Production of antibodies against the coenzyme pyrrolequinoline quinone

Rat intestinal epithelial cells were isolated and the activity of the enzyme diacylglycerol lipase (DG lipase, EC 3.1.1.3) was investigated. When cells were treated with Escherichia coli heat-stable toxin (ST) liberation of endogenous glycerol and fatty acids was observed. The enzyme responsible for this effect could be demonstrated to be a DG lipase by using specific substrates. It was found that the activity of DG lipase was increased 5–6-fold with the substrates diolein and 1,2-dioleyl-rac-glycerol and triolein being neutral lipid insensitive to DG lipase. ST had no direct effect on the DG lipase. The enzyme DG lipase was activated via a chain reaction due to the hydrolysis of phosphatidylinositol (PI) by the enzyme PI-specific phospholipase C stimulated by ST.     

Lipase-catalyzed esterification of glycerol and polyunsaturated fatty acids from fish and microalgae oils

This paper reports on the synthesis of triglycerides by enzymatic esterification of polyunsaturated fatty acids (PUFA) with glycerol. The lipase Novozym 435 (Novo Nordisk, A/S) from Candida antarctica was used to catalyze this reaction. The main factors influencing the degree of esterification and triglyceride yield were the amount of enzyme, water content, temperature and glycerol/fatty acid ratio. The optimum reaction conditions were established as: 100 mg of lipase; 9 ml hexane; 50°C; glycerol/PUFA concentrate molar ratio 1.2:3; 0% initial water; 1 g molecular sieves added at the start of reaction; and an agitation rate of 200 rpm. Under these conditions, a triglyceride yield of 93.5% was obtained from cod liver oil PUFA concentrate; the product contained 25.7% eicosapentaenoic acid and 44.7% docosahexaenoic acid. These optimized conditions were used to study esterification from a PUFA concentrate of the microalgae Phaeodactylum tricornutum and Porphyridium cruentum. With the first, a triglyceride yield of 96.5%, without monoglycerides and very few diglycerides, was obtained after 72 h of reaction; the resulting triglycerides had 42.5% eicosapentaenoic acid. A triglyceride yield of 89.3% was obtained from a P. cruentum PUFA concentrate at 96 h of reaction, which contained 43.4% arachidonic acid and 45.6% EPA. These high triglyceride yields were also achieved when the esterification reaction was scaled up 5-fold.     

The Influence of Fatty Acid and Glycerol on the Kinetics of Fat Hydrolysis by Candida Rugosa Lipase in a Membrane Reactor

The kinetics of lipid-hydrolysis by Candida rugosa lipase was investigated in a membrane reactor and in an emulsion system. Two models were chosen to describe the kinetics of the enzyme:

(1) The hydrolysis of triglycerides to fatty acids was considered to be a chain reaction with the intermediary products di- and mono-glyceride; each step was assumed to be a reversible second-order reaction. The reaction rate constants were determined from batch experiments. The experimental results could be described with this model.

(2) For process optimization and control, a model based on the power law was developed. For this model, the rate of hydrolysis was measured as a function of fatty acid and glycerol concentrations. Relations for the initial rate and equilibrium ester fraction as a function of the glycerol concentration were determined. Further, the reaction rate could be described with the power-law model with a power of 1.75 in the hydrolyzable ester fraction for a wide range of glycerol concentrations. The model with power 1.75 gave much better results when compared to a similar first order model. Although simpler, the first order model can not be used. The power law model was applied in the simulation of a reactor composed of three modules. The fatty acid production rate was calculated for this reactor system as a function of the outgoing glycerol concentration at different conditions.     

Lipase-Catalyzed Resolution of Isopropylidene Glycerol: Effect of Co-Solvents on Enantioselectivity

The resolution of 1,2-O-isopropylidene glycerol via enzyme catalyzed hydrolysis of the corresponding benzoic ester was investigated. Using lipase PS from Pseudomonas cepacia, we determined the influence of organic co-solvents on the activity and enantioselectivity of the enzyme. The performance of the lipase was correlated to the nature (logP, ε,μ and the percentage of the organic media. The highest enzymatic activity was found in solvents completely miscible or completely immiscible in water. The enzyme stereoselectivity was inversely related to the logP of the solvent.     

Production of acidic lipase by a mutant of Aspergillus niger NCIM 1207 in submerged fermentation

Mutants of Aspergillus niger NCIM 1207, isolated by subjecting conidia to UV-irradiation, were tested for the production of lipase (glycerol ester hydrolase EC 3.1.1.3). Mutants UV-10 and ANCR-1 showed seven fold and five fold enhanced productivity of enzyme, respectively, over the wild strain in shake flask culture when grown in SOB medium containing 1% olive oil. Maximum lipase activity (41 IU/ml) was obtained in the culture broth when UV-10 was grown in medium supplemented with 0.5% Triton X-100. A higher concentration of oil in the medium did not help lipase production in the case of mutant UV-10. Similarly no increase in enzyme levels was observed when mutant UV-10 was grown in medium supplemented with glucose. However, the addition of glucose in the medium resulted in increased levels of lipase production by wild strain, Aspergillus niger NCIM 1207.     

Purification and characterization of an extracellular lipase from Clostridium tetanomorphum

Summary The strictly anaerobic bacterium Clostridium tetanomorphum formed an extracellular lipase when the growth medium contained glycerol in addition to fermentable substrates such as l-glutamate or glucose. The lipase was purified from the concentrated culture supernatant and exhibited a final specific activity of 900 U/mg. The purified lipase had a Stokes’ radius of 5.0 nm and a sedimentation coefficient of 5.7S. The native molecular mass calculated from these values was 118,000 Da, which is considerably higher than the molecular mass calculated by PAGE (70,000 Da). With p-nitrophenyl esters of different fatty acids as substrates enzyme activity was highest when the acyl chain was short (C₂). The purified lipase showed no protease or thioesterase activity.     

Synthesis of monoacylglycerol containing pinolenic acid via stepwise esterification using a cold active lipase

High purity monoacylglycerol (MAG) containing pinolenic acid was synthesized via stepwise esterification of glycerol and fatty acids from pine nut oil using a cold active lipase from Penicillium camembertii as a biocatalyst. Effects of temperature, molar ratio, water content, enzyme loading, and vacuum on the synthesis of MAG by lipase‐catalyzed esterification of glycerol and fatty acid from pine nut oil were investigated. Diacylglycerol (DAG) as well as MAG increased significantly when temperature was increased from 20 to 40°C. At a molar ratio of 1:1, MAG content decreased because of the significant increase in DAG content. Water has a profound influence on both MAG and DAG content through the entire course of reaction. The reaction rate increased significantly as enzyme loading increased up to 600 units. Vacuum was an effective method to reduce DAG content. The optimum temperature, molar ratio, water content, enzyme loading, vacuum, and reaction time were 20°C, 1:5 (fatty acid to glycerol), 2%, 600 units, 5 torr, and 24 h, respectively. MAG content further increased via lipase‐catalyzed second step esterification at subzero temperature. P. camembertii lipase exhibited esterification activity up to ?30°C. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012     

Fatty Acid Specificity in Lipase-Catalyzed Synthesis of Glucoside Esters

The fatty acid specificity of the B-lipase derived from Candida antarctica was investigated in the synthesis of esters of ethyl D-glucopyranoside. The specificity was almost identical with respect to straight-chain fatty acids with 10 to 18 carbon atoms. However, lower fatty acids such as hexanoic and octanoic acid and the unsaturated 9-cis-octadecenoic acid were found to be poor substrates of the enzyme. As a consequence of this selectivity, these fatty acids were accumulated in the unconverted fraction when ethyl D-glucopyranoside was esterified with an excess of a mixture of fatty acids. This accumulation can reduce the overall effectiveness of the process as the activity of the lipase was found to be reduced when exposed to high concentrations of short-chain fatty acids. Finally, using a simplified experimental set-up, the specificity of the C. antarctica B-lipase was compared to the specificity of lipases derived from C. rugosa, Mucor miehei, Humicola, and Pseudomonas. Apart from the C. rugosa lipase, which exhibited a very poor performance, all the enzymes showed a very similar specificity with respect to fatty acids longer than octanoic acid while only the C. antarctica B-lipase showed activity towards sort-chain fatty acids.     

Lipase catalyzed production of monoacylglycerols by the esterification of fish oil fatty acids with glycerol

In this study, we attempted the efficient production of monoacylglycerols (MAG) via the lipase-catalyzed esterification of glycerol with fatty acids obtained from sardine oil. The reaction factors that influenced MAG synthesis were the glycerol to fatty acid mole ratio, amount of enzyme, organic solvent, temperature, and the type of lipase used. Porcine pancreas lipase was selected to catalyze this reaction. The optimum conditions we determined for MAG synthesis were a glycerol to fatty acid mole ratio of 1∶6, 100 mg/mL of lipase, and 30°C in dioxane. Under these conditions, the MAG content was 68% (w/w) after 72 h of reaction. The MAGs synthesized via the lipase-catalyzed esterification of glycerol with fatty acids included monomyristin, monopamiltin, and monoolein, as identified by GCMS.     

The lid is a structural and functional determinant of lipase activity and selectivity

In several lipases access to the enzyme active site is regulated by the position of a mobile structure named the lid. The role of this region in modulating lipase function is reviewed in this paper analysing the results obtained with three different recombinant lipases modified in the lid sequence: Candida rugosa lipase isoform 1 (CRL1), Pseudomonas fragi lipase (PFL) and Bacillus subtilis lipase A (BSLA). A CRL chimera enzyme obtained by replacing its lid with that of another C. rugosa lipase isoform (CRL1LID3) was found to be affected in both activity and enantioselectivity in organic solvent. Variants of the PFL protein in which three polar lid residues were replaced with amino acids strictly conserved in homologous lipases displayed altered chain length preference profile and increased thermostability. On the other hand, insertion of lid structures from structurally homologous enzymes into BSLA, a lipase that naturally does not possess such a lid structure, caused a reduction in the enzyme activity and an altered substrate specificity. These results strongly support the concept that the lid plays an important role in modulating not only activity but also specifity, enantioselectivity and stability of lipase enzymes.     

Glyceride Structure and Biosynthesis of Natural Fats

In order to study specificity of pancreatic lipase, a number of synthetic triglycerides were hydrolyzed by the enzyme under an improved condition. The proportions of isomers of the derived mono- and diglycerides, and the fatty acid compositions of the derived free acids and monoglycerides were determined. The hydrolyzing rate of fatty acids in glycerides depended on the position esterified in the glycerol, carbon number of the acid, and structure of the glyceride. Positional specificity of the enzyme was markedly displayed for symmetrical triglyceridcs composed of long chain acids, but at somewhat lower rate for glycerides containing short chain or highly unsaturated acids.     

Conversion of Soybean Oil to Biodiesel Fuel Using Lipozyme TL IM in a Solvent-free Medium

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.     

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.     

Cloning, expression and characterization of a novel thermal stable and short-chain alcohol tolerant lipase from Burkholderia cepacia strain G63

A lipase gene lipA and its chaperone gene lipB were cloned from Burkholderia cepacia strain G63. The lipA was composed of 1092 bp, encoding 363 amino acid residues, and the lipB composed of 1035 bp, corresponding to 344 amino acid residues. The significant amino acid similarity with Pseudomonas cepacia lipase revealed that this enzyme could be classified into the lipolytic subfamily I.2. The lipA and lipB genes were cloned into pBBR1Tp vector and conjugated into B. cepacia strains G63 with the help of pRK2013. The recombinant strain was fermented in 10 l bioreactor and the lipase was purified by a combination of ammonium sulfate fractionation, DEAE ion-exchange chromatography and gel filtration. The purified lipase kept stable at a temperature range of 40–70 °C. After incubated at 70 °C, the optimal temperature of this enzyme, for 10 h it remained 86.1% of its activity. The enzyme was also highly tolerant to a series of organic solution. Incubated in 50% methanol solution up to 48 h, the enzyme still kept 98.3% of its activity. The transesterification activity of soybean oil to fatty acid methyl esters (FAMEs) reached 87.8% after 72 h, indicating that it is a potential biocatalyzer for biodiesel production.     

Biochemical properties and potential applications of an organic solvent-tolerant lipase isolated from Serratia marcescens ECU1010

An extracellular lipase was purified to homogeneity with a purification factor of 5.5-fold from a bacterial strain Serratia marcescens ECU1010. The purified lipase is a dimer with two homologous subunits, of which the molecular mass is 65 kDa, and the pI is 4.2. The pH and temperature optima were shown to be pH 8.0 and 45 °C, respectively. Among p-nitrophenyl esters of fatty acids with varied chain length, the lipase showed the maximum activity on p-nitrophenyl myristate (C₁₄). The lipase was activated by some surfactants such as Gum Arabic, polyvinyl alcohol (PVA) and Pg350me, but not by Ca²⁺. The enzyme displayed pretty high stability in many water miscible and immiscible solvents. This is a unique property of the enzyme which makes it extremely suitable for chemo-enzymatic applications in non-aqueous phase organic synthesis including enantiomeric resolution. Several typical chiral compounds were tested for kinetic resolution with this lipase, consequently giving excellent enantioselectivities (E = 83 >100) for glycidyl butyrate (GB), 4-hydroxy-3-methyl-2-(2-propenyl)-2-cyclopenten-1-one acetate (HMPCA), naproxen methyl ester (NME) and trans-3-(4′-methoxyphenyl) glycidic acid methyl ester (MPGM).     

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.     

Kinetic characterisation of enzymatic esterification in a solvent system: adsorptive control of water with molecular sieves

The kinetics of enzymatic esterification of glycerol with oleic acid, in equimolar ratio, catalyzed by immobilized Mucor miehei lipase in a solvent system in the presence of the molecular sieves was carried out at 37°C at different Lipozym and solvent (n-hexane) concentrations and the molecular sieve contents were studied in a batch stirred-tank reactor (BSTR). The reactions were followed by the determination of reaction conversions during 45 h. The experimental data of enzymatic esterification of glycerol with oleic acid in a solvent system in the presence of molecular sieves showed minimal deviation from the calculated value in the irreversible second order kinetic model. On the basis of the experimental data, we found an empirical correlation between concentrations of Lipozym, concentrations of solvent (n-hexane), contents of the molecular sieve and the reaction rate constant, k₁.     

基于响应面设计脂肪酶Novo435催化合成甘油二酯的工艺优化

以甘油、油酸为原料,优化在无溶剂体系中以固定化脂肪酶Novo435催化合成甘油二酯（diglyceride,DAG）的工艺。系统考察底物摩尔比（油酸／甘油）、反应温度、时间和加酶量等因素对油酸转化率和甘油二酯含量影响的基础上,利用响应面试验设计优化各主效因子,并经回归分析获得最优的工艺条件。所得最优条件：油酸与甘油底物摩尔比2．27、反应温度48．14℃、反应时间6．3h、加酶量1．68％。在此条件下,实验测得油酸转化率为45．42％,甘油二酯质量分数为70．01％,与响应面模型预测值吻合。     

利用麻风树油驯化筛选高活性脂肪酶产生菌及其催化酯化反应

以1株分解麻风树油的脂肪酶产生菌Pseudomonas sp. LP-1为出发菌株, 通过麻疯树油定向驯化筛选获得1株酶活较高且产酶稳定的菌株P. sp. X-2-45, 其水解酶活为29.79 U/mL, 比原始菌株提高了288%。对P. sp. X-2-45生长与产酶特征、对植物油脂水解能力及在有机相中催化脂肪酸和有机醇间的酯化反应研究发现, 该菌株生长速率和产酶速率明显加快, 培养30 h时生物量和酶活达到最大, 稳定期延长, 培养过程中脂肪酶在培养基中的稳定性提高。以麻疯树油诱导合成的P. sp. X-2-45脂肪酶对麻疯树油的水解能力比原始菌株提高了378%, 说明采用麻风树油定向驯化可提高脂肪酶对相应底物的水解能力。X-2-45脂肪酶可以催化月桂酸与正丁醇、正辛醇、月桂醇和丙三醇之间, 棕榈酸、硬脂酸与甲醇、正辛醇、月桂醇和丙三醇之间, 油酸与甲醇、正丁醇、正辛醇、月桂醇和丙三醇之间发生酯化反应。     

Immobilization of lipases for non-aqueous synthesis

Immobilization of lipases involves many levels of complications relating to the structure of the active site and its interactions with the immobilization support. Interaction of the so called hydrophobic ‘lid’ with the support has been reported to affect synthetic activity of an immobilized lipase. In this work we evaluate and compare the synthetic activity of lipases from different sources immobilized on different kinds of supports with varying hydrophobicity. Humicola lanuginosa lipase, Candida antarctica lipase B and Rhizomucor miehei lipase were physically adsorbed onto two types of hydrophobic carriers, namely hydrophilic carriers with conjugated hydrophobic ligands, and supports with base matrix hydrophobicity. The prepared immobilized enzymes were used for acylation of n-butanol with oleic acid as acyl donor in iso-octane with variable water content (0–2.8%, v/v) as reaction medium. Enzyme activity and effect of water on the activity of the immobilized derivatives were compared with those of respective soluble lipases and a commercial immobilized lipase Novozyme 435. Both R. miehei and H. lanuginosa immobilized lipases showed maximum activity at 1.39% (v/v) added water concentration. Sepabeads, a methacrylate based hydrophilic support with conjugated octadecyl chain showed highest immobilized esterification (synthetic) activity for all three enzymes, and of the three R. miehei lipase displayed maximum esterification activity comparable to the commercial enzyme.