毕赤酵母高密度发酵产β-甘露聚糖酶的工艺优化

Optimization Technology of β-Mannanase Fermented by Pichia pastoris

以表达β-甘露聚糖酶的毕赤酵母工程菌为研究对象,采用摇瓶发酵确定碳源、接种量、温度、p H基础条件,通过30 L发酵罐高密度发酵,探究菌体浓度、甲醇浓度、甲醇补料方式、溶氧量等条件对目的蛋白产量的影响,并通过正交试验优化发酵工艺条件。结果表明,最佳产酶条件为接种量10%,初始葡萄糖质量浓度30 g/L,诱导温度28℃,p H 5.0,溶氧量10%~20%。在此发酵条件下,最终细胞干重135 g/L,目的蛋白表达量5.04 g/L,最高酶活力29 600 U/m L,较优化前提高24倍,已满足工业化要求。

The fermentation conditions of genetically engineered Pichia pastoris for β-Mannanase were studied. Basic conditions of the carbon source, immunization rates, temperature, pH were optimized in shake-flask. The tests in 30 L fermenter was applied to study the effects of DCW,methanol concentration and DO on enzyme production. Through the orthogonal design to optimize the fermentation conditions. The results indicated that the optimum conditions were as follows ：10% inoculum size,30 g/L initial glucose concentration, 10%-20% DO,pH 5.0 at 28 ℃. The dry cell weight was 135 g/L,the target protein concen- tration could reach to 5.04 g/L. And the β-Mannanase activity got to be 29 600 U/mL,which was 24 times of that the results in the shake flask under these conditions.