Abstract:

In order to improve the efficiency of cellulase production， the fermentation medium and culture conditions of Bacillus subtilis engineered strains C36， CM， KF and GH5 were optimized by single factor test and orthogonal test. The results showed that the enzyme production efficiency of engineered strains C36， CM， KF and GH5 was significantly improved under the optimal fermentation conditions. After optimizing the fermentation conditions， the cellulase activity of the engineered strain CM was the highest. Under the conditions of 7% of aged tobacco leaves， 0.8% of peptone， pH 6.1， fermentation temperature 37 ℃， rotation speed 220 r·min^-1， liquid medium volume 40 mL and fermentation for 96 h， the enzyme activities of carboxymethylcellulose （CMC） and filter paper activity （FPA） of the engineered strain CM were 504.95 and 222.11 U·mL^-1， respectively. After optimizing the fermentation conditions， the increase of cellulase activity of the engineered strain GH5 was the highest. Under the conditions of 6% of aged tobacco leaves， 0.9% of peptone， pH 6.1， fermentation temperature 37 ℃， rotation speed 220 r·min^-1， liquid medium volume 40 mL and fermentation for 96 h， the enzyme activities of CMC and FPA was 28.55 and 9.68 times higher than that before optimization.

Key words: cellulase, engineered strains, fermentation conditions, orthogonal design

摘要：

为提高工程菌株纤维素酶产酶效率，采用单因素试验和正交试验法对高产纤维素酶枯草芽孢杆菌（Bacillus subtilis）工程菌株C36、CM、KF、GH5的发酵培养基和培养条件进行优化。结果表明，工程菌株C36、CM、KF和GH5在最优发酵条件下的产酶效率显著提高。发酵条件优化后，工程菌株CM的纤维素酶活最高，在醇化烟叶添加量7%、蛋白胨添加量0.8%、pH 6.1、发酵温度37 ℃、转速220 r·min^-1、装液量40 mL及发酵时间96 h的条件下，其羧甲基纤维素（carboxymethylcellulose, CMC）酶活和滤纸酶活（filter paper activity, FPA）分别达到504.95和222.11 U·mL^-1。发酵条件优化后，工程菌株GH5的纤维素酶活提升倍数最高，在醇化烟叶添加量6%、蛋白胨添加量0.9%、pH 6.1、发酵温度37 ℃、转速220 r·min^-1、装液量40 mL及发酵时间96 h的条件下，其CMC和FPA酶活分别是优化前的28.55和9.68倍。

关键词: 纤维素酶, 工程菌株, 发酵条件, 正交设计