圆弧青霉碱性脂肪酶在毕赤酵母中的高效表达

High Expression of Alkaline Lipase from Penicillium cyclopium in Pichia pastoris

目的在毕赤酵母(Pichia pastoris)中高效表达圆弧青霉碱性脂肪酶(Alkaline lipase,LipⅠ)。方法采用RT-PCR法从圆弧青霉PG37中扩增lipⅠ基因的cDNA片段,克隆入表达质粒pPIC9K中,构建重组表达质粒pPIC9K-lipⅠ,转化入毕赤酵母GS115,筛选高拷贝重组子GS115/lipⅠ,甲醇诱导表达,并对诱导表达条件进行初步优化。结果 GS115/lipⅠ在培养基初始pH6.0,甲醇添加量1.0%的BMMY培养基中,于30℃诱导96h,发酵液上清中LipⅠ的活性为10.5U/ml。在培养基初始pH9.0,甲醇添加量1.0%的BMMY培养基中,24℃诱导120h,GS115/lipⅠ发酵液上清中LipⅠ的活性最高,达407U/ml。结论在毕赤酵母GS115中高效表达了具有活性的圆弧青霉LipⅠ。

Objective To highly express the alkaline lipase（LipⅠ）from Penicillium cyclopium in Pichia pastoris.Methods The cDNA fragment of lipⅠgene was amplified from Penicillium cyclopium PG37 by RT-PCR and cloned into expression vector pPIC9K.The constructed recombinant plasmid pPIC9K-lip Ⅰ was transformed to Pichia pastoris GS115.High copy recombinant GS115/lipⅠwas screened for expression under induction of methanol,based on which the condition for expression was preliminarily optimized.Results After GS115/lipⅠ was cultured in the BMMY medium,at an original pH of 6.0,containing 1.0% methanol at 30℃ for 96 h,the LipⅠactivity in fermentation supernatant was 10.5 U/ml.However,after culture in the BMMY medium containing 1.0% methanol at 24℃ for 120 h,at an original pH of 9.0,the LipⅠactivity reached 407 U/ml.Conclusion The LipⅠ from Penicillium cyclopium was highly expressed in Pichia pastoris.