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Showing papers on "Lipase published in 2004"


Journal ArticleDOI
TL;DR: The latest trend in lipase research is the development of novel and improved lipases through molecular approaches such as directed evolution and exploring natural communities by the metagenomic approach.
Abstract: Lipases, triacylglycerol hydrolases, are an important group of biotechnologically relevant enzymes and they find immense applications in food, dairy, detergent and pharmaceutical industries. Lipases are by and large produced from microbes and specifically bacterial lipases play a vital role in commercial ventures. Some important lipase-producing bacterial genera include Bacillus, Pseudomonas and Burkholderia. Lipases are generally produced on lipidic carbon, such as oils, fatty acids, glycerol or tweens in the presence of an organic nitrogen source. Bacterial lipases are mostly extracellular and are produced by submerged fermentation. The enzyme is most commonly purified by hydrophobic interaction chromatography, in addition to some modern approaches such as reverse micellar and aqueous two-phase systems. Most lipases can act in a wide range of pH and temperature, though alkaline bacterial lipases are more common. Lipases are serine hydrolases and have high stability in organic solvents. Besides these, some lipases exhibit chemo-, regio- and enantioselectivity. The latest trend in lipase research is the development of novel and improved lipases through molecular approaches such as directed evolution and exploring natural communities by the metagenomic approach.

1,077 citations


Journal ArticleDOI
TL;DR: Methyl acetate, a novel acyl acceptor for biodiesel production, seems very promising for lipase-catalyzed large-scale production of biodiesel, and the by-product triacetylglycerol is an important chemical with a higher value than glycerol.
Abstract: Methyl acetate, a novel acyl acceptor for biodiesel production has been developed, and a comparative study on Novozym 435-catalyzed transesterification of soybean oil for biodiesel production with different acyl acceptors was conducted and reported in this paper. Methanol has a serious negative effect on enzymatic activity. A molar ratio of methanol to oil of above 1:1 leads to serious inactivation of the enzyme. However, when methyl acetate was used as the acyl acceptor, a yield of 92% of methyl ester could be obtained with a molar ratio of methyl acetate to oil of 12:1, and methyl acetate showed no negative effect on enzymatic activity. Additionally, with crude soybean oil as the oil source and methanol as acyl acceptor, a much lower methyl ester yield was obtained than that with refined soybean oil, while with methyl acetate as acyl acceptor, an equally high yield of methyl ester (92%) was achieved for both soybean oils. Lipase loses its activity very rapidly during repeated experiments with methanol as the acyl acceptor, while there is almost no detected loss in lipase activity, even after being continuously used for 100 batches, when methyl acetate was used for biodiesel production. Moreover, the by-product triacetylglycerol is an important chemical with a higher value than glycerol, and this novel acyl acceptor seems very promising for lipase-catalyzed large-scale production of biodiesel.

492 citations


Journal ArticleDOI
TL;DR: A method for immobilization of Candida rugosa lipase to two types of chitosan beads by activating the hydroxyl groups of ch itosan using carbodiimide coupling agent has been successfully developed.

371 citations


Journal ArticleDOI
TL;DR: Immobilization of lipase (Chromobacterium viscosum) on Celite-545 enhanced the biodiesel yield to 71% from 62% yield obtained by using free tuned enzyme preparation with a process time of 8 h at 40 °C.
Abstract: Alkyl esters of long chain fatty acid are called biodiesel. These esters can be obtained from vegetable oils by transesterification with methanol/ethanol. The transesterification can be carried out chemically or enzymatically. In the present work three different lipases (Chromobacterium viscosum, Candida rugosa, and Porcine pancreas) were screened for a transesterification reaction of Jatropha oil in a solvent-free system to produce biodiesel; only lipase from Chromobacterium viscosum was found to give appreciable yield. Immobilization of lipase (Chromobacterium viscosum) on Celite-545 enhanced the biodiesel yield to 71% from 62% yield obtained by using free tuned enzyme preparation with a process time of 8 h at 40 °C. Further addition of water to the free (1%, w v-1) and immobilized (0.5%, w v-1) enzyme preparations enhanced the yields to 73 and 92%, respectively. Immobilized Chromobacterium viscosum lipase can be used for ethanolysis of oil. It was seen that immobilization of lipases and optimization of...

364 citations


Journal ArticleDOI
TL;DR: It is now clear that HSL (hormone-sensitive lipase) is expressed in multiple tissues and plays a number of roles in lipid metabolism, including that of a neutral cholesteryl ester hydrolase.
Abstract: Although described initially as an intracellular adipocyte-specific triacylglycerol lipase, it is now clear that HSL (hormone-sensitive lipase) is expressed in multiple tissues and plays a number of roles in lipid metabolism, including that of a neutral cholesteryl ester hydrolase The major isoform is a single polypeptide with a moleclar mass of approx 84 kDa and which comprises three major domains: a catalytic domain, a regulatory domain encoding several phosphorylation sites and an N-terminal domain involved in protein–protein and protein–lipid interactions The activity of HSL is regulated acutely by several mechanisms, including reversible phosphorylation by a number of different protein kinases, translocation to different sites within the cell and interaction with a number of proteins, some of which may serve to direct the inhibitory products of HSL away from the protein It is also apparent from work with HSL null mice that more than one enzyme species may be classified as a hormone-sensitive lipase The possible presence of HSL in macrophages remains controversial, and the role of the protein in pancreatic β-cells has yet to be fully elucidated Altered expression of HSL in different cell types may be associated with a number of pathological states, including obesity, atherosclerosis and Type II diabetes

246 citations


Journal ArticleDOI
01 Jan 2004
TL;DR: Response surface methodology was employed to study the effects of carbon source (soy oil, olive oil and glucose) and nitrogen source concentrations (corn steep liquor and NH(4)NO(3)) on the lipase production by Geotrichum sp.
Abstract: Response surface methodology was employed to study the effects of carbon source (soy oil, olive oil and glucose) and nitrogen source concentrations (corn steep liquor and NH(4)NO(3)) on the lipase production by Geotrichum sp. The experiment included a 2(4) central composite rotatable design (CCRD) and four others 2(3) CCRD. According to the responses from the experimental designs, the effects of each variable were calculated and the interactions between them were determined. The response surface methodology was applied for the optimization of the nutrient concentrations in the culture medium for the enzyme production, at 30 degrees C. The optimum medium composition for lipase production by Geotrichum sp. was ammonium nitrate 2.1-2.5%, corn steep liquor 13-15% and soy oil 0.6% as carbon source, which lead to a lipase activity of about 20 U/ml. Using olive oil as carbon source, the optimum composition was ammonium nitrate 0.8-1%, corn steep liquor 13-15% and olive oil 0.6%, leading to an activity of 17 U/ml.

243 citations


Journal ArticleDOI
TL;DR: In this paper, a support for enzyme immobilization was prepared by functionalization of mesoporous silica with octyltriethoxysilane, which enabled the adsorption of lipase from Candida antarctica B via strong hydrophobic interactions.
Abstract: A support for enzyme immobilization was prepared by functionalization of mesoporous silica with octyltriethoxysilane. The features of the surface enables the adsorption of lipase from Candida antarctica B via strong hydrophobic interactions, enhancing the stability of the adsorbed enzyme molecules. Derivatives with a high enzyme loading (200 mg protein/g of silica) can be obtained due to the high porosity and surface properties of the support while the immobilization occurs in a monolayer fashion. The lack of inactive enzyme aggregates, together with the high enzyme loading, are responsible for the high catalytic activity achieved by these species. Derivatives were prepared with different lipase loading, and the activities were tested and compared to the commercial derivative Novozym 435. The stability of the catalyst and hence its industrial applicability were tested by performing subsequent reaction cycles of acylation of ethanolamine with lauric acid in acetonitrile. Conversion was quantitative even after 15 reaction cycles.

224 citations


Journal Article
TL;DR: All these aspects provide convincing support for the possibility of using biotechnologically remodeled lipids with specific physicochemical properties for health benefits.
Abstract: Fatty acid bioavailability can be managed through the physicochemical properties of lipid such as lipid-droplet size, lipid-droplet ultrastructure (lipids organization between core and surface), structure of triglycerides and of phospholipids. The lipid-droplet size exhibits a major effect on lipase activity during lipid digestion. The lipid-droplet ultrastructure is a dynamic factor controling lipase interaction at the lipid interface via the surface phospholipid layer, and also lipase activity via the proportion of triglyceride molecules able to locate at the surface. Triglyceride structure affects in a strong manner digestion, absorption and fatty acid metabolism. Finally, optimal fatty acid transport to specific tissues is dependent on the vehicle molecule (triglyceride or ethyl ester or phospholipid). All these aspects provide convincing support for the possibility of using biotechnologically remodeled lipids with specific physicochemical properties for health benefits.

222 citations


Journal ArticleDOI
TL;DR: The concept of hepatic lipase as mainly a lipolytic enzyme that reduces atherogenic risk has evolved into that of a complex protein with multiple functions that, depending on genetic background and sites of expression, can have a variable effect on atherosclerosis.
Abstract: The role of hepatic lipase as a multifunctional protein that modulates lipoprotein metabolism and atherosclerosis has been extensively documented over the last decade. Hepatic lipase functions as a lipolytic enzyme that hydrolyzes triglycerides and phospholipids present in circulating plasma lipoproteins. Hepatic lipase also serves as a ligand that facilitates lipoprotein uptake by cell surface receptors and proteoglycans, thereby directly affecting cellular lipid delivery. Recently, another process by which hepatic lipase modulates atherogenic risk has been identified. Bone marrow transplantation studies demonstrate that hepatic lipase present in aortic lesions markedly alters aortic lesion formation even in the absence of changes in plasma lipids. These multiple functions of hepatic lipase, which facilitate not only plasma lipid metabolism but also cellular lipid uptake, can be anticipated to have a major and complex impact on atherogenesis. Consistently, human and animal studies support proatherogenic and antiatherogenic roles for hepatic lipase. The concept of hepatic lipase as mainly a lipolytic enzyme that reduces atherogenic risk has evolved into that of a complex protein with multiple functions that, depending on genetic background and sites of expression, can have a variable effect on atherosclerosis.

196 citations


Journal ArticleDOI
TL;DR: A new enzymatic process is described that widens the applicability of these biocatalysts in organic synthesis by catalyse Michael-type addition of secondary amines to acrylonitrile.

166 citations


Journal ArticleDOI
TL;DR: Functional proteomics was used to detect non-HSL lipase(s) in mouse WAT and discovered two peaks of esterase activity using p-nitrophenyl butyrate as a substrate, which contained most of the lipase activity.

Journal ArticleDOI
TL;DR: To analyse the influence of nitrogen and carbon sources on extracellular lipase production by Yarrowia lipolytica‐overproducing mutant in order to optimize its production in large‐scale bioreactors.
Abstract: Aims: To analyse the influence of nitrogen and carbon sources on extracellular lipase production by Yarrowia lipolytica-overproducing mutant in order to optimize its production in large-scale bioreactors. Methods and Results: The level of lipase production and LIP2 induction, measured using an LIP2–LacZ reporter gene, were compared for different carbon and nitrogen sources and for different concentrations. The localization of the enzyme during growth was also determined by Western blotting analysis using a six-histidine-tagged lipase. Significance and Impact of the Study: Tryptone N1 and oleic acid are the most suitable nitrogen and carbon sources for the production of the extracellular lipase by the Y. lipolytica mutant. Higher levels of lipase production were obtained as the tryptone concentration increased in the culture medium. Such a positive correlation was not observed with oleic acid media where the highest lipolytic productivities were obtained in the presence of low concentration. We also demonstrate that in the presence of oleic acid, lipase is cell-bound during the growth phase before being released in the media. Conclusions: This work provides a better understanding of the mechanism controlling LIP2 expression and, thus, extracellular lipase production in the yeast Y. lipolytica.

Journal ArticleDOI
TL;DR: In this paper, Crude Rhizopus oryzae lipase was used for enzymatic esterification between oleic acid and butanol at 37°C with shaking (200-rpm) in two systems: n-hexane and solvent-free.

Journal ArticleDOI
TL;DR: Immobilized lipase-catalyzed synthesis of tetrahydrofurfuryl butyrate is reported, which shows that esterification is a better method compared to transesterification.

Journal ArticleDOI
TL;DR: Promising results with the crude preparation of Penicillium aurantiogriseum lipolytic preparation justify the undertaking of purification studies and the use of the pure enzyme in a more in-depth investigation for its potential in biocatalysis in organic solvents.


Journal ArticleDOI
TL;DR: DNA family shuffling was used to create chimeric lipase B proteins with improved activity toward the hydrolysis of diethyl 3-(3',4'-dichlorophenyl)glutarate (DDG) and the stability characteristics of several highly active chimeric proteins were improved.
Abstract: DNA family shuffling was used to create chimeric lipase B proteins with improved activity toward the hydrolysis of diethyl 3-(3',4'-dichlorophenyl)glutarate (DDG). Three homologous lipases from Candida antarctica ATCC 32657, Hyphozyma sp. CBS 648.91 and Crytococcus tsukubaensis ATCC 24555 were cloned and shuffled to generate a diverse gene library. A high-throughput screening assay was developed and used successfully to identify chimeric lipase B proteins having a 20-fold higher activity toward DDG than lipase B from C.antarctica ATCC 32657 and a 13-fold higher activity than the most active parent derived from C.tsukubaensis ATCC 24555. In addition, the stability characteristics of several highly active chimeric proteins were also improved as a result of family shuffling. For example, the half-life at 45 degrees C and melting point (T(m)) of one chimera exceeded those of lipase B from C.antarctica ATCC 32657 by 11-fold and 6.4 degrees C, respectively, which closely approached the stability characteristics of the most thermostable parent derived from Hyphozyma sp. CBS 648.91.

Journal ArticleDOI
TL;DR: Sequence comparisons reveal that families of OBL1-like proteins are present in many species, and it is likely that they play an important role in regulating lipolysis.

Journal ArticleDOI
TL;DR: The lipase from Bacillus thermocatenulatus cloned in Escherichia coli was selectively adsorbed on this immobilized enzyme, enabling a very simple purification strategy and the enzyme was also able to adsorb other lipases.

Journal ArticleDOI
TL;DR: Olive oil was shown to increase lipase production in both free and immobilized cells, and different concentrations of alginate were tried to study their effect onlipase production.

Journal ArticleDOI
TL;DR: In this article, the conversion of soybean oil to biodiesel fuel was investigated in the presence of a lipase from Thermomyces lanuginosus (commercially called Lipozyme TL IM) in a solvent-free medium.
Abstract: The conversion of soybean oil to biodiesel fuel was investigated in the presence of a lipase from Thermomyces lanuginosus (commercially called Lipozyme TL IM) in a solvent-free medium. The lipase was inactivated when more than 1.5 molar equivalent of methanol was added to the oil mixture. To fully convert the oil to its corresponding methyl esters, the reaction was performed successfully by a three-step addition of 1 molar equivalent of methanol and under the optimized conditions (40°C, 150 rpm, 10% enzyme quantity based on oil weight), the maximum methyl ester (ME) yield was 98% after 12 h reaction. By-product glycerol had a negative effect on enzymatic activity and iso-propanol was found to be effective for glycerol removal, in the presence of which lipase expressed relatively high activity and more than 94% of the ME yield was maintained after being used repeatedly for 15 batches.

Journal ArticleDOI
TL;DR: In this paper, the cellulose fiber surfaces were reacted with polyethylene glycol (PEG) diacylchloride to simultaneously add amphiphilic spacers and reactive end groups for coupling with a lipase enzyme.
Abstract: Ultra-high specific surface cellulose fibers with an average diameter of 500 nm were generated from electrospinning and alkaline hydrolysis of cellulose acetate and used as porous supports for enzyme immobilization. The cellulose fiber surfaces were reacted with polyethylene glycol (PEG) diacylchloride to simultaneously add amphiphilic spacers and reactive end groups for coupling with a lipase enzyme. The quantity of reactive carboxylic acid on the fiber surfaces could be readily controlled by COCl/OH molar ratios and PEG lengths. The highest free acid (COOH) content of 1.0 mmol per gram of cellulose was obtained at 10 COCl/OH ratio with the 600-Da PEG diacylchloride. Enzyme coupling on such PEG-attached cellulose was optimal in the presence of a water-soluble carbodiimide [1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC)] at a very low EDC/COOH molar ratio of 0.2 under acidic condition and at ambient temperature. Whereas the free lipase retained only 25% of its original activity, the fiber-bound lipase possessed much superior retention of catalytic activity after exposure to cyclohexane (81%) and toluene (62%) and hexane (34%). The fiber-bound lipase also exhibited significantly higher catalytic activity at elevated temperatures than the free form, that is, 10 times at 70 °C. The ultra-fine, fibrous, and porous structures were retained throughout alkaline hydrolysis, activation, coupling, and activity assays. © 2004 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 42: 4289–4299, 2004

Journal ArticleDOI
TL;DR: There was a remarkable acceleration when the lipase was coated with this novel ionic liquid and used as catalyst for transesterification in i-Pr2O or in hexane.

Journal ArticleDOI
TL;DR: Observations could be explained in terms of a decrease in active-site flexibility brought about by the mutation and were consistent with the hypothesis that cold activity and thermolability arise from local flexibility around the active site of the enzyme.

Journal ArticleDOI
TL;DR: The ordered bi–bi mechanism with inhibition by both R(−)-methyl mandelate (substrate) and R( (−)-mandelic acid (product) was found to fit the initial rate data very well and the kinetic constants were determined.

Journal ArticleDOI
01 Jun 2004-Lipids
TL;DR: Protein engineering through lid swapping and rationally designed site-directed mutagenesis will continue to lead to the production of CRL isoforms with improved catalytic power, thermostability, enantioselectivity, and substrate specificity, while providing evidence for the mechanisms of actions of the various isoforms.
Abstract: Commercial preparations of Candida rugosa lipase (CRL) are mixtures of lipase isoforms used for the hydrolysis and synthesis of various esters. The presence of variable isoforms and the amount of lipolytic protein in the crude lipase preparations lead to a lack of reproducibility of biocatalytic reactions. Purification of crude CRL improve their substrate specificity, enantioselectivity, stability, and specific activities. The expression of the isoforms is governed by culture or fermentation conditions. Unfortunately, the nonsporogenic yeast C. rugosa does not utilize the universal codon CTG for leucine; therefore, most of the CTG codons were converted to universal serine triplets by site-directed mutagenesis to gain expression of functional lipase in heterologous hosts. Recombinant expressions by multiple-site mutagenesis or complete synthesis of the lipase gene are other possible ways of obtaining pure and different CRL isoforms, in addition to culture engineering. Protein engineering of purified CRL isoforms allows the tailoring of enzyme function. This involves computer modeling based on available 3-D structures of lipase isoforms. Lid swapping and DNA shuffling techniques can be used to improve the enantioselectivity, thermostability, and substrate specificity of CRL isoforms and increase their biotechnological applications. Lid swapping can result in chimera proteins with new functions. The sequence of the lid can affect the activity and specificity of recombinant CRL isoforms. Candida rugosa lipase is toxicologically safe for food applications. Protein engineering through lid swapping and rationally designed site-directed mutagenesis will continue to lead to the production of CRL isoforms with improved catalytic power, thermostability, enantioselectivity, and substrate specificity, while providing evidence for the mechanisms of actions of the various isoforms.

Journal ArticleDOI
TL;DR: This study demonstrates that the PFLD1 promoter is at least as efficient as the PAOX1 for extracellular expression of heterologous proteins in P. pastoris bioreactor cultures and provides a first basis for the further design of methanol-free high cell density fed-batch cultivation strategies for controlled overproduction of foreign proteins in the organism.

Journal ArticleDOI
TL;DR: The results show that CAL-B can be employed as a robust biocatalyst in esterification reactions due to the high conversions obtained in the synthesis of short-chain flavor esters in an organic solvent, although this enzyme exhibited modest enantioselectivity with chiral short- chain carboxylic acids.
Abstract: Candida antarctica lipase fraction B (CAL-B) showed substrate specificity in the synthesis of esters in hexane involving reactions of short-chain acids having linear (acetic and butyric acids) and branched chain (isovaleric acid) structures, an unsaturated (tiglic acid) fatty acid, and phenylacetic acid with n-butanol and geraniol. The variation in the conversion to the esters was ca. 10%. Similar results were observed in a study of the alcohol specificity of the enzyme for esterification of acetic and butyric acids with four alcohols: n-butyl, isopentyl, 2-phenylethyl, and geraniol. Enantioselectivity of CAL-B in hexane with a range of chiral α-substituted or β-substituted carboxylic acids and n-butyl alcohol was analyzed. The results show that CAL-B can be employed as a robust biocatalyst in esterification reactions due to the high conversions obtained in the synthesis of short-chain flavor esters in an organic solvent, although this enzyme exhibited modest enantioselectivity with chiral short-chain carboxylic acids.

Journal ArticleDOI
TL;DR: In this article, a response surface methodology based on a five-level, five-variable design was employed, firstly for studying the interactive effects of various parameters on the reactions and secondly, for their optimization.

Journal ArticleDOI
TL;DR: To stabilize the lipase activity of Rhizopus oryzae cells as whole-cell biocatalysts, the effect of cell membrane fatty acid composition on biodiesel-fuel production was investigated and both methanolysis activity and enzymatic stability were maintained at significantly elevated levels.