为了筛选高产蛋白酶活性细菌，以大豆及其发酵制品为目标样品，采用酪蛋白平皿筛选到一株既可产中性蛋白酶又可产碱性蛋白酶的细菌菌株W1，经形态学及16S rDNA基因序列分析鉴定为枯草芽孢杆菌枯草亚种(Bacillus subtilis subsp. Subtilis)，其产中性蛋白酶和碱性蛋白酶活力分别为28.99 U/mL和33.19 U/mL。通过单因素试验对其产蛋白酶的培养条件进行初步优化，并在此基础上以总蛋白酶活力为指标对其产蛋白酶条件进一步进行正交试验优化，最佳条件为培养基初始pH 9.0，接种量4%，培养温度35 ℃，培养时间48 h，此条件下中性蛋白酶活力为40.39 U/mL，碱性蛋白酶活力为52.02 U/mL，总蛋白酶活力为92.41 U/mL，优化后相应酶活力分别提高了39.3%、56.7%和48.6%，这将为枯草芽孢杆菌W1菌株蛋白酶的酶学性质研究提供帮助，同时也是对蛋白酶活性枯草芽孢杆菌资源库的丰富。

To obtain the bacteria strain with high protease activity, high-protease-producing strain W1 was isolated from soybean products by casein plate method, and identified as Bacillus subtilis subsp. subtilis by phenotypical characteristics and 16S rDNA sequences. The strain W1 exhibited both neutral protease activity and alkaline protease activity, which were 28.99 U/mL and 33.19 U/mL, respectively. The single factor experiment was selected for preliminary optimization. The orthogonal experimental design was used to optimize the culture condition of Bacillus subtilis W1 strain based on the single factor results and the total protease activity was selected as an indicator. The optimal conditions were as follow: the initial pH 9.0 in the medium, 4% inoculation, culture temperature 37 ℃, and culture time 60 h. In the above conditions, the activity of neutral protease, alkaline protease and total protease were 40.39 U/mL, 52.02 U/mL and 92.41 U/mL, respectively. Thought optimization, the corresponding protease activities increased by 39.3%, 56.7% and 48.6%, respectively. These results will be helpful to further study the protease characteristics of the Bacillus subtilis W1 strain, and also enrich the resource library of Bacillus subtilis with protease activity.

黑龙江省教育厅面上项目(12541761)