Purification And Gene Cloing Of Esterase From Morganella Morganii ZJB09203

As a new generation of anti-epileptic drug Pregabalin with more toleration and higher safety,was used more extensively in the treatment of neuropathic pain and epilepsy.The production of Pregabalin through chemoenzymatic route was a hot issue.(3S)-2-carboxyethyl-3-cyano-5-methylhexanoic acid was a valuable synthetic intermediate for Pregabalin.Morgarella morganii ZJB-09203 performed a good effect on kinetic resolution of 2-carboxyethyl-3-cyano-5-methylhexanoic acid ethyl ester(CNDE)to(3S)-2-carboxyethyl-3-cyano-5-methylhexanoic acid.In this study,esterase from M.morganii ZJB-09203 was purified,and characterized.Additionally,We were trying to clone the gene of the esterase.These results provided a basis for the easterase further cloning,expression and applications.Firstly,the key enzyme was purified from M.morganii ZJB-09203 by a four-step purification,included ammonium sulfate precipitation,Phenyl Sepharose 6 FF,DEAE-Sepharose A-50,and Bio-Scale CHT column chromatography.The overall yield of purified esterase was 6.5%and the maximal sepecific activity was 15.61 U/mg.SDS-PAGE showed that the esterase,comprised of one subunits with a molecular weights of 66 kDa.Exclusion chromatography analysis revealed that the esterase molecular weight was around 68 kDa.Furthermore,the enzymatic property of the purified M.morganii ZJB-09203 esterase was investigated.The purified enzyme exhibited maximurn activity at pH 8.9 and 45?.The enzyme showed good stablity when pH range of 7.0-9.0,and the temperature below 45 ?.Enzyme activity was strongly inhibited in the presence of Co2+,Fe3+,Ni2+,Zn2+,Ba2+and EDTA,while it promoted by Ca2+,Cu2+ and Mn2+.Substrate specificity of M.morganii ZJB-09203 esterase was evaluated by hydrolyzing p-nitrophenyl esters with different acyl chain lengths,the highest activity was observed in the presence of 4-Nitrophenyl Hexanoate.The activity of esterase was inhibited by the addition of an organic solvent such as methanol,ethanol,isopropanol and acetone,but the stereoselectivity maintain a higher level.The non-ionic detergents,such as Tween-80,Tween-20 and cosolvent DMSO have slight inhibited effect on the esterase activity.The kinetic parameters for the esterase towards CNDE was determined.The esterase was found to have a Km value of 12.14 mmol/L,and the Vmax.value of 29.41,?mol/mg-min.The reaction progress of CNDE to(3S)-2-carboxyethyl-3-cyano-5-methylhexanoic acid used the purifed enzyme as biocatalysts was studied.The result showed that the(3S)-2-carboxyethyl-3-cyano-5-methylhexanoic acid with 45%conversion and 96%e.e.p in 6 h with a substrate concentration of 60 mmol/L.The LC-MS-MS analysis of the purified esterase showed that the esterase contains a peptide with amino acid sequences of R.SRDGLTLVSYLTLPR.E.The partial gene sequence(about 400 bp)was cloned from genomie DNA of M.morganii ZJB-09203 by PCR,which using the primers designed based on the LC-MS-MS results.And the partial sequence was verified through Blast in NCBI.