菌株TP-3中基因pgmG的克隆、表达及酶活性分析  

Cloning,Expression and Analysis Activity of Gene pgmG from Strain TP - 3

作　　者：王生秀[1] 黄海东[1] 李晓雁[2] 刘梦虹[1] 

机构地区：[1]天津农学院农学系,天津300384 [2]天津农学院食品科学系,天津300384

出　　处：《生物技术》2013年第3期20-24,共5页Biotechnology

基　　金：天津市科委基础重点项目("新型鞘氨醇胶合成关键基因的研究";11JCZDJC16600);天津农学院大学生科技创新活动项目("鞘氨醇单胞菌TP-3中葡萄糖磷酸变位酶的基因克隆;表达及酶活性分析";B12-2)资助~~

摘　　要：目的:克隆得到鞘氨醇单胞菌TP-3的葡萄糖磷酸变位酶基因全长,表达并验证其酶活性,为下一步菌株TP-3的基因工程改造奠定基础。方法:利用Tail PCR的方法扩增得到菌株TP-3的pgmG基因全长,通过pET-28a(+)大肠杆菌表达系统表达目的基因,优化诱导表达条件,并测定酶活性。结果:扩增得到1 383bp的pgmG基因全长,编码460个氨基酸,诱导表达后得到一个分子量约为49.8kDa的目的蛋白,纯化后的酶活为5 985 U/g。结论:首次得到新菌Sphingomonas sanxanigenenssp.nov.TP-3的pgmG基因全长,其编码蛋白PgmG与温轮胶合成菌Sphingomonas sp.NX-3的葡萄糖磷酸变位酶同源性最高,达81%,在大肠杆菌中表达目的基因并验证了葡萄糖磷酸变位酶活性,经Ni-NTA纯化后酶收率为29.3%。Objective： Cloning the full - length gene of phosphoglucomutase from Sphingomonas sanxanigenens sp. nov. TP - 3, and confir- ming the enzyme activity for the gene, that was the foundation for further genetic reconstruction research of Strain TP - 3. Method ：The pg- mG gene of TP -3 was obtained by Tail PCR, and was expressed in Escherichia coli by prokaryotie expression system, pET- 28a（ ＋ ）.Expressing conditions was optimized. At last, its enzyme activity was assayed. Result： The pgmG gene coding region which includes 1 383bp and encodes 460 amino acids was obtained from S. sanxanigenens sp. nov. TP - 3, and a 49. 8kDa protein was successfully ex- pressed. The purified enzyme activity is 5 985 U/g. Conclusion：The full -length gene ofpgmG was obtained from S. sartxanigenens sp. TP -3 for the first time. PgmG from S. sanxanigenens showed highest 81% sequence identity with phosphoglucomutase from Sphingomonas sp. NX - 3. Phosphoglucomutase activity was confirmed in E. coli overexpressed PgmG, and the enzyme was purified by Ni - NTA with a 29. 3% yield.

关 键 词：鞘氨醇单胞菌 鞘氨醇胶 葡萄糖磷酸变位酶 克隆表达 

分 类 号：Q812[生物学—生物工程]