产亚硝酸还原酶基因工程菌pET-28a (+)-<i>nir</i>-BL21的培养基优化

彭鑫; 黄燕燕; 赵珊; 孙丽娜; 陈思敏; 肖弘毅; 刘冬梅

doi:10.13386/j.issn1002-0306.2020.10.014

产亚硝酸还原酶基因工程菌pET-28a (+)-nir-BL21的培养基优化

Optimization of Culture Medium for Nitrite Reductase Produced by Genetic Engineering Bacteria E.coli pET-28a(+)-nir-BL21

摘要

摘要: 从豆瓣酱中筛选的一株能高效降解亚硝酸盐的蜡样芽孢杆菌(Bacillus cereus)LJ01,将其nir基因克隆至相应的表达载体上,构建了重组菌pET-28a(+)-nir-BL21。为提高基因工程菌pET-28a(+)-nir-BL21的产亚硝酸盐还原酶(NiR)水平,对重组菌pET-28a(+)-nir-BL21的培养基组成通过单因素实验、Plackett-Burman和Box-Behnken试验优化。结果表明,获得最佳培养基为:以液体LB培养基为基础培养基,再添加葡萄糖3.72 g·L^-1、甘油2.63 g·L^-1和细菌学蛋白胨8.70 g·L^-1,活菌数预测值为3.14×10⁸ CFU·mL^-1,通过验证得3.02×10⁸ CFU·mL^-1,与预测值接近。本实验构建的高产亚硝酸盐还原酶的菌株,将为日后降解食品中的亚硝酸盐进行工业化应用提供理论基础。

Abstract: Bacillus cereus LJ01,isolated from bean paste with efficient nitrite degradation was used to construct the recombinant strain pET-28a(+)-nir-BL21 with the corresponding expression vectors of nir gene. In order to improve the production of nitrite reductase(NiR)of pET-28a(+)-nir-BL21,the composition of culture medium was preliminarily optimized. The optimum medium conditions were obtained by single factor test,Plackett-Burman and Box-Behnken experiment. The results showed that the optimum medium were:The liquid LB medium as the base medium,followed by 3.72 g·L^-1 glucose,2.63 g·L^-1 glycerol and 8.70 g·L^-1 peptone. The verified number of viable cells was 3.02×10⁸ CFU·mL^-1,which was close to the predicted value(3.14×10⁸ CFU·mL^-1). The high-yield nitrite reductase strain constructed in this experiment would provide a theoretical basis for industrial application of nitrite in food degradation in the future.