重组共表达大肠杆菌细胞从D-葡萄糖一锅法生产D-甘露糖

【背景】D-甘露糖的酶促转化方法已受到相当大的关注。【目的】研究D-葡萄糖异构酶(D-glucoseisomerase,D-GIase)和D-来苏糖异构酶(D-lyxoseisomerase,D-LIase)共表达于大肠杆菌细胞生产D-甘露糖的工艺条件。【方法】将D-GIase和D-LIase基因片段合成后酶切连接到载体p CDFDuet-1上,构建p CDFDuet-Acce-DGI/Peba-DLI重组质粒并导入到大肠杆菌BL21(DE3)中共表达,通过摇瓶培养得到产D-GIase和D-LIase的菌体,测定该共表达细胞体系的反应条件。【结果】添加1 mmol/L Co~(2+),共表达体系酶的最适温度和p H分别为70°C和6.0。以浓度分别为100、300、500 g/L的D-葡萄糖为底物生产D-甘露糖,平衡后D-甘露糖质量浓度分别为13.8、38.1、62.6 g/L,相应的转化率分别为13.8%、12.7%、12.5%,D-葡萄糖、D-果糖和D-甘露糖的平衡比约为50:37.5:12.5。【结论】D-GIase和D-LIase在大肠杆菌细胞中组成的共表达体系通过一锅法可利用D-葡萄糖为底物生产D-甘露糖。

One-pot production of D-mannose from D-glucose by recombinant co-expressing Escherichia coli cells

[Background] Enzymatic transformation for production of D-mannose has attracted considerable attention. [Objective] The production conditions of D-mannose were investigated using the co-expressed E. coli cells harboring D-glucose isomerase (D-GIase) and D-lyxose isomerase (D-LIase). [Methods] The synthesized D-GIase and D-LIase gene fragments were digested and ligated into the vector pCDFDuet-1. Then, the resultant recombinant plasmid pCDFDuet-Acce-DGI/Peba-DLI was transformated into E. coli BL21(DE3) strain to co-express the two enzymes. After collecting the cells co-expressing D-GIase and D-LIase by shake flask culture, the reaction conditions of the co-expression cells were determined. [Results] The optimal temperature and pH of enzymes in the co-expressing system were 70 °C and 6.0 in the presence of Co2+ (1 mmol/L), respectively. After the reaction reached equilibrium, 13.8, 38.1 and 62.6 g/L of D-mannose were obtained from 100, 300 and 500 g/L of D-glucose, which corresponded to a conversion of 13.8%, 12.7% and 12.5%, respectively, and the equilibrium ratio of D-glucose, D-fructose and D-mannose was about 50?37.5?12.5. [Conclusion] A co-expression system consisted of D-GIase and D-LIase in E. coli cells can be used for one-pot production of D-mannose from D-glucose.