在50 L发酵罐水平对影响毕赤酵母表面展示米黑根毛霉脂肪酶(Rhizomucor miehei lipase,RML)发酵因素进行了研究。最适初始菌体密度(OD600)约为250,最佳诱导pH为6.0;降低诱导温度能有效提高单位甲醇转化率,诱导温度为20℃和25℃时的转化率分别是30℃的1.56倍和1.32倍。在以上最适条件下单位干菌体展示酶活及生产强度分别达275.0 U/g和462.5 U/(L.h),比优化前提高54.5%。研究发现,山梨醇和甲醇混合补料有效地提高了诱导前期(t=12h)的产酶效率,当山梨醇和甲醇混合比例为1∶4及1∶5是以甲醇为单一碳源时酶活力的1.7倍。

Several key factors which affect the production of Rhizomucor miehei lipase(RML) displayed on cell surface of Pichia pastoris were studied by 50 L scale fermentation.Results indicated that the optimum initial induction cell density(OD600) was around 250;the optimum pH during induction phase was 6.0;reducing temperature during induction phase resulted in increase of specific productivity by 1.56 and 1.32 folds at 20℃ and 25℃ respectively,compared to that of 30℃.Under the above optimum conditions,the RML activity and productivity reached 275.0 U/g and 462.5 U/(L·h),increased by 54.5% compared to that of initial conditions.Methanol/Sorbitol co-feeding strategy was also studied.By implementing this strategy and setting the sorbitol and methanol mix rate at 1∶ 4 and 1∶ 5,the RML productivity at 12h was 1.7 times of that by sole carbon resource.