Substrate Specificity and Mode of Action of the Lipase of Thermophilic Fungus Humicola lanuginosa S-38

Substrate specificity of the lipase of thermophilic fungus, Humicola lanuginosa S–38, was investigated. It was found that the lipolytic activity was greatly influenced by the structure of both fatty acid and alcohol moieties of the substrate. It was concluded that the hydrolysis of both water soluble and water insoluble ester was catalyzed by the Humicola lipase itself. The Humicola lipase showed no positional specificity and split ester bonds on all positions of triolein at about the same rate. Both palmitic acid (α) and linoleic acid (β) ester bonds of phosphatidyl-ethanolamine were split indicating no positional specificity of fatty acid ester bonds. From above results, it was made clear that mode of action of Humicola lipase on triolein and on phosphatidyl-ethanolamine is identical. The Humicola lipase had no activity of lipoprotein lipase.     

Quantitative analysis of enzymatic fractionation of multiple substrate mixtures

The enzymatic conversion of mixtures of multiple substrates was studied quantitatively, based on established methodology used for the enzymatic kinetic resolution of racemic mixtures, involving the use of competitive factors: ratios of specificity constants (k_cat/K_M) of substrate pairs. The competitive factors of the substrates were defined in relation to a reference substrate. These competitive factors were used to predict the composition of the reaction mixture as a function of the degree of conversion of the reaction. The methodology was evaluated using three different lipases to hydrolyze a model mixture of four fatty acid methyl esters and for the esterification of a mixture of the same fatty acids in free form with ethanol. In most cases, the competitive factors determined from the initial phase of the reactions predicted the product composition during the rest of the reaction very well. The slowest reacting fatty acid was erucic acid (both in free form and as methyl ester), which was thus enriched in the remaining substrate fraction, while the other fatty acids: lauric acid, palmitic acid and oleic acid were converted faster. Simulations of the compositions of reaction mixtures with different values of the competitive factors were carried out to provide an overview of what could be achieved using enzymatic enrichment. Possible applications include reactions involving homologous substrates and mixtures of multiple isomers. The analysis presented provides guidelines that can be useful in the screening and development of enzymes for enzymatic enrichment applications. Biotechnol. Bioeng. 2013; 110: 78–86. © 2012 Wiley Periodicals, Inc.     

Acid lipase and carboxylesterases in EBV-transformed lymphoid cell line from Wolman's disease: influence of fatty acid structure of substrate

Lymphoid cell lines established by Epstein-Barr virus transformation of blood B lymphocytes from a patient with Wolman's disease exhibited the acid lipase deficiency characteristic for this disease. Comparison of hydrolysis by normal and Wolman's cells of 4-methylumbelliferyl-acyl esters with variable chain length demonstrates that: (1) the best substrates for acid lipase were characterized by an acyl chain length of 12-18 carbon atoms; (2) the acid residual activity in Wolman's cells showed a slightly different substrate specificity and this is probably due to an acid carboxylesterase different from the lysosomal acid lipase, and (3) the 'nonspecific' carboxylesterases (at pH 6.0 and 8.0) not inhibited by taurocholate showed a characteristic substrate specificity for short-chain fatty acids. In the used assay conditions (optimal for acid lipase), methylumbelliferyl-palmitate, -elaidate and -lignocerate are the most accurate synthetic substrates for the diagnostic of Wolman's disease.     

Positional specificity and substrate preference of purified Staphylococcus aureus lipase

We have studied the substrate preference and specificity, including positional specificity, of a lipase purified from Staphylococcus aureus (strain FN 37). This extracellular bacterial enzyme is relatively insensitive to product inhibition, and hydrolyzes tri-, di- and monooleoylglycerol in emulsified and micellar form at similar rates and without marked substrate preference. The lipase lacks positional specificity, and the hydrolysis of triacylglycerol proceeds rapidly to free fatty acid and glycerol without accumulation of intermediary products.     

Blocking the tunnel: engineering of Candida rugosa lipase mutants with short chain length specificity

The molecular basis of chain length specificity of Candida rugosa lipase 1 was investigated by molecular modeling and site-directed mutagenesis. The synthetic lip1 gene and the lipase mutants were expressed in Pichia pastoris and assayed for their chain length specificity in single substrate assays using triglycerides as well as in a competitive substrate assay using a randomized oil. Mutation of amino acids at different locations inside the tunnel (P246F, L413F, L410W, L410F/S300E, L410F/S365L) resulted in mutants with a different chain length specificity. Mutants P246F and L413F have a strong preference for short chain lengths whereas substrates longer than C10 are hardly hydrolyzed. Increasing the bulkiness of the amino acid at position 410 led to mutants that show a strong discrimination of chain lengths longer than C14. The results obtained can be explained by a simple mechanical model: the activity for a fatty acid sharply decreases as it becomes long enough to reach the mutated site. In contrast, a mutation at the entrance of the tunnel (L304F) has a strong impact on C4 and C6 substrates. This mutant is nevertheless capable of hydrolyzing chain lengths longer than C8.     

脂肪酶催化有机硅化合物的生物转化

C.cylindracea脂肪酶可催化有机介质中有机硅醇与脂肪酸的酯化反应。微水有机介质比水-水不溶有机介质更有利于酶的反应,有机硅醇是比其碳结构类似物更好的酰基受体。对不同有机硅醇底物,当其空间障碍大时,不利于酶催化酯化反应,对不同脂肪酸底物,有机硅醇未影响该脂肪酶的脂肪酸底物特异性。     

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.     

Multi-competitive enzymatic reactions in organic media: Application to the determination of lipase alcohol specificity

Multicompetitive reactions catalyzed by lipases in organic media were used for the determination of lipase specificity towards alcohols. The competitive factors (, defined as the ratio of the kinetic powers, k_cat/K_m, for two substrates in competition for the enzyme active site) were estimated in a one-step experiment and a scale of specificity was easily deduced. The specificity towards the alcohol chain length and degree of substitution of seventeen commercial lipase preparations was investigated. The results show that, like fatty acids, alcohols greatly influence the reaction rates of lipase catalyzed reactions in organic solvents. Five groups of alcohol specificity are proposed after using the statistical method of principal component analysis.     

Characterization of a partially purified diacylglycerol lipase from bovine aorta

A partially-purified diacylglycerol (DG) lipase from bovine aorta has been characterized with respect to the effects of lipid metabolites and two lipase inhibitors, phenylboronic acid and tetrahydrolipstatin (THL). DG lipase activity was determined by the hydrolysis of the sn-1 position of 1-[1-⁴C]palmitoyl-2-oleoyl-sn-glycerol. The products of the lipase reaction, 2-monoacylglycerol (2-monoolein) and non-esterified fatty acids (oleate, arachidonate) produced a concentration-dependent (20–200 μM) inhibition of DG lipase activity. Oleoyl-CoA and dioleoylphosphatidic acid also inhibited aortic DG lipase activity, but lysophosphatidylcholine had little or no effect. The inhibition of aortic DG lipase by phenylboronic acid was competitive, with a K_i of approx. 4 mM. THL was a very potent inhibitor of aortic DG lipase; the concentration required for inhibition to 50% of control was 2–6 nM. THL was a very potent inhibitor of concentration of substrate in the assay was increased. Attempts to identify the aortic DG lipase by covalent-labelling with [¹⁴C]THL were unsuccessful. Immunoblotting experiments revealed that hormone-sensitive triacylglycerol lipase (HSL) could not be detected in bovine aorta.     

Synthesis of glycerides containing n-3 fatty acids and conjugated linoleic acid by solvent-free acidolysis of fish oil

Menhaden oil, a rich source of n-3 fatty acids, was interesterified with conjugated linoleic acid (CLA) in a reaction medium composed solely of substrates and either free or immobilized commercial lipase preparations. Of five lipases tested, an immobilized preparation from Mucor miehei provided the fastest rate of incorporation of CLA into fish oil acylglycerols; however, and as observed with most of the lipases utilized, a significant proportion of the n-3 fatty acid residues were liberated in the process. A soluble lipase from Candida rugosa converted free CLA to acylglycerol residues while leaving the n-3 fatty acid residues virtually untouched. Even though the reaction rate was slower for this enzyme than for the other four lipase preparations, the specificity of the free C. rugosa lipase gives it the greatest potential for commercial use in preparing fish oils enriched in CLA residues but still retaining their original n-3 fatty acid residues.     

脂肪酶催化合成生物柴油的研究

生物柴油是用动植物油脂或长链脂肪酸与甲醇等低碳醇合成的脂肪酸甲酯,是一种替代能源。这里探讨了生物法制备生物柴油的过程,采用脂肪酶酯化和酯交换两条工艺路线进行催化合成。深入研究制备过程中,不同脂肪酶、酶的用量和纯度、有机溶剂、低碳醇的抑制作用、吸水剂的作用、反应时间和进程、底物的特异性和底物摩尔比等参数对酯化过程的影响。试验结果表明,采用最佳酯化反应参数和分批加入甲醇并用硅胶作脱水剂的工艺过程,酯化率可以达到92％,经分离纯化后的产品GC分析的纯度可达98％以上,固定化酶的使用半衰期可达到360h。同时对酯交换制备生物柴油过程中,甲醇的用量和甲醇的加入方式对脂肪酶催化过程的影响作了初步研究,优化后的酯交换率可达到83％。     

由扩展青霉(Penicillium expansum)PF868产生脂肪酶催化油脂水解的研究

研究了由扩展青霉（Ｐｅｎｉｃｉｌｉｕｍｅｘｐａｎｓｕｍ）ＰＦ８６８产生脂肪酶催化水解三种油脂（橄榄油、豆油、鱼油）的影响因素与工艺条件,其中包括：水解时间、温度、ｐＨ、酶量、油水比及添加剂,并用气相色谱对产品脂肪酸进行了分析鉴定,初步分析其催化水解的脂肪酸的特异性     

Purification and characterization of an extracellular lipase from a thermophilic Rhizopus oryzae strain isolated from palm fruit

We have isolated a lipolytic strain from palm fruit that was identified as a Rhizopus oryzae. Culture conditions were optimized and highest lipase production amounting to 120 U/ml was achieved after 4 days of cultivation. The extracellular lipase was purified 1200-fold by ammonium sulfate precipitation, sulphopropyl-Sepharose chromatography, Sephadex G 75 gel filtration and a second sulphopropyl-Sepharose chromatography. The specific activity of the purified enzyme was 8800 U/mg. The lipolytic enzyme has a molecular mass of 32 kDa by SDS-polyacrylamide gel electrophoresis and gel filtration. The enzyme exhibited a single band in active polyacrylamide gel electrophoresis and its isoelectric point was 7.6. Analysis of Rhizopus oryzae lipase by RP-HPLC confirmed the homogeneity of the enzyme preparation. Determination of the N-terminal sequence over 19 amino acid residues showed a high homology with lipases of the same genus. The optimum pH for enzyme activity was 7.5. Lipase was stable in the pH range from 4.5 to 7.5. The optimum temperature for lipase activity was 35 degrees C and about 65% of its activity was retained after incubation at 45 degrees C for 30 min. The lipolytic enzyme was inhibited by Triton X100, SDS, and metal ions such as Fe(3+), Cu(2+), Hg(2+) and Fe(2+). Lipase activity against triolein was enhanced by sodium cholate or taurocholate. The purified lipase had a preference for the hydrolysis of saturated fatty acid chains (C(8)-C(18)) and a 1, 3-position specificity. It showed a good stability in organic solvents and especially in long chain-fatty alcohol. The enzyme poorly hydrolyzed triacylglycerols containing n-3 polyunsaturated fatty acids, and appeared as a suitable biocatalyst for selective esterification of sardine free fatty acids with hexanol as substrate. About 76% of sardine free fatty acids were esterified after 30 h reaction whereas 90% of docosahexaenoic acid (DHA) was recovered in the unesterified fatty acids.     

Geotrichum candidum 4013: Extracellular lipase versus cell-bound lipase from the single strain

Two types of lipases (extracellular and cell-bound) were produced by Geotrichum candidum 4013 in liquid medium and were used as biocatalysts in blackcurrant oil hydrolysis. Reaction products were analysed for the degree of conversion from which enzyme activity was evaluated, and the composition of free fatty acids was compared to the composition of oil substrate. The enzyme activity was measured also before and after the reaction in SC-CO₂. The fatty acid composition of the acids liberated from oil by hydrolysis suggests a specificity of the cell-bound and extracellular enzymes from Geotrichum candidum 4013. The extracellular lipase displays low selectivity to the polyunsaturated fatty acids, and the cell-bound lipase possesses selectivity to the saturated fatty acids. Enantioselectivity of the tested processes achieved with both induced enzymes was high (from 43 to 242). The activity of all enzymes has markedly increased after their exposure to SC-CO₂. The treatment of enzymes by SC-CO₂ could be easy-to-use approaches to improve the efficiency of enzymatic reactions.     

Diacylglycerol lipase and kinase activities in rat brain microvessels

Diacylglycerols can accumulate transiently in intact cells as a consequence of the degradation of phosphatidylinositol by phospholipase C, but little information is available concerning their metabolic fate in the vascular endothelium. Diacylglycerol lipase and kinase activities were measured in rat brain microvessel preparations. Lipase activity, measured by the release of free fatty acids, was much greater at pH 4.5 than at pH 7. The acid lipase was predominantly particulate and likely originated in lysosomes, whereas the neutral lipase was mainly soluble. The fatty acid at the sn-1 position of the diacylglycerol substrate was hydrolyzed faster than that at the sn-2 position at both pH 4.5 and 7. The 2-monoacylglycerol accumulated at pH 4.5 but not at 7 due to the presence of a monoacylglycerol lipase activity with a neutral pH optimum. The formation of phosphatidic acid (kinase activity) was also measured in microvessels. When lipase and kinase activities were measured simultaneously, the formation of phosphatidic acid from a 1-palmitoyl-2-[1-14C]oleoyl-sn-glycerol substrate was 4-fold greater than the release of fatty acid (oleate) from the sn-2 position. Introduction of arachidonic acid to the sn-2 position of the diacylglycerol substrate increased kinase activity but reduced lipase activity. The release of fatty acids from the sn-2 position of phosphatidic acid could not be detected.     

Fatty acid preference of mycelium-bound lipase from a locally isolated strain of <Emphasis Type="Italic">Geotrichum candidum</Emphasis>

Mycelium-bound lipase (MBL) was prepared using a strain of Geotrichum candidum isolated from local soil. At the time of maximum lipase activity (54 h), the mycelia to which the lipase was bound were harvested by filtration and centrifugation. Dry MBL was prepared by lyophilizing the mycelia obtained. The yield of MBL was 3.66 g/l with a protein content of 44.11 mg/g. The lipase activity and specific lipase activity were 22.59 and 510 U/g protein, respectively. The moisture content of the MBL was 3.85%. The activity of free (extracellular) lipase in the culture supernatant (after removal of mycelia) was less than 0.2 U/ml. The MBL showed selectivity for oleic acid over palmitic acid during hydrolysis of palm olein, indicating that the lipase from G. candidum displayed high substrate selectivity for unsaturated fatty acid 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.     

Triacylglycerol catabolism in the prawn Macrobrachium borellii (Crustacea: Palaemoniade)

While invertebrates store neutral lipids as their major energy source, little is known about triacylglycerol (TAG) class composition and their differential catabolism in aquatic arthropods. This study focuses on the composition of the main energy source and its catabolism by lipase from the midgut gland (hepatopancreas) of the crustacean Macrobrachium borellii. Silver-ion thin-layer chromatography of prawn large TAG deposit (80% of total lipids) and its subsequent fatty acid analysis by gas chromatography allowed the identification of 4 major fractions. These are composed of fatty acids of decreasing unsaturation and carbon chain length, the predominant being 18:1n-9. Fraction I, the most unsaturated one, contained mainly 20:5n-3; fraction II 18:2n-6; fraction III 18:1n-9 while the most saturated fraction contained mostly 16:0. Hepatopancreas main lipase (Mr 72 kDa) cross-reacted with polyclonal antibodies against insect lipase, was not dependent on the presence of Ca²⁺ and had an optimum activity at 40 °C and pH 8.0. Kinetic analysis showed a Michaelis–Menten behavior. A substrate competition assay evidenced lipase specificity following the order: 18:1n-9-TAG > PUFA-enriched-TAG > 16:0-TAG different from that in vertebrates. These data indicate there is a reasonable correspondence between the fatty acid composition of TAG and the substrate specificity of lipase, which may be an important factor in determining which fatty acids are mobilized during lipolysis for oxidation in crustaceans.     

Hydrolysis of tri- and monoacylglycerol by lipoprotein lipase: evidence for a common active site.

The relationship between triacylglycerol and monoacylglycerol hydrolyzing activities of purified rat heart lipoprotein lipase was studied using emulsified trioleoylglycerol and micellar or albumin-bound monooleoylglycerol as substrates. The maximal reaction rates obtained with the two substrates were similar (650 and 550 nmol of fatty acid released per min per mg of protein, respectively). Addition of apolipoprotein C-II or serum increased the maximal reaction rate for the trioleolyglycerol hydrolyzing activity about four-fold, but had no effect on the monooleolyglycerol hydrolyzing activity. Hydolysis of the two substrates apparently takes place at the same active site of the enzyme since (1) mutual competitive inhibition between the substrates could be demonstrated; (2) the rate of inactivation of enzymatic activity with the two substrates in 1.2 M NaCl was the same; (3) similar losses of hydrolytic activity with tri- and monooleoylglycerol were observed in the presence of low concentrations of n-butyl (p-nitrophenyl) carbamide; (4) inhibition of both hydrolytic activities by this compound could be prevented by prior exposure of lipoprotein lipase to either substrate.     

Human lipoprotein lipase: the loop covering the catalytic site is essential for interaction with lipid substrates.

Lipoprotein lipase (LPL), a key enzyme which initiates the hydrolysis of triglycerides present in chylomicrons and very low density lipoproteins, consists of multiple functional domains which are necessary for normal activity. The catalytic domain of LPL mediates the esterase function of the enzyme but separate lipid binding sites have been proposed to be involved in the interaction of LPL with emulsified lipid substrates at the water-lipid interface. Like pancreatic lipase (PL), LPL contains a surface loop covering the catalytic pocket that may modulate access of the substrate to the active site of the enzyme. Secondary structural analysis of this loop reveals a helix-turn-helix motif with two short amphipathic helices that have hydrophobic moments of 0.64 and 0.68. In order to investigate the role of the loop in the initial interaction of LPL with its substrate, we utilized site-directed mutagenesis to generate eight constructs in which the amphipathic properties of the loop were altered and expressed them in human embryonal kidney-293 cells. Reducing the amphiphilicity without changing the predicted secondary structure of the loop abolished the ability of the lipase to hydrolyze emulsified, long chain fatty acid triglycerides (triolein) but not the water soluble substrate tributyrin. Replacing the loop of LPL with the loop of hepatic lipase, which differs in 15 of 22 amino acids but is also amphiphilic, led to the expression of an enzyme that retained both triolein and tributyrin hydrolyzing activity. Substitution of the LPL loop by a short four amino acid peptide, which may allow more direct access to the active site than the 22 amino acid loop, enhanced hydrolysis of short chain fatty acid triglycerides by more than 2-fold, while the ability to hydrolyze emulsified substrates was abolished. Thus, disruption of the amphipathic structure of the LPL loop selectively decreases the hydrolysis of emulsified lipid substrate without affecting the esterase or catalytic function of the enzyme. These studies establish that the loop with its two amphipathic helices is essential for hydrolysis of long chain fatty acid substrate by LPL providing new insight into the role of the LPL loop in lipid-substrate interactions. We propose that the interaction between the lipoprotein substrates and the amphipathic helices within this loop may in part determine lipase substrate specificity.     

Characteristics of immobilized lipase on hydrophobic superparamagnetic microspheres to catalyze esterification

A novel immobilized lipase (from Candida rugosa) on hydrophobic and superparamagnetic microspheres was prepared and used as a biocatalyst to catalyze esterification reactions in diverse solvents and reaction systems. The results showed that the immobilized lipase had over 2-fold higher activities in higher log P value solvents. An exponential increase of lipase activity against log P of two miscible solvent mixtures was observed for the first time. Both free and immobilized lipase achieved its maximum activity at the range of water activity (a(w)) 0.5-0.8 or higher. At a(w) 0.6, the immobilized lipase exhibited markedly higher activities in heptane and a solvent-free system than did the native lipase. In multicompetitive reactions, the alcohol specificity of the lipase showed a strong chain-length dependency, and the immobilized enzyme exhibited more preference for a longer-chain alcohol, which is different from previous reports. The immobilized lipase showed higher specificities for butyric acid and the medium-chain-length fatty acids (C(8)-C(12)). Then, the immobilized lipase was extended to solvent-free synthesis of glycerides from glycerol and fatty acids. Recovered by magnetic separation, the immobilized lipase exhibited good reusability in repeated batch reaction, indicating its promising feature for biotechnology application.