摘要
通过PCR扩增E.coliDH5α的γ-ggt基因,产物经纯化后用KpnI和XhoI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.coliBL21中,获得工程菌。工程菌株经0.05mol/LIPTG,32℃诱导表达,湿菌体的酶活达到2.0U/g,大约是出发菌株E.coliDH5α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29.40g/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.coliDH5α提高了100多倍。
γ-ggt was cloned by PCR from E.coli DH5a. 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 0.05 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 DH5a. Under the catalysis of cells of the engineered strain induced with IPTG, the yield of theanine from L-GIn and ethylamine was 29.40 g/L and the conversion rate of L-GIn as to theanine being 48.22%; it is about 100 times higher than that of E.coli DH5a.
出处
《茶叶科学》
CAS
CSCD
北大核心
2007年第1期61-66,共6页
Journal of Tea Science
关键词
Γ-谷氨酰转肽酶
茶氨酸
工程菌
构建
γ-Glutamyltranspeptidase, theanine, engineered strain, construction