产腈水解酶基因工程菌的产酶诱导条件及培养基优化

摘要

In this study, the production of nitrilase by using recombinant strain E. coli BL21 (DE3) –pETNYNit was improved by optimization of induced conditions and fermentation medium. The results indicated that the optimal medium were as follows:glucose 0. 2%, glycerol 0. 7% (v/v), peptone 1. 2%, yeast extract 0. 8%, NaCl 0. 3%, (NH4)2SO4 0. 3%, NH4Cl and 0. 13%, Na2HPO4 ·12H2O 1. 04%, KH2PO4 0. 39%, MgSO4 ·7H2O 0. 03%, pH 7. 2; the optimal induced conditions were the following: 0. 5 mmol/L IPTG induced expression after fermented for 4 hours, and then cultured for 14 hours to 16 hours at 28℃ and 240 r/min. After optimization, the nitrilase activity of the recombinant strain increased to (1~0. 9) ×105 U. Compared with that of the wild strain, the enzyme activity increased by more than 50%. At the same time, the culture time of the recombinant strain was about 24 hours, reduced more than 50 hours.%本文通过对产酶诱导条件及发酵培养基进行优化,成功提高了产腈水解酶基因工程菌E. coli BL21(DE3)-pETNYNit的产酶水平。研究结果显示,最佳发酵培养基为：葡萄糖0.2%、甘油0.7%(v/v)、蛋白胨1.2%、酵母膏0.8%、NaCl 0.3%、(NH4)2SO40.3%、NH4Cl 0.13%、Na2 HPO4·12H2 O 1.04%、KH2 PO40.39%、MgSO4·7H2 O 0.03%,pH 7.2。最佳产酶诱导条件为：发酵4 h时加入0.5 mmol/L IPTG,然后在28℃、240 r/min下诱导腈水解酶基因表达14 h~16 h。采用优化方案,重组菌产酶水平可提升至0.9~1×105 U,与野生菌株的产酶水平相比,提高幅度超过50%。同时重组菌培养仅需24 h,培养周期缩短超过50 h。