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Showing papers on "Lipase published in 1990"


Journal ArticleDOI
22 Feb 1990-Nature
TL;DR: The X-ray structure of the Mucor miehei triglyceride lipase is reported and the atomic model obtained reveals a Ser .. His .. Asp trypsin-like catalytic triad with an active serine buried under a short helical fragment of a long surface loop.
Abstract: True lipases attach triacylglycerols and act at an oil-water interface; they constitute a ubiquitous group of enzymes catalysing a wide variety of reactions, many with industrial potential. But so far the three-dimensional structure has not been reported for any lipase. Here we report the X-ray structure of the Mucor miehei triglyceride lipase and describe the atomic model obtained at 3.1 A resolution and refined to 1.9 A resolution. It reveals a Ser..His..Asp trypsin-like catalytic triad with an active serine buried under a short helical fragment of a long surface loop.

1,149 citations


Journal ArticleDOI
TL;DR: In this paper, the results of lipase-catalyzed, nonaqueous alcoholysis of sunflower oil under anhydrous conditions were examined and the overall content of tri-, di- and monoglycerides, as well as the corresponding alkyl esters, was measured.
Abstract: Lipase-catalyzed alcoholysis of sunflower oil under anhydrous conditions was examined. Lipases fromPseudomonas fluorescens and 2 immobilized enzymes fromMucor miehei and aCandida sp. gave sufficient conversion with petroleum ether as the solvent, even when methanol and ethanol were used. The overall content of tri-, di- and monoglycerides, as well as the corresponding alkyl esters, was measured. BecausePseudomonas lipase led to almost quantitative esterification, further studies were carried out with that enzyme varying the amounts of enzyme or the alcohols. Acceptable conversions were achieved even without solvent. Reaction rates of alcoholysis with 5 homologous alcohols, with or without the addition of water, were measured, and in all cases the reaction rates increased with higher chain length of the alcohol. In the case of methanol the highest rate was obtained without any addition of water, but a significantly higher rate was observed with 96% ethanol as opposed to absolute ethanol. The main advantages of lipasecatalyzed, nonaqueous alcoholysis as compared to classical procedures are the mild reaction conditions, the isolation of glycerin without further purification and without the formation of chemical waste, and the ability of lipases to catalyze the esterification of free fatty acids.

297 citations


Journal ArticleDOI
TL;DR: It is suggested that in situations when fatty acids are generated more rapidly by LPL than they are used by the local tissue, they cause dissociation of the enzyme from its binding to endothelial heparin sulfate and are themselves released into circulation.
Abstract: Lipoprotein lipase (LPL) catalyzes the flux-generating step in transport of fatty acids from lipoprotein triacylglycerols into tissues for use in metabolic reactions. In vitro studies have shown that fatty acids can bind to the enzyme and impede its other interactions. In this study we have searched for evidence of fatty acid control of LPL in vivo by rapid infusion of a triacylglycerol emulsion to healthy volunteers. During infusion the activity of LPL but not of hepatic lipase increased in plasma, but to different degrees in different individuals. The time course for the increase in LPL activity differed from that for triacylglycerols but followed the plasma levels of free fatty acids. This was true during infusions and when the emulsion was given as a bolus injection. In particular there were several instances when plasma triacylglycerol levels were very high but free fatty acids and LPL activity remained low. Model studies with bovine LPL showed that fatty acids displace the enzyme from heparin-agarose. We suggest that in situations when fatty acids are generated more rapidly by LPL than they are used by the local tissue, they cause dissociation of the enzyme from its binding to endothelial heparin sulfate and are themselves released into circulation.

236 citations


Journal ArticleDOI
TL;DR: The peparative synthesis of 35 short chain flavour esters by lipases from Mucor miehi, Aspergillus sp.
Abstract: The peparative synthesis of 35 short chain flavour esters by lipases fromMucor miehi, Aspergillus sp.,Candida rugosa andRhizopus arrhizus was investigated in organic media. Acetic, propionic, butyric, valeric and caproic acids, as well as methanol, ethanol, butanol, i-pentanol, hexanol, citronellol and geraniol were used as substrates. Most of the esters were synthesized in good yield by at least one of the lipase preparations tested. Different conversion yields were observed according to the lipase specificity toward the acid or the alcohol moiety of the ester. Methyl- and ethyl acetates were also produced by changing the organic solvent. Enzymatic catalysis in organic solvent is thought to be a valuable method for preparative synthesis of flavour esters.

213 citations


Journal ArticleDOI
TL;DR: It is concluded that digestion of human milk triacylglycerol depends on three lipases with unique, only partly overlapping, functions, whose concerted action results in complete digestion with free glycerol and fatty acids as final products.
Abstract: Gastric lipase, pancreatic colipase-dependent lipase, and bile salt-stimulated lipase all have potential roles in digestion of human milk triacylglycerol. To reveal the function of each lipase, an in vitro study was carried out with purified lipases and cofactors, and with human milk as substrate. Conditions were chosen to resemble those of the physiologic environment in the gastrointestinal tract of breast-fed infants. Gastric lipase was unique in its ability to initiate hydrolysis of milk triacylglycerol. Activated bile salt-stimulated lipase could not on its own hydrolyze native milk fat globule triacylglycerol, whereas a limited hydrolysis by gastric lipase triggered hydrolysis by bile salt-stimulated lipase. Gastric lipase and colipase-dependent lipase, in combination, hydrolyzed about two thirds of total ester bonds, with monoacylglycerol and fatty acids being the end products. Addition of bile salt-stimulated lipase resulted in hydrolysis also of monoacylglycerol. When acting together with colipase-dependent lipase, bile salt-stimulated lipase contributed also to digestion of tri- and diacylglycerol. We conclude that digestion of human milk triacylglycerol depends on three lipases with unique, only partly overlapping, functions. Their concerted action results in complete digestion with free glycerol and fatty acids as final products.

195 citations


Journal ArticleDOI
TL;DR: The detection of two well-defined pH optima, one at slightly acidic and the other at alkaline pH for both the intestinal and pancreatic lipases suggests a physiological versatility for lipid digestion in milkfish.

189 citations



Journal ArticleDOI
TL;DR: Differences in solubility between phospholipid-stabilized emulsions of medium-chain (MCT) versus long-chain triacylglycerols (LCT) found that both purified bovine milk lipoprotein lipase and human hepatic lipase hydrolyzed MCT at rates at least 2-fold higher than for LCT.
Abstract: To explore how enzyme affinities and enzyme activities regulate hydrolysis of water-insoluble substrates, we compared hydrolysis of phospholipid-stabilized emulsions of medium-chain (MCT) versus long-chain triacylglycerols (LCT). Because substrate solubility at the emulsion surface might modulate rates of hydrolysis, the ability of egg yolk phosphatidylcholine to solubilize MCT was examined by NMR spectroscopy. Chemical shift measurements showed that 11 mol % of [13C]carbonyl enriched trioctanoin was incorporated into phospholipid vesicles as a surface component. Similar methods with [13C]triolein showed a maximum solubility in phospholipid bilayers of 3 mol % (Hamilton & Small, 1981). Line widths of trioctanoin surface peaks were half that of LCT, and relaxation times, T1, were also shorter for trioctanoin, showing greater mobility for MCT in phospholipid. In assessing the effects of these differences in solubility on lipolysis, we found that both purified bovine milk lipoprotein lipase and human hepatic lipase hydrolyzed MCT at rates at least 2-fold higher than for LCT. With increasing concentrations of MCT, saturation was not reached, indicating low affinities of lipase for MCT emulsions, but with LCT emulsion incubated with lipoprotein lipase, saturation was reached at relatively low concentration, demonstrating higher affinity of lipase for LCT emulsions. Differences in affinity were also demonstrated in mixed incubations where increasing amounts of LCT emulsion resulted in decreased hydrolysis of MCT emulsions. Increasing MCT emulsion amounts had little or no effect on LCT emulsion hydrolysis.(ABSTRACT TRUNCATED AT 250 WORDS)

178 citations


Journal ArticleDOI
TL;DR: It is worth noting that rabbit gastriclipase, unlike human gastric lipase, became more stereoselective for the triglyceride with shorter acyl chains (trioctanoin), one of the most striking catalytic differences observed between these two Gastric lipases.

171 citations


Journal ArticleDOI
01 Jan 1990
TL;DR: Applications of lipase-catalyzed reactions, such as hydrolysis of fats for the production of fatty acids and esterification or interesterification of fats and other lipids for the preparation of diverse products in food and non-food industries, are reviewed.
Abstract: Applications of lipase-catalyzed reactions, such as hydrolysis of fats for the production of fatty acids and esterification or interesterification of fats and other lipids for the preparation of diverse products in food and non-food industries, are reviewed. At present, the application of lipases in biotechnological processes seems to be economically feasible and appropriate mainly for the preparation of specific products of high commercial value, which cannot be prepared conveniently by chemical synthesis. For example, polyunsaturated fatty acids that can be used in dietetic products are prepared under mild conditions by hydrolysis of marine oils and certain plant oils with non-specific triacylglycerol lipases. Very long chain monounsaturated fatty acids (gadoleic, erucic and nervonic) that are of value in oleochemical industry can be prepared by partial hydrolysis of cruciferous oils with sn-1,3-specific lipases. Lipase-catalyzed esterification yields a variety of products, such as monoacylglycerols tha...

171 citations


Journal ArticleDOI
TL;DR: In this paper, the peroxycarboxylic acids formed continuously in situ by lipase-catalysed perhydrolysis of the corresponding carboxyric acids were used for alkenes.
Abstract: Epoxidation of alkenes was achieved under extremely mild conditions by employing peroxycarboxylic acids formed continuously in situ by lipase-catalysed perhydrolysis of the corresponding carboxylic acids.

Journal ArticleDOI
TL;DR: The dietary hyperlipidemia caused by the high-fructose diet was improved by Spirulina feeding, accompanied by a significant increase in the lipoprotein lipase activity in post-heparin plasma.
Abstract: The effects of Spirulina platensis on lipoprotein lipase activity and hepatic triglyceride lipase activity in post-heparin plasma were studied in fructose-induced hyperlipidemic rats. Male Wistar rats aged 3 weeks old (body weight, 54 g) were fed on the high-fructose diet (68%) or the high-fructose diets containing Spirulina at the level of 5, 10, and 15%, respectively, for 4 weeks. The dietary hyperlipidemia caused by the high-fructose diet was improved by Spirulina feeding, accompanied by a significant increase in the lipoprotein lipase activity in post-heparin plasma.

Journal ArticleDOI
TL;DR: Lipase immobilized on PVC exhibited higher thermal stability than that on agarose and the specific activity of the immobilized lipase was affected by the pore size but not by the chain length of the hydrocarbon attached to the support.
Abstract: Six different types of materials including PVC, chitosan, chitin, agarose, Sepharose, and Trisacryl were evaluated for their lipase-coupling efficiencies. Among those tested, chitosan yielded the highest amount of lipase (79 mg/mL packed gel) immobilized but with lowest oil hydrolytic activity (0.03 mg eq/mL gel). The amount of lipase immobilized was affected by the length of the hydrocarbon chain attached to the PVC matrix but not by the pore size of the supports used. On the other hand, the specific activity of the immobilized lipase was affected by the pore size but not by the chain length of the hydrocarbon attached to the support. After immobilization, the optimal reaction pH was shifted from 7.5 to 8.5 and the optimal reaction temperature from 35 to 45-55 degrees C. Lipase immobilized on PVC exhibited higher thermal stability than that on agarose. The half-life of the PVC immobilized lipase operating at 30 degrees C in a packed-bed reactor was estimated to be about 400 h.

Journal ArticleDOI
TL;DR: In this article, the triacylglycerol lipase preparations were evaluated for their suitability as catalysts in the interesterification of palm oil mid fraction and ethyl stearate to form a cocoa butter equivalent.
Abstract: Twelve commercially available triacylglycerol lipase preparations were screened for their suitability as catalysts in the interesterification of palm oil mid fraction and ethyl stearate to form a cocoa butter equivalent. Five fungal lipase preparations were found to be suitable. The hydrolytic activity of the commercial lipase preparations was tested with sunflower seed oil and was independent of their interesterification activity. The operational stability of three of the preparations most suited for production of cocoa butter equivalents was examined. The amount of a commercial lipase preparation loaded onto a support was surveyed for optimum short-term catalytic activity.

Journal ArticleDOI
TL;DR: Comparisons with other enzymes show that the lipase is a new member of the supergene family of serine hydrolases, not only closely related to lysophospholipase from rat pancreas and cholesterol esterase from bovine Pancreas, but also shows a high degree of similarity to several esterases.
Abstract: We have isolated and sequenced cDNA clones covering the entire coding sequence of human-milk bile-salt-stimulated lipase, as well as 996 nucleotides of the 3' end of the pancreatic enzyme carboxylic ester hydrolase. The deduced amino acid sequence of the lipase starts with a 23-residue leader peptide. The open reading frame continues with 722 amino acid residues. The sequence contains in the C-terminal part a proline-rich repeat, 16 repeats of 11 amino acid residues each. The mRNA was estimated to be approximately 2500 nucleotides from Northern blot and of similar size in mammary and pancreatic tissues. Data obtained indicate that the lipase and the carboxylesterase are identical and coded for by the same gene. The cDNA is 2428 bases long, which indicates that a near full-length copy of the transcript has been isolated. Comparisons with other enzymes show that the lipase is a new member of the supergene family of serine hydrolases. It is not only closely related (and in its N-terminal half virtually identical) to lysophospholipase from rat pancreas and cholesterol esterase from bovine pancreas, but also shows a high degree of similarity to several esterases, e.g. acetylcholine esterase. In contrast, no such similarity could be found to typical lipases.

Journal ArticleDOI
TL;DR: It was observed that the structure of the media, as dictated by the type and concentration of the substrates and products and by the water/AOT ratio, w0, had a strong impact on enzyme activity.
Abstract: The activities of lipase from Candida cylindracea and Rhizopus delemar have been investigated in water/AOT/iso-octane reverse micellar media through the use of two esterification reactions: fatty acid-alcohol esterification and glyceride synthesis. Such media promotes the occurrence of these two lipase-catalyzed reactions due to its low water content. The effect of various parameters on the activity of lipase from C. cylindracea in reverse micelles was determined and compared to results where alternate media were employed. It was observed that the structure of the media, as dictated by the type and concentration of the substrates and products and by the water/AOT ratio, w0, had a strong impact on enzyme activity. Strong deactivation of both typase types occurred in reverse micelles, especially in the absence of substrates and for w0 values greater than 3.0. Glyceride synthesis was realized with lipase from R. delemar, but not with that from C. cylindracea; the temperature and concentration of substrates and water strongly dictated the reaction rate and the percent conversion.

Journal ArticleDOI
TL;DR: Immobilized Mucor miehei lipase catalyzes synthesis reactions between glycerol and oleic acid, which allows for the preferential synthesis of one of the glycerides.
Abstract: Immobilized Mucor miehei lipase catalyzes synthesis reactions between glycerol and oleic acid. No organic solvent is necessary to solubilize the substrates, which allows for the use of a reaction medium solely composed of the necessary substrates. Water produced in the reaction evaporates due to the high temperature used for the process. A conversion of 86% of oleic acid into triolein is obtained when using the substrates in stoichiometric amounts. Varying the ratio of glycerol over oleic acid allows for the preferential synthesis of one of the glycerides. Some batch reactors have been set up using different means of removing the water: spontaneous evaporation, molecular sieves, vacuum, and dry air bubbling.

Journal ArticleDOI
TL;DR: It is concluded that in the presence of a large excess of lipase the liberated fatty acids would approach the composition of the dietary alkyl or glyceryl esters, as observed during lumenal lipolysis.

Journal Article
TL;DR: Lingual and gastric lipase activity has been found in subjects with cystic fibrosis and appears to continue in the upper small intestine in these patients, perhaps replacing some of the missing pancreatic lipase.

Journal ArticleDOI
TL;DR: Candida cylindracea lipase seems the most promising with respect to recovery of triacylglycerol and Aspergillus niger lipase increased not only the docosahexaenoic acid (DHA) content but also eicosapentaenoic acids (EPA) and docosapentenoic Acid (DPA), although the absolute value of the latter was quite low.
Abstract: Six lipases were examined for concentrating the n-3 polyunsaturated fatty acid (n-3 PUFA) of two kinds of fish oil (cod liver oil and refined sardine oil). Although all lipases could increase the n-3 PUFA content of the remaining glycerides, Candida cylindracea and Aspergillus niger lipases gave glycerides with a more than two-fold increase in n-3 PUFA content over the original fish oils.Candida cylindracea lipase seems the most promising with respect to recovery of triacylglycerol. Aspergillus niger lipase increased not only the docosahexaenoic acid (DHA) content but also eicosapentaenoic acid (EPA) and docosapentaenoic acid (DPA), although the absolute value of the latter was quite low. The effects of temperature (15–40 °C) on the concentration of n-3 PUFA were investigated. Lower temperatures did not improve the concentration of n-3 PUFA, but prevented the development of an unpleasant odor in the product.

Journal ArticleDOI
TL;DR: Southern blot analysis of the Geotrichum candidum genome with a cloned lipase cDNA as the probe indicated the existence of two genes on the chromosome of the fungus which are homologous to the cDNA.
Abstract: Southern blot analysis of the Geotrichum candidum genome with a cloned lipase cDNA as the probe indicated the existence of two genes on the chromosome of the fungus which are homologous to the cDNA. As expected, two forms of lipase (lipases I and II) were actually isolated by hydrophobic interaction chromatography after a multistep procedure including ammonium sulfate fractionation, anion exchange chromatography, and gel filtration of the culture filtrate. Lipase I, the first eluted fraction, was the predominant form, and more than 80% of the total activity was attributed to this form. Amino acid sequence analysis of the amino and carboxyl termini of these two enzyme preparations indicated that lipase I was the product of the lipase gene whose cDNA had previously been cloned and sequenced [Shimada et al. (1989) J. Biochem. 106, 383-388]. Lipase II, on the other hand, had similar amino acid composition, but different terminal sequences which were not found in the primary structure of lipase I deduced from the cDNA sequence. These results gave lines of evidence for the expression of truely different lipase genes and ruled out the possibility that the observed multiple forms are caused by proteolytic digestion. The molecular mass estimated by SDS-PAGE and the isoelectric point of lipase I were 64 kDa and 4.3, while those of lipase II were 66 kDa and 4.3, respectively. The two lipases had essentially the same specific activities, substrate specificities, pH stabilities, and optimal temperatures, but different pH optima and thermal stabilities.

Journal ArticleDOI
TL;DR: Stability of the lipase has been proved to be correct and similar in both solvents and inhibition by ethanol excess has been found but is greater in n-hexane, which can explain the higher initial velocities obtained in supercritical carbon dioxide for the highest ethanol concentrations.
Abstract: Oleic acid esterification by ethanol has been performed by an immobilized lipase fromMucor miehei in supercritical carbon dioxide and in n-hexane as solvents. In both media, determination of apparent kinetic constants has been achieved and influence of water content has been shown to be different due to various rates of water solubilities. Stability of the lipase has been proved to be correct and similar in both solvents. Inhibition by ethanol excess has been found but is greater in n-hexane. That can explain the higher initial velocities obtained in supercritical carbon dioxide for the highest ethanol concentrations.

Journal ArticleDOI
TL;DR: The results presented here demonstrate the absolute necessity to consider the polarity of every substrate, because of its ability to modify the water partition between the solid phase (enzyme preparation) and the liquid phase (substrate and product), which results in drastic changes in enzyme activity.
Abstract: The reaction rate of two lipase-catalysed reactions, esterification and transesterification, were studied in a liquid/solid two-phase system in order to investigate the effect of water partition between the enzyme preparation and the liquid phase composed of only the reactants, i.e. without the conventional solvents. Lipase from Candida cylindracea was used for these studies. The enzyme was inactive in dehydrated systems. In the case of monoester synthesis, the reaction rate increased with increasing water activity. The reaction rates of the non-specific C. cylindracea lipase-catalysed reactions were very sensitive to the nature of the substrates in this unusual system. For instance, the transesterification reaction rate of ethyl propionate was 48 times higher with nonanol than heptanol in the case of dehydrated substrates, but only 2.2 times higher in the case of watersaturated substrates. The results presented here demonstrate the absolute necessity to consider the polarity of every substrate, because of its ability to modify the water partition between the solid phase (enzyme preparation) and the liquid phase (substrate and product), which results in drastic changes in enzyme activity. Contrary to esterification, which is known to be activated by the water produced, the rate of transesterification remained constant at the beginning of the reaction. However, when transesterification and esterification were carried out in the same liquid phase, the transesterification reaction rate was controlled by the water produced by the concomitant esterification. Activation effects of the water molecules produced during the enzymatic reaction were of exactly the same order of magnitude for both reactions.

Journal ArticleDOI
TL;DR: Results clearly show that hepatic lipase may strongly promote chylomicron remnant cholesterol ether uptake by the liver.

Journal ArticleDOI
TL;DR: In this paper, the authors used the monolayer technique to study protein-lipid interactions and found a good correlation between the amount of lipase bound to the lipid monolayers and the surface pressure increase.

Journal ArticleDOI
TL;DR: It is concluded that DNA variation at or adjacent to the lipoprotein lipase gene may contain genetic determinants for the occurrence of premature coronary artery disease.

Journal ArticleDOI
TL;DR: Properties of lipases excreted by 4 psychrotropic Moraxella strains from antarctic sea water have investigated, finding that one of the selected strain is able to express the antibiotic resistances carried by RP4, at both 4 and 25°C.
Abstract: Properties of lipases excreted by 4 psychrotropic Moraxella strains from antarctic sea water have investigated. Despite an optimal cell generation time at 25°C, maximal lipase excretion essentially occurs at low temperatures. These enzymes are characterized by a shift of the apparent optimal activity towards low temperatures, by a reduction of the activation energy value and by a decrease in heat stability. These lipases are associated with lipopolysaccharides, forming high molecular weight complexes. One of the selected strain is able to express the antibiotic resistances carried by RP4, at both 4 and 25°C.

Journal ArticleDOI
TL;DR: It is deduced that the following three factors are required for a pure enzyme to exhibit its full activity in a water‐immiscible organic solvent: optimum moisture content, disperser (support particles having enough surface area on which the enzyme is thinly deposited), and activity enhancer (additive) at optimum concentration.
Abstract: Various factors affecting the catalytic activity of pure lipase of Pseudomonas fluorescens in microaqueous benzene were investigated with respect to lactonization of 15-hydroxypentadecanoic acid. Without deposition of the enzyme or of the enzyme plus activity enhancer (additive) on celite powder, the pure enzyme was very poorly dispersed in the microaqueous benzene, resulting in very low activity. The enzyme immobilized on celite powder exhibited the highest activity at a free water content of ca. 0.083%. When a sugar alcohol such as erythritol, arabitol, or sorbitol was added before lyophilization with approximate proportion of 3 g/g enzyme, marked increases in the enzyme activity were observed at a shifted optimal free water content, i.e., 0.04%. Inclusion of phosphotidylcholine resulted in a somewhat higher activity than in the system of enzyme plus celite only. Addition of lactose, bovine serum albumin, casein, dextran, polyvinyl alcohol, phosphate, or NaCl all caused a decrease in the enzyme activity. From the effects of the additives examined, it is deduced that the following three factors are required for a pure enzyme to exhibit its full activity in a water-immiscible organic solvent: (1) optimum moisture content, (2) disperser (support particles having enough surface area on which the enzyme is thinly deposited), and (3) activity enhancer (additive) at optimum concentration The importance of noting the purity of the enzyme preparation is emphasized when its catalysis in an organic solvent is investigated.

Journal ArticleDOI
TL;DR: These enzyme-catalyzed kinetic resolutions constitute an economical method of preparing enantiomerically pure 2-substituted esters and acids on a large scale and are important intermediates in the synthesis of bioactive compounds in the areas of prostanoids, angiotensin-converting enzyme inhibitors, and leukotriene antagonists.
Abstract: Enantioselective hydrolyses of a variety of 2-substituted racemic esters catalyzed by a bacterial lipase have been effected in high enantiomeric excess. These enzyme-catalyzed kinetic resolutions constitute an economical method of preparing enantiomerically pure 2-substituted esters and acids on a large scale. Several of the resolved enantiomers are important intermediates in the synthesis of bioactive compounds in the areas of prostanoids, angiotensin-converting enzyme inhibitors, and leukotriene antagonists

Journal ArticleDOI
TL;DR: It is demonstrated that duodenal deliveries of lipase and amylase activities increase when intraluminal protease activity is decreased and suggested that this increase is not caused by slower proteolytic destruction of enzyme protein but by stimulation of pancreatic secretion, which may in part be independent of cholecystokinin.