Effects of lipidic carbon sources on the extracellular lipolytic activity of a newly isolated strain of Bacillus subtilis

An isolate exhibiting high extracellular lipolytic activity was identified as Bacillus subtilis by 16S rRNA gene sequence analysis. The enzyme activity of the isolate was improved by using different concentrations of lipidic carbon sources such as vegetable oils, fatty acids and triglycerides. Lipolytic activity was assayed spectrophotometrically using p-nitrophenyl palmitate. One percent (v/v) of sesame oil provided the highest activity with 80 and 98% enhancements with respect to 1% (v/v) concentrations of linoleic acid and triolein as the favored fatty acid and triglyceride, respectively. Glucose presented a repressive effect on lipase production. Lipase secreted by B. subtilis was partially purified by ultrafiltration and anion exchange chromatography; and the purified enzyme was tested for its residual activity in the presence of EDTA, SDS, Triton X-100, Tween 20, Tween 80 and protease. The present work reports, for the first time, that the lipolytic activity of a B. subtilis strain can be improved by using inexpensive vegetable oils; and also that B. subtilis lipase is suitable for use in detergents.     

Production of recombinant Rhizopus oryzae lipase by the yeast Yarrowia lipolytica results in increased enzymatic thermostability

The gene encoding Rhizopus oryzae lipase (ROL) was expressed in the non-conventional yeast Yarrowia lipolytica under the control of the strong inducible XPR2 gene promoter. The effects of three different preprosequence variants were examined: a preprosequence of the Y. lipolytica alkaline extracellular protease (AEP) encoded by XPR2, the native preprosequence of ROL, and a hybrid variant of the presequence of AEP and the prosequence of ROL. Lipase production was highest (7.6 U/mL) with the hybrid prepropeptide. The recombinant protein was purified by ion-exchange chromatography. The ROL included 28 amino acids of the C-terminal region of the prosequence, indicating that proteolytic cleavage occurred below the KR site through the activity of the Kex2-like endoprotease. The optimum temperature for recombinant lipase activity was between 30 and 40 °C, and the optimum pH was 7.5. The enzyme was shown not to be glycosylated. Furthermore, recombinant ROL exhibited greater thermostability than previously reported, with the enzyme retaining 64% of its hydrolytic activity after 30 min of incubation at 55 °C.     

Partial purification of a lipolytic enzyme from Escherichia coli.

An enzyme with phospholipase Al activity was purified some 500-fold from Escherichia coli cell homogenates. Lipase, phospholipase A2, and lysophospholipase copurified with phospholipase A1 and the four activities displayed similar susceptibility to heat treatment. The phospholipase A and lipase activities were recovered in a single band when partially purified preparations were subjected to SDS gel electrophoresis. Phospholipase, lysophospholipase, and lipase all required Ca2+ for activity. Phosphatidylcholine, phosphatidylethanolamine, and their lyso analogues were all hydrolysed at equivalent rates and these were substantially greater than the rate of methylpalmitate or tripalmitoylglycerol hydrolyses under similar incubation conditions. Evidence for a direct but slow hydrolysis of the ester at position 2 of phosphoglyceride was obtained; however, release of fatty acid from this position is mostly indirect involving acyl migration to position 1 and subsequent release of the translocated fatty acid. Escherichia coli, therefore, appears to possess a lipolytic enzyme of broad substrate specificity acting mainly at position 1 but also at position 2 of phosphoglycerides and on triacylglycerols and methyl fatty-acid esters.     

Purification and general properties of a metal-insensitive lipase from Rhizopus japonicus NR 400

Lipase [triacylglycerol lipase, EC 3.1.1.3] has been purified to homogeneity from Rhizopus japonicus NR 400 by chromatography on hydroxylapatite, octyl-Sepharose and Sephacryl S-200. It showed a molecular weight of about 30,000 by SDS-PAGE and a specific activity of 68,900 units/mg protein. The enzyme catalyzed the hydrolysis of tricapryn and tricaprylin rapidly in comparison with other triglycerides. This lipase had an optimum pH of around 5, and albumin enhanced its activity between pH 3 and 8. The composition of fatty acids liberated from linseed oil by the lipase was similar to that in the case of pancreatic lipase. The lipase activity was not affected by the addition of 1 mM metal ions or bile salts. Stimulation of the lipase activity was observed upon addition of albumin to the reaction mixture. Immunotitration experiments were also performed with antibodies raised against the purified lipase.     

Preparation of pancreatin and purification of lipase from hog pancreas

Pancreatin containing high activities of proteolytic enzymes, amylase and lipase was prepared from optimally autolyzed hog pancreas. About one hundred grams of pancreatin were obtained from one kilogram of hog pancreas. Lipase was purified from the pancreatin preparation through steps of mild alkaline solution extraction, removing proteolytic enzymes by affinity adsorption, first ammonium sulfate fractionation, Sephacryl S-200 gel filtration, and secondary ammonium sulfate fractionation. By these steps, the purity of the enzyme increased 14 fold and the recovery of the enzyme activity was 33%. The purified lipase was not homogeneous and contained several contaminating proteins when examined by disc polyacrylamide gel electrophoresis.     

Characterization of lysosomal acid lipase purified from rabbit liver

Lysosomal acid lipase from rabbit liver was solubilized with digitonin and purified 25,000-fold by Bio-Gel A-1.5 m, DEAE Bio-Gel A and phenyl Sepharose column chromatographies, preparative slab gel electrophoresis and finally Affi-Gel Blue affinity column chromatography. The purified enzyme gave a single protein band on polyacrylamide gel electrophoresis both in the presence and absence of sodium dodecyl sulfate. The molecular weight of the acid lipase was estimated to be 42,000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis and 40,000 by gel filtration on Bio-Gel A-0.5 m. The enzyme was a hydrophobic glycoprotein with an isoelectric point of 5.15-5.90. The purified enzyme hydrolyzed tri-, di-, and monoolein and cholesterol oleate, with apparent Vmax values of 5.41, 56.1, 21.7, and 3.25 mumol/min/mg protein, and Km values of 50, 70, 200, and 40 microM, respectively. It hydrolyzed 4-methylumbelliferyl esters with fatty acids of different lengths in the order, medium length chains greater than long chains much greater than short chains. It did not hydrolyze dipalmitoylphosphatidylcholine. Its activity was inhibited by micromolar concentrations of p-chloromercuriphenyl sulfonic acid and p-bromophenacyl bromide and millimolar concentrations of Cu2+ and diethylpyrocarbonate. The activities of the enzyme towards the five substrates listed above showed almost identical thermal stabilities, mobilities on polyacrylamide gel electrophoresis and inhibition by several inhibitors. These findings support the idea that one enzyme is involved in the hydrolysis of both acylglycerols and cholesterol esters in lysosomes.     

Studies on lipase from Mucor javanicus. I. Purification and properties.

1. Lipase produced by a mold, Mucor javanicus, was purified about 180-fold from the ethanol precipitate of the culture filtrate. Purification was achieved by acid precipitation followed by gel filtrations on Sephadex G-200 (at low ionic strength) and Sephadex G-75 (at a high ionic strength). The purified enzyme preparation showed unusual behavior on polyacrylamide gel electrophoresis. The molecular weight was estimated to be 21 000. The enzyme had a positional specificity towards the position 1 and 3 of triacylglycerols. 2. Lipase in the crude preparation takes an aggregated form. aggregated form was achieved by raising the ionic strength of the medium. 3. The purified lipase preparation from Mucor javanicus exhibits phospholipase A1 activity, hydrolyzing the carboxyl ester at the 1-position of phosphatidylcholine. This activity seems to be due to the action of the lipase itself and not due to any other specific phospholipases.     

Sunflower seed lipase: extraction, purification, and characterization

A simple procedure for the extraction of the lipolytic activity from sunflower seed has been developed. Various conditions of extraction have been optimized in order to obtain maximum yield of lipase. A new lipase enzyme was purified by the fractional salt precipitation from the supernatant, dialysis on a cellulose membrane, and gel column chromatography on Sephadex G-75. The lipase was monomeric, with an apparent Mr of 22 kDa and a pI of 8, with the electrophoretic analysis. Kinetics of the enzyme activity versus substrate concentration showed typical lipase behavior, with Km and Vmax, values of 1.33 mM and 555 U/mg. All triglycerides were efficiently hydrolyzed by the enzyme, but this showed a preference towards triglycerides of natural mono unsaturated fatty acids. The optimum temperature, pH, and incubation time for lipolytic activity were 50 degrees C, 7.5, and 5 min, respectively. The stability of the sunflower lipase was investigated under operational and storage conditions. It was found that this enzyme preserved its lipolytic activity at temperatures between at 35-50 degrees C, alkaline pH, and for a period of about four months.     

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.     

Pancreatic bile salt dependent lipase from cod (Gadus morhua): purification and properties.

The enzymatic basis for cod digestive lipolysis has been investigated. Lipase activity was found in aqueous extracts from pyloric caeca as well as in pancreatic tissue surrounding the caeca and the bile duct. A bile salt-dependent lipase (BSDL) was purified from either defatted powder of cod pyloric caeca or aqueous pancreatic extracts by combined affinity chromatography on cholate-Sepharose and gel filtration on Sephacryl S-200 HR. By SDS-PAGE analysis the molecular weight of purified cod BSDL was estimated to 60 kDa. The enzyme was totally dependent on bile salts for hydrolysis of insoluble fatty acid esters. Antiserum raised against purified cod BSDL reacted specifically with selected mammalian pancreatic BSDLs by Western blot analysis. Results presented in this paper strongly suggest that the bile salt-dependent lipase is the only pancreatic enzyme involved in lipid digestion in cod. The enzyme has been characterized and compared to human pancreatic BSDL with respect to substrate specificity, temperature- and pH-dependence and inhibitors. Both soluble and insoluble fatty acid esters were hydrolysed and the enzyme was 1,3-specific in hydrolysis of triolein. The enzyme was inhibited by di-isopropyl fluorophosphate and phenyl boronic acid, but not significantly by phenyl methyl sulfonyl fluoride. The cod BSDL is probably homologous to mammalian pancreatic BSDLs.     

Isolation and partial characterization of an amphiphilic 56-kDa fatty acid binding protein from rat renal basolateral membrane

We have identified a 56-kDa fatty acid binding protein in rat renal basolateral membrane and purified it by extraction in nonionic detergent (Triton X-100), followed by gel filtration, DEAE-cellulose chromatography, and affinity chromatography. The purified protein was homogeneous on polyacrylamide gel electrophoresis in the presence of Triton X-100 or SDS. It showed amphiphilic properties on gel filtration, polyacrylamide gel electrophoresis, and oleate-Sepharose 4B chromatography. Its molecular mass was estimated to be 56 kDa by SDS-polyacrylamide gel electrophoresis. The protein showed optimal binding activity at pH 7.5 and 37 degrees C. The apparent Kd for palmitic acid was 0.79 microM. It was immunologically clearly distinct from renal cytosolic fatty acid binding protein.     

Kinetic studies of Rhizopus oryzae lipase using monomolecular film technique

Rhizopus oryzae lipase (ROL) was found to be a true lipase. This enzyme presents the interfacial activation phenomenon. The N-terminal amino acid sequence of ROL was compared to those of rhizopus lipases. Purified ROL possesses the same N-terminal sequence as the mature Rhizopus niveus lipase (RNL). This sequence was found in the last 28 amino acids of the propeptide sequence derived from the cDNA of Rhizopus delemar lipase (RDL). Using the baro-stat method, we have measured the hydrolysis rate of dicaprin films by ROL as a function of surface pressure. Our results show that Rhizopus oryzae lipase is markedly stereoselective of the sn-3 position of the 2,3 enantiomer of dicaprin. Polyclonal antibodies (PAB) directed against ROL have been produced and purified by immunoaffinity. The effects of these PAB on the interfacial behavior of ROL were determined. The immunoblot analysis with polyclonal antibodies anti-ROL (PAB anti-ROL) and various lipases shows a cross-immunoreactivity between the lipase from the rhizopus family (Rhizopus delemar lipase and Rhizopus arrhizus lipase).     

Synthesis activity-based zymography for detection of lipases and esterases

A new zymography method for lipases and esterases was developed on the basis of the esterification reaction between fatty acids and alcohols. The enzymes were separated by SDS-PAGE and native PAGE. The gel was washed and then incubated in an aqueous solution containing fatty acids (oleic acid 18:1 or caprylic acid 8:0) and dodecanol. Synthesis was visualized by in situ precipitation of water-insoluble and non-diffusible fatty acid esters, such as dodecyl oleate and dodecyl octanoate. The synthesis activity-based zymography was confirmed with different enzyme samples, including commercial lipase preparations, purified recombinant lipase and cutinase, and crude culture supernatants of lipolytic enzyme-producing soil bacteria.     

Production,purification and characterization of an extracellular lipase from Aureobasidium pullulans HN2.3 with potential application for the hydrolysis of edible oils

Lipase production (8.02 ± 0.24 U/ml) by the yeast Aureobasidium pullulans HN2.3 isolated from sea saltern was carried by using time-dependent induction strategy. The lipase in the supernatant of the yeast cell culture was purified to homogeneity with a 3.4-fold increase in specific lipase activity as compared to that in the supernatant by ammonium sulfate fractionation, gel filtration chromatography and anion-exchange chromatography. According to the data on SDS polyacrylamide gel electrophoresis, the molecular mass of the purified enzyme was estimated to be 63.5 kDa. The optimal pH and temperature of the purified enzyme were 8.5 and 35 °C, respectively. The enzyme was greatly inhibited by Hg²⁺, Fe²⁺ and Zn²⁺. The enzyme was strongly inhibited by phenylmethanesulphonyl fluoride, not inhibited by ethylene diamine tetraacetic acid (EDTA), but weakly inhibited by iodoacetic acid. It was found that the purified lipase had the highest hydrolytic activity towards peanut oil.     

Purification and characterization of two distinct lipases from Geotrichum candidum

Lipase, an enzyme that hydrolyzes triacylglycerol, has been purified and characterized. The purification procedure includes ethanol precipitation and chromatographies on Sephacryl-200 HR, high resolution anion-exchange (mono Q) and Polybuffer exchanger 94. With this procedure, two forms of lipases from Geotrichum candidum were obtained. Lipase I (main enzyme) and lipase II (minor enzyme) were purified 35-fold with a 62% recovery in activity and 94-fold with a 18% recovery in activity, respectively. Their molecular weights have been estimated by polyacrylamide gel electrophoresis under denaturing conditions and by molecular sieving under native conditions at 56,000. Lipase I and II had optimum pH values of 6.0 and 6.8 and isoelectric points of 4.56 and 4.46, respectively. The enzymes are stable at a pH range of 6.0 to 8.0. Monovalent ions had little effect on both enzyme activities, while divalent ions at concentrations above 50 mM inhibited the lipase activities in a concentration-dependent manner. Sodium dodecyl sulfate at a concentration lower than 10 mM completely inhibited the lipase activity.     

Purification and characterization of the fatty acid synthase from Bugula neritina

The fatty acid synthase from Bugula neritina has been purified 100-fold using ammonium sulfate precipitation, ion-exchange and size exclusion chromatography. The purified enzyme has a molecular weight of approximately 382,000 Da, as judged by gel filtration. Polyacrylamide gel electrophoresis under denaturing conditions in the presence of SDS revealed one major protein band of approximately 190,000 Da suggesting that the enzyme is a homodimer. The size of the enzyme, together with the observation that the FAS activity is independent of the concentration of acyl carrier protein, indicate that the FAS from Bugula neritina is a type I. A detailed analysis of the products of the purified FAS indicated that palmitic acid is the primary product and longer chain fatty acids are not produced.     

Purification and characterization of a lipase from Pichia burtonii

An extracellular lipase from Pichia burtonii was purified to homogeneity by a combination of DEAE-Sephadex A-50 ion-exchange chromatography, Sephadex G-100 gel filtration, and isoelectric focusing. The purified enzyme preparation showed a single protein band corresponding to a molecular mass of 51 kDa on sodium dodecyl sulphate/polyacrylamide gel electrophoresis. The molecular mass of the enzyme was estimated to be 47 kDa on Superdex 200 gel filtration, suggesting that the enzyme was a monomeric protein. The pI was about 5.8. The optimum pH and temperature for the hydrolysis of olive oil were about 6.5 and 45°C respectively. Rapid loss of the enzyme activity was observed above 30°C in the absence of olive oil, but the addition of olive oil or trimethylolpropane diallyl ether greatly stabilized the enzyme. At 30°C, the enzyme hydrolysed Spans and Tweens as well as simple triglycerides of short- and middle-chain fatty acids. Although the enzyme cleaved all the ester bonds of triolein, it showed some preference for the outer ester bonds.     

Purification and characterization of fatty acyl-acyl carrier protein synthetase from Vibrio harveyi.

A Vibrio harveyi enzyme which catalyzes the ATP-dependent ligation of fatty acids to acyl carrier protein (ACP) has been purified 6,000-fold to apparent homogeneity by anion-exchange, gel filtration, and ACP-Sepharose affinity chromatography. Purified acyl-ACP synthetase migrated as a single 62-kDa band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and as an 80-kDa protein by gel filtration under reducing conditions. Activity of the purified enzyme was lost within hours in the absence of glycerol and low concentrations of Triton X-100. Acyl-ACP synthetase exhibited Kms for myristic acid, ACP, and ATP of 7 microM, 18 microM, and 0.3 mM, respectively. The enzyme was specific for adenine-containing nucleotides, and AMP was the product of the reaction. No covalent acyl-enzyme intermediate was observed. Enzyme activity was stimulated up to 50% by iodoacetamide but inhibited > 80% by N-ethylmaleimide: inhibition by the latter was prevented by ATP and ACP but not myristic acid. Dithiothreitol and sulfhydryl-directed reagents also influenced enzyme size, activity, and elution pattern on anion-exchange resins. The function of acyl-ACP synthetase has not been established, but it may be related to the capacity of V. harveyi to elongate exogenous fatty acids by an ACP-dependent mechanism.     

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.     

Butyrate kinase from Clostridium acetobutylicum

Crude extracts of Clostridium acetobutylicum contain a butyrate kinase of high specific activity (5.2 mumol/min/mg of protein). The enzyme has been purified 77-fold in a six-step procedure to a specific activity of 402 mumol/min/mg of protein. The purified butyrate kinase showed a single band with a molecular weight of 85,000 on nondenaturing polyacrylamide gradient gel electrophoresis. Separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the enzyme is a dimer of two apparently identical subunits with molecular weights of 39,000. The pH optimum for the reaction in the butyryl phosphate-forming direction is 7.5, and the pI of the kinase is 5.6. The amino acid composition of the enzyme is also reported. It contains no tryptophan and is low in sulfur-containing amino acids. The kinase has a broad substrate specificity and exhibits its highest relative activities with butyrate and valerate. Butyrate kinase is rapidly inactivated at 50 degrees C in the absence of a fatty acid substrate. Although a reducing agent was required for maximum activity, treatment with several sulfhydryl-modifying agents failed to inhibit the enzyme.