产γ聚谷氨酸菌株的诱变选育及其种子液工艺优化

摘要

Bacillus amyloliquefaciens C1,isolated by our laboratory as aγ⁃polyglutamic acid producer,was treated with composite mutation method of ultraviolet and nitrosoguanidine. The liquid seed medium was also optimized by single⁃factor and orthogonal experiments. The resulted mutant strain C1⁃6 was screened with a γ⁃polyglutamic acid productivity of 24�2 g/L, increased by 31�5% compared with the original strain C1. C1⁃6 had a good genetic stability after 8 generations by transfer of culture experiment. Corn flour and soybean cake were selected as the carbon and nitrogen source respectively for C1⁃6 through single⁃factor experiments. Finally,the results of orthogonal experiments showed that the optimized liquid seed medium component was K2 HPO4 1�0 g/L,MgSO4 0�5 g/L,soybean cake 15�0 g/L and corn flour 5�0 g/L. In 150 mL shake flask,the optimal temperature,original pH and medium volume were 37 ℃, pH 6�5 and 30 mL,respectively,the larger biomass is obtained,the OD600 is 6�31.%以已筛选的1株产γ多聚谷氨酸解淀粉芽胞杆菌C1为出发菌株，对其进行紫外线亚硝基胍（ NTG）复合诱变，并运用单因素试验和正交试验设计对诱变菌株的种子培养工艺进行优化。通过复合诱变选育得到1株能够稳定遗传的正突变菌株C16，其摇瓶发酵生产γ PGA的产量由18�4 g／L提高到24�2 g／L，增加了31�5％，且传代8次后仍能保持稳定。通过单因子试验筛选到玉米粉和黄豆粉作为C16生长的C源和N源。正交试验后， C16在成分为K2HPO41�0 g／L、MgSO40�5 g／L、黄豆粉15�0 g／L、玉米粉5�0 g／L，pH 6�5的培养基中，37℃、装液量1／5（150 mL三角瓶装液30 mL）的培养条件下可获得较大的生物量，OD600达到6�31。