茶树CCoAOMT原核表达及酶促条件优化

Prokaryotic expression of CCoAOMT from Camellia sinensis and optimization of enzymatic conditions

优化了重组茶树咖啡酰辅酶A–O–甲基转移酶基因(CCoAOMT)在大肠杆菌中的表达,采用Ni–NTA亲和层析柱对重组蛋白进行纯化。以表没食子儿茶素没食子酸酯(EGCG)为底物,进行体外酶促反应,采用HPLC测定反应产物EGCG3"Me的生成量。结果表明,CCoAOMT在E.coliBL21中能够高效表达,最优条件为诱导温度37℃,诱导时间4 h,异丙基–β–D–硫代半乳糖苷(IPTG)浓度0.5 mmol/L;重组蛋白经Ni–NTA亲和层析柱梯度洗脱达到了较好的纯化效果;重组酶在体外能够催化EGCG,合成EGCG3"Me,底物EGCG最佳浓度为0.05 mmol/L,最适温度为37℃,最适pH值为4。

The expression of the recombinant caffeinyl-coenzyme A O-methyltransferase gene(CCoAOMT)in E.coli cells was optimized and the enzyme was purified by Ni-NTA affinity chromatography.The enzymatic reaction was carried out in vitro with epigallocatechin gallate(EGCG)as substrate,and EGCG3"Me,the reaction products,were analyzed by HPLC.As a result,CCoAOMT was highly expressed in E.coli BL21 and the best induction conditions of recombinant CCoAOMT was 0.5 mmol/L IPTG at 37℃for 4 h.The recombinant protein was purified by Ni-NTA affinity chromatography.The recombinant CCoAOMT could catalyze EGCG to EGCG3"Me in vitro and the optimal reaction conditions include substrate EGCG of 0.05 mmol/L,pH of 4 and temperature of 37℃.