产中性蛋白酶工程菌的构建及其发酵条件的初步优化

为构建1株产中性蛋白酶的工程菌株并对其发酵条件进行优化,以中性蛋白酶工业生产菌株枯草芽孢杆菌AS1.398的基因组为模板,采用PCR技术扩增获得中性蛋白酶基因npr,与高拷贝质粒pWB980连接并转入蛋白酶缺陷型菌株枯草芽孢杆菌WB600中得到重组工程菌WB600/pWB980-npr.在初筛平板上工程菌蛋白酶活力明显高于工业生产菌AS1.398,工程菌发酵活力可达1,398.3,U/mL.通过对其发酵工艺进一步优化,在接种量5%、装液量100,mL/500,mL、摇床转速180,r/min、温度34,℃的条件下发酵48,h,发酵液的酶活力可达2,487.5,U/mL,比优化前提高了78%.

Construction and Study of Fermentation Conditions of Engineering Strain for Producing Neutral Protease

The neutral protease gene was cloned and expressed in Bacillus subtilis WB600. The fermentation conditions of the engineering strain were also studied in this research. The genome of Bacillus subtilis AS 1.398 producing neutral protease was selected as a template to obtain npr gene by PCR. The gene was then expressed using pWB980 as the vector in Bacillus subtilis WB600, and the neutral protease activity, which reached 1 398.3 U/mL, is significantly higher than Bacillus subtilis AS1.398 on the primary screening medium. Then the fermentation conditions were studied. The optimal culture conditions were the inoculum volume was 5%, poured solution volume 100 mL/500 mL, temperature 34 ~C, rotation speed 180 r/min, and the fermentation time was 48 h. Under the optimized conditions, the maximum enzyme activity reached 2 487.5 U/mL, which was increased by 78%.