大肠杆菌来源的喹啉酸磷酸核糖转移酶和烟酸磷酸核糖转移酶的表达纯化及酶活性的初步检测

摘要

[Objective] The expression, purification of quinolinic acid phosphoribosyl trans-ferase (QPRT) and nicotinic acid phosphoribosyl transferase (NaPPT) from Escherichia coli (E. Coli) were undertaken in prokaryotic expression system and activity detection of QPRT and NaPPT in vitro. [Methods] The NadC encoding QPRT and PncB encoding NaPPT were cloned and then ligated into pET28a and Prsetb to generate the expression plasmid pET28a-iNadC and Prsetb-PncB, respectively. The plasmids were expressed in E. Coli BL21(DE3) and enzymes were purified in vitro. Enzymes activity were detected by analyzing the reaction mixture on a high performance liquid chromatography (HPLC) system. [Results] QPRT and NaPPT were expressed and purified, the results indicate that quinolinic acid can be transformed into nicotinic acid through regioselective decarboxylation catalyzed by recombinant QPRT and NaPPT sequentially.%[目的] 在原核表达体系中实现大肠杆菌来源的喹啉酸磷酸核糖转移酶(Quinolinic acid phosphoribosyl transferase,QPRT)和烟酸磷酸核糖转移酶(Nicotinic acid phosphoribosyl transferase,NaPPT)的表达与纯化,并利用酶的生物催化作用实现2,3-二羧酸喹啉的2位选择性脱羧得到烟酸.[方法] 通过PCR扩增分别得到编码QPRT和NaPPT 的基因片段,构建成原核表达质粒pET28a-NadC和pRSETB-PncB,在Escherichia coli (E.coli)中对其进行表达,在体外对目标蛋白进行纯化并利用高效液相色谱法(HPLC)检测酶催化反应的发生.[结果] 成功表达纯化得到QPRT和NaPPT,检测结果表明在这两个酶的生物催化作用下可实现喹啉酸的2位选择性脱羧.