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构建了一株能高效表达嘌呤核苷磷酸化酶(PNPase)的重组大肠埃希菌Escherichia coli BL21(p ET-His-pup G)。选择不同包埋剂进行细菌细胞固定化,结果显示明胶的包埋效果较好。进一步确定明胶包埋细胞的最适反应条件:温度55℃,菌体包埋浓度200 g/L,固定化细胞用量20 g/L,催化周期20 h。在p H 7.6的磷酸盐缓冲液中,底物鸟苷和1,2,4-三唑-3-甲酰胺(TCA)浓度均为100 mmol/L,采用上述固定化细胞催化合成利巴韦林,连续反应8次后,固定化细胞仍具有较高的酶活力。
A recombinant Escherichia coli BL21 (p ET-His-pup G) was constructed to express purine nucleoside phosphorylase (PNPase) efficiently. Select different embedding agent for bacterial cell immobilization, the results show that gelatin embedding better. The optimal reaction conditions of gelatin-embedded cells were further confirmed: the temperature was 55 ℃, the concentration of bacterial cells was 200 g / L, the amount of immobilized cells was 20 g / L, and the catalytic cycle was 20 h. The concentration of substrate guanosine and 1,2,4-triazole-3-carboxamide (TCA) was 100 mmol / L in p H 7.6 phosphate buffer. The immobilized cells were used to synthesize ribavirin Lin, continuous reaction 8 times, the immobilized cells still have a high enzyme activity.