Production, Expression And Characterization Of A Heat-Stable Organic Solvent Tolerant Lipase From Bacillus Sp, Strain 42

Ninety two bacterial strains were isolated from oil palm effluent from Bangi,Selangor; Kluang, Johor; Alor Gajah hot spring (up to 54 OC) Melaka and Slim River hot spring (up to 9I0C) Perak. An enrichment culture technique was used to isolate bacteria utilizing olive oil as a substrate. Cultures were incubated at 60°C to select for the thermophilic bacteria. Eight isolates showed lipolytic activity on tributyrin and triolein agar plates. In order to screen for highest lipase producer, six production media were used. Isolate 42 was observed to produce the highest level (0.059 Ulml) after 72h. Its crude lipase retained its full activity when preincubated at 70°C for 30 min. It also showed high stability in several organic solvents (25% vlv). Furthermore, its activity was enhanced in benzene, hexane and hexadecane while, completely inhibited by butanol. Isolate 42 was identified as Bacillus sp. Strain 42 using 16s rDNA. The nucleotide sequence deposited at GenBank under accession number AY 7631 18. Further optimization studies were done in order to determine the best lipase production condition. lnoculum size of 3% proved to be the best for lipase production, with an optimum temperature of 50°C when, grown under shaking condition of 150 rpm. A combination of tryptone and yeast extract was the best nitrogen source. Lipase production was stimulated by olive oil. The lipase gene was amplified by polymerase chain reaction using consensus primers based on multiple aligned sequences of thermophilic genes from other thermophilic Bacillus species. Nucleotide sequence comparison shared high homology with the thermostable genes in Geobacillus sp., Bacillus stearothermophilus and Bacillus thermoleovorans. Nucleotide sequence deposited at GenBank under accession number AY 787835. The amplified gene was successfully cloned using a pQE-30 UA expression vector and induced by IPTG at the optimum concentration of 0.75 mM. The recombinant lipase was facilitated by the fusion of 6-histidine and this allowed a one-step purification of the lipase enzyme using Ni-NTA affinity chromatography. The histidine-tagged lipase was purified 6-fold with a yield of 21.7%. Purified lipase migrated as a single band with a molecular mass of -43 KDa on SDS-PAGE. The purified lipase showed high activity at 70°C with its optimum at pH 8.0. The enzyme was stable over a broad range of pH from 6.0 to 10.0. It also showed high stability with half-lives of 315 min at 60°C, 120 min at 65OC, and 45 min at 70°C. Preincubation enzyme activity was stimulated with Na+, K' and ca2+. While, zn2+, ~ n ' + and Fe *+ at high concentration (10 mM) were greatly inhibitory. Protease inhibitors Bestatin and pepstatin stimulated the lipase activity while, phenylmethylsulfonyl fluoride (PMSF) completely inhibited the lipase activity. Tween 80 (0.1%) enhanced the lipase activity while higher concentration ( I %) dramatically decreased the lipase activity. The activity of preincubated enzyme in heptanol (log P 2.4) and octanol (log P 2.9) was slightly enhanced while, remains very stable with other organic solvents tested. Solvents such as ethylbenzene (log P 3.1) and dodecane (log P 6.6) reduced the lipase activity up to 35% and 38%, respectively. The highest specificity was observed towards tricaprylin (CtJ, followed by tricaprin (Clo). Its hydrolyzed all the natural oils tested, with highest hydrolysis rate on olive oil

Overexpression and characterization of strep-tagged thermostable organic solvent-stable lipase from Bacillus sp. strain 42

In addition to stability at high temperatures, thermophilic enzymes also possess great resistance against proteolysis, detergents and chaotropic agents (Sellek and Chaudhuri, 1999). For this reasons, there was more attention to consider for their future use in organic solvent. Bacillus sp. strain 42 producing a solventstable thermostable lipase was isolated from Malaysian palm oil mill effluent. The 1.2 kb gene (AY 787835) code for lipase was amplified using consensus primers based on multiple sequence alignment with thermostable genes. The gene was cloned into pQE-30UA and pET51b expression vectors. An overexpression was achieved in heterologous system using pET51b vector with Escherichia coli host strain BL21(DE3)pLysS. The optimum expression was after 24 h incubation at 37 ◦C and lipase activity was at 80.0 U/ml culture (160.0 U/mg protein) after induction with 0.5mM IPTG. Under strong T7 promoter system, expression using pET51b/BL21(DE3)pLysS host-vector system is 11.5 fold higher compared to pQE-30UA/M15(pREP4) host-vector system which achieved at 17 U/ml culture (34 U/mg protein). The fusion lipase in pET51b contained Strep-tag II affinity tag that in one step of purification, the lipase was purified to homogeneity using Strep-tag II agarose column with 1.3-fold purification factor and 70% recovery. Sodium-dodecyl sulphate polyachrylamide gel electrophoresis (SDS-phage) analysis showed that the molecular weight of fusion lipasewas about 43 kDa. The purified lipasewasmost active at 70 ◦C and pH 8.0 and was stable in a broad pH range 7–10. The lipase showed high stability with half life of 315 min at 60 ◦C, for 125 min at 65 ◦C and 45 min at 70 ◦C. By 30 min incubation in 25% (v/v) solvents with shaking rate at 150 strokes per min, the solvent stability of the enzyme was different depending on solvents and temperatures. The lipase was more stable in polar organic solvent such as DMSO, DMF, acetone, methanol, heptanol and octanol. In thiswork, thermostable and organic solvent stable lipase gene from Bacillus sp. strain 42 was successfully identified and overexpressed into high expression vector pET51b using host BL21(DE3)pLysS, under the control of T7 expression mechanism. The fusion enzyme was successfully purifie to homogeneity using Strep-tag affinity tag, and characterized. Stability of enzyme in organic solvent with low partition coefficient value (log P) will enable its future industrial use, for instance in biodiesel production

Characterization and solvent stable features of Strep-tagged purified recombinant lipase from thermostable and solvent tolerant Bacillus sp. strain 42

A 1.2 kb lipase gene (AY 78735) from solvent stable and thermostableBacillus sp. strain 42 was overexpressed in a heterologous system that allowed for an extensive characterization of its solvent stability and thermostability. An overexpression was achieved using pET51b vector withEscherichia coli host strain BL21(DE3)pLysS, in which optimum expression was at 22–24 h incubation at 37°C, with lipase activity reached at 80.0 U mL−1 (specific activity 160.0 U mg−1), after induction by 0.5 mM IPTG. This expression was 11.5 fold higher and superseded the pQE-30UA/M15 (pREP4) host-vector system, which only achieved at 17.0 U mL−1 (34.0 U mg−1). The fusion lipase contains N-terminal Strep-tag II affinity tag that in one step of purification, the lipase was purified to homogeneity using Strep-tag II agarose column. The lipase was purified at 1.3 fold and 70% recovery with the elution fraction gave a band of 43 kDa in SDS-PAGE. The purified fusion lipase was most active at 70°C and pH 8.0, and was stable in a broad pH range of 7–10. It showed hydrolysis preference towards olive, sunflower and corn oils. Based on solvent stability studies in 30 min pre-incubation in 25% v/v solvents with a shaking rate at 150 strokes per min, the purified Lip 42 showed a different residual activity profiles depending on solvents and temperatures. Lip 42 was found be stable in polar organic solvents such as DMSO, DMF, acetone, methanol, heptanol and octanol, which could make it as a potential biocatalyst for the use in industrial biodiesel production

Characterization and solvent stability of strep-tagged purified recombinant lipase from thermostable and solvent tolerant Bacillus sp. strain 42.

A 1.2 kb lipase gene (AY 78735) from solvent stable and thermostable Bacillus sp. strain 42 was overexpressed in a heterologous system that allowed for an extensive characterization of its solvent stability and thermostability. An overexpression was achieved using pET51b vector with Escherichia coli host strain BL21(DE3)pLysS, in which optimum expression was at 22-24 h incubation at 37 °C, with lipase activity reached at 80.0 U mL-1 (specific activity 160.0 U mg-1), after induction by 0.5 mM IPTG. This expression was 11.5 fold higher and superseded the pQE-30UA/M15(pREP4) host-vector system, which only achieved at 17.0 U mL-1 (34.0 U mg-1). The fusion lipase contains N-terminal Strep-tag II affinity tag that in one step of purification, the lipase was purified to homogeneity using Strep-tag II agarose column. The lipase was purified at 1.3 fold and 70% recovery with the elution fraction gave a band of 43 kDa in SDS-PAGE. The purified fusion lipase was most active at 70 °C and pH 8.0, and was stable in a broad pH range of 7-10. It showed hydrolysis preference towards olive, sunflower and corn oils. Based on solvent stability studies in 30 min pre-incubation in 25% v/v solvents with a shaking rate at 150 strokes per min, the purified Lip 42 showed a different residual activity profiles depending on solvents and temperatures. Lip 42 was found be stable in polar organic solvents such as DMSO, DMF, acetone, methanol, heptanol and octanol, which could make it as a potential biocatalyst for the use in industrial biodiesel production

Overexpression and characterization of strep-tagged thermostable organic solvent-stable lipase from Bacillus sp. strain 42

In addition to stability at high temperatures, thermophilic enzymes also possess great resistance against proteolysis, detergents and chaotropic agents (Sellek and Chaudhuri, 1999). For this reasons, there was more attention to consider for their future use in organic solvent. Bacillus sp. strain 42 producing a solventstable thermostable lipase was isolated from Malaysian palm oil mill effluent. The 1.2 kb gene (AY 787835) code for lipase was amplified using consensus primers based on multiple sequence alignment with thermostable genes. The gene was cloned into pQE-30UA and pET51b expression vectors. An overexpression was achieved in heterologous system using pET51b vector with Escherichia coli host strain BL21(DE3)pLysS. The optimum expression was after 24 h incubation at 37 ◦C and lipase activity was at 80.0 U/ml culture (160.0 U/mg protein) after induction with 0.5mM IPTG. Under strong T7 promoter system, expression using pET51b/BL21(DE3)pLysS host-vector system is 11.5 fold higher compared to pQE-30UA/M15(pREP4) host-vector system which achieved at 17 U/ml culture (34 U/mg protein). The fusion lipase in pET51b contained Strep-tag II affinity tag that in one step of purification, the lipase was purified to homogeneity using Strep-tag II agarose column with 1.3-fold purification factor and 70% recovery. Sodium-dodecyl sulphate polyachrylamide gel electrophoresis (SDS-phage) analysis showed that the molecular weight of fusion lipasewas about 43 kDa. The purified lipasewasmost active at 70 ◦C and pH 8.0 and was stable in a broad pH range 7–10. The lipase showed high stability with half life of 315 min at 60 ◦C, for 125 min at 65 ◦C and 45 min at 70 ◦C. By 30 min incubation in 25% (v/v) solvents with shaking rate at 150 strokes per min, the solvent stability of the enzyme was different depending on solvents and temperatures. The lipase was more stable in polar organic solvent such as DMSO, DMF, acetone, methanol, heptanol and octanol. In thiswork, thermostable and organic solvent stable lipase gene from Bacillus sp. strain 42 was successfully identified and overexpressed into high expression vector pET51b using host BL21(DE3)pLysS, under the control of T7 expression mechanism. The fusion enzyme was successfully purifie to homogeneity using Strep-tag affinity tag, and characterized. Stability of enzyme in organic solvent with low partition coefficient value (log P) will enable its future industrial use, for instance in biodiesel production