Lipase activity in the human aorta

The hydrolysis of triglycerides by grossly normal male human aortas has been studied in vitro. The tissue contains an acid lipase (pH optimum, 5.4) and an alkaline lipase (pH optimum, 8.8). Both lipases catalyze the hydrolysis of saturated triglycerides; the rate decreases with increasing fatty acyl chain from C(10) to C(18). Glycerol trioleate, trilinoleate, and trilinolenate are hydrolyzed at similar rates. Alkaline lipase is inhibited about 50% at 7.2 mm glycerol trioleate, while acid lipase is unaffected at this concentration. Both lipases are activated by Ca(++) ions. The acid lipase is easily inactivated by deionized water used either as a homogenizing or dialyzing medium. Acid lipase is strongly inhibited by BSA, sodium deoxycholate, and sodium taurocholate; alkaline lipase is unaffected by BSA and is activated about twofold by bile salts. The products of hydrolysis of glycerol trioleate by aortic lipases are predominantly oleic acid and glycerol 1,2-dioleate with a small accumulation of glycerol monooleate. The aortic preparations appear to contain inhibitors for both the acid and alkaline lipase. The substance which inhibits alkaline lipase also inhibits pancreatic lipase; it is heat-stable and dialyzable. The inhibitor of the acid lipase is also heat-stable but is nondialyzable.     

Specificity and glyceride synthesis by mycelial lipases of Rhizopus delemar

Mycelial lipase activity of the mould Rhizopus delemar was purified by gel filtration chromatography to three distinct proteins of notable lipase activity. The three enzymes were designated A′, B′ and C′, according to elution volumes from a Sephadex G150 column. The capacity of the three lipases to catalyse glyceride synthesis from free fatty acids and glycerol indicated a tendency towards short-chain and unsaturated fatty acids in preference to long-chain saturated fatty acids. The postional specificity of all lipases involved in such synthetic reactions indicated the formation of ester bonds at positions 1 and 3 of glycerol.     

Substrate specificity of Staphylococcus aureus (TEN5) lipases with isomeric oleoyl-sn-glycerol ethers as substrates

For the first time fully protected substrates with only one hydrolyzable ester bond have been used to analyze the substrate specificity of microbial lipases. In these substrates the ester is attached to the glycerol molecule in a precisely defined position. The use of three different substituents generates chirality and thus allows the analysis of positional specificities of individual lipases. Therefore, these new substrates have been used to study the enzymatic activities of two closely related lipases isolated from Staphylococcus aureus (TEN5) designated the 44 and 43 kDa lipase. The lipases, especially the 44 kDa molecule, show a high specificity for the hydrolysis of the ester in the sn-1 position (S-configuration), which is hydrolyzed by a factor of ten faster than that in the sn-3 position. In addition, the study demonstrates for the first time that the rate of hydrolysis of a fatty acid ester attached to the sn-2 position of glycerol by microbial lipases depends on the configuration of the substrate molecule.     

Lipase specificity towards eicosapentaenoic acid and docosahexaenoic acid depends on substrate structure

The fatty acid specificity of five lipases towards eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) was evaluated in the hydrolysis of fish oil, squid oil and a model system. The model system contained methyl esters of EPA, DHA and palmitic acid. All the investigated lipases discriminated against both EPA and DHA more in the model system than in the natural oils. Thus both EPA and DHA were more easily hydrolysed from a glyceride than from a methyl ester. In the model system, the lipase from Candida rugosa showed the highest discrimination against DHA, while the lipases from Pseudomonas fluorescens and Pseudomonas cepacia discriminated against EPA the most. In a glyceride, the fatty acid specificity of lipases towards EPA and DHA was affected by the positional distribution of the fatty acids and the glyceride structure due to the regiospecificity and triglyceride specificity of the lipase. In the oils, the Pseudomonas lipases also discriminated against EPA the most, while DHA was initially discriminated the most by the lipase from Thermomyces lanuginosus. However, after longer reaction times the enrichment of DHA in the glyceride fraction of the oils was greatest for the lipase from C. rugosa.     

Various lipases for producing products enriched with polyenic acids in fish fat hydrolysis]

The enzymic hydrolysis of fish with lipases from various sources was studied. The lipase from the fungus Rhizopus microsporus preferentially removes saturated fatty acids, while lipase from the pyloric caeca of salmon unsaturated fatty acids upon hydrolysis of fish fats. The enzymes can be used to obtain fatty products enriched with eicosanopentaenoic acid, mono- and diacylglycerols by enzymic hydrolysis of the ivasi fat.     

Hormone-sensitive lipase and monoacylglycerol lipase are both required for complete degradation of adipocyte triacylglycerol

The respective roles of monoacylglycerol lipase and hormone-sensitive lipase in the sequential hydrolysis of adipose tissue triacylglycerols have been examined. An adipose tissue preparation, containing both lipases in approximately the same proportion as in the intact tissue, hydrolyzed emulsified tri- or dioleoylglycerol to fatty acids and glycerol, with little accumulation of di- or monooleoylglycerol. Selective removal of the monoacylglycerol lipase by immunoprecipitation markedly reduced the glycerol release. Isolated hormone-sensitive lipase hydrolyzed acylglycerols with a marked accumulation of monoacylglycerol in accordance with the positional specificity of this enzyme (Fredrikson, G. and Belfrage, P. (1983) J. Biol. Chem. 258, 14253-14256). Addition of increasing amounts of isolated monoacylglycerol lipase led to a corresponding increase in glycerol release, due to hydrolysis of the monoacylglycerols formed. The reaction proceeded to completion when the relative proportion of the two lipases was similar to that in the intact tissue. These findings indicate that hormone-sensitive lipase catalyzes the hydrolysis of triacylglycerol in the rate-limiting step of adipose tissues lipolysis, and of the resulting diacylglycerol, whereas the action of monoacylglycerol lipase is required in the final hydrolysis of the 2-monoacylglycerols produced.     

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

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

Substrate specificities of lipases from corn and other seeds

Lipases from several seed species were shown to be relatively specific on triacylglycerols containing the major fatty acid components of the storage triacylglycerols in the same species. In a direct comparison using individual triacylglycerol as well as mixed triacylglycerol preparations, highest activities were observed in corn lipase on trilinolein and triolein, castor bean lipase on triricinolein, rapeseed lipase on trierucin, and elm seed lipase on tricaprin. This pattern of fatty acyl specificity was also observed on diacylglycerols, monoacylglycerols, and fatty acyl 4-methylumbelliferone, although the pattern became less distinct. The seed lipases were inactive on lecithins. Corn lipase was more active on tri- than di- or monolinolein, and released linoleic acids from both primary and secondary positions. As judged from the kinetics of hydrolysis of rac-glyceryl-2,3-stearate-1-oleate and rac-glyceryl-1,3-stearate-2-oleate, and of trilinolein and dilinoleins, corn lipase exerted some degree of preference in releasing fatty acid from the primary than the secondary position of a triacylglycerol. At the primary position, corn lipase was more active on oleyl ester than stearyl ester.     

Acidolysis between triolein and short-chain fatty acid by lipase in organic solvents

Ten kinds of lipases were examined as biocatalysts for the incorporation of short-chain fatty acids (acetic, propionic, and butyric acids) into triolein in order to produce one kind of reduced-calorie structured lipids. Trans-esterification (acidolysis) was successfully done in n-hexane by several microbial lipases. Among them, lipase from Aspergillus oryzae was used to investigate the effects of incubation time, substrate molar ratio, and water content on acidolysis. Finally, more than 80% of triolein was incorporated by butyric acid (molar ratio of triolein to butyric acid, 1:10) in the dried n-hexane at 52 degrees C for 72 h. More than 90% of the products was monosubstituent, which was esterified with this short chain fatty acid at the 1-position of the glycerol moiety of triolein. These results suggest that A. oryzae lipase would be a powerful biocatalyst for the synthesis of low caloric oil, such as triacylglycerol containing a mixture of long- and short-chain aliphatic acids.     

Inhibition of lipases from Chromobacterium viscosum and Rhizopus oryzae by tetrahydrolipstatin

Tetrahydrolipstatin is known as an inhibitor for pancreatic lipase but not for microbial lipases. In this paper we demonstrate that in the presence of water-insoluble substrates like tributyrin or olive oil, tetrahydrolipstatin inhibits the lipases of Chromobacterium viscosum and Rhizopus oryzae, although with different potency. In contrast to porcine pancreatic lipase, which forms an irreversible and covalent enzyme-inhibitor complex with tetrahydrolipstatin, the inhibition of the microbial lipases is reversible as the inhibitor can be removed from the enzyme-inhibitor complex by solvent extraction. Moreover, after inhibition of Chromobacterium viscosum lipase tetrahydrolipstatin remains chemically unchanged.     

Substrate specificity of Geotrichum candidum lipase preparations

Summary Lipases with different fatty acid specificity were produced byGeotrichum candidum depending on growth condition. The hydrolysis of olive oil was inhibited by glycerol tributyrate and was dependent on Ca-ions for running at maximal rate.     

Lipase applications in oil hydrolysis with a case study on castor oil: a review

Lipase (triacylglycerol acylhydrolase) is a unique enzyme which can catalyze various types of reactions such as hydrolysis, esterification, alcoholysis etc. In particular, hydrolysis of vegetable oil with lipase as a catalyst is widely studied. Free lipase, lipase immobilized on suitable support, lipase encapsulated in a reverse micelle and lipase immobilized on a suitable membrane to be used in membrane reactor are the most common ways of employing lipase in oil hydrolysis. Castor oil is a unique vegetable oil as it contains high amounts (90%) of a hydroxy monounsaturated fatty acid named ricinoleic acid. This industrially important acid can be obtained by hydrolysis of castor oil. Different conventional hydrolysis processes have certain disadvantages which can be avoided by a lipase-catalyzed process. The degree of hydrolysis varies widely for different lipases depending on the operating range of process variables such as temperature, pH and enzyme loading. Immobilization of lipase on a suitable support can enhance hydrolysis by suppressing thermal inactivation and estolide formation. The presence of metal ions also affects lipase-catalyzed hydrolysis of castor oil. Even a particular ion has different effects on the activity of different lipases. Hydrophobic organic solvents perform better than hydrophilic solvents during the reaction. Sonication considerably increases hydrolysis in case of lipolase. The effects of additives on the same lipase vary with their types. Nonionic surfactants enhance hydrolysis whereas cationic and anionic surfactants decrease it. A single variable optimization method is used to obtain optimum conditions. In order to eliminate its disadvantages, a statistical optimization method is used in recent studies. Statistical optimization shows that interactions between any two of the following pH, enzyme concentration and buffer concentration become significant in presence of a nonionic surfactant named Span 80.     

Interesterification of phosphatidylcholine with lipases in organic media

Summary 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. Offprint requests to: I. Svensson     

Enzymatic hydrolysis of soybean oil using lipase from different sources to yield concentrated of polyunsaturated fatty acids

The ability of three commercially available lipases to mediate the hydrolysis of the soybean oil to yield concentrated of essential fatty acids was evaluated. The tested lipases were from microbial (Candida rugosa and Thermomyces lanuginosa) and animal cells (Porcine pancreatic lipase). In terms of free fatty acids, microbial lipases were more effective to promote the enzymatic hydrolysis of the soybean oil (over 70%) than the porcine pancreatic lipase (24%). In spite of this, porcine pancreatic lipase (PPL) showed the most satisfactory specificity towards both essential fatty acids and was, therefore, chosen to carry out additional studies. An experimental design was performed taking into consideration the enzyme and NaCl amounts as independent variables. The main effects were fitted by multiple regression analysis to a linear model and maximum fatty acids concentration could be obtained using 3.0 wt% of lipase and 0.08 wt% of NaCl. The mathematical model representing the hydrolysis degree was found to describe adequately the experimental results. Under these conditions, concentrations of 29.5 g/L and 4.6 g/L for linoleic and linolenic acids, respectively, were obtained.     

Simple screening method for lipase for transesterification in organic solvent

We investigated lipase-catalyzed hydrolysis in water and dioxane—water with a simple colorimetric method. We screened 24 lipases for the ability to hydrolyze p-nitrophenyl esters as chromogenic substrates. Their hydrolytic activities were varied by adding dioxane. Most of the lipases showed high activity in hydrolysis in water, but some showed activity in 50% dioxane—water several tens times higher than those in water. Moreover, several lipases with hydrolytic abilities in 50% dioxane—water also catalyzed the transesterification of p-nitrophenol using fatty acid vinyl esters. We found it possible that a useful lipase for transesterification can be selected by measuring the hydrolysis activity of p-nitrophenyl ester in 50% dioxane—water.     

Pancreatic lipase activity as influenced by unconjugated bile acids and pH,measured in vitro and in vivo

The relation between pancreatic lipase activity, unconjugated bile acids and pH was studied in vitro and in vivo. Lipase activity was assayed in vitro using automatic titration, where the fatty acids liberated from the hydrolysis of glycerol tributyrate (GTB) were measured. The lipase activity was determined at different ratios of conjugated to unconjugated bile acids (100:0, 75:25, 50:50, 25:75, 0:100) in response to pH 6.6, 6.8, 7.0 and 7.5. The in vivo study involved 96 one-day-old male broiler chickens. The chickens were assigned randomly, in pens of six animals, into two dietary treatments (8 replicate blocks), composing a non-supplemented diet (A(-)) and a diet supplemented (A(+)) with avilamycin (10 mg/kg feed) and salinomycin (40 mg/kg feed). After 35 days, the chickens were killed and content of the proximal part of the small intestine was collected and analyzed for bacterial counts, pH, bile acid concentration, and lipase activity. Evidence for a significant pH-dependent inhibition of lipase activity by unconjugated bile acids was provided in vitro and confirmed in vivo. Due to a reduction in nutrient fermentation, the pH in the small intestine of antibiotic-fed chickens was significantly higher than in chickens fed the non-supplemented diet. The high pH in the small intestine of chickens fed the A(+)diet was accompanied by a significant increase in lipase activity, and coincided with a significantly lower concentration of unconjugated bile acids and a higher ratio of conjugated to unconjugated bile acids. This study emphasizes the important influence of unconjugated bile acids on lipase activity at physiological pH-values.     

Inhibition of human pancreatic lipase by tetrahydrolipstatin: Further kinetic studies showing its reversibility

Tetrahydrolipstatin (THL, Orlistat) is a potent inhibitor of gastrointestinal lipases. Using the pH-stat technique we report that, in the absence of substrate, THL (at a molar excess of 100) inhibits rapidly (after few minutes of incubation) human pancreatic lipase (HPL). Bile salts over their critical micellar concentration (CMC) were found to accelerate the inhibition process.At variance with the generally accepted model of a covalent and quasi-irreversible acyl-lipase complex, we showed here that the inhibition of HPL could be rapidly and partially reversed in the presence of an emulsion of short- or long-chain triacylglycerols, as indicated by a kinetic reactivation process. The presence of bile salts in the incubation medium, containing THL and HPL, was found to stabilise the covalent complex as reflected by a decrease in the reactivation rate. Paradoxically, the presence of bile salts in the lipase assay enhanced this reactivation process probably by forming mixed micelles between bile salts and THL, which accelerates the deacylation phenomenon.On the basis of this kinetic study, a general model is proposed to describe the inhibition of lipases by THL in the aqueous phase as well as its partial reactivation process at the lipid–water interface.     

Acidolysis between Triolein and Short-Chain Fatty Acid by Lipase in Organic Solvents

Ten kinds of lipases were examined as biocatalysts for the incorporation of short-chain fatty acids (acetic, propionic, and butyric acids) into triolein in order to produce one kind of reduced-calorie structured lipids. Trans-esterification (acidolysis) was successfully done in n-hexane by several microbial lipases. Among them, lipase from Aspergillus oryzae was used to investigate the effects of incubation time, substrate molar ratio, and water content on acidolysis. Finally, more than 80% of triolein was incorporated by butyric acid (molar ratio of triolein to butyric acid, 1:10) in the dried n-hexane at 52 °C for 72 h. More than 90% of the products was monosubstituent, which was esterified with this short chain fatty acid at the 1-position of the glycerol moiety of triolein. These results suggest that A. oryzae lipase would be a powerful biocatalyst for the synthesis of low caloric oil, such as triacylglycerol containing a mixture of long- and short-chain aliphatic acids.     

Inhibition of lipase-catalyzed hydrolysis of sunflower oil in AOT/isooctane reversed micelles

The hydrolysis of sunflower oil using Candida cylindracea lipase in reversed micelles of AOT/isooctane was investigated. The inhibition caused by substrate and hydrolysis products has been found in the process of reaction. It was revealed that the extent of inhibition caused by oleic acid was higher than that caused by glycerol, and was much more serious in the case of the mixture of hydrolysis products. Moreover, with the initial addition of glycerol into the reaction mixture, the stability of lipase could be increased during the hydrolysis of sunflower oil in reversed micelles. We thank the National Natural Science Foundation of China for the financial support of this work. We also thank Prof. Xu, Jia-li for his contributions to this work.     

X-ray structure of Candida antarctica lipase A shows a novel lid structure and a likely mode of interfacial activation

In nature, lipases (EC 3.1.1.3) catalyze the hydrolysis of triglycerides to form glycerol and fatty acids. Under the appropriate conditions, the reaction is reversible, and so biotechnological applications commonly make use of their capacity for esterification as well as for hydrolysis of a wide variety of compounds. In the present paper, we report the X-ray structure of lipase A from Candida antarctica, solved by single isomorphous replacement with anomalous scattering, and refined to 2.2-Å resolution. The structure is the first from a novel family of lipases. Contrary to previous predictions, the fold includes a well-defined lid as well as a classic α/β hydrolase domain. The catalytic triad is identified as Ser184, Asp334 and His366, which follow the sequential order considered to be characteristic of lipases; the serine lies within a typical nucleophilic elbow. Computer docking studies, as well as comparisons to related structures, place the carboxylate group of a fatty acid product near the serine nucleophile, with the long lipid tail closely following the path through the lid that is marked by a fortuitously bound molecule of polyethylene glycol. For an ester substrate to bind in an equivalent fashion, loop movements near Phe431 will be required, suggesting the primary focus of the conformational changes required for interfacial activation. Such movements will provide virtually unlimited access to solvent for the alcohol moiety of an ester substrate. The structure thus provides a basis for understanding the enzyme's preference for acyl moieties with long, straight tails, and for its highly promiscuous acceptance of widely different alcohol and amine moieties. An unconventional oxyanion hole is observed in the present structure, although the situation may change during interfacial activation.