微生物谷氨酰胺转胺酶的乳糖诱导表达与纯化

Lactose Induction Expression and Purification of Microbial Transglutaminase

对已构建的工程菌株pET22b-MTG/E.coli BL21(DE3)进行质粒酶切和PCR鉴定,优化诱导温度、时间、pH和乳糖浓度等反应条件,采用乳糖自诱导培养基培养重组菌.并将重组菌上清液经凝胶过滤,离子交换及镍柱亲和层析后,对谷氨酰胺转胺酶纯化和SDS-PAGE电泳分析.结果表明:谷氨酰胺转胺酶在28℃,添加乳糖为1.2g/L诱导18 h,pH为7.0,培养11.5 h时升温至50℃诱导1 h,再放至28℃培养,表达效果较好,超过IPTG诱导效果.纯化后酶活为7.5 U/mL,比活力为10.9 U/mg.

An engineering strain pET22b-MTG/E.coli BL21（DE3） was constructed and identified by plasmid digestion and PCR.The lactose inducing temperature,time,pH,concentration and other conditions were optimized to cultivate recombinant strain.Then its supernatant was purified by gel filtration,ion exchange and Ni-affinity column chromatography and analysised by SDS-PAGE electrophoresis.The results indicateded that transglutaminase was expressed more than induced by IPTG under such condition：28℃,1.2 g/L lactose,inducing 18h,pH 7.0,at 11.5 h up to 50℃ for 1 h,then down to 28℃.The activity of transglutaminase reached 7.5 U/mL and specific activity was 10.9 U/mg after purification.