通过PCR扩增E.coli DH5α的γ-ggt基因,产物经纯化后用Kpn I和Xho 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0.05βmol/L IPTG,32℃诱导表达,湿菌体的酶活达到2.0βU/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29.40βg/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。
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 Xho 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 0.05βmmol/L IPTG in 32℃ incubation. The activity of 1βg fresh cells of engineered strain was 2.0βU/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 29.40βg/L and the conversion rate of L-Gln as to theanine being 48.22%; it is about 100 times higher than that of E.coli DH5α．
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