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  

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

作　　者：吴昊 黄嘉苇 张文立 沐万孟[1,2] WU Hao;HUANG Jia-Wei;ZHANG Wen-Li;MU Wan-Meng(State Key Laboratory of Food Science and Technology,Jiangnan University,Wuxi,Jiangsu 214122,China;International Joint Laboratory on Food Safety,Jiangnan University,Wuxi,Jiangsu 214122,China)

机构地区：[1]江南大学食品科学与技术国家重点实验室,江苏无锡214122 [2]江南大学食品安全国际合作联合实验室,江苏无锡214122

出　　处：《微生物学通报》2020年第7期2151-2160,共10页Microbiology China

基　　金：国家自然科学基金(31922073,31801583);江苏省自然科学基金(BK20180607)。

摘　　要：【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 Co2+,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。[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.

关 键 词：一锅法 异构酶 D-葡萄糖 D-甘露糖 共表达 大肠杆菌 

分 类 号：TS218[轻工技术与工程—粮食、油脂及植物蛋白工程]