重组青霉素G酰化酶在枯草芽孢杆菌中的表达条件优化

 为获得巨大芽孢杆菌青霉素 G酰化酶 (PGA)的高产菌株和条件 ,构建了分泌表达 PGA的基因工程枯草杆菌菌株 ,对表达条件进行了优化 .以 LB作为初始培养基 ,考察了温度、苯乙酸、装液量、碳源对于工程菌 PGA产量的影响 .实验发现重组细胞产酶不再需要变温和苯乙酸诱导 .充足的通气量和适当浓度的淀粉可使细胞密度及 PGA表达量大为提高 .表达条件优化后 ,菌体 A60 0由 3提高到 2 0 ,PGA的表达量由 3～ 6U/ml提高到 35～ 40 U/ml,为目前生产用巨大芽孢杆菌表达量的 6倍 .

Optimization of Recombinant Penicillin G Acylase Expression in Bacillus subtilis

Recombinant penicillin G acylase (PGA) was produced in Bacillus subtilis. The best recombinant B.subtilis strain with PGA overproductivity was selected and designated as SIBAS205.In order to enhance the expression level and optimize growth conditions some ferment parameters,such as temperature,phenylacetic acid,volumn of medium,carbon sources were experimented.In contrast to the parent cells that had to be induced by phenylacetic acid (PAA) and cultured at 28℃ and 25℃ successively to produce PGA,the recombinant cells needed no induction nor thermoregulation during fermentation at 37℃.PGA production was repressed by high concentration of glucose (≥2\^5 g/L) and however remarkably enhanced by starch.PGA was secreted out of cells and reached an expression level of 40 U/ml under optimized conditions.After optimization WB600(pEES102) was grown at 37℃ with vigorous aeration for 72 hours and about 40 U/ml PGA was obtained from the culture broth.