useful new approach to enzyme nanoimmobilization susceptible to be industrially exploited. PMID:27148295

  2. Nanonets Derived from Turnip Mosaic Virus as Scaffolds for Increased Enzymatic Activity of Immobilized Candida antarctica Lipase B.

    PubMed

    Cuenca, Sol; Mansilla, Carmen; Aguado, Marta; Yuste-Calvo, Carmen; Sánchez, Flora; Sánchez-Montero, Jose M; Ponz, Fernando

    2016-01-01

    Elongated flexuous plant viral nanoparticles (VNPs) represent an interesting platform for developing different applications in nanobiotechnology. In the case of potyviruses, the virion external surface is made up of helically arrayed domains of the viral structural coat protein (CP), repeated over 2000 times, in which the N- and C-terminal domains of each CP are projected toward the exterior of the external virion surface. These characteristics provide a chemical environment rich in functional groups susceptible to chemical conjugations. We have conjugated Candida antarctica lipase B (CALB) onto amino groups of the external surface of the potyvirus turnip mosaic virus (TuMV) using glutaraldehyde as a conjugating agent. Using this approach, TuMV virions were transformed into scaffolds for CALB nanoimmobilization. Analysis of the resulting structures revealed the formation of TuMV nanonets onto which large CALB aggregates were deposited. The functional enzymatic characterization of the CALB-bearing TuMV nanonets showed that CALB continued to be active in the nanoimmobilized form, even gaining an increased relative specific activity, as compared to the non-immobilized form. These novel virus-based nanostructures may provide a useful new approach to enzyme nanoimmobilization susceptible to be industrially exploited. PMID:27148295

  3. Mathematical modeling of lipase and protease production by Penicillium restrictum in a batch fermenter.

    PubMed

    Freire, D M; Sant'Anna, G L; Alves, T L

    1999-01-01

    This work presents a mathematical model that describes time course variations of extracellular lipase and protease activities for the batch fermentation of the fungus Penicillium restrictum, a new and promising strain isolated from soil and wastes of a Brazilian babassu coconut oil industry. The fermentation process was modeled by an unstructured model, which considered the following dependent variables: cells, fat acid, dissolved oxygen concentrations, lipase and protease activities, and cell lysate concentration. The last variable represents the amount of cells that has been lysed by the shear stress and natural cell death. Proteases released to the medium, as consequence of this process, enhance lipase inactivation. The model is able to predict the effects of some operation variables such as air flow rate and agitation speed. The mathematical model was validated against batch-fermentation data obtained under several operating conditions. Because substrate concentration has antagonistic effects on lipase activity, a typical optimization scheme should be developed in order to minimize these deleterious effects while maximizing lipase activity. PMID:15304703

  4. PHARMACOLOGICAL ACTIVITIES OF PROTOCATECHUIC ACID.

    PubMed

    Khan, Abida Kalsoom; Rashid, Rehana; Fatima, Nighat; Mahmood, Sadaf; Mir, Sadullah; Khan, Sara; Jabeen, Nyla; Murtaza, Ghulam

    2015-01-01

    Protocatechuic acid (3,4-dihydroxybenzoic acid, PCA) is a simple phenolic acid. It is found in a large variety of edible plants and possesses various pharmacological activities. This article aims to review the modern trends in phytochemical isolation and extraction of PCA from plants and other natural resources. Moreover, this article also encompasses pharmacological and biological activities of PCA. It is well known to have anti-inflammatory, antioxidant, anti-hyperglycemia, antibacterial, anticancer, anti-ageing, anti-athro- genic, anti-tumoral, anti-asthma, antiulcer, antispasmodic and neurological properties. PMID:26647619

  5. Effects of antibodies and glycerol as potential inhibitors of ruminal lipase activity

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Ruminant-derived foods contain high proportions of saturated fats, a result of ruminal biohydrogenation, which rapidly saturates and thus limits the availability of free unsaturated fatty acids for assimilation. Strategies to enrich ruminant-derived foods with unsaturated fatty acids are desired as...

  6. Role of Pseudomonas aeruginosa lipase in inflammatory mediator release from human inflammatory effector cells (platelets, granulocytes, and monocytes.

    PubMed Central

    König, B; Jaeger, K E; Sage, A E; Vasil, M L; König, W

    1996-01-01

    Previously, we have shown that Pseudomonas aeruginosa lipase and phospholipase C (PLC), two extracellular lipolytic enzymes, interact with each other during 12-hydroxyeicosatetraenoic acid (HETE) generation from human platelets. In this regard. the addition of purified P. aeruginosa lipase to PLC-containing crude P. aeruginosa culture supernatants enhances the generation of the chemotactically active 12-HETE from human platelets. Therefore, we analyzed the interaction of purified P. aeruginosa lipase and purified hemolytic P. aeruginosa PLC with regard to inflammatory mediator release from human platelets, neutrophilic and basophilic granulocytes, and monocytes. Purified P. aeruginosa PLC, but not purified lipase by itself, induced 12-HETE generation from human platelets, the generation of leukotriene B4 (LTB4) and oxygen metabolites, enzyme release from human neutrophils, and histamine release from basophils but diminished interleukin-8 (IL-8) release from human monocytes in a dose-dependent manner. The addition of purified lipase enhanced PLC-induced 12-HETE and LTB4 generation, did not influence enzyme, histamine, or IL-8 release, but diminished the PLC-induced chemiluminescent response. Similar results were obtained when the hemolytic PLC from Clostridium perfringens was used instead of P. aeruginosa PLC. For further comparison, we used the well-defined calcium ionophore A23187 and phorbol-12-myristate-13-acetate (PMA) as stimuli. Lipase enhanced calcium ionophore-induced LTB4 generation and beta-glucuronidase release but reduced calcium ionophore-induced and PMA-induced chemiluminescence. In parallel, we analyzed the role of lipase in a crude P. aeruginosa culture supernatant containing PLC and lipase. Lipase activity in the P. aeruginosa culture supernatant was inhibited by treatment with the lipase-specific inhibitor hexadecylsulfonyl fluoride, leaving the activity of PLC unaffected. The capacity of "lipase-inactivated culture supernatant" to induce 12-HETE

  7. AMP-Activated Kinase Regulates Lipid Droplet Localization and Stability of Adipose Triglyceride Lipase in C. elegans Dauer Larvae

    PubMed Central

    Xie, Meng; Roy, Richard

    2015-01-01

    Animals have developed diverse mechanisms to adapt to their changing environment. Like many organisms the free-living nematode C. elegans can alternate between a reproductive mode or a diapause-like "dauer" stage during larval development to circumvent harsh environmental conditions. The master metabolic regulator AMP-activated protein kinase (AMPK) is critical for survival during the dauer stage, where it phosphorylates adipose triglyceride lipase (ATGL-1) at multiple sites to block lipid hydrolysis and ultimately protect the cellular triglyceride-based energy depot from rapid depletion. However, how the AMPK-mediated phosphorylation affects the function of ATGL-1 has not been characterised at the molecular level. Here we show that AMPK phosphorylation leads to the generation of 14-3-3 binding sites on ATGL-1, which are recognized by the C. elegans 14-3-3 protein orthologue PAR-5. Physical interaction of ATGL-1 with PAR-5 results in sequestration of ATGL-1 away from the lipid droplets and eventual proteasome-mediated degradation. In addition, we also show that the major AMPK phosphorylation site on ATGL-1, Ser 303, is required for both modification of its lipid droplet localization and its degradation. Our data provide mechanistic insight as to how AMPK functions to enhance survival through its ability to protect the accumulated triglyceride deposits from rapid hydrolysis to preserve the energy stores during periods of extended environmental duress. PMID:26098762

  8. A Novel Halophilic Lipase, LipBL, Showing High Efficiency in the Production of Eicosapentaenoic Acid (EPA)

    PubMed Central

    Pérez, Dolores; Martín, Sara; Fernández-Lorente, Gloria; Filice, Marco; Guisán, José Manuel; Ventosa, Antonio; García, María Teresa; Mellado, Encarnación

    2011-01-01

    Background Among extremophiles, halophiles are defined as microorganisms adapted to live and thrive in diverse extreme saline environments. These extremophilic microorganisms constitute the source of a number of hydrolases with great biotechnological applications. The interest to use extremozymes from halophiles in industrial applications is their resistance to organic solvents and extreme temperatures. Marinobacter lipolyticus SM19 is a moderately halophilic bacterium, isolated previously from a saline habitat in South Spain, showing lipolytic activity. Methods and Findings A lipolytic enzyme from the halophilic bacterium Marinobacter lipolyticus SM19 was isolated. This enzyme, designated LipBL, was expressed in Escherichia coli. LipBL is a protein of 404 amino acids with a molecular mass of 45.3 kDa and high identity to class C β-lactamases. LipBL was purified and biochemically characterized. The temperature for its maximal activity was 80°C and the pH optimum determined at 25°C was 7.0, showing optimal activity without sodium chloride, while maintaining 20% activity in a wide range of NaCl concentrations. This enzyme exhibited high activity against short-medium length acyl chain substrates, although it also hydrolyzes olive oil and fish oil. The fish oil hydrolysis using LipBL results in an enrichment of free eicosapentaenoic acid (EPA), but not docosahexaenoic acid (DHA), relative to its levels present in fish oil. For improving the stability and to be used in industrial processes LipBL was immobilized in different supports. The immobilized derivatives CNBr-activated Sepharose were highly selective towards the release of EPA versus DHA. The enzyme is also active towards different chiral and prochiral esters. Exposure of LipBL to buffer-solvent mixtures showed that the enzyme had remarkable activity and stability in all organic solvents tested. Conclusions In this study we isolated, purified, biochemically characterized and immobilized a lipolytic enzyme from

  9. Telmisartan increases lipoprotein lipase expression via peroxisome proliferator-activated receptor-alpha in HepG2 cells.

    PubMed

    Yin, Shi Nan; Liu, Min; Jing, Dan Qing; Mu, Yi Ming; Lu, Ju Ming; Pan, Chang Yu

    2014-01-01

    In addition to their hypotensive properties, angiotensin receptor blockers (ARBs) have been shown to exert clinical antidyslipidemic effects. The mechanism underlying these ARB lipid metabolic effects remains unclear. Some ARBs, for example, telmisartan, activate peroxisome proliferator-activated activated receptor-gamma (PPAR-gamma). We hypothesized that PPAR-gamma-activating ARBs might exert antidyslipidemic effects via PPAR-alpha. In this study, we assessed the effect of telmisartan on the expression of PPAR-alpha and lipoprotein lipase (LPL). PPAR-alpha expression was detected by reverse-transcription polymerase chain reaction and Western blot in HepG2 hepatocytes as well as differentiated C2C12 myocytes treated with increasing concentrations of telmisartan (0.1-10 μmol/L) for 48 h. Results showed that 1 μmol/L and 10 μmol/L telmisartan significantly increased the expression of PPAR-alpha mRNA and protein in HepG2 cells (p < 0.01). No effect was shown in differentiated C2C12 cells. Similarly, 1 µmol/L and 10 μmol/L telmisartan significantly increased the expression of LPL mRNA and protein in HepG2 cells (p < 0.01), and this increase was significantly (p < 0.01) inhibited by the PPAR-alpha-specific antagonist MK886. These results indicate that certain of the antidyslipidemic effects of telmisartan might be mediated via increased PPAR-alpha-dependent induction of LPL expression. PMID:24067162

  10. 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. PMID:25575887

  11. From Structure to Catalysis: Recent Developments in the Biotechnological Applications of Lipases

    PubMed Central

    Anobom, Cristiane D.; Pinheiro, Anderson S.; De-Andrade, Rafael A.; Aguieiras, Erika C. G.; Andrade, Guilherme C.; Moura, Marcelo V.; Almeida, Rodrigo V.; Freire, Denise M.

    2014-01-01

    Microbial lipases are highly appreciated as biocatalysts due to their peculiar characteristics such as the ability to utilize a wide range of substrates, high activity and stability in organic solvents, and regio- and/or enantioselectivity. These enzymes are currently being applied in a variety of biotechnological processes, including detergent preparation, cosmetics and paper production, food processing, biodiesel and biopolymer synthesis, and the biocatalytic resolution of pharmaceutical derivatives, esters, and amino acids. However, in certain segments of industry, the use of lipases is still limited by their high cost. Thus, there is a great interest in obtaining low-cost, highly active, and stable lipases that can be applied in several different industrial branches. Currently, the design of specific enzymes for each type of process has been used as an important tool to address the limitations of natural enzymes. Nowadays, it is possible to “order” a “customized” enzyme that has ideal properties for the development of the desired bioprocess. This review aims to compile recent advances in the biotechnological application of lipases focusing on various methods of enzyme improvement, such as protein engineering (directed evolution and rational design), as well as the use of structural data for rational modification of lipases in order to create higher active and selective biocatalysts. PMID:24783219

  12. A highly regioselective route to arbutin esters by immobilized lipase from Penicillium expansum.

    PubMed

    Yang, Rong-Ling; Li, Ning; Li, Ri-Feng; Smith, Thomas J; Zong, Min-Hua

    2010-01-01

    Immobilized lipase from Penicillium expansum, a novel and inexpensive enzyme preparation that we immobilized in our laboratory, was an excellent catalyst for highly regioselective acylation of arbutin with fatty acid vinyl esters. For the enzymatic butanoylation of arbutin, under the optimal conditions, initial reaction rate was 75.1 mM/h, and substrate conversion and regioselectivity were greater than 99%. In addition, a variety of 6'-esters of arbutin were prepared with high conversion (>99%) and excellent regioselectivity (>99%). It was found that the enzymatic reaction rate varied widely with different acyl donors, presumably owing to their different interactions with the active site of the lipase. The immobilized lipase from P. expansum displayed highest catalytic activity with medium-length straight-chain acyl donors. Acyl donors bearing a substituent or a conjugate double bond gave reduced reaction rates. PMID:19695875

  13. Effect of environmental conditions on extracellular lipases production and fungal morphology from Aspergillus niger MYA 135.

    PubMed

    Colin, Veronica Leticia; Baigori, Mario Domingo; Pera, Licia Maria

    2010-02-01

    Under the current assay conditions, lipase production in mineral medium was only detected in the presence of vegetable oils, reaching the highest specific activity with olive oil. In this way, effect of different environmental conditions on fungal morphology and olive oil-induced extracellular lipases production from Aspergillus niger MYA 135 was studied. It was observed that addition of 1.0 g l(-1) FeCl(3)to the medium encouraged filamentous growth and increased the specific activity 6.6 fold after 4 days of incubation compared to the control. However, major novelty of this study was the satisfactory production of an acidic lipase at initial pH 3 of the culture medium (1.74 +/- 0.06 mU microg(-1)), since its potencial applications in food and pharmaceutical industry are highly promising. PMID:20082373

  14. Covalent immobilization of porcine pancreatic lipase on carboxyl-activated magnetic nanoparticles: characterization and application for enzymatic inhibition assays.

    PubMed

    Zhu, Yuan-Ting; Ren, Xiao-Yun; Liu, Yi-Ming; Wei, Ying; Qing, Lin-Sen; Liao, Xun

    2014-05-01

    Using carboxyl functionalized silica-coated magnetic nanoparticles (MNPs) as carrier, a novel immobilized porcine pancreatic lipase (PPL) was prepared through the 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride/N-hydroxysuccinimide (EDC/NHS) coupling reaction. Transmission electron microscopic images showed that the synthesized nanoparticles (Fe3O4-SiO2) possessed three dimensional core-shell structures with an average diameter of ~20 nm. The effective enzyme immobilization onto the nanocomposite was confirmed by atomic force microscopic (AFM) analysis. Results from Fourier-transform infrared spectroscopy (FT-IR), Bradford protein assay, and thermo-gravimetric analysis (TGA) indicated that PPL was covalently attached to the surface of magnetic nanoparticles with a PPL immobilization yield of 50mg enzyme/g MNPs. Vibrating sample magnetometer (VSM) analysis revealed that the MNPs-PPL nanocomposite had a high saturation magnetization of 42.25 emu·g(-1). The properties of the immobilized PPL were investigated in comparison with the free enzyme counterpart. Enzymatic activity, reusability, thermo-stability, and storage stability of the immobilized PPL were found significantly superior to those of the free one. The Km and the Vmax values (0.02 mM, 6.40 U·mg(-1) enzyme) indicated the enhanced activity of the immobilized PPL compared to those of the free enzyme (0.29 mM, 3.16 U·mg(-1) enzyme). Furthermore, at an elevated temperature of 70 °C, immobilized PPL retained 60% of its initial activity. The PPL-MNPs nanocomposite was applied in the enzyme inhibition assays using orlistat, and two natural products isolated from oolong tea (i.e., EGCG and EGC) as the test compounds. PMID:24656379

  15. Adipose Triglyceride Lipase, Not Hormone-Sensitive Lipase, Is the Primary Lipolytic Enzyme in Fasting Elephant Seals (Mirounga angustirostris).

    PubMed

    Fowler, Melinda A; Costa, Daniel P; Crocker, Daniel E; Shen, Wen-Jun; Kraemer, Fredric B

    2015-01-01

    Little is known about the mechanisms that allow capital breeders to rapidly mobilize large amounts of body reserves. Northern elephant seals (Mirounga angustirostris) utilize fat reserves for maternal metabolism and to create high fat milk for the pup. Hormone-sensitive lipase (HSL) has been hypothesized to be an important lipolytic enzyme in fasting seals, but the activity of HSL and adipose triglyceride lipase (ATGL) has not been quantified in fasting adult seals, nor has their relationship to milk lipid content been assessed. Blubber and milk samples were obtained from 18 early lactation and 19 late lactation females, as well as blubber from five early and five late molting female seals. Blubber lipolytic activity was assessed with radiometric assays. HSL activity was negligible in seal blubber at all fasting stages. Total triglyceride lipase activity was stable among early and late lactation and early molt but increased in late molting seals. Relative abundance of ATGL protein increased across fasting, but neither activity nor relative protein levels were related to circulating nonesterified fatty acids or milk lipid content, suggesting the possibility of other regulatory pathways between lipolytic activity and milk lipid content. These results demonstrate that HSL is not the primary lipolytic enzyme in fasting adult female seals and that ATGL contributes more to lipolysis than HSL. PMID:25860827

  16. Studies on the inhibition of pancreatic and carboxylester lipases by protamine.

    PubMed

    Tsujita, T; Matsuura, Y; Okuda, H

    1996-07-01

    The basic protein protamine strongly inhibited hydrolysis of triolein emulsified with soybean phosphatidylcholine (PC) by pancreatic and carboxylester lipases; 10 micrograms/ml protamine, about 1000 times lower than the concentration of bovine serum albumin for the same effect, inhibited triolein hydrolysis completely. This inhibition was not affected by the incubation pH or bile salt concentration. Two other basic proteins, histone and purothionin, also inhibited hydrolysis of triolein emulsified with soybean PC, but they did not inhibit triolein hydrolysis by gastric lipase. When gum arabic was used as an emulsifier instead of soybean PC, these basic proteins did not affect triolein hydrolysis by pancreatic or carboxylester lipases. The effects of protamine on triolein hydrolysis by pancreatic and carboxylester lipases was studied using various phospholipids as emulsifiers. Protamine (10 micrograms/ml) did not inhibit hydrolysis of triolein emulsified with dicaproyl PC (DCPC), phosphatidic acid (PA), or phosphatidylserine (PS) by pancreatic and carboxylester lipases. Conversely, protamine at high concentrations slightly stimulated hydrolysis of triolein emulsified with DCPC or PA. Hydrolysis of triolein-phosphatidylethanolamine (PE) emulsion was inhibited slightly by protamine. The profiles of protamine inhibition of triolein-phosphatidyl-N,N-dimethyl ethanolamine (PDME) and triolein-phosphatidyl-N-monomethyl ethanolamine (PMME) emulsions were intermediate between those of PC and PE emulsions. These results suggest that the phospholipid species, especially choline moieties and fatty acid chain length, affect the lipase inhibitory activity of protamine profoundly. In vivo, oral administration of protamine to rats reduced and delayed the peak plasma triacylglycerol concentration, but neither bovine serum albumin nor an amino acid mixture with an amino acid composition identical to protamine affected plasma triacylglycerol levels. PMID:8827520

  17. Lipase-catalyzed synthesis of hyperbranched poly-L-lactide in an ionic liquid.

    PubMed

    Mena, María; López-Luna, Alberto; Shirai, Keiko; Tecante, Alberto; Gimeno, Miquel; Bárzana, Eduardo

    2013-03-01

    Hyperbranched poly-L-lactides have been synthesized by eROP in [C4MIM][PF6] media. The bis(hydroxymethyl)butyric acid molecule was used as the AB2 core co-monomer and immobilized lipase B from Candida antarctica as biocatalyst. The degree of branching could be controlled by the reaction conditions, with the maximum achieved being 0.21. The successful achievement of the hyperbranched structure is attributed to the high solvent power of substrates and products in the ionic liquid besides sustained lipase activity. PMID:22869004

  18. Expression of a lipid-inducible, self-regulating form of Yarrowia lipolytica lipase LIP2 in Saccharomyces cerevisiae

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The Yarrowia lipolytica lipase 2 gene (YlLIP2) was cloned into galactose- and fatty acid-inducible Saccharomyces cerevisiae expression vectors and used to generate yeast strains that secrete active LIP2 enzyme activity, as evidenced by results from gene expression analysis and tributyrin turbidity c...

  19. Genetic mutations in adipose triglyceride lipase and myocardial up-regulation of peroxisome proliferated activated receptor-γ in patients with triglyceride deposit cardiomyovasculopathy.

    PubMed

    Hirano, Ken-ichi; Tanaka, Tatsuya; Ikeda, Yoshihiko; Yamaguchi, Satoshi; Zaima, Nobuhiro; Kobayashi, Kazuhiro; Suzuki, Akira; Sakata, Yasuhiko; Sakata, Yasushi; Kobayashi, Kunihisa; Toda, Tatsushi; Fukushima, Norihide; Ishibashi-Ueda, Hatsue; Tavian, Daniela; Nagasaka, Hironori; Hui, Shu-Ping; Chiba, Hitoshi; Sawa, Yoshiki; Hori, Masatsugu

    2014-01-10

    Adipose triglyceride lipase (ATGL, also known as PNPLA2) is an essential molecule for hydrolysis of intracellular triglyceride (TG). Genetic ATGL deficiency is a rare multi-systemic neutral lipid storage disease. Information regarding its clinical profile and pathophysiology, particularly for cardiac involvement, is still very limited. A previous middle-aged ATGL-deficient patient in our institute (Case 1) with severe heart failure required cardiac transplantation (CTx) and exhibited a novel phenotype, "Triglyceride deposit cardiomyovasculopathy (TGCV)". Here, we tried to elucidate molecular mechanism underlying TGCV. The subjects were two cases with TGCV, including our second case who was a 33-year-old male patient (Case 2) with congestive heart failure requiring CTx. Case 2 was homozygous for a point mutation in the 5' splice donor site of intron 5 in the ATGL, which results in at least two types of mRNAs due to splicing defects. The myocardium of both patients (Cases 1 and 2) showed up-regulation of peroxisome proliferated activated receptors (PPARs), key transcription factors for metabolism of long chain fatty acids (LCFAs), which was in contrast to these molecules' lower expression in ATGL-targeted mice. We investigated the intracellular metabolism of LCFAs under human ATGL-deficient conditions using patients' passaged skin fibroblasts as a model. ATGL-deficient cells showed higher uptake and abnormal intracellular transport of LCFA, resulting in massive TG accumulation. We used these findings from cardiac specimens and cell-biological experiments to construct a hypothetical model to clarify the pathophysiology of the human disorder. In patients with TGCV, even when hydrolysis of intracellular TG is defective, the marked up-regulation of PPARγ and related genes may lead to increased uptake of LCFAs, the substrates for TG synthesis. This potentially vicious cycle of LCFAs could explain the massive accumulation of TG and severe clinical course for this rare

  20. Inhibition of Monoacylglycerol Lipase Activity Decreases Glucose-Stimulated Insulin Secretion in INS-1 (832/13) Cells and Rat Islets

    PubMed Central

    Burritt, Nathan E.; Corkey, Barbara E.; Deeney, Jude T.

    2016-01-01

    Lipid signals derived from lipolysis and membrane phospholipids play an important role in glucose-stimulated insulin secretion (GSIS), though the exact secondary signals remain unclear. Previous reports have documented a stimulatory role of exogenously added mono-acyl-glycerol (MAG) on insulin secretion from cultured β-cells and islets. In this report we have determined effects of increasing intracellular MAG in the β-cell by inhibiting mono-acyl-glycerol lipase (MGL) activity, which catalyzes the final step in triacylglycerol breakdown, namely the hydrolysis of MAG to glycerol and free fatty acid (FA). To determine the role of MGL in GSIS, we used three different pharmacological agents (JZL184, MJN110 and URB602). All three inhibited GSIS and depolarization-induced insulin secretion in INS-1 (832/13). JZL184 significantly inhibited both GSIS and depolarization-induced insulin secretion in rat islets. JZL184 significantly decreased lipolysis and increased both mono- and diacyglycerol species in INS-1 cells. Analysis of the kinetics of GSIS showed that inhibition was greater during the sustained phase of secretion. A similar pattern was observed in the response of Ca2+ to glucose and depolarization but to a lesser degree suggesting that altered Ca2+ handling alone could not explain the reduction in insulin secretion. In addition, a significant reduction in long chain-CoA (LC-CoA) was observed in INS-1 cells at both basal and stimulatory glucose following inhibition of MGL. Our data implicate an important role for MGL in insulin secretion. PMID:26867016

  1. Conversion of a Mono- and Diacylglycerol Lipase into a Triacylglycerol Lipase by Protein Engineering.

    PubMed

    Lan, Dongming; Popowicz, Grzegorz Maria; Pavlidis, Ioannis V; Zhou, Pengfei; Bornscheuer, Uwe T; Wang, Yonghua

    2015-07-01

    Despite the fact that most lipases are believed to be active against triacylglycerides, there is a small group of lipases that are active only on mono- and diacylglycerides. The reason for this difference in substrate scope is not clear. We tried to identify the reasons for this in the lipase from Malassezia globosa. By protein engineering, and with only one mutation, we managed to convert this enzyme into a typical triacylglycerol lipase (the wild-type lipase does not accept triacylglycerides). The variant Q282L accepts a broad spectrum of triacylglycerides, although the catalytic behavior is altered to some extent. From in silico analysis it seems that specific hydrophobic interactions are key to the altered substrate specificity. PMID:25955297

  2. 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

  3. Production of lipase by clinical isolates of Pseudomonas cepacia.

    PubMed Central

    Lonon, M K; Woods, D E; Straus, D C

    1988-01-01

    Ten clinical isolates of Pseudomonas cepacia from the sputum of cystic fibrosis patients were examined for the ability to produce lipase. Lipase substrates used included egg yolk agar, four different polyoxyethylene sorbitans (Tweens), and p-nitrophenylphosphorylcholine, a chromogenic substrate used to assay for phospholipase C. Lipase activity was detected in the filtrates of organisms grown to the exponential phase in either tryptose minimal medium or chemically defined medium. Lipase activity increased in the filtrates if the cultures were allowed to proceed into the stationary phase. None of the isolates produced phospholipase C. Lipase activity on Tween 20 ranged from 41.6 X 10(-3) to 640.0 X 10(-3) U/micrograms of protein. The activity was similar or slightly lower when Tween 40, 60, or 80 was used as the substrate. There was no correlation between lipase activity on Tween and that demonstrated on egg yolk agar. Lipase activity increased as pH increased from 7.0 to 9.0. Boiling for 5 min resulted in 66% loss of enzyme activity. The remaining activity continued to decrease with increasing boiling time. The enzyme was purified by gel filtration on Sephadex G-200, and the resultant preparation, when subjected to polyacrylamide gel electrophoresis, resulted in a single protein band (molecular weight, approximately 25,000) from which lipase activity could be eluted. The purified lipase was not cytotoxic to HeLa cells, nor was it toxic when injected intravenously into mice. PMID:3384918

  4. 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. PMID:25879679

  5. Covalent immobilization of Pseudomonas cepacia lipase on semiconducting materials

    NASA Astrophysics Data System (ADS)

    Fernandez, Renny Edwin; Bhattacharya, Enakshi; Chadha, Anju

    2008-05-01

    Lipase from Pseudomonas cepacia was covalently immobilized on crystalline silicon, porous silicon and silicon nitride surfaces. The various stages of immobilization were characterized using FTIR (Fourier transform infrared) spectroscopy. The surface topography of the enzyme immobilized surfaces was investigated using scanning electron microscopy (SEM). The quantity of the immobilized active enzyme was estimated by the para-nitrophenyl palmitate (pNPP) assay. The immobilized lipase was used for triglyceride hydrolysis and the acid produced was detected by a pH sensitive silicon nitride surface as a shift in the C- V (capacitance-voltage) characteristics of an electrolyte-insulator-semiconductor capacitor (EISCAP) thus validating the immobilization method for use as a biosensor.

  6. 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. PMID:27268114

  7. Efficient biocatalyst by encapsulating lipase into nanoporous gold

    NASA Astrophysics Data System (ADS)

    Du, Xiaoyu; Liu, Xueying; Li, Yufei; Wu, Chao; Wang, Xia; Xu, Ping

    2013-04-01

    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.

  8. [Rapid improvement of lipase production in Penicillium expansum by genome shuffling].

    PubMed

    Lin, Jun; Shi, Bi-Hong; Shi, Qiao-Qin;