Cloing,Expression And Characterization Of Lipase/esterase And Its Biocatalysis Research

Through analysis and screening of the microbial genome database,this research dedicated to developing new lipase/esterase gene with high catalytic activity.Then based on the development and practical application,the recombinant enzyme was used in biological catalysis applications.Using enrichment procedures,a lipolytic strain was isolated from the stinky tofu brine and was identified as Bacillus amyloliquefaciens(named Bacillus amyloliquefaciens Nsic-8)by morphological,physiological,biochemical tests and 16S rDNA sequence analysis.Meanwhile,the key enzyme gene(named lipBA)involved in ester metabolism was obtained from Nsic-8 with the assistance of homology analysis.The novel gene has an open reading frame of 645 bp,and encoding 214-amino-acid lipase(LipBA).The deduced amino acid sequence shows the highest identity with the lipase from Bacillus amyloliquefaciens IT-45(NCBI database)and belongs to the family of triacylglycerol lipase(EC 3.1.1.3).The lipase gene was expressed in E.coli BL21(DE3)using plasmid pET-28a.The enzyme activity and specific activity were 250±16 U/mL and 1750±153 U/mg,respectively.The optimum pH and temperature of the recombinant enzyme were 9.0 and 40? respectively.LipBA showed much higher stability under alkaline conditions and was stable at pH 7.0-11.0.The Km and Vmax values of purified LipBA using 4-nitrophenyl palmitate as the substrate were 1.04±0.06 mM and 119.05±7.16?mol/(mL min),respectively.After purification,recombinant lipase was immobilized with the optimal conditions(immobilization time 3 h at 30?,with 92%enzyme recovery)and the immobilized enzyme was applied in biodiesel production.The current work also focuses on the synthesis of cinnamyl acetate from cinnamyl alcohol and vinyl acetate,including screening optimization of reaction conditions such as organic solvents,temperature,catalyst loading and mole ratio.Conversion(93%)was achieved after 4 h when transesterification was carried out at vinyl acetate/cinnamyl alcohol 2:1,4.0 g L-1 of lipase loading,and at 40? in hexane as solvent.Also the catalytic behaviors of lipase LipBA for synthesis different carbon chain lengths of cinnamyl esters were determined.Among the different acyl donors employed,vinyl propionate was found to be the best acyl donor which presents a 96%conversion.The gene(EstPS1),which encodes a novel carboxylesterase of Pseudomonas synxantha PS1 isolated from oil well-produced water,was cloned and sequenced.EstPSl has an open reading frame of 1923 bp and encodes the 640 amino-acid carboxylesterase(EstPSl),which contains an autotransporter(AT)domain(357-640 aa).Homology analysis revealed that EstPSl shared the highest identity(88%)with EstA from Pseudomonas fluorescens A506(NCBI database)and belongs to the carboxylesterase family(EC 3.1.1.1).The optimum pH and temperature of recombinant EstPS1 is 8.0 and 60?.EstPS1 showed remarkable thermostability,and the half-lives(T1/2 thermal inactivation)were 14 h,2 h,31 min,10 min,and 1 min at 60,70,80,90,and 100?,respectively.To understand the role of the AT domain in the carboxylesterase of P.synxantha PS1,AT domain-truncated carboxylesterase(EstPS1AAT)was generated.EstPS1AAT showed a clearly decreased secretion rate,as the AT domain strongly improved secretory expression even in the heterogeneous system.EstPS1 degraded a wide range of pyrethroid pesticides,and hydrolysis efficiencies were dependent on the pyrethroid molecular structure.EstPS1 not only degraded all pyrethroid pesticides tested but also hydrolyzed p-nitrophenyl esters of medium-short chain fatty acids,indicating that EstPSl is an esterase with broad specificity.Also the genome database of Geobacillus sp.was analysis,dozens of lipase/esterase gene were success cloned and expressed in e.coli.Homology analysis revealed that these enzymes amino acid sequence shared the identity(60%-95%)with lipid hydrolase(NCBI database).The enzyme EstGSU753 with high activity towards tributyrin was studied in pending further research.