转氨酶高效表达重组工程菌的培养基及诱导条件优化

Optimization of Culture Conditions of Recombinant Engineered Bacteria with High Expression of Transaminase

通过构建重组菌株PE-ATS(BL21)表达可催化底物合成西格列汀的转氨酶,采用摇瓶正交和单因素实验确定发酵培养基的最佳条件为甘油9.0 g/L,酵母粉16.0 g/L、硫酸铵4.0 g/L、氯化钠6.0 g/L、磷酸氢二钠2.0 g/L、磷酸二氢钾3.0 g/L;最佳表达条件为36℃、0.6 mmol/L IPTG诱导4 h。该条件下,摇瓶培养菌液OD600=6.63,SDS-PAGE分析显示酶表达量约占可溶蛋白总量的50%,酶活为758 U/L。

Through the construction of recombinant strain PE-ATS(BL21) expression of a catalytic substrate for the synthesis of siglitine transaminase, the optimal conditions for fermentation medium were determined by shaking bottle orthogonal and single factor experiment, namely 9.0 g/L of glycerol, 16.0 g/L of peptone, 4.0 g/L of ammonium sulfate, 6.0 g/L of sodium chloride, 2.0 g/L of disodium phosphate, and 3.0 g/L of potassium dihydrogen phosphate. Best expression condition of 36 ℃, tendency for 0.6 mmol/L IPTG induction 4 h. Under the condition, the bacteria solution in shaking bottle was OD600 = 6.63. SDS-PAGE analysis showed that the soluble expression of transaminase accounted for about50% of the total soluble protein, and the enzyme activity was 758 U/L.