Purification of lipase from Cunninghamella verticillata by stepwise precipitation and optimized conditions for crystallization

Lipase from the oil-mill waste isolate Cunninghamella verticillata was purified by stepwise precipitation using acetone, as a sequel to our earlier conventional column chromatographic method [Gopinath et al. (2002)World Journal of Microbiology and Biotechnology 18, 449–458]. The yield of purified lipase was approx. 4-fold higher than by the previous method and the purified lipase was obtained with 70–80% acetone saturations. The enzyme was resolved as a single band with homogeneity both by native and by SDS–PAGE. The optimum condition for the lipase to crystallize was 5 g of enzyme in 0.05 M sodium phosphate buffer (pH 6.5) with 5 mM FeCl₂ and 10% 2-methyl 2,4-pentanediol (MPD).These authors equally contributed to this work     

Stability and structure of Penicillium chrysogenum lipase in the presence of organic solvents

Abstract

The present work describes the enzymatic properties of Penicillium chrysogenum lipase and its behavior in the presence of organic solvents. The temperature and pH optima of the purified lipase was found to be 55?°C and pH 8.0 respectively. The lipase displayed remarkable stability in both polar and non-polar solvents upto 50% (v/v) concentrations for 72?h. A structural perspective of the purified lipase in different organic solvents was gained by using circular dichroism and intrinsic fluorescence spectroscopy. The native lipase consisted of a predominant α-helix structure which was maintained in both polar and non-polar solvents with the exception of ethyl butyrate where the activity was decreased and the structure was disrupted. The quenching of fluorescence intensity in the presence of organic solvents indicated the transformation of the lipase microenviroment P. chrysogenum lipase offers an interesting system for understanding the solvent stability mechanisms which could be used for rationale designing of engineered lipase biocatalysts for application in organic synthesis in non-aqueous media.     

Aggregation properties of cold-active lipase produced by a psychrotolerant strain of Pseudomonas palleroniana (GBPI_508)

Abstract

The present study aims to exploit microbial potential from colder region to produce lipase enzyme stable at low temperatures. A newly isolated bacterium GBPI_508 from Himalayan environment, was investigated for the production of cold-active lipase emphasizing on its aggregation properties. Plate based assays followed by quantitative production of enzyme was estimated under different culture conditions. Further characterization of partially purified enzyme was done for molecular weight determination and activity and stability under varying conditions of pH, temperature, and in presence of organic solvents, inhibitors, and metal ions. The psychrotolerant bacterium was identified as Pseudomonas palleroniana following 16S rRNA gene sequencing. Maximum lipase production by GBPI_508 was recorded in 7?days at 25?°C utilizing yeast extract as nitrogen source and olive oil as substrate in the lipase production medium. Triton X-100 (1%) in the medium as emulsifier significantly enhanced the lipase production. Lipase produced by bacterium showed aggregation which was confirmed by dynamic light scattering and native PAGE. SDS-PAGE followed by zymogram analysis of partially purified enzyme showed two active bands of ～50?kDa and ～54?kDa. Optimum activity of partially purified enzymatic preparation was recorded at 40?°C while the activity remained nearly consistent from pH 7.0 to 12.0, whereas, maximum stability was recorded at pH values 7.0 and 11.0 at 25?°C. Interestingly, lipase in the partially purified fraction retained 60% enzyme activity at 10?°C. Medium chain pNP ester (C10) was the most preferred substrate for the lipase of GBPI_508. The lipase possessed >50% residual activity when incubated with different organic solvents (25% v/v) except toluene and dichloromethane which inhibited the activity below 50%. Partially purified enzyme was also stable in the presence of metal ions and inhibitors. The study suggests applicability of GBPI_508 lipase in low temperature conditions such as cold-active detergent formulations and cold bioremediation.     

Isolation and characterization of a new alkali-thermostable lipase cloned from a metagenomic library

The construction of a cosmid library from the biomass produced in an enriched Sequencing Fed-Batch Reactor allowed the isolation of a new lipase by functional screening. The open reading frame of 928 bp encoded a polypeptide of 308 amino acids with a molecular mass of 32.6 kDa. The amino acid sequence analysis revealed the presence of the conserved pentapeptide GXSXG essential for lipase activity. Alignment with known sequences of proteins showed no more than 52% identity with different lipases, confirming the discovery of a novel gene sequence. The lipase was cloned and expressed in Streptomyces lividans and further purified by a combination of hydrophobic interaction and size-exclusion chromatography. Spectrophotometric assays with different p-nitrophenyl esters demonstrated a preference for long-length acyl chains, especially p-nitrophenylmyristate (C14). Moreover, the enzyme presented an optimal activity at 60°C and at alkaline pH of 10.5.     

Production and characterization of a cold-active and n-hexane activated lipase from a newly isolated Serratia grimesii RB06-22

Abstract

A lipase-producing bacterium isolated from raw milk was identified as Serratia grimesii based on 16S rRNA sequence analysis. The extracellular lipase was partially purified by ammonium sulfate precipitation and ultrafiltration. Maximal activity was observed at 10°C, the optimum pH was 8.0 and the enzyme was stable at 5–30°C for 1 h. The K_m and V_max values were 1.7 mM and 0.3 mM/min respectively. It was found that the lipase had the highest hydrolytic activity towards sunflower oil and soybean oil. CaCl₂ had a stimulatory effect on lipase activity, while EDTA and iodoacetic acid slightly inhibited the lipase activity and the enzyme was strongly inhibited by PMSF. The enzyme was compatible with various non-ionic surfactants as well as sodium cholate and saponin. In addition, the enzyme was relatively stable towards oxidizing agents. This lipase exhibited maximum activity in 35% n-hexane retaining about 2191% activity for 1 h.     

Lipase from Candida paralipolytica

The lipase from Candida paralipolytica was purified, as judged by disc electrophoresis. The purification was about 132 fold, based on protein, with a recovery of 32% from the acetone precipitate of the cultivated broth.

After purification, modification of the enzyme was performed by dialyzing its solution against 1 m sodium chloride in acetate buffer at room temperature and by separating the modified enzyme from an unknown substance(s) with a Sephadex G–75 column.

The optimum pH for lipolysis of the purified lipase was 8.0, while that of the modified one was 7.0. Sodium taurocholate was required essentially by the purified enzyme, but not by the modified one. The purified lipase was stable below 37°C and in the pH range from 3.5 to 9.0 at 5°C.     

Purification and characterization of an extracellular lipase from Clostridium tetanomorphum

Summary The strictly anaerobic bacterium Clostridium tetanomorphum formed an extracellular lipase when the growth medium contained glycerol in addition to fermentable substrates such as l-glutamate or glucose. The lipase was purified from the concentrated culture supernatant and exhibited a final specific activity of 900 U/mg. The purified lipase had a Stokes’ radius of 5.0 nm and a sedimentation coefficient of 5.7S. The native molecular mass calculated from these values was 118,000 Da, which is considerably higher than the molecular mass calculated by PAGE (70,000 Da). With p-nitrophenyl esters of different fatty acids as substrates enzyme activity was highest when the acyl chain was short (C₂). The purified lipase showed no protease or thioesterase activity.     

An organic solvent-tolerant alkaline lipase from <Emphasis Type="Italic">Streptomyces</Emphasis> sp. CS268 and its application in biodiesel production

In an effort to identify a microbial lipase that can catalyze transesterification reactions used in biodiesel production, an organic solvent-tolerant lipase was purified from Streptomyces sp. CS268. The molecular weight of the purified lipase was estimated to be 37.5 kDa by SDS-PAGE. The lipase showed highest activity at a temperature of 30°C and pH 8.0 while it was stable in the pH range 4.0 ∼ 9.0 and at temperatures ≤ 50°C. It showed the highest hydrolytic activity towards medium-length acyl chain p-nitrophenyl decanoate with K _m and V _max values of 0.59 mM and 319.5 mmol/mg/min, respectively. Also, the lipase showed non-position specificity for triolein hydrolysis. The purified lipase catalyzed transesterification reaction of soybean oil with methanol, suggesting that it can be a potential enzymatic catalyst for biodiesel production.     

Inhibition of hepatic lipase by m-aminophenylboronate application of phenylboronate affinity chromatography for purification of human postheparin plasma lipases

Phenylboronates are competitive inhibitors of serine hydrolases including lipases. We studied the effect of m-aminophenylboronate on triglyceride-hydrolyzing activity of hepatic lipase (EC 3.1.1.3). m-Aminophenylbo ronate inhibited hepatic lipase activity with a K₁ value of 55 μM. Furthermore, m-aminophenylboronate protected hepatic lipase activity from inhibition by di-isopropyl fluorophosphate, an irreversible active site inhibitor of serine hydrolases. Inhibition of hepatic lipase activity by m-aminophenylboronate was pH-dependent. The inhibition was maximal at pH 7.5, while at pH 10 it was almost non-existent. These data were used to develop a purification procedure for postheparin plasma hepatic lipase and lipoprotein lipase. The method is a combination of m-aminophenylboronate and heparin-Sepharose affinity chromatographies. Hepatic lipase was purified to homogeneity as analyzed on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The specific activity of purified hepatic lipase was 5.46 mmol free fatty acids h⁻¹ mg⁻¹ protein with a total purification factor of 14 400 and a final recovery of approximately 20%. The recovery of hepatic lipase activity in m-aminophenylboronate affinity chromatography step was 95%. The purified lipoprotein lipase was a homogeneous protein with a specific activity of 8.27 mmol free fatty acids h⁻¹ mg⁻¹ The purification factor was 23 400 and the final recovery approximately 20%. The recovery of lipoprotein lipase activity in the m-aminophenylboronate affinity chromatography step was 87%. The phenylboronate affinity chromatography step can be used for purification of serine hydrolases which interact with boronates.     

Screening,partial purification and characterization of the hyper-thermophilic lipase produced by a new isolate of Bacillus subtilis LP2

Abstract

Lipases are one of the most important catalysts for several industries such as detergent, dairy, and textile industry due to their bio-catalytic ability in aqueous and non-aqueous media. Stability to extreme conditions is an important property since it makes enzymes suitable to several industrial processes. In this study, lipase producing soil bacteria were screened and identified with 16S rDNA sequencing. A new hyper-thermophilic lipase named as Bacillus subtilis LP2 isolate was partially purified by ammonium sulphate precipitation with 17.8-fold purification and 583?U/mg specific activity. Maximum activity was exhibited at pH 7 and 80?°C with the substrate tween 80?K_M and V_max values were calculated as 18.3?mM and 680?U/mg with a catalytic efficiency (k_cat/K_M) of 307?s^?1M^?1. These results indicate that lipase from Bacillus subtilis LP2 can be a valuable candidate for industrial applications such as organic synthesis and fats and oils industry due to their efficient catalysis in higher temperatures.     

Characterization and Expression of a GDSL-Like Lipase Gene from Brassica napus in Nicotiana benthamiana

The GDSL esterase and lipase families play important roles in abiotic stress, pathogen defense, seed development and lipid metabolism. Identifying the lipase activity of the putative GDSL lipase is the prerequisite for dissecting its function. According to the sequence similarity and the conserved domains, we cloned the Brassica napus BnGLIP gene, which encodes a GDSL-like protein. We failed to identify the BnGLIP lipase activity in the bacterium and yeast expression systems. In this paper, we expressed the BnGLIP gene by fusing a 6× His tag in Nicotiana benthamiana and purified the recombinant protein. The extraction buffer contained 1 % (v/v) n-caprylic acid and was able to remove most of the protein impurities. About 50 μg of recombinant BnGLIP was obtained from 1 g of N. benthamiana leaves. The lipase activity was tested with the purified BnGLIP and the maximum enzyme activity reached 17.7 mM/mg. In conclusion, this study found that the recombinant protein BnGLIP expressed in tobacco system was effectively purified and was detected as a GDSL lipase.     

Purification of Chromobacterium viscosum lipases using reversed micelles

Summary Two upases produced by Chromobacterium viscosum were isolated and purified by liquid-liquid extraction using organic solvents containing reversed micelles. One lipase (lipase A) was purified from the original crude enzyme preparation by 4,3-fold with a recovery of 91% and the other (lipase B) by 3,7-fold with a recovery of 76%.     

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.     

Conversion of a pentane-1,3,5-triol derivative using lipases as chiral catalysts and possible function of the lid for the regulation of substrate selectivity and enantioselectivity

Abstract

The enantioselective desymmetrization of a prochiral 3-O-silyl protected pentane-1,3,5-triol derivative was achieved by lipase-catalysed hydrolysis. The lipase from B. cepacia led to 95.4% enantiomeric excess (ee)of the (R)-configured compound (R)-4 at a theoretical yield of 79% and was isolated with 98.2% ee and 27% yield. Furthermore, it was found that the ee switched from an excess of (R)-4 to an excess of (S)-4 during the course of the reaction using crude lipase from C. rugosa under the same conditions. This finding was investigated in detail and compared to the change of substrate selectivity known for the lipase from M. miehei. It is supposed that both phenomena may result from a movement of the lid.     

Characterization of two novel lipase genes isolated directly from environmental sample

Two novel lipase genes (lipJ02, lipJ03) were isolated directly from environmental DNA via genome-walking method. Lipase gene lipJ02 contained an open reading frame (ORF) of 1,425 bp and encoded a 474-amino acids lipase protein, while lipase gene lipJ03 contained an ORF of 1,413 bp and encoded a 470-amino acids lipase protein. The lipase genes were cloned into expression vector pPIC9K and successfully integrated into a heterologous fungal host, Pichia pastoris KM71, and the recombinant P. pastoris were screened via a high-throughput method. The recombinants were induced by methanol to secrete active lipases into cultural medium. The recombinant lipases were also purified and characterized. The optimum temperature for the purified lipase LipJ02 and LipJ03 was 30 and 35°C, respectively, at pH 8.0. They exhibited similar thermostability, but LipJ02 exhibited better pH stability than LipJ03.     

Characterisation of a thermo-alkali-stable lipase from oil-contaminated soil using a metagenomic approach

Lipases are widely used for a variety of biotechnological applications. Screening these industrial enzymes directly from environmental microorganisms is a more efficient and practical approach than conventional cultivation-dependent methods. Combined with activity-based functional screening, six clones with lipase activity were detected and a gene (termed lipZ01) isolated from a target clone with the highest lipase activity was cloned from an oil-contaminated soil-derived metagenomic library and then sequenced. Gene lipZ01 was expressed in Pichia pastoris GS115 and the molecular weight of the recombinant lipase LipZ01 was estimated by electrophoresis analysis to be approximately 50 kDa. The maximum activity of the purified lipase was 42 U/mL, and the optimum reaction temperature and pH value were 45 °C and 8.0, respectively. The enzyme was highly stable in the temperature range 35–60 °C and under alkaline conditions (pH 7–10). The presence of Ca²⁺ and Mn²⁺ ions could significantly enhance the activity of the lipase. The purified lipase preferentially hydrolysed triacylglycerols with acyl chain lengths ≥8 carbon atoms, and the conversion degree of biodiesel production was nearly 92% in a transesterification reaction using olive oil and methanol. Some attractive properties suggested that the recombinant lipase may be valuable in industrial applications.     

Stereoselective enzymatic esterification of 3-benzoylthio-2-methylpropanoic acid

Summary A key intermediate, S-(–)-3-benzoylthio-2-methylpropanoic acid (1) was made in high optical purity by the lipase-catalyzed stereoselective esterification of racemic 1 with methanol in an organic solvent system. Among various lipases evaluated, Amano P-30 lipase from Pseudomonas sp. efficiently catalyzed the esterification of 1 to yield R-(+) methyl ester and unreacted S-(–) 1. A reaction yield of 40 mol% and an optical purity of 97.2% were obtained for compound 1 at a substrate concentration of 0.1 m (22 mg/ml). Lipase P-30 was immobilized on Accurel polypropylene (PP) and the immobilized enzyme was reused (23 cycles) in the esterification reaction without loss of enzyme acitivity, productivity or optical purity. Among various solvents evaluated, toluene was found to be the most suitable organic solvent and methanol was the best alcohol for the esterification of racemic 1 by immobilized lipase. Substrate concentrations as high as 1.0 m were used in the esterification reaction. When the temperature was increased from 28° C to 60° C, the reaction time required for the esterification of 0.1 m substrate decreased from 16 h to 2 h. On increasing the methanol to substrate molar ratio from 1:1 to 4:1, the rate of esterification decreased. A lipase fermentation using Pseudomonas sp. ATCC 21 808 was developed. In the batch-fermentation process, 56 units/ml of extracellular lipase activity was obtained. A fed-batch process using soybean oil gave a significant increase in the lipase activity (126 units/ml). Crude lipase recovered from the filtrate by ethanol precipitation and immobilized on Accurel PP was also effective: S-(–) compound 1 was obtained in 35 mol% yield and 95% optical purity. Offsprint requests to: R. N. Patel     

Production,partial purification and characterization of organic solvent tolerant lipase from Burkholderia multivorans V2 and its application for ester synthesis

Burkholderia multivorans V2 (BMV2) isolated from soil was found to produce an extracellular solvent tolerant lipase (6.477 U/mL). This lipase exhibited maximum stability in n-hexane retaining about 97.8% activity for 24 h. After performing statistical optimization of medium components for lipase production, a 2.2-fold (14 U/mL) enhancement in the lipase production was observed. The crude lipase from BMV2 was partially purified by ultrafiltration and gel permeation chromatography with 24.64-fold purification. The K_m and V_max values for partially purified BMV2 lipase were found to be 1.56 mM and 5.62 μmoles/mg min. The metal ions Ca²⁺, Mg²⁺ and Mn²⁺ had stimulatory effect on lipase activity, whereas Cu²⁺, Fe²⁺ and Zn²⁺ strongly inhibited the lipase activity. EDTA and PMSF at 10 mM concentration strongly inhibited the lipase activity. Non-ionic and anionic surfactants stimulated the lipase activity. BMV2 lipase was proved to be efficient in synthesis of ethyl butyrate ester under non-aqueous environment.     

Isolation and purification of Mucor circinelloides intracellular chitosanolytic enzymes

This study aimed at isolation, purification and characterization of a chitosanase from Mucor circinelloides mycelium. The latter contains also a mycelium-bound lipase and lipids. The chitosanase and lipase were extracted from defatted M. circinelloides mycelium with a detergent and purified through a two-step procedure comprising chromatography on bacitracin–CNBr-Sepharose 4B and gel filtration on Sephadex G-100. Purification degree of the chitosanase (endo-type enzyme) and lipase was 23 and 12, respectively. These enzymes were optimally active at pH of 5.5–6.0 (chitosanase) and 7.2 (lipase in olive oil hydrolysis) and at 37 °C. Both purified enzymes were activated by Ca²⁺ and Mg²⁺ ions. The preferred substrates of chitosanase were chitosan preparations with a high degree of deacetylation. This enzyme showed no activity for colloidal chitin, Na-CMC and starch. SDS–PAGE of both purified enzymes showed two bands with molecular masses of 42 and 43 kDa. Our results suggest that M. circinelloides synthesizes an oligomeric (bifunctional) lipase which also efficiently depolymerizes chitosan.     

Purification of lipase from Cunninghamella verticillata and optimization of enzyme activity using response surface methodology

The fungus Cunninghamella verticillata was selected from isolates of oil-mill waste as a potent lipase producer as determined by the Rhodamine-B plate method. The lipase was purified from C. verticillata by ammonium sulphate fractionation, ion exchange chromatography and gel filtration. The purified enzyme was formed from a monomeric protein with molecular masses of 49 and 42 kDa by SDS–PAGE and gel filtration, respectively. The optimum pH at 40 °C was 7.5 and the optimum temperature at pH 7.5 was 40 °C. The enzyme was stable between a pH range of 7.5 and 9.0 at 30 °C for 24 h. The enzyme activity was strongly inhibited by AgNO₃, NiCl₂, HgCl₂, CdCl₂ and EDTA. However, the presence of Ca²⁺, Mn²⁺ and Ba²⁺ ions enhanced the activity of the enzyme. The activity of purified lipase with respect to pH, temperature and salt concentration was optimized using a Box–Behnken design experiment. A polynomial regression model used in analysing this data, showed a significant lack of fitness. Therefore, quadratic terms were incorporated in the regression model through variables. Maximum lipase activity (100%) was observed with 2 mM CaCl₂, (pH 7.5) at a temperature of 40 °C. Regression co-efficient correlation was calculated as 0.9956.