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Sample records for acid lipase lal

  1. Deletion of sterol O-acyltransferase 2 (SOAT2) function in mice deficient in lysosomal acid lipase (LAL) dramatically reduces esterified cholesterol sequestration in the small intestine and liver

    PubMed Central

    Lopez, Adam M.; Posey, Kenneth S.; Turley, Stephen D.

    2014-01-01

    Sterol O-acyltransferase 2 (SOAT2), also known as ACAT2, is the major cholesterol esterifying enzyme in the liver and small intestine (SI). Esterified cholesterol (EC) carried in certain classes of plasma lipoproteins is hydrolyzed by lysosomal acid lipase (LAL) when they are cleared from the circulation. Loss-of-function mutations in LIPA, the gene that encodes LAL, result in Wolman disease (WD) or cholesteryl ester storage disease (CESD). Hepatomegaly and a massive increase in tissue EC levels are hallmark features of both disorders. While these conditions can be corrected with enzyme replacement therapy, the question arose as to what effect the loss of SOAT2 function might have on tissue EC sequestration in LAL-deficient mice. When weaned at 21 days, Lal−/−:Soat2+/+ mice had a whole liver cholesterol content (mg/organ) of 24.7 mg vs. 1.9 mg in Lal+/+:Soat2+/+ littermates, with almost all the excess sterol being esterified. Over the next 31 days, liver cholesterol content in the Lal−/−:Soat2+/+ mice increased to 145 ± 2 mg but to only 29 ± 2 mg in their Lal−/−:Soat2−/− littermates. The level of EC accumulation in the SI of the Lal−/−:Soat2−/− mice was also much less than in their Lal−/−:Soat2+/+ littermates. In addition, there was a >70% reduction in plasma transaminase activities in the Lal−/−:Soat2−/− mice. These studies illustrate how the severity of disease in a mouse model for CESD can be substantially ameliorated by elimination of SOAT2 function. PMID:25450374

  2. 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 ...

  3. 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

  4. 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

  5. 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

  6. 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

  7. 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

  8. 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

  9. 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

  10. 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

  11. 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

  12. 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

  13. 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

  14. Critical role of the mTOR pathway in development and function of myeloid-derived suppressor cells in lal-/- mice.

    PubMed

    Ding, Xinchun; Du, Hong; Yoder, Mervin C; Yan, Cong

    2014-02-01

    Lysosomal acid lipase (LAL) is essential for the hydrolysis of cholesteryl esters and triglycerides to generate cholesterol and free fatty acids in cellular lysosomes. Ablation of the lal gene (lal(-/-)) systemically increased expansion of cluster of differentiation molecule 11b (CD11b), lymphocyte antigen 6G (Ly6G) myeloid-derived suppressor cells (MDSCs) that caused myeloproliferative neoplasms in mice. Study of lal(-/-) bone marrow Ly6G(+) MDSCs via transcriptional profiling showed increases in mammalian target of rapamycin (mTOR) signaling pathway transcripts. Injection of mTOR pharmacologic inhibitors into lal(-/-) mice significantly reduced bone marrow myelopoiesis and systemic CD11b(+)Ly6G(+) cell expansion. Rapamycin treatment of lal(-/-) mice stimulated a shift from immature CD11b(+)Ly6G(+) cells to CD11b(+) single-positive cells in marrow and tissues and partially reversed the increased cell proliferation, decreased apoptosis, increased ATP synthesis, and increased cell cycling of bone marrow CD11b(+)Ly6G(+) cells obtained from lal(-/-) mice. Pharmacologic and siRNA suppression of mTOR, regulatory-associated protein of mTOR, rapamycin-insensitive companion of mTOR, and Akt1 function corrected CD11b(+)Ly6G(+) cell in lal(-/-) mice development from Lin(-) progenitor cells and reversed the immune suppression on T-cell proliferation and function in association with decreased reactive oxygen species production, and recovery from impairment of mitochondrial membrane potential compared with control mutant cells. These results indicate a crucial role of LAL-regulated mTOR signaling in the production and function of CD11b(+)Ly6G(+) cells. The mTOR pathway may serve as a novel target to modulate the emergence of MDSCs in those pathophysiologic states in which these cells play an immunosuppressive role. PMID:24287405

  15. 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

  16. 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

  17. 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...

  18. 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

  19. PRD125, a potent and selective inhibitor of sterol O-acyltransferase 2 markedly reduces hepatic cholesteryl ester accumulation and improves liver function in lysosomal acid lipase-deficient mice.

    PubMed

    Lopez, Adam M; Chuang, Jen-Chieh; Posey, Kenneth S; Ohshiro, Taichi; Tomoda, Hiroshi; Rudel, Lawrence L; Turley, Stephen D

    2015-11-01

    In most organs, the bulk of cholesterol is unesterified, although nearly all possess a varying capability of esterifying cholesterol through the action of either sterol O-acyltransferase (SOAT) 1 or, in the case of hepatocytes and enterocytes, SOAT2. Esterified cholesterol (EC) carried in plasma lipoproteins is hydrolyzed by lysosomal acid lipase (LAL) when they are cleared from the circulation. Loss-of-function mutations in LIPA, the gene that encodes LAL, result in Wolman disease or cholesteryl ester storage disease (CESD). Hepatomegaly and a massive increase in tissue EC levels are hallmark features of both disorders. While these conditions can be corrected with enzyme replacement therapy, the question arose as to whether pharmacological inhibition of SOAT2 might reduce tissue EC accretion in CESD. When weaned at 21 days, Lal(-/-) mice, of either gender, had a whole liver cholesterol content that was 12- to 13-fold more than that of matching Lal(+/+) littermates (23 versus 1.8 mg, respectively). In Lal(-/-) males given the selective SOAT2 inhibitor PRD125 1,11-O-o-methylbenzylidene-7-O-p-cyanobenzoyl-1,7,11-trideacetylpyripyropene A in their diet (∼10 mg/day per kg body weight) from 21 to 53 days, whole liver cholesterol content was 48.6 versus 153.7 mg in untreated 53-day-old Lal(-/-) mice. This difference reflected a 59% reduction in hepatic EC concentration (mg/g), combined with a 28% fall in liver mass. The treated mice also showed a 63% reduction in plasma alanine aminotransferase activity, in parallel with decisive falls in hepatic mRNA expression levels for multiple proteins that reflect macrophage presence and inflammation. These data implicate SOAT2 as a potential target in CESD management. PMID:26283692

  20. 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. ...

  1. 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

  2. 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

  3. 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

  4. 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.

  5. 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

  6. Cholestane-3β,5α,6β-triol: high levels in Niemann-Pick type C, cerebrotendinous xanthomatosis, and lysosomal acid lipase deficiency.

    PubMed

    Pajares, Sonia; Arias, Angela; García-Villoria, Judit; Macías-Vidal, Judit; Ros, Emilio; de las Heras, Javier; Girós, Marisa; Coll, Maria J; Ribes, Antonia

    2015-10-01

    Niemann-Pick type C (NPC) is a progressive neurodegenerative disease characterized by lysosomal/endosomal accumulation of unesterified cholesterol and glycolipids. Recent studies have shown that plasma cholestane-3β,5α,6β-triol (CT) and 7-ketocholesterol (7-KC) could be potential biomarkers for the diagnosis of NPC patients. We aimed to know the sensitivity and specificity of these biomarkers for the diagnosis of NPC compared with other diseases that can potentially lead to oxysterol alterations. We studied 107 controls and 122 patients including 16 with NPC, 3 with lysosomal acid lipase (LAL) deficiency, 8 with other lysosomal diseases, 5 with galactosemia, 11 with cerebrotendinous xanthomatosis (CTX), 3 with Smith-Lemli-Opitz, 14 with peroxisomal biogenesis disorders, 19 with unspecific hepatic diseases, 13 with familial hypercholesterolemia, and 30 with neurological involvement and no evidence of an inherited metabolic disease. CT and 7-KC were analyzed by HPLC-ESI-MS/MS as mono-dimethylglycine derivatives. Levels of 7-KC were high in most of the studied diseases, whereas those of CT were only high in NPC, LAL, and CTX patients. Consequently, although CT is a sensitive biomarker of NPC disease, including those cases with doubtful filipin staining, it is not specific. 7-KC is a very unspecific biomarker. PMID:26239048

  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. 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

  9. 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

  10. 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

  11. 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

  12. 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

  13. 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

  14. [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

  15. 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

  16. 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

  17. 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

  18. [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

  19. 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

  20. 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) (...

  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. 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

  3. 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

  4. 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

  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. 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

  7. 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

  8. 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

  9. 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

  10. [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

  11. 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

  12. 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

  13. 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

  14. 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

  15. 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

  16. 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...

  17. 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

  18. 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

  19. 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

  20. 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

  1. 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

  2. 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

  3. 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

  4. 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

  5. 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

  6. 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

  7. 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

  8. 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

  9. 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

  10. 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

  11. 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

  12. 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

  13. 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.

  14. Successful long-term outcome of liver transplantation in late-onset lysosomal acid lipase deficiency.

    PubMed

    Sreekantam, S; Nicklaus-Wollenteit, I; Orr, J; Sharif, K; Vijay, S; McKiernan, P J; Santra, S

    2016-09-01

    Late-onset LAL deficiency, previously referred to as cholesteryl ester storage disorder, is a rare lysosomal storage disorder characterized by accumulation of cholesteryl esters. It has a heterogeneous clinical phenotype including abdominal pain, poor growth, hyperlipidemia with vascular complications and hepatosplenomegaly. End-stage liver disease may occur, but there are few reports of successful LT. There are also concerns that systemic manifestations of the disease might persist post-LT. We report a case with excellent outcome eight yr following LT. The subject was noted to have asymptomatic hepatosplenomegaly during an intercurrent illness, and LAL deficiency was confirmed with compound heterozygosity in the LIPA. Despite dietary fat restriction, he developed signs of progressive liver disease and subsequently developed hepatopulmonary syndrome. He underwent cadaveric LT at the age of nine and a half yr and recovered with prompt resolution of hepatopulmonary syndrome. Eight yr post-transplant he has normal growth, normal lipid profile, and liver and renal function tests. Liver histology showed no evidence of disease recurrence at this stage. LT in this subject resulted in an excellent functional correction of late-onset LAL deficiency. PMID:27392817

  15. 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

  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. 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. 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

  19. 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

  20. 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

  1. 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

  2. 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.

  3. 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

  4. 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

  5. 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

  6. 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

  7. 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

  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. 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

  11. 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

  12. 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.

  13. 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.

  14. 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

  15. 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

  16. 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.

  17. 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

  18. 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

  19. 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

  20. 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

  1. 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

  2. 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

  3. 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

  4. 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

  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. 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

  7. 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

  8. 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.

  9. 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

  10. 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

  11. 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

  12. 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. ...

  13. 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 ...

  14. 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

  15. 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

  16. 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

  17. 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

  18. 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

  19. 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

  20. 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

  1. 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

  2. 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

  3. 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...

  4. 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

  5. Genomics and genetics of Sulfolobus islandicus LAL14/1, a model hyperthermophilic archaeon

    PubMed Central

    Jaubert, Carole; Danioux, Chloë; Oberto, Jacques; Cortez, Diego; Bize, Ariane; Krupovic, Mart; She, Qunxin; Forterre, Patrick; Prangishvili, David; Sezonov, Guennadi

    2013-01-01

    The 2 465 177 bp genome of Sulfolobus islandicus LAL14/1, host of the model rudivirus SIRV2, was sequenced. Exhaustive comparative genomic analysis of S. islandicus LAL14/1 and the nine other completely sequenced S. islandicus strains isolated from Iceland, Russia and USA revealed a highly syntenic common core genome of approximately 2 Mb and a long hyperplastic region containing most of the strain-specific genes. In LAL14/1, the latter region is enriched in insertion sequences, CRISPR (clustered regularly interspaced short palindromic repeats), glycosyl transferase genes, toxin–antitoxin genes and MITE (miniature inverted-repeat transposable elements). The tRNA genes of LAL14/1 are preferential targets for the integration of mobile elements but clusters of atypical genes (CAG) are also integrated elsewhere in the genome. LAL14/1 carries five CRISPR loci with 10 per cent of spacers matching perfectly or imperfectly the genomes of archaeal viruses and plasmids found in the Icelandic hot springs. Strikingly, the CRISPR_2 region of LAL14/1 carries an unusually long 1.9 kb spacer interspersed between two repeat regions and displays a high similarity to pING1-like conjugative plasmids. Finally, we have developed a genetic system for S. islandicus LAL14/1 and created ΔpyrEF and ΔCRISPR_1 mutants using double cross-over and pop-in/pop-out approaches, respectively. Thus, LAL14/1 is a promising model to study virus–host interactions and the CRISPR/Cas defence mechanism in Archaea. PMID:23594878

  6. 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

  7. 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

  8. 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

  9. 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

  10. 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.

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

    PubMed

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

    2016-09-01

    Part of medium chain fatty acids (MCFAs) coming from dietary triglycerides (TGs) can be directly absorbed through the gastric mucosa after the action of preduodenal lipase (lingual lipase in the rat). MCFA gastric absorption, particularly that of octanoic acid (C8:0), may have a physiological importance in the octanoylation of ghrelin, the orexigenic gastric peptide acting as an endogenous ligand of the hypothalamic growth hormone secretagogue receptor 1a (GHSR-1a). However, the amount of C8:0 absorbed in the stomach and its metabolic fate still haven't been clearly characterized. The purpose of the present study was to further characterize and quantify the importance of preduodenal lipase activity on the release and gastric absorption of dietary C8:0 and on the subsequent ghrelin octanoylation in the stomach mucosa. Fifteen days old rats received fat emulsions containing triolein or [1,1,1-(13)C]-Tri-C8:0 and a specific inhibitor of preduodenal lipase, 5-(2-(benzyloxy)ethoxy)-3-(3-phenoxyphenyl)-1,3,4-oxadiazol-2(3H)-one or BemPPOX. The fate of the (13)C-C8:0 was followed in rat tissues after 30 and 120min of digestion and octanoylated ghrelin was measured in the plasma. This work (1) demonstrates that part of C8:0 coming from Tri-C8:0 is directly absorbed at the gastric level, (2) allows the estimation of C8:0 gastric absorption level (1.3% of the (13)C-C8:0 in sn-3 position after 30min of digestion), as well as (3) the contribution of rat lingual lipase to total lipolysis and to duodenal absorption of dietary FAs (at least 30%), (4) shows no short-term effect of dietary Tri-C8:0 consumption and subsequent increase of C8:0 gastric tissue content on plasma octanoylated ghrelin concentration. PMID:27317984

  12. 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

  13. 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

  14. 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

  15. 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.

  16. 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

  17. 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

  18. 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

  19. 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

  20. 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

  1. Detection and quantitative evaluation of endotoxin contamination in nanoparticle formulations by LAL-based assays.

    PubMed

    Neun, Barry W; Dobrovolskaia, Marina A

    2011-01-01

    Bacterial endotoxin or lipopolysaccharide (LPS) is a membrane component of all Gram-negative bacteria. The administration of products contaminated with bacterial endotoxin can cause fever, shock, and even death. Accordingly, the FDA sets limits on the number of endotoxin units (EU) that may be present in a drug or device product. Limulus amoebocyte lysate (LAL) is the extract from amoebocytes of the horseshoe crab Limulus polyphemus, which reacts with bacterial endotoxin. Detection of the products of this reaction is an effective means of quantifying the EU present in a drug formulation. However, nanoparticles frequently interfere with the reactivity of endotoxin, the LAL reaction, or the detection of the reaction products. This interference can be manifested as either an enhancement or an inhibition, causing a respective overestimation or underestimation of the EU in the sample. Here, we present two methods for the detection and quantification of endotoxin in nanoparticle preparations: one is based on an end-point chromogenic LAL assay, and the second approach is based on measuring the turbidity of the LAL extract. PMID:21116960

  2. 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

  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. 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

  5. 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

  6. 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

  7. 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

  8. 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...

  9. 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.

  10. 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

  11. 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

  12. 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

  13. 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

  14. 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

  15. 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

  16. 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

  17. 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.

  18. 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

  19. Continuous Production of Alkyl Esters Using an Immobilized Lipase Bioreactor

    Technology Transfer Automated Retrieval System (TEKTRAN)

    An immobilized lipase packed-bed bioreactor was developed for esterifying the free fatty acids in greases as a pretreatment step in the production of their simple alkyl esters for use as biodiesel. The immobilized lipases used in the study were immobilized preparations of Candida antarctica (C. a.)...

  20. 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

  1. Production of 7, 10-dihydroxy-8(E)-octadecenoic acid from triolein via lipase induction by Pseudomonas aeruginosa PR3

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Hydroxy fatty acids have gained important attentions because of their special properties such as higher viscosity and reactivity compared with other non-hydroxy fatty acids. The new bacterial isolate Pseudomonas aeruginosa (PR3) had been reported to produce mono-, di-, and tri-hydroxy fatty acids f...

  2. Lipase-induced Production of 7, 10-dihydroxy-8(E)-octadecenoic Acid from Triolein by Pseudomonas aeruginosa PR3

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Hydroxy fatty acids have gained attention because of their special properties such as higher viscosity and reactivity compared with other non-hydroxy fatty acids. The bacterial isolate Pseudomonas aeruginosa (PR3) had been reported to produce mono-, di-, and tri-hydroxy fatty acids from different f...

  3. Lipases and their industrial applications: an overview.

    PubMed

    Houde, Alain; Kademi, Ali; Leblanc, Danielle

    2004-01-01

    Lipases (triacylglycerol acylhydrolase, EC 3.1.1.3) are part of the family of hydrolases that act on carboxylic ester bonds. The physiologic role of lipases is to hydrolyze triglycerides into diglycerides, monoglycerides, fatty acids, and glycerol. These enzymes are widely found throughout the animal and plant kingdoms, as well as in molds and bacteria. Of all known enzymes, lipases have attracted the most scientific attention. In addition to their natural function of hydrolyzing carboxylic ester bonds, lipases can catalyze esterification, interesterification, and transesterification reactions in nonaqueous media. This versatility makes lipases the enzymes of choice for potential applications in the food, detergent, pharmaceutical, leather, textile, cosmetic, and paper industries. The most significant industrial applications of lipases have been mainly found in the food, detergent, and pharmaceutical sectors. Limitations of the industrial use of these enzymes have mainly been owing to their high production costs, which may be overcome by molecular technologies, enabling the production of these enzymes at high levels and in a virtually purified form. PMID:15304746

  4. Lipase-catalyzed acidolysis of tripalmitin with capric acid in organic solvent medium: Analysis of the effect of experimental conditions through factorial design and analysis of multiple responses.

    PubMed

    Foresti, María Laura; Ferreira, María Luján

    2010-05-01

    The acidolysis of tripalmitin with capric acid catalyzed by an immobilized form of a 1,3-positionally selective lipase (Rhizomucor miehei) showed to be effective for the synthesis of structured lipids of the MLL and MLM type. The effects that reaction parameters such as substrate molar ratio (N), biocatalyst load (E), and reaction temperature (T) have on selected responses variables (i.e. total conversion of tripalmitin, selectivity and yield of the desired structured lipid, hydrolysis yield, and acyl migration importance), were evaluated by use of an experimental factorial design of three factors and three levels with two central points and with a confidence level of 95%. The range of each parameter was selected as follows: N=3-9, E=5-15wt%, T=50-70°C. The statistical analysis of results was addressed by use of both simple linear models and more complicated quadratic models using specific commercial software. The results obtained showed that a proper selection of reaction conditions is needed in order to maximize not only the yield of the desired structured lipid, but also to minimize the generation of hydrolysis and acyl migration by-products. PMID:25919616

  5. Enzymatic Analysis of Positional Fatty Acid Distributions in Triacylglycerols by 1(3)-Selective Transesterification with Candida antarctica Lipase B: a Collaborative Study.

    PubMed

    Watanabe, Yomi; Sato, Shinichi; Asada, Mihoko; Arishima, Toshiharu; Iida, Yasuhiro; Imagi, Jun; Saito, Katsuyoshi; Sano, Takashi; Sasaki, Akiko; Sasaki, Ryo; Sato, Chiemi; Shibuya, Tadahisa; Tsukahara, Yuki; Nagai, Toshiharu; Fukazawa, Toru; Hori, Ryuji; Homma, Rika; Miyazaki, Yosuke; Yamashita, Atsushi; Yoshinaga, Kazuaki; Watanabe, Shimpei

    2015-01-01

    The positional distributions of fatty acids (FAs) in fats and oils are principally analyzed by selectively transesterifying the target triacylglycerols (TAGs) at the 1(3) position using Pseudozyma (Candida) antarctica lipase, followed by recovering the resulting 2-monoacylglycerols (MAGs) by chromatography. FA compositions were measured by gas chromatography (GC) after methylating target TAGs and 2-MAGs. The method was collaboratively evaluated by 12 laboratories by analyzing the positional FA distributions in soybean, palm, and sardine oils. The maximum reproducibility relative standard deviations for the major FAs and those at the sn-2 positions of soybean, palm, and sardine oils were 4.41% and 3.92% (18:3n-3), 4.48% and 3.82% (18:0), and 8.93 and 8.24% (14:0), respectively. The values at the sn-2 position were always low. Therefore, these results indicated that the variations were mainly caused by the FA analysis procedure, i.e., the methylation and GC analyses, rather than the enzymatic transesterification and chromatography utilized to prepare 2-MAGs from the target oil. PMID:26521812

  6. 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.

  7. 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...

  8. Optimising the use of commercial LAL assays for the analysis of endotoxin contamination in metal colloids and metal oxide nanoparticles.

    PubMed

    Li, Yang; Italiani, Paola; Casals, Eudald; Tran, Ngoc; Puntes, Victor F; Boraschi, Diana

    2015-05-01

    Engineered nanoparticles (NP) are generally contaminated by bacterial endotoxin, a ubiquitous bacterial molecule with significant toxic and inflammatory effects. The presence of endotoxin, if not recognised, can be responsible for many of the in vitro and in vivo effects attributed to NPs. The Limulus Amoebocyte Lysate (LAL) assay, the test requested by regulatory authorities for assessing endotoxin contamination in products for human use, is not immediately applicable for testing endotoxin in NP preparations, mainly due to the possible interference of NPs with the assay readouts and components. In this study, we have compared different commercially available LAL assays for detecting endotoxin in gold, silver and iron oxide NPs. Different NP chemistry, concentrations and surface coatings could differently interfere with the LAL assays' results. After accurate testing of the possible interaction/interference of NPs with the various assay components, the modified chromogenic LAL assay proved the most suitable assay for measuring endotoxin in NP samples, provided the appropriate controls are performed. Thus, endotoxin determination can be performed in NP preparation with commercial LAL assays only after assay validation, i.e. once possible interference of NPs with the assay components and readouts has been excluded. PMID:25119419

  9. Studies of reaction variables for lipase-catalyzed production of alpha-linolenic acid enriched structured lipid and oxidative stability with antioxidants.

    PubMed

    Mitra, Kanika; Shin, Jung-Ah; Lee, Jeung-Hee; Kim, Seong-Ai; Hong, Soon-Taek; Sung, Chang-Keun; Xue, Cheng Lian; Lee, Ki-Teak

    2012-01-01

    Alpha-linolenic acid (ALA) enriched structured lipid (SL) was produced by lipase-catalyzed interesterification from perilla oil (PO) and corn oil (CO). The effects of different reaction conditions (substrate molar ratio [PO/CO 1:1 to 1:3], reaction time [0 to 24 h], and reaction temperature [55 to 65 °C]) were studied. Lipozyme RM IM from Rhizomucor miehei was used as biocatalyst. We obtained 32.39% of ALA in SL obtained under the optimized conditions (molar ratio-1:1 [PO:CO], temperature-60 °C, reaction time-15 h). In SL, the major triacylglycerol (TAG) species (linolenoyl-linolenoyl-linolenoyl glycerol [LnLnLn], linolenoyl-linolenoyl-linoleoyl glycerol [LnLnL]) mainly from PO and linoleoyl-linoleoyl-oleoyl glycerol (LLO), linoleoyl-oleoyl-oleoyl glycerol (LOO), palmitoyl-linoleoyl-oleoyl glycerol (PLO) from CO decreased while linolenoyl-linolenoyl-oleoyl glycerol (LnLnO) (18.41%), trilinolein (LLL) (9.06%), LLO (16.66%), palmitoyl-linoleoyl-linoleoyl glycerol (PLL) (9.69%) were increased compared to that of physical blend. Total tocopherol content (28.01 mg/100 g), saponification value (SV) (192.2), and iodine value (IV) (161.9) were obtained. Furthermore, oxidative stability of the SL was also investigated by addition of 3 different antioxidants (each 200 ppm of rosemary extract [SL-ROS], BHT [SL-BHT], catechin [SL-CAT]) was added into SL and stored in 60 °C oven for 30 d. 2-Thiobabituric acid-reactive substances (TBARS) value was 0.16 mg/kg in SL-CAT and 0.18 mg/kg in SL-ROS as compared with 0.22 mg/kg in control (SL) after oxidation. The lowest peroxide value (POV, 200.9 meq/kg) and longest induction time (29.88 h) was also observed in SL-CAT. PMID:22122200

  10. 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

  11. 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

  12. 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

  13. 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

  14. Lipase-catalyzed production of a bioactive fatty amide derivative of 7,10-dihydroxy-8(E)-octadecenoic acid

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Fatty amides are of considerable interest due to their wide ranging industrial applications in detergents, shampoo, cosmetics and surfactant formulations. They are produced commercially from fatty acids by reacting with anhydrous ammonia at approximately 200 deg C and 345-690 KPa pressure. We inve...

  15. 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

  16. Influence of the degree of unsaturation of the acyl side chain upon the interaction of analogues of 1-arachidonoylglycerol with monoacylglycerol lipase and fatty acid amide hydrolase

    SciTech Connect

    Vandevoorde, Severine; Saha, Bijali; Mahadevan, Anu; Razdan, Raj K.; Pertwee, Roger G.; Martin, Billy R.; Fowler, Christopher J. . E-mail: cf@pharm.umu.se

    2005-11-11

    Little is known as to the structural requirements of the acyl side chain for interaction of acylglycerols with monoacylglycerol lipase (MAGL), the enzyme chiefly responsible for the metabolism of the endocannabinoid 2-arachidonoylglycerol (2-AG) in the brain. In the present study, a series of twelve analogues of 1-AG (the more stable regioisomer of 2-AG) were investigated with respect to their ability to inhibit the metabolism of 2-oleoylglycerol by cytosolic and membrane-bound MAGL. In addition, the ability of the compounds to inhibit the hydrolysis of anandamide by fatty acid amide hydrolase (FAAH) was investigated. For cytosolic MAGL, compounds with 20 carbon atoms in the acyl chain and 2-5 unsaturated bonds inhibited the hydrolysis of 2-oleoylglycerol with similar potencies (IC{sub 50} values in the range 5.1-8.2 {mu}M), whereas the two compounds with a single unsaturated bond were less potent (IC{sub 50} values 19 and 21 {mu}M). The fully saturated analogue 1-monoarachidin did not inhibit the enzyme, whereas the lower side chain analogues 1-monopalmitin and 1-monomyristin inhibited the enzyme with IC{sub 50} values of 12 and 32 {mu}M, respectively. The 22-carbon chain analogue of 1-AG was also potent (IC{sub 50} value 4.5 {mu}M). Introduction of an {alpha}-methyl group for the C20:4, C20:3, and C22:4 compounds did not affect potency in a consistent manner. For the FAAH and the membrane-bound MAGL, there was no obvious relationship between the degree of unsaturation of the acyl side chain and the ability to inhibit the enzymes. It is concluded that increasing the number of unsaturated bonds on the acyl side chain of 1-AG from 1 to 5 has little effect on the affinity of acylglycerols for cytosolic MAGL.

  17. Full Fatty Acid Amide Hydrolase Inhibition Combined with Partial Monoacylglycerol Lipase Inhibition: Augmented and Sustained Antinociceptive Effects with Reduced Cannabimimetic Side Effects in Mice

    PubMed Central

    Ghosh, Sudeshna; Kinsey, Steven G.; Liu, Qing-song; Hruba, Lenka; McMahon, Lance R.; Grim, Travis W.; Merritt, Christina R.; Wise, Laura E.; Abdullah, Rehab A.; Selley, Dana E.; Sim-Selley, Laura J.; Cravatt, Benjamin F.

    2015-01-01

    Inhibition of fatty acid amide hydrolase (FAAH) or monoacylglycerol lipase (MAGL), the primary hydrolytic enzymes for the respective endocannabinoids N-arachidonoylethanolamine (AEA) and 2-arachidonylglycerol (2-AG), produces antinociception but with minimal cannabimimetic side effects. Although selective inhibitors of either enzyme often show partial efficacy in various nociceptive models, their combined blockade elicits augmented antinociceptive effects, but side effects emerge. Moreover, complete and prolonged MAGL blockade leads to cannabinoid receptor type 1 (CB1) receptor functional tolerance, which represents another challenge in this potential therapeutic strategy. Therefore, the present study tested whether full FAAH inhibition combined with partial MAGL inhibition would produce sustained antinociceptive effects with minimal cannabimimetic side effects. Accordingly, we tested a high dose of the FAAH inhibitor PF-3845 (N-​3-​pyridinyl-​4-​[[3-​[[5-​(trifluoromethyl)-​2-​pyridinyl]oxy]phenyl]methyl]-​1-​piperidinecarboxamide; 10 mg/kg) given in combination with a low dose of the MAGL inhibitor JZL184 [4-nitrophenyl 4-(dibenzo[d][1,3]dioxol-5-yl(hydroxy)methyl)piperidine-1-carboxylate] (4 mg/kg) in mouse models of inflammatory and neuropathic pain. This combination of inhibitors elicited profound increases in brain AEA levels (>10-fold) but only 2- to 3-fold increases in brain 2-AG levels. This combination produced significantly greater antinociceptive effects than single enzyme inhibition and did not elicit common cannabimimetic effects (e.g., catalepsy, hypomotility, hypothermia, and substitution for Δ9-tetrahydrocannabinol in the drug-discrimination assay), although these side effects emerged with high-dose JZL184 (i.e., 100 mg/kg). Finally, repeated administration of this combination did not lead to tolerance to its antiallodynic actions in the carrageenan assay or CB1 receptor functional tolerance. Thus, full FAAH inhibition combined

  18. Familial lipoprotein lipase deficiency

    MedlinePlus

    ... and white-colored blood vessels in the retinas Pancreatitis that keeps returning Yellowing of the eyes and ... discuss your diet needs with a registered dietitian. Pancreatitis that is related to lipoprotein lipase deficiency responds ...

  19. Mechanism of acetaldehyde-induced deactivation of microbial lipases

    PubMed Central

    2011-01-01

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

  20. Lipase-catalyzed ethanolysis of borage oil: a kinetic study.

    PubMed

    Torres, Carlos F; Hill, Charles G; Otero, Cristina

    2004-01-01

    Ethanolysis of borage oil catalyzed by two commercial lipases (from Pseudomonas cepacia and Candida antarctica) was studied using two different methodologies. Multiresponse models derived from a generalized Michaelis-Menten mechanism were utilized to describe the rates of formation of ethyl esters of the primary fatty acids present in the precursor oil. The relative rate constants determined for each of the fatty acid residues indicated that both lipases discriminate against release of gamma-linolenic acid residues under the reaction conditions studied. However, both lipases also released some of the residues located at the sn-2 position, indicating that for the experimental conditions studied, both lipases are nonspecific. Moreover, inactivation of Novozym 435 was rapid. Because the half-life of this enzyme (ca. 2.2 h) is comparable to the half-life of the reaction, the intrinsic reaction rate and enzyme deactivation must both be considered in modeling the kinetics. PMID:15176879

  1. 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

  2. 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

  3. Links for Academic Learning (LAL): A Conceptual Model for Investigating Alignment of Alternate Assessments Based on Alternate Achievement Standards

    ERIC Educational Resources Information Center

    Flowers, Claudia; Wakeman, Shawnee; Browder, Diane M.; Karvonen, Meagan

    2009-01-01

    This article describes an alignment procedure, called Links for Academic Learning (LAL), for examining the degree of alignment of alternate assessments based on alternate achievement standards (AA-AAS) to grade-level content standards and instruction. Although some of the alignment criteria are similar to those used in general education…

  4. 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

  5. Polyphenolic Compounds as Pancreatic Lipase Inhibitors.

    PubMed

    Buchholz, Tina; Melzig, Matthias F

    2015-07-01

    Obesity and its associated diseases such as diabetes mellitus and coronary heart diseases are a major challenge for our society. An important target for the treatment of obesity includes the development of inhibitors of nutrient digestion and absorption. Inhibition of pancreatic lipase and the associated reduction of lipid absorption is an attractive approach for the discovery of potent agents. Currently, the only clinically approved pharmacologic agent as pancreatic lipase inhibitor is Orlistat. However, its usage is compromised by unpleasant gastrointestinal adverse reactions (oily stools, oily spotting, flatulence). The use of botanical materials as a potential source of new drugs is of increasing importance and application. Natural products that are interesting for obesity treatment are generally considered to have less toxic and side effects than totally synthetic drugs. One of the most important sources of potential pancreatic lipase inhibitors represents the class of polyphenols. This article summarizes most studied subclasses of polyphenols including flavonoids, hydroxycinnamic acids, hydroxybenzoic acids and lignans with pancreatic lipase inhibitory effects. A structural comparison of potent inhibitors shows an increased inhibitory effect depending on number and position of phenolic hydroxyl groups, degree of polymerization and elimination of glycosylation during digestion. PMID:26132857

  6. 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

  7. 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

  8. 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

  9. The use of immobilised digestive lipase from Chinook salmon (Oncorhynchus tshawytscha) to generate flavour compounds in milk.

    PubMed

    Kurtovic, Ivan; Marshall, Susan N; Cleaver, Helen L; Miller, Matthew R

    2016-05-15

    The aim of this research was to determine the potential of immobilised digestive lipase from Chinook salmon (Oncorhynchus tshawytscha) to generate flavour compounds in milk. The lipase was immobilised on hydrophobic resin (Toyopearl® Butyl) and used to hydrolyse milk lipids in a batch reactor. The lipase was stable when immobilised and there was no significant resin fouling or enzyme inhibition between cycles. Eight cycles were achieved before the hydrolysis rate dropped significantly because of physical losses of the immobilised lipase. The immobilised lipase showed the highest specificity towards short-chain fatty acids butanoic and hexanoic acids, the main dairy product flavour and odour compounds. Based on the performance of the reactor, and the ability of the lipase to alter free fatty acid composition and sensory characteristics of milk, the immobilised salmon lipase has potential applications in developing dairy products with unique flavours. PMID:26775978

  10. 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

  11. 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

  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. 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

  14. 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

  15. 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

  16. 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

  17. 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

  18. Performances of the Alpha-X RF gun on the PHIL accelerator at LAL

    NASA Astrophysics Data System (ADS)

    Vinatier, T.; Bruni, C.; Roux, R.; Brossard, J.; Chancé, S.; Cayla, J. N.; Chaumat, V.; Xu, G.; Monard, H.

    2015-10-01

    The Alpha-X RF-gun was designed to produce an ultra-short (<100 fs rms), 100 pC and 6.3 MeV electron beam with a normalized rms transverse emittance of 1π mm mrad for a gun peak accelerating field of 100 MV/m. Such beams will be required by the Alpha-X project, which aims to study a laser-driven plasma accelerator with a short wavelength accelerating medium. It has been demonstrated on PHIL (Photo-Injector at LAL) that the coaxial RF coupling, chosen to preserve the gun field cylindrical symmetry, is perfectly understood and allows reaching the required peak accelerating field of 100 MV/m giving beam energy of 6.3 MeV. Moreover, a quite low beam rms relative energy spread of 0.15% at 3.8 MeV has been measured, completely agreeing with simulations. Dark current, quantum efficiencies and dephasing curves measurements have also been performed. They all show high values of the field enhancement factor β, which can be explained by the preparation of the photocathodes. Finally, measurements on the transverse phase-space have been carried out, with some limitations given by the difficult modelization of one of the PHIL solenoid magnets and by the enlargement of the beam transverse dimensions due to the use of YAG screens. These measurements give a normalized rms transverse emittance around 5π mm mrad, which does not fulfill the requirement for the Alpha-X project.

  19. 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

  20. 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...

  1. 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...

  2. 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

  3. 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

  4. 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

  5. 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

  6. 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

  7. 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

  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. 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

  10. 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

  11. 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

  12. 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.

  13. 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

  14. Enthalpic and entropic contributions in the transesterification of sucrose: computational study of lipases and subtilisin.

    PubMed

    Fuentes, Gloria; Ballesteros, Antonio; Verma, Chandra S

    2007-10-01

    Transesterification of sucrose with fatty acids catalyzed by subtilisin Carlsberg occurs with regioselectivity that is different from that in lipases. Thermomyces lanuginosus lipase (TlL) and Candida antarctica lipase B (CALB) catalyze synthesis at positions 6 and 6', with differing abilities, while subtilisin catalysis leads to the 1'-acylated sucrose. The catalytic machinery in lipases is approximately mirrored in subtilisins but different pocket morphologies including size, shape, and rearrangement of the catalytic elements underlies the differing regioselectivities. The thermodynamic consequences of these differences on the above reactions have been explored systematically using computational methods, determining the free energies of interaction of the putative transition-state adducts. Analysis of the conformers with the lowest transition state energies (protein-ligand interactions and vibrational entropy contributions) indicates that enthalpic factors control specificities in lipases while entropic factors are more important in subtilisin. PMID:17718593

  15. 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

  16. 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

  17. Lipase-catalyzed polyester synthesis – A green polymer chemistry

    PubMed Central

    Kobayashi, Shiro

    2010-01-01

    This article is a short comprehensive review describing in vitro polyester synthesis catalyzed by a hydrolysis enzyme of lipase, most of which has been developed for these two decades. Polyesters are prepared by repeated ester bond-formation reactions; they include two major modes, ring-opening polymerization (ROP) of cyclic monomers such as cyclic esters (lactones) and condensation polymerization via the reaction between a carboxylic acid or its ester group and an alcohol group. Polyester synthesis is, therefore, a reaction in reverse way of in vivo lipase catalysis of ester bond-cleavage with hydrolysis. The lipase-catalyzed polymerizations show very high chemo-, regio-, and enantio-selectivities and involve various advantageous characteristics. Lipase is robust and compatible with other chemical catalysts, which allows novel chemo-enzymatic processes. New syntheses of a variety of functional polyesters and a plausible reaction mechanism of lipase catalysis are mentioned. The polymerization characteristics are of green nature currently demanded for sustainable society, and hence, desirable for conducting ‘green polymer chemistry’. PMID:20431260

  18. Bioscouring of cotton using lipase from marine bacteria Bacillus sonorensis.

    PubMed

    Nerurkar, Madhura; Joshi, Manasi; Adivarekar, Ravindra

    2015-01-01

    Bioscouring refers to the enzymatic removal of impurities from cotton fabric, which imparts it with improved hydrophilicity for further wet processes. In the present study, the efficacy of lipase from newly isolated marine bacteria Bacillus sonorensis isolated from marine clams Paphia malabarica collected from Kalbadevi estuary, Mumbai, India, has been evaluated for scouring of cotton fabric and compared with conventional alkaline scouring of cotton. As a scouring agent for cotton fabrics, the lipase from B. sonorensis was capable of removing substantial amount of wax from the cotton surface and hydrolyzing it into fatty acids. Bioscouring carried out with lipase at a concentration of 8 % on the weight of the fabric (owf) at pH 9, temperature 60 °C for 120 min showed maximum weight loss and hydrophilicity. The Fourier transform infrared spectroscopy (FTIR) and scanning electron microscopy (SEM) studies revealed that the lipase-scoured fabric showed smooth surface indicating no damage to the fabric whereas the surface of the alkaline-scoured fabric appeared rough causing damage to the fabric. Evaluation of fabric properties such as wettability, whiteness, dyeing behaviour, tensile strength and bending rigidity revealed that the bioscouring using lipase from B. sonorensis is as effective as conventional alkaline treatment. PMID:25256798

  19. 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

  20. Production of 7,10-dihydroxy-8(E)-octadecenoic Acid from Triolein via Lipase Induction by Pseudomonas aeruginosa PR3

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Hydroxy fatty acids (HFA) have gained important attention due to special properties such as higher viscosity and reactivity compared with other non-hydroxy fatty acids. Pseudomonas aeruginosa PR3 has been previously reported to produce mono-, di-, and tri-hydroxy fatty acids from different unsatura...

  1. Mono-estolide synthesis from trans-8-hydroxy fatty acids by lipases in solvent-free media and their physical properties

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Pseudomonas aeruginosa 42A2 is known to produce two hydroxy-fatty acids, 10(S)-hydroxy-8(E)-octadecenoic and 7,10(S,S)-dihydroxy-8(E)-octadecenoic acids, when cultivated in a mineral medium using oleic acid as a single carbon source. These compounds were purified, 91 and 96 % respectively, to produc...

  2. Application of lipases to regiospecific interesterification of exotic oils from an Amazonian area.

    PubMed

    Speranza, Paula; Ribeiro, Ana Paula Badan; Macedo, Gabriela Alves

    2016-01-20

    Enzymatic interesterification may favor the development of lipid fractions from Amazonian oils with greater application potential. In this study, the Amazonian buriti oil and murumuru fat were subjected to enzymatic interesterification using two lipases in three different enzyme systems: one with a commercial lipase from Thermomyces lanuginosa, a second with the lipase produced by Rhizopus sp., and a third with a mixture of both lipases. The three enzyme systems were able to catalyze the reaction, but the enzymes showed different specificities. The commercial lipase was specific for unsaturated fatty acids, whereas the Rhizopus sp. lipase was specific for both unsaturated fatty acids and the positions sn -1 and sn -3 of the fatty acid on the triacylglycerol. The mixture of both lipases showed no synergistic effect: the results were intermediate between the two enzymes applied alone. Interesterification reduced the levels of trisaturated and triunsaturated triacylglycerols and increased the levels of diunsaturated-monosaturated and monounsaturated-disaturated triacylglycerols. The thermal melting behavior indicated the formation of a single endothermic region with more homogeneous triacylglycerols. The content of the bioactive β-carotene was preserved after the interesterification reaction with all three-enzyme systems. The interesterified lipids obtained, because of the characteristics of the oils, may be applied to the formulation of cosmetics and pharmaceuticals. PMID:26657709

  3. Safety evaluation of lipase produced from Rhizopus oryzae: summary of toxicological data.

    PubMed

    Flood, Michael T; Kondo, Mitsuru

    2003-04-01

    The toxicity of Lipase D, an enzyme preparation, was evaluated in a series of studies. Lipase D selectively hydrolyzes triglycerides of fatty acids. It also catalyzes the interesterification of edible fats and oils. In a 13-week gavage study, Sprague-Dawley rats received Lipase D at levels of 0, 500, 1000, or 2000 mg/kg body wt./day. A dose dependent decrease in urinary pH was observed, but there were no effects on electrolyte balance, kidney weight, or histology of the kidney. The no-observed-adverse-effect level in rats was 1000 mg/kg body wt./day. In common with other enzyme preparations, Lipase D was not genotoxic. Lipase D was tested in the Ames assay, the mouse lymphoma forward mutation assay, and the chromosome aberration assay. Finally, the particular strain of Rhizopus oryzae used to prepare Lipase D was shown to have low to moderate pathogenicity when injected into the tail vein of mice at doses up to 1.3 x 10(6) colony-forming units (CFU) per animal. No effects were observed when mice received up to 2.2 x 10(5) CFU by gavage or in their diets daily for 28 days. The results indicate that this particular strain can be handled using ordinary safety practices current in the fermentation industry. These studies support a conclusion that Lipase D is safe when used as described in the processing of dietary fatty acids and glycerides of fatty acids. PMID:12726758

  4. [Rapid and high throughput measurement of lipase thermo-stability through ANS fluorescence signal assay].

    PubMed

    Feng, Weizong; Lin, Junhan; Cai, Shaoli; Zou, Youtu; Chen, Guoren; Huang, Ping; Lin, Yajing; Wang, Bingbing; Lin, Lin

    2011-04-01

    We have developed a rapid and high throughput lipase-ANS (8-Anilino-l-naphthalenesulfonic acid) assay to evaluate the thermo-stability of lipases based on the ANS fluorescence signal's increasing and shifting when this small fluorescence probes binds to lipase. The testing lipase samples were incubated at a temperature range of 25 degrees C to 65 degrees C for 30 min before mixed with ANS solution (0.20 mg/mL lipase and 0.05 mmol/L ANS in the buffer of 20 mmol/L Tris-HCl, 100 mmol/L NaCl, pH 7.2) in a cuvette or microplate. Fluorescence signals of the samples were measured at EX 378 nm, EM 465 nm with a fluorescence photometer or a plate reader, and Tm was calculated with the software of GraphPad Prism5.0. The Tm values of several mutants of Penicillium expansum lipase (PEL) were measured with this ANS assay and conventional method simultaneously and the results show that Tm values are comparative and consistent between these methods, suggesting that the lipase-ANS assay is a reliable, rapid and high throughput method for lipase thermo-stability measurement. PMID:21847993

  5. 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

  6. 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

  7. Lipases in Medicine: An Overview.

    PubMed

    Loli, Heni; Narwal, Sunil Kumar; Saun, Nitin Kumar; Gupta, Reena

    2015-01-01

    Lipases are part of the family of hydrolases that act on carboxylic ester bonds. They are involved in catalyzing the hydrolysis of triglycerides (TG) into chylomicrons and very low density lipoprotein (VLDL) particles. Uses of lipases are evolving rapidly and currently they are reported to show high potential in medicine. Intensive study and investigations have led researchers to explore lipases for their use in substitution therapy, where in enzyme deficiency during diseased conditions is compensated by their external administration. In our body, they are used to break down fats present in food so that they can be absorbed in the intestine and deficiency of lipases leads to malabsorption of fats and fat-soluble vitamins. Lipases help a person who has cystic fibrosis, Alzheimer's disease, atherosclerosis and act as a candidate target for cancer prevention and therapy. They act as diagnostic tool and their presence or increasing levels can indicate certain infection or disease. Obesity causes metabolic disease and is a serious health problem around the world. Thus inhibiting digestive lipase to reduce fat absorption has become the main pharmacological approach to the treatment of obesity in recent years. PMID:26156413

  8. 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

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  10. 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

  11. Assessing endotoxins in equine-derived snake antivenoms: Comparison of the USP pyrogen test and the Limulus Amoebocyte Lysate assay (LAL).

    PubMed

    Solano, Gabriela; Gómez, Aarón; León, Guillermo

    2015-10-01

    Snake antivenoms are parenterally administered; therefore, endotoxin content must be strictly controlled. Following international indications to calculate endotoxin limits, it was determined that antivenom doses between 20 mL and 120 mL should not exceed 17.5 Endotoxin Units per milliliter (EU/mL) and 2.9 EU/mL, respectively. The rabbit pyrogen test (RPT) has been used to evaluate endotoxin contamination in antivenoms, but some laboratories have recently implemented the LAL assay. We compared the capability of both tests to evaluate endotoxin contamination in antivenoms, and we found that both methods can detect all endotoxin concentrations in the range of the antivenom specifications. The acceptance criteria of RPT and LAL must be harmonized by calculating the endotoxin limit as the quotient of the threshold pyrogenic dose and the therapeutic dose and the dose administered to rabbits as the quotient of the threshold pyrogenic dose and the endotoxin limit. Since endotoxins from Gram-negative bacteria exert different pyrogenicity, if contamination occurred, antivenom batches that induce pyrogenic reactions may be found in spite of passing LAL specifications. Although LAL assay can be used to assess endotoxin content throughout the antivenom manufacturing process, we recommend that the release of final products be based on the results of both methods. PMID:26325294

  12. 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

  13. Comparative studies of vertebrate lipoprotein lipase: a key enzyme of very low density lipoprotein metabolism.

    PubMed

    Holmes, Roger S; Vandeberg, John L; Cox, Laura A

    2011-06-01

    Lipoprotein lipase (LIPL or LPL; E.C.3.1.1.34) serves a dual function as a triglyceride lipase of circulating chylomicrons and very-low-density lipoproteins (VLDL) and facilitates receptor-mediated lipoprotein uptake into heart, muscle and adipose tissue. Comparative LPL amino acid sequences and protein structures and LPL gene locations were examined using data from several vertebrate genome projects. Mammalian LPL genes usually contained 9 coding exons on the positive strand. Vertebrate LPL sequences shared 58-99% identity as compared with 33-49% sequence identities with other vascular triglyceride lipases, hepatic lipase (HL) and endothelial lipase (EL). Two human LPL N-glycosylation sites were conserved among seven predicted sites for the vertebrate LPL sequences examined. Sequence alignments, key amino acid residues and conserved predicted secondary and tertiary structures were also studied. A CpG island was identified within the 5'-untranslated region of the human LPL gene which may contribute to the higher than average (×4.5 times) level of expression reported. Phylogenetic analyses examined the relationships and potential evolutionary origins of vertebrate lipase genes, LPL, LIPG (encoding EL) and LIPC (encoding HL) which suggested that these have been derived from gene duplication events of an ancestral neutral lipase gene, prior to the appearance of fish during vertebrate evolution. Comparative divergence rates for these vertebrate sequences indicated that LPL is evolving more slowly (2-3 times) than for LIPC and LIPG genes and proteins. PMID:21561822

  14. 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

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

    NASA Astrophysics Data System (ADS)

    Nurhasanah, Nurbaiti, Santi; Madayanti, Fida; Akhmaloka

    2015-09-01

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

  16. Overview of fungal lipase: a review.

    PubMed

    Singh, Abhishek Kumar; Mukhopadhyay, Mausumi

    2012-01-01

    Lipases (triacylglycerolacyl hydrolases, EC3.1.1.3) are class of enzymes which catalyze the hydrolysis of long-chain triglycerides. In this review paper, an overview regarding the fungal lipase production, purification, and application is discussed. The review describes various industrial applications of lipase in pulp and paper, food, detergent, and textile industries. Some important lipase-producing fungal genera include Aspergillus, Penicillium, Rhizopus, Candida, etc. Current fermentation process techniques such as batch, fed-batch, and continuous mode of lipase production in submerged and solid-state fermentations are discussed in details. The purification of lipase by hydrophobic interaction chromatography is also discussed. The development of mathematical models applied to lipase production is discussed with special emphasis on lipase engineering. PMID:22072143

  17. 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

  18. 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

  19. 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

  20. Origin, Microbiology, Nutrition, and Pharmacology of D-Amino Acids

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Exposure of food proteins to certain processing conditions induces two major chemical changes: racemization of all L-amino acids (LAA) to D-amino acids (DAA) and concurrent formation of crosslinked amino acids such as lysinoalanine (LAL). The diet contains both processing-induced and naturally-form...

  1. Lipase-catalyzed highly enantioselective kinetic resolution of boron-containing chiral alcohols.

    PubMed

    Andrade, Leandro H; Barcellos, Thiago

    2009-07-16

    The first application of enzymes as catalysts to obtain optically pure boron compounds is described. The kinetic resolution of boron-containing chiral alcohols via enantioselective transesterification catalyzed by lipases was studied. Aromatic, allylic, and aliphatic secondary alcohols containing a boronate ester or boronic acid group were resolved by lipase from Candida antartica (CALB), and excellent E values (E > 200) and high enantiomeric excesses (up to >99%) of both remaining substrates and acetylated product were obtained. PMID:19552446

  2. The Saccharomyces cerevisiae YLL012/YEH1, YLR020/YEH2, and TGL1 genes encode a novel family of membrane-anchored lipases that are required for steryl ester hydrolysis.

    PubMed

    Köffel, René; Tiwari, Rashi; Falquet, Laurent; Schneiter, Roger

    2005-03-01

    Sterol homeostasis in eukaryotic cells relies on the reciprocal interconversion of free sterols and steryl esters. The formation of steryl esters is well characterized, but the mechanisms that control steryl ester mobilization upon cellular demand are less well understood. We have identified a family of three lipases of Saccharomyces cerevisiae that are required for efficient steryl ester mobilization. These lipases, encoded by YLL012/YEH1, YLR020/YEH2, and TGL1, are paralogues of the mammalian acid lipase family, which is composed of the lysosomal acid lipase, the gastric lipase, and four novel as yet uncharacterized human open reading frames. Lipase triple-mutant yeast cells are completely blocked in steryl ester hydrolysis but do not affect the mobilization of triacylglycerols, indicating that the three lipases are required for steryl ester mobilization in vivo. Lipase single mutants mobilize steryl esters to various degrees, indicating partial functional redundancy of the three gene products. Lipase double-mutant cells in which the third lipase is expressed from the inducible GAL1 promoter have greatly reduced steady-state levels of steryl esters, indicating that overexpression of any of the three lipases is sufficient for steryl ester mobilization in vivo. The three yeast enzymes constitute a novel class of membrane-anchored lipases that differ in topology and subcellular localization. PMID:15713625

  3. Screening of lipases for the synthesis of xylitol monoesters by chemoenzymatic esterification and the potential of microwave and ultrasound irradiations to enhance the reaction rate.

    PubMed

    Rufino, Alessandra R; Biaggio, Francisco C; Santos, Julio C; de Castro, Heizir F

    2010-07-01

    Lipases from different sources, Pseudomonas fluorescens (AK lipase), Burkholderia cepacia (PS lipase), Penicillium camembertii (lipase G) and Porcine pancreas lipase (PPL), previously immobilized on epoxy SiO(2)-PVA, were screened for the synthesis of xylitol monoesters by esterification of the protected xylitol using oleic acid as acyl donor group. Among all immobilized derivatives, the highest esterification yield was achieved by P. camembertii lipase, showing to be attractive alternative to bulk chemical routes to satisfy increasing commercial demands. Further experiments were performed to determine the influence of fatty acids chain size on the reaction yield and the feasibility of using non-conventional heating systems (microwave and ultrasound irradiations) to enhance the reaction rate. PMID:20420851

  4. 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

  5. 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

  6. 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

  7. 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

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

    PubMed

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

    2013-08-01

    Macrophages express lipoprotein lipase (LPL) and endothelial lipase (EL) within atherosclerotic plaques; however, little is known about how lipoprotein hydrolysis products generated by these lipases might affect macrophage cell signalling pathways. We hypothesized that hydrolysis products affect macrophage cell signalling pathways associated with atherosclerosis. To test our hypothesis, we incubated differentiated THP-1 macrophages with products from total lipoprotein hydrolysis by recombinant LPL or EL. Using antibody arrays, we found that the phosphorylation of six receptor tyrosine kinases and three signalling nodes--most associated with atherosclerotic processes--was increased by LPL derived hydrolysis products. EL derived hydrolysis products only increased the phosphorylation of tropomyosin-related kinase A, which is also implicated in playing a role in atherosclerosis. Using electrospray ionization-mass spectrometry, we identified the species of triacylglycerols and phosphatidylcholines that were hydrolyzed by LPL and EL, and we identified the fatty acids liberated by gas chromatography-mass spectrometry. To determine if the total liberated fatty acids influenced signalling pathways, we incubated differentiated THP-1 macrophages with a mixture of the fatty acids that matched the concentrations of liberated fatty acids from total lipoproteins by LPL, and we subjected cell lysates to antibody array analyses. The analyses showed that only the phosphorylation of Akt was significantly increased in response to fatty acid treatment. Overall, our study shows that macrophages display potentially pro-atherogenic signalling responses following acute treatments with LPL and EL lipoprotein hydrolysis products. PMID:23794138

  9. 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

  10. 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

  11. 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

  12. Adipocyte lipases and defect of lipolysis in human obesity.

    PubMed

    Langin, Dominique; Dicker, Andrea; Tavernier, Geneviève; Hoffstedt, Johan; Mairal, Aline; Rydén, Mikael; Arner, Erik; Sicard, Audrey; Jenkins, Christopher M; Viguerie, Nathalie; van Harmelen, Vanessa; Gross, Richard W; Holm, Cecilia; Arner, Peter

    2005-11-01

    The mobilization of fat stored in adipose tissue is mediated by hormone-sensitive lipase (HSL) and the recently characterized adipose triglyceride lipase (ATGL), yet their relative importance in lipolysis is unknown. We show that a novel potent inhibitor of HSL does not inhibit other lipases. The compound counteracted catecholamine-stimulated lipolysis in mouse adipocytes and had no effect on residual triglyceride hydrolysis and lipolysis in HSL-null mice. In human adipocytes, catecholamine- and natriuretic peptide-induced lipolysis were completely blunted by the HSL inhibitor. When fat cells were not stimulated, glycerol but not fatty acid release was inhibited. HSL and ATGL mRNA levels increased concomitantly during adipocyte differentiation. Abundance of the two transcripts in human adipose tissue was highly correlated in habitual dietary conditions and during a hypocaloric diet, suggesting common regulatory mechanisms for the two genes. Comparison of obese and nonobese subjects showed that obesity was associated with a decrease in catecholamine-induced lipolysis and HSL expression in mature fat cells and in differentiated preadipocytes. In conclusion, HSL is the major lipase for catecholamine- and natriuretic peptide-stimulated lipolysis, whereas ATGL mediates the hydrolysis of triglycerides during basal lipolysis. Decreased catecholamine-induced lipolysis and low HSL expression constitute a possibly primary defect in obesity. PMID:16249444

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

    PubMed

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

    2013-12-13

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

  14. Lipase-mediated selective oxidation of furfural and 5-hydroxymethylfurfural.

    PubMed

    Krystof, Monika; Pérez-Sánchez, María; Domínguez de María, Pablo

    2013-05-01

    Furfural and 5-hydroxymethylfurfural (HMF) are important biomass-derived platform chemicals that can be obtained from the dehydration of lignocellulosic sugars. A possible route for the derivatization of furanics is their oxidation to afford a broad range of chemicals with promising applications (e.g., diacids, hydroxyl acids, aldehyde acids, monomers for novel polymers). Herein we explore the organic peracid-assisted oxidation of furanics under mild reaction conditions. Using lipases as biocatalysts, alkyl esters as acyl donors, and aqueous solutions of hydrogen peroxide (30 % v/v) added stepwise, peracids are formed in situ, which subsequently oxidize the aldehyde groups to afford carboxylic acids with high yields and excellent selectivities. Furthermore, the use of an immobilized silica-based 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) affords the selective oxidation of the hydroxymethyl group of HMF to afford 2,5-diformylfuran. That product can be subsequently oxidized using again lipases for the in situ peracid formation to yield 2,5-furandicarboxylic acid, which is considered to be a key building block for biorefineries. These lipase-mediated reactions proceeded efficiently even with high substrate loadings under still non-optimized conditions. Overall, a proof-of-concept for the oxidation of furanics (based on in situ formed organic peracids as oxidants) is provided. PMID:23576295

  15. 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

  16. 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

  17. The Role of Mannosylated Enzyme and the Mannose Receptor in Enzyme Replacement Therapy

    PubMed Central

    Du, Hong; Levine, Mark; Ganesa, Chandrashekar; Witte, David P.; Cole, Edward S.; Grabowski, Gregory A.

    2005-01-01

    Lysosomal acid lipase (LAL) is the critical enzyme for the hydrolysis of triglycerides (TGs) and cholesteryl esters (CEs) in lysosomes. LAL defects cause Wolman disease (WD) and CE storage disease (CESD). An LAL null (lal−/−) mouse model closely mimics human WD/CESD, with hepatocellular, Kupffer cell and other macrophage, and adrenal cortical storage of CEs and TGs. The effect on the cellular targeting of high-mannose and complex oligosaccharide–type oligosaccharide chains was tested with human LAL expressed in Pichia pastoris (phLAL) and CHO cells (chLAL), respectively. Only chLAL was internalized by cultured fibroblasts, whereas both chLAL and phLAL were taken up by macrophage mannose receptor (MMR)–positive J774E cells. After intraperitoneal injection into lal−/− mice, phLAL and chLAL distributed to macrophages and macrophage-derived cells of various organs. chLAL was also detected in hepatocytes. Ten injections of either enzyme over 30 d into 2- and 2.5-mo-old lal−/− mice produced normalization of hepatic color, decreased liver weight (50%–58%), and diminished hepatic cholesterol and TG storage. Lipid accumulations in macrophages were diminished with either enzyme. Only chLAL cleared lipids in hepatocytes. Mice double homozygous for the LAL and MMR deficiences (lal−/−;MMR−/−) showed phLAL uptake into Kupffer cells and hepatocytes, reversal of macrophage histopathology and lipid storage in all tissues, and clearance of hepatocytes. These results implicate MMR-independent and mannose 6-phosphate receptor–independent pathways in phLAL uptake and delivery to lysosomes in vivo. In addition, these studies show specific cellular targeting and physiologic effects of differentially oligosaccharide-modified human LALs mediated by MMR and that lysosomal targeting of mannose-terminated glycoproteins occurs and storage can be eliminated effectively without MMR. PMID:16380916

  18. Monoglycerides and Diglycerides Synthesis in a Solvent-Free System by Lipase-Catalyzed Glycerolysis

    NASA Astrophysics Data System (ADS)

    Fregolente, Patricia Bogalhos Lucente; Fregolente, Leonardo Vasconcelos; Pinto, Gláucia Maria F.; Batistella, Benedito César; Wolf-Maciel, Maria Regina; Filho, Rubens Maciel

    Five lipases were screened (Thermomyces lanuginosus free and immobilized forms, Candida antarctica B, Candida rugosa, Aspergillus niger, and Rhizomucor miehei) to study their ability to produce monoglycerides (MG) and diglycerides (DG) through enzymatic glycerolysis of soybean oil. Lipase from C. antarctica was further studied to verify the enzyme load (wt% of oil mass), the molar ratio glycerol/oil, and the water content (wt% of glycerol) on the glycerolysis reaction. The best DG and MG productions were in the range 45-48% and 28-30% (w/w, based on the total oil), respectively. Using immobilized lipases, the amount of free fatty acids (FFA) produced was about 5%. However, the amount of FFA produced when using free lipases, with 3.5% extra water in the system, is equivalent to the MG yield, about 23%. The extra water content provides a competition between hydrolysis and glycerolysis reactions, increasing the FFA production.

  19. 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

  20. 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

  1. Estolides synthesis catalyzed by immobilized lipases.

    PubMed

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

    2011-01-01

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

  2. Estolides Synthesis Catalyzed by Immobilized Lipases

    PubMed Central

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

    2011-01-01

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

  3. Dieselzymes: development of a stable and methanol tolerant lipase for biodiesel production by directed evolution

    PubMed Central

    2013-01-01

    Background Biodiesels are methyl esters of fatty acids that are usually produced by base catalyzed transesterification of triacylglyerol with methanol. Some lipase enzymes are effective catalysts for biodiesel synthesis and have many potential advantages over traditional base or acid catalyzed transesterification. Natural lipases are often rapidly inactivated by the high methanol concentrations used for biodiesel synthesis, however, limiting their practical use. The lipase from Proteus mirabilis is a particularly promising catalyst for biodiesel synthesis as it produces high yields of methyl esters even in the presence of large amounts of water and expresses very well in Escherichia coli. However, since the Proteus mirabilis lipase is only moderately stable and methanol tolerant, these properties need to be improved before the enzyme can be used industrially. Results We employed directed evolution, resulting in a Proteus mirabilis lipase variant with 13 mutations, which we call Dieselzyme 4. Dieselzyme 4 has greatly improved thermal stability, with a 30-fold increase in the half-inactivation time at 50°C relative to the wild-type enzyme. The evolved enzyme also has dramatically increased methanol tolerance, showing a 50-fold longer half-inactivation time in 50% aqueous methanol. The immobilized Dieselzyme 4 enzyme retains the ability to synthesize biodiesel and has improved longevity over wild-type or the industrially used Brukholderia cepacia lipase during many cycles of biodiesel synthesis. A crystal structure of Dieselzyme 4 reveals additional hydrogen bonds and salt bridges in Dieselzyme 4 compared to the wild-type enzyme, suggesting that polar interactions may become particularly stabilizing in the reduced dielectric environment of the oil and methanol mixture used for biodiesel synthesis. Conclusions Directed evolution was used to produce a stable lipase, Dieselzyme 4, which could be immobilized and re-used for biodiesel synthesis. Dieselzyme 4 outperforms

  4. 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

  5. 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

  6. Contribution of Adipose Triglyceride Lipase and Hormone-sensitive Lipase to Lipolysis in hMADS Adipocytes*

    PubMed Central

    Bezaire, Véronic; Mairal, Aline; Ribet, Carole; Lefort, Corinne; Girousse, Amandine; Jocken, Johan; Laurencikiene, Jurga; Anesia, Rodica; Rodriguez, Anne-Marie; Ryden, Mikael; Stenson, Britta M.; Dani, Christian; Ailhaud, Gérard; Arner, Peter; Langin, Dominique

    2009-01-01

    Lipolysis is the catabolic pathway by which triglycerides are hydrolyzed into fatty acids. Adipose triglyceride lipase (ATGL) and hormone-sensitive lipase (HSL) have the capacity to hydrolyze in vitro the first ester bond of triglycerides, but their respective contributions to whole cell lipolysis in human adipocytes is unclear. Here, we have investigated the roles of HSL, ATGL, and its coactivator CGI-58 in basal and forskolin-stimulated lipolysis in a human white adipocyte model, the hMADS cells. The hMADS adipocytes express the various components of fatty acid metabolism and show lipolytic capacity similar to primary cultured adipocytes. We show that lipolysis and fatty acid esterification are tightly coupled except in conditions of stimulated lipolysis. Immunocytochemistry experiments revealed that acute forskolin treatment promotes HSL translocation from the cytosol to small lipid droplets and redistribution of ATGL from the cytosol and large lipid droplets to small lipid droplets, resulting in enriched colocalization of the two lipases. HSL or ATGL overexpression resulted in increased triglyceride-specific hydrolase capacity, but only ATGL overexpression increased whole cell lipolysis. HSL silencing had no effect on basal lipolysis and only partially reduced forskolin-stimulated lipolysis. Conversely, silencing of ATGL or CGI-58 significantly reduced basal lipolysis and essentially abolished forskolin-stimulated lipolysis. Altogether, these results suggest that ATGL/CGI-58 acts independently of HSL and precedes its action in the sequential hydrolysis of triglycerides in human hMADS adipocytes. PMID:19433586

  7. Contribution of adipose triglyceride lipase and hormone-sensitive lipase to lipolysis in hMADS adipocytes.

    PubMed

    Bezaire, Véronic; Mairal, Aline; Ribet, Carole; Lefort, Corinne; Girousse, Amandine; Jocken, Johan; Laurencikiene, Jurga; Anesia, Rodica; Rodriguez, Anne-Marie; Ryden, Mikael; Stenson, Britta M; Dani, Christian; Ailhaud, Gérard; Arner, Peter; Langin, Dominique

    2009-07-01

    Lipolysis is the catabolic pathway by which triglycerides are hydrolyzed into fatty acids. Adipose triglyceride lipase (ATGL) and hormone-sensitive lipase (HSL) have the capacity to hydrolyze in vitro the first ester bond of triglycerides, but their respective contributions to whole cell lipolysis in human adipocytes is unclear. Here, we have investigated the roles of HSL, ATGL, and its coactivator CGI-58 in basal and forskolin-stimulated lipolysis in a human white adipocyte model, the hMADS cells. The hMADS adipocytes express the various components of fatty acid metabolism and show lipolytic capacity similar to primary cultured adipocytes. We show that lipolysis and fatty acid esterification are tightly coupled except in conditions of stimulated lipolysis. Immunocytochemistry experiments revealed that acute forskolin treatment promotes HSL translocation from the cytosol to small lipid droplets and redistribution of ATGL from the cytosol and large lipid droplets to small lipid droplets, resulting in enriched colocalization of the two lipases. HSL or ATGL overexpression resulted in increased triglyceride-specific hydrolase capacity, but only ATGL overexpression increased whole cell lipolysis. HSL silencing had no effect on basal lipolysis and only partially reduced forskolin-stimulated lipolysis. Conversely, silencing of ATGL or CGI-58 significantly reduced basal lipolysis and essentially abolished forskolin-stimulated lipolysis. Altogether, these results suggest that ATGL/CGI-58 acts independently of HSL and precedes its action in the sequential hydrolysis of triglycerides in human hMADS adipocytes. PMID:19433586

  8. 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

  9. The realm of microbial lipases in biotechnology.

    PubMed

    Pandey, A; Benjamin, S; Soccol, C R; Nigam, P; Krieger, N; Soccol, V T

    1999-04-01

    In this review, a comprehensive and illustrious survey is made of the applied aspects of microbial lipases in modern biotechnological practices. Lipases are the most versatile biocatalyst and bring about a range of bioconversion reactions such as hydrolysis, interesterification, esterification, alcoholysis, acidolysis and aminolysis. After a brief description of the microbial sources of lipases, the pivotal role of lipases in the processes and products of the food and flavourings industry is illustrated. An illustration is presented of biomedical applications. The panorama of lipases in the manufacture of fine chemicals is depicted with special emphasis on pharmaceuticals, pesticides, cosmetics, biosensors and detergents. Widening applications such as those in waste management and improved tanning techniques are other novel aspects of lipase utilization that are discussed in this review. PMID:10075908

  10. 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

  11. 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

  12. 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

  13. Lipase-nanoporous gold biocomposite modified electrode for reliable detection of triglycerides.

    PubMed

    Wu, Chao; Liu, Xueying; Li, Yufei; Du, Xiaoyu; Wang, Xia; Xu, Ping

    2014-03-15

    For triglycerides biosensor design, protein immobilization is necessary to create the interface between the enzyme and the electrode. In this study, a glassy carbon electrode (GCE) was modified with lipase-nanoporous gold (NPG) biocomposite (denoted as lipase/NPG/GCE). Due to highly conductive, porous, and biocompatible three-dimensional structure, NPG is suitable for enzyme immobilization. In cyclic voltammetry experiments, the lipase/NPG/GCE bioelectrode displayed surface-confined reaction in a phosphate buffer solution. Linear responses were obtained for tributyrin concentrations ranging from 50 to 250 mg dl(-1) and olive oil concentrations ranging from 10 to 200 mg dl(-1). The value of apparent Michaelis-Menten constant for tributyrin was 10.67 mg dl(-1) and the detection limit was 2.68 mg dl(-1). Further, the lipase/NPG/GCE bioelectrode had strong anti-interference ability against urea, glucose, cholesterol, and uric acid as well as a long shelf-life. For the detection of triglycerides in human serum, the values given by the lipase/NPG/GCE bioelectrode were in good agreement with those of an automatic biochemical analyzer. These properties along with a long self-life make the lipase/NPG/GCE bioelectrode an excellent choice for the construction of triglycerides biosensor. PMID:24121205

  14. Effect of lipase addition on hydrolysis and biomethane production of Chinese food waste.

    PubMed

    Meng, Ying; Li, Sang; Yuan, Hairong; Zou, Dexun; Liu, Yanping; Zhu, Baoning; Li, Xiujin

    2015-03-01

    The lipase obtained from Aspergillums niger was applied to promote the hydrolysis of food waste for achieving high biomethane production. Two strategies of lipase additions were investigated. One (Group A) was to pre-treat food waste to pre-decompose lipid to fatty acids before anaerobic digestion, and another one (Group B) was to add lipase to anaerobic digester directly to degrade lipid inside digester. The lipase was used at the concentrations of 0.1%, 0.5%, and 1.0% (w/v). The results showed that Group A achieved higher biomethane production, TS and VS reductions than those of Group B. At 0.5% lipase concentration, Group A obtained experimental biomethane yield of 500.1 mL/g VS(added), 4.97-26.50% higher than that of Group B. The maximum Bd of 73.8% was also achieved in Group A. Therefore, lipase pre-treatment strategy is recommended. This might provide one of alternatives for efficient biomethane production from food waste and mitigating environmental impact associated. PMID:25575204

  15. 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

  16. Spectroscopic investigations of the chiral interactions between lipase and the herbicide dichlorprop.

    PubMed

    Wen, Yue-Zhong; Yuan, Yu-Li; Shen, Chen-Si; Liu, Hui-Jun; Liu, Wei-Ping

    2009-03-01

    The enantioselective interaction between penicillium expansum alkaline lipase and chiral phenoxypropionic acid herbicide dichlorprop was studied by using UV differential spectrophotometry and fluorescence spectrophotometry in the presence of a pH 8, phosphate buffer solution. Chiral differences in the UV absorption and fluorescence spectra of lipase with dichlorprop were detected. (R)-Dichlorprop interacted the strongest with lipase as measured by both UV absorption and fluorescence spectrophotometry, followed by (Rac)-dichlorprop, while (S)-dichlorprop had the weakest interaction. The hydrophobic interaction seem to play the dominant role in the interactions and the (R)-enantiomer needed the minimum put of energy to drive the endothermic reaction, while the Rac-type and S-type compounds needed more for the reaction to take place. In the meantime, the catalytic hydrolysis of FDA with lipase show that (R)-DCPP could inhibit lipase the most strongly relatively at the same condition, perhaps because (R)-DCPP had a stronger combining effect and high enantiomeric selectivity on lipase than (Rac)-DCPP and (S)-DCPP. PMID:18570309

  17. 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

  18. 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

  19. 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

  20. 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.

  1. 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

  2. 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

  3. 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

  4. 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

  5. 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

  6. Identification and characterization of a new true lipase isolated through metagenomic approach

    PubMed Central

    2011-01-01

    stability and stability in water-miscible organic solvents and at high salt concentrations. These characteristics suggest that this lipase has potential to perform well in biocatalytic processes, such as for hydrolysis and synthesis reactions involving long-chain triacylglycerols and fatty acid esters. PMID:21762508

  7. Cloning, sequence, and expression of a lipase gene from Pseudomonas cepacia: lipase production in heterologous hosts requires two Pseudomonas genes.

    PubMed Central

    Jørgensen, S; Skov, K W; Diderichsen, B

    1991-01-01

    The lipA gene encoding an extracellular lipase from Pseudomonas cepacia was cloned and sequenced. Downstream from the lipase gene an open reading frame was identified, and the corresponding gene was named limA. lipA was well expressed only in the presence of limA. limA exerts its effect both in cis and in trans and therefore produces a diffusible gene product, presumably a protein of 344 amino acids. Replacement of the lipA expression signals (promoter, ribosome-binding site, and signal peptide-coding sequences) by heterologous signals from gram-positive bacteria still resulted in limA-dependent lipA expression in Escherichia coli, Bacillus subtilis, and Streptomyces lividans. Images PMID:1987151

  8. 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

  9. 21 CFR 184.1415 - Animal lipase.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... ed. (1981), p. 110, which is incorporated by reference in accordance with 5 U.S.C. 552(a) and 1 CFR... Substances Affirmed as GRAS § 184.1415 Animal lipase. (a) Animal lipase (CAS Reg. No. 9001-62-1) is an enzyme... tissue. The enzyme preparation may be produced as a tissue preparation or as an aqueous extract....

  10. 21 CFR 184.1415 - Animal lipase.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... ed. (1981), p. 110, which is incorporated by reference in accordance with 5 U.S.C. 552(a) and 1 CFR... Substances Affirmed as GRAS § 184.1415 Animal lipase. (a) Animal lipase (CAS Reg. No. 9001-62-1) is an enzyme... tissue. The enzyme preparation may be produced as a tissue preparation or as an aqueous extract....

  11. 21 CFR 184.1415 - Animal lipase.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... ed. (1981), p. 110, which is incorporated by reference in accordance with 5 U.S.C. 552(a) and 1 CFR... Substances Affirmed as GRAS § 184.1415 Animal lipase. (a) Animal lipase (CAS Reg. No. 9001-62-1) is an enzyme... tissue. The enzyme preparation may be produced as a tissue preparation or as an aqueous extract....

  12. 21 CFR 184.1415 - Animal lipase.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... ed. (1981), p. 110, which is incorporated by reference in accordance with 5 U.S.C. 552(a) and 1 CFR... Substances Affirmed as GRAS § 184.1415 Animal lipase. (a) Animal lipase (CAS Reg. No. 9001-62-1) is an enzyme... tissue. The enzyme preparation may be produced as a tissue preparation or as an aqueous extract....

  13. 21 CFR 184.1415 - Animal lipase.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    .... 110, which is incorporated by reference in accordance with 5 U.S.C. 552(a) and 1 CFR part 51. Copies....1415 Animal lipase. (a) Animal lipase (CAS Reg. No. 9001-62-1) is an enzyme preparation obtained from edible forestomach tissue of calves, kids, or lambs, or from animal pancreatic tissue. The...

  14. 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

  15. [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

  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. Numerical simulation of natural convection in a spherical container due to cooling at the center (idealization of the Lal/Kroes experiment)

    NASA Technical Reports Server (NTRS)

    Robertson, S. J.

    1981-01-01

    Natural convection in a spherical container with cooling at the center was numerically simulated using a numerical fluid dynamics computer program. The numerical analysis was simplified by assuming axisymmetric flow in the spherical container, with the symmetry axis being a sphere diagonal parallel to the gravity vector. This axisymmetric spherical geometry was intended as an idealization of the proposed Lal/Kroes crystal growing experiment to be performed on Spacelab. Results were obtained for a range of Rayleigh numbers from 25 to 10,000. The computed velocities were found to be approximately proportional to the Rayleigh number over the range of Rayleigh numbers investigated.

  18. Preliminary studies on immobilization of lipase using chicken eggshell

    NASA Astrophysics Data System (ADS)

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

    2016-06-01

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

  19. Interesterification and synthesis by Candida cylindracea lipase in microemulsions.

    PubMed

    Bello, M; Thomas, D; Legoy, M D

    1987-07-15

    Unusual reactions of interesterification and synthesis catalyzed by Candida cylindracea lipase have been tested in reverse microemulsions. The microemulsions used are made of fatty acids or triglycerides, the enzyme dissolved in a very low water quantity, Brij 35 used as surfactant and an alcoholic cosurfactant. In such a system, fats and alcohols are both the substrates of the enzyme and the microemulsion components. Incidentally, non specific Candida cylindracea lipase does not catalyze interesterification of short chain triglycerides, revealing a specificity for the chain length. Interesterification reactions tested in the presence of a given water quantity but with varying water activities show that it is the water activity and not the water quantity which is a fundamental parameter of the system. The effect of the surfactant (Brij 35) on the interesterification reaction is studied. Heptyl-oleate synthesis catalyzed by non-specific lipase is obtained in microemulsions at a 98% yield. Synthesis of glycerol esters is also tested in monophasic medium and mono and diglycerides are obtained. PMID:3606623

  20. Discrimination against diacylglycerol ethers in lipase-catalysed ethanolysis of shark liver oil.

    PubMed

    Fernández, Óscar; Vázquez, Luis; Reglero, Guillermo; Torres, Carlos F

    2013-01-15

    Lipase-catalysed ethanolysis of squalene-free shark liver oil was investigated. The mentioned shark liver oil was comprised mainly of diacylglycerol ether and triacylglycerols. In order to test discrimination against diacylglycerol ether, up to 10 different lipases were compared. The ratio of oil to ethanol and lipase stability were also evaluated. Surprisingly, lipase from Pseudomonas stutzeri was the fastest biocatalyst among all assayed, although poor discrimination against diacylglycerol ether was observed. The best results in terms of selectivity and stability were obtained with immobilised lipase from Candida antarctica (Novozym 435). Ethanolysis reaction after 24h in the presence of Novozym 435 produced total disappearance of triacylglycerol and a final reaction mixture comprised mainly of diacylglycerol ethers (10.6%), monoacylglycerol ethers (32.9%) and fatty acid ethyl esters (46.0%). In addition, when an excess of ethanol was used, diacylglycerol ethers completely disappeared after 15 h, giving a final product mainly composed of monoacylglycerol ethers (36.6%) and fatty acid ethyl esters (46.4%). PMID:23122085

  1. Biodiesel production using lipase immobilized on epoxychloropropane-modified Fe3O4 sub-microspheres.

    PubMed

    Zhang, Qian; Zheng, Zhong; Liu, Changxia; Liu, Chunqiao; Tan, Tianwei

    2016-04-01

    Superparamagnetic Fe3O4 sub-microspheres with diameters of approximately 200 nm were prepared via a solvothermal method, and then modified with epoxychloropropane. Lipase was immobilized on the modified sub-microspheres. The immobilized lipase was used in the production of biodiesel fatty acid methyl esters (FAMEs) from acidified waste cooking oil (AWCO). The effects of the reaction conditions on the biodiesel yield were investigated using a combination of response surface methodology and three-level/three-factor Box-Behnken design (BBD). The optimum synthetic conditions, which were identified using Ridge max analysis, were as follows: immobilized lipase:AWCO mass ratio 0.02:1, fatty acid:methanol molar ratio 1:1.10, hexane:AWCO ratio 1.33:1 (mL/g), and temperature 40 °C. A 97.11% yield was obtained under these conditions. The BBD and experimental data showed that the immobilized lipase could generate biodiesel over a wide temperature range, from 0 to 40 °C. Consistently high FAME yields, in excess of 80%, were obtained when the immobilized lipase was reused in six replicate trials at 10 and 20 °C. PMID:26803008

  2. 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

  3. 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

  4. 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

  5. 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

  6. 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

  7. 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

  8. Endothelial lipase: Its role in cardiovascular disease

    PubMed Central

    Paradis, Marie-Eve; Lamarche, Benoit

    2006-01-01

    Endothelial lipase (EL) has recently been identified as a new member of the triglyceride lipase gene family. EL shares a relatively high degree of homology with lipoprotein lipase and hepatic lipase, but it appears to be more specific at hydrolyzing phospholipids than lipoprotein lipase and hepatic lipase. EL is also the only identified lipase that is synthesized and expressed by endothelial cells. Data from in vitro and in vivo animal studies have suggested that EL may play a key role in modulating the metabolism of high density lipoproteins. Data are less consistent in clarifying how EL contributes to the metabolism of apolipoprotein B-containing lipoproteins. Investigations in humans are scarce. To date, increased plasma EL concentrations have been associated with a deteriorated lipoprotein-lipid profile along with elevated plasma triglyceride and apolipoprotein B concentrations, as well as with smaller low density lipoprotein particle size. Elevated proinflammatory cytokine concentrations and an increased prevalence of the metabolic syndrome have also been observed among individuals with elevated plasma EL concentrations. Taken together, data suggest that EL is one of several key regulatory enzymes of lipoprotein-lipid metabolism and that a proinflammatory state, such as the metabolic syndrome, may be implicated in the processes relating plasma EL concentrations and lipoprotein concentrations. EL should thus be considered to play an important role in the pathophysiology of cardiovascular disease. PMID:16498510

  9. 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.

  10. 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

  11. 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

  12. 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

  13. Alleviation of Proteolytic Sensitivity To Enhance Recombinant Lipase Production in Escherichia coli▿

    PubMed Central

    Narayanan, Niju; Chou, C. Perry

    2009-01-01

    Two amino acids, Leu149 and Val223, were identified as proteolytically sensitive when Pseudozyma antarctica lipase (PalB) was heterologously expressed in Escherichia coli. The functional expression was enhanced using the double mutant for cultivation. However, the recombinant protein production was still limited by PalB misfolding, which was resolved by DsbA coexpression. PMID:19542329

  14. 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...

  15. Lipase catalyzed interesterification of Amazonian patauá oil and palm stearin for preparation of specific-structured oils.

    PubMed

    Speranza, Paula; Ribeiro, Ana Paula Badan; Macedo, Gabriela Alves

    2015-12-01

    This study showed that enzymatic interesterification of Amazonian oils could be an important tool in order to produce new oils with physicochemical properties that improve the applications of these raw materials. Structured oils of Amazonian patauá oil and palm stearin using two lipases were produced in three different enzymatic systems: first, a crude lipase from the fungus Rhizopus sp (a microorganism isolated in our laboratory); second, a commercial lipase; and third, to check any synergistic effect, a mixture of both lipases (Rhizopus sp and commercial). The lipase from Rhizopus sp was specific in the incorporation of oleic acid at the sn-1,3 positions of the triacylglycerol, resulting in an oil richer in saturated fatty acid in the sn-2 position. This enzyme, produced by solid-state fermentation, even though crude, was fatty acid and positional specific and able to operate at low concentration (2.5 %, w/w). In the second enzyme system, the commercial lipase from Thermomyces lanuginosus was not specific in the tested conditions; there was no change in the distribution of saturated and unsaturated fatty acids in the three positions of the triacylglycerol profile, there was only a replacement by the type of fatty acid at the same position. In the third enzyme system, the mixture of both lipases shows no synergic effect. The structured oils retained the concentration of bioactive α- and γ- tocopherol in the three enzyme systems. Triacylglycerol classes and Thermal behavior tests indicated the formation of more homogeneous triacylglycerols, especially the mono and di-unsaturated. PMID:26604403

  16. Cocoa pod husk: A new source of CLEA-lipase for preparation of low-cost biodiesel: An optimized process.

    PubMed

    Khanahmadi, Soofia; Yusof, Faridah; Chyuan Ong, Hwai; Amid, Azura; Shah, Harmen

    2016-08-10

    Enzymatic reactions involving lipases as catalyst in transesterification can be an excellent alternative to produce environmental-friendly biodiesel. In this study, lipase extracted from Cocoa Pod Husk (CPH) and immobilized through cross linked enzyme aggregate (CLEA) technology catalysed the transesterification of Jatropha curcas oil successfully. Face centered central composite design (FCCCD) under response surface methodology (RSM) was used to get the optimal conditions of 3% (w/w) enzyme loading, 4h reaction time and 1:6 oil/ethanol ratio to achieve the highest conversion of free fatty acid and glycerides into biodiesel (93%). The reusability of CLEA-lipase was tested and after seven cycles, the conversion percentage reduced to 58%. The results revealed that CLEA lipase from CPH is a potential catalyst for biodiesel production. PMID:27184429

  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. 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

  19. 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

  20. 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

  1. 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

  2. The role of tetracycline in chronic blepharitis. Inhibition of lipase production in staphylococci.

    PubMed

    Dougherty, J M; McCulley, J P; Silvany, R E; Meyer, D R

    1991-10-01

    Tetracycline administered in low doses can be effective in the long-term management of patients with meibomian keratoconjunctivitis (MKC). However, the mechanism of action does not appear to be a reduction of bacteria. Seventy-five percent of the ocular staphylococci in such patients are resistant to tetracycline. An alternative mechanism of action could be the inhibition of production of extracellular enzymes by the ocular flora. Inhibition of lipase production could result in lowered levels of toxic hydrolysis products (free fatty acids), which may exacerbate the disease process. The authors tested this hypothesis by examining the differential effect of tetracycline on growth and lipase production in a tetracycline-resistant and tetracycline-sensitive strain of Staphylococcus epidermidis and S. aureus isolated from patients with MKC and Staphylococcal blepharitis. Tetracycline caused significant decreases in the production of lipase in the sensitive and resistant strains of S. epidermidis without concominant decreases in growth. In contrast, S. aureus strains showed parallel decreases in both lipase production and inhibition of growth. The authors propose that the sensitivity of lipase production to tetracycline, in tetracycline-resistant S. epidermidis, may partially explain the clinical improvement observed in MKC patients. PMID:1917401

  3. Separation and immobilization of lipase from Penicillium simplicissimum by selective adsorption on hydrophobic supports.

    PubMed

    Cunha, Aline G; Fernández-Lorente, Gloria; Gutarra, Melissa L E; Bevilaqua, Juliana V; Almeida, Rodrigo V; Paiva, Lúcia M C; Fernández-Lafuente, Roberto; Guisán, Jose M; Freire, Denise M G

    2009-05-01

    Lipases are an enzyme class of a great importance as biocatalysts applied to organic chemistry. However, it is still necessary to search for new enzymes with special characteristics such as good stability towards high temperatures, organic solvents, and high stereoselectivity presence. The present work's aim was to immobilize the lipases pool produced by Penicillium simplissicimum, a filamentous fungi strain isolated from Brazilian babassu cake residue. P. simplissicimum lipases were separated into three different fractions using selective adsorption method on different hydrophobic supports (butyl-, phenyl-, and octyl-agarose) at low ionic strength. After immobilization, it was observed that these fractions' hyperactivation is in the range of 131% to 1133%. This phenomenon probably occurs due to enzyme open form stabilization when immobilized onto hydrophobic supports. Those fractions showed different thermal stability, specificity, and enantioselectivity towards some substrates. Enantiomeric ratio for the hydrolysis of (R,S) 2-O-butyryl-2-phenylacetic acid ranged from 1 to 7.9 for different immobilized P. simplissicimum lipase fractions. Asymmetry factor for diethyl 2-phenylmalonate hydrolysis ranged from 11.8 to 16.4 according to the immobilized P. simplissicimum lipase fractions. Those results showed that sequential adsorption methodology was an efficient strategy to obtain new biocatalysts with different enantioselectivity degrees, thermostability, and specificity prepared with a crude extract produced by a simple and low-cost technology. PMID:19037600

  4. 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

  5. Lipase coated clusters of iron oxide nanoparticles for biodiesel synthesis in a solvent free medium.

    PubMed

    Mukherjee, Joyeeta; Gupta, Munishwar Nath

    2016-06-01

    Methyl or ethyl esters of long chain fatty acids are called biodiesel. Biodiesel is synthesized by the alcoholysis of oils/fats. In this work, lipase from Thermomyces lanuginosus was precipitated over the clusters of Fe3O4 nanoparticles. This biocatalyst preparation was used for obtaining biodiesel from soybean oil. After optimization of both immobilization conditions and process parameters, complete conversion to biodiesel was obtained in 3h and on lowering the enzyme amount, as little as 1.7U of lipase gave 96% conversion in 7h. The solvent free media with oil:ethanol (w/w) of 1:4 and 40°C with 2% (w/w) water along with 20% (w/w) silica (for facilitating acyl migration) were employed for reaching this high % of conversion. The biocatalyst design enables one to use a rather small amount of lipase. This should help in switching over to a biobased production of biodiesel. PMID:26967340

  6. [Rapid improvement of lipase production in Penicillium expansum by genome shuffling].

    PubMed

    Lin, Jun; Shi, Bi-Hong; Shi, Qiao-Qin; He, Yun-Xia; Wang, Ming-Zi

    2007-07-01

    In the present study, the genome shuffling was used to improve lipase production of Penicillium expansum. A lipase producing mutant strain-Penicillium expansum FS8486 and a wild type of Aspergillus Tamarii FS-132 isolated from soil of a volcano in Xinjiang were used as the parental strains. After two rounds of genome shuffling, several elite daughter strains were screened. The lipase activity in one of the daughter strains was increased 317% over the starting strain FS8486. Comparisons of the morphology, RAPD (Random Amplification of Polymorphic DNA) polymorphism and the fatty acid compositions between the daughter and the parental strains suggested that the filial generation were generated by genome shuffling. In this study, the genome shuffling used successfully first time in eukaryotic microorganism and increases the production of the desired metabolite in short time, the study will be useful to spread the genome shuffling in eukaryotic microbial breeding. PMID:17822042

  7. 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

  8. 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

  9. 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

  10. [Lipases in catalytic reactions of organic chemistry].

    PubMed

    Bezborodov, A M; Zagustina, N A

    2014-01-01

    Aspects of enzymatic catalysis in lipase-catalyzed reactions of organic synthesis are discussed in the review. The data on modern methods of protein engineering and enzyme modification allowing a broader range of used substrates are briefly summarized. The application of lipase in the preparation of pharmaceuticals and agrochemicals containing no inactive enantiomers and in the synthesis of secondary alcohol enantiomers and optically active amides is demonstrated. The subject of lipase involvement in the C-C bond formation in the Michael reaction is discussed. Data on the enzymatic synthesis of construction materials--polyesters, siloxanes, etc.--are presented. Examples demonstrating the application of lipase enzymatic catalysis in industry are given. PMID:25707112

  11. [Water binding of adsorptive immobilized lipases].

    PubMed

    Loose, S; Meusel, D; Muschter, A; Ruthe, B

    1990-01-01

    It is supposed that not only the total water content of lipase preparations but more their state of water binding is of technological importance in enzymatic interesterification reactions in systems nearly free from water. The isotherms at 65 degrees C of two microbial lipases immobilized on various adsorbents as well as different adsorbents themselves are shown. The water binding capacity in the range of water content of technological interest decreases from the anion exchange resin Amberlyst A 21 via nonpolar adsorbent Amberlite XAD-2 to kieselguhr Celite 545. It is demonstrated that water binding by lipases is depending on temperature but is also affected by adsorptive immobilization. Adsorptive immobilized lipases show hysteresis, which is very important for preparing a definite water content of the enzyme preparations. PMID:2325750

  12. Monoglyceride lipase: Structure and inhibitors.

    PubMed

    Scalvini, Laura; Piomelli, Daniele; Mor, Marco

    2016-05-01

    Monoglyceride lipase (MGL), the main enzyme responsible for the hydrolytic deactivation of the endocannabinoid 2-arachidonoyl-sn-glycerol (2-AG), is an intracellular serine hydrolase that plays critical roles in many physiological and pathological processes, such as pain, inflammation, neuroprotection and cancer. The crystal structures of MGL that are currently available provide valuable information about how this enzyme might function and interact with site-directed small-molecule inhibitors. On the other hand, its conformational equilibria and the contribution of regulatory cysteine residues present within the substrate-binding pocket or on protein surface remain open issues. Several classes of MGL inhibitors have been developed, from early reversible ones, such as URB602 and pristimerin, to carbamoylating agents that react with the catalytic serine, such as JZL184 and more recent O-hexafluoroisopropyl carbamates. Other inhibitors that modulate MGL activity by interacting with conserved regulatory cysteines act through mechanisms that deserve to be more thoroughly investigated. PMID:26216043

  13. Immobilization and characterization of a thermostable lipase.

    PubMed

    Song, Chongfu; Sheng, Liangquan; Zhang, Xiaobo

    2013-12-01

    Lipases have found a number of commercial applications. However, thermostable lipase immobilized on nanoparticle is not extensively characterized. In this study, a recombinant thermostable lipase (designated as TtL) from Thermus thermophilus WL was expressed in Escherichia coli and immobilized onto 3-APTES-modified Fe3O4@SiO2 supermagnetic nanoparticles. Based on analyses with tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis, X-ray diffraction, transmission electron microscopy, and vibrating sample magnetometer observation, the diameter of immobilized lipase nanoparticle was 18.4 (± 2.4) nm, and its saturation magnetization value was 52.3 emu/g. The immobilized lipase could be separated from the reaction medium rapidly and easily in a magnetic field. The biochemical characterizations revealed that, comparing with the free one, the immobilized lipase exhibited better resistance to temperature, pH, metal ions, enzyme inhibitors, and detergents. The K m value for the immobilized TtL (2.56 mg/mL) was found to be lower than that of the free one (3.74 mg/mL), showing that the immobilization improved the affinity of lipase for its substrate. In addition, the immobilized TtL exhibited good reusability. It retained more than 79.5 % of its initial activity after reusing for 10 cycles. Therefore, our study presented that the possibility of the efficient reuse of the thermostable lipase immobilized on supermagnetic nanoparticles made it attractive from the viewpoint of practical application. PMID:23748908

  14. Bacterial lipases and chronic blepharitis.

    PubMed

    Dougherty, J M; McCulley, J P

    1986-04-01

    Eyelids and conjunctivae of 36 normal individuals and 60 patients from six clinical groups of chronic blepharitis were cultured for aerobic and anaerobic bacteria. The most common species isolated were coagulase-negative staphylococci (C-NS) and Propionibacterium acnes. All strains of these species, and all Staphylococcus aureus strains isolated were tested for the ability to break down triglycerides, cholesterol esters, and fatty waxes. Each strain was incubated independently with appropriate substrates in nutrient media. Each medium was then extracted and assayed for the presence of substrate hydrolysis products by thin-layer chromatography. The percentage of strains capable of hydrolyzing a particular substrate was determined for each individual. S. aureus was a consistent and strong lipase producer, able to hydrolyze all three substrates. P. acnes was able to hydrolyze triolein and behenyl oleate but not cholesteryl oleate. No differences were observed among groups for P. acnes or S. aureus. C-NS showed a high degree of strain variability. Eighty-three percent of C-NS strains could hydrolyze triolein, 82% behenyl oleate, and 40% cholesteryl oleate. Significant group differences were seen in the percentage of lipase positive C-NS strains isolated per individual. Patients in the mixed staphylococcal/seborrheic, meibomian seborrheic, secondary meibomitis, and the meibomian keratoconjunctivitis (MKC) groups harbored significantly more C-NS strains capable of hydrolyzing cholesteryl oleate than did normal individuals. Patients in the meibomian seborrheic, secondary meibomitis, and MKC groups harbored significantly more C-NS strains capable of hydrolyzing behenyl oleate than did normals. No group differences were seen among groups with triolein hydrolyzing C-NS strains. PMID:3957566

  15. Lipase

    MedlinePlus

    ... indigestion, heartburn, allergy to gluten in wheat products (celiac disease), Crohn's disease, and cystic fibrosis. ... cystic fibrosis.Allergy to gluten in wheat products (celiac disease). Crohn's disease. Indigestion. Heartburn. Other conditions. More evidence ...

  16. Homogenization and lipase treatment of milk and resulting methyl ketone generation in blue cheese.

    PubMed

    Cao, Mingkai; Fonseca, Leorges M; Schoenfuss, Tonya C; Rankin, Scott A

    2014-06-25

    A specific range of methyl ketones contribute to the distinctive flavor of traditional blue cheeses. These ketones are metabolites of lipid metabolism by Penicillium mold added to cheese for this purpose. Two processes, namely, the homogenization of milk fat and the addition of exogenous lipase enzymes, are traditionally applied measures to control the formation of methyl ketones in blue cheese. There exists little scientific validation of the actual effects of these treatments on methyl ketone development. The present study evaluated the effects of milk fat homogenization and lipase treatments on methyl ketone and free fatty acid development using sensory methods and the comparison of selected volatile quantities using gas chromatography. Initial work was conducted using a blue cheese system model; subsequent work was conducted with manufactured blue cheese. In general, there were modest effects of homogenization and lipase treatments on free fatty acid (FFA) and methyl ketone concentrations in blue cheese. Blue cheese treatments involving Penicillium roqueforti lipase with homogenized milk yielded higher FFA and methyl ketone levels, for example, a ∼20-fold increase for hexanoic acid and a 3-fold increase in 2-pentanone. PMID:24460517

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

    PubMed Central

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

    2008-01-01

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

  18. Therapeutic potential of monoacylglycerol lipase inhibitors.

    PubMed

    Mulvihill, Melinda M; Nomura, Daniel K

    2013-03-19

    Marijuana and aspirin have been used for millennia to treat a wide range of maladies including pain and inflammation. Both cannabinoids, like marijuana, that exert anti-inflammatory action through stimulating cannabinoid receptors, and cyclooxygenase (COX) inhibitors, like aspirin, that suppress pro-inflammatory eicosanoid production have shown beneficial outcomes in mouse models of neurodegenerative diseases and cancer. Both cannabinoids and COX inhibitors, however, have untoward effects that discourage their chronic usage, including cognitive deficits and gastrointestinal toxicity, respectively. Recent studies have uncovered that the serine hydrolase monoacylglycerol lipase (MAGL) links the endocannabinoid and eicosanoid systems together through hydrolysis of the endocannabinoid 2-arachidonoylglycerol (2-AG) to provide the major arachidonic acid (AA) precursor pools for pro-inflammatory eicosanoid synthesis in specific tissues. Studies in recent years have shown that MAGL inhibitors elicit anti-nociceptive, anxiolytic, and anti-emetic responses and attenuate precipitated withdrawal symptoms in addiction paradigms through enhancing endocannabinoid signaling. MAGL inhibitors have also been shown to exert anti-inflammatory action in the brain and protect against neurodegeneration through lowering eicosanoid production. In cancer, MAGL inhibitors have been shown to have anti-cancer properties not only through modulating the endocannabinoid-eicosanoid network, but also by controlling fatty acid release for the synthesis of protumorigenic signaling lipids. Thus, MAGL serves as a critical node in simultaneously coordinating multiple lipid signaling pathways in both physiological and disease contexts. This review will discuss the diverse (patho)physiological roles of MAGL and the therapeutic potential of MAGL inhibitors in treating a vast array of complex human diseases. PMID:23142242

  19. 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

  20. 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

  1. 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

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

    PubMed

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

    2016-07-01

    A lipase-producing bacterial strain was isolated from oil-well-produced water in Shengli oilfield (Shandong province, China) and was identified as Pseudomonas synxantha by 16S rDNA sequence analysis (named Pseudomonas synxantha PS1). Strain PS1 showed a maximum lipase activity of 10.8 U/mL after culturing for 48 h at 30 °C, with lactose (4 g/L) as carbon source, tryptone (8 g/L) as nitrogen source, olive oil (0.5%, v/v) as inductor, and the initial pH 8.0. Meanwhile, the lipase gene from P. synxantha PS1 was cloned and expressed in Escherichia coli BL21 with the vector pET28a. The novel gene (lipPS1) has an open reading frame of 1425 bp and encodes a 474 aa lipase (LipPS1) sharing the most identity (87%) with the lipase in Pseudomonas fluorescens. LipPS1 preferably acted on substrates with a long chain (C10-C18) of fatty acids. The optimum pH and temperature of the recombinant enzyme were 8.0 and 40 °C, respectively, towards the optimum substrate p-nitrophenyl palmitate. The LipPS1 showed remarkable stability under alkaline conditions and was stable at pH 7.0-10.0 (retaining more than 60% activity). From the organic solvents tests, the lipase was activated by 15% (v/v) methanol (112%), 15% ethanol (127%), and 15% n-butyl alcohol (116%). LipPS1 presented strong biodegradability of waste grease; 93% of waste grease was hydrolyzed into fatty acid after 12 h at 30 °C. This is the first report of the lipase activity and lipase gene obtained from P. synxantha (including wild strain and recombinant strain) and of the recombinant LipPS1 with the detailed enzymatic properties. Also a preliminary study of the biodegradability of waste greases shows the potential value in industry applications. PMID:27321682

  3. 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

  4. Thermomyces lanuginosus lipase-catalyzed hydrolysis of the lipid cubic liquid crystalline nanoparticles.

    PubMed

    Barauskas, Justas; Anderberg, Hanna; Svendsen, Allan; Nylander, Tommy

    2016-01-01

    In this study well-ordered glycerol monooleate (GMO)-based cubic liquid crystalline nanoparticles (LCNPs) have been used as substrates for Thermomyces lanuginosus lipase in order to establish the relation between the catalytic activity, measured by pH-stat titration, and the change in morphology and nanostructure determined by cryogenic transmission electron microscopy and synchrotron small angle X-ray diffraction. The initial lipase catalyzed LCNP hydrolysis rate is approximately 25% higher for large 350nm nanoparticles compared to the small 190nm particles, which is attributed to the increased number of structural defects on the particle surface. At pH 8.0 and 8.4 bicontinuous Im3m cubic LCNPs transform into "sponge"-like assemblies and disordered multilamellar onion-like structures upon exposure to lipase. At pH 6.5 and 7.5 lipolysis induced phase transitions of the inner core of the particles, following the sequence Im3m cubic → reversed hexagonal → reversed micellar Fd3m cubic → reversed micelles. These transitions to the liquid crystalline phases with higher negative curvature of the lipid/water interface were found to trigger protonation of the oleic acid produced during lipase catalyzed reaction. The increase curvature of the reversed discrete micellar cubic phase was suggested to cause an increase in the oleic acid pKa to a larger value observed by pH-stat titration. PMID:26047576

  5. Partial purification and characterization of lipase from Geobacillus stearothermophilus AH22.

    PubMed

    Ekinci, Arife Pınar; Dinçer, Barbaros; Baltaş, Nimet; Adıgüzel, Ahmet

    2016-01-01

    The lipase was partially purified by ion exchange chromatography and gel filtration column chromatography, and was characterized from Geobacillus stearothermophilus AH22 strain. The lipase was purified 18.3-folds with 19.7% recovery. The lipase activity was determined by using p-nitrophenyl esters (C2-C12) as substrates. The Km values of the enzyme for these substrates were found as 0.16, 0.02, 0.19 and 0.55 mM, respectively, while Vmax values were 0.52, 1.03, 0.72 and 0.15 U mg(-1). The enzyme showed maximum activity at 50 °C and between pH 8.0 and 9.0. The enzyme was found to be quite stable at pH range of 4.0-10.0, and thermal stability between 50 and 60 °C. It was found that the best inhibitory effect of the enzyme activity was of Hg(2+). The inhibitory effect as orlistat, catechin, propyl paraben, p-coumaric acid, 3,4-dihydroxy hydro-cinnamic acid was examined. These results suggest that G. stearothermophilus AH22 lipase presents very suitable properties for industrial applications. PMID:25798692

  6. Cloning of an alkaline lipase gene from Penicillium cyclopium and its expression in Escherichia coli.

    PubMed

    Wu, Minchen; Qian, Zhikang; Jiang, Peihong; Min, Taishan; Sun, Chongrong; Huang, Weida

    2003-03-01

    The gene encoding an alkaline lipase of Penicillium cyclopium PG37 was cloned with four steps of PCR amplification based on different principles. The cloned gene was 1,480 nucleotides in length, consisted of 94 bp of promoter region, and had 6 exons and 5 short introns ranging from 50 to 70 nucleotides. The open reading frame encoded a protein of 285 amino acid residues consisting of a 27-AA signal peptide and a 258-AA mature peptide, with a conserved motif of Gly-X-Ser-X-Gly shared by all types of alkaline lipases. However, this protein had a low homology with lipases of P. camembertii (22.9%), Humicola lanuginosa (25.6%), and Rhizomucor miehei (22.3%) at the amino acid level. The mature peptide-encoding cDNA was cloned and expressed in Escherichia coli on pET-30a for confirmation. A distinct band with a M.W. of 33 kDa was detected on SDS-PAGE. Results of a Western blot analysis and an enzyme activity assay verified the recombinant 33-kDa protein as an alkaline lipase. Its catalytic properties were not changed when compared with its natural counterpart. PMID:12784858

  7. Isolation and biochemical characterization of two lipases from a metagenomic library of China Holstein cow rumen.

    PubMed

    Liu, Kailang; Wang, Jiaqi; Bu, Dengpan; Zhao, Shengguo; McSweeney, Chris; Yu, Ping; Li, Dan

    2009-08-01

    Two novel lipase genes RlipE1 and RlipE2 which encoded 361- and 265-amino acid peptides, respectively, were recovered from a metagenomic library of the rumen microbiota of Chinese Holstein cows. A BLAST search revealed a high similarity (90%) between RlipE2 and a carboxylesterase from Thermosinus carboxydivorans Nor1, while there was a low similarity (below 50%) between RlipE1 and other lipases. Phylogenetic analysis indicated that RlipE2 clustered with the lipolytic enzymes from family V while RlipE1 clustered with six other putative bacterial lipases which might constitute a new subfamily. The recombinant lipases were thermally unstable and retained 60% activity over a pH range of 6.5-8.5. Substrate specificity assay indicated that both enzymes had higher hydrolytic activity toward laurate (C(12)), palmitate (C(16)) and stearate (C(18)). The novel phylogenetic affiliation and high specificity of both enzymes for long-chain fatty acid make them interesting targets for manipulation of rumen lipid metabolism. PMID:19486892

  8. 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

  9. Lipase production by diverse phylogenetic clades of Aureobasidium pullulans

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Thirty-nine strains representing 12 diverse phylogenetic clades of Aureobasidium pullulans were surveyed for lipase production using a quantitative assay. Strains in clades 4 and 10 produced 0.2-0.3 U lipase/ml, while color variant strain NRRL Y-2311-1 in clade 8 produced 0.54 U lipase/ml. Strains i...

  10. 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

  11. 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

  12. Effective production of Pro-Gly by mutagenesis of l-amino acid ligase.

    PubMed

    Kino, Haruka; Nakajima, Shota; Arai, Toshinobu; Kino, Kuniki

    2016-08-01

    l-Amino acid ligase (Lal) catalyzes dipeptide synthesis from unprotected l-amino acids by hydrolysis ATP to ADP. Each Lal displays unique substrate specificity, and many different dipeptides can be synthesized by selecting suitable Lal. We have already successfully synthesized Met-Gly selectively by replacing the Pro85 residues of Lal from Bacillus licheniformis (BL00235). From these results, we deduced that the amino acid residue at position 85 had a key role in enzyme activity, and applied these findings to other Lals. When Pro and Gly were used as substrates, TabS from Pseudomonas syringae, synthesized the salt taste enhancing dipeptide Pro-Gly and other three dipeptides (Gly-Pro, Pro-Pro, and Gly-Gly) was hardly synthesized from its substrate specificity. However, the amount of Pro-Gly was low. Therefore, to alter the substrate specificity and increase the amount of Pro-Gly, we selected amino acid residues that might affect the enzyme activity, Ser85 corresponding to Pro85 of BL00235, and His294 on the results from previous studies and the predicted structure of TabS. These residues were replaced with 20 proteogenic amino acids, and Pro-Gly synthesizing reactions were conducted. The S85T and the H294D mutants synthesized more Pro-Gly than wild-type. Furthermore, the S85T/H294D double mutant synthesized considerably more Pro-Gly than the single mutant did. These results showed that the amino acid position 85 of TabS affect the enzyme activity similarly to BL00235. In addition, replacing the amino acid residue positioning around the N-terminal substrate and constructing the double mutant led to increase the amount of Pro-Gly. PMID:27017332

  13. 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

  14. 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

  15. A novel method for beef bone protein extraction by lipase-pretreatment and its application in the Maillard reaction.

    PubMed

    Song, Shiqing; Li, Sisi; Fan, Li; Hayat, Khizar; Xiao, Zuobing; Chen, Lihua; Tang, Qi

    2016-10-01

    Five beef bone hydrolysates were obtained by different enzyme treatment schemes, including papain (M), combination of porcine pancreatic lipase and papain (Z+M, combination of lipase and papain (Y+M), Protamex (F), combination of porcine pancreatic lipase and Protamex (Z+F). The degree of hydrolysis (DH), free amino acids and molecular weight distribution of these hydrolysates were evaluated. To further explore the differences between these five hydrolysates, Maillard reaction products (MRPs) were prepared using a xylose/cysteine/hydrolysate model. It was found that the DH, content of low molecular weight peptides and amino acids of hydrolysates increased significantly after lipase pre-treatment. GC-MS showed that the total content of furans, pyrroles and thioethers in MRPs Y+M increased by 78.0% compared with MRPs M, while in MRPs Z+F, pyrazines increased by 44.1% compared with MRPs F. Examining the sensory characteristics of the MRPs, the MRP from the hydrolysate of Y+M had the best mouthful, umami and meaty characteristics. The correlation analysis further confirmed that an appropriate lipase pre-treatment could improve the flavour of MRPs. PMID:27132826

  16. Solvent-Free Lipase-Catalyzed Synthesis of Diacylgycerols as Low-Calorie Food Ingredients.

    PubMed

    Vázquez, Luis; González, Noemí; Reglero, Guillermo; Torres, Carlos

    2016-01-01

    Problems derived from obesity and overweight have recently promoted the development of fat substitutes and other low-calorie foods. On the one hand, fats with short- and medium-chain fatty acids are a source of quick energy, easily hydrolyzable and hardly stored as fat. Furthermore, 1,3-diacylglycerols are not hydrolyzed to 2-monoacylglycerols in the gastrointestinal tract, reducing the formation of chylomicron and lowers the serum level of triacylglycerols by decreasing its resynthesis in the enterocyte. In this work, these two effects were combined to synthesize short- and medium-chain 1,3-diacylglycerols, leading to a product with great potential as for their low-calorie properties. Lipase-catalyzed transesterification reactions were performed between short- and medium-chain fatty acid ethyl esters and glycerol. Different variables were investigated, such as the type of biocatalyst, the molar ratio FAEE:glycerol, the adsorption of glycerol on silica gel, or the addition of lecithin. Best reaction conditions were evaluated considering the percentage of 1,3-DAG produced and the reaction rate. Except Novozym 435 (Candida antarctica), other lipases required the adsorption of glycerol on silica gel to form acylglycerols. Lipases that gave the best results with adsorption were Novozym 435 and Lipozyme RM IM (Rhizomucor miehei) with 52 and 60.7% DAG at 32 h, respectively. Because of its specificity for sn-1 and sn-3 positions, lipases leading to a higher proportion of 1,3-DAG vs. 1,2-DAG were Lipozyme RM IM (39.8 and 20.9%, respectively) and Lipase PLG (Alcaligenes sp.) (35.9 and 19.3%, respectively). By adding 1% (w/w) of lecithin to the reaction with Novozym 435 and raw glycerol, the reaction rate was considerably increased from 41.7 to 52.8% DAG at 24 h. PMID:26904539

  17. Solvent-Free Lipase-Catalyzed Synthesis of Diacylgycerols as Low-Calorie Food Ingredients

    PubMed Central

    Vázquez, Luis; González, Noemí; Reglero, Guillermo; Torres, Carlos

    2016-01-01

    Problems derived from obesity and overweight have recently promoted the development of fat substitutes and other low-calorie foods. On the one hand, fats with short- and medium-chain fatty acids are a source of quick energy, easily hydrolyzable and hardly stored as fat. Furthermore, 1,3-diacylglycerols are not hydrolyzed to 2-monoacylglycerols in the gastrointestinal tract, reducing the formation of chylomicron and lowers the serum level of triacylglycerols by decreasing its resynthesis in the enterocyte. In this work, these two effects were combined to synthesize short- and medium-chain 1,3-diacylglycerols, leading to a product with great potential as for their low-calorie properties. Lipase-catalyzed transesterification reactions were performed between short- and medium-chain fatty acid ethyl esters and glycerol. Different variables were investigated, such as the type of biocatalyst, the molar ratio FAEE:glycerol, the adsorption of glycerol on silica gel, or the addition of lecithin. Best reaction conditions were evaluated considering the percentage of 1,3-DAG produced and the reaction rate. Except Novozym 435 (Candida antarctica), other lipases required the adsorption of glycerol on silica gel to form acylglycerols. Lipases that gave the best results with adsorption were Novozym 435 and Lipozyme RM IM (Rhizomucor miehei) with 52 and 60.7% DAG at 32 h, respectively. Because of its specificity for sn-1 and sn-3 positions, lipases leading to a higher proportion of 1,3-DAG vs. 1,2-DAG were Lipozyme RM IM (39.8 and 20.9%, respectively) and Lipase PLG (Alcaligenes sp.) (35.9 and 19.3%, respectively). By adding 1% (w/w) of lecithin to the reaction with Novozym 435 and raw glycerol, the reaction rate was considerably increased from 41.7 to 52.8% DAG at 24 h. PMID:26904539

  18. Lipase from Pseudomonas fragi CRDA 323: partial purification, characterization and interesterification of butter fat.

    PubMed

    Pabai, F; Kermasha, S; Morin, A

    1995-04-01

    Several strains of Pseudomonas were screened for lipase production using the rhodamine agar diffusion test. On the basis of the diameter of the halo produced, P. fragi CRDA 323 and P. putida ATCC 795 were considered to be good and weak lipase producers respectively. P. fragi, cultured in a 2-l fermenter, produced a maximal amount of lipase after 3-4 days of incubation at 27 degrees C. The lipase extract of P. fragi was obtained by acidification of culture supernatant at pH 4.0 and partially purified with ammonium sulphate precipitation. The majority of lipase activity (42%) was located in fraction IV, precipitated at 20%-40% of saturation, with a 19-fold enzyme purification. The Km and Vmax values for the partially purified enzymatic extract (fraction IV) were 0.70 mg/ml and 0.97 x 10(-3) U/min respectively. Fraction IV, which showed an optimum activity at pH 8.5, was used for the interesterification of butter fat in a microemulsion free co-surfactant system containing Span 60 (sorbitol monostearate) and Tween 60 (polyoxyethylene sorbitan monostearate) in the ratio 48:52 (v/v). The results showed that the interesterification of butter fat resulted in a considerable decrease in long-chain saturated fatty acids (C12:0, C14:0 and C16:0) with a concomitant increase in C18:0 and C18:1 at the sn-2 position of selected triacylglycerols. In addition, the results demonstrated an increase in the fatty acids (C12:0, C14:0 and 16:0) among the 1 and 3 positions of the triacylglycerol molecules of modified buffer fat accompanied by a decrease in C18:0 and C18:1. PMID:7766135

  19. A new alkaline lipase obtained from the metagenome of marine sponge Ircinia sp.

    PubMed

    Su, Jing; Zhang, Fengli; Sun, Wei; Karuppiah, Valliappan; Zhang, Guangya; Li, Zhiyong; Jiang, Qun

    2015-07-01

    Microorganisms associated with marine sponges are potential resources for marine enzymes. In this study, culture-independent metagenomic approach was used to isolate lipases from the complex microbiome of the sponge Ircinia sp. obtained from the South China Sea. A metagenomic library was constructed, containing 6568 clones, and functional screening on 1 % tributyrin agar resulted in the identification of a positive lipase clone (35F4). Following sequence analysis 35F4 clone was found to contain a putative lipase gene lipA. Sequence analysis of the predicted amino acid sequence of LipA revealed that it is a member of subfamily I.1 of lipases, with 63 % amino acid similarity to the lactonizing lipase from Aeromonas veronii (WP_021231793). Based on the predicted secondary structure, LipA was predicted to be an alkaline enzyme by sequence/structure analysis. Heterologous expression of lipA in E. coli BL21 (DE3) was performed and the characterization of the recombinant enzyme LipA showed that it is an alkaline enzyme with high tolerance to organic solvents. The isolated lipase LipA was active in the broad alkaline range, with the highest activity at pH 9.0, and had a high level of stability over a pH range of 7.0-12.0. The activity of LipA was increased in the presence of 5 mM Ca(2+) and some organic solvents, e.g. methanol, acetone and isopropanol. The optimum temperature for the activity of LipA is 40 °C and the molecular weight of LipA was determined to be ~30 kDa by SDS-PAGE. LipA is an alkaline lipase and shows good tolerance to some organic solvents, which make it of potential utility in the detergent industry and enzyme mediated organic synthesis. The result of this study has broadened the diversity of known lipolytic genes and demonstrated that marine sponges are an important source for new enzymes. PMID:25921581

  20. High-level soluble expression of Serratia marcescens H30 lipase in Escherichia coli.

    PubMed

    Su, Erzheng; Xu, Jingjing; Wu, Xiangping

    2015-01-01

    Serratia marcescens lipase (SmL) is an important biocatalyst used to enantioselectively hydrolyze (±)-trans-3-(4-methoxyphynyl) glycidic acid methyl ester. However, the economically justified level recombinant soluble expression of SmL in Escherichia coli has not been established. Thus, fusion genes of lipase from S. marcescens H30 with different fusion tags were constructed and expressed in E. coli. The effects of fusion tags were revealed. A significant increase in recombinant lipase solubility showed that E. coli BL21 (DE3)/pET32a-SmL was a suitable choice for SmL production. To optimize the performance of recombinant SmL production, changes in culture medium compositions and induction conditions were systematically tested. Finally, the recombinant SmL activity and productivity reached approximately 23,000 U/L and 1,278 U/L/H in shake flasks, respectively. This value is the highest SmL activity attained by heterogeneous recombinant expression in E. coli. Lipase activity and productivity reached 19,650 U/L and 1,228 U/L/H, respectively, by scaling up SmL production in a 7.0 L fermenter. The existence of the Trx tag did not influence the chiral selectivity of recombinant SmL. These findings indicate a possibility for soluble and economical SmL expression in E. coli to meet industrial needs. PMID:24852607

  1. An alkaline lipase from organic solvent tolerant Acinetobacter sp. EH28: Application for ethyl caprylate synthesis.

    PubMed

    Ahmed, Eltayib Hassan; Raghavendra, Tripti; Madamwar, Datta

    2010-05-01

    A mesophilic bacterium producing a thermostable alkaline lipase was isolated from oil rich soil sample and identified as Acinetobacter sp. EH28. The lipase was partially purified by ammonium sulphate precipitation followed by hydrophobic interaction chromatography with 24.2-fold purification and 57.1U/ml specific activity. The partially purified enzyme exhibited maximum activity at pH 10.0 and at 50 degrees C and was highly stable at 50 degrees C retaining 100% of its activity up to 90min. It was highly stable and retained more than 80% of its initial activity upon exposure to various organic solvents. The EH28 lipase was used for synthesis of the flavor ester ethyl caprylate in organic solvents, thus providing a concept of application of Acinetobacter sp. lipase in non-aqueous catalysis. Reaction parameters best suited for this esterification reaction were 40 degrees C reaction temperature, 1.3:1 ratio of caprylic acid to ethanol and cyclohexane as the medium. PMID:20096565

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

    NASA Astrophysics Data System (ADS)

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

    2013-12-01

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

  3. Biodiesel production from Nannochloropsis gaditana lipids through transesterification catalyzed by Rhizopus oryzae lipase.

    PubMed

    Navarro López, Elvira; Robles Medina, Alfonso; González Moreno, Pedro Antonio; Esteban Cerdán, Luis; Martín Valverde, Lorena; Molina Grima, Emilio

    2016-03-01

    Biodiesel (fatty acid methyl esters, FAMEs) was produced from saponifiable lipids (SLs) extracted from wet Nannochloropsis gaditana biomass using methanolysis catalyzed by Rhizopus oryzae intracellular lipase. SLs were firstly extracted with ethanol to obtain 31 wt% pure SLs. But this low SL purity also gave a low biodiesel conversion (58%). This conversion increased up to 80% using SLs purified by crystallization in acetone (95 wt% purity). Polar lipids play an important role in decreasing the reaction velocity - using SLs extracted with hexane, which have lower polar lipid content (37.4% versus 49.0% using ethanol), we obtained higher reaction velocities and less FAME conversion decrease when the same lipase batch was reused. 83% of SLs were transformed to biodiesel using a 70 wt% lipase/SL ratio, 11:1 methanol/SL molar ratio, 10 mL t-butanol/g SLs after 72 h. The FAME conversion decreased to 71% after catalyzing three reactions with the same lipase batch. PMID:26735878

  4. Efficient production of biodiesel from waste grease: one-pot esterification and transesterification with tandem lipases.

    PubMed

    Yan, Jinyong; Li, Aitao; Xu, Yi; Ngo, Thao P N; Phua, Szechao; Li, Zhi

    2012-11-01

    A novel concept and efficient method for producing biodiesel (FAME) from grease (15-40wt% free fatty acid, FFA) were developed by using tandem lipases for one-pot esterification of FFA and transesterification of triglyceride with methanol in a solvent-free system. Combining immobilized Candida antarctica lipase B (CALB) (Novozyme 435) favoring the esterification and immobilized Thermomyces lanuginosus lipase (TLL) (Lipozyme TLIM) preferring the transesterification at 2:8 (wt/wt) gave FAME in 80% yield, being better than that with Novozyme 435 or Lipozyme TLIM. Recombinant Escherichia coli (Calb/Tll) co-expressing CALB and TLL was engineered as a more efficient tandem-lipases system. Using wet or dry cells (4wt%) gave FAME in 87% or 95% yield, which is much better than that with E. coli cells expressing either CALB or TLL alone. Cells of E. coli (Calb/Tll) were recycled for five times and retained 75% productivity, thus being practical for producing biodiesel from grease. PMID:22940338

  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. Strategies for production of butanol and butyl-butyrate through lipase-catalyzed esterification.

    PubMed

    Xin, Fengxue; Basu, Anindya; Yang, Kun-Lin; He, Jianzhong

    2016-02-01

    In this study, a fermentation process for production of butanol and butyl-butyrate by using Clostridium sp. strain BOH3 is developed. This strain is able to produce butyric acid and butanol when it ferments 60 g/L xylose. Meanwhile, it also excreted indigenous lipases (induced by olive oil) which naturally convert butyric acid and butanol into 1.2 g/L of butyl-butyrate. When Bio-OSR was used as both an inducer for lipase and extractant for butyl-butyrate, the butyl-butyrate concentration can reach 6.3 g/L. To further increase the yield, additional lipases and butyric acid are added to the fermentation system. Moreover, kerosene was used as an extractant to remove butyl-butyrate in situ. When all strategies are combined, 22.4 g/L butyl-butyrate can be produced in a fed-batch reactor spiked with 70 g/L xylose and 7.9 g/L butyric acid, which is 4.5-fold of that in a similar system (5 g/L) with hexadecane as the extractant. PMID:26710347

  9. [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

  10. Data on hepatic lipolysis, adipose triglyceride lipase, and hormone-sensitive lipase in fasted and non-fasted C57BL/6J female mice.

    PubMed

    Marvyn, Phillip M; Mardian, Emily B; Bradley, Ryan M; A Marks, Kristin; Duncan, Robin E

    2016-06-01

    Liver homogenates produced from fasted and non-fasted C57BL/6J female mice were assayed for total lipolytic activity measured as hydrolysis of [9,10-(3)H(N)]-triolein into [(3)H] free fatty acids (FFA). Liver homogenates were also used for immunoblotting to determine levels of the lipolytic enzymes adipose-triglyceride lipase (ATGL) and hormone-sensitive lipase (HSL), as well as site specific phosphorylation at the 14-3-3 binding site of ATGL and the serine 565 and serine 660 sites of HSL. Significantly higher triolein hydrolysis activity was observed in fasted liver samples, as well as a significant increase in total ATGL and a significant decrease in HSL phosphorylation at the S565 site. PMID:27054184

  11. Data on hepatic lipolysis, adipose triglyceride lipase, and hormone-sensitive lipase in fasted and non-fasted C57BL/6J female mice

    PubMed Central

    Marvyn, Phillip M.; Mardian, Emily B.; Bradley, Ryan M.; A. Marks, Kristin; Duncan, Robin E.

    2016-01-01

    Liver homogenates produced from fasted and non-fasted C57BL/6J female mice were assayed for total lipolytic activity measured as hydrolysis of [9,10-3H(N)]-triolein into [3H] free fatty acids (FFA). Liver homogenates were also used for immunoblotting to determine levels of the lipolytic enzymes adipose-triglyceride lipase (ATGL) and hormone-sensitive lipase (HSL), as well as site specific phosphorylation at the 14-3-3 binding site of ATGL and the serine 565 and serine 660 sites of HSL. Significantly higher triolein hydrolysis activity was observed in fasted liver samples, as well as a significant increase in total ATGL and a significant decrease in HSL phosphorylation at the S565 site. PMID:27054184

  12. 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

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

    PubMed Central

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

    2012-01-01

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

  14. Direct transesterification of Oedogonium sp. oil be using immobilized isolated novel Bacillus sp. lipase.

    PubMed

    Sivaramakrishnan, Ramachandran; Muthukumar, Karuppan

    2014-01-01

    This work emphasizes the potential of the isolated Bacillus sp. lipase for the production of fatty acid methyl ester by the direct transesterification of Oedogonium sp. of macroalgae. Dimethyl carbonate was used as the extraction solvent and also as the reactant. The effect of solvent/algae ratio, water addition, catalyst, temperature, stirring and time on the direct transesterification was studied. The highest fatty acid methyl ester yield obtained under optimum conditions (5 g Oedogonium sp. powder, 7.5 ml of solvent (dimethyl carbonate)/g of algae, 8% catalyst (%wt/wt of oil), distilled water 1% (wt/wt of algae), 36 h, 55°C and 180 rpm) was 82%. Final product was subjected to thermogravimetric analysis and (1)H NMR analysis. The results showed that the isolated enzyme has good potential in catalyzing the direct transesterification of algae, and the dimethyl carbonate did not affect the activity of the isolated lipase. PMID:23890544

  15. Efficient regioselective acylation of quercetin using Rhizopus oryzae lipase and its potential as antioxidant.

    PubMed

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

    2016-10-01

    The present investigation describes the regioselective enzymatic acylation of quercetin with ferulic acid using Rhizopus oryzae lipase. Optimization of reaction parameters resulted in 93.2% yield of the ester synthesized using 750IU of lipase in cyclo-octane at a temperature of 45°C. The reaction was successfully carried out upto 25g scale. The ester synthesized was analyzed by (1)H Nuclear magnetic resonance spectroscopy. The ester synthesized (quercetin ferulate) showed higher antiradical activity as compared to ascorbic acid using the 2,2-diphenyl-1-picrylhydrazyl radical method. These results on enzyme-catalyzed acylation of quercetin might be used to prepare and scale-up other flavonoids derivatives. PMID:27372535

  16. 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

  17. New Extremophilic Lipases and Esterases from Metagenomics

    PubMed Central

    López-López, Olalla; Cerdán, Maria E; González Siso, Maria I

    2014-01-01

    Lipolytic enzymes catalyze the hydrolysis of ester bonds in the presence of water. In media with low water content or in organic solvents, they can catalyze synthetic reactions such as esterification and transesterification. Lipases and esterases, in particular those from extremophilic origin, are robust enzymes, functional under the harsh conditions of industrial processes owing to their inherent thermostability and resistance towards organic solvents, which combined with their high chemo-, regio- and enantioselectivity make them very attractive biocatalysts for a variety of industrial applications. Likewise, enzymes from extremophile sources can provide additional features such as activity at extreme temperatures, extreme pH values or high salinity levels, which could be interesting for certain purposes. New lipases and esterases have traditionally been discovered by the isolation of microbial strains producing lipolytic activity. The Genome Projects Era allowed genome mining, exploiting homology with known lipases and esterases, to be used in the search for new enzymes. The Metagenomic Era meant a step forward in this field with the study of the metagenome, the pool of genomes in an environmental microbial community. Current molecular biology techniques make it possible to construct total environmental DNA libraries, including the genomes of unculturable organisms, opening a new window to a vast field of unknown enzymes with new and unique properties. Here, we review the latest advances and findings from research into new extremophilic lipases and esterases, using metagenomic approaches, and their potential industrial and biotechnological applications. PMID:24588890

  18. Design of ionic liquids for lipase purification.

    PubMed

    Ventura, Sónia P M; Sousa, Sílvia G; Freire, Mara G; Serafim, Luísa S; Lima, Alvaro S; Coutinho, João A P

    2011-09-15

    Aqueous two-phase systems (ATPS) are considered as efficient downstream processing techniques in the production and purification of enzymes, since they can be considered harmless to biomolecules due to their high water content and due to the possibility of maintaining a neutral pH value in the medium. A recent type of alternative ATPS is based on hydrophilic ionic liquids (ILs) and salting-out inducing salts. The aim of this work was to study the lipase (Candida antarctica lipase B - CaLB) partitioning in several ATPS composed of ionic liquids (ILs) and inorganic salts, and to identify the best IL for the enzyme purification. For that purpose a wide range of IL cations and anions, and some of their combinations were studied. For each system the enzyme partitioning between the two phases was measured and the purification factors and enzyme recoveries were determined. The results indicate that the lipase maximum purification and recovery were obtained for cations with a C(8) side alkyl chain, the [N(CN)(2)] anion and ILs belonging to the pyridinium family. However, the highest purification parameters were observed for 1-methyl-3-octylimidazolium chloride [C(8)mim]Cl, suggesting that the IL extraction capability does not result from a cumulative character of the individual characteristics of ILs. The results indicate that the IL based ATPS have an improved performance in the lipase purification and recovery. PMID:21852207

  19. Structural characterization of MAPLE deposited lipase biofilm

    NASA Astrophysics Data System (ADS)

    Aronne, Antonio; Ausanio, Giovanni; Bloisi, Francesco; Calabria, Raffaela; Califano, Valeria; Fanelli, Esther; Massoli, Patrizio; Vicari, Luciano R. M.

    2014-11-01

    Lipases (triacylglycerol ester hydrolases) are enzymes used in several industrial applications. Enzymes immobilization can be used to address key issues limiting widespread application at industrial level. Immobilization efficiency is related to the ability to preserve the native conformation of the enzyme. MAPLE (Matrix Assisted Pulsed Laser Evaporation) technique, a laser deposition procedure for treating organic/polymeric/biomaterials, was applied for the deposition of lipase enzyme in an ice matrix, using near infrared laser radiation. Microscopy analysis showed that the deposition occurred in micrometric and submicrometric clusters with a wide size distribution. AFM imaging showed that inter-cluster regions are uniformly covered with smaller aggregates of nanometric size. Fourier transform infrared spectroscopy was used for both recognizing the deposited material and analyzing its secondary structure. Results showed that the protein underwent reversible self-association during the deposition process. Actually, preliminary tests of MAPLE deposited lipase used for soybean oil transesterification with isopropyl alcohol followed by gas chromatography-mass spectrometry gave results consistent with undamaged deposition of lipase.

  20. 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...

  1. A multisubstrate assay for lipases/esterases: assessing acyl chain length selectivity by reverse-phase high-performance liquid chromatography.

    PubMed

    Divakar, K; Gautam, Pennathur

    2014-03-01

    Lipases and esterases are hydrolytic enzymes and are known to hydrolyze esters with unique substrate specificity and acyl chain length selectivity. We have developed a simple competitive multiple substrate assay for determination of acyl chain length selectivity of lipases/esterases using RP-HPLC with UV detection. A method for separation and quantification of 4-nitrophenyl fatty acid esters (C4-C18) was developed and validated. The chain length selectivity of five lipases and two esterases was determined in a multisubstrate reaction system containing equimolar concentrations of 4-nitrophenyl esters (C4-C18). This assay is simple, reproducible, and a useful tool for determining chain length selectivity of lipases/esterases. PMID:24316114

  2. Strategy to Overcome Effect of Raw Materials on Enzymatic Process of Biodiesel from Non-edible Oils Using Candida sp. 99-125 Lipase.

    PubMed

    Nie, Kaili; Wang, Fang; Tan, Tianwei; Liu, Luo

    2015-11-01

    Non-edible oils are preferred raw materials for biodiesel production. However, the properties of raw materials significantly affect the synthesis process, leading to difficulties to design one process suitable for any kind of raw material. In this study, the composition of five typical non-edible oils was analyzed. The major difference was the content of free fatty acids, reflected from their acid values. The influence of different oils was investigated by using lipase from Candida sp. 99-125. At low lipase dosage and low water content, the conversion was found proportional to the acid value. However, by increasing the water content or lipase dosage, we observed that the conversions for all kinds of oils used in this study could exceed 80%. Time course analysis indicates that the lipase used in this study catalyzed hydrolysis followed by esterification, rather than direct transesterification. Accumulation of free fatty acids at the very beginning was necessary. A high water content facilitated the hydrolysis of oils with low acid value. This lipase showed capability to transform all the oils by controlling the water content. PMID:26280803

  3. 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.

  4. Selection of lipase-producing microorganisms through submerged fermentation.

    PubMed

    Colla, Luciane Maria; Primaz, Andreiza Lazzarotto; Benedetti, Silvia; Loss, Raquel Aparecida; de Lima, Marieli; Reinehr, Christian Oliveira; Bertolin, Telma Elita; Costa, Jorge Alberto Vieira

    2010-01-01

    Lipases are enzymes used in various industrial sectors such as food, pharmaceutical and chemical synthesis industries. The selection of microorganisms isolated from soil or wastewater is an alternative to the discovery of new species with high enzymes productivity and with different catalytic activities. In this study, the selection of lipolytic fungi was carried out by submerged fermentation. A total of 27 fungi were used, of which 20 were isolated from dairy effluent and 7 from soil contaminated with diesel oil. The largest producers were the fungi Penicillium E-3 with maximum lipolytic activity of 2.81 U, Trichoderma E-19 and Aspergillus O-8 with maximum activities of 2.34 and 2.03 U where U is the amount of enzyme that releases 1 micromol of fatty acid per min per mL of enzyme extract. The fungi had maximum lipolytic activities on the 4th day of fermentation. PMID:20737918

  5. Placental lipases in pregnancies complicated by gestational diabetes mellitus (GDM).

    PubMed

    Barrett, Helen L; Kubala, Marta H; Scholz Romero, Katherin; Denny, Kerina J; Woodruff, Trent M; McIntyre, H David; Callaway, Leonie K; Nitert, Marloes Dekker

    2014-01-01

    Infants of women with gestational diabetes mellitus (GDM) are more likely to be born large for gestational age with a higher percentage body fat. Elevated maternal lipids may contribute to this. Placental lipases such as lipoprotein lipase (LPL), endothelial lipase (EL) and hormone sensitive lipase (HSL) are involved in transferring lipids from mother to fetus. Previous studies of expression of these lipases in placentae in women with diabetes in pregnancy have reported divergent results. Intracellular lipases such as adipose triglyceride lipase (ATGL), and HSL are central to lipid droplet metabolism. The activities of these lipases are both influenced by Perilipin 1, and ATGL is also activated by a co-factor comparative gene identification-58 (CGI-58) and inhibited by G0/G1 switch gene 2 (GS02). None of these modifying factors or ATGL have been examined previously in placenta. The purpose of this study was therefore to examine the expression of ATGL, HSL, LPL, EL, as well as Perilipin 1, GS02 and CGI-58 in term pregnancies complicated by GDM. mRNA and protein expression of the lipases were measured in placentae from 17 women with GDM and 17 normoglycaemic pregnancies, matched for maternal BMI and gestational age of delivery. ATGL mRNA expression was increased and HSL mRNA expression reduced in placentae from GDM although there was no differences in protein expression of any of the lipases. All lipases were localised to trophoblasts and endothelial cells. The expression of Perilipin 1 and CGI-58 mRNA was increased and GS02 not altered in GDM. These results suggest that there is no difference in expression in these four lipases between GDM and normoglycaemic placentae, and therefore altered lipid transfer via these lipases does not contribute to large for gestational age in infants of women with GDM. PMID:25118138

  6. Combined effect of enterocin and lipase from Enterococcus faecium NCIM5363 against food borne pathogens: mode of action studies.

    PubMed

    Ramakrishnan, Vrinda; Narayan, Bhaskar; Halami, Prakash M

    2012-08-01

    Food borne diseases have a major impact on public health whose epidemiology is rapidly changing. The whole cells of pathogens involved or their toxins/metabolites affect the human health apart from spoiling sensory properties of the food products finally affecting the food industry as well as consumer health. With pathogens developing mechanisms of antibiotic resistance, there has been an increased need to replace antibiotics as well as chemical additives with naturally occurring bacteriocins. Bacteriocins are known to act mainly against Gram-positive pathogens and with little or no effect towards Gram-negative enteric bacteria. In the present study, combination effect of lipase and bacteriocin produced by Enterococcus faecium NCIM5363, a highly lipolytic lactic acid bacterium against various food pathogens was assessed. The lipase in combination with enterocin exhibited a lethal effect against Gram-negative pathogens. Scanning electron microscopy studies carried out to ascertain the constitutive mode of action of lipase and enterocin revealed that the lipase degrades the cell wall of Gram-negative bacteria and creates a pore through which enterocin enters thereby resulting in cell death. The novelty of this work is the fact that this is the first report revealing the synergistic effect of lipase with enterocin against Gram-negative bacteria. PMID:22580888

  7. Purification and characterization of solvent tolerant lipase from Bacillus sp. for methyl ester production from algal oil.

    PubMed

    Sivaramakrishnan, Ramachandran; Incharoensakdi, Aran

    2016-05-01

    Lipase from Bacillus sp. isolated from the oil contaminated soil was purified by ammonium sulphate precipitation and ion-exchange chromatography with a 5.1-fold purification and 10.5% yield. SDS-PAGE analysis of the enzyme revealed the molecular mass of 24 kDa. The optimum pH and temperature for lipase activity were 6.5 and 37°C, respectively. The isolated lipase was stimulated by pretreatment with methanol and ethanol as well as by divalent metal ions Ca(2+), Mg(2+) and Mn(2+). The enzyme showed high activity towards oleic rich oils. The enzyme immobilized on celite could retain 90% lipase activity after eight cycles. Transesterification of Botryococcus sp. oil using the immobilized enzyme for 40 h resulted in 80% yield of fatty acid methyl esters which had good properties for use as biodiesel. Overall results suggested that the solvent tolerant Bacillus lipase can be a potential biocatalyst for methyl ester production. PMID:26467697

  8. Biodiesel production from sunflower, soybean, and waste cooking oils by transesterification using lipase immobilized onto a novel microporous polymer.

    PubMed

    Dizge, Nadir; Aydiner, Coskun; Imer, Derya Y; Bayramoglu, Mahmut; Tanriseven, Aziz; Keskinler, Bülent

    2009-03-01

    This study aims at carrying out lipase-catalyzed synthesis of fatty acid methyl esters (biodiesel) from various vegetable oils using lipase immobilized onto a novel microporous polymeric matrix (MPPM) as a low-cost biocatalyst. The research is focused on three aspects of the process: (a) MPPM synthesis (monolithic, bead, and powder forms), (b) microporous polymeric biocatalyst (MPPB) preparation by immobilization of lipase onto MPPM, and (c) biodiesel production by MPPB. Experimental planning of each step of the study was separately carried out in accordance with design of experiment (DoE) based on Taguchi methodology. Microporous polymeric matrix (MPPM) containing aldehyde functional group was synthesized by polyHIPE technique using styrene, divinylbenzene, and polyglutaraldehyde. Thermomyces lanuginosus lipase was covalently attached onto MPPM with 80%, 85%, and 89% immobilization efficiencies using bead, powder, and monolithic forms, respectively. Immobilized enzymes were successfully used for the production of biodiesel using sunflower, soybean, and waste cooking oils. It was shown that immobilized enzymes retain their activities during 10 repeated batch reactions at 25 degrees C, each lasting 24h. Since the developed novel method is simple yet effective, it could have a potential to be used industrially for the production of chemicals requiring immobilized lipases. PMID:19028094

  9. Medium-based optimization of an organic solvent-tolerant extracellular lipase from the isolated halophilic Alkalibacillus salilacus.

    PubMed

    Samaei-Nouroozi, Amene; Rezaei, Shahla; Khoshnevis, Nika; Doosti, Mahmoud; Hajihoseini, Reza; Khoshayand, Mohammad Reza; Faramarzi, Mohammad Ali

    2015-09-01

    A haloalkaliphilic solvent-tolerant lipase was produced from Alkalibacillus salilacus within 48 h of growth in liquid medium. An overall 4.9-fold enhanced production was achieved over unoptimized media after medium optimization by statistical approaches. Plackett-Burman screening suggested lipase production maximally influenced by olive oil, KH2PO4, NaCl, and glucose; and response surface methodology predicted the appropriate levels of each parameter. Produced lipase was highly active and stable over broad ranges of temperature (15-65 °C), pH (4.0-11.0), and NaCl concentration (0-30 %) showing excellent thermostable, pH-stable, and halophilic properties. The enzyme was optimally active at pH 8.0 and 40 °C. Majority of cations, except some like Co(2+) and Al(3+) were positive signals for lipase activity. In addition, the presence of chemical agents and organic solvents with different log P ow was well tolerated by the enzyme. Finally, efficacy of lipase-mediated esterification of various alcohols with oleic acid in organic solvents was studied. PMID:26198037

  10. Effectiveness of immobilized lipase Thermomyces lanuginosa in catalyzing interesterification of palm olein in batch reaction.

    PubMed

    Saw, Mei Huey; Siew, Wai Lin

    2014-01-01

    Lipase Thermomyces lanuginosa has shown potential in modifying oils and fats through interesterification. Analyzing the physicochemical properties of the modified oils is important to determine the effectiveness of lipase in catalyzing interesterification. In this study, the effectiveness of the immobilized lipase (Lipozyme(®) TL IM) in catalyzing interesterification of palm olein in pilot-scale batch reactor was determined. The evaluation was done by analyzing the changes of triacylglycerol (TAGs) composition, sn-2 position fatty acids composition and the physical properties of the palm olein after the interesterifications. The pilot-scale batch reaction was conducted for 8 hours with 5 %w/w enzyme dosage based on the results of TAGs composition of the laboratory-scale interesterified products. The pilot-scale results showed that Lipozyme(®) TL IM act as an effective enzyme in converting TAGs, in which 4.5% of trisaturated TAGs (PPP and PPS) were produced in the batch reaction. The formation of these new TAGs had also altered the thermal and physical properties of the palm olein. These interesterified products showed a broad peak and shoulder at high temperature, ranging from 10°C to 40°C, indicating the formation of some new TAGs with high melting points. However, the enzyme did not perform perfectly as a 1,3-specific enzyme in the reaction as a significant reduction of oleic acid and an increment of palmitic acid at the sn-2 position was observed. PMID:24492381

  11. A Novel Lipase as Aquafeed Additive for Warm-Water Aquaculture

    PubMed Central

    Yang, Yalin; Huang, Lu; Zhou, Zhigang

    2015-01-01

    A novel Acinetobacter lipase gene lipG1was cloned from DNA extracted from intestinal sample of common carp (Cyprinus carpio), and expressed in E. coli BL21. The encoded protein was 406 amino acids in length. Phylogenetic analysis indicated that LipG1 and its relatives comprised a novel group of true lipases produced by Gram-negative bacteria. LipG1 showed maximal activity at 40℃ and pH 8.0 when pNP decanoate (C10) was used as the substrate, and remained high activity between 20℃ and 35℃. Activity of the lipase was promoted by Ca2+ and Mg2+, and inhibited by Zn2+ and Cu2+. Moreover, LipG1 is stable with proteases, most commercial detergents and organic solvents. Substrate specificity test indicated that LipG1can hydrolyse pNP esters with acyl chain length from C2 to C16, with preference for medium-chain pNP esters (C8, C10). Lastly, LipG1was evaluated as an aquafeed additive for juvenile common carp (Cyprinus carpio). Results showed that supplementation of LipG1significantly improved the gut and heptaopancreas lipase activity of fish fed with palm oil diet. Consistently, improved feed conversion ratio and growth performance were recorded in the LipG1 feeding group, to levels comparable to the group of fish fed with soybean oil diet. Collectively, LipG1 exhibited good potential as an aquafeed additive enzyme, and deserves further characterization as the representative of a novel group of lipases. PMID:26147311

  12. Enhancing trimethylolpropane esters synthesis through lipase immobilized on surface hydrophobic modified support and appropriate substrate feeding methods.

    PubMed

    Tao, Yifeng; Cui, Caixia; Shen, Huaqing; Liu, Luo; Chen, Biqiang; Tan, Tianwei

    2014-05-10

    Candida sp. 99-125 lipase immobilized on surface hydrophobic modified support and appropriate substrate feeding methods were used to improve the synthesis of tri-substituted trimethylolpropane (TMP) esters, which can be used as raw materials for biodegradable lubricants. The proposed novel production method is environmentally friendly. Lipase was adsorbed on surface hydrophobic silk fibers that were pretreated by amino-modified polydimethylsiloxane. A 5-level-4-factors central composite model, including reaction time, temperature, enzyme amount, and molar ratio of fatty acid to TMP, was designed to evaluate the interaction of process variables in the enzymatic esterification. The water activity was kept constant using a LiCl-saturated salt solution. Under the optimum conditions with 30% enzyme amount and substrates molar ratio 8.4 at 45°C for 47h, the total conversion of caprylic acid is 97.3% and the yield of tri-substituted TMP esters is 95.5%. The surface hydrophobic treatment resulted in less cluster water accumulated on the surface immobilized lipase, which was demonstrated by near-infrared spectra. Consequently, the optimum temperature and water tolerance of immobilized lipase were increased. Two TMP-feeding methods were used to maintain high molar ratio of fatty acid to TMP, and increase the final tri-substituted TMP esters content exceeding 85% (w/w) in reactant. PMID:24731826

  13. Lipidomic analyses of female mice lacking hepatic lipase and endothelial lipase indicate selective modulation of plasma lipid species.

    PubMed

    Yang, Yanbo; Kuwano, Takashi; Lagor, William R; Albert, Carolyn J; Brenton, Siobhan; Rader, Daniel J; Ford, David A; Brown, Robert J

    2014-06-01

    Hepatic lipase (HL) and endothelial lipase (EL) share overlapping and complementary roles in lipoprotein metabolism. The deletion of HL and EL alleles in mice raises plasma total cholesterol and phospholipid concentrations. However, the influence of HL and EL in vivo on individual molecular species from each class of lipid is not known. We hypothesized that the loss of HL, EL, or both in vivo may affect select molecular species from each class of lipids. To test this hypothesis, we performed lipidomic analyses on plasma and livers from fasted female wild-type, HL-knockout, EL-knockout, and HL/EL-double knockout mice. Overall, the loss of HL, EL, or both resulted in minimal changes to hepatic lipids; however, select species of CE were surprisingly reduced in the livers of mice only lacking EL. The loss of HL, EL, or both reduced the plasma concentrations for select molecular species of triacylglycerol, diacylglycerol, and free fatty acid. On the other hand, the loss of HL, EL, or both raised the plasma concentrations for select molecular species of phosphatidylcholine, cholesteryl ester, diacylglycerol, sphingomyelin, ceramide, plasmanylcholine, and plasmenylcholine. The increased plasma concentration of select ether phospholipids was evident in the absence of EL, thus suggesting that EL might exhibit a phospholipase A2 activity. Using recombinant EL, we showed that it could hydrolyse the artificial phospholipase A2 substrate 4-nitro-3-(octanoyloxy)benzoic acid. In summary, our study shows for the first time the influence of HL and EL on individual molecular species of several classes of lipids in vivo using lipidomic methods. PMID:24777581

  14. Acinetobacter lipases: molecular biology, biochemical properties and biotechnological potential.

    PubMed

    Snellman, Erick A; Colwell, Rita R

    2004-10-01

    Lipases (EC 3.1.1.3) have received increased attention recently, evidenced by the increasing amount of information about lipases in the current literature. The renewed interest in this enzyme class is due primarily to investigations of their role in pathogenesis and their increasing use in biotechnological applications. Also, many microbial lipases are available as commercial products, the majority of which are used in detergents, cosmetic production, food flavoring, and organic synthesis. Lipases are valued biocatalysts because they act under mild conditions, are highly stable in organic solvents, show broad substrate specificity, and usually show high regio- and/or stereo-selectivity in catalysis. A number of lipolytic strains of Acinetobacter have been isolated from a variety of sources and their lipases possess many biochemical properties similar to those that have been developed for biotechnological applications. This review discusses the biology of lipase expression in Acinetobacter, with emphasis on those aspects relevant to potential biotechnology applications. PMID:15378387

  15. Novel lipase purification methods - a review of the latest developments.

    PubMed

    Tan, Chung Hong; Show, Pau Loke; Ooi, Chien Wei; Ng, Eng-Poh; Lan, John Chi-Wei; Ling, Tau Chuan

    2015-01-01

    Microbial lipases are popular biocatalysts due to their ability to catalyse diverse reactions such as hydrolysis, esterification, and acidolysis. Lipases function efficiently on various substrates in aqueous and non-aqueous media. Lipases are chemo-, regio-, and enantio-specific, and are useful in various industries, including those manufacturing food, detergents, and pharmaceuticals. A large number of lipases from fungal and bacterial sources have been isolated and purified to homogeneity. This success is attributed to the development of both conventional and novel purification techniques. This review highlights the use of these techniques in lipase purification, including conventional techniques such as: (i) ammonium sulphate fractionation; (ii) ion-exchange; (iii) gel filtration and affinity chromatography; as well as novel techniques such as (iv) reverse micellar system; (v) membrane processes; (vi) immunopurification; (vi) aqueous two-phase system; and (vii) aqueous two-phase floatation. A summary of the purification schemes for various bacterial and fungal lipases are also provided. PMID:25273633

  16. 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

  17. Structure-Guided Modification of Rhizomucor miehei Lipase for Production of Structured Lipids

    PubMed Central

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

    2013-01-01

    To improve the performance of yeast surface-displayed Rhizomucor miehei lipase (RML) in the production of human milk fat substitute (HMFS), we mutated amino acids in the lipase substrate-binding pocket based on protein hydrophobicity, to improve esterification activity. Five mutants: Asn87Ile, Asn87Ile/Asp91Val, His108Leu/Lys109Ile, Asp256Ile/His257Leu, and His108Leu/Lys109Ile/Asp256Ile/His257Leu were obtained and their hydrolytic and esterification activities were assayed. Using Discovery Studio 3.1 to build models and calculate the binding energy between lipase and substrates, compared to wild-type, the mutant Asp256Ile/His257Leu was found to have significantly lower energy when oleic acid (3.97 KJ/mol decrease) and tripalmitin (7.55 KJ/mol decrease) were substrates. This result was in accordance with the esterification activity of Asp256Ile/His257Leu (2.37-fold of wild-type). The four mutants were also evaluated for the production of HMFS in organic solvent and in a solvent-free system. Asp256Ile/His257Leu had an oleic acid incorporation of 28.27% for catalyzing tripalmitin and oleic acid, and 53.18% for the reaction of palm oil with oleic acid. The efficiency of Asp256Ile/His257Leu was 1.82-fold and 1.65-fold that of the wild-type enzyme for the two reactions. The oleic acid incorporation of Asp256Ile/His257Leu was similar to commercial Lipozyme RM IM for palm oil acidolysis with oleic acid. Yeast surface-displayed RML mutant Asp256Ile/His257Leu is a potential, economically feasible catalyst for the production of structured lipids. PMID:23844120

  18. L-amino acid ligase from Pseudomonas syringae producing tabtoxin can be used for enzymatic synthesis of various functional peptides.

    PubMed

    Arai, Toshinobu; Arimura, Yasuhiro; Ishikura, Shun; Kino, Kuniki

    2013-08-01

    Functional peptides are expected to be beneficial compounds that improve our quality of life. To address the growing need for functional peptides, we have examined peptide synthesis by using microbial enzymes. l-Amino acid ligase (Lal) catalyzes the condensation of unprotected amino acids in an ATP-dependent manner and is applicable to fermentative production. Hence, Lal is a promising enzyme to achieve cost-effective synthesis. To obtain a Lal with novel substrate specificity, we focused on the putative Lal involved in the biosynthesis of the dipeptidic phytotoxin designated tabtoxin. The tabS gene was cloned from Pseudomonas syringae NBRC14081 and overexpressed in Escherichia coli cells. The recombinant TabS protein produced showed the broadest substrate specificity of any known Lal; it detected 136 of 231 combinations of amino acid substrates when dipeptide synthesis was examined. In addition, some new substrate specificities were identified and unusual amino acids, e.g., l-pipecolic acid, hydroxy-l-proline, and β-alanine, were found to be acceptable substrates. Furthermore, kinetic analysis and monitoring of the reactions over a short time revealed that TabS showed distinct substrate selectivity at the N and C termini, which made it possible to specifically synthesize a peptide without by-products such as homopeptides and heteropeptides with the reverse sequence. TabS specifically synthesized the following functional peptides, including their precursors: l-arginyl-l-phenylalanine (antihypertensive effect; yield, 62%), l-leucyl-l-isoleucine (antidepressive effect; yield, 77%), l-glutaminyl-l-tryptophan (precursor of l-glutamyl-l-tryptophan, which has antiangiogenic activity; yield, 54%), l-leucyl-l-serine (enhances saltiness; yield, 83%), and l-glutaminyl-l-threonine (precursor of l-glutamyl-l-threonine, which enhances saltiness; yield, 96%). Furthermore, our results also provide new insights into tabtoxin biosynthesis. PMID:23770908

  19. 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

  20. Biosensor Applications of MAPLE Deposited Lipase

    PubMed Central

    Califano, Valeria; Bloisi, Francesco; Aronne, Antonio; Federici, Stefania; Nasti, Libera; Depero, Laura E.; Vicari, Luciano R. M.

    2014-01-01

    Matrix Assisted Pulsed Laser Evaporation (MAPLE) is a thin film deposition technique derived from Pulsed Laser Deposition (PLD) for deposition of delicate (polymers, complex biological molecules, etc.) materials in undamaged form. The main difference of MAPLE technique with respect to PLD is the target: it is a frozen solution or suspension of the (guest) molecules to be deposited in a volatile substance (matrix). Since laser beam energy is mainly absorbed by the matrix, damages to the delicate guest molecules are avoided, or at least reduced. Lipase, an enzyme catalyzing reactions borne by triglycerides, has been used in biosensors for detection of β-hydroxyacid esters and triglycerides in blood serum. Enzymes immobilization on a substrate is therefore required. In this paper we show that it is possible, using MAPLE technique, to deposit lipase on a substrate, as shown by AFM observation, preserving its conformational structure, as shown by FTIR analysis. PMID:25587426

  1. Efficient lipase purification using reverse micellar extraction.

    PubMed

    Gaikaiwari, Raghavendra P; Wagh, Shilpa A; Kulkarni, Bhaskar D

    2012-03-01

    Reverse micellar extraction (RME) of enzyme provides an attractive option for conventional method with the potential to achieve purification and concentration in a single step with high yield. This study presents a methodology for optimization of RME with Pseudomonas lipase as model system. Fold-purification, percent recovery and extraction time were the objective functions while the type and concentration of surfactant, contact time, pH, ionic strength, and the ratio of organic to aqueous phase were the decision variables. Under optimized conditions, the AOT (Aerosol OT (bis 2-ethylhexyl) sodium sulfosuccinate)-isooctane system gave a 15-fold purification, 80% recovery and 2.5-fold concentration of the Pseudomonas lipase with process time of 45 min. PMID:22230773

  2. Structural, functional and chemical changes in Pseudozyma antarctica lipase B on exposure to hydrogen peroxide.

    PubMed

    Törnvall, Ulrika; Hedström, Martin; Schillén, Karin; Hatti-Kaul, Rajni

    2010-12-01

    The effect on primary, secondary, tertiary and quaternary structure of Pseudozyma (formerly Candida) antarctica lipase B (PalB) on exposure to hydrogen peroxide was investigated using nano-electrospray ionization-mass spectrometry (nano-ESI-MS), liquid chromatography tandem mass spectrometry (LC/MS/MS), circular dichroism (CD), and dynamic light scattering (DLS). Treatment with hydrogen peroxide generated heavier protein variants, with a mass gain that increased with increasing incubation time. Furthermore, elevated concentration of H(2)O(2) was shown to result in partial fragmentation of the protein. Proteolytic digestion of the enzyme gave primary sequence coverage of more than 90%, revealing oxidation of methionine, tryptophan and cystine residues. The active site histidine was not observed in oxidized form in any of the experiments. However, oxidation of cystine to cysteic acid indicated disruption of disulphide bridges, and CD evaluations confirmed that severe changes to the secondary structure towards random coil had occurred. The structural changes could be an effect of the observed amino acid side chain oxidations, and was correlated with deactivation of the lipase. From DLS experiments, it was seen that the lipase exposed to both high temperature and H(2)O(2) formed large and intermediate sized aggregates, not observed for the heat-treated enzyme. The findings reported here could lay the basis for developing enzyme variants with higher oxidative stability. PMID:20654682

  3. Lipase-catalyzed esterification of 2-monoricinolein for 1,2 (2,3)-diricinolein synthesis.

    PubMed

    Turner, Charlotta; Wani, Seiji; Wong, Rosalind; Lin, Jiann-Tsyh; McKeon, Thomas

    2006-01-01

    The purpose of this investigation was to develop conditions for producing 2-monoricinoleoyl DAG. We used lipase-catalyzed hydrolysis of triricinolein to obtain 2-monoricinolein and thereafter synthesized 1,2(2,3)-diricinolein through esterification of 2-monoricinolein, using ricinoleic acid as the acyl donor. Five different 1,3-specific immobilized lipases were tested for the initial methanolysis reaction: Candida antarctica type B, Rhizomucor miehei, Rhizopus oryzae (ROL), Thermomyces lanuginosus, and Aspergillus niger. For the second esterification reaction, we investigated these five lipases plus Pseudomonas cepacia, Penicillium roquefortii, Candida rugosa, and Pseudomonas fluorescence. Toluene and diisopropyl ether (DIPE) were examined as reaction media at a water activity of 0.11. ROL in DIPE gave the highest yield of 2-monoricinolein from triricinolein, 78% after 3 h of reaction. The isolated 2-monoricinolein was esterified with ricinoleic acid for synthesis of 1,2(2,3)-diricinolein. ROL in DIPE gave the highest yield of 1,2(2,3)-diricinolein, 58% after 1 h of reaction, and NMR analysis showed that the purity was 97.2%. This methodology can be used for synthesizing radiolabeled 1,2(2,3)-diricinolein to study lipid biosynthesis in castor and other oilseeds. PMID:16555475

  4. Application of a Burkholderia cepacia lipase-immobilized silica monolith to batch and continuous biodiesel production with a stoichiometric mixture of methanol and crude Jatropha oil

    PubMed Central

    2011-01-01

    Background The enzymatic production of biodiesel through alcoholysis of triglycerides has become more attractive because it shows potential in overcoming the drawbacks of chemical processes. In this study, we investigate the production of biodiesel from crude, non-edible Jatropha oil and methanol to characterize Burkholderia cepacia lipase immobilized in an n-butyl-substituted hydrophobic silica monolith. We also evaluate the performance of a lipase-immobilized silica monolith bioreactor in the continuous production of biodiesel. Results The Jatropha oil used contained 18% free fatty acids, which is problematic in a base-catalyzed process. In the lipase-catalyzed reaction, the presence of free fatty acids made the reaction mixture homogeneous and allowed bioconversion to proceed to 90% biodiesel yield after a 12 hour reaction time. The optimal molar ratio of methanol to oil was 3.3 to 3.5 parts methanol to one part oil, with water content of 0.6% (w/w). Further experiments revealed that B. cepacia lipase immobilized in hydrophobic silicates was sufficiently tolerant to methanol, and glycerol adsorbed on the support disturbed the reaction to some extent in the present reaction system. The continuous production of biodiesel was performed at steady state using a lipase-immobilized silica monolith bioreactor loaded with 1.67 g of lipase. The yield of 95% was reached at a flow rate of 0.6 mL/h, although the performance of the continuous bioreactor was somewhat below that predicted from the batch reactor. The bioreactor was operated successfully for almost 50 days with 80% retention of the initial yield. Conclusions The presence of free fatty acids originally contained in Jatropha oil improved the reaction efficiency of the biodiesel production. A combination of B. cepacia lipase and its immobilization support, n-butyl-substituted silica monolith, was effective in the production of biodiesel. This procedure is easily applicable to the design of a continuous flow

  5. Sensitive impedimetric biosensor for direct detection of diazinon based on lipases

    NASA Astrophysics Data System (ADS)

    Jaffrezic-Renault, Nicole; Zehani, Nedjla; Dzyadevych, Sergei; Kherrat, Rochdi

    2014-07-01

    Two novel impedimetric biosensors for highly sensitive and rapid quantitative detection of diazinon in an aqueous medium were developed using two types of lipase, from Candida Rugosa (microbial source) (CRL) and from porcine pancreas (animal source) (PPL) immobilized onto a functionalized gold electrode. The lipase is characterized to specifically catalyze the hydrolysis of ester functions leading to the transformation of diazinon into diethyl phosphorothioic acid (DETP) and 2-isopropyl-4-methyl-6-hydroxypyrimidine (IMHP). The developed biosensors both presented a large wide range of linearity up to 50µM with a detection limit of 10 nM for the CRL biosensor and 0.1 µM for the PPL biosensor. A comparative study was carried out between the two biosensors and results showed higher sensitivity for the CRL sensor. Moreover, it presented good accuracy and reproducibility, and had very good storage and multiple use stability for 25 days when stored at 4°C.

  6. Sensitive impedimetric biosensor for direct detection of diazinon based on lipases

    PubMed Central

    Zehani, Nedjla; Dzyadevych, Sergei V.; Kherrat, Rochdi; Jaffrezic-Renault, Nicole J.

    2014-01-01

    Two novel impedimetric biosensors for highly sensitive and rapid quantitative detection of diazinon in aqueous medium were developed using two types of lipase, from Candida Rugosa (microbial source) (CRL) and from porcine pancreas (animal source) (PPL) immobilized on functionalized gold electrode. Lipase is characterized to specifically catalyze the hydrolysis of ester functions leading to the transformation of diazinon into diethyl phosphorothioic acid (DETP) and 2-isopropyl-4-methyl-6-hydroxypyrimidine (IMHP). The developed biosensors both presented a wide range of linearity up to 50 μM with a detection limit of 10 nM for Candida Rugosa biosensor and 0.1 μM for porcine pancreas biosensor. A comparative study was carried out between the two biosensors and results showed higher efficiency of Candida Rugosa sensor. Moreover, it presented good accuracy and reproducibility, had very good storage and multiple use stability for 25 days when stored at 4°C. PMID:25072052

  7. Residue Val237 is critical for the enantioselectivity of Penicillium expansum lipase.

    PubMed

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

    2014-03-01

    The shape of the hydrophobic tunnel leading to the active site of Penicillium expansum lipase (PEL) was redesigned by single-point mutations, in order to better understand enzyme enantioselectivity towards naproxen. A variant with a valine-to-glycine substitution at residue 237 exhibited almost no enantioselectivity (E = 1.1) compared with that (E = 104) of wild-type PEL. The function of the residue, Val237, in the hydrophobic tunnel was further analyzed by site-directed mutagenesis. For each of these variants a significant decrease of enantioselectivity (E < 7) was observed compared with that of wild-type enzyme. Further docking result showed that Val237 plays the most important role in stabilizing the correct orientation of (R)-naproxen. Overall, these results indicate that the residue Val237 is the key amino acid residue maintaining the enantioselectivity of the lipase. PMID:24338160

  8. Characterization of a novel testicular form of human hormone-sensitive lipase.

    PubMed

    Mairal, Aline; Melaine, Nathalie; Laurell, Henrik; Grober, Jacques; Holst, Lena Stenson; Guillaudeux, Thierry; Holm, Cecilia; Jégou, Bernard; Langin, Dominique

    2002-02-22

    Hormone-sensitive lipase (HSL) is an esterase and lipase, which are essential for spermatogenesis. Two HSL mRNAs are expressed in human testis. A long form is encoded by a testis-specific exon and nine exons common to testis and adipocyte HSL. Here we show that the short-form 3.3-kb mRNA possesses a unique 5' end that is transcribed from a novel testis-specific exon. The corresponding protein is similar to the 775-amino-acid-long adipocyte HSL. Immunohistochemistry experiments on human testis sections revealed that the long form is strictly expressed in haploid germ cells whereas the short form is expressed in interstitial and tubular somatic cells as well as premeiotic germ cells. PMID:11846402

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

    PubMed

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

    2011-01-01

    The objective of this work was to select the most suitable procedure to immobilize lipase from Penicillium camembertii (Lipase G). Different techniques and supports were evaluated, including physical adsorption on hydrophobic supports octyl-agarose, poly(hydroxybutyrate) and Amberlite resin XAD-4; ionic adsorption on the anionic exchange resin MANAE-agarose and covalent attachment on glyoxyl-agarose, MANAE-agarose cross-linked with glutaraldehyde, MANAE-agarose-glutaraldehyde, and epoxy-silica-polyvinyl alcohol composite. Among the tested protocols, the highest hydrolytic activity (128.2 ± 8.10 IU·g(-1) of support) was achieved when the lipase was immobilized on epoxy-SiO(2)-PVA using hexane as coupling medium. Lipase immobilized by ionic adsorption on MANAE-agarose also gave satisfactory result, attaining 55.6 ± 2.60 IU·g(-1) of support. In this procedure, the maximum loading of immobilized enzyme was 9.3 mg·g(-1) of gel, and the highest activity (68.8 ± 2.70 IU·g(-1) of support) was obtained when 20 mg of protein·g(-1) was offered. Immobilization carried out in aqueous medium by physical adsorption on hydrophobic supports and covalent attachment on MANAE-agarose-glutaraldehyde and glyoxyl-agarose was shown to be unfeasible for Lipase G. Thermal stability tests revealed that the immobilized derivative on epoxy-SiO(2)-PVA composite using hexane as coupling medium had a slight higher thermal stability than the free lipase. PMID:21811674

  10. Production of lipase by soil fungi and partial characterization of lipase from a selected strain (Penicillium wortmanii).

    PubMed

    Costa, M A; Peralta, R M

    1999-01-01

    Filamentous fungi from soil were screened for their ability to produce lipase. Among 56 filamentous fungi tested, one strain identified as Penicillium wortmanii was selected as the highest lipase producer. Maximum lipase production (12.5 U/ml) was obtained in 7-days cultures utilizing 5% (w/v) olive oil as the carbon source. Optimum pH and temperature for crude lipase were 7.0 and 45 degrees C, respectively. The enzyme was stable at 40 and 45 degrees C and it retained about 55% of its activity when heated at 50 degrees C for 1 hour. PMID:10071862

  11. Detergent compatible alkaline lipase produced by marine Bacillus smithii BTMS 11.

    PubMed

    Lailaja, V P; Chandrasekaran, M

    2013-08-01

    Bacillus smithii BTMS 11, isolated from marine sediment, produced alkaline and thermostable lipase. The enzyme was purified to homogeneity by ammonium sulfate precipitation and ion exchange chromatography which resulted in 0.51 % final yield and a 4.33 fold of purification. The purified enzyme was found to have a specific activity of 360 IU/mg protein. SDS-PAGE analyses, under non-reducing and reducing conditions, yielded a single band of 45 kDa indicating the single polypeptide nature of the enzyme and zymogram analysis using methylumbelliferyl butyrate as substrate confirmed the lipolytic activity of the protein band. The enzyme was found to have 50 °C and pH 8.0 as optimum conditions for maximal activity. However, the enzyme was active over wide range of temperatures (30-80 °C) and pH (7.0-10.0). Effect of a number of metal salts, solvents, surfactants, and other typical enzyme inhibitors on lipase activity was studied to determine the novel characteristics of the enzyme. More than 90 % of the enzyme activity was observed even after 3 h of incubation in the presence of commercial detergents Surf, Sunlight, Ariel, Henko, Tide and Ujala indicating the detergent compatibility of B. smithii lipase. The enzyme was also found to be efficient in stain removal from cotton cloths. Further it was observed that the enzyme could catalyse ester synthesis between fatty acids of varying carbon chain lengths and methanol with high preference for medium to long chain fatty acids showing 70 % of esterification. Results of the study indicated scope for application of this marine bacterial lipase in various industries. PMID:23443894

  12. Production and characterization of an extracellular lipase from Candida guilliermondii

    PubMed Central

    Oliveira, Anne Caroline Defranceschi; Fernandes, Maria Luiza; Mariano, André Bellin

    2014-01-01

    Extracellular lipases from the endophytic yeast Candida guilliermondii isolated from castor leaves (Ricinus communis L.) were produced using low-cost raw materials such as agro-industrial residues and applying them in the esterification of oleic acid for evaluating their potential use in biodiesel production. After partial purification using ammonium sulfate, the enzyme was characterized and presented higher activity (26.8 ± 1.5 U mL−1) in the presence of 5 mmol L−1 NaCl at 30 °C and pH 6.5. The production through submerged fermentation was formerly performed in 150 mL erlenmeyer flasks and, once the enzyme production was verified, assays in a 14 L bioreactor were conducted, obtaining 18 ± 1.4 U mL−1. The produced enzyme was applied in the oleic acid esterification under different solvents: hexane, cyclohexane or cyclohexanone) and different acid:alcohol molar ratios. Higher ester conversion rate (81%) was obtained using hexane and the molar ratio of 1:9 was the best conditions using methanol. The results suggest the potential for development of endophytic yeast in the production of biocatalyst through submerged fermentation using agroindustrial residues as culture medium. PMID:25763060

  13. Production and characterization of an extracellular lipase from Candida guilliermondii.

    PubMed

    Oliveira, Anne Caroline Defranceschi; Fernandes, Maria Luiza; Mariano, André Bellin

    2014-01-01

    Extracellular lipases from the endophytic yeast Candida guilliermondii isolated from castor leaves (Ricinus communis L.) were produced using low-cost raw materials such as agro-industrial residues and applying them in the esterification of oleic acid for evaluating their potential use in biodiesel production. After partial purification using ammonium sulfate, the enzyme was characterized and presented higher activity (26.8 ± 1.5 U mL(-1)) in the presence of 5 mmol L(-1) NaCl at 30 °C and pH 6.5. The production through submerged fermentation was formerly performed in 150 mL erlenmeyer flasks and, once the enzyme production was verified, assays in a 14 L bioreactor were conducted, obtaining 18 ± 1.4 U mL(-1). The produced enzyme was applied in the oleic acid esterification under different solvents: hexane, cyclohexane or cyclohexanone) and different acid:alcohol molar ratios. Higher ester conversion rate (81%) was obtained using hexane and the molar ratio of 1:9 was the best conditions using methanol. The results suggest the potential for development of endophytic yeast in the production of biocatalyst through submerged fermentation using agroindustrial residues as culture medium. PMID:25763060

  14. Monoacylglycerol lipase inhibition by organophosphorus compounds leads to elevation of brain 2-arachidonoylglycerol and the associated hypomotility in mice

    SciTech Connect

    Quistad, Gary B.; Klintenberg, Rebecka; Caboni, Pierluigi; Liang, Shannon N.; Casida, John E. . E-mail: ectl@nature.berkeley.edu

    2006-02-15

    Three components of the cannabinoid system are sensitive to selected organophosphorus (OP) compounds: monoacylglycerol (MAG) lipase that hydrolyzes the major endogenous agonist 2-arachidonoylglycerol (2-AG); fatty acid amide hydrolase (FAAH) that cleaves the agonist anandamide present in smaller amounts; the CB1 receptor itself. This investigation considers which component of the cannabinoid system is the most likely contributor to OP-induced hypomotility in mice. Structure-activity studies by our laboratory and others rule against major involvement of a direct toxicant-CB1 receptor interaction for selected OPs. Attention was therefore focused on the OP sensitivities of MAG lipase and FAAH, assaying 19 structurally diverse OP chemicals (pesticides, their metabolites and designer compounds) for in vitro inhibition of both enzymes. Remarkably high potency and low selectivity is observed with three O-alkyl (C{sub 1}, C{sub 2}, C{sub 3}) alkylphosphonofluoridates (C{sub 8}, C{sub 12}) (IC50 0.60-3.0 nM), five S-alkyl (C{sub 5}, C{sub 7}, C{sub 9}) and alkyl (C{sub 1}, C{sub 12}) benzodioxaphosphorin oxides (IC50 0.15-5.7 nM) and one OP insecticide metabolite (chlorpyrifos oxon, IC50 34-40 nM). In ip-treated mice, the OPs at 1-30 mg/kg more potently inhibit brain FAAH than MAG lipase, but FAAH inhibition is not correlated with hypomotility. However, the alkylphosphonofluoridate-treated mice show dose-dependent increases in severity of hypomotility, inhibition of MAG lipase activity and elevation of 2-AG. Moderate to severe hypomotility is accompanied by 64 to 86% MAG lipase inhibition and about 6-fold elevation of brain 2-AG level. It therefore appears that OP-induced MAG lipase inhibition leads to elevated 2-AG and the associated hypomotility.

  15. Production of thermostable lipase by Thermomyces lanuginosus on solid-state fermentation: selective hydrolysis of sardine oil.

    PubMed

    Avila-Cisneros, Nayeli; Velasco-Lozano, Susana; Huerta-Ochoa, Sergio; Córdova-López, Jesús; Gimeno, Miquel; Favela-Torres, Ernesto

    2014-11-01

    A naturally immobilized biocatalyst with lipase activity was produced by Thermomyces lanuginosus on solid-state fermentation with perlite as inert support. Maxima lipase activities (22 and 120 U/g of dry matter, using p-nitrophenyl octanoate and trioctanoine, respectively, as substrates) were obtained after 72 h of solid culture, remaining nearly constant up to 120 h. Maxima lipase activity was found at 60 to 85 °C and pH 10. The biocatalyst was stable at 60 °C for at least 4 h of incubation and a pH from 7 to 10. Energy values of activation and deactivation of lipase were of 26 and 6.7 kJ/mol, respectively. The biocatalyst shows high selectivity for the release of the omega-3 polyunsaturated fatty acids, eicosapentaenoic (EPA) and docosahexaenoic acids (DHA), during the hydrolysis of sardine oil. The EPA/DHA ratio (16:6) released by this biocatalyst was superior to that obtained with the commercial preparations of T. lanuginosus. PMID:25149456

  16. Characteristic of fungal lipases and their application in synthesis of structered lipids.

    PubMed

    Adamczak, M

    2003-01-01

    The experiments showed the possibility of the MLM type structured triacylglycerol synthesis. Such sTAGs should have polyene acids positioned in sn-2 (in this case evening primrose oil was the source of polyene acids) and caprylic acid in the sn-1 and sn-3 positions. The sTAG synthesis was carried out in two-stages: 1. alcoholysis and synthesis of 2-MAG; 2. 2-MAG esterification with caprylic acid. For further experiments 4 lipase preparations were selected: MCL, RDL, Lipozyme RM and TL. The lipase preparations synthesized 2-MAG with the yields of 85.7%,. 87.5%,. 54.3%,. 65.2%, respectively. The 2-MAGs separated by crystallization contained over 70% of C18:1 acid and over 7% of C18:3, n-6 acid. The two-staged sTAG synthesis was shown to be more efficient by analyzing the products composition. The two- stage process gave over 70% of MLM type triacylglycerols and about 3% of side-products, whereas the acidolysis or interesterification produced only about 30% MLM or MLL type triacylglycerols. PMID:24757817

  17. Production and Use of Lipases in Bioenergy: A Review from the Feedstocks to Biodiesel Production

    PubMed Central

    Ribeiro, Bernardo Dias; de Castro, Aline Machado; Coelho, Maria Alice Zarur; Freire, Denise Maria Guimarães

    2011-01-01

    Lipases represent one of the most reported groups of enzymes for the production of biofuels. They are used for the processing of glycerides and fatty acids for biodiesel (fatty acid alkyl esters) production. This paper presents the main topics of the enzyme-based production of biodiesel, from the feedstocks to the production of enzymes and their application in esterification and transesterification reactions. Growing technologies, such as the use of whole cells as catalysts, are addressed, and as concluding remarks, the advantages, concerns, and future prospects of enzymatic biodiesel are presented. PMID:21785707

  18. Adipose Triglyceride Lipase and Hormone-Sensitive Lipase Are Involved in Fat Loss in JunB-Deficient Mice

    PubMed Central

    Pinent, Montserrat; Prokesch, Andreas; Hackl, Hubert; Voshol, Peter J.; Klatzer, Ariane; Walenta, Evelyn; Panzenboeck, Ute; Kenner, Lukas; Trajanoski, Zlatko; Hoefler, Gerald

    2011-01-01

    Proteins of the activator protein-1 family are known to have roles in many physiological processes such as proliferation, apoptosis, and inflammation. However, their role in fat metabolism has yet to be defined in more detail. Here we study the impact of JunB deficiency on the metabolic state of mice. JunB knockout (JunB-KO) mice show markedly decreased weight gain, reduced fat mass, and a low survival rate compared with control mice. If fed a high-fat diet, the weight gain of JunB-KO mice is comparable to control mice and the survival rate improves dramatically. Along with normal expression of adipogenic marker genes in white adipose tissue (WAT) of JunB-KO mice, this suggests that adipogenesis per se is not affected by JunB deficiency. This is supported by in vitro data, because neither JunB-silenced 3T3-L1 cells nor mouse embryonic fibroblasts from JunB-KO mice show a change in adipogenic potential. Interestingly, the key enzymes of lipolysis, adipose triglyceride lipase and hormone-sensitive lipase, were significantly increased in WAT of fasted JunB-KO mice. Concomitantly, the ratio of plasma free fatty acids per gram fat mass was increased, suggesting an elevated lipolytic rate under fasting conditions. Furthermore, up-regulation of TNFα and reduced expression of perilipin indicate that this pathway is also involved in increased lipolytic rate in these mice. Additionally, JunB-KO mice are more insulin sensitive than controls and show up-regulation of lipogenic genes in skeletal muscle, indicating a shuttling of energy substrates from WAT to skeletal muscle. In summary, this study provides valuable insights into the impact of JunB deficiency on the metabolic state of mice. PMID:21540289

  19. A Novel Cold-Adapted Lipase from Sorangium cellulosum Strain So0157-2: Gene Cloning, Expression, and Enzymatic Characterization

    PubMed Central

    Cheng, Yuan-Yuan; Qian, Yun-Kai; Li, Zhi-Feng; Wu, Zhi-Hong; Liu, Hong; Li, Yue-Zhong

    2011-01-01

    Genome sequencing of cellulolytic myxobacterium Sorangium cellulosum reveals many open-reading frames (ORFs) encoding various degradation enzymes with low sequence similarity to those reported, but none of them has been characterized. In this paper, a predicted lipase gene (lipA) was cloned from S. cellulosum strain So0157-2 and characterized. lipA is 981-bp in size, encoding a polypeptide of 326 amino acids that contains the pentapeptide (GHSMG) and catalytic triad residues (Ser114, Asp250 and His284). Searching in the GenBank database shows that the LipA protein has only the 30% maximal identity to a human monoglyceride lipase. The novel lipA gene was expressed in Escherichia coli BL21 and the recombinant protein (r-LipA) was purified using Ni-NTA affinity chromatography. The enzyme hydrolyzed the p-nitrophenyl (pNP) esters of short or medium chain fatty acids (≤C10), and the maximal activity was on pNP acetate. The r- LipA is a cold-adapted lipase, with high enzymatic activity in a wide range of temperature and pH values. At 4 °C and 30 °C, the Km values of r-LipA on pNP acetate are 0.037 ± 0.001 and 0.174 ± 0.006 mM, respectively. Higher pH and temperature conditions promoted hydrolytic activity toward the pNP esters with longer chain fatty acids. Remarkably, this lipase retained much of its activity in the presence of commercial detergents and organic solvents. The results suggest that the r-LipA protein has some new characteristics potentially promising for industrial applications and S. cellulosum is an intriguing resource for lipase screening. PMID:22072918

  20. Novel Strategy of Using Methyl Esters as Slow Release Methanol Source during Lipase Expression by mut+ Pichia pastoris X33

    PubMed Central

    Kumari, Arti; Gupta, Rani

    2014-01-01

    One of the major issues with heterologous production of proteins in Pichia pastoris X33 under AOX1 promoter is repeated methanol induction. To obviate repeated methanol induction, methyl esters were used as a slow release source of methanol in lipase expressing mut+ recombinant. Experimental design was based on the strategy that in presence of lipase, methyl esters can be hydrolysed to release their products as methanol and fatty acid. Hence, upon break down of methyl esters by lipase, first methanol will be used as a carbon source and inducer. Then P. pastoris can switch over to fatty acid as a carbon source for multiplication and biomass maintenance till further induction by methyl esters. We validated this strategy using recombinant P. pastoris expressing Lip A, Lip C from Trichosporon asahii and Lip11 from Yarrowia lipolytica. We found that the optimum lipase yield under repeated methanol induction after 120 h was 32866 U/L, 28271 U/L and 21978 U/L for Lip C, Lip A and Lip 11 respectively. In addition, we found that a single dose of methyl ester supported higher production than repeated methanol induction. Among various methyl esters tested, methyl oleate (0.5%) caused 1.2 fold higher yield for LipA and LipC and 1.4 fold for Lip11 after 120 h of induction. Sequential utilization of methanol and oleic acid by P. pastoris was observed and was supported by differential peroxisome proliferation studies by transmission electron microscopy. Our study identifies a novel strategy of using methyl esters as slow release methanol source during lipase expression. PMID:25170843

  1. Novel strategy of using methyl esters as slow release methanol source during lipase expression by mut+ Pichia pastoris X33.

    PubMed

    Kumari, Arti; Gupta, Rani

    2014-01-01

    One of the major issues with heterologous production of proteins in Pichia pastoris X33 under AOX1 promoter is repeated methanol induction. To obviate repeated methanol induction, methyl esters were used as a slow release source of methanol in lipase expressing mut+ recombinant. Experimental design was based on the strategy that in presence of lipase, methyl esters can be hydrolysed to release their products as methanol and fatty acid. Hence, upon break down of methyl esters by lipase, first methanol will be used as a carbon source and inducer. Then P. pastoris can switch over to fatty acid as a carbon source for multiplication and biomass maintenance till further induction by methyl esters. We validated this strategy using recombinant P. pastoris expressing Lip A, Lip C from Trichosporon asahii and Lip11 from Yarrowia lipolytica. We found that the optimum lipase yield under repeated methanol induction after 120 h was 32866 U/L, 28271 U/L and 21978 U/L for Lip C, Lip A and Lip 11 respectively. In addition, we found that a single dose of methyl ester supported higher production than repeated methanol induction. Among various methyl esters tested, methyl oleate (0.5%) caused 1.2 fold higher yield for LipA and LipC and 1.4 fold for Lip11 after 120 h of induction. Sequential utilization of methanol and oleic acid by P. pastoris was observed and was supported by differential peroxisome proliferation studies by transmission electron microscopy. Our study identifies a novel strategy of using methyl esters as slow release methanol source during lipase expression. PMID:25170843

  2. 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

  3. 21 CFR 862.1465 - Lipase test system.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Lipase test system. 862.1465 Section 862.1465 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry Test Systems § 862.1465 Lipase test system. (a) Identification....

  4. Lipase production by diverse phylogenetic clades of Aureobasidium pullulans.

    PubMed

    Leathers, Timothy D; Rich, Joseph O; Anderson, Amber M; Manitchotpisit, Pennapa

    2013-10-01

    Thirty-nine strains representing 12 diverse phylogenetic clades of Aureobasidium pullulans were surveyed for lipase production using a quantitative assay. Strains in clades 4 and 10 produced 0.2-0.3 U lipase/ml, while color variant strain NRRL Y-2311-1 in clade 8 produced 0.54 U lipase/ml. Strains in clade 9, which exhibit a dark olivaceous pigment, produced the highest levels of lipase, with strain NRRL 62034 yielding 0.57 U lipase/ml. By comparison, Candida cylindracea strain NRRL Y-17506 produced 0.05 U lipase/ml under identical conditions. A. pullulans strain NRRL 62034 reached maximal lipase levels in 5 days on lipase induction medium, while A. pullulans strain NRRL Y-2311-1 and strains in clades 4 and 10 were highest after 6 days. A. pullulans strain NRRL Y-2311-1 and strains in clade 9 produced two extracellular proteins in common, at >50 and <37 kDa. PMID:23801121

  5. 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

  6. Unraveling the rationale behind organic solvent stability of lipases.

    PubMed

    Chakravorty, Debamitra; Parameswaran, Saravanan; Dubey, Vikash Kumar; Patra, Sanjukta

    2012-06-01

    Organic solvent-stable lipases have pronounced impact on industrial economy as they are involved in synthesis by esterification, interesterification, and transesterification. However, very few of such natural lipases have been isolated till date. A study of the recent past provided few pillars to rely on for this work. The three-dimensional structure, inclusive of the surface and active site, of 29 organic solvent-stable lipases was analyzed by subfamily classification and protein solvent molecular docking based on fast Fourier transform correlation approach. The observations revealed that organic solvent stability of lipases is their intrinsic property and unique with respect to each lipase. In this paper, factors like surface distribution of charged, hydrophobic, and neutral residues, interaction of solvents with catalytically immutable residues, and residues interacting with essential water molecules required for lipase activity, synergistically and by mutualism contribute to render a stable lipase organic solvent. The propensity of surface charge in relation to stability in organic solvents by establishing repulsive forces to exclude solvent molecules from interacting with the surface and prohibiting the same from gaining entry to the protein core, thus stabilizing the active conformation, is a new finding. It was also interesting to note that lipases having equivalent surface-exposed positive and negative residues were stable in a wide range of organic solvents, irrespective of their LogP values. PMID:22562495

  7. 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

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

    PubMed Central

    Zhao, Minjie; Fu, Guanwen; Lai, Jia; Feng, Fengqin

    2015-01-01

    The enzymatic synthesis of 1,3-dioleoyl-2-palmitoylglycerol (OPO), one of the main components of human milk fats, has been hindered by the relatively high cost of sn-1,3-specific lipases and the deficiency in biocatalyst stability. The sn-1,3-specific lipase from Aspergillus oryzae (AOL) is highly and efficiently immobilized with the polystyrene-based hydrophobic resin D3520, with a significant 49.54-fold increase in specific lipase activity compared with the AOL powder in catalyzing the synthesis of OPO through the acidolysis between palm stearin and oleic acid (OA). The optimal immobilization conditions were investigated, including time course, initial protein concentration and solution pH. The sn-1,3 specificity of lipases under different immobilization conditions was evaluated and identified as positively associated with the lipase activity, and the pH of the immobilization solution influenced the regiospecificity and synthetic activity of these lipases. Immobilized AOL D3520, as the biocatalyst, was used for the enzymatic synthesis of the structured lipid OPO through the acidolysis between palm stearin and OA. The following conditions were optimized for the synthesis of structured lipid OPO: 65 °C temperature; 1:8 substrate molar ratio between palm stearin and OA; 8% (w/w) enzyme load; 3.5% water content of the immobilized lipase; and 1 h reaction time. Under these conditions, highly efficient C52 production (45.65%) was achieved, with a tripalmitin content of 2.75% and a sn-2 palmitic acid (PA) proportion of 55.08% in the system. PMID:26218640

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

    PubMed

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

    2015-01-01

    The enzymatic synthesis of 1,3-dioleoyl-2-palmitoylglycerol (OPO), one of the main components of human milk fats, has been hindered by the relatively high cost of sn-1,3-specific lipases and the deficiency in biocatalyst stability. The sn-1,3-specific lipase from Aspergillus oryzae (AOL) is highly and efficiently immobilized with the polystyrene-based hydrophobic resin D3520, with a significant 49.54-fold increase in specific lipase activity compared with the AOL powder in catalyzing the synthesis of OPO through the acidolysis between palm stearin and oleic acid (OA). The optimal immobilization conditions were investigated, including time course, initial protein concentration and solution pH. The sn-1,3 specificity of lipases under different immobilization conditions was evaluated and identified as positively associated with the lipase activity, and the pH of the immobilization solution influenced the regiospecificity and synthetic activity of these lipases. Immobilized AOL D3520, as the biocatalyst, was used for the enzymatic synthesis of the structured lipid OPO through the acidolysis between palm stearin and OA. The following conditions were optimized for the synthesis of structured lipid OPO: 65 °C temperature; 1:8 substrate molar ratio between palm stearin and OA; 8% (w/w) enzyme load; 3.5% water content of the immobilized lipase; and 1 h reaction time. Under these conditions, highly efficient C52 production (45.65%) was achieved, with a tripalmitin content of 2.75% and a sn-2 palmitic acid (PA) proportion of 55.08% in the system. PMID:26218640

  10. Lipase-catalyzed synthesis of ascorbyl oleate in acetone: optimization of reaction conditions and lipase reusability.

    PubMed

    Stojanović, Marija; Velićković, Dušan; Dimitrijević, Aleksandra; Milosavić, Nenad; Knežević-Jugović, Zorica; Bezbradica, Dejan

    2013-01-01

    Lipase-catalyzed ascorbyl oleate synthesis is eco-friendly and selective way of production of liposoluble biocompatible antioxidants, but still not present on an industrial level due to the high biocatalyst costs. In this study, response surface methodology was applied in order to estimate influence of individual experimental factors, identify interactions among them, and to determine optimum conditions for enzymatic synthesis of ascorbyl oleate in acetone, in terms of limiting substrate conversion, product yield, and yield per mass of consumed enzyme. As a biocatalyst, commercial immobilized preparation of lipase B from Candida antarctica, Novozym 435, was used. In order to develop cost-effective process, at reaction conditions at which maximum amount of product per mass of biocatalyst was produced (60°C, 0.018 % (v/v) of water, 0.135 M of vitamin C, substrates molar ratio 1:8, and 0.2 % (w/v) of lipase), possibilities for further increase of ester yield were investigated. Addition of molecular sieves at 4(th) hour of reaction enabled increase of yield from 16.7 mmol g⁻¹ to 19.3 mmol g⁻¹. Operational stability study revealed that after ten reaction cycles enzyme retained 48 % of its initial activity. Optimized synthesis with well-timed molecular sieves addition and repeated use of lipase provided production of 153 mmol per gram of enzyme. Further improvement of productivity was achieved using procedure for the enzyme reactivation. PMID:23985489

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

    PubMed

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

    2011-10-01

    Members of the genus Nepenthes are carnivorous plants that use the pitfall method of insect capture as a supplementary nutritional source. We extracted metagenomic DNA from the microbial community found in the pitcher fluid of Nepenthes and constructed a plasmid-based metagenomic library. An activity-based screening method enabled the isolation of two lipase genes, lip1 and lip2. Both Lip1 and Lip2 belong to a novel family or subfamily of lipases and show lipase activities in acidic conditions, such as those found in pitcher fluid. This study was conducted under the assumption that the secreted Lip1 and Lip2 were capable of enzymatic activity in the acidic pitcher fluid. PMID:21778111

  12. Genomic organization of the murine CTL lipase gene

    SciTech Connect

    Kaplan, M.H.; Boyer, S.N.; Grusby, M.J.

    1996-08-01

    Murine cytotoxic T-lymphocyte (CTL) lipase was originally identified as an IL-4-inducible gene in CD8-positive T cells. To further our understanding of both the function and the regulation of CTL lipase in T cells, we have cloned and characterized the murine gene. Two overlapping phage clones spanning 29 kb contain the entire CTL lipase gene. The exon structure in similar to those characterized for the human and canine pancreatic lipase-related protein 1 genes, with notable differences in the 5{prime} end. Transcripts initiate from a site that matches a consensus for an initiator sequence. Potential cis-regulatory elements in the CTL lipase 5{prime} regulatory region that would confer dual tissue specificity in exocrine pancreas and cytotoxic T lymphocytes are identified. The implications of this promoter organization are discussed. 27 refs., 2 figs.

  13. Monoolein production by triglycerides hydrolysis using immobilized Rhizopus oryzae lipase.

    PubMed

    Ghattas, Nesrine; Abidi, Ferid; Galai, Said; Marzouki, M Nejib; Salah, Abderraouf Ben

    2014-07-01

    Lipase extracted from Rhizopus oryzae was immobilized in alginate gel beads. The effects of the immobilization conditions, such as, alginate concentration, CaCl2 concentration and amount of initial enzyme on retained activity (specific activity ratio of entrapped active lipase to free lipase) were investigated. The optimal conditions for lipase entrapment were determined: 2% (w/v) alginate concentration, 100mM CaCl2 and enzyme ratio of 2000IU/mL.In such conditions, immobilized lipase by inclusion in alginate showed a highest stability and activity, on olive oil hydrolysis reaction where it could be reused for 10 cycles. After 15min of hydrolysis reaction, the mass composition of monoolein, diolein and triolein were about 78%, 10% and 12%. Hydrolysis' products purification by column chromatography lead to a successful separation of reaction compounds and provide a pure fraction of monoolein which is considered as the widest used emulsifier in food and pharmaceutical industries. PMID:24755261

  14. ROG1 encodes a monoacylglycerol lipase in Saccharomyces cerevisiae.

    PubMed

    Vishnu Varthini, Lakshmanaperumal; Selvaraju, Kandasamy; Srinivasan, Malathi; Nachiappan, Vasanthi

    2015-01-01

    Lipid metabolism is extensively studied in Saccharomyces cerevisiae. Here, we report that revertant of glycogen synthase kinase mutation-1 (Rog1p) possesses monoacylglycerol (MAG) lipase activity in S. cerevisiae. The lipase activity of Rog1p was confirmed in two ways: through analysis of a strain with a double deletion of ROG1 and monoglyceride lipase YJU3 (yju3Δrog1Δ) and by site-directed mutagenesis of the ROG1 lipase motif (GXSXG). Rog1p is localized in both the cytosol and the nucleus. Overexpression of ROG1 in a ROG1-deficient strain resulted in an accumulation of reactive oxygen species. These results suggest that Rog1p is a MAG lipase that regulates lipid homeostasis. PMID:25433290

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

    PubMed

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

    2014-01-01

    In this work, lipase from Candida rugosa was immobilized onto chitosan/graphene oxide beads. This was to provide an enzyme-immobilizing carrier with excellent enzyme immobilization activity for an enzyme group requiring hydrophilicity on the immobilizing carrier. In addition, this work involved a process for the preparation of an enzymatically active product insoluble in a reaction medium consisting of lauric acid and oleyl alcohol as reactants and hexane as a solvent. This product enabled the stability of the enzyme under the working conditions and allowed the enzyme to be readily isolated from the support. In particular, this meant that an enzymatic reaction could be stopped by the simple mechanical separation of the "insoluble" enzyme from the reaction medium. Chitosan was incorporated with graphene oxide because the latter was able to enhance the physical strength of the chitosan beads by its superior mechanical integrity and low thermal conductivity. The X-ray diffraction pattern showed that the graphene oxide was successfully embedded within the structure of the chitosan. Further, the lipase incorporation on the beads was confirmed by a thermo-gravimetric analysis. The lipase immobilization on the beads involved the functionalization with coupling agents, N-hydroxysulfosuccinimide sodium (NHS) and 1-ethyl-(3-dimethylaminopropyl) carbodiimide (EDC), and it possessed a high enzyme activity of 64 U. The overall esterification conversion of the prepared product was 78% at 60 °C, and it attained conversions of 98% and 88% with commercially available lipozyme and novozyme, respectively, under similar experimental conditions. PMID:25127038

  16. Extraordinary enantiospecificity of lipase catalysis in organic media induced by purification and catalyst engineering.

    PubMed

    Tsai, S W; Dordick, J S

    1996-10-20

    A purified lipase preparation from Candida rugosa was compared to its crude counterpart in anhydrous and slightly hydrated hydrophobic organic solvents. The purified lipase preparation was less active than the crude enzyme in dry n-heptane, whereas the presence of small concentrations of added water dramatically activated the purified enzyme but not the crude enzyme. Thus, in the presence of as little as 0.25 microL/mL of added water in n-heptane, the purified enzyme is over 230-fold more active and 6-fold more enantioselective than the dry enzyme suspension in the esterification of racemic 2-(4-chlorophenoxy)propionic acid with n-butanol. The reactivity and selectivity of this biocatalyst, however, was affected by coalescence of the enzyme preparation suspended in the wet organic solvent. Engineering the biocatalyst environment by dissolving the purified lipase in aqueous buffer and then adding this solution to n-heptane resulted in a precipitated enzyme preparation with smaller particle sizes that did not coalesce severely. In the presence of 5 microL/mL of water added with the enzyme, this pretreatment resulted in an activation over the dry, purified enzyme preparation of over 1800-fold and nearly enantiospecific catalysis (E > 100). Hence, by simply modifying the way enzymes are hydrated, dramatic activation of catalytic competency can be achieved. PMID:18629896

  17. Optimized production of biodiesel from waste cooking oil by lipase immobilized on magnetic nanoparticles.

    PubMed

    Yu, Chi-Yang; Huang, Liang-Yu; Kuan, I-Ching; Lee, Shiow-Ling

    2013-01-01

    Biodiesel, a non-toxic and biodegradable fuel, has recently become a major source of renewable alternative fuels. Utilization of lipase as a biocatalyst to produce biodiesel has advantages over common alkaline catalysts such as mild reaction conditions, easy product separation, and use of waste cooking oil as raw material. In this study, Pseudomonas cepacia lipase immobilized onto magnetic nanoparticles (MNP) was used for biodiesel production from waste cooking oil. The optimal dosage of lipase-bound MNP was 40% (w/w of oil) and there was little difference between stepwise addition of methanol at 12 h- and 24 h-intervals. Reaction temperature, substrate molar ratio (methanol/oil), and water content (w/w of oil) were optimized using response surface methodology (RSM). The optimal reaction conditions were 44.2 °C, substrate molar ratio of 5.2, and water content of 12.5%. The predicted and experimental molar conversions of fatty acid methyl esters (FAME) were 80% and 79%, respectively. PMID:24336109

  18. Plastic fats from sal, mango and palm oil by lipase catalyzed interesterification.

    PubMed

    Shankar Shetty, Umesha; Sunki Reddy, Yella Reddy; Khatoon, Sakina

    2014-02-01

    Speciality plastic fats with no trans fatty acids suitable for use in bakery and as vanaspati substitute were prepared by interesterification of blends of palm stearin (PSt) with sal and mango fats using Lipozyme TLIM lipase as catalyst. The blends containing PSt/sal or PSt/mango showed short melting range and hence are not suitable as bakery shortenings. Lipase catalysed interesterification extended the plasticity or melting range of all the blends. The blends containing higher proportion of PSt with sal fat (50/50) were harder having high solids at and above body temperature and hence cannot be used as bakery shortenings. The blends with PSt/sal (30-40/60-70) after interesterification showed melting profiles similar to those of commercial hydrogenated bakery fats. Similarly, the blends containing PSt/mango (30-40/60-70) after interesterification also showed melting profiles similar to those of commercial hydrogenated shortenings. The slip melting point and solidification characteristics also confirm the plastic nature of these samples. The improvement in plasticity after interesterification is due to formation of higher melting as well as lower melting triglycerides during lipase catalysed interesterification. PMID:24493889

  19. Lipases Immobilization for Effective Synthesis of Biodiesel Starting from Coffee Waste Oils

    PubMed Central

    Ferrario, Valerio; Veny, Harumi; De Angelis, Elisabetta; Navarini, Luciano; Ebert, Cynthia; Gardossi, Lucia

    2013-01-01

    Immobilized lipases were applied to the enzymatic conversion of oils from spent coffee ground into biodiesel. Two lipases were selected for the study because of their conformational behavior analysed by Molecular Dynamics (MD) simulations taking into account that immobilization conditions affect conformational behavior of the lipases and ultimately, their efficiency upon immobilization. The enzymatic synthesis of biodiesel was initially carried out on a model substrate (triolein) in order to select the most promising immobilized biocatalysts. The results indicate that oils can be converted quantitatively within hours. The role of the nature of the immobilization support emerged as a key factor affecting reaction rate, most probably because of partition and mass transfer barriers occurring with hydrophilic solid supports. Finally, oil from spent coffee ground was transformed into biodiesel with yields ranging from 55% to 72%. The synthesis is of particular interest in the perspective of developing sustainable processes for the production of bio-fuels from food wastes and renewable materials. The enzymatic synthesis of biodiesel is carried out under mild conditions, with stoichiometric amounts of substrates (oil and methanol) and the removal of free fatty acids is not required. PMID:24970178

  20. 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

  1. Tetrahydrolipstatin Inhibition, Functional Analyses, and Three-dimensional Structure of a Lipase Essential for Mycobacterial Viability

    SciTech Connect

    Crellin, Paul K.; Vivian, Julian P.; Scoble, Judith; Chow, Frances M.; West, Nicholas P.; Brammananth, Rajini; Proellocks, Nicholas I.; Shahine, Adam; Le Nours, Jerome; Wilce, Matthew C.J.; Britton, Warwick J.; Coppel, Ross L.; Rossjohn, Jamie; Beddoe, Travis

    2010-09-17

    The highly complex and unique mycobacterial cell wall is critical to the survival of Mycobacteria in host cells. However, the biosynthetic pathways responsible for its synthesis are, in general, incompletely characterized. Rv3802c from Mycobacterium tuberculosis is a partially characterized phospholipase/thioesterase encoded within a genetic cluster dedicated to the synthesis of core structures of the mycobacterial cell wall, including mycolic acids and arabinogalactan. Enzymatic assays performed with purified recombinant proteins Rv3802c and its close homologs from Mycobacterium smegmatis (MSMEG{_}6394) and Corynebacterium glutamicum (NCgl2775) show that they all have significant lipase activities that are inhibited by tetrahydrolipstatin, an anti-obesity drug that coincidently inhibits mycobacterial cell wall biosynthesis. The crystal structure of MSMEG{_}6394, solved to 2.9 {angstrom} resolution, revealed an {alpha}/{beta} hydrolase fold and a catalytic triad typically present in esterases and lipases. Furthermore, we demonstrate direct evidence of gene essentiality in M. smegmatis and show the structural consequences of loss of MSMEG{_}6394 function on the cellular integrity of the organism. These findings, combined with the predicted essentiality of Rv3802c in M. tuberculosis, indicate that the Rv3802c family performs a fundamental and indispensable lipase-associated function in mycobacteria.

  2. Cloning, expression and characterization of a lipase encoding gene from human oral metagenome.

    PubMed

    Preeti, Arivaradarajan; Hemalatha, Devaraj; Rajendhran, Jeyaprakash; Mullany, Peter; Gunasekaran, Paramasamy

    2014-09-01

    The human oral metagenomic DNA cloned into plasmid pUC19 was used to construct a DNA library in Escherichia coli. Functional screening of 40,000 metagenomic clones led to identification of a clone LIP2 that exhibited halo on tributyrin agar plate. Sequence analysis of LIP2 insert DNA revealed a 939 bp ORF (omlip1) which showed homology to lipase 1 of Acinetobacter junii SH205. The omlip1 ORF was cloned and expressed in E. coli BL21 (DE3) using pET expression system. The recombinant enzyme was purified to homogeneity and the biochemical properties were studied. The purified OMLip1 hydrolyzed p-nitrophenyl esters and triacylglycerol esters of medium and long chain fatty acids, indicating the enzyme is a true lipase. The purified protein exhibited a pH and temperature optima of 7 and 37 °C respectively. The lipase was found to be stable at pH range of 6-7 and at temperatures lower than 40 °C. Importantly, the enzyme activity was unaltered, by the presence or absence of many divalent cations. The metal ion insensitivity of OMLip1offers its potential use in industrial processes. PMID:24891735

  3. Optimal production and biochemical properties of a lipase from Candida albicans.

    PubMed

    Lan, Dongming; Hou, Shulin; Yang, Ning; Whiteley, Chris; Yang, Bo; Wang, Yonghua

    2011-01-01

    Lipases from microorganisms have multi-faceted properties and play an important role in ever-growing modern biotechnology and, consequently, it is of great significance to develop new ones. In the present work, a lipase gene from Candida albicans (CaLIP10) was cloned and two non-unusual CUG serine codons were mutated into universal codons, and its expression in Pichia pastoris performed optimally, as shown by response surface methodology. Optimal conditions were: initial pH of culture 6.86, temperature 25.53 °C, 3.48% of glucose and 1.32% of yeast extract. The corresponding maximal lipolytic activity of CaLIP10 was 8.06 U/mL. The purified CaLIP10 showed maximal activity at pH 8.0 and 25 °C, and a good resistance to non-ionic surfactants and polar organic solvent was noticed. CaLIP10 could effectively hydrolyze coconut oil, but exhibited no obvious preference to the fatty acids with different carbon length, and diacylglycerol was accumulated in the reaction products, suggesting that CaLIP10 is a potential lipase for the oil industry. PMID:22072943

  4. Optimal Production and Biochemical Properties of a Lipase from Candida albicans

    PubMed Central

    Lan, Dongming; Hou, Shulin; Yang, Ning; Whiteley, Chris; Yang, Bo; Wang, Yonghua

    2011-01-01

    Lipases from microorganisms have multi-faceted properties and play an important role in ever-growing modern biotechnology and, consequently, it is of great significance to develop new ones. In the present work, a lipase gene from Candida albicans (CaLIP10) was cloned and two non-unusual CUG serine codons were mutated into universal codons, and its expression in Pichia pastoris performed optimally, as shown by response surface methodology. Optimal conditions were: initial pH of culture 6.86, temperature 25.53 °C, 3.48% of glucose and 1.32% of yeast extract. The corresponding maximal lipolytic activity of CaLIP10 was 8.06 U/mL. The purified CaLIP10 showed maximal activity at pH 8.0 and 25 °C, and a good resistance to non-ionic surfactants and polar organic solvent was noticed. CaLIP10 could effectively hydrolyze coconut oil, but exhibited no obvious preference to the fatty acids with different carbon length, and diacylglycerol was accumulated in the reaction products, suggesting that CaLIP10 is a potential lipase for the oil industry. PMID:22072943

  5. Enhanced Biocatalytic Esterification with Lipase-Immobilized Chitosan/Graphene Oxide Beads

    PubMed Central

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

    2014-01-01

    In this work, lipase from Candida rugosa was immobilized onto chitosan/graphene oxide beads. This was to provide an enzyme-immobilizing carrier with excellent enzyme immobilization activity for an enzyme group requiring hydrophilicity on the immobilizing carrier. In addition, this work involved a process for the preparation of an enzymatically active product insoluble in a reaction medium consisting of lauric acid and oleyl alcohol as reactants and hexane as a solvent. This product enabled the stability of the enzyme under the working conditions and allowed the enzyme to be readily isolated from the support. In particular, this meant that an enzymatic reaction could be stopped by the simple mechanical separation of the “insoluble” enzyme from the reaction medium. Chitosan was incorporated with graphene oxide because the latter was able to enhance the physical strength of the chitosan beads by its superior mechanical integrity and low thermal conductivity. The X-ray diffraction pattern showed that the graphene oxide was successfully embedded within the structure of the chitosan. Further, the lipase incorporation on the beads was confirmed by a thermo-gravimetric analysis. The lipase immobilization on the beads involved the functionalization with coupling agents, N-hydroxysulfosuccinimide sodium (NHS) and 1-ethyl-(3-dimethylaminopropyl) carbodiimide (EDC), and it possessed a high enzyme activity of 64 U. The overall esterification conversion of the prepared product was 78% at 60°C, and it attained conversions of 98% and 88% with commercially available lipozyme and novozyme, respectively, under similar experimental conditions. PMID:25127038

  6. A green approach for starch modification: Esterification by lipase and novel imidazolium surfactant.

    PubMed

    Adak, Sunita; Banerjee, Rintu

    2016-10-01

    Starch being one of the most abundant polysaccharides in nature has been subjected to modification to enhance its applicability. Modification by esterification involves acylation of hydroxyl groups of glucose units to form starch esters. Lipases, as catalysts have emerged as a promising alternative to chemical processes. Although ionic liquids and microwave assisted heating are emerging as green technology yet their use along with lipases for starch modification has not been probed. In the present study esterification of corn starch employing Rhizopus oryzae lipase, microwave irradiation and novel imidazolium surfactants has been attempted. At 80% irradiation, 1:3 starch/oleic acid molar ratio, 150 IU enzyme, and 50μmol of [C16-3-C16im]Br2 maximum degree of substitution (DS=2.75) was attained. The modified starch showed better hydrophobicity and thermoplasticity with corresponding structural changes depicted by FTIR, XRD and SEM. These properties advocate the usefulness of the modified starch in food and biopolymer sectors. PMID:27312646

  7. Peroxisome extensions deliver the Arabidopsis SDP1 lipase to oil bodies.

    PubMed

    Thazar-Poulot, Nelcy; Miquel, Martine; Fobis-Loisy, Isabelle; Gaude, Thierry

    2015-03-31

    Lipid droplets/oil bodies (OBs) are lipid-storage organelles that play a crucial role as an energy resource in a variety of eukaryotic cells. Lipid stores are mobilized in the case of food deprivation or high energy demands--for example, during certain developmental processes in animals and plants. OB degradation is achieved by lipases that hydrolyze triacylglycerols (TAGs) into free fatty acids and glycerol. In the model plant Arabidopsis thaliana, Sugar-Dependent 1 (SDP1) was identified as the major TAG lipase involved in lipid reserve mobilization during seedling establishment. Although the enzymatic activity of SDP1 is associated with the membrane of OBs, its targeting to the OB surface remains uncharacterized. Here we demonstrate that the core retromer, a complex involved in protein trafficking, participates in OB biogenesis, lipid store degradation, and SDP1 localization to OBs. We also report an as-yet-undescribed mechanism for lipase transport in eukaryotic cells, with SDP1 being first localized to the peroxisome membrane at early stages of seedling growth and then possibly moving to the OB surface through peroxisome tubulations. Finally, we show that the timely transfer of SDP1 to the OB membrane requires a functional core retromer. In addition to revealing previously unidentified functions of the retromer complex in plant cells, our work provides unanticipated evidence for the role of peroxisome dynamics in interorganelle communication and protein transport. PMID:25775518

  8. Optimized Production of Biodiesel from Waste Cooking Oil by Lipase Immobilized on Magnetic Nanoparticles

    PubMed Central

    Yu, Chi-Yang; Huang, Liang-Yu; Kuan, I-Ching; Lee, Shiow-Ling

    2013-01-01

    Biodiesel, a non-toxic and biodegradable fuel, has recently become a major source of renewable alternative fuels. Utilization of lipase as a biocatalyst to produce biodiesel has advantages over common alkaline catalysts such as mild reaction conditions, easy product separation, and use of waste cooking oil as raw material. In this study, Pseudomonas cepacia lipase immobilized onto magnetic nanoparticles (MNP) was used for biodiesel production from waste cooking oil. The optimal dosage of lipase-bound MNP was 40% (w/w of oil) and there was little difference between stepwise addition of methanol at 12 h- and 24 h-intervals. Reaction temperature, substrate molar ratio (methanol/oil), and water content (w/w of oil) were optimized using response surface methodology (RSM). The optimal reaction conditions were 44.2 °C, substrate molar ratio of 5.2, and water content of 12.5%. The predicted and experimental molar conversions of fatty acid methyl esters (FAME) were 80% and 79%, respectively. PMID:24336109

  9. Studies on immobilization and partial characterization of lipases from wheat seeds (Triticum aestivum).

    PubMed

    Pierozan, Morgana Karin; Oestreicher, Enrique Guillermo; Oliveira, J Vladimir; Oliveira, Débora; Treichel, Helen; Cansian, Rogério Luís

    2011-09-01

    The objective of this study was to provide some features on immobilization and partial characterization of lipases from wheat seeds. The optimum pH and temperature were found to be 5.5 and 32-37 °C, respectively. The stability of the concentrated enzymatic extract to high temperatures (25, 35, 45, and 55 °C) showed that the incubation of the extract at 55 °C led to its complete inactivation. The concentrated enzymatic extract kept 90% of its hydrolytic and esterification activities until 70 and 40 days of storage at 4 °C, respectively. The extract presented higher hydrolytic specificity to substrates of medium and long chains and higher esterification affinity to fatty acids of short and medium chains and alcohols with two and three carbon atoms. After the immobilization process using activated coal and sodium alginate as supports, an enhancement of about threefold in lipase activity was observed. The development of the present work permitted us to point out some characteristics of lipases from wheat seeds necessary for the proposition of new future industrial applications for this important biocatalyst. PMID:21468634

  10. Lipase production from a novel thermo-tolerant and extreme acidophile Bacillus pumilus using palm oil as the substrate and treatment of palm oil-containing wastewater.

    PubMed

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

    2014-03-01

    The thermo-tolerant and extreme acidophilic microorganism Bacillus pumilus was isolated from the soil collected from a commercial edible-oil extraction industry. Optimisation of conditions for the lipase production was conducted using response surface methodology. The optimum conditions for obtaining the maximum activity (1,100 U/mL) of extremely acidic thermostable lipase were fermentation time, 96 h; pH, 1; temperature, 50 °C; and 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 thermo-tolerant acidophilic lipase (TAL) was 55 kDa. The predominant amino acid in the TAL was glycine. The functional groups of lipase were determined by Fourier transform infrared spectroscopy. TAL exhibited enhanced activity (114 %) with dimethyl sulphoxide (20 %, v/v), and it showed a moderate activity with methanol, hexane and benzene. The optimum conditions for the treatment of palm oil in wastewater using the TAL were found to be time, 3 h; pH, 1; temperature, 50 °C with pseudo second-order kinetic constant of 1.88 × 10(-3) L mol(-1) min(-1). The Michaelis-Menten enzyme kinetic model and the nonlinear kinetic model were evaluated for the TAL. TAL established hydrolysis efficiency of 96 % for palm oil in wastewater at 50 °C. PMID:24293300

  11. 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.

  12. Unique regulation of adipose triglyceride lipase (ATGL) by perilipin 5, a lipid droplet-associated protein.

    PubMed

    Wang, Hong; Bell, Ming; Sreenivasan, Urmila; Sreenevasan, Urmilla; Hu, Hong; Liu, Jun; Dalen, Knut; Londos, Constantine; Yamaguchi, Tomohiro; Rizzo, Mark A; Coleman, Rosalind; Gong, Dawei; Brasaemle, Dawn; Sztalryd, Carole

    2011-05-01

    Lipolysis is a critical metabolic pathway contributing to energy homeostasis through degradation of triacylglycerides stored in lipid droplets (LDs), releasing fatty acids. Neutral lipid lipases act at the oil/water interface. In mammalian cells, LD surfaces are coated with one or more members of the perilipin protein family, which serve important functions in regulating lipolysis. We investigated mechanisms by which three perilipin proteins control lipolysis by adipocyte triglyceride lipase (ATGL), a key lipase in adipocytes and non-adipose cells. Using a cell culture model, we examined interactions of ATGL and its co-lipase CGI-58 with perilipin 1 (perilipin A), perilipin 2 (adipose differentiation-related protein), and perilipin 5 (LSDP5) using multiple techniques as follows: anisotropy Forster resonance energy transfer, co-immunoprecipitation, [(32)P]orthophosphate radiolabeling, and measurement of lipolysis. The results show that ATGL interacts with CGI-58 and perilipin 5; the latter is selectively expressed in oxidative tissues. Both proteins independently recruited ATGL to the LD surface, but with opposite effects; interaction of ATGL with CGI-58 increased lipolysis, whereas interaction of ATGL with perilipin 5 decreased lipolysis. In contrast, neither perilipin 1 nor 2 interacted directly with ATGL. Activation of protein kinase A (PKA) increased [(32)P]orthophosphate incorporation into perilipin 5 by 2-fold, whereas neither ATGL nor CGI-58 was labeled under the incubation conditions. Cells expressing both ectopic perilipin 5 and ATGL showed a 3-fold increase in lipolysis following activation of PKA. Our studies establish perilipin 5 as a novel ATGL partner and provide evidence that the protein composition of perilipins at the LD surface regulates lipolytic activity of ATGL. PMID:21393244

  13. Molecular and biochemical characterizations of the monoacylglycerol lipase gene family of Arabidopsis thaliana.

    PubMed

    Kim, Ryeo Jin; Kim, Hae Jin; Shim, Donghwan; Suh, Mi Chung

    2016-03-01

    Monoacylglycerol lipase (MAGL) catalyzes the last step of triacylglycerol breakdown, which is the hydrolysis of monoacylglycerol (MAG) to fatty acid and glycerol. Arabidopsis harbors over 270 genes annotated as 'lipase', the largest class of acyl lipid metabolism genes that have not been characterized experimentally. In this study, computational modeling suggested that 16 Arabidopsis putative MAGLs (AtMAGLs) have a three-dimensional structure that is similar to a human MAGL. Heterologous expression and enzyme assays indicated that 11 of the 16 encoded proteins indeed possess MAG lipase activity. Additionally, AtMAGL4 displayed hydrolase activity with lysophosphatidylcholine and lysophosphatidylethanolamine (LPE) substrates and AtMAGL1 and 2 utilized LPE as a substrate. All recombinant AtMAGLs preferred MAG substrates with unsaturated fatty acids over saturated fatty acids and AtMAGL8 exhibited the highest hydrolase activities with MAG containing 20:1 fatty acids. Except for AtMAGL4, -14 and -16, all AtMAGLs showed similar activity with both sn-1 and sn-2 MAG isomers. Spatial, temporal and stress-induced expression of the 16 AtMAGL genes was analyzed by transcriptome analyses. AtMAGL:eYFP fusion proteins provided initial evidence that AtMAGL1, -3, -6, -7, -8, -11, -13, -14 and -16 are targeted to the endoplasmic reticulum and/or Golgi network, AtMAGL10, -12 and -15 to the cytosol and AtMAGL2, -4 and -5 to the chloroplasts. Furthermore, AtMAGL8 was associated with the surface of oil bodies in germinating seeds and leaves accumulating oil bodies. This study provides the broad characterization of one of the least well-understood groups of Arabidopsis lipid-related enzymes and will be useful for better understanding their roles in planta. PMID:26932457

  14. Clinical efficacy of serum lipase subtype analysis for the differential diagnosis of pancreatic and non-pancreatic lipase elevation

    PubMed Central

    Bang, Chang Seok; Kim, Jin Bong; Park, Sang Hyun; Baik, Gwang Ho; Su, Ki Tae; Yoon, Jai Hoon; Kim, Yeon Soo; Kim, Dong Joon

    2016-01-01

    Background/Aims: Non-pancreatic elevations of serum lipase have been reported, and differential diagnosis is necessary for clinical practice. This study aimed to evaluate the clinical efficacy of serum lipase subtype analysis for the differential diagnosis of pancreatic and non-pancreatic lipase elevation. Methods: Patients who were referred for the serum lipase elevation were prospectively enrolled. Clinical findings and serum lipase subtypes were analyzed and compared by dividing the patients into pancreatitis and non-pancreatitis groups. Results: A total of 34 patients (12 pancreatitis vs. 22 non-pancreatitis cases) were enrolled. In univariate analysis, the fraction of pancreatic lipase (FPL) in the total amount of serum lipase subtypes was statistically higher in patients with pancreatitis ([median, 0.004; interquartile range [IQR], 0.003 to 0.011] vs. [median, 0.002; IQR, 0.001 to 0.004], p = 0.04). Based on receiver operating characteristic curve analysis for the prediction of acute pancreatitis, FPL was the most valuable predictor (area under the receiver-operating characteristic curve [AUROC], 0.72; 95% confidence interval [CI], 0.54 to 0.86; sensitivity, 83.3%; specificity, 63.6%; positive predictive value, 55.6%; negative predictive value, 97.5%). In multivariate analysis, a cut-off value higher than 0.0027 for the FPL was associated with acute pancreatitis (odds ratio, 8.3; 95% CI, 1.3 to 51.7; p = 0.02). Conclusions: The results did not support that serum lipase subtype analysis could replace standard lipase measurement for the diagnosis of acute pancreatitis. However, the test demonstrated adequate sensitivity for use in triage or as an add-on test for serum lipase elevation. PMID:27243230

  15. Gene cloning and molecular characterization of the Talaromyces thermophilus lipase catalyzed efficient hydrolysis and synthesis of esters.

    PubMed

    Romdhane, Ines Belhaj-Ben; Frikha, Fakher; Maalej-Achouri, Inès; Gargouri, Ali; Belghith, Hafedh

    2012-02-15

    A genomic bank from Talaromyces thermophilus fungus was constructed and screened using a previously isolated fragment lipase gene as probe. From several clones isolated, the nucleotide sequence of the lipase gene (TTL gene) was completed and sequenced. The TTL coding gene consists of an open reading frame (ORF) of 1083bp encoding a protein of 269 Aa with an estimated molecular mass of 30kDa. The TTL belongs to the same gene family as Thermomyces lanuginosus lipase (TLL, Lipolase®), a well known lipase with multiple applications. The promoter sequence of the TTL gene showed the conservation of known consensus sequences PacC, CreA, Hap2-3-4 and the existence of a particular sequence like the binding sites of Oleate Response Element (ORE) and Fatty acids Responsis Element (FARE) which are similar to that already found to be specific of lipolytic genes in Candida and Fusarium, respectively. Northern blot analysis showed that the TTL expression was much higher on wheat bran than on olive oil as sole carbon source. Compared to the Lipolase®, this enzyme was found to be more efficient for the hydrolysis and the synthesis of esters; and its synthetic efficiency even reached 91.6% from Waste Cooking Oil triglycerides. PMID:22178764

  16. 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

  17. Highly efficient solvent-free synthesis of 1,3-diacylglycerols by lipase immobilised on nano-sized magnetite particles.

    PubMed

    Meng, Xiao; Xu, Gang; Zhou, Qin-Li; Wu, Jian-Ping; Yang, Li-Rong

    2014-01-15

    Recently, 1,3-DAGs (1,3-diacylglycerols) have attracted considerable attention as healthy components of food, oil and pharmaceutical intermediates. Generally, 1,3-DAG is prepared by lipase-mediated catalysis in a solvent free system. However, the system's high reaction temperature (required to reach the reactants' melting point), high substrate concentration and high viscosity severely reduce the lipase's activity, selectivity and recycling efficiency. In this report, MjL (Mucor javanicus lipase) was found to have the best performance in the solvent-free synthesis of 1,3-DAGs of several common commercial lipases. By covalent binding to amino-group-activated NSM (nano-sized magnetite) particles and cross-linking to form an enzyme aggregate coat, MjL's specific activity increased 10-fold, and was able to be reused for 10 cycles with 90% residual activity at 55°C. 1,3-DAGs of lauric, myristic, palmitic, stearic, oleic and linoleic acid were prepared using the resulting immobilised enzyme, all with yields greater than 90%, and the reaction time was also greatly reduced. PMID:24054246

  18. Biodiesel production from crude Jatropha oil catalyzed by non-commercial immobilized heterologous Rhizopus oryzae and Carica papaya lipases.

    PubMed

    Rodrigues, J; Canet, A; Rivera, I; Osório, N M; Sandoval, G; Valero, F; Ferreira-Dias, S

    2016-08-01

    The aim of this study was to evaluate the feasibility of biodiesel production by transesterification of Jatropha oil with methanol, catalyzed by non-commercial sn-1,3-regioselective lipases. Using these lipases, fatty acid methyl esters (FAME) and monoacylglycerols are produced, avoiding the formation of glycerol as byproduct. Heterologous Rhizopus oryzae lipase (rROL) immobilized on different synthetic resins and Carica papaya lipase (rCPL) immobilized on Lewatit VP OC 1600 were tested. Reactions were performed at 30°C, with seven stepwise methanol additions. For all biocatalysts, 51-65% FAME (theoretical maximum=67%, w/w) was obtained after 4h transesterification. Stability tests were performed in 8 or 10 successive 4h-batches, either with or without rehydration of the biocatalyst between each two consecutive batches. Activity loss was much faster when biocatalysts were rehydrated. For rROL, half-life times varied from 16 to 579h. rROL on Lewatit VPOC 1600 was more stable than for rCPL on the same support. PMID:26980626

  19. Inhibition of lipase and inflammatory mediators by Chlorella lipid extracts for antiacne treatment

    PubMed Central

    Sibi, G.

    2015-01-01

    Acne vulgaris is a chronic inflammatory disease, and its treatment is challenging due to the multifactorial etiology and emergence of antibiotic-resistant Propionibacterium acnes strains. This study was focused to reduce antibiotics usage and find an alternate therapeutic source for treating acne. Lipid extracts of six Chlorella species were tested for inhibition of lipase, reactive oxygen species (ROS) production, cytokine production using P. acnes (Microbial Type Culture Collection 1951). Lipase inhibitory assay was determined by dimercaprol Tributyrate - 5, 5'- dithiobis 2-nitrobenzoic acid method and ROS production assay was performed using nitro-blue tetrazolium test. The anti-inflammatory activity of algal lipid extracts was determined by in vitro screening method based on inhibition of pro-inflammatory cytokines, tumor necrosis factor-alpha (TNF-α) produced by human peripheral blood mononuclear cells. Minimum inhibitory concentration (MIC) values of lipid extracts were determined by microdilution method, and the fatty acid methyl esters (FAME) were analyzed by gas chromatography-mass spectroscopy. Chlorella ellipsoidea has the highest lipase inhibitory activity with 61.73% inhibition, followed by Chlorella vulgaris (60.31%) and Chlorella protothecoides (58.9%). Lipid extracts from C. protothecoides and C. ellipsoidea has significantly reduced the ROS production by 61.27% and 58.34% respectively. Inhibition of pro-inflammatory cytokines TNF-α showed the inhibition ranging from 58.39% to 78.67%. C. vulgaris has exhibited the MICvalue of 10 μg/ml followed by C. ellipsoidea, C. protothecoides and Chlorella pyrenoidosa (20 μg/ml). FAME analysis detected 19 fatty acids of which 5 were saturated fatty acids, and 14 were unsaturated fatty acids ranging from C14 to C24. The results suggest that lipid extracts of Chlorella species has significant inhibitory activity on P. acnes by inhibiting lipase activity. Further, anti-inflammatory reaction caused by the

  20. Phospholipase C and diacylglycerol lipase in human gallbladder and hepatic bile.

    PubMed

    Pattinson, N R; Willis, K E

    1990-12-01

    A phospholipase C in bile, free of bacterial infection, has recently been identified from cholesterol gallstone patients. Because of the importance of phosphatidylcholine in solubilizing cholesterol in bile, this study further investigates the metabolism of phosphatidylcholine in delipidated gallbladder and common bile duct biles. Phospholipase C activity, as measured by the release of phosphoryl[3H]choline from the substrate 1,2-dipalmitoyl-sn-glycero-3-phospho [N-methyl-3H]choline, was identified in both hepatic and gallbladder biles. Similar levels of activity (nmol.h-1.mg-1 of delipidated protein) were found in common bile duct (11.25 +/- 14.23) and gallbladder bile (19.07 +/- 22.24), although per milliliter of bile, the mean gallbaldder levels were 6.4 times greater than those found in common duct bile. With the tow substrates, 1-palmitoyl-2[9,10-3H] palmitoyl-sn-glycero-3-phosphocholine and 1,2(1-14C) dipalmitoyl-sn-glycero-3-phosphocholine, the majority of organically extracted label, after thin-layer chromatography, was recovered as radiolabeled diglyceride, confirming the presence of phospholipase C. Diglyceride levels were found to be closely correlated with [3H]choline (slope, 0.9820; r = 0.9844). In addition to diglyceride, both radiolabeled free fatty acid and monoglyceride were identified in common bile duct and gallbladder biles, although their levels were an order of magnitude less than measurable phospholipase C activity. To determine whether the free fatty acid release was due to either a diacylglycerol-lipase or a phospholipase A2, the effect of adding unlabeled diglyceride on free fatty acid formation from the substrate [14C]DPPC was examined. As the concentration of unlabeled diglyceride was increased, the amount of free fatty acid and monoglyceride released were both reduced in parallel. Direct measurement of diacylglycerol-lipase activity by incubating the diglyceride, sn-2[3H]dipalmitoyl, resulted in release of both products in a ratio

  1. Biocatalytic synthesis of starch esters by immobilized lipase on magnetic microparticles.

    PubMed

    Prasertpornsakun, Nattanun; Raita, Marisa; Laosiripojana, Navadol; Champreda, Verawat

    2015-01-01

    Enzymatic esterification is an efficient approach for modifying starch to functionalized biomaterials. In this study, conversion of cassava starch to fatty acid acyl esters using immobilized Thermomyces lanuginosus lipase on Fe3O4 microparticles modified with 3-aminopropyltriethoxysilane and covalently linked by 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide and N-hydroxysuccinimide (Fe3O4-AP-ED-lipase) in a solvent-free system was studied. An optimized reaction containing 5% w/v gelatinized starch, 1% v/v Triton X-100, and 1% w/v biocatalyst with 2.5% w/v of fatty acids (palmitic, oleic, or linoleic acid) resulted in esterified products with a degree of substitution (DS) of 0.12-0.14, while a slightly lower DS was observed using crude palm fatty acid distillate as the acyl donor with 42.9-59.6% recovery yield. Increasing DS led to lower glass transition temperature and higher viscosity of the esterified products. The enzyme showed high operational stability with 85% retaining in activity after recycling in three consecutive batches with simple separation by magnetization, leading to improved process economics. PMID:26107973

  2. An Antibody to the Castor Bean Glyoxysomal Lipase (62 kD) also Binds to a 62 kD Protein in Extracts from Many Young Oilseed Plants.

    PubMed

    Hills, M J; Beevers, H

    1987-12-01

    An antibody raised against purified glyoxysomal lipase (triacylglycerol hydrolase EC 3.1.1.3.) from castor bean (relative molecular weight of 62,000) also binds to a protein with a relative molecular weight of 62,000 in extracts of food reserve tissues from many young oilseed plants. These plants include Brassica napus L., Zea mays L., Arachis hypogaea L., Glycine max L., Gossipium hirsutum L., Cucurbita pepo L., Helianthus annuus L., Pisum sativum L., and Cicer arietinum L. The antibody caused inhibition of triacylglycerol hydrolysis by the lipases in extracts from seedlings of corn, oilseed rape, castor bean, soybean, and peanut. The pattern of antilipase binding to the 62 kilodalton protein in subcellular fractions from these other seedlings was consistent with the patterns of lipase activity reported in the literature and it is suggested that lipases from these oil seeds all have a subunit with a molecular weight of 62,000. The protein was only found in the food reserve tissues and was not present in extracts of roots and leaves of mature plants. In addition, the immunoreactive 62 kilodalton polypeptide was not detectable in lima beans and only at very low levels in kidney beans. Both these seeds are known to contain very little storage lipid and would not be expected to contain lipase. With the exception of the acid lipase of castor bean, ungerminated seeds do not generally contain active lipases. The immunoreactive 62 kilodalton protein could not be detected in the ungerminated seeds of most plants and only at very low low levels in others. PMID:16665808

  3. Dimethyl carbonate-mediated lipid extraction and lipase-catalyzed in situ transesterification for simultaneous preparation of fatty acid methyl esters and glycerol carbonate from Chlorella sp. KR-1 biomass.

    PubMed

    Jo, Yoon Ju; Lee, Ok Kyung; Lee, Eun Yeol

    2014-04-01

    Fatty acid methyl esters (FAMEs) and glycerol carbonate were simultaneously prepared from Chlorella sp. KR-1 containing 40.9% (w/w) lipid using a reactive extraction method with dimethyl carbonate (DMC). DMC was used as lipid extraction agent, acyl acceptor for transesterification of the extracted triglycerides, substrate for glycerol carbonate synthesis from glycerol, and reaction medium for the solvent-free reaction system. For 1g of biomass, 367.31 mg of FAMEs and 16.73 mg of glycerol carbonate were obtained under the optimized conditions: DMC to biomass ratio of 10:1 (v/w), water content of 0.5% (v/v), and Novozyme 435 to biomass ratio of 20% (w/w) at 70°C for 24h. The amount of residual glycerol was only in the range of 1-2.5mg. Compared to conventional method, the cost of FAME production with the proposed technique could be reduced by combining lipid extraction with transesterification and omitting the extraction solvent recovery process. PMID:24583221

  4. Obtaining lipases from byproducts of orange juice processing.

    PubMed

    Okino-Delgado, Clarissa Hamaio; Fleuri, Luciana Francisco

    2014-11-15

    The presence of lipases was observed in three byproducts of orange juice processing: peel, core and frit. The enzymes were characterised biochemically over a wide pH range from neutral (6-7) to alkaline (8-9). The optimal temperature for the activity of these byproducts showed wide range at 20°C to 70°C, indicating fairly high thermostability. The activities were monitored on p-NP-butyrate, p-NP-laurate and p-NP-palmitate. For the first time, lipase activity was detected in these residues, reaching 68.5 lipase U/g for the crude extract from fractions called frit. PMID:24912703

  5. Discovery of XEN445: a potent and selective endothelial lipase inhibitor raises plasma HDL-cholesterol concentration in mice.

    PubMed

    Sun, Shaoyi; Dean, Richard; Jia, Qi; Zenova, Alla; Zhong, Jing; Grayson, Celene; Xie, Clark; Lindgren, Andrea; Samra, Pritpaul; Sojo, Luis; van Heek, Margaret; Lin, Linus; Percival, David; Fu, Jian-Min; Winther, Michael D; Zhang, Zaihui

    2013-12-15

    Endothelial lipase (EL) activity has been implicated in HDL metabolism and in atherosclerotic plaque development; inhibitors are proposed to be efficacious in the treatment of dyslipidemia related cardiovascular disease. We describe here the discovery of a novel class of anthranilic acids EL inhibitors. XEN445 (compound 13) was identified as a potent and selective EL inhibitor, that showed good ADME and PK properties, and demonstrated in vivo efficacy in raising plasma HDLc concentrations in mice. PMID:24211162

  6. The 3D model of the lipase/acyltransferase from Candida parapsilosis, a tool for the elucidation of structural determinants in CAL-A lipase superfamily.

    PubMed

    Subileau, Maeva; Jan, Anne-Hélène; Nozac'h, Hervé; Pérez-Gordo, Marina; Perrier, Véronique; Dubreucq, Eric

    2015-10-01

    Because lipids are hydrophobic, the development of efficient bioconversions in aqueous media free of organic solvents is particularly challenging for green oleochemistry. Within this aim, enzymes exhibiting various abilities to catalyze acyltransfer reaction in water/lipid systems have been identified. Among these, CpLIP2 from Candida parapsilosis has been characterized as a lipase/acyltransferase, able to catalyze acyltransfer reactions preferentially to hydrolysis in the presence of particularly low acyl acceptor concentration and high thermodynamic activity of water (aw>0.9). Lipase/acyltransferases are thus of great interest, being able to produce new esters at concentrations above the thermodynamic equilibrium of hydrolysis/esterification with limited to no release of free fatty acids. Here, we present a 3D model of CpLIP2 based on homologies with crystallographic structures of Pseudozyma antarctica lipase A. Indeed, the two enzymes have 31% of identity in their primary sequence, yielding a same general structure, but different catalytic properties. The quality of the calculated CpLIP2 model was confirmed by several methods. Limited proteolysis confirmed the location of some loops at the surface of the protein 3D model. Directed mutagenesis also supported the structural model constructed on CAL-A template: the functional properties of various mutants were consistent with their structure-based putative involvement in the oxyanion hole, substrate specificity, acyltransfer or hydrolysis catalysis and structural stability. The CpLIP2 3D model, in comparison with CAL-A 3D structure, brings insights for the elucidation and improvement of the structural determinants involved in the exceptional acyltransferase properties of this promising biocatalyst and of homologous enzymes of the same family. PMID:26123263

  7. 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

  8. Bioremediation of waste cooking oil using a novel lipase produced by Penicillium chrysogenum SNP5 grown in solid medium containing waste grease.

    PubMed

    Kumar, Sunil; Mathur, Anisha; Singh, Varsha; Nandy, Suchismita; Khare, Sunil Kumar; Negi, Sangeeta

    2012-09-01

    The aim of present work was to bioremediate the waste cooking oil using a novel lipase produced in solid medium containing waste grease and wheat bran by Penicillium chrysogenum. Enzyme extracted with phosphate buffer was purified 10.6 and 26.28-fold after 90% ammonium sulfate precipitation and ion-exchange chromatography, respectively. The partial characterization of enzyme revealed its K(m) and V(max) value for p-nitrophenolpamitate as 0.4mM and 47.61 U/ml, respectively. The relative molecular mass of lipase was 40 kDa by SDS-PAGE and confirmed by zymogram. Purified lipase was most stable at 40°C and at 8.0 pH. Lipase activity was enhanced by metal ions such as Mg(2+), Fe(2+), Ca(2+) and non-ionic surfactant TritonX-100, while suppressed in the presence of SDS. Crude lipase was applied on cooking oil waste and the acid value was 26.92 mg/g. This showed that the enzyme could be employed for the bioremediation of used cooking oil. PMID:22770974

  9. Use of continuous culture to screen for lipase-producing microorganisms and interesterification of butter fat by lipase isolates.

    PubMed

    Pabai, F; Kermasha, S; Morin, A

    1996-05-01

    The continuous cultivation technique was used to investigate the screening for lipase-producing microorganisms from four commercial starters suitable for the degradation of domestic wastes. Using this technique, three strains of lipase-producing bacteria were isolated and identified: Pantoea agglomerans (BB96CC1, BB168CC2) and Pseudomonas fluorescens (BW96CC1). In addition, butter fat induced more lipase production when present in the growth medium. Interesterification of butter fat triacylglycerols by enzymatic extracts of the isolated strains of microorganisms resulted in an appreciable interesterification yield, implying that hydrolysis was suppressed and interesterification of butter fat triacylglycerols was maximized in a microemulsion free-cosurfactant system. PMID:8640605

  10. Cloning and characterization of a novel lipase from Stenotrophomonas maltophilia GS11: The first member of a new bacterial lipase family XVI.

    PubMed

    Li, Mu; Yang, Li-Rong; Xu, Gang; Wu, Jian-Ping

    2016-06-20

    Bacterial lipases are an important group of enzymes that offer enormous potential in organic synthesis, and there is considerable interest in identifying and developing novel bacterial lipases. In previous studies, strains of the genus Stenotrophomonas were proved to be potential source of lipases, but there is little genetic information describing lipase from the genus Stenotrophomonas. We have cloned and characterized a novel lipase (LipSM54), the first lipase described from the genus Stenotrophomonas. Enzymatic study showed that LipSM54 was a cold-active, solvent-tolerant and alkaline lipase. Using bioinformatics tools, LipSM54 was found to be related only to several putative lipases from different bacterial origins, none of which could be assigned to any previously described bacterial lipase family. LipSM54 and these related putative lipases share four conserved motifs around the catalytic residues. These motifs clearly distinguish them from the known bacterial lipase families. Consequently, LipSM54 is the first characterized member of the novel bacterial lipase family. PMID:27117245

  11. Cellulose binding domain assisted immobilization of lipase (GSlip-CBD) onto cellulosic nanogel: characterization and application in organic medium.

    PubMed

    Kumar, Ashok; Zhang, Shaowei; Wu, Gaobing; Wu, Cheng Chao; Chen, JunPeng; Baskaran, R; Liu, Ziduo

    2015-12-01

    A cbd gene was cloned into the C-terminal region of a lip gene from Geobacillus stearothermophilus. The native lipase (43.5 kDa) and CBD-Lip fusion protein (60.2 kDa) were purified to homogeneity by SDS-PAGE. A highly stable cellulosic nanogel was prepared by controlled hydrolysis of microcrystalline cellulose onto which the CBD-lip fusion protein was immobilized through bio-affinity based binding. The nanogel-bound lipase showed optimum activity at 55 °C, and it remains stable and active at pH 10-10.5. Furthermore, the immobilized lipase showed an over two-fold increase of relative activity in the presence of DMSO, isopropanol, isoamyl alcohol and n-butanol, but a mild activity decrease at a low concentration of methanol and ethanol. The immobilized biocatalyst retained ~50% activity after eight repetitive hydrolytic cycles. Enzyme kinetic studies of the immobilized lipase showed a 1.24 fold increase in Vmax and 5.25 fold increase in kcat towards p-NPP hydrolysis. Additionally, the nanogel bound lipase was tested to synthesize a biodiesel ester, ethyl oleate in DMSO. Kinetic analysis showed the km 100.5 ± 4.3 mmol and Vmax 0.19 ± 0.015 mmolmin(-1) at varied oleic acid concentration. Also, the values of km and Vmax at varying concentration of ethanol were observed to be 95.9 ± 13.9 mmol and 0.22 ± 0.013 mmolmin(-1) respectively. The maximum yield of ethyl oleate 111.2 ± 1.24 mM was obtained under optimized reaction conditions in organic medium. These results suggest that this immobilized biocatalyst can be used as an efficient tool for the biotransformation reactions on an industrial scale. PMID:26590897

  12. Stability of a Lipase Extracted from Seeds of Pachira aquatica in Commercial Detergents and Application Tests in Poultry Wastewater Pretreatment and Fat Particle Hydrolysis.

    PubMed

    Polizelli, Patrícia Peres; Facchini, Fernanda Dell Antonio; Bonilla-Rodriguez, Gustavo Orlando

    2013-01-01

    A protein extract containing a plant lipase from oleaginous seeds of Pachira aquatica was tested using soybean oil, wastewater from a poultry processing plant, and beef fat particles as substrate. The hydrolysis experiments were carried out at a temperature of 40°C, an incubation time of 90 minutes, and pH 8.0-9.0. The enzyme had the best stability at pH 9.0 and showed good stability in the alkaline range. It was found that P. aquatica lipase was stable in the presence of some commercial laundry detergent formulations, and it retained full activity up to 0.35% in hydrogen peroxide, despite losing activity at higher concentrations. Concerning wastewater, the lipase increased free fatty acids release by 7.4 times and promoted the hydrolysis of approximately 10% of the fats, suggesting that it could be included in a pretreatment stage, especially for vegetable oil degradation. PMID:24455209

  13. Gene cloning and characterization of a novel highly organic solvent tolerant lipase from Proteus sp. SW1 and its application for biodiesel production.

    PubMed

    Whangsuk, Wirongrong; Sungkeeree, Pareenart; Thiengmag, Sirinthra; Kerdwong, Jarunee; Sallabhan, Ratiboot; Mongkolsuk, Skorn; Loprasert, Suvit

    2013-01-01

    Proteus sp. SW1 was found to produce an extracellular solvent tolerant lipase. The gene, lipA, encoding a bacterial lipase, was cloned from total Proteus sp. SW1 DNA. lipA was predicted to encode a 287 amino acid protein of 31.2 kDa belonging to the Group I proteobacterial lipases. Purified His-tagged LipA exhibited optimal activity at pH 10.0 and 55°C. It was highly stable in organic solvents retaining 112% of its activity in 100% isopropanol after 24 h, and exhibited more than 200% of its initial activity upon exposure to 60% acetone, ethanol, and hexane for 18 h. Biodiesel synthesis reactions, using a single step addition of 13% an acyl acceptor ethanol, showed that LipA was highly effective at converting palm oil into biodiesel. PMID:22371263

  14. Stability of a Lipase Extracted from Seeds of Pachira aquatica in Commercial Detergents and Application Tests in Poultry Wastewater Pretreatment and Fat Particle Hydrolysis

    PubMed Central

    Polizelli, Patrícia Peres; Facchini, Fernanda Dell Antonio

    2013-01-01

    A protein extract containing a plant lipase from oleaginous seeds of Pachira aquatica was tested using soybean oil, wastewater from a poultry processing plant, and beef fat particles as substrate. The hydrolysis experiments were carried out at a temperature of 40°C, an incubation time of 90 minutes, and pH 8.0-9.0. The enzyme had the best stability at pH 9.0 and showed good stability in the alkaline range. It was found that P. aquatica lipase was stable in the presence of some commercial laundry detergent formulations, and it retained full activity up to 0.35% in hydrogen peroxide, despite losing activity at higher concentrations. Concerning wastewater, the lipase increased free fatty acids release by 7.4 times and promoted the hydrolysis of approximately 10% of the fats, suggesting that it could be included in a pretreatment stage, especially for vegetable oil degradation. PMID:24455209

  15. 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

  16. Lipase-catalyzed synthesis of xylitol monoesters: solvent engineering approach.

    PubMed

    Castillo, E; Pezzotti, F; Navarro, A; López-Munguía, A

    2003-05-01

    A solvent engineering strategy was applied to the lipase-catalyzed synthesis of xylitol-oleic acid monoesters. The different esterification degrees for this polyhydroxylated molecule were examined in different organic solvent mixtures. In this context, conditions for high selectivity towards monooleoyl xylitol synthesis were enhanced from 6 mol% in pure n-hexane to 73 mol% in 2-methyl-2-propanol/dimethylsulfoxide (DMSO) 80:20 (v/v). On the contrary, the highest production of di- and trioleoyl xylitol, corresponding to 94 mol%, was achieved in n-hexane. Changes in polarity of the reaction medium and in the molecular interactions between solvents and reactants were correlated with the activity coefficients of products. Based on experimental results and calculated thermodynamic activities, the effect of different binary mixtures of solvents on the selective production of xylitol esters is reported. From this analysis, it is concluded that in the more polar conditions (100% dimethylsulfoxide (DMSO)), the synthesis of xylitol monoesters is favored. However, these conditions are unfavorable in terms of enzyme stability. As an alternative, binary mixtures of solvents were proposed. Each mixture of solvents was characterized in terms of the quantitative polarity parameter E(T)(30) and related with the activity coefficients of xylitol esters. To our knowledge, the characterization of solvent mixtures in terms of this polarity parameter and its relationship with the selectivity of the process has not been previously reported. PMID:12730008

  17. 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

  18. Structural insights of a hormone sensitive lipase homologue Est22

    PubMed Central

    Huang, Jing; Huo, Ying-Yi; Ji, Rui; Kuang, Siyun; Ji, Chaoneng; Xu, Xue-Wei; Li, Jixi

    2016-01-01

    Hormone sensitive lipase (HSL) catalyzes the hydrolysis of triacylglycerols into fatty acids and glycerol, thus playing key roles in energy homeostasis. However, the application of HSL serving as a pharmaceutical target and an industrial biocatalyst is largely hampered due to the lack of high-resolution structural information. Here we report biochemical properties and crystal structures of a novel HSL homologue esterase Est22 from a deep-sea metagenomic library. Est22 prefers short acyl chain esters and has a very high activity with substrate p-nitrophenyl butyrate. The crystal structures of wild type and mutated Est22 with its product p-nitrophenol are solved with resolutions ranging from 1.4 Å to 2.43 Å. The Est22 exhibits a α/β-hydrolase fold consisting with a catalytic domain and a substrate-recognizing cap domain. Residues Ser188, Asp287, and His317 comprise the catalytic triad in the catalytic domain. The p-nitrophenol molecule occupies the substrate binding pocket and forms hydrogen bonds with adjacent residues Gly108, Gly109, and Gly189. Est22 exhibits a dimeric form in solution, whereas mutants D287A and H317A change to polymeric form, which totally abolished its enzymatic activities. Our study provides insights into the catalytic mechanism of HSL family esterase and facilitates the understanding for further industrial and biotechnological applications of esterases. PMID:27328716

  19. Transcriptional regulation of adipocyte hormone-sensitive lipase by glucose.

    PubMed

    Smih, Fatima; Rouet, Philippe; Lucas, Stéphanie; Mairal, Aline; Sengenes, Coralie; Lafontan, Max; Vaulont, Sophie; Casado, Marta; Langin, Dominique

    2002-02-01

    Hormone-sensitive lipase (HSL) catalyzes the rate-limiting step in the mobilization of fatty acids from adipose tissue, thus determining the supply of energy substrates in the body. HSL mRNA was positively regulated by glucose in human adipocytes. Pools of stably transfected 3T3-F442A adipocytes were generated with human adipocyte HSL promoter fragments from -2,400/+38 to -31/+38 bp linked to the luciferase gene. A glucose-responsive region was mapped within the proximal promoter (-137 bp). Electromobility shift assays showed that upstream stimulatory factor (USF)-1 and USF2 and Sp1 and Sp3 bound to a consensus E-box and two GC-boxes in the -137-bp region. Cotransfection of the -137/+38 construct with USF1 and USF2 expression vectors produced enhanced luciferase activity. Moreover, HSL mRNA levels were decreased in USF1- and USF2-deficient mice. Site-directed mutagenesis of the HSL promoter showed that the GC-boxes, although contributing to basal promoter activity, were dispensable for glucose responsiveness. Mutation of the E-box led to decreased promoter activity and suppression of the glucose response. Analogs and metabolites were used to determine the signal metabolite of the glucose response. The signal is generated downstream of glucose-6-phosphate in the glycolytic pathway before the triose phosphate step. PMID:11812735

  20. Structural insights of a hormone sensitive lipase homologue Est22.

    PubMed

    Huang, Jing; Huo, Ying-Yi; Ji, Rui; Kuang, Siyun; Ji, Chaoneng; Xu, Xue-Wei; Li, Jixi

    2016-01-01

    Hormone sensitive lipase (HSL) catalyzes the hydrolysis of triacylglycerols into fatty acids and glycerol, thus playing key roles in energy homeostasis. However, the application of HSL serving as a pharmaceutical target and an industrial biocatalyst is largely hampered due to the lack of high-resolution structural information. Here we report biochemical properties and crystal structures of a novel HSL homologue esterase Est22 from a deep-sea metagenomic library. Est22 prefers short acyl chain esters and has a very high activity with substrate p-nitrophenyl butyrate. The crystal structures of wild type and mutated Est22 with its product p-nitrophenol are solved with resolutions ranging from 1.4 Å to 2.43 Å. The Est22 exhibits a α/β-hydrolase fold consisting with a catalytic domain and a substrate-recognizing cap domain. Residues Ser188, Asp287, and His317 comprise the catalytic triad in the catalytic domain. The p-nitrophenol molecule occupies the substrate binding pocket and forms hydrogen bonds with adjacent residues Gly108, Gly109, and Gly189. Est22 exhibits a dimeric form in solution, whereas mutants D287A and H317A change to polymeric form, which totally abolished its enzymatic activities. Our study provides insights into the catalytic mechanism of HSL family esterase and facilitates the understanding for further industrial and biotechnological applications of esterases. PMID:27328716

  1. Characterization of a Hyperthermostable Alkaline Lipase from Bacillus sonorensis 4R.

    PubMed

    Bhosale, Hemlata; Shaheen, Uzma; Kadam, Tukaram

    2016-01-01

    Hyperthermostable alkaline lipase from Bacillus sonorensis 4R was purified and characterized. The enzyme production was carried out at 80°C and 9.0 pH in glucose-tween inorganic salt broth under static conditions for 96 h. Lipase was purified by anion exchange chromatography by 12.15 fold with a yield of 1.98%. The molecular weight of lipase was found to be 21.87 KDa by SDS-PAGE. The enzyme activity was optimal at 80°C with t 1/2 of 150 min and at 90°C, 100°C, 110°C, and 120°C; the respective values were 121.59 min, 90.01 min, 70.01 min, and 50 min. The enzyme was highly activated by Mg and t 1/2 values at 80°C were increased from 150 min to 180 min when magnesium and mannitol were added in combination. The activation energy calculated from Arrhenius plot was 31.102 KJ/mol. At 80-120°C, values of ΔH and ΔG were in the range of 28.16-27.83 KJ/mol and 102.79 KJ/mol to 111.66 KJ/mol, respectively. Lipase activity was highest at 9.0 pH and stable for 2 hours at this pH at 80°C. Pretreatment of lipase with MgSO4 and CaSO4 stimulated enzyme activity by 249.94% and 30.2%, respectively. The enzyme activity was greatly reduced by CoCl2, CdCl2, HgCl2, CuCl2, Pb(NO3)2, PMSF, orlistat, oleic acid, iodine, EDTA, and urea. PMID:26904276

  2. Characterization of a Hyperthermostable Alkaline Lipase from Bacillus sonorensis 4R

    PubMed Central

    Bhosale, Hemlata; Shaheen, Uzma

    2016-01-01

    Hyperthermostable alkaline lipase from Bacillus sonorensis 4R was purified and characterized. The enzyme production was carried out at 80°C and 9.0 pH in glucose-tween inorganic salt broth under static conditions for 96 h. Lipase was purified by anion exchange chromatography by 12.15 fold with a yield of 1.98%. The molecular weight of lipase was found to be 21.87 KDa by SDS-PAGE. The enzyme activity was optimal at 80°C with t1/2 of 150 min and at 90°C, 100°C, 110°C, and 120°C; the respective values were 121.59 min, 90.01 min, 70.01 min, and 50 min. The enzyme was highly activated by Mg and t1/2 values at 80°C were increased from 150 min to 180 min when magnesium and mannitol were added in combination. The activation energy calculated from Arrhenius plot was 31.102 KJ/mol. At 80–120°C, values of ΔH and ΔG were in the range of 28.16–27.83 KJ/mol and 102.79 KJ/mol to 111.66 KJ/mol, respectively. Lipase activity was highest at 9.0 pH and stable for 2 hours at this pH at 80°C. Pretreatment of lipase with MgSO4 and CaSO4 stimulated enzyme activity by 249.94% and 30.2%, respectively. The enzyme activity was greatly reduced by CoCl2, CdCl2, HgCl2, CuCl2, Pb(NO3)2, PMSF, orlistat, oleic acid, iodine, EDTA, and urea. PMID:26904276

  3. 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

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

    PubMed

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

    2002-04-01

    High conversions in lipase-catalyzed syntheses of esters from free acyl donors and an alcohol requires efficient removal of water preferentially at temperatures compatible to enzyme activity. Using a lipase B from Candida antarctica (CAL-B)-mediated synthesis of sugar fatty-acid esters, we show that a mixture of ethyl methylketone (EMK) and hexane (best ratio: 4:1, vo/vo) allows efficient removal of water generated during esterification. Azeotropic distillation of the solvent mixture (composition: 26% EMK, 55% hexane, 19% water) takes place at 59 degrees C, which closely matches the optimum temperature reported for CAL-B. Water is then removed from the azeotrope by membrane vapor permeation. In case of glucose stearate, 93% yield was achieved after 48 h using an equimolar ratio of glucose and stearic acid. CAL-B could be reused for seven reaction cycles, with 86% residual activity after 14 d total reaction time at 59 degrees C. A decrease in fatty-acid chain length as well as increasing temperatures (75 degrees C) resulted in lower conversions. In addition, immobilization of CAL-B on a magnetic polypropylene carrier (EP 100) facilitated separation of the biocatalyst. PMID:11857278

  5. Normal lipase drug-induced pancreatitis: a novel finding.

    PubMed

    Shafqet, Muhammad A; Brown, Teresa V; Sharma, Ranita

    2015-03-01

    Acute pancreatitis (AP) in the setting of a normal serum amylase has been previously reported in the literature. Serum lipase on the other hand has a negative predictive value approaching 100% and therefore is an excellent test to rule out AP in the emergency department. The occurrence of AP with a normal lipase is extremely rare and has never been reported in the setting of drug-induced pancreatitis. Thiazide diuretics have been implicated as a cause of pancreatic injury via a number of proposed mechanisms. However, all such cases have been in the setting of elevated serum amylase or lipase. We report the first case of radiographically proven hydrochlorothiazide-induced pancreatitis with a normal lipase. PMID:25227976

  6. 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

  7. Preparation of 3-deacetyl cephalosporins by Aspergillus niger lipase.

    PubMed

    Carrea, G; Corcelli, A; Palmisano, G; Riva, S

    1996-12-20

    Lipase from Aspergillus niger was used for the selective hydrolysis of the 3-O-acetate of cephalosporin C to give an intermediate useful for further chemical elaborations. This lipase was purified to homogeneity and its properties compared with previously published data that present some discrepancies. The lipase proved to be very effective in catalyzing 3-O-acetate hydrolysis and versatile toward substitution on the beta-lactamic ring. In fact, as an example, two other cephalosporinic derivatives, cephalotin and cefotaxime, were efficiently deacetylated. The lipase was immobilized on Eupergit C and employed continuously in either a column or a batch reactor for 2 months without appreciable loss of activity. (c) 1996 John Wiley & Sons, Inc. PMID:18629943

  8. 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

  9. Characterization of lipase-deficient mutants of Acinetobacter calcoaceticus BD413: identification of a periplasmic lipase chaperone essential for the production of extracellular lipase.

    PubMed Central

    Kok, R G; van Thor, J J; Nugteren-Roodzant, I M; Vosman, B; Hellingwerf, K J

    1995-01-01

    Acinetobacter calcoaceticus BD413 produces an extracellular lipase, which is encoded by the lipA gene. Five lipase-deficient mutants have been generated via random insertion mutagenesis. Phenotypic characterization of these mutants revealed the presence of as many as four lipolytic enzymes in A. calcoaceticus. Biochemical evidence classified four of the mutants as export mutants, which presumably are defective in translocation of the lipase across the outer membrane. The additional mutant, designated AAC302, displays a LipA- phenotype, and yet the mutation in this strain was localized 0.84 kbp upstream of lipA. Sequence analysis of this region revealed an open reading frame, designated lipB, that is disrupted in AAC302. The protein encoded by this open reading frame shows extensive similarity to a chaperone-like helper protein of several pseudomonads, required for the production of extracellular lipase. Via complementation of AAC302 with a functional extrachromosomal copy of lipA, it could be determined that LipB is essential for lipase production. As shown by the use of a translational LipB-PhoA fusion construct, the C-terminal part of LipB of A. calcoaceticus BD413 is located outside the cytoplasm. Sequence analysis further strongly suggests that A. calcoaceticus LipB is N terminally anchored in the cytoplasmic membrane. Therefore, analogous to the situation in Pseudomonas species, however, lipB in A. calcoaceticus is located upstream of the structural lipase gene. lipB and lipA form a bicistronic operon, and the two genes are cotranscribed from an Escherichia coli sigma 70-type promoter. The reversed order of genes, in comparison with the situation in Pseudomonas species, suggests that LipA and LipB are produced in equimolar amounts. Therefore, the helper protein presumably does not only have a catalytic function, e.g., in folding of the lipase, but is also likely to act as a lipase-specific chaperone. A detailed model of the export route of the lipase of A

  10. Characterization of lipase-deficient mutants of Acinetobacter calcoaceticus BD413: identification of a periplasmic lipase chaperone essential for the production of extracellular lipase.

    PubMed

    Kok, R G; van Thor, J J; Nugteren-Roodzant, I M; Vosman, B; Hellingwerf, K J

    1995-06-01

    Acinetobacter calcoaceticus BD413 produces an extracellular lipase, which is encoded by the lipA gene. Five lipase-deficient mutants have been generated via random insertion mutagenesis. Phenotypic characterization of these mutants revealed the presence of as many as four lipolytic enzymes in A. calcoaceticus. Biochemical evidence classified four of the mutants as export mutants, which presumably are defective in translocation of the lipase across the outer membrane. The additional mutant, designated AAC302, displays a LipA- phenotype, and yet the mutation in this strain was localized 0.84 kbp upstream of lipA. Sequence analysis of this region revealed an open reading frame, designated lipB, that is disrupted in AAC302. The protein encoded by this open reading frame shows extensive similarity to a chaperone-like helper protein of several pseudomonads, required for the production of extracellular lipase. Via complementation of AAC302 with a functional extrachromosomal copy of lipA, it could be determined that LipB is essential for lipase production. As shown by the use of a translational LipB-PhoA fusion construct, the C-terminal part of LipB of A. calcoaceticus BD413 is located outside the cytoplasm. Sequence analysis further strongly suggests that A. calcoaceticus LipB is N terminally anchored in the cytoplasmic membrane. Therefore, analogous to the situation in Pseudomonas species, however, lipB in A. calcoaceticus is located upstream of the structural lipase gene. lipB and lipA form a bicistronic operon, and the two genes are cotranscribed from an Escherichia coli sigma 70-type promoter. The reversed order of genes, in comparison with the situation in Pseudomonas species, suggests that LipA and LipB are produced in equimolar amounts. Therefore, the helper protein presumably does not only have a catalytic function, e.g., in folding of the lipase, but is also likely to act as a lipase-specific chaperone. A detailed model of the export route of the lipase of A

  11. Immobilization and characterization of a new regioselective and enantioselective lipase obtained from a metagenomic library.

    PubMed

    Alnoch, Robson Carlos; Martini, Viviane Paula; Glogauer, Arnaldo; Costa, Allen Carolina dos Santos; Piovan, Leandro; Muller-Santos, Marcelo; de Souza, Emanuel Maltempi; de Oliveira Pedrosa, Fábio; Mitchell, David Alexander; Krieger, Nadia

    2015-01-01

    In previous work, a new lipase and its cognate foldase were identified and isolated from a metagenomic library constructed from soil samples contaminated with fat. This new lipase, called LipG9, is a true lipase that shows specific activities that are comparable to those of well-known industrially-used lipases with high activity against long-chain triglycerides. In the present work, LipG9 was co-expressed and co-immobilized with its foldase, on an inert hydrophobic support (Accurel MP1000). We studied the performance of this immobilized LipG9 (Im-LipG9) in organic media, in order to evaluate its potential for use in biocatalysis. Im-LipG9 showed good stability, maintaining a residual activity of more than 70% at 50 °C after incubation in n-heptane (log P 4.0) for 8 h. It was also stable in polar organic solvents such as ethanol (log P -0.23) and acetone (log P -0.31), maintaining more than 80% of its original activity after 8 h incubation at 30 °C. The synthesis of ethyl esters was tested with fatty acids of different chain lengths in n-heptane at 30 °C. The best conversions (90% in 3 h) were obtained for medium and long chain saturated fatty acids (C8, C14 and C16), with the maximum specific activity, 29 U per gram of immobilized preparation, being obtained with palmitic acid (C16). Im-LipG9 was sn-1,3-specific. In the transesterification of the alcohol (R,S)-1-phenylethanol with vinyl acetate and the hydrolysis of the analogous ester, (R,S)-1-phenylethyl acetate, Im-LipG9 showed excellent enantioselectivity for the R-isomer of both substrates (E> 200), giving an enantiomeric excess (ee) of higher than 95% for the products at 49% conversion. The results obtained in this work provide the basis for the development of applications of LipG9 in biocatalysis. PMID:25706996

  12. Immobilization and Characterization of a New Regioselective and Enantioselective Lipase Obtained from a Metagenomic Library

    PubMed Central

    Alnoch, Robson Carlos; Martini, Viviane Paula; Glogauer, Arnaldo; Costa, Allen Carolina dos Santos; Piovan, Leandro; Muller-Santos, Marcelo; de Souza, Emanuel Maltempi; de Oliveira Pedrosa, Fábio; Mitchell, David Alexander; Krieger, Nadia

    2015-01-01

    In previous work, a new lipase and its cognate foldase were identified and isolated from a metagenomic library constructed from soil samples contaminated with fat. This new lipase, called LipG9, is a true lipase that shows specific activities that are comparable to those of well-known industrially-used lipases with high activity against long-chain triglycerides. In the present work, LipG9 was co-expressed and co-immobilized with its foldase, on an inert hydrophobic support (Accurel MP1000). We studied the performance of this immobilized LipG9 (Im-LipG9) in organic media, in order to evaluate its potential for use in biocatalysis. Im-LipG9 showed good stability, maintaining a residual activity of more than 70% at 50°C after incubation in n-heptane (log P 4.0) for 8 h. It was also stable in polar organic solvents such as ethanol (log P -0.23) and acetone (log P -0.31), maintaining more than 80% of its original activity after 8 h incubation at 30°C. The synthesis of ethyl esters was tested with fatty acids of different chain lengths in n-heptane at 30 °C. The best conversions (90% in 3 h) were obtained for medium and long chain saturated fatty acids (C8, C14 and C16), with the maximum specific activity, 29 U per gram of immobilized preparation, being obtained with palmitic acid (C16). Im-LipG9 was sn-1,3-specific. In the transesterification of the alcohol (R,S)-1-phenylethanol with vinyl acetate and the hydrolysis of the analogous ester, (R,S)-1-phenylethyl acetate, Im-LipG9 showed excellent enantioselectivity for the R-isomer of both substrates (E> 200), giving an enantiomeric excess (ee) of higher than 95% for the products at 49% conversion. The results obtained in this work provide the basis for the development of applications of LipG9 in biocatalysis. PMID:25706996

  13. 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

  14. Domain-structure analysis of recombinant rat hormone-sensitive lipase.

    PubMed Central

    Osterlund, T; Danielsson, B; Degerman, E; Contreras, J A; Edgren, G; Davis, R C; Schotz, M C; Holm, C

    1996-01-01

    Hormone-sensitive lipase (HSL) plays a key role in lipid metabolism and overall energy homoeostasis, by controlling the release of fatty acids from stored triglycerides in adipose tissue. Lipases and esterases form a protein superfamily with a common structural fold, called the alpha/beta-hydrolase fold, and a catalytic triad of serine, aspartic or glutamic acid and histidine. Previous alignments between HSL and lipase 2 of Moraxella TA144 have been extended to cover a much larger part of the HSL sequence. From these extended alignments, possible sites for the catalytic triad and alpha/beta-hydrolase fold are suggested. Furthermore, it is proposed that HSL contains a structural domain with catalytic capacity and a regulatory module attached, as well as a structural N-terminal domain unique to this enzyme. In order to test the proposed domain structure, rat HSL was overexpressed and purified to homogeneity using a baculovirus/insect-cell expression system. The purification, resulting in > 99% purity, involved detergent solubilization followed by anion-exchange chromatography and hydrophobic-interaction chromatography. The purified recombinant enzyme was identical to rat adipose-tissue HSL with regard to specific activity, substrate specificity and ability to serve as a substrate for cAMP-dependent protein kinase. The recombinant HSL was subjected to denaturation by guanidine hydrochloride and limited proteolysis. These treatments resulted in more extensive loss of activity against phospholipid-stabilized lipid substrates than against water-soluble substrates, suggesting that the hydrolytic activity can be separated from recognition of lipid substrates. These data support the concept that HSL has at least two major domains. PMID:8912675

  15. Genetic and structure-function studies of missense mutations in human endothelial lipase.