The Cloning, Expression, Characterization And Application Of A Recombinant Thermostable Lipase From Geobacillus Stearothermophilus Jc

A thermophilic lipase gene of Geobacillus stearothermophilus JC was cloned and sequenced,the lipase gene shows an open reading frame of 1251 nucleotides coding a 28-amino-acid signal sequence and a mature sequence of 388 amino acids.The lipase was only weakly expressed when the prelipase gene was directly subcloned into pET 28a(+) expression vector,while removal of the signal peptide greatly improved the expression level.High yield purification of JC lipase was achieved through one-step purification by IMAC with a final specific activity and yield of 515.6U/mg and 40.5%,respectively.The purified JC lipase had an optimum temperature and pH of 55℃and pH 9,respectively.The recombinant lipase was stable in enzyme inhibitors such as EDTA,β-mercaptoethanol,PMSF,DTT,Triton X-100 significantly enhanced JC lipase.Its catalytic function was enhanced in the presence of K⁺,Na⁺,Mg²⁺,Ca²⁺,Mn²⁺,but inhibited by Fe²⁺,Zn²⁺,Cu²⁺.The highest activity was found with p-nitrophenyl -caprate(C12) among different p-nitrophenyl esters.It was stable in organic solvents such as dimethylsulfoxide(DMSO),methanol,ethanol, acetone,isopropanol,acetonitrile.Furthermore,it showed high enantioselectivity when (RS)-1-phenylethyl acetate was selected as substrates,generating optically-pure(R)-1-phenylethanol in short time.The reaction conversion catalyzed by JC lipase could reach 46%,the ee_p reached 97.7%and E value reached 236.6.