Interfacial catalysis by lipases

We designed a convenient, specific, sensitive and continuous lipase activity assay using natural long-chain triacylglycerols (TAGs). Oil was extracted from Parinari glaberrimum seed kernels and the purified TAGs used as a substrate for detecting low levels of lipase activities. The purified TAGs are naturally fluorescent. The presence of detergents above their critical micellar concentration dramatically increases the fluorescence of the parinaric acid released by various lipases. This increase is linear with time and proportional to the amount of lipase added. Quantities as low as 0.1 ng of pure pancreatic lipase could be detected under standard conditions (pH 8).

The interfacial activation of human pancreatic lipase (HPL) probably involves the motion of a lid covering the active site of the enzyme. We observed that the presence of either bile salts or a small proportion of water-miscible organic solvents (called activator compounds) considerably enhances the enzymatic activity of HPL on a monomeric solution of tripropionin. This finding suggests that the activator compounds may favor the opening of the lid. This hypothesis was checked by comparing the immunoreactivity of HPL and HPL with a mini-lid (HPL(-lid)) towards anti-HPL monoclonal antibodies (mAbs), in the presence and absence of the activator compounds.     

Assaying Arabidopsis lipase activity

A low lipase activity from a crude extract of Arabidopsis seedlings was assayed using three sensitive methods (radiolabelled triacylglycerols, commercial resorufin ester and triacylglycerols containing the naturally fluorescent parinaric acid as substrates). The specific activity of the extract was found to be similar using the three methods. However, the plant lipase activity measured using the radioactivity and the fluorescence assays could be abolished by heating the extract, contrary to the apparent activity measured using the commercial colorimetric assay. Unlike the radioactivity assay, the fluorescence assay can be monitored continuously. The parinaric acid-based method is therefore the only one to provide a sensitive, specific and continuous assay.     

An ultraviolet spectrophotometric assay for measuring lipase activity using long-chain triacyglycerols from Aleurites fordii seeds

In this study, we designed a specific, continuous, and sensitive UV spectrophotometric lipase assay using natural triacylglycerols (TAGs) from the Aleurites fordii seed oil (tung oil). alpha-Eleostearic acid (9,11,13-cis, trans,trans-octadecatrienoic acid) is the main fatty acid component (it accounts for up to 70%) of the TAGs from tung oil. The conjugated triene present in alpha-eleostearic acid constitutes an intrinsic chromophore, which confers strong UV absorption properties on both the free fatty acid and the TAGs from tung oil. The lipase assay is based on the difference between the apparent molar extinction coefficients of the two types of alpha-eleostearic acid present, that which is esterified into TAGs and that which is released into the reaction medium. This difference is responsible for the variations in the UV absorption spectrum of the reaction medium occurring upon enzymatic TAGs hydrolysis. Using the purified lipase from Thermomyces lanuginosa (TLL) and the detergent sodium taurodeoxycholate (NaTDC, 4 mM), it was established that the most suitable method of measuring lipolysis consisted of monitoring the decrease in the OD at 292 nm, which was linear with time and proportional to the amount of lipase added. In order to be able to estimate the specific activity of TLL, we determined an apparent molar extinction coefficient of alpha-eleostearic acid (epsilon = 13,900 M(-1) cm(-1)) under the assay conditions. Amounts of pure TLL as small as 1 ng can be easily detected in the presence of 4 mM NaTDC. Interestingly, the NaTDC concentration can be decreased as far as 0.05 mM. In comparison with other well-known methods of lipase assay, the detection limit of this new method is 100-fold lower than with the pH-stat method and similar to that of a fluorescent assay recently developed at our laboratory.     

An ultraviolet spectrophotometric assay for the screening of sn-2-specific lipases using 1,3-O-dioleoyl-2-O-α-eleostearoyl-sn-glycerol as substrate

In the present study, we propose a continuous assay for the screening of sn-2 lipases by using triacylglycerols (TAGs) from Aleurites fordii seed (tung oil) and a synthetic TAG containing the α-eleostearic acid at the sn-2 position and the oleic acid (OA) at the sn-1 and sn-3 positions [1,3-O-dioleoyl-2-O-α-eleostearoyl-sn-glycerol (sn-OEO)]. Each TAG was coated into a microplate well, and the lipase activity was measured by optical density increase at 272 nm due to transition of α-eleostearic acid from the adsorbed to the soluble state. The sn-1,3-regioselective lipases human pancreatic lipase (HPL), LIP2 lipase from Yarrowia lipolytica (YLLIP2), and a known sn-2 lipase, Candida antarctica lipase A (CALA) were used to validate this method. TLC analysis of lipolysis products showed that the lipases tested were able to hydrolyze the sn-OEO and the tung oil TAGs, but only CALA hydrolyzed the sn-2 position. The ratio of initial velocities on sn-OEO and tung oil TAGs was used to estimate the sn-2 preference of lipases. CALA was the enzyme with the highest ratio (0.22 ± 0.015), whereas HPL and YLLIP2 showed much lower ratios (0.072 ± 0.026 and 0.038 ± 0.016, respectively). This continuous sn-2 lipase assay is compatible with a high sample throughput and thus can be applied to the screening of sn-2 lipases.     

Lipid peroxidation and its inhibition in low density lipoproteins: quenching of cis-parinaric acid fluorescence.

The fluorescent polyunsaturated parinaric acid incorporated in LDL particles is highly sensitive to the concentration of peroxyl radicals in the aqueous medium, undergoing rapidly oxidative degradation, as detected by a quenching of fluorescence, without delay after radical generation in solution. Ascorbate, cysteine, and urate suppress the parinaric acid fluorescence decay promoted by peroxyl radicals generated at a constant rate (thermal decomposition of 2,2'-azo-bis(2-amidino-propane hydrochloride)) in a concentration-dependent manner. The chain-breaking efficiencies of these antioxidants are evaluated from the time interval (inhibition period) of parinaric acid protection from oxidative degradation. The results correlate with the inhibition periods of LDL oxidation as monitored by O2 consumption. Therefore, the sensitive and simple parinaric acid assay can be used as a semiquantitative screening test for the detection of potentially important water-soluble chain-breaking antioxidants. Conversely to O2 consumption, the absence of any initial lag phase of probe degradation attests to the sensitivity of the assay. An improved methodology based on second-derivative spectroscopy to follow the formation of conjugated diene isomers directly in the preparation without the need for lipid extraction also confirms the sensitivity of this assay. To assess the usefulness of parinaric acid assay, strong chain-breaking activities of caffeic and chlorogenic acids are reported.     

A broad pH range indicator-based spectrophotometric assay for true lipases using tributyrin and tricaprylin

A continuous assay is proposed for the screening of acidic, neutral, or alkaline lipases using microtiter plates, emulsified short- and medium-chain TGs, and a pH indicator. The lipase activity measurement is based on the decrease of the pH indicator optical density due to protonation which is caused by the release of FFAs during the hydrolysis of TGs and thus acidification. Purified lipases with distinct pH optima and an esterase were used to validate the method. The rate of lipolysis was found to be linear with time and proportional to the amount of enzyme added in each case. Specific activities measured with this microplate assay method were lower than those obtained by the pH-stat technique. Nevertheless, the pH-dependent profiles of enzymatic activity were similar with both assays. In addition, the substrate preference of each enzyme tested was not modified and this allowed discriminating lipase and esterase activities using tributyrin (low water solubility) and tricaprylin (not water soluble) as substrates. This continuous lipase assay is compatible with a high sample throughput and can be applied for the screening of lipases and lipase inhibitors from biological samples.     

A novel method for measuring human lipoprotein lipase and hepatic lipase activities in postheparin plasma

The objective of this study was to establish a new lipoprotein lipase (LPL) and hepatic lipase (HL) activity assay method. Seventy normal volunteers were recruited. Lipase activities were assayed by measuring the increase in absorbance at 546 nm due to the quinoneine dye. Reaction mixture-1 (R-1) contained dioleoylglycerol solubilized with lauryldimethylaminobetaine, monoacylglycerol-specific lipase, glycerolkinase, glycerol-3-phosphate oxidase, peroxidase, ascorbic acid oxidase, and apolipoprotein C-II (apoC-II). R-2 contained Tris-HCl (pH 8.7) and 4-aminoantipyrine. Automated assay of lipase activities was performed with an automatic clinical analyzer. In the assay for HL + LPL activity, 160 microl R-1 was incubated at 37 degrees C with 2 microl of sample for 5 min, and 80 microl R-2 was added. HL activities were measured under the same conditions without apoC-II. HL and LPL activities were also measured by the conventional isotope method and for HL mass by ELISA. Lipase activity detected in a 1.6 M NaCl-eluted fraction from a heparin-Sepharose column was enhanced by adding purified apoC-II in a dose-dependent manner, whereas that eluted by 0.8 M NaCl was not. Postheparin plasma-LPL and HL activities measured in the present automated method had high correlations with those measured by conventional activity and mass methods. This automated assay method for LPL and HL activities is simple and reliable and can be applied to an automatic clinical analyzer.     

Flow cytometric measurement of lipid peroxidation in vital cells using parinaric acid.

A method for measuring lipid peroxidation using time resolved flow cytometry is described. Because of its chemical nature, the naturally fluorescent fatty acid cis-parinaric acid is readily consumed in lipid peroxidation reactions. It could be loaded into Chinese hamster ovary cells in a time and concentration dependent manner at 37 degrees C, with 5 microM for 60' giving consistent, bright fluorescence without evidence of cytotoxicity. Examination of cells by fluorescence microscopy showed diffuse staining of surface and internal membranes. Cells were maintained at 37 degrees C while being examined in an Epics Elite flow cytometer equipped with a 325 nm HeCd laser, and parinaric acid fluorescence at 405 nm was measured over time. Addition of the oxidant tert-butyl hydroperoxide resulted in a burst of intracellular oxidation, shown by simultaneously loading the cells with dichlorofluorescein, and loss of parinaric fluorescence over time. This was followed by cell death, indicated by loss of forward light scatter and uptake of propidium iodide. Pretreatment of the cells with the antioxidant alpha-tocopherol, 200 microM, reduced the rate of loss of parinaric acid fluorescence and delayed the onset of cell death. Simultaneous biochemical determination of the lipid peroxidation breakdown product malondialdehyde confirmed a close temporal relationship with loss of parinaric acid fluorescence, both with and without alpha-tocopherol pretreatment and suggested that the flow cytometric assay for lipid peroxidation is of comparable sensitivity. The mitochondrial stain dodecyl acridine orange and the cyanine dye DiOC(6)3 were combined with cis-parinaric acid staining and could be excited by the latter using resonance energy transfer.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation and properties of extracellular lipase of native (B-10) and mutant (M-1) Serratia marcescens strains

The cultivation conditions of wild-type strain V-10 and mutant strain M-1 (overproducer of endonuclease and chitinase) of Serratia marcescens optimal for extracellular lipase biosynthesis were determined. The strain V-10 displayed the maximal lipase yield (840 AU/ml) after 10-12 h of cultivation; the strain M-1 (33 AU/ml), after 25-30 h. The data showed that extracellular lipases from V-10 and M-1 can be precipitated in a weakly acid medium (pH 5.0 and 4.5, respectively). This property was used to obtain partially purified lipase preparations. The effect of the ionic composition of the reaction mixture on the activities of these enzymatic preparations was studied. Both preparations displayed highest activities in weakly alkaline media (pH 8.0); however, the wild-type strain lipase displayed a higher thermal stability and stability at alkaline pH compared with M-1 lipase. Both lipases were activated by various anionic and nonionic surfactants and inactive in the presence of cetyltrimethylammonium bromide.     

Physiological regulation and optimization of lipase activity in Pseudomonas aeruginosa EF2.

Physiological regulation of extracellular lipase activity by a newly-isolated, thermotolerant strain of Pseudomonas aeruginosa (strain EF2) was investigated by growing the organism under various conditions in batch, fed-batch and continuous culture. Lipase activity, measured as the rate of olive oil (predominantly triolein) hydrolysis, was weakly induced by general carbon and/or energy limitation, strongly induced by a wide range of fatty acyl esters including triglycerides, Spans and Tweens, and repressed by long-chain fatty acids including oleic acid. The highest lipase activities were observed during the stationary phase of batch cultures grown on Tween 80, and with Tween 80-limited fed-batch and continuous cultures grown at low specific growth rates. The lipase activity of Tween 80-limited continuous cultures was optimized with respect to pH and temperature using response surface analysis; maximum activity occurred during growth at pH 6.5, 35.5 degrees C, at a dilution rate of 0.04 h-1. Under these conditions the culture exhibited a lipase activity of 39 LU (mg cells)-1 and a specific rate of lipase production (qLipase) of 1.56 LU (mg cells)-1 h-1 (1 LU equalled 1 mumol fatty acid released min-1). Esterase activity, measured with p-nitrophenyl acetate as substrate, varied approximately in parallel with lipase activity under all growth conditions, suggesting that a single enzyme may catalyse both activities.     

Isolation and characterization of extracellular lipase preparations from wild-type (V-10) and mutant (M-1)Serratia marcescens strains

—The cultivation conditions of wild-type strain V-10 and mutant strain M-l (overproducer of endonuclease and chitinase) ofSerratia marcescens optimal for extracellular lipase biosynthesis were determined. The strain V-10 displayed the maximum lipase yield (840 AU/ml) after 10–12 h of cultivation; the strain M-l (330 AU/ml), after 25–30 h. The data showed that extracellular lipases from V-10 and M-1 can be precipitated in a weakly acidic medium (pH 5.0 and 4.5, respectively). This property was used to obtain partially purified lipase preparations. The effect of the ionic composition of the reaction mixture on the activities of these enzymatic preparations was studied. Both preparations displayed the highest activities in weakly alkaline media (pH 8.0); however, the wild-type strain lipase displayed higher thermal stability and stability at alkaline pH compared with M-1 lipase. Both lipases were activated by various anionic and nonionic surfactants and were inactive in the presence of cetyltrimethylammonium bromide.     

A novel method for measuring human hepatic lipase activity in postheparin plasma

The objective of this study was to establish a hepatic lipase (HL) assay method that can be applied to automatic clinical analyzers. Seventy-four hyperlipidemic subjects (men/women 45/29) were recruited. Lipase activity was assayed measuring the increase in absorbance at 546 nm due to quinonediimine dye production. Reaction mixture R-1 contained 50 mM Tris-HCl (pH 9.5), 0.5 mM glycerol-1,2-dioleate, 0.4% (unless otherwise noted) polyoxyethylene-nonylphenylether, 3 mM ATP, 3 mM MgCl(2), 1.5 mM CaCl(2), monoacylglycerol-specific lipase, glycerol kinase, glycerol-3-phosphate oxidase, 0.075% N,N-bis-(4-sulfobutyl)-3-methylaniline-2 Na, peroxidase, ascorbic acid oxidase. Reaction mixture R-2 contained 50 mM Tris-HCl (pH9.5), 0.15% 4-aminoantypirine. Automated assay for activity was performed with a Model 7080 Hitachi analyzer. In the lipase assay, 160 microl of R-1 was incubated at 37 degrees C with 3 microl of samples for 5 min, and 80 microl of R-2 was added. Within-run coefficient of variations was 0.9-1.0%. Calibration curve of lipase activity was linear (r = 0.999) between 0 and 320 U/l. Analytical recoveries of purified HL added to plasma were 96.6-99.8%. HL activity in postheparin plasma measured in this method had a closer correlation with HL mass by a sandwich ELISA (r = 0.888, P < 0.0001) than those in the conventional method using [(14)C-]triolein (r = 0.730, P < 0.0001). This assay method for HL activity can be applied to an automatic clinical analyzer.     

Characterization of an extracellular lipase in Burkholderia sp. HY-10 isolated from a longicorn beetle

Burkholderia sp. HY-10 isolated from the digestive tracts of the longicorn beetle, Prionus insularis, produced an extracellular lipase with a molecular weight of 33.5 kDa estimated by SDS-PAGE. The lipase was purified from the culture supernatant to near electrophoretic homogenity by a one-step adsorption-desorption procedure using a polypropylene matrix followed by a concentration step. The purified lipase exhibited highest activities at pH 8.5 and 60 degrees . A broad range of lipase substrates, from C4 to C18 rho-nitrophenyl esters, were hydrolyzed efficiently by the lipase. The most efficient substrate was rho-nitrophenyl caproate (C6). A 2485 bp DNA fragment was isolated by PCR amplification and chromosomal walking which encoded two polypeptides of 364 and 346 amino acids, identified as a lipase and a lipase foldase, respectively. The N-terminal amino acid sequence of the purified lipase and nucleotide sequence analysis predicted that the precursor lipase was proteolytically modified through the secretion step and produced a catalytically active 33.5 kDa protein. The deduced amino acid sequence for the lipase shared extensive similarity with those of the lipase family I.2 of lipases from other bacteria. The deduced amino acid sequence contained two Cystein residues forming a disulfide bond in the molecule and three, well-conserved amino acid residues, Ser131, His330, and Asp308, which composed the catalytic triad of the enzyme.     

Biochemical and molecular characterisation of a thermoactive, alkaline and detergent-stable lipase from a newly isolated Staphylococcus aureus strain

A newly soil-isolated Staphylococcus aureus strain secretes a non-induced lipase in the culture medium. The extracellular lipase from S. aureus (SAL3) is purified to homogeneity. The purified enzyme is a tetrameric protein (180 kDa) corresponding to the association of four lipase molecules. The 15 N-terminal amino acid residues showed a high degree of homology with other staphylococcal lipase sequences. The part of the gene encoding the mature SAL3 is cloned and sequenced. The deduced polypeptide sequence, corresponding to the mature SAL3, was very similar to the mature Staphylococcus simulans lipase sequence with two additional amino acid residues (LK) at the N-terminus of SAL3. The lipase activity is maximal at pH 9.5 and 55 °C. The specific activity of about 4200 U/mg or 3500 U/mg was measured using tributyrin or olive oil emulsion as substrate, respectively, at pH 9.5 and 55 °C.In contrast to other staphylococcal lipases previously characterised, SAL3 is found to be stable between pH 5 and 12 after 24 h incubation. The enzyme retained 50% of its activity after 60 min incubation at 60 °C. This novel lipase is able to hydrolyse its substrate in presence of various oxidizing agents as well as some surfactants and some commercial detergents, then SAL3 can be considered as a good candidate for industrial and biotechnological applications.     

Biochemical characterization of a lipase from olive fruit (Olea europaea L.)

Lipase (triacylglycerol acylhydrolase; EC 3.1.1.3) is the first enzyme of the degradation path of stored triacylglycerols (TAGs). In olive fruits, lipase may determine the increase of free fatty acids (FFAs) which level is an important index of virgin olive oil quality. However, despite the importance of virgin olive oil for nutrition and human health, few studies have been realized on lipase activity in Olea europaea fruits. In order to characterize olive lipase, fruits of the cv. Ogliarola, widely diffused in Salento area (Puglia, Italy), were harvested at four stages of ripening according to their skin colour (green, spotted I, spotted II, purple). Lipase activity was detected in the fatty layer obtained after centrifugation of the olive mesocarp homogenate. The enzyme exhibited a maximum activity at pH 5.0. The addition of calcium in the lipase assay medium leads to an increment of activity, whereas in the presence of copper the activity was reduced by 75%. Furthermore, mesocarp lipase activity increases during olive development but declined at maturity (purple stage). The data represent the first contribution to the biochemical characterization of an olive fruit lipase associated to oil bodies.     

Purification and properties of a phospholipase A2/lipase preferring phosphatidic acid,bis(monoacylglycerol) phosphate,and monoacylglycerol from rat testis

Phospholipase A(2) (PLA(2)) was purified to homogeneity from the supernatant fraction of rat testis homogenate. The purified 63-kDa enzyme did not require Ca(2+) ions for activity and exhibited both phosphatidic acid-preferring PLA(2) and monoacylglycerol lipase activities with a modest specificity toward unsaturated acyl chains. Anionic detergents enhanced these activities. Serine-modifying irreversible inhibitors, (p-amidinophenyl) methanesulfonyl fluoride and methylarachidonyl fluorophosphonate, inhibited both activities to a similar extent, indicating a single active site is involved in PLA(2) and lipase activities. The sequence of NH(2)-terminal 12 amino acids of purified enzyme was identical to that of a carboxylesterase from rat liver. The optimal pH for PLA(2) activity (around 5.5) differed from that for lipase activity (around 8.0). At pH 5.5 the enzyme also hydrolyzed bis(monoacylglycerol) phosphate, or lysobisphosphatidic acid (LBPA), that has been hitherto known as a secretory PLA(2)-resistant phospholipid and a late endosome marker. LBPA-enriched fractions were prepared from liver lysosome fractions of chloroquine-treated rats, treated with excess of pancreatic PLA(2), and then used for assaying LBPA-hydrolyzing activity. LBPA and the reaction products were identified by microbore normal phase high performance liquid chromatography/electrospray ionization ion-trap mass spectrometry. These enzymatic properties suggest that the enzyme can metabolize phosphatidic and lysobisphosphatidic acids in cellular acidic compartments.     

Purification and characterization of a lipase from the glycolipid-producing yeast Kurtzmanomyces sp. I-11

An extracellular lipase produced by the glycolipid-producing yeast Kurtzmanomyces sp. I-11 was purified by ammonium sulfate precipitation and column chromatographies on DEAE-Sephadex A-25, SP-Sephadex C-50, and Sephadex G-100. Based on the analysis of the purified lipase on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the purified lipase was judged to be homogeneous and its molecular mass was estimated to be approximately 49 kDa. The optimum temperature for the activity was 75 degrees C, and the activity was very stable at temperatures below 70 degrees C. The active pH range of this lipase was 1.9-7.2, and the activity was stable at pH below 7.1. The lipase showed a preference for C18 acyl groups by measurements with p-nitrophenyl esters and triglycerides as substrates. The lipase was very stable in the presence of various organic solvents at a concentration of 40%. Although the N-terminal sequence of the Kurtzmanomyces lipase was very similar to that of lipase A from Candida antarctica, the pH profiles of the two lipases were significantly different.     

Comparison of hydrolysis and esterification behavior of Humicola lanuginosa and Rhizomucor miehei lipases in AOT-stabilized water-in-oil microemulsions: I. Effect of pH and water content on reaction kinetics

Lipolase and Lipozyme are produced in large quantities (as a result of genetic engineering and overexpression) for the detergents market and provide a cheap source of highly active biocatalysts. Humicola lanuginosa lipase (HIL) and Rhizomucor miehei lipase (RmL) have been isolated in partially purified form from commercial preparations of Lipolase and Lipozyme, respectively. These lipases were solubilized in Aerosol-OT (AOT)-stabilized water-in-oil (w/o) microemulsions in n-heptane. HIL and RmL activity in these microemulsions was assayed by spectrophotometric measurement of the initial rate of p-nitophenyl butyrate hydrolysis, and by chromatographic determination of the initial rate of octyl decanoate synthesis from 1-octanol and decanoic acid. The hydrolytic activity of HIL in microemulsions measured as a function of buffer pH prior to dispersal, followed a sigmoidal profile with the highest activities observed at alkaline pHs. This broadly matches the pH-activity profile for tributyrin hydrolysis by Lipolase in an aqueous emulsion assay. The hydrolytic activity of RmL in the same microemulsions, measured as a function of pH, gave a bell-shaped profile with a maximum activity at pH 7.5. Again, the observed pH-activity profile was similar to that reported for a purified RmL in a tributyrin-based aqueous emulsion assay. In contrast, the esterification activity exhibited by both HIL and RmL in AOT microemulsions over the available range pH 6.1 to 10.4, decreases as the pH increases, most likely reflecting the effect of substrate ionization. The dependence of the hydrolytic and condensation activity of HIL on R, the mole ratio of water to surfactant, were similar with both profiles exhibiting a maximum at R = 5. The hydrolytic and esterification activities of RmL followed similar R-dependent profiles, but the profiles in this case exhibited a maximum at R = 10. The water activities at these R values were directly measured as 0.78 and 0.9, respectively. Measured water activities were unperturbed by the presence of lipase at the concentrations used in these studies. (c) 1995 John Wiley & Sons, Inc.     

A novel organic solvent tolerant lipase from Bacillus sphaericus 205y: extracellular expression of a novel OST-lipase gene

An organic solvent tolerant (OST) lipase gene from Bacillus sphaericus 205y was successfully expressed extracellularly. The expressed lipase was purified using two steps purification; ultrafiltration and hydrophobic interaction chromatography (HIC) to 8-fold purity and 32% recovery. The purified 205y lipase revealed homogeneity on denaturing gel electrophoresis and the molecular mass was at approximately 30 kDa. The optimum pH for the purified 205y lipase was 7.0-8.0 and its stability showed a broad range of pH value between pH 5.0 to 13.0 at 37 degrees C. The purified 205y lipase exhibited an optimum temperature of 55 degrees C. The activity of the purified lipase was stimulated in the presence of Ca2+ and Mg2+. Ethylenediaminetetraacetic acid (EDTA) has no effect on its activity; however inhibition was observed with phenylmethane sulfonoyl fluoride (PMSF) a serine hydrolase inhibitor. Organic solvents such as dimethylsulfoxide (DMSO), methanol, p-xylene and n-decane enhanced the activity. Studies on the effect of oil showed that the lipase was most active in the presence of tricaprin (C10). The lipase exhibited 1,3 positional specificity. Keywords: Bacter