嘌呤核苷磷酸化酶温敏型表达载体的构建、表达及应用于腺苷转化的研究

Construction and Expression of Purine Nucleoside Phosphorylase Temperature Inducible Expression Vector and Study on Its Application to Conversion of Adenosine

通过双酶切方法将重组质粒上大肠杆菌嘌呤核苷磷酸化酶(PNPase)基因克隆到pBV220表达载体上,构建了温敏型PNPase的表达载体pBV 220-PNP,转化大肠杆菌DH5α并使之表达.SDS-PAGE电泳结果显示出明显的特异表达条带,其分子量约为26 kDa.在此基础上研究了不同诱导条件对蛋白表达量的影响.分别以肌苷和尿苷为核糖供体研究重组菌转化腺苷的条件,发现肌苷最适合作为核糖供体,在30 mmol.L-1pH 7.5的Na2HPO4-KH2PO4缓冲液中,以30 mmol.L-1肌苷和10 mmol.L-1腺嘌呤作底物,加入0.5%基因工程湿菌体60℃反应3 h,腺苷的转化率为85.57%.

The gene of purine nucleoside phosphorylase （PNPase） of E. coli was subcloned into the temperature inducible expression vector pBV 220 to construct the recombinant plasmid pBV 220-PNP and transformed and expressed in DH5α. The optimum culturing and inducing times were determined by SDS-PAGE analysis. The construction of recombinant PNPase provided a basis for the research on biocatalysed synthesis of adenosine. In comparison with uridine, inosine was the better donar of pentose -1- phosphorate. In the reaction mixture composed of 30 mmol ·L^-1inosine, 10 mmol ·L^-1 adenine, and 30 mmol ·L^-1 pH 7.5 phosphate buffer and 0.5% wet ceils gave a high yield of 85.57% at 60℃ in 3 h.