米根霉(Rhizopus oryzae)脂肪酶基因ProROL在毕赤酵母(Pichia pastoris)中的高效表达

摘要

米根霉(Rhizopus oryzae)脂肪酶在食品和油脂加工等方面具有广泛的应用前景.本研究通过PCR技术获得了米根霉脂肪酶前肽基因序列与成熟肽基因序列(ProROL),并将该基因克隆到表达载体pPIC9K上.线性化表达质粒后电转化感受态毕赤酵母菌株(Pichia pastoris)GS115,通过筛选得到了阳性重组菌.摇瓶及7L罐发酵结果显示,以p-NPP为底物,上清胞外酶活分别为10.6 U/mL和126.8 U/mL.酶学性质研究发现,该酶的最适温度为40℃左右,在低于40℃条件下,保温30 min后,能够保存60％以上的酶活力；其最适pH为8.5左右,在pH 6.0-10.0范围内保持1h后,均能保留60％以上的酶活力.本研究构建的基因重组菌株将为该酶的工业化生产与应用提供一定的理论基础.%The Rhizopus oryzae lipase prosequsenee and mature ROL were cloned into the expression vector pPIC9K. After the linearized expression plasmid was transformed into Pichia pastoris GS115, the recombinant strain ( Mut + His+ ) was obtained by preliminary screening and molecular validation. The lipase activity of the supernatant was 10.6 U/mL towards p-NPP in shake flask, while in 7-liter fennenter it reached 126. 8 U/mL. The optimal temperature of the enzyme was about 40 ℃ and the optimal pH was about 8.5. The residual activity was more than 60% after incubation at pH 6.0 - 10.0 for 1 h or keeping below 40 ℃ for 30 min. The recombinant strain was very promising in the industrial application.