摩氏摩根菌ZJB-09203酯酶的分离纯化及性质

Purification and characterization of esterase from Morganella morganii ZJB-09203

摘要:

立体选择性水解2-羧乙基-3-氰基-5-甲基己酸乙酯 (CNDE) 是化学-酶法合成重大疾病治疗药物普瑞巴林的关键步骤。分离纯化了能够高效水解S-型CNDE的摩氏摩根菌ZJB-09203胞内酯水解酶，并进行酶学性质研究。采用硫酸铵分级沉淀、Phenyl Sepharose 6 FF疏水柱层析、DEAE Sephadex A-50 阴离子交换和羟基磷灰石Bio-Scale CHT层析，纯化得到电泳纯的酯水解酶。SDS-PAGE和凝胶过滤HPLC分析确定该酶为单亚基蛋白，分子量为68 kDa。不同碳链长度p-硝基苯酚酯特异性酶解结果表明，该酶为酯酶，酶促合成普瑞巴林手性中间体(S)-2-羧乙基-3-氰基-5-甲基己酸的最适 pH 为9.0，最适温度为45 ℃，且在 pH 7.0?9.0和40 ℃条件下具有良好的稳定性。Ca2+、Cu2+、Mn2+对酶活有一定的促进作用，Co2+、Fe3+、Ni2+及EDTA对酶活有较强的抑制作用。此外，考察了该酯酶水解p-硝基苯酚酯的动力学参数，及CNDE浓度对转化率的影响。首次报道了能够立体选择性水解CNDE的酯酶，相关酶学性质研究将为该酶催化合成普瑞巴林手性中间体的工业化应用提供重要依据。

Abstract:

Enantioselective hydrolysis of 2-carboxyethyl-3-cyano-5-methylhexanoic acid (CNDE) is the key step in chemoenzymatic synthesis of pregabalin. We purified an intracellular carboxyl esterase from Morganella morganii ZJB-09203, which exhibited high enantioselectivity and activity towards CNDE. The carboxyl esterase was purified to electrophoretic homogeneity by ammonium sulfate fraction precipitation, Phenyl Sepharose 6 FF hydrophobic interaction chromatography, anion exchange with DEAE Sephadex A-50 and Bio-Scale CHT column. The purified enzyme was a monomer with molecular mass of 68 kDa determined by SDS-PAGE and gel chromatography. Substrate specificity of the enzyme towards p-nitrophenyl esters suggested that the purified enzyme was an esterase. The optimal reaction pH for CNDE hydrolysis was 9.0, and optimal temperature was 45 ℃. The esterase was stable between pH 7.0 and 9.0, and at 40 ℃. The enzyme activity was enhanced by Ca2+, Cu2+ and Mn2+, whereas strongly inhibited by Co2+, Fe3+, Ni2+ and EDTA. Meanwhile, we investigated the kinetic parameters of the esterase towards p-nitrophenyl esters and effect of CNDE concentration on conversion.The present study reported the esterase capable of stereospecific hydrolysis of CNDE for the first time. Our research will provide foundations for industrial production of Pregabalin using the new biocatalyst.