中文摘要：

[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pWB980连接，构建表达质粒pWB—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pH、接种量对发酵产酶的影响。[结果]获得基因工程菌B．subtilis WB600／pWB—pul324，SDS—PAGE电泳结果显示在89kDa处有特异性条带，发酵初始酶活为12．34U／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50g/L和30g／L。Mn2＋、Fe2＋、Fe2＋和Tween-80的添加能提高发酵产酶活力。在最适初始pH6．5，以最适5％接种量接种于优化后的培养基中，摇瓶发酵80h普鲁兰酶的酶活达到414．48U／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414．48U／ml，是之前研究结果（20．16U／m1）的20倍。

英文摘要：

[ 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.