米根霉脂肪酶在毕赤酵母中表达条件的优化及其高密度发酵

Optimization of conditions for expression of Rhizopus oryzae lipase in Pichia pastoris and its high-density fermentation

从pH、甲醇浓度、诱导时间、诱导温度和培养基氮源等方面对表达重组脂肪酶的毕赤酵母的摇瓶诱导培养条件进行了优化,研究了毕赤酵母在10 L发酵罐中表达脂肪酶的高密度发酵。结果表明:摇瓶诱导培养时,pH适宜范围为5.5～6.0,甲醇适宜质量分数为1%,诱导最佳时间为96 h,诱导适宜温度为28～30℃,可以用2%玉米浆粉替代传统的酵母浸提物和蛋白胨作为氮源。在10 L发酵罐高密度发酵中,以恒溶氧反馈调控补加甘油和梯度补加甲醇诱导的分批补料方式,每升发酵液中菌体干质量达到120.1 g,总蛋白质量达到2.5 g。经甲醇诱导54 h后,最高酶活达564μmol/(min.mL),约为摇瓶培养最高酶活的2倍。

The expression conditions of lipase from genetically engineering Pichia pastoris were optimized and its high-density fermentation in a 10 L fermenter was investigated.The results showed that the optimal pH was in range of 5.5-6.0,suitable methanol dosage was 1%,the optimal fermentation time was 96 h after induction with methanol and temperature was 28-30 ℃,and it was also indicated that 2% of corn steep powder could substitute yeast extract and peptone as nitrogen source.By using glycerol feeding strategy with DO control in high-density fermentation,the dry cell weight was up to 120.1 g/L,and the total protein concentration reached at 2.5 g/L in the fed batch fermentation phase.54 hours after the induction with methanol,the lipase activity got to be 564 μmol/（min·mL）,which is as much as two times of the lipase activity in the shake flask fermentation.