茶氨酸生物合成工程菌构建.pdfVIP

茶 叶 科 学 2007 ，27 （1 ）：61~66 Journal of Tea Science 茶氨酸生物合成工程菌构建 王贤波，王丽鸳，成浩* ，周健，林智 （农业部茶叶化学工程重点实验室，中国农业科学院茶叶研究所，浙江 杭州 310008 ） 摘要：通过 PCR 扩增 E.coli DH5α 的γ-ggt 基因，产物经纯化后用 Kpn I 和 Xh o I 双酶切，回收γ-谷氨酰转肽酶 基因目的片断，并与经相同双酶切的表达载体 pET-32a 连接，得到重组质粒 pET-GGT 。将重组质粒转化到 E.coli BL21 中，获得工程菌。工程菌株经 0.05 mol/L IPTG ，32 ℃诱导表达，湿菌体的酶活达到 2.0 U/g ，大约是出发 菌株 E.coli DH5α 的 15 倍。工程菌催化 L-谷氨酰胺和盐酸乙胺反应生成茶氨酸的产量达到 29.40 g/L ，L-Gln 的转化率为 48.22% ，其催化 L-谷氨酰胺和盐酸乙胺反应生成茶氨酸的能力比出发菌株 E.coli DH5α提高了 100 多倍。 关键词：γ-谷氨酰转肽酶；茶氨酸；工程菌；构建 中图分类号：S571.1 ；Q523 文献标识码：A 文章编号：1000-369X （2007 ）01-061-06 The Construction of the Engineered Escherichia coli Strain for the Biosynthesis of Theanine * WANG Xian-bo, WANG Li-yuan, CHENG Hao , ZHOU Jian, LIN Zhi (Key Lab of Tea Chemical Engineering, Ministry of Agriculture; Tea Research Institute, Chinese Acade my of Agricultural Sciences, Hangzhou 310008, China) Abstract : γ-ggt was cloned by PCR from E.coli DH5α. Then, the PCR product of γ-ggt digested with two restriction enzymes, Kpn I and Xh o I, was purified and ligated with the pET-32a vector digested with the same enzymes by T4 DNA ligase. Then the ligation product was transformed to E.coli BL21 and the engineered Escherichia coli strain was constructed successfully. The γ-Glutamyltranspeptidase was expressed with induction of 0.05 mmol/L IPTG in 32 ℃ incubation. The activity of 1 g fresh cells of engineered strain was 2.0 U/g ，it is about 15 times higher than that of E.coli DH5α ．Under the catalysis of cells of the engineered strain induced with IPTG, the yield of theanine from L-Gln and ethylamine was 29.40 g/L and the conversion rate of L-Gln as to thea