重组巴斯德毕赤酵母产米根霉α-淀粉酶发酵条件的研究

Studies on conditions for the expression of Rhizopus oryzae-amylase in recombinant Pichia pastoris strain

为了提高重组菌的淀粉酶表达量,以可分泌表达米根霉α-淀粉酶的甲醇快速利用型巴斯德毕赤酵母重组菌为基础,采用摇瓶发酵方式对影响重组菌表达淀粉酶的多个因素进行了研究和优化。摇瓶发酵条件确定为:温度为30℃,pH值为6.0,接种量为2.0(OD_(600)),甲醇补加方式采用前72 h发酵时间内每隔12h添加至终浓度为1.0%,72 h以后每隔24 h添加至终浓度为1.0%,在此条件下获得的淀粉酶最高表达量为47.5 U/mL,且在无机盐培养基中和有机氮源培养基中获得的淀粉酶发酵单位相当。以摇瓶发酵数据为基础确定15 L发酵罐放大实验条件为:无机盐培养基,温度为30℃,pH值为6.0,接种量为10%,甲醇流加方式采用DO—Start法控制,在此发酵条件下获得的淀粉酶表达量为440 U/mL,约为摇瓶发酵方式获得的淀粉酶表达量的9倍。

In order to increase the Rhizopus oryzae a-amylase expression level in a genetic modified Pichia pastoris which was a methanol utilization plus phenotype strain, a number of influence factors on the expression of amylase that involved in the fermentation process were investigated and optimized by shake flask fermentation. The optimized shake flask fermentation conditions were as follows： 30℃, pH 6.0, inoculum size 2 0 （OD600 ）. The methanol was added to a final concentration of 1.0% by every 12 h in the first 72 h fermentation stage and after 72 h to a final concentration of 1.0% by every 24 h, and the highest a-amylase expression level observed under the optimized conditions was 47.5 U/mL. The amount of expressed amylase was almost equal between the cultures of basal salts medium and organic nitrogen based medium. According to the data obtained in shake flask fermentation, ferraentation conditions based on basal salts medium were scaled up in a 15 L fermenter and adjusted to 30℃, pH 6.0, inoculum size 10%. The flow rate of methanol was controlled by DO-Start methanol, and the amylase expression level was 440 U/mL, which was about nine times higher than that achieved in shake flask eultures.