从赤子爱胜蚓体内克隆得到蚓激酶基因lks2,并成功在毕赤酵母GS115中表达,酶活为254.4 U/mL。经摇瓶培养条件优化,确定最佳诱导条件为:初始OD600=1.5,pH 5.5、温度28 ℃,此条件下诱导84 h,重组菌GS115-pPIC9K-lks2蚓激酶活峰值为397.6 U/mL。利用10 L发酵罐进行放大培养,经优化确定最佳菌体接种浓度为9±0.5 g/L,此条件下高密度发酵使前期培养时间缩短了11.5 h,蚓激酶活力达到1 098.2 U/mL,具有十分可观的工业应用潜力。
The encoding gene lks2 of lumbrokinase was cloned from Eisenia foetida and successfully expressed in Pichia pastoris GS115 with an activity of 254 U/mL. Optimization of culture conditions in shake flask showed that the best induction conditions were as follows: initial OD600=1.5, pH 5.5, temperature 28 ℃. Under these conditions, the activity of lumbrokinase peaked 397.6 U/mL at 84 h. Moreover, the amplification culture of recombinant P. pastoris GS115-pPIC9K-lks2 was occurred in a 10 L fermenter, of which the optimized inoculation concentration was 9±0.5 g/L. Under this high-density fermentation condition, the pre-culture time was shortened by 11.5 h and the activity of lumbrokinase was raised to 1 098.2 U/mL, which has a considerable potential for industrial application. In this study, the yield of lumbrokinase from P. pastoris was effectively increased by the optimization of induction conditions and the high-density fermentation, laying a foundation for the industrial production and application of lumbrokinase.