中性蛋白酶高产菌株的诱变选育

Mutation and Screening of Neutral Protease High-yield Strain

对产中性蛋白酶的枯草芽孢杆菌（Bacillus subtilis） SHB 2010-1进行物理和化学诱变以筛选高产菌株。考察了6种筛选培养基在分辨中性蛋白酶高产菌株上的效率，菌株在脱脂牛奶培养基上生长状态最佳，水解圈易于观察。以紫外线、硫酸二乙酯为诱变剂，研究了两者的致死率曲线，选择致死率为90~95%的剂量进行两轮复合诱变，诱变菌的发酵液酶活依次较出发菌提高1.49%、10.99%、38.77%和59.68%，复合诱变表现出较单一诱变更强的效应，并能利用弱诱变剂紫外线积累的隐性突变。最终筛选得到的菌株命名为DES-59，在50 mL发酵培养基中培养60 h酶活达到7307 U/mL，相应的生物量为2.205×1010 CFU/mL，表现出典型的生长偶联型。突变菌的nprE基因经克隆及测序与数据库上的序列一致，酶活性的提高并非由中性蛋白酶A结构改变引起，突变出现在生产蛋白酶的其他位点上，有利于潜在性能的提高。

The biological effects of physical and chemical mutagenesis inducements on neutral protease produced by Bacillus subtilis SHB 2010-1 were investigated. Six selective media were studied on screening the efficiency of high-yield strain. Skim milk agar was proved to be the excellent cell culture medium, with which the hydrolysis zone was easily observed. On the base of the corresponding death curves, death rate at 90-95% was selected to take two-round ultraviolet-diethyl sulfate mutation. The enzyme activities of mutant strains from each step were 1.49%, 10.99%, 38.77% and 59.68%,respectively, higher than the original strain. The results showed that complex mutagenesis performed a more significant effect than single factor. The mutant strain named DES-59 were finally screened with the maximum enzyme activity of 7307 U/mL in 50 mL fermentation medium after 60 h, and the cell density of 2.205×1010 CFU/mL. The nprE gene of the mutant DES-59 was the same as sequence in database, indicating that the improvement was not contributed by the structural modification of neutral protease A, but by some unstated sites.