促进氰钴胺素转化为腺苷钴胺素的基因表达与鉴定

辅酶B12作为甘油脱水酶(GDHt)的辅酶参与到Klebsiella pneumoniae代谢甘油生成1,3-丙二醇(1,3-PD)和3-羟基丙酸(3-HP)的途径中.前期研究表明底物甘油可以导致辅酶B12分子中Co—C键的断裂,进而引起GDHt的失活.为进一步研究非活性的维生素B12(CNCbI)转化为具有活性的辅酶B12(AdoCbI)的过程,从K.pneumoniae中克隆得到了ATP:钴(I)胺素腺苷转移酶(ACA)基因btuR和还原酶基因yciK,并成功构建了双启动子表达质粒pUC18-tac-btuR-tac-yciK,将表达质粒转化Escherichia coli JM109获得了一株重组菌.利用改进的分析方法,结果表明:1)重组蛋白具有腺苷转移酶的活性;2)重组菌可以很好地将非活性的钴胺素,如维生素B12,转化生成具有活性的辅酶B12.

Facilitated Expression and Function Identification of Key Genes Converting Cyanocobalamin to Adenosylcobalamin

Klebsiella pneumoniae degrades glycerol generating 1,3-propanediol(1,3-PD) and 3-hydroxypropionic acid(3-HP) by a pathway dependent on coenzyme B12.Previous studies showed that glycerol could cause the irreversible cleavage of Co— C of coenzyme B12,inactivating glycerol dehydratase(GDHt).For further study on the course of converting inactive vitamin B12(CNCbI) to active coenzyme B12(AdoCbI),ATP:cob(I) alamin adenosyltransferase(ACA) gene btuR and dehydrogenase gene yciK from K.pneumoniae were cloned and a dual-promoter expression plasmid pUC18-tac-btuR-tac-yciK,including gene btuR and yciK,were successfully constructed.Finally,a strain of recombinant bacterium was obtained by transferring the plasmid into Escherichia coli JM109.The results of the improved analytical methods showed that(i) overexpressed proteins had cob(I) alamin adenosyltransferase activity;(ii) the recombinant E.coli JM109 could convert inactive cobalamin,such as vitamin B12,to active coenzyme B12very well.