毕赤酵母高密度发酵产脂肪酶条件的研究

Lipase production by high cell-density culture of Pichia pastoris

以毕赤酵母基因工程菌为材料，通过单因素试验在15L发酵罐中对发酵的pH、通气量、接种量、搅拌转速及诱导剂的量进行优化；进而对影响明显的因素在700L发酵罐中进行三因素三水平正交试验，最终确定最佳的发酵条件为：pH为5．0，温度为30℃，转速为250r／min，接种量为5％，溶氧为20％~30％，诱导剂甲醇的量为129L，培养时间为160~170h，在开始诱导后分3次（48h／次）向发酵液中添加0．5％的酵母膏（w／v）和1％的蛋白胨（w／v）可以显著提高发酵液酶活．酶活可以达到18U／mL。

The production of lipase by recombinant Pichia pastoris was studied in 15 L fermenter. The single factor test was firstly carried out to optimize pH, aeration, inoculurn, stirring speed and inducer concentration. The most important factors reflected from the single factor test were then se- lected for three levels orthogonal test in 700 L fermenter. The optimum fermentation conditions were pH 5.0, fermentation temperature 30 ℃, stirring speed 250 r/min, inoculum volume 5%, dis- solved oxygen 20%-30%, methanol 129 L and fermentation time 160-170 h. The results showed that the addition of 0.5% yeast extract （w/v） and 1% peptone （w/v） in 3 portions （once every 48 hours） after induction could significantly improve the lipase activity and the maximum enzyme ac- tivity reached 18 U/mL.