Extracellular cold-active lipase of Microbacterium luteolum isolated from Gangotri glacier,western Himalaya: Isolation,partial purification and characterization

A psychrophilic bacterium producing cold-active lipase upon growth at low temperature was isolated from the soil samples of Gangotri glacier and identified as Microbacterium luteolum. The bacterial strain produced maximum lipase at 15 °C, at a pH of 8.0. Beef extract served as the best organic nitrogen source and ammonium nitrate as inorganic for maximum lipase production. Castor oil served as an inducer and glucose served as an additional carbon source for production of cold-active lipase. Ferric chloride as additional mineral salt in the medium, highly influenced the lipase production with an activity of 8.01 U ml^?1. The cold-active lipase was purified to 35.64-fold by DEAE-cellulose column chromatography. It showed maximum activity at 5 °C and thermostability up to 35 °C. The purified lipase was stable between pH 5 and 9 and the optimal pH for enzymatic hydrolysis was 8.0. Lipase activity was stimulated in presence of all the solvents (5%) tested except with acetonitrile. Lipase activity was inhibited in presence of Mn²⁺, Cu²⁺, and Hg²⁺; whereas Fe⁺, Na⁺ did not have any inhibitory effect on the enzyme activity. The purified lipase was stable in the presence of SDS; however, EDTA and dithiothreitol inhibited enzyme activity. Presence of Ca²⁺ along with inhibitors stabilized lipase activity. The cold active lipase thus exhibiting activity and stability at a low temperature and alkaline pH appears to be practically useful in industrial applications especially in detergent formulations.     

Purification and properties of enantioselective lipase from a newly isolated Bacillus cereus C71

A lipase from Bacillus cereus C71 was purified to homogeneity by ammonium sulfate precipitation, followed by Phenyl-Sepharose chromatography, DEAE ion exchange chromatography and CIM^® QA chromatography. This purification procedure resulted in a 1092-fold purification of lipase with 18% yield. The molecular mass of the purified enzyme was determined to be approximately 42 kDa by SDS-PAGE and mass spectrometer. The lipase was stable in the pH range of 8.5–10.0, with the optimum pH 9.0. The enzyme exhibited maximum activity at 33 °C and retained 92% of original activity after incubation at 35 °C for 3 h. The protein hydrolyzed p-nitrophenyl esters with acyl chain lengths between C4 and C12. Enzyme activity was strongly inhibited in the presence of Cu²⁺ and Zn²⁺ but promoted by non-ionic surfactants. The lipase demonstrated higher enantioselectivity toward R-isomer of ethyl 2-arylpropanoate than the commercial lipases, and can be used potentially as a catalyst to prepare optically pure pharmaceuticals.     

Thermophilic lipase from Thermomyces lanuginosus: Gene cloning,expression and characterization

An extracellular lipase gene ln1 from thermophilic fungus Thermomyces lanuginosus HSAUP₀₃80006 was cloned through RT-PCR and RACE amplification. Its coding sequence predicted a 292 residues protein with a 17 amino acids signal peptide. The deduced amino acids showed 78.4% similarity to another lipase lgy from T. lanuginosus while shared low similarity with other fungi lipases. Higher frequencies hydrophobic amino acids related to lipase thermal stability, such as Ala, Val, Leu and Gly were observed in this lipase (named LN). The sequence, -Gly-His-Ser-Leu-Gly-, known as a lipase-specific consensus sequence of mould, was also found in LN. High level expression for recombinant lipase was achieved in Pichia pastoris GS115 under the control of strong AOX1 promoter. It was purified to homogeneity through only one step DEAE-Sepharose anion exchange chromatography and got activity of 1328 U/ml. The molecular mass of one single band of this lipase was estimated to be 33 kDa by SDS-PAGE. The enzyme was stable at 60 °C and kept 65% enzyme activity after 30 min incubation at 70 °C. It kept half-activity after incubated for 40 min at 80 °C. The optimum pH for enzyme activity was 9.0 and the lipase was stable from pH 8.0 to 12.0. Lipase activity was enhanced by Ca²⁺ and inhibited by Fe²⁺, Zn²⁺, K⁺, and Ag⁺. The cell-free enzyme hydrolyzed and synthesized esters efficiently, and the synthetic efficiency even reached 81.5%. The physicochemical and catalytic properties of the lipase are extensively investigated for its potential industrial applications.     

Optimization of culture conditions and properties of lipase from Penicillium camembertii Thom PG-3

The culture medium including nitrogen source, carbon source and metal ions, for lipase from Penicillium camembertii Thom PG-3 was optimized and the optimal medium consisted of soybean meal (fat free) 4%, Jojoba oil 0.5%, (NH₄)₂HPO₄, 0.1% Tween 60, initial pH 6.4 and the inoculation was at 28 °C for 96 h. The lipase activity produced was enhanced 3.9-fold and reached 500 U/ml. The lipase was purified 19.8-fold by pH precipitation, ethanol precipitation and ammonium sulphate precipitation as well as DEAE-cellulose chromatography. The purified lipase showed one polypeptide band in SDS-polyacrylamide gel electrophoreses (SDS-PAGE) with molecular weight 28.18 kDa. The optimal pH and temperature for activity of lipase were 6.4 and 48 °C, respectively, which are higher than those lipases from other penicillium sources. The P. camembertii Thom lipase is 1,3-positional specificity for hydrolysis of triglyceride and hydrolyses plant oil preferentially to animal oil. The lipase can be used in short chain ester synthesis with an esterification degree of 95%.     

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.     

Immobilization of acidic lipase derived from Pseudomonas gessardii onto mesoporous activated carbon for the hydrolysis of olive oil

Mesoporous activated carbon (MAC) derived from rice husk is used for the immobilization of acidic lipase (ALIP) produced from Pseudomonas gessardii. The purified acidic lipase had the specific activity and molecular weight of 1473 U/mg and 94 kDa respectively. To determine the optimum conditions for the immobilization of lipase onto MAC, the experiments were carried out by varying the time (10–180 min), pH (2–8), temperature (10–50 °C) and the initial lipase activity (49 × 10³, 98 × 10³, 147 × 10³ and 196 × 10³ U/l in acetate buffer). The optimum conditions for immobilization of acidic lipase were found to be: time—120 min; pH 3.5; temperature—30 °C, which resulted in achieving a maximum immobilization of 1834 U/g. The thermal stability of the immobilized lipase was comparatively higher than that in its free form. The free and immobilized enzyme kinetic parameters (K_m and V_max) were found using Michaelis–Menten enzyme kinetics. The K_m values for free enzyme and immobilized one were 0.655 and 0.243 mM respectively. The immobilization of acidic lipase onto MAC was confirmed using Fourier Transform-Infrared Spectroscopy, X-ray diffraction analysis and scanning electron microscopy.     

Purification and characterization of SDG-β-d-glucosidase hydrolyzing secoisolariciresinol diglucoside to secoisolariciresinol from Aspergillus oryzae

The SDG-β-d-glucosidase that hydrolyzes the glucopyranoside bond of secoisolariciresinol diglucoside (SDG) to release secoisolariciresinol (SECO) was isolated from Aspergillus oryzae 39 strain and the enzyme was purified and characterized. The enzyme was purified to one spot in SDS polyacrylamide gel electrophoresis, and its molecular weight was about 64.9 kDa. The optimum temperature of the SDG-β-d-glucosidase was 40 °C, and the optimum pH was 5.0. The SDG-β-d-glucosidase was stable at less than 65 °C, and pH 4.0–6.0. Ca²⁺, K⁺, Mg²⁺ and Na⁺ ions have no significant effect on enzyme activity, Zn²⁺ and Cu²⁺ ions have weakly effect on enzyme activity, but Fe³⁺ ion inhibits enzyme activity strongly. The K_m value of SDG-β-d-glucosidase was 0.14 mM for SDG.     

Purification,characterization and application of acidic lipase from Pseudomonas gessardii using beef tallow as a substrate for fats and oil hydrolysis

Beef tallow, a slaughter house waste was used as a substrate for lipase production, employing Pseudomonas gessardii. The strain, P. gessardii was isolated from the beef tallow acclimatized soil. The crude lipase activity at 139 U/ml by volume was obtained at optimized conditions of pH 5.0 and temperature of 37 °C. After purification, a 7.59-fold purity of lipase with specific activity of 1120 U/mg protein and molecular mass of 92 kDa was obtained. The purified lipase showed maximum activity and stability at pH 5.0 and 30 °C. Ca²⁺ had a stimulatory effect on the lipase activity compared to the other metal ions studied. The relative activity was enhanced with the addition of Triton X-100 with lower hydrophilic–lipophilic balance (HLB) value as 13.0 and DMSO with the lowest partition coefficient (log P) value, as 1.378. The amino acid composition and the functional groups of lipase were confirmed by HPLC and FT-IR spectroscopy. The purified lipase had the highest hydrolytic activity towards slaughterhouse wastes and vegetable oils. This work provides a potential biocatalyst for the wide applications in oleochemical and biotechnological industries.     

An organic solvent and thermally stable lipase from Burkholderia ambifaria YCJ01: Purification,characteristics and application for chiral resolution of mandelic acid

A solvent-tolerant bacterium Burkholderia ambifaria YCJ01 was newly isolated by DMSO enrichment of the medium. The lipase from the strain YCJ01 was purified to homogeneity with apparent molecular mass of 34 kDa determined by SDS-PAGE. The purified lipase exhibited maximal activity at a temperature of 60 °C and a pH of 7.5. The lipase was very stable below 55 °C for 7 days (remaining 80.3% initial activity) or at 30 °C for 60 days. PMSF significantly inhibited the lipase activity, while EDTA had no effect on the activity. Strikingly, the lipase showed distinct super-stability to the most tested hydrophilic and hydrophobic solvents (25%, v/v) for 60 days, and different optimal pH in contrast with the alkaline lipase from B. cepacia S31. The lipase demonstrated excellent enantioselective transesterification toward the S-isomer of mandelic acid with a theoretical conversion yield of 50%, ee_p of 99.9% and ee_s of 99.9%, which made it an exploitable biocatalyst for organic synthesis and pharmaceutical industries.     

Evaluating aqueous two-phase systems for Yarrowia lipolytica extracellular lipase purification

In this study an aqueous two-phase system (ATPS) composed of polyethylene glycol (PEG) and potassium phosphate was tested for the purification of lipase from Yarrowia lipolytica IMUFRJ 50682. Ultrafiltration and precipitation with acetone and kaolin were also used as traditional comparison methods Ultrafiltration was a good method with a purification factor of 6.55, but protease was also purified in this extract. For the precipitation with acetone and kaolin lower values of lipase and protease activity were found in relation to the original crude enzyme extract. Under the best conditions of ATPS (pH 6 and 4 °C), the purification fold was greater than 40 and selectivity was almost 500. Lipase was recovered in the salty phase which makes it easier to purify it. The optimum pH and temperature ranges for purified lipase with this system was 6–7 and 35–40 °C, respectively. Lipase thermostability was increased in relation to crude extract after the purification with the PEG/phosphate buffer system for temperatures lower than 50 °C. All enzyme extracts showed good stability to a wide pH range. Y. lipolytca lipase was successfully purified by using ATPS in a single downstream processing step and presented good process characteristics after this treatment.     

Characterization of a spore surface lipase from the biocontrol agent Metarhizium anisopliae

A Metarhizium anisopliae spore surface lipase (MASSL) strongly bound to the fungal spore surface has been purified by ion exchange chromatography on DEAE sepharose followed by ultrafiltration and hydrophobic interaction chromatography on phenyl sepharose. Electrophoretic analyses showed that the molecular weight of this lipase is ～66 kDa and pI is 5.6. Protein sequencing revealed that identified peptides in MASSL shared identity with several lipases or lipase-related sequences. The enzyme was able to hydrolyze triolein, the animal lipid cholesteryl stearate and all ρNP ester substrates tested with some preference for esters with a short acyl chain. The values of K_m and V_max for the substrates ρNP palmitate and ρNP laurate were respectively 0.474 mM and 1.093 mMol min^?1 mg^?1 and 0.712 mM and 5.696 mMol min^?1 mg^?1. The optimum temperature of the purified lipase was 30 °C and the enzyme was most stable within the most acid pH range (pH 3–6). Triton X-100 increased and SDS reduced enzyme lipolytic activity. MASSL activity was stimulated by Ca²⁺, Mg²⁺ and Co²⁺ and inhibited by Mn²⁺. The inhibitory effect on activity exerted by EDTA and EGTA was limited, while the lipase inhibitor Ebelactone B completely inhibited MASSL activity as well as PMSF. Methanol 0.5% apparently did not affect MASSL activity while β-mercaptoethanol activated the enzyme.     

Highly soluble expression and molecular characterization of an organic solvent-stable and thermotolerant lipase originating from the metagenome

A novel organic solvent-stable and thermotolerant lipase gene (designated ostl28) was cloned from a metagenomic library and overexpressed in Escherichia coli BL21 (DE3) in soluble form. OSTL28 contained 262 amino acids with relative molecular mass 30.1 kDa and isoelectric point 9.7. The optimum pH and temperature of the OSTL28 were 7.5 and 60 °C, respectively. OSTL28 was stable in the pH range of 4.5–9.5 and at temperatures below 65 °C. The enzyme could hydrolyze a wide range of ρ-nitrophenyl esters, but its best substrate is ρ-nitrophenyl laurate with the highest activity of 236 U/mg (54,000 U/L). The recombinant OSTL28 was highly resisted to organic solvents, especially glycerol and methanol. The metal ions, with the exception of Hg²⁺ and Ag⁺, did not have any influence on enzyme activity, whereas non-ionic surfactants and Al³⁺ slightly activated the enzyme. These features indicate that it is a potential biocatalyst for biodiesel production.     

Purification and characterization of an organic solvent-tolerant lipase from Pseudomonas aeruginosa LX1 and its application for biodiesel production

An organic solvent-tolerant lipase from newly isolated Pseudomonas aeruginosa LX1 has been purified by ammonium sulfate precipitation and ion-exchange chromatography leading to 4.3-fold purification and 41.1% recovery. The purified lipase from P. aeruginosa LX1 was homogeneous as determined by SDS-PAGE, and the molecular mass was estimated to be 56 kDa. The optimum pH and temperature for lipase activity were found to be 7.0 and 40 °C, respectively. The lipase was stable in the pH range 4.5–12.0 and at temperatures below 50 °C. Its hydrolytic activity was found to be highest towards p-nitrophenyl palmitate (C16) among the various p-nitrophenol esters investigated. The lipase displayed higher stability in the presence of various organic solvents, such as n-hexadecane, isooctane, n-hexane, DMSO, and DMF, than in the absence of an organic solvent. The immobilized lipase was more stable in the presence of n-hexadecane, tert-butanol, and acetonitrile. The transesterification activity of the lipase from P. aeruginosa LX1 indicated that it is a potential biocatalyst for biodiesel production.     

Purification of keratinase from poultry farm isolate-Scopulariopsis brevicaulis and statistical optimization of enzyme activity

The fungus Scopulariopsis brevicaulis was isolated from poultry farm soil at Namakkal, India. The extracellular keratinase from this fungus was purified to homogeneity by ammonium sulphate precipitation and procedure involving DEAE-Cellulose and Sephadex G-100 chromatographic techniques. The purified enzyme was formed from a monomeric protein with molecular masses of 39 and 36 kDa by SDS–PAGE and gel filtration, respectively. The optimum pH at 40 °C was 8.0 and the optimum temperature at pH 8.0 was 40 °C. The activity of purified keratinase with respect to pH, temperature and salt concentration was optimized by Box–Behnken design experiment. It was shown that a second-order polynominal regression model could properly interpret the experimental data with an R²-value of 0.9957 and an F-value of 178.32, based on the maximum enzyme activity examined. Calculated optimum conditions were predicted to confer a 100% yield of keratinase activity with 5 mM CaCl₂, pH 8.0 and at a temperature of 40 °C. The enzyme was strongly inhibited by PMSF, which suggests a serine residue at or near an active site. The purified keratinase was examined with its potential for dehairing the skin.     

An organic solvent–tolerant lipase from Streptomyces sp. CS133 for enzymatic transesterification of vegetable oils in organic media

The enzymatic route for biodiesel production has been noted to be cost ineffective due to the high cost of biocatalysts. Reusing the biocatalyst for successive transesterification cycles is a potential solution to address such cost inefficiency. However, when organic solvent like methanol is used as acyl-acceptor in the reaction, the biocatalyst (lipase) gets severely inactivated due to the inhibitory effect of undissolved methanol in the reaction medium. Thus, organic solvent–tolerant lipase is highly desirable for enzymatic transesterification. In response to such desirability, a lipase (LS133) possessing aforesaid characteristic was extracted from Streptomyces sp. CS133. Relative molecular mass of the purified LS133 was estimated to be 39.8 kDa by SDS-PAGE. Lipase LS133 was stable in pH range 5.0–9.0 and at temperature lower than 50 °C while its optimum lipolytic activity was achieved at pH 7.5 and 40 °C. It showed the highest hydrolytic activity towards long chain p-nitrophenyl palmitate with K_m and V_max values of 0.152 mM and 270.2 mmol min^?1 mg^?1, respectively. It showed non-position specificity for triolein hydrolysis. The first 15 amino acid residues of its N-terminal sequence, AIPLRQTLNFQAXYQ, were noted to have partial similarity with some of the previously reported microbial lipases. Its catalytic involvement in biodiesel production process was confirmed by performing enzymatic transesterification of vegetable oils with methanol.     

Purification and characterization of piceid-β-d-glucosidase from Aspergillus oryzae

The piceid-β-d-glucosidase that hydrolyzes the β-d-glucopyranoside bond of piceid to release resveratrol was isolated from Aspergillus oryzae sp.100 strain, and the enzyme was purified and characterized. The enzyme was purified to one spot in SDS polyacrylamide gel electrophoresis, and its molecular weight was about 77 kDa. The optimum temperature of the piceid-β-d-glucosidase was 60 °C, and the optimum pH was 5.0. The piceid-β-d-glucosidase was stable at less than 60 °C, and pH 4.0–5.0. Ca²⁺, Mg²⁺ and Zn²⁺ ions have no significant effect on enzyme activity, but Cu²⁺ ion inhibits enzyme activity strongly. The K_m value was 0.74 mM and the V_max value was 323 nkat mg⁻¹ for piceid.     

Purification and characterization of an extracellular cholesterol oxidase from a Bordetella species

A soil-isolated bacterium (strain B4) was identified as a species of Bordetella and deposited with the China General Microbiological Culture Collection (code, CGMCC 2229). The bacterium grew in a mineral medium, on cholesterol as a sole source of carbon and energy. Only one metabolite of cholesterol was accumulated in detectable amounts during the strain growth. It was identified as 4-cholesten-3-one. Cholesterol oxidase (COD) (EC 1.1.3.6), which catalyzes cholesterol into this metabolite, was evidenced from the strain. The conditions of the bacterium growth were optimized for extracellular enzyme production, which then reached around 1700 UL⁻¹ within 24 h culturing. The enzyme was purified from the spent medium of the strain to homogeneity on SDS-PAGE, and characterized. Its molecular mass, as estimated by this technique, was 55 kDa. COD showed an optimum activity at pH 7.0. It was completely stable at pH 5.0 and 4 °C for 48 h, and retained 80% at least of its initial activity at pH 4.0 or at a pH of 6.0–10.0. The optimum temperature for its reaction was 37 °C. The thermal stability of COD was appreciable, as 90% or 80% of its initial activity was recovered after 1 h or 2 h incubation at 50 °C. Ag⁺ or Hg⁺ at 1 mM, was inhibitor of COD activity, while Cu²⁺, at the same concentration, was activator. The COD K_m, determined at 37 °C and pH 7.0, was 0.556 mM. The enzyme was stable at pH 7.0 and 37 °C during 24 h mechanical shaking in the presence of 33% (v/v) of either of the solvents, dimethylsulfoxide, ethyl acetate, butanol, chloroform, benzene, xylene or cyclohexane.     

Hen egg white as a feeder protein for lipase immobilization

Enzyme stabilization via immobilization is one of the preferred processes as it provides the advantages of recovery and reusability. In this study, Thermomyces lanuginosus lipase has been immobilized through crosslinking using 2% glutaraldehyde and hen egg white, as an approach towards CLEA preparation. The immobilization efficiency and the properties of the immobilized enzyme in terms of stability to pH, temperature, and denaturants was studied and compared with the free enzyme. Immobilization efficiency of 56% was achieved with hen egg white. The immobilized enzyme displayed a shift in optimum pH towards the acidic side with an optimum at pH 4.0 whereas the pH optimum for free enzyme was at pH 6.0. The immobilized enzyme was stable at higher temperature retaining about 83% of its maximum activity as compared to the free enzyme retaining only 41% activity at 70 °C. The denaturation of lipase in free form was rapid with a half-life of 2 h at 60 °C and 58 min at 70 °C as compared to 12 h at 60 °C and 2 h at 70 °C for the immobilized enzyme. The effect of denaturants, urea and guanidine hydrochloride on the free and immobilized enzyme was studied and the immobilized enzyme was found to be more stable towards denaturants retaining 74% activity in 8 M urea and 98% in 6 M GndHCl as compared to 42% and 33% respectively in the case of free enzyme. The apparent K_m (2.08 mM) and apparent V_max (0.95 μmol/min) of immobilized enzyme was lower as compared to free enzyme; K_m (8.0 mM) and V_max (2.857 μmol/min). The immobilized enzyme was reused several times for the hydrolysis of olive oil.     

Calcium dependent,alkaline detergent-stable trypsin from the viscera of Goby (Zosterisessor ophiocephalus): Purification and characterization

An alkaline calcium dependent trypsin from the viscera of Goby (Zosterisessor ophiocephalus) was purified to homogeneity with a 16-fold increase in specific activity and 20% recovery. The purified trypsin appeared as a single band on sodium dodecyl sulphate-polyacrylamide gel (SDS-PAGE) and native-PAGE. The enzyme had an estimated molecular weight of 23.2 kDa.The optimum pH was 9.0, and the enzyme was extremely stable in various pH buffers between pH 7.0 and 11.0. The optimum temperature for enzyme activity was 60 °C, and the activity and stability of trypsin was highly dependent on the presence of calcium ion. At 60 °C, Ca²⁺ (5 mM) stimulated the protease activity by 220%. The trypsin kinetic constants, K_m and k_cat, were 0.312 mM and 2.03 s^?1.The enzyme showed high stability towards non-ionic surfactants and oxidizing agent. In addition, the enzyme showed excellent stability and compatibility with some commercial solid and liquid detergents.     

Refolding,purification and characterization of an organic solvent-tolerant lipase from Serratia marcescens ECU1010

Expression of recombinant proteins as inclusion bodies in bacteria is one of the most efficient ways to produce cloned proteins, as long as the inclusion bodies can be successfully refolded. In this study, the different parameters were investigated and optimized on the refolding of denatured lipase. The maximum lipase activity of 5000 U/L was obtained after incubation of denatured enzyme in a refolding buffer containing 20 mM Tris–HCl (pH 7.0), 1 mM Ca²⁺ at 20 °C. Then, the refolded lipase was purified to homogeneity by anion exchange chromatography. The purified refolded lipase was stable in broad ranges of temperatures and pH values, as well as in a series of water-miscible organic solvents. In addition, some water-immiscible organic solvents, such as petroleum ether and isopropyl ether, could reduce the polarity and increase the nonpolarity of the refolding system. The results of Fourier transform infrared (FT-IR) microspectroscopy were the first to confirm that lipase refolding could be further improved in the presence of organic solvents. The purified refolded lipase could enantioselectively hydrolyze trans-3-(4-methoxyphenyl) glycidic acid methyl ester [(±)-MPGM]. These features render the lipase attraction for biotechnological applications in the field of organic synthesis and pharmaceutical industry.