产γ-谷氨基甲酰胺合成酶菌株的筛选及产酶条件研究

Studies on the mutation breeding of γ-glutamylmethylamide synthetase-producing strain of Pseudomonas sp.and the optimum conditions for the production of γ-glutamylmethylamide synthetase

以假单胞菌(Pseudomonas sp.)为出发菌株,通过紫外诱变筛选得到一株γ-谷氨基甲酰胺合成酶高产菌株UV-19,其酶活提高32.54%。以突变株UV-19为供试菌株,对γ-谷氨基甲酰胺合成酶的发酵条件进行优化。首先利用Plackett-Burman设计筛选出影响较大的4个因素:葡萄糖、蛋白胨、起始pH值、装液量。在此基础上再利用CCD响应面分析法进行优化,得到最佳产酶培养条件为(g/L):葡萄糖15、蛋白胨12、NaCl 5.0、MgSO4.7H2O 0.2、K2HPO4.3H2O 0.5、甲胺盐酸盐1.0g/L、起始pH值6.5、装液量72mL/250mL。该优化条件下进行产酶培养,假单胞菌发酵产γ-谷氨基甲酰胺合成酶酶活力可达32.68U/mL。

Pseudomonas sp.was used as initial strain to isolate the high activity strain by means of mutation under the UV-light treatment.One strain UV-19 with higher enzymatic activity of γ-glutamylmethylamide synthetase increased by 32.54% was obtained.The culture medium components and enzyme production conditions of mutant strain UV-19 were optimized by response surface methodology.Initially,Plackett-Burman design was used to evaluate the process variables which were relevant to the enzyme production.Four statistically significant parameters including glucose,peptone,initial pH and liquid volume were selected and utilized in order to optimize the process.The optimized levels of these factors were defined by response surface analysis（RSA）.The optimized medium and conditions of producing enzyme were obtained.When the strain was cultured with the medium containing 15g/L glucose,12 g/L peptone,5g/L NaCl,0.2 g/L MgSO4·7H2O,0.5g/L K2HPO4·3H2O,1g/L CH5N·HCl,initial pH6.5,enzyme activity was 32.68 U/mL.