East China University of Science and Technology, Shanghai, People's Republic of China.
Appl Microbiol Biotechnol. 2011 Feb;89(3):665-72. doi: 10.1007/s00253-010-2866-y. Epub 2010 Sep 23.
The production of a recombinant nitrilase expressed in Escherichia coli JM109/pNLE was optimized in the present work. Various culture conditions and process parameters, including medium composition, inducer, induction condition, pH and temperature, were systematically examined. The results showed that nitrilase production in E. coli JM109/pNLE was greatly affected by the pH condition and the temperature in batch culture, and the highest nitrilase production was obtained when the fermentation was carried out at 37°C, initial pH 7.0 without control and E. coli was induced with 0.2 mM isopropyl-β-D-thiogalactoside at 4.0 h. Furthermore, enzyme production could be significantly enhanced by adopting the glycerol feeding strategy with lower flow rate. The enzyme expression was also authenticated by sodium dodecyl phosphate polyacrylamide gel electrophoresis analysis. Finally, under the optimized conditions for fed-batch culture, cell growth, specific activity and nitrilase production of the recombinant E. coli were increased by 9.0-, 5.5-, and 50-fold, respectively.
本工作优化了在大肠杆菌 JM109/pNLE 中表达的重组腈水解酶的生产。系统研究了各种培养条件和工艺参数，包括培养基组成、诱导剂、诱导条件、pH 值和温度。结果表明，大肠杆菌 JM109/pNLE 中的腈水解酶生产受分批培养中 pH 值条件和温度的影响很大，当发酵在 37°C、初始 pH 值为 7.0 且不进行控制以及大肠杆菌在 4.0 h 时用 0.2 mM 异丙基-β-D-硫代半乳糖苷诱导时，腈水解酶的产量最高。此外，采用较低流速的甘油补料策略可以显著提高酶的产量。通过十二烷基硫酸钠聚丙烯酰胺凝胶电泳分析也验证了酶的表达。最后，在补料分批培养的优化条件下，重组大肠杆菌的细胞生长、比活和腈水解酶的产量分别提高了 9.0 倍、5.5 倍和 50 倍。
