重组球孢白僵菌表达目标产物类枯草杆菌蛋白酶的发酵特性研究  

Optimization of the Fermentation Conditions to Increase the Production of the Subtilisin-like Protease in A Recombinant Strain of Beauveria bassiana

作　　者：罗志兵[1] 张永军[1] 金凯[1] 彭荣[1] 方卫国[1] 裴炎[1] 

机构地区：[1]西南农业大学农业部农业生物技术与作物品质改良重点实验室,重庆市农业生物技术重点实验室,西南农业大学生物技术中心,重庆400716

出　　处：《农业生物技术学报》2005年第3期372-376,共5页Journal of Agricultural Biotechnology

基　　金：国家高技术研究与发展计划(863)项目(No.2002AA245021;2004AA214121);国家自然科学基金(No.30200183)资助。

摘　　要：采用单因子筛选和正交试验,研究了碳源、氮源、微量元素及无机盐等营养成分对重组球孢白僵菌(Beauveria bassiana)菌株Bb0062-15-4-CDEP-1产生目标产物类枯草杆菌蛋白酶CDEP-1的影响,并通过摇瓶发酵和30L发酵罐发酵对其发酵特性进行了研究。结果表明,其优化发酵培养基为2.0%玉米粉,1.0%麦麸,0.25%豆粕粉,0.4%NaNO3,0.1%MgSO·47H2O。摇瓶发酵结果表明,在对数生长期,随着菌体生物量的增加,CDEP-1产量上升,二者呈一定的平行关系;当菌体生长到稳定期,CDEP-1产量继续上升,并达到产酶高峰;此后,随着菌体生长的衰退,CDEP-1产量出现快速下降趋势。扩大培养时,30L发酵罐中60h产酶量最高,比摇瓶培养(168h)缩短了108h,单位体积的产酶量比摇瓶培养提高80%。结果对研究和利用该球孢白僵菌重组菌株的代谢产物,提高真菌杀虫剂的杀虫效果奠定了基础。To increase the yield of the subtilisin-like protease, CDEP-1, produced by a recombinant strain of Beauveria bassiana overexpressing the CDEP-1 gene, the medium compositions, including carbon and nitrogen sources, trace elements and salts, were optimized using single-factor searching and orthogonal test designs, and the fermentation characters were investigated in the flask shaking culture and in the stirred tank bioreactor （30 L） culture, respectively. The result suggested that the optimal medium compositions for the subtilisin-like protease production were 2.0% corn flour, 1.0% wheat gluten, 0.25% soybean draft, 0.4% NaNO3 and 0.1% MgSO4·7H2O. In flask shaking culture, the output of CDEP-1 increased in parallel with the accumulation of biomass during the logarithmic growth phase and reached the peak value in stationary growth phase, then fleetly decreased in decay period. In the culture of the stirred tank bioreactor, the peak production of CDEP-1 appeared at 60h, 108h ahead than that in the flask shaking culture. The maximum activity of the enzyme per volume of the fermentation broth in the stirred tank bioreactor was 283.456 U/μL, 80% increased over than that in the flask shaking culture. The results paved a way for utilizing the cultural product of the recombinant strain to improve the efficacy of mycoinsecticide against target pest.

关 键 词：球孢白僵菌 类枯草杆菌蛋白酶 重组菌株 液体发酵 发酵特性 代谢产物 特性研究 CDEP 摇瓶发酵 菌体生长 

分 类 号：TQ925[轻工技术与工程—发酵工程] S476.12[农业科学—农业昆虫与害虫防治]