碱性果胶酯裂解酶基因工程菌Pichia pastoris诱导表达条件初步优化

Optimization of Culture Conditions for Alkaline Pectate Lyase Production by Recombinant Pichia pastoris

在摇瓶水平上,采用单因素试验方法,对影响碱性果胶酯基因工程菌Pichia pastoris表达的主要因素进行研究,确定初始甲醇浓度、甲醇补加量和氮源酵母氮基(YNB)浓度3个主要影响因素的初步优化结果为5%、0.8%和1.3%.在此基础上,利用响应面分析法进一步优化.经优化,表达条件为初始甲醇浓度4.35%、甲醇补加量0.75%以及YNB浓度1.31%,优化后碱性果胶酯裂解酶(PL)表达量达到40.6U/mL,较初始培养条件提高约2.3倍.同时,利用SDS-PAGE电泳对优化前后基因工程菌表达情况进行分析,结果同样表明胞外目标蛋白PL表达量有明显增加,进一步证实条件优化后对目标蛋白PL分泌表达具有十分明显的促进作用.

Three factors for producing alkaline pectate lyase （PL） by recombinant strain Pichia pastoris： initial concentration of CH3OH, adding amount of CH3OH and concentration of yeast nitrogen base （YNB）, were investigated. The optimized factors were determined by single factor analysis experiments. The primary results were obtained as the initial concentrations of CH3OH 5%, the adding amount of CH3OH 0.8% per 24 h and YNB 1.3%. The response surface method was further applied to optimize the fermentation condition of the recombinant strain. Then, a mathematical model was developed to show the effect of each factor and their interactions on PL production by recombinant P. pastoris. A maximal yield of PL could be estimated by the model when the initial concentration of CH3OH, the adding amount of CH3OH and YNB were set at 4.35%, 0.75% and 1.31%, respectively. The yield under the optimal conditions could reach 40.6 U/mL, which was increased by 230% compared to that under the original fermentation conditions. SDS-PAGE analysis was used to verify the availability of the recombinant strain under the optimal fermentation condition, and the result proved that the optimal fermentation condition was very helpful for over-expressing PL. Fig 4, Tab 5, Ref 18