米黑根毛霉脂肪酶的克隆表达及发酵条件优化

米黑根毛霉脂肪酶RML是一种重要的工业用酶,具有非常广泛的应用前景。本研究将人工合成的RML基因rml克隆到毕赤酵母表达载体pPIC9K中,构建pPIC9K-rml诱导型表达载体。表达载体经线性化后转化巴斯德毕赤酵母GS115,并使用抗生素G418筛选、三丁酸甘油酯-MM板及PCR方法筛选阳性重组工程菌。工程菌发酵液经SDS-PAGE分析、三丁酸甘油酯板鉴定,表明脂肪酶RML在毕赤酵母中获得高效表达,且蛋白分子大小与预期一致。进一步优化了发酵条件,包括对培养基组分、诱导剂浓度和发酵温度等的优化。优化后的培养基组成为：甘油4.0%,（NH4）2SO45.000 g/L,CaSO40.465 g/L,K2SO49.100 g/L,MgSO4.7H2O7.450 g/L;培养条件为：pH值6.0,生长阶段温度30℃,诱导阶段采用22℃低温诱导,诱导期每24 h补加甲醇浓度为3%。优化后,发酵液中的RML活力最高达到116 U/ml,比优化前提高了3.14倍。

Secreted Gene Expression and Fermentation Optimization of Rhizomucor miehei Lipase in Pichia pastoris

Rhizomucor miehei lipase(RML) as an important biocatalyst is widely used in many industrial processes.In this study,the gene rml encoding RML was synthesized and cloned into Picha pastoris expression vector pPIC9K to generate pPIC9K-rml.The linearized plasmid pPIC9K-rml was transformed into HIS4 mutant Pichia pastoris yeast GS115.The transformants secreting RML were indentified by using tributyrin plate method.The SDS-PAGE analysis indicated that the RML have been successfully expressed in yeast with the apparent molecular mass of 36 kDa.Moreover,the optimum fermentation conditions,including medium elements,cultural temperature and inducer concentration,were investigated.The maximal yield of RML(116 U/ml) could be obtained when the recombinant strain was cultured in the synthetic medium containing 4.0% glycerol,5.00 g/L(NH4)2SO4,0.465 g/L CaSO4,9.100 g/L K2SO4,7.450 g/L MgSO4·7H2O,under the optimized cultivation conditions of pH6,30 ℃ in growing period and 22 ℃ in induction period with the addtion of 3% methanol per day.The activity of lipase was up to 3.14 times of the original.