CAJ | 학술논문

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%．
위구건1주산중성단백매적공정균주병대기발효조건진행우화，이중성단백매공업생산균주고초아포간균AS1.398적기인조위모판，채용PCR기술확증획득중성단백매기인npr，여고고패질립pWB980련접병전입단백매결함형균주고초아포간균WB600중득도중조공정균WB600/pWB980-npr．재초사평판상공정균단백매활력명현고우공업생산균 AS1.398，공정균발효활력가체1,398.3,U/mL ．통과대기발효공예진일보우화，재접충량5%、장액량100,mL/500,mL、요상전속180,r/min、온도34,℃적조건하발효48,h，발효액적매활력가체2,487.5,U/mL，비우화전제고료78%．
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 AS1.398,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,℃,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%.