枯草芽孢杆菌工程菌株产普鲁兰酶发酵条件的优化

Optimization of Fermentation Conditions for the Pullulanase Production by Engineered Bacillus subtilis

利用基因工程手段获得的产长野芽孢杆菌普鲁兰酶的枯草芽孢杆菌基因工程菌株WB600/pWB-pulB,通过单因素筛选及正交试验进行发酵培养基优化,得到产酶最佳配方为玉米淀粉3.0%,酵母膏2.0%,CaCl20.03%,Na2HPO41.0%。同时对重组菌株摇瓶条件下液体发酵的主要影响因素初始pH、接种量、装液量、转速、温度等进行探讨,确定了产酶最佳培养条件为,以3%接种量接种于优化后培养基,初始pH7.0,装液量30 mL/250 mL,37℃,200 r/min摇床培养36 h。在此优化条件下,普鲁兰酶活力达到20.16 U/mL,是之前研究结果(10.94 U/mL)的1.84倍。

The single factor comparative experiments and orthogonal experiments were adopted to optimize the liquid fermentation medium and conditions in shaking flasks of Bacillus subtilis WB600/pWB-pulB for producing the pullulanase. The composition of the best medium was corn starch 3.0 %, yeast extract 2.0 %, CaCl2 0.03 %, Na2HPO4 1.0 %. In addition, the optimal fermentation conditions in shaking flasks were the combination of the inoculums size 3 %, initial pH 7.0, medium volume 30 mL/250 mL, temperature 37 ℃, and shaking at 200 r/min for 36 h. Under these conditions, the activity of recombinant pullulanase reached 20.16 U/mL, which was 1.84 times of before result （10.94 U/mL）.