Purification of rat kidney carboxylesterase and its comparison with other tissue esterases

Carboxylesterase was purified from rat kidney in an electrophoretically homogeneous form by acetone precipitation, followed by successive chromatographies on DEAE-cellulose and hydroxyapatite and then isoelectric focusing. The purified enzyme catalyzed the hydrolyses of monoacylglycerols and short-chain triacylglycerols, such as tributyrin, but not the hydrolysis of long-chain triacylglycerol. Its optimum pH with methyl butyrate as a substrate was 8.0. The relation of its activity to the methyl butyrate concentration differed from those for pancreatic lipase and liver esterase, and also from those for lipolytic enzymes from various other tissues. The relations of methyl butyrate-hydrolyzing activity with methyl butyrate concentration were compared among various carboxylester hydrolyzing enzymes. Based on the results, these enzymes were classified into four classes.     

Purification and some properties of intracellular esterase from Pseudomonas fluorescens

A novel esterase was found in Pseudomonas fluorescens cells and purified to homogeneity as determined by polyacrylamide gel electrophoresis. The esterase was extracted from the cells by freeze-thawing and hypotonic treatment. Purification was achieved by ammonium sulfate precipitation, followed by successive chromatographies on DEAE-cellulose and benzylamine-agarose and then electrophoresis. The enzyme catalyzed the hydrolysis of methyl esters, such as methyl butyrate, but its hydrolyzing activity decreased with increase in the chain length of the alcohol moiety, and it did not catalyze the hydrolysis of triacylglycerols, such as triacetin. In contrast, the enzyme acted on various acyl residues in a series of methyl esters, such as dimethyl succinate, methyl methacrylate, and dimethyl malate. The optimum pH for activity of this enzyme with methyl butyrate was 7.0-8.5. The enzyme was inhibited by phenylmethylsulfonylfluoride. Its molecular weight was estimated as 48,000 by molecular sieve electrophoresis and gel filtration on Sephadex G-150.     

Separation of a bradykinin-releasing enzyme from the proteolytic complex of Levantine viper venom

Two serine hydrolases have been separated from the proteolytic complex of the venom of Levantine viper, Vipera lebetina turanica. The enzyme with mol. weight of 50,000 +/- 5,000, pH-optimum of 8.5 and isoelectric point in the range of 5.6--6.6 had proteolytic activity against casein and hydrolyzed benzoyl-arginine p-nitroanilide. The other enzyme with mol. weight of 37,000 +/- 2,000, pH optimum of 9 and isoelectric point in the range of 4.1--4.5 had no effect on benzoylarginine p-nitroanilide, casein or hemoglobin, but possessed a bradykinin-releasing activity. Both enzymes were stereoselective against L-arginine, hydrolyzing tosyl-L-arginine methyl ester without having any effect on D-arginine ester. The interaction of the enzymes with a number of N(alpha)-arylsulfonylarginine methyl esters has been studied. The influence of the substitute X in the arylsulfonyl part of the substrates upon their hydrolysis by the bradykinin-releasing enzyme has been described by the Hammett equation of rho omicron = 1.14 +/- 0.33 (r = 0.974).     

A new irreversible enzyme-aided esterification method in organic solvents

A new irreversible esterification method for carboxylic acids catalyzed by a lipase from Candida antarctica (Novozyme 435) in organic solvents has been developed. The water produced during the process is chemically destroyed by a corresponding ester of acetoacetate, which acts as a sacrificial substrate in this reaction. The flavour esters isobutyl acetate, methyl butyrate, ethyl butyrate and benzyl butyrate were synthesized either in small scale (0.05 mol) or large scale (1 mol). The yields range from 82 to 92% within 24 h at 52°C. Optimal molar ratios of reactants were 1:1:1 (carboxylic acid:alcohol:acetoacetate).     

Evaluation of strains of Geotrichum candidum for lipase production and fatty acid specificity

Summary Three strains of Geotrichum candidum (ATCC 34614, NRRL Y-552 and NRRL Y-553) were examined for lipase production and activity. Variables including medium, pH, temperature, agitation rate and incubation time were examined to define the optimal culture conditions. Growth on oil in complex medium at 30°C, 300 rpm, and pH 7 produced maximal lipase activity. Fatty acid specificity of these strains and of two crude G. candidum enzyme preparations (lipase 26557 RP, Rhône Poulenc and lipase GC-4, Amano) was measured using equimolar mixtures of methyl or butyl esters of palmitic and oleic acids. The lipase from NRRL Y-553 and lipase 26557 RP displayed preferential specificity for hydrolyzing oleic acid esters, while the lipases from ATCC 34614, NRRL Y-552 and lipase GC-4 failed to discriminate between plamitic and oleic acids.     

Lipase of Mucor pusillus

Lipase of Mucor pusillus NRRL 2543 was recovered with ammonium sulfate precipitation, gel filtration on Sephadex G-75, and anion-exchange chromatography on diethylaminoethyl-Sephadex A-50. Maximal glycerol ester hydrolase (lipase) activity was observed at pH 5.0 to 5.5 and 50 C when trioctanoin and olive oil were used as substrates. The enzyme also showed esterase activity; it hydrolyzed, with the exception of methyl butyrate, all methyl esters tested. A minimum chain length of six carbons appeared to be a requirement for esterase activity, which was maximal at about pH 5.5 with methyl dodecanoate (C(12)) as the substrate. Neither the glycerol ester hydrolase (lipase) nor the esterase activity of the enzyme appeared to be affected by thiol group inhibitors, chelating agents, and reducing compounds. On the other hand, hydrolysis of triolein and methyl dodecanoate was arrested to the same extent in the presence of diisopropyl fluorophosphate, which suggested the involvement of serine in the active center of the enzyme. The enzyme remained stable during a 30-day storage at - 10 C.     

On the Substrate Specificity of the Lipase Produced by Candida paralipolytica

The lipase from Candida paralipolytica required activating factors for the hydrolysis of synthetic triglycerides, methyl esters of fatty acid and so on. Of the saturated monoacid triglycerides tested, tricaprylin was hydrolyzed most quickly. On the other hand, the lipase was hardly able to hydrolyze methyl butyrate, methyl caproate, monoolein, Tween 20 and Span 20.

Human blood plasma did not act as an activator, but act rather as an inhibitor of the lipase. Therefore, it seems that the lipase does not belong to the group of lipoprotein lipase.     

Effects of a polyoxyethylene detergent (Brij 58) on platelet aggregation, release and clotting activity

Low concentrations of a polyoxyethylene detergent, Brij 58, inhibited the secondary phase of platelet aggregation induced by ADP in human citrated platelet-rich plasma but had no effect on primary aggregation. Thrombin-induced aggregation of washed human platelets suspended in Tyrode's buffer was inhibited after incubation of cells with 4.10(-6) M detergent. Efflux of [14C]serotonin, 45Ca2+ and labile aorta contracting substance (thromboxane A2) and development of prothrombin-converting activity (platelet factor 3) were abolished concomitantly. Aggregation of washed platelets either by sodium arachidonate or by collagen was also inhibited by the same concentration of Brij 58 which inhibited thrombin aggregation. This concentration did not itself produce any release of a cytoplasmic marker, lactate dehydrogenase, from platelets. Higher concentrations of Brij 58, exceeding 4.10(-5) M, lysed the cells liberating lactate dehydrogenase, serotonin and Ca2+. When albumin was included as a platelet stabilizer in the suspending medium the concentration of detergent required for the inhibitory effects was increased ten-fold. This could be attributed to competitive binding of the detergent to albumin, demonstrated with [14C]acetylated Brij 58. A variety of other polyoxyethylene detergents, at concentrations from 8.10(-4) to 5.10(-3) M, also inhibited platelet aggregation induced by thrombin. It is concluded that low concentrations of Brij 58 stabilize the platelets against the action of aggregating agents, while higher concentrations produce membrane destabilization and cell lysis.     

Biocatalytic potential of lipase from Staphylococcus sp. MS1 for transesterification of jatropha oil into fatty acid methyl esters

An extracellular lipase producing isolate Staphylococcus sp. MS1 was optimized for lipase production and its biocatalytic potential was assessed. Medium with tributyrin (0.25 %) and without any exogenous inorganic nitrogen source was found to be optimum for lipase production from Staphylococcus sp. MS1. The optimum pH and temperature for lipase production were found to be pH 7 and 37 °C respectively, showing lipase activity of 37.91 U. It showed good lipase production at pH 6–8. The lipase was found to be stable in organic solvents like hexane and petroleum ether, showing 98 and 88 % residual activity respectively. The biotransformation using the concentrated enzyme in petroleum ether resulted in the synthesis of fatty acid methyl esters like methyl oleate, methyl palmitate and methyl stearate. Thus, the lipase under study has got the potential to bring about transesterification of oils into methyl esters which can be exploited for various biotechnological applications.     

Overexpression of Serratia marcescens lipase in Escherichia coli for efficient bioresolution of racemic ketoprofen

Lipase from Serratia marcescens ECU1010 was cloned and overexpressed in E. coli. After optimization, the maximum lipase activities reached 5000–6000 U/l and this recombinant lipase could enantioselectively hydrolyze (S)-ketoprofen esters into (S)-ketoprofen. Among six alkyl esters of racemic ketoprofen investigated, this lipase showed the best enantioselectivity for the kinetic resolution of ketoprofen ethyl ester, with an ee_p (enantiomeric excess of product) of 91.6% and E-value of 63 obtained at 48.2% conversion. Twelve nonionic surfactants were tested for enhancing the enantioselectivity of this lipase in the bioresolution of ketoprofen ethyl ester. A very high E-value of 1084 was achieved, with an optical purity of >99% ee_p and a yield of 42.6% in the presence of 3% Brij 92V. Further studies showed that the selectivity of the lipase was improved with the increase of Brij 92V concentration. The substrate (ketoprofen ethyl ester) does not inhibit the lipase activity, while the product (S)-ketoprofen inhibits the lipase activity to some extent. These results indicate that the S. marcescens lipase is very useful for biocatalytic production of chiral profens such as (S)-ketoprofen.     

Purification and characterization of the extracellular lipase Lip2 from Yarrowia lipolytica

An extracellular lipase from Yarrowia lipolytica (YlLip2) has been purified by ion exchange chromatography on Q sepharose FF, followed by hydrophobic interaction chromatography on butyl sepharose FF. SDS-PAGE showed that the molecular weight of this lipase is about 38 kDa. N-terminal amino acid sequencing and MALDI-TOF mass spectral analysis showed that this lipase is encoded by gene LIP2 (GenBank accession no. AJ012632). Enzymatic deglycosylation showed that this lipase is a glycosylated protein which contains about 12% sugar. The corresponding deglycosylated lipase remained 88% specific activity of untreated lipase. There was a high amino acid sequence identity (91%) between YlLip2 and Candida deformans lipase CdLip1 (GenBank accession no. AJ428393). The optima temperature and pH for the purified lipase was 40 °C and 8.0, respectively. The lipase showed a preference for long chain fatty acid methyl esters (C12–C16), with the highest activity toward methyl myristate (C14). Lipase activity was stimulated by Ca²⁺ and Mg²⁺ and inhibited by Zn²⁺, Ni²⁺ and Cu²⁺, whereas EDTA had no effect on its activity. A 0.1% of Tween 80 and Span 65 increased slightly the enzyme activity and SDS inhibited it.     

Lipase of Mucor pusillus

Lipase of Mucor pusillus NRRL 2543 was recovered with ammonium sulfate precipitation, gel filtration on Sephadex G-75, and anion-exchange chromatography on diethylaminoethyl-Sephadex A-50. Maximal glycerol ester hydrolase (lipase) activity was observed at pH 5.0 to 5.5 and 50 C when trioctanoin and olive oil were used as substrates. The enzyme also showed esterase activity; it hydrolyzed, with the exception of methyl butyrate, all methyl esters tested. A minimum chain length of six carbons appeared to be a requirement for esterase activity, which was maximal at about pH 5.5 with methyl dodecanoate (C₁₂) as the substrate. Neither the glycerol ester hydrolase (lipase) nor the esterase activity of the enzyme appeared to be affected by thiol group inhibitors, chelating agents, and reducing compounds. On the other hand, hydrolysis of triolein and methyl dodecanoate was arrested to the same extent in the presence of diisopropyl fluorophosphate, which suggested the involvement of serine in the active center of the enzyme. The enzyme remained stable during a 30-day storage at - 10 C.     

Ionic liquids as a reaction medium for lipase-catalyzed methanolysis of sunflower oil

Ionic liquids, 1-butyl-3-methyl imidazolium hexafluorophosphate ([BMIm][PF₆]) and 1-ethyl-3-methyl imidazolium hexafluorophosphate ([EMIm][PF₆]), were used for the methanolysis of sunflower oil using Candida antarctica lipase (Novozyme 435) and gave yields of fatty acid methyl esters at 98–99% within 10 h. The optimum conditions of methanolysis in hydrophobic ionic liquids are 2% (w/w) lipase, 1:1 (w/w) oil/ionic liquid and 1:8 (mol/mol) oil/methanol at 58–60°C. Methanolysis using hydrophilic ionic liquids, 3-methyl imidazolium tetrafluoroborate ([HMIm][BF₄]) and 1-butyl-3-methyl imidazolium tetrafluoroborate ([BMIm][BF₄]), gave very poor yields. A hydrophobic ionic liquid thus protects the lipase from methanol. Recovered ionic liquids and lipase were used for four successive reaction cycles without any significant loss of activity.     

Interesterification and synthesis by Candida cylindracea lipase in microemulsions

Unusual reactions of interesterification and synthesis catalyzed by Candida cylindracea lipase have been tested in reverse microemulsions. The microemulsions used are made of fatty acids or triglycerides, the enzyme dissolved in a very low water quantity, Brij 35 used as surfactant and an alcoholic cosurfactant. In such a system, fats and alcohols are both the substrates of the enzyme and the microemulsion components. Incidentally, non specific Candida cylindracea lipase does not catalyze interesterification of short chain triglycerides, revealing a specificity for the chain length. Interesterification reactions tested in the presence of a given water quantity but with varying water activities show that it is the water activity and not the water quantity which is a fundamental parameter of the system. The effect of the surfactant (Brij 35) on the interesterification reaction is studied. Heptyl-oleate synthesis catalyzed by non-specific lipase is obtained in microemulsions at a 98% yield. Synthesis of glycerol esters is also tested in monophasic medium and mono and diglycerides are obtained.     

Age-dependent effects of sodium butyrate and hydrocortisone on acetylation of high mobility group proteins of rat liver

The in vitro acetylation of high mobility group (HMG) proteins and its modulation by sodium butyrate and hydrocortisone have been studied using liver slices of young (13-) and old (114-week-old) rats. Acetylation of total HMG proteins was significantly higher in young than old rats. HMG 1, in particular, showed greater acetylation than others. Whereas acetylation of HMG 1 and 2 decreased drastically, that of HMG 14 and 17 increased in old age. In young rats, sodium butyrate and hydrocortisone stimulated acetylation of HMG 14 and 17, and decreased that of HMG 2. Butyrate had no effect on HMG 1, but hydrocortisone decreased it. In old rats, butyrate and hydrocortisone decreased acetylation of all HMGs, except HMG 17, which was stimulated to a slight extent by butyrate.     

Subcellular fractionation, partial purification and characterization of neutral triacylglycerol lipase from pig liver.

The subcellular distributions of acidic (pH 4.5) and neutral (pH 7.5) longchain triacylglycerol lipases (glycerol ester hydrolase, EC 3.1.1.3) of pig liver have been determined. The distribution of the acidic lipase closely paralleled that of the lysosomal marker enzyme, cathepsin D. Approx. 60% of the neutral lipolytic activity resided in the soluble fraction;the distribution of this activity failed to parallel that of marker enzymes for mitochondria, lysosomes, microsomes, or plasma membranes. A method has been developed for purification of the neutral lipase from the soluble fraction by ultracentrifugation. An approximate 90-fold purification was achieved, with recovery of 16% of the initial activity. The partially purified neutral lipase exhibited a pH optimum between 7.25 and 7.5. It required 30 mM emulsified triolein for optimal activity and ceased to liberate fatty acids after 30 min of incubation. The enzymatic activity was destroyed by heating at 60 degrees C. Neutral lipase was inhibited by sodium deoxycholate, Triton X-100 and iodoacetamide. The activity was not inhibited by sodium taurocholate, EDTA, heparin and diethyl-p-nitrophenyl phosphate. Neutral lipase failed to exhibit activity in assay systems specific for lipoprotein lipase, monoolein hydrolase, tributyrinase, and methyl butyrate esterase and showed little or no capacity to hydrolyze chyle chylomicrons or plasma very low density lipoproteins. It is suggested that the function of neutral lipase may be to supply the liver with fatty acids liberated from endogenously synthesized or stored triacylglycerols.     

Hepatic lipase purification and characterization

Hepatic lipase has been purified to homogeneity from rat liver homogenates. The purified enzyme exhibits a single band on SDS-polyacrylamide gel electrophoresis. The molecular size of the native hepatic lipase is 200000, while on SDS-polyacrylamide gel electrophoresis the apparent minimum molecular weight of the enzyme is 53000, suggesting that the active enzyme is composed of four subunits. The relationship between triacylglycerol, monoacylglycerol and phospholipid hydrolyzing activities of the purified rat liver enzyme was studied. All three activities had a pH optimum of 8.5. The maximal reaction rates obtained with triolein, monoolein and dipalmitoylphosphatidylcholine were 55000, 66000 and 2600 μmol fatty acid/mg per h with apparent Michaelis constant (K_m) values of 0.4, 0.25 and 1.0 mM, respectively. Hydrolysis of triolein and monoolein probably takes place at the same site on the enzyme molecule, since competitive inhibition between these two substrates was observed, and a similar loss of hydrolytic activity occurred in the presence of diisopropylfluorophosphate. Addition of apolipoproteins C-II and C-I had no effect on the hydrolytic activity of the enzyme with the three substrates tested. However, the triacylglycerol hydrolyzing activity was inhibited by the addition of apolipoprotien C-III. Monospecific antiserum to the pure hepatic lipase has been raised in a rabbit.     

Improved activity and stability of Rhizopus oryzae lipase via immobilization for citronellol ester synthesis in supercritical carbon dioxide

In present work, Rhizopus oryzae lipase immobilized on a film prepared using blend of hydroxylpropyl methyl cellulose (HPMC) and polyvinyl alcohol (PVA) was investigated for synthesis of citronellol esters with supercritical carbon dioxide (Sc-CO₂) as a reaction medium. The transesterification reaction was optimized for various reaction parameters like effect of molar ratio, acyl donor, time, temperature, enzyme concentration, effect of pressure and co-solvent to achieve the maximum yield of desired product. The results obtained signify remarkable increment (about eightfold) in the yield of citronellol acetate (91%) as compared to that of free lipase (11%) in Sc-CO₂. The developed biocatalytic methodology provides a substantial advantage of low biocatalyst loading (1.5%, w/v), lower reaction temperature (45 °C) and lower pressure (8 MPa) as compared to previous reports. The immobilization method has significantly enhanced the operational stability of lipase for ester synthesis under Sc-CO₂ conditions. The developed methodology was successfully applied for synthesis of three different industrially important citronellol esters namely citronellol acetate (91%), citronellol butyrate (98%), citronellol laurate (99%) with excellent yields using vinyl esters as acyl donor under Sc-CO₂ conditions. In addition, the immobilized biocatalyst was effectively recycled for three consecutive recycles.     

Partial purification and characterization of almond seed lipase

A lipase was partially purified from the almond (Amygdalus communis L.) seed by ammonium sulfate fractionation and dialysis. Kinetics of the enzyme activity versus substrate concentration showed typical lipase behavior, with K(m) and V(max) values of 25 mM and 113.63 micromol min(-1) mg(-1) for tributyrin as substrate. All triglycerides were efficiently hydrolyzed by the enzyme. The partially purified almond seed lipase (ASL) was stable in the pH range of 6-9.5, with an optimum pH of 8.5. The enzyme was stable between 20 and 90 degrees C, beyond which it lost activity progressively, and exhibited an optimum temperature for the hydrolysis of soy bean oil at 65 degrees C. Based on the temperature activity data, the activation energy for the hydrolysis of soy bean oil was calculated as -5473.6 cal/mol. Soy bean oil served as good substrate for the enzyme and hydrolytic activity was enhanced by Ca(2+), Fe(2+), Mn(2+), Co(2+), and Ba(2+), but strongly inhibited by Mg(2+), Cu(2+), and Ni(2+). The detergents, sodiumdeoxicholate and Triton X-100 strongly stimulated enzyme activity while CTAB, DTAB, and SDS were inhibitors. Triton X-405 had no effect on lipase activity. The partially purified enzyme retained its activity for more than 6 months at -20 degrees C, beyond which it lost activity progressively.     

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.