共表达磷脂酶C促进葡萄糖异构酶在大肠杆菌中的胞外表达

Co-Expression of Phospholipase C and Glucose Isomerase Which Promoting the Extracellular Expression of Glucose Isomerase in E.coli

DOI：10.3969/j.issn.1673-1689.2017.03.002

中文关键词: 磷脂酶C，葡萄糖异构酶，共表达，胞外表达

英文关键词: phospholipase C,glucose isomerase,co-expression,released into culture medium

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中文摘要:

磷脂酶C（PLC）能够水解细胞膜主要成分磷脂，使细胞膜通透性增强，从而能够释放出胞内物质。作者构建了PLC与天然胞内定位蛋白葡萄糖异构酶（GI）共表达的重组大肠杆菌，摇瓶发酵胞外上清液中GIC酶活达到3.4 U/mL，占胞外和胞内总酶活的93%，表明GIC成功实现了胞外表达。将胞外上清液中的GIC进行分离纯化和酶学定性，发现其比活为12.1 U/mg，最适反应温度为80 ℃，最适pH为10，均与对照菌单独表达的GIO性质基本一致。在此基础上，对上述重组菌进行3 L发酵罐培养，发酵周期为24 h，酶活达到17.7 U/mL，表明其良好的工业化放大生产前景。

英文摘要:

Phospholipase C（PLC） hydrolyzes the phospholipids in the membrane. The hydrolysis was partial，which would enhance the cell membrane permeability，and then proteins in the cytoplasm were released into culture medium. In this study，PLC was co-expressed with glucose isomerase （GI）. In shake flask fermentation of recombinant E. coli BL21 （DE3），the activity of GIC in culture supernatant was 3.4 U/mL，which was 93% of the total enzyme activity in culture supernatant and cytoplasm. GIC was released into culture medium. The GIC was purified and characterized. The specific activity of GIC was 12.1 U/mg，the optimum temperature was 80 ℃，and the activity of GIC was maximal at pH 10. The characteristics of GIC were similar to GIO，In addition，The enzyme activity reached 17.7 U/mL in 24 hours by utilizing fed-batch strategy in 3 L fermentor.

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