重组枯草芽孢杆菌发酵廉价原料生产普鲁兰酶  

Recombinant Bacillus subtilis producing pullulanase by the cheap raw materials

作　　者：黄艳燕[1] 李检秀[1] 刘祺霞 陈先锐 李晓明 陆迪 谢能中[1] 王青艳[1] 

机构地区：[1]广西科学院,非粮生物质酶解国家重点实验室,国家非粮生物质能源工程技术研究中心,广西南宁530007 [2]南宁中诺生物工程有限责任公司,广西南宁530007

出　　处：《生物技术》2016年第6期606-612,592,共8页Biotechnology

基　　金：国家自然科学基金项目(“普鲁兰酶的理性设计及其在枯草芽孢杆菌中的分泌表达研究”,No.31400079;“基于基因组学的多粘类芽孢杆菌合成(2R,3R)-2,3-丁二醇研究”,No.21466007);广西科技计划项目(“生物质高效转化光学纯(R,R)-2,3-丁二醇关键技术引进与合作研究”,No.桂科合14125008-2-22;“糖苷键水解酶催化机制和酶分子改造的NMR(核磁共振)方法研究”,No.桂科合14123001-19)

摘　　要：[目的]构建长野芽孢杆菌普鲁兰酶突变体枯草芽孢杆菌工程菌株,优化发酵条件,筛选廉价的培养基原料生产普鲁兰酶。[方法]利用分子生物学手段,将基因pul324和表达载体p WB980连接,构建表达质粒p WB-pul324并转化Bacillus subtilis WB600;对表达产物进行SDS-PAGE分析和初始酶活的测定。进一步优化发酵条件,对不同碳源和氮源进行发酵培养基的筛选,同时研究不同金属离子的添加和培养基初始p H、接种量对发酵产酶的影响。[结果]获得基因工程菌B.subtilis WB600/p WB-pul324,SDS-PAGE电泳结果显示在89 k Da处有特异性条带,发酵初始酶活为12.34 U/ml;筛选得到玉米淀粉水解液和玉米浆干粉为培养基最适碳源和氮源,其最适浓度分别为50 g/L和30 g/L。Mn^(2+)、Fe^(3+)、Fe^(2+)和Tween-80的添加能提高发酵产酶活力。在最适初始p H 6.5,以最适5%接种量接种于优化后的培养基中,摇瓶发酵80 h普鲁兰酶的酶活达到414.48 U/ml。[结论]实现了普鲁兰酶突变体在枯草芽孢杆菌中的高效表达,筛选获得的培养基主要原料经济低廉,经过发酵条件优化后,重组菌酶活达到414.48 U/ml,是之前研究结果(20.16U/ml)的20倍。[ Objective ] To study the optimize cheap raw fermentation materials and conditions in shaking flasks of the recombi- nant Bacillus subtilis WB600/pWB - pu1324 for producing the pullulanase. [ Methods ] The expression vector pWB - pu1324 was constructed by inserting the mutant pu1324 gene into plasmid pWB -980 and then transformed into the B. subtilis WB600. The single factor comparative experiments were adopted to optimize the liquid fermentation medium and conditions in shaking flasks of B. subtilis WB600/pWB - pu1324 for producing the pullulanase. [ Results ] The recombinant WB600/pWB - pu1324 was obtained. SDS -PAGE anlysis showed a band with moleeuler weight of 89 kDa, and the initial enzymy activity was 12.34 U/ml. The optimal carbon source corn starch hydrolysate and nitrogen source corn extract powder were screened and the optimal concentrations of them were 50 g/L and 30 g/L, respectively. Furthermore, the yield of pullulanase was increased by the addi- tion of Mn2＋ , Fe3 ＋ , Fe2 ＋ and Tween - 80 . Under the optimal fermentation condition in shaking flasks of the initial pH 6.5 and the inoculums 5%, the activity of recombinant pullulanase reached 414.48 U/ml. [ Conclusion] Firstly, pullulanase mutant gene pu1324 was successfully over - expressed in B. Subtilis. Secondly, the economical industrial mediums were optimized for high -level expression of pullulanase mutant in Bo subtilis and the activity of recombinant pullulanase reached 414.48 U/ml which was 20 times of before result.

关 键 词：普鲁兰酶 长野芽孢杆菌 枯草芽孢杆菌 玉米淀粉水解液 玉米浆干粉 发酵条件优化 

分 类 号：TQ925[轻工技术与工程—发酵工程]