E.coli酪氨酸转氨酶的纯化与非天然氨基酸的底物特异性检测  

Purification of E.coli tyrosine transferase and detection of unnatural amino acid substrate specificity

作　　者：陈茹[1] 王行国[1] 何婷[1] 张萍萍[1] 

机构地区：[1]湖北大学生命科学学院,湖北武汉430062

出　　处：《湖北大学学报（自然科学版）》2010年第2期210-213,共4页Journal of Hubei University：Natural Science

基　　金：国家自然科学基金(30570009);湖北大学重点基金资助项目资助

摘　　要：构建重组菌株E.coliBL21(DE3)转pET23a-tyrB,IPTG诱导其大量表达,然后依次通过盐析、离子交换层析、疏水层析、凝胶过滤层析获得纯酶,运用薄层层析技术(TLC)检测,结果表明纯化的TyrAT转氨酶对自身底物酪氨酸在37℃和50℃具有较高活性.分别加入非天然氨基酸L-正缬氨酸、L-新戊基甘氨酸、L-叔亮氨酸、D-正亮氨酸作为底物,检测到TyrAT转氨酶对L-叔亮氨酸在50℃下有活性,对D-正亮氨酸在37℃和50℃均有催化活性,且50℃下效果更好.E.coli tyrosine transferase gene tyrB was cloned,then transformed it into E.coli BL21（DE3） via vector pET23a,under the induction of IPTG and lysis of cell,the crude enzyme TyrAT was gotten.Then via salting out,ion exchange chromatography,hydrophobic interaction chromatography,gel filtration chromatography orderly,pure active transaminase was obtained.By using thin-layer chromatography,the transaminase activity was detected with its own substrate at 37 ℃ and 50 ℃,then added L-norvaline,L-neopentylglycine,L-tertary leucine and D-norleucine as substrates.The pure TyrAT could catalyze L-tertary leucine and D-norleucine at 50 ℃,the pure TyrAT also catalyzed D-norleucine at 37 ℃,although was weaker than that at 50 ℃.This result laid the foundation for construction of tyrosine transferase engineering bacteria with unnatural amino acid activity.

关 键 词：酪氨酸转氨酶 纯化 非天然氨基酸 薄层层析 

分 类 号：Q786[生物学—分子生物学] Q814.1