Immobilization of Yarrowia lipolytica Lipase—a Comparison of Stability of Physical Adsorption and Covalent Attachment Techniques

Lipase immobilization offers unique advantages in terms of better process control, enhanced stability, predictable decay rates and improved economics. This work evaluated the immobilization of a highly active Yarrowia lipolytica lipase (YLL) by physical adsorption and covalent attachment. The enzyme was adsorbed on octyl–agarose and octadecyl–sepabeads supports by hydrophobic adsorption at low ionic strength and on MANAE–agarose support by ionic adsorption. CNBr–agarose was used as support for the covalent attachment immobilization. Immobilization yields of 71, 90 and 97% were obtained when Y. lipolytica lipase was immobilized into octyl–agarose, octadecyl–sepabeads and MANAE–agarose, respectively. However, the activity retention was lower (34% for octyl–agarose, 50% for octadecyl–sepabeads and 61% for MANAE–agarose), indicating that the immobilized lipase lost activity during immobilization procedures. Furthermore, immobilization by covalent attachment led to complete enzyme inactivation. Thermal deactivation was studied at a temperature range from 25 to 45°C and pH varying from 5.0 to 9.0 and revealed that the hydrophobic adsorption on octadecyl–sepabeads produced an appreciable stabilization of the biocatalyst. The octadecyl–sepabeads biocatalyst was almost tenfold more stable than free lipase, and its thermal deactivation profile was also modified. On the other hand, the Y. lipolytica lipase immobilized on octyl–agarose and MANAE–agarose supports presented low stability, even less than the free enzyme.     

Immobilization of Pseudomonas fluorescens Lipase onto Magnetic Nanoparticles for Resolution of 2-Octanol

The lipase from Pseudomonas fluorescens (Lipase AK, AKL) was immobilized onto the magnetic Fe₃O₄ nanoparticles via hydrophobic interaction. Enzyme loading and immobilization yield were determined as 21.4?±?0.5?mg/g and 49.2?±?1.8?%, respectively. The immobilized AKL was successfully used for resolution of 2-octanol with vinyl acetate used as acyl donor. Effects of organic solvent, water activity, substrate ratio, and temperature were investigated. Under the optimum conditions, the preferred isomer for AKL is the (R)-2-octanol and the highest enantioselectivity (E?=?71.5?±?2.2) was obtained with a higher enzyme activity (0.197?±?0.01???mol/mg/min). The results also showed that the immobilized lipase could be easily separated from reaction media by the magnetic steel and remained 89?% of its initial activity as well as the nearly unchanged enantioselectivity after five consecutive cycles, indicating a high stability in practical operation.     

Expression and Characterization of a Novel Enantioselective Lipase from Aspergillus fumigatus

A 1,080-bp cDNA (CGMCC 2873) encoding of a cold-active lipase of Aspergillus fumigatus (AFL67) was cloned and expressed in Escherichia coli for the first time. The new lipase, AFL67, was one-step purified by 8.30 folds through Ni?CNTA affinity chromatography with a recovery of 86.8?%. The specific activity of purified AFL67 was 449?U?mg^?1 on p-NP hexanoate. AFL67 preferentially hydrolyzed p-nitrophenyl esters of short- and medium-chain fatty acids, with p-nitrophenyl hexanoate the maximum. The optimum temperature and pH was 15?°C and 7.5, respectively. The purified AFL67 was stable at 10?C25?°C for 30?min, and in the pH range of 6.0?C9.0 for 16?h (at 4?°C). Its activity was increased by 47 and 50?%, in the presence of 10?% (v/v) ethanol and isopropanol, respectively. The new lipase AFL67 highly enantioselectively deacylated (S)-??-acetoxyphenylacetic acid (APA) and o-Cl-APA, m-Cl-APA, and p-Cl-APA to (S)-mandelic acid and its derivates. These features render this cold-active novel lipase AFL67 attractive for biotechnological applications in the field of enantioselective synthesis of chiral mandelic acids, o-acylated mandelic acids, and their derivates and detergent additives.     

Synthesis of SBA-15 from low cost silica precursor obtained from sugarcane leaf ash and its application as a support matrix for lipase in biodiesel production

Ordered mesoporous silica material was synthesized from a low-cost precursor, sugarcane leaf ash, was used as a support matrix for lipase for the production of biodiesel. The mesoporous samples were characterized using Fourier transform infra red spectroscopy. The surface topography and morphology of the mesoporous materials were studied using scanning electron microscope. The pore diameter, pore volume, Brunauer Emmett and Teller surface area of the mesoporous material were determined by N₂ gas adsorption technique. Different pore size Santa Barbara Acid-15 (SBA-15) samples were synthesized and their lipase immobilization capacity and specific enzyme activity of immobilization lipase were determined and compared. Lipase from Candida Antarctica immobilized on SBA-15 (C) had shown maximum percentage immobilization and specific enzyme activity. The immobilized lipase mesoporous matrix was used for biodiesel production from crude non-edible Calophyllum inophyllum oil. The percentage yield of fatty acid methyl ester, 97.6 % was obtained under optimized conditions: 100 mg of lipase immobilized on SBA-15, 6:1 methanol to oil molar ratio, the reaction of 2 g C. inophyllum oil with methanol.     

An Enhanced Process for the Production of a Highly Purified Extracellular Lipase in the Non-conventional Yeast <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis>

Yarrowia lipolytica LgX64.81 is a non-genetically modified mutant that was previously identified as a promising microorganism for extracellular lipase production. In this work, the development of a fed-batch process for the production of this enzyme in this strain was described. A lipolytic activity of 2,145 U/mL was obtained after 32 h of batch culture in a defined medium supplemented with 10 g/L of tryptone, an enhancer of lipase expression. To maximize the volumetric productivity, two different fed-batch strategies had been investigated. In comparison to batch process, the intermittent fed-batch strategy had not improved the volumetric lipase productivity. In contrast, the stepwise feeding strategy combined with uncoupled cell growth and lipase production phases resulted in a 2-fold increase in the volumetric lipase productivity, namely, the lipase activity reached 10,000 U/mL after 80 h of culture. Furthermore, this lipase was purified to homogeneity by anion exchange chromatography on MonoQ resin followed by gel filtration on Sephacryl S-100. This process resulted in an overall yield of 72% and a 3.5-fold increase of the specific lipase activity. The developed process offers a great potential for an economic production of Lip2 at large scale in Y. lipolytica LgX64.81.     

Triacylglycerol lipase from rape (Brassica napus L.) suitable for biotechnological purposes

Triacylglycerol lipase (EC 3.1.1.3) from rape (Brassica napus L. cv Ceres) is quite easily prepared from the 100,000 x g supernatant of cotyledon homogenates. The lipase is present in a high-molecular-mass fraction ( > 1.5 x 10⁶ dalton by gel filtration), but it can be rapidly extracted from the 100,000 xg supernatant by precipitation with polyethyleneglycol 8000 (4%, w/v) and MgCl₂ (40 mM) giving about a 10-fold purification. After delipidation, the lipase has an M_r of about 300,000. It hydrolyzes triacylglycerols to fatty acids and glycerol, although the fatty acids from the sn-1 or -3 positions are hydrolyzed first to yield 1,2(2,3)-diaclyglycerols. Lipase immobilized onto Celite by precipitation with acetone at-20°C catalyzes the esterification of oleic acid with butanol dissolved in hexane.     

Activation and Stabilization of Olive Recombinant 13-Hydroperoxide Lyase Using Selected Additives

The stabilization of olive recombinant hydroperoxide lyases (rHPLs) was investigated using selected chemical additives. Two rHPLs were studied: HPL full-length and HPL with its chloroplast transit peptide deleted (matured HPL). Both olive rHPLs are relatively stable at 4 °C, and enzyme activity can be preserved (about 100% of the rHPL activities are maintained) during 5 weeks of storage at ?20 or at ?80 °C in the presence of glycerol (10%, v/v). Among the additives used in this study, glycine (2.5% w/v), NaCl (0.5 M), and Na₂SO₄ (0.25 M) provided the highest activation of HPL full-length activity, while the best matured HPL activity was obtained with Na₂SO₄ (0.25 M) and NaCl (1 M). Although the inactivation rate constants (k) showed that these additives inactivate both rHPLs, their use is still relevant as they strongly increase HPL activity. Results of C6-aldehyde production assays also showed that glycine, NaCl, and Na₂SO₄ are appropriate additives and that NaCl appears to be the best additive, at least for hexanal production.     

Physical and Covalent Immobilization of <Emphasis Type="Italic">Lipase</Emphasis> onto Amine Groups Bearing Thiol-Ene Photocured Coatings

In this study, amine groups containing thiol-ene photocurable coating material for lipase immobilization were prepared. Lipase (EC 3.1.1.3) from Candida rugosa was immobilized onto the photocured coatings by physical adsorption and glutaraldehyde-activated covalent bonding methods, respectively. The catalytic efficiency of the immobilized and free enzymes was determined for the hydrolysis of p-nitrophenyl palmitate and also for the synthesis of p-nitrophenyl linoleate. The storage stability and the reusability of the immobilized enzyme and the effect of temperature and pH on the catalytic activities were also investigated. The optimum pH for free lipase and physically immobilized lipase was determined as 7.0, while it was found as 7.5 for the covalent immobilization. After immobilization, the optimum temperature increased from 37 °C (free lipase) to 50–55 °C. In the end of 15 repeated cycles, covalently bounded enzyme retained 60 and 70 % of its initial activities for hydrolytic and synthetic assays, respectively. While the physically bounded enzyme retained only 56 % of its hydrolytic activity and 67 % of its synthetic activity in the same cycle period. In the case of hydrolysis V _max values slightly decreased after immobilization. For synthetic assay, the V _max value for the covalently immobilized lipase was found as same as free lipase while it decreased dramatically for the physically immobilized lipase. Physically immobilized enzyme was found to be superior over covalent bonding in terms of enzyme loading capacity and optimum temperature and exhibited comparable re-use values and storage stability. Thus, a fast, easy, and less laborious method for lipase immobilization was developed.     

Characterization of a New Cold-adapted Lipase from Pseudomonas sp. TK-3

A psychrotrophic Pseudomonas sp. TK-3 was isolated from dirty and cool stream water in Toyama, Japan from which we cloned and characterized the bacterial lipase LipTK-3. The sequenced DNA fragment contains an open reading frame of 1,428?bp that encoded a protein of 476 amino acids with an estimated molecular mass of 50,132?Da. The lipase showed high sequence similarity to those of subfamily ??.3 lipase and had a conserved GXSXG motif around the catalytic Ser residue. Its optimal temperature was 20?C25?°C, lower than in most other subfamily ??.3 lipases. The lipase exhibited about 30?% of maximal activity at 5?°C. The optimal pH value was 8.0. The activity was strongly inhibited by EDTA and was highly dependent on Ca²⁺. Tricaprylin and p-nitrophenyl caprylate were the most favorable substrates among the triglycerides and p-nitrophenyl esters, respectively. LipTK-3 also showed high activity towards natural substrates including edible vegetable oils and animal fats. Furthermore, LipTK-3 was very active and stable in the presence of several detergents, metal ions, and organic solvents. This cold-adapted lipase may prove useful for future applications.     

Lipase immobilized on poly(VP-co-HEMA) hydrogel for esterification reaction

Lipase from Candida rugosa was immobilized by entrapment on poly(N-vinyl-2-pyrrolidone-co-2-hydroxyethyl methacrylate)(poly[VP-co-HEMA]) hydrogel, and divinylbenzene was the crosslinking agent. The immobilized enzymes were used in the esterification reaction of oleic acid and butanol in hexane. The activities of the immobilized enzymes and the leaching ability of the enzyme from the support with respect to the different compositions of the hydrogels were investigated. The thermal, solvent, and storage stability of the immobilized lipases was also determined. Increasing the percentage of composition of VP from 0 to 90, which corresponds to the increase in the hydrophilicity of the hydrogels, increased the activity of the immobilized enzyme. Lipase immobilized on VP(%):HEMA(%) 90∶10 exhibited the highest activity. Lipase immobilized on VP(%):HEMA(%) 50∶50 showed the highest thermal, solvent, storage, and operational stability compared to lipase immobilized on other compositions of hydrogels as well as the native lipase.     

Enantioselective enzymatic resolution of racemic alcohols by lipases in green organic solvents

The effects of two eco-friendly solvents, 2-methyltetrahydrofuran (MeTHF) and cyclopentyl methyl ether (CPME), on the enzyme activity and enantioselectivity of Novozym 435, Candida rugosa lipase (CRL), Porcine pancreas lipase (PPL), Lipase AK, Lipase PS, and Lipozyme, a series of commercial lipases, in the enantioselective transesterfications of racemic menthol, racemic sulcatol and racemic α-cyclogeraniol were studied. Vinyl acetate was chosen as the acyl donor and the reactions were carried out at water activity 0.06. The activity of lipases in CPME was similar to that observed in other largely employed organic solvents [toluene and tert-butyl methyl ether (MTBE)], and was slightly lower in MeTHF. However, for most of the lipases tested, the enantioselectivity was higher in the eco-friendly solvents. Lipase AK exhibited a high enantioselectivity (E = 232) for the resolution of racemic menthol but the reaction rate was low. Lipase formulation (the enzyme was frozen and lyophilized in potassium phosphate buffer without and with 5% (w/v) of sucrose, d-mannitol, or methoxy poly(ethylene glycol)) was tested with this lipase in order to improve its activity, which increased up to 4.5 times, compared to the untreated enzyme. CALB was found to be a useful biocatalyst for the resolution of racemic sulcatol, where high activity and enantioselectivity were obtained (E ≥ 1000). For the resolution of the racemic primary alcohol α-cyclogeraniol, most of the lipases tested were active but not enantioselective, except lipase PS which displayed a moderate enantioselectivity (E = 19). The effect of the presence of a low percentage of two ionic liquids (ILs) 1-Butyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide ([BMIM][TFSI]) (5% (v/v)) and 1-Butyl-3-methylimidazoliumtetrafluoroborate ([BMIM][BF4]) (1% (v/v)) in the medium was also investigated. Only in the case of CRL the ILs slightly increased the enantioselectivity from E = 91 to E = 103 and E = 120 for [BMIM][TFSI] and [BMIM][BF4], respectively. However, in all cases ILs caused a decrease of enzyme activity.     

Activation and Stabilization of The Hydroperoxide Lyase Enzymatic Extract from Mint Leaves (Mentha spicata) Using Selected Chemical Additives

The effects of selected lyoprotecting excipients and chemical additives on the specific activity and the thermal stability of the hydroperoxide lyase (HPL) enzymatic extract from mint leaves were investigated. The addition of KCl (5%, w/w) and dextran (2.5%, w/w) to the enzymatic extract, prior to lyophilization, increased the HPL specific activity by 2.0- and 1.2-fold, respectively, compared to the control lyophilized extract. From half-life time (t _1/2), it can be seen that KCl has enhanced the HPL stability by 1.3- to 2.3-fold, during long-period storage at ?20 °C and 4 °C. Among the selected additives used throughout this study, glycine appeared to be the most effective one. In addition to the activation effect conferred by glycine, it also enhanced the HPL thermal stability. In contrast, polyhydroxyl-containing additives were not effective for stabilizing the HPL enzymatic extract. On the other hand, there was no signification increase in HPL activity and its thermal stability with the presence of Triton X-100. The results also showed that in the presence of glycine (10%), the catalytic efficiency of HPL was increased by 2.45-fold than that without additive.     

Comparison of <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis> Lipase Immobilization Yield of Entrapment,Adsorption, and Covalent Bond Techniques

The purpose of this study was to immobilize lipase from Yarrowia lipolytica using three methods including inclusion, adsorption, and covalent bond to study enzyme leaching, storage, and catalytic properties. Sodium alginate and chitosan were the polymers selected to immobilize lipase by inclusion. The beads of each polymer were dried by freeze drying and fluidization. The results show that chitosan was more adapted to the inclusion of lipase. Even though freeze dried, bead activity was low compared to that of fluidized beads. The freeze-drying process seems to produce suitable beads for storage at 4 and 20 degrees C. The immobilization by adsorption was carried out on both celite and silica gel. Maximum immobilization yield of 76% was obtained with celite followed by 43% in silica gel. The enzyme adsorbed on the two supports exhibited greater stability at a certain temperature (50 degrees C) and in no polar solvents (Isooctane, n-heptane, and n-hexane). In addition, the lipase immobilized by covalent bond retained residual activity equitable to 70%. It was demonstrated that the enzyme immobilized by covalent bond showed greater activity (80%) after 5 months of storage.     

Immobilized Lipase from Candida sp. 99–125 on Hydrophobic Silicate: Characterization and Applications

Lipase Candida sp. 99–125 has been proved to be quite effective in catalyzing organic synthesis reactions and is much cheaper than commercial lipases. Mesoporous silicates are attractive materials for the immobilization of enzymes due to their unique structures. The present research designed a hydrophobic silicate with uniform pore size suitable for the comfort of lipase Candida sp. 99–125 for improving its activity and stability. The resulting immobilized lipase (LP@PMO) by adsorption was employed to catalyze hydrolysis, esterification, and transesterification reactions, and the performances were compared with the lipase immobilized on hydrophilic silicate (LP@PMS) and native lipase. The LP@PMO showed as high activity as that of native lipase in hydrolysis and much increased catalytic activity and reusability in the reactions for biodiesel production. Besides, LP@PMO also possessed better organic stability. Such results demonstrate that immobilization of lipase onto hydrophobic supports is a promising strategy to fabricate highly active and stable biocatalysts for applications.     

Preparation and characterization of stable chitosan nanofibrous membrane for lipase immobilization

Nanofibrous membrane with a fiber diameter of 80-150 nm was fabricated from mixed chitosan/poly(vinyl alcohol) (PVA) solution by an electrospinning process. Field emission scanning electron microscope and transmission electron microscope were used to characterize the morphology of the nanofibrous membrane. It was found that chitosan nanofibrous membrane with stabilized morphology could be prepared through removing most of PVA from the nascent one with 0.5 M NaOH aqueous solution. This treatment also resulted in an obvious decrease in fiber diameter. The stabilized chitosan nanofibrous membrane was explored as support for enzyme immobilization due to the characteristics of excellent biocompatibility, high surface/volume ratio, and large porosity. Lipase from Candida rugosa was immobilized on the nanofibrous membrane using glutaraldehyde (GA) as coupling reagent. The properties of the immobilized lipase were assayed and compared with the free one. Results showed that, the observed lipase loading on this nanofibrous membrane was up to 63.6 mg/g and the activity retention of the immobilized lipase was 49.8% under the optimum condition. The pH and thermal stabilities of lipase were improved after it was immobilized on the chitosan nanofibrous membrane. In addition, the experimental results of reusability and storage stability indicated that the residual activities of the immobilized lipase were 46% after 10 cycles and 56.2% after 30 days, which were obviously higher than that of the free one.     

Production and Characterization of a Halo-, Solvent-, Thermo-tolerant Alkaline Lipase by Staphylococcus arlettae JPBW-1, Isolated from Rock Salt Mine

Studies on lipase production and characterization were carried out with a bacterial strain Staphylococcus arlettae JPBW-1 isolated from rock salt mine, Darang, HP, India. Higher lipase activity has been obtained using 10 % inoculum with 5 % of soybean oil as carbon source utilizing a pH 8.0 in 3 h at 35 °C and 100 rpm through submerged fermentation. Partially purified S. arlettae lipase has been found to be active over a broad range of temperature (30–90 °C), pH (7.0–12.0) and NaCl concentration (0–20 %). It has shown extreme stability with solvents such as benzene, xylene, n-hexane, methanol, ethanol and toluene up to 30 % (v/v). The lipase activity has been found to be inhibited by metal ions of K⁺, Co²⁺ and Fe ²⁺ and stimulated by Mn²⁺, Ca²⁺ and Hg²⁺. Lipase activity has been diminished with denaturants, but enhanced effect has been observed with surfactants, such as Tween 80, Tween 40 and chelator EDTA. The K _m and V _max values were found to be 7.05 mM and 2.67 mmol/min, respectively. Thus, the lipase from S. arlettae may have considerable potential for industrial application from the perspectives of its tolerance towards industrial extreme conditions of pH, temperature, salt and solvent.     

Screening,isolation and selection of a potent lipase producing microorganism and its use in the kinetic resolution of drug intermediates

Lipases are ubiquitous enzymes that belong to family of serine hydolases with a wide variety of industrial applications. This study reports isolation, screening and identification of enantioselctive lipase producing microorganism for kinetic resolution of racemic alcohols. For this, we collected soil samples from different oil rich environments and we performed primary screening that was by carried out by using MSM-tributryin clear zone assay. The selected samples from first screen were subjected to secondary screening to distinguish lipase producing strains from esterase producing strains using p-nitrophenyl palmitate lipase assay. In tertiary screening, 16 lipase producing strains that were identified in secondary screening were employed for resolution of 5 different (RS)-alcohols. Out of all 16 lipase producing strains, only one strain selectively converted 3 racemic alcohols. Based on morphological, biochemical and physiological characteristics, and 16S rRNA gene sequencing, the strain was identified as Pseudomonas beteli. The strain was found to be S-selective and there been no reports on use of Pseudomonas beteli lipase for kinetic resolution of alcohols. The lipase activity was further increased by media optimization and by improving growth conditions, and production of lipase in shake flask study as well as in laboratory scale fermenter. The optimum time for enzyme production by Pseudomonas beteli was 96 ​h whereas cell mass growth was highest at 72 ​h. Optimum temperature and pH were 30 ​°C and 6, respectively. Beef extract (5 ​g/L), peptone (5 ​g/L), sodium chloride (5 ​g/L), yeast extract (1 ​g/L) and glucose (5 ​g/L) were found as optimum nutrition sources for the cell mass growth and lipase production by Pseudomonas sp. Overall, 3.4 times higher enzyme activity and 2.75 times higher cell mass growth were achieved in bioreactor in comparison to the shake flask study. Lipase having high titer was employed successfully for the kinetic resolution of several drug intermediates.     

Direct Conversion of Pretreated Straw Cellulose into Citric Acid by Co-cultures of Yarrowia lipolytica SWJ-1b and Immobilized Trichoderma reesei Mycelium

The immobilized cellulase-producing mycelium of Trichoderma reesei was found to produce 2.9 U/ml of cellulase activity within 144 h while 2.1 U/ml of cellulase activity was produced within 120 h by the free mycelium of the same strain. When the immobilized mycelium of T. reesei was co-cultivated with the free cells of Yarrowia lipolytica SWJ-1b in flask, Y. lipolytica SWJ-1b could yield 10.7 g/l of citric acid and 3.9 g/l of isocitric acid from 40.0 g/l pretreated straw within 240 h. Under the similar conditions, Y. lipolytica SWJ-1b could yield 32.8 g/l of citric acid and 4.7 g/l of isocitric acid from 40.0 g/l pretreated straw supplemented with 20.0 g/l glucose within 288 h. When the co-cultures were grown in 10-l fermentor, Y. lipolytica SWJ-1b could yield 83.4 g/l of citric acid and 8.7 g/l of isocitric acid from 100.0 g/l of pretreated straw supplemented with 50.0 g/l glucose within 312 h.     

Lipase Production in Solid-State Fermentation Monitoring Biomass Growth of Aspergillus niger Using Digital Image Processing

The aim of this study was to monitor the biomass growth of Aspergillus niger in solid-state fermentation (SSF) for lipase production using digital image processing technique. The strain A. niger 11T53A14 was cultivated in SSF using wheat bran as support, which was enriched with 0.91% (m/v) of ammonium sulfate. The addition of several vegetable oils (castor, soybean, olive, corn, and palm oils) was investigated to enhance lipase production. The maximum lipase activity was obtained using 2% (m/m) castor oil. In these conditions, the growth was evaluated each 24 h for 5 days by the glycosamine content analysis and digital image processing. Lipase activity was also determined. The results indicated that the digital image process technique can be used to monitor biomass growth in a SSF process and to correlate biomass growth and enzyme activity. In addition, the immobilized esterification lipase activity was determined for the butyl oleate synthesis, with and without 50% v/v hexane, resulting in 650 and 120 U/g, respectively. The enzyme was also used for transesterification of soybean oil and ethanol with maximum yield of 2.4%, after 30 min of reaction.     

Transesterification of Waste Cooking Oil by an Organic Solvent-Tolerant Alkaline Lipase from Streptomyces sp. CS273

The aim of this present study was to produce a microbial enzyme that can potentially be utilized for the enzymatic transesterification of waste cooking oil. To that end, an extracellular lipase was isolated and purified from the culture broth of Streptomyces sp. CS273. The molecular mass of purified lipase was estimated to be 36.55 kDa by SDS PAGE. The optimum lipolytic activity was obtained at alkaline pH 8.0 to 8.5 and temperature 40 °C, while the enzyme was stable in the pH range 7.0?～?9.0 and at temperature ≤40 °C. The lipase showed highest hydrolytic activity towards p-nitrophenyl myristate (C14). The lipase activity was enhanced by several salts and detergents including NaCl, MnSo₄, and deoxy cholic acid, while phenylmethylsulfonyl fluoride at concentration 10 mM inhibited the activity. The lipase showed tolerance towards different organic solvents including ethanol and methanol which are commonly used in transesterification reactions to displace alcohol from triglycerides (ester) contained in renewable resources to yield fatty acid alkyl esters known as biodiesel. Applicability of the lipase in transesterification of waste cooking oil was confirmed by gas chromatography mass spectrometry analysis.