【摘要】 目的:脂肪酶是世界主要工业用酶制剂之一,用基因工程方法克隆与表达白地霉(Geotrichum candidum)ch-3菌株的低温脂肪酶基因。方法:用PCR方法从白地霉ch-3的总DNA中扩增得到一种低温脂肪酶基因lip,将PCR产物连接到pBluescriptⅡSK(+)载体上,测序证明正确后再将基因lip插入到巴斯德毕赤酵母表达载体pPIC9α中,含有目的基因的重组质粒在毕赤酵母中进行诱导表达。结果:毕赤酵母成功表达了lip基因,其活性为37U/mL。SDS-PAGE分析显示表达的脂肪酶蛋白相对分子质量约62kDa,比推测分子量大,lip的表达产物可能被糖基化修饰。初步研究表明:重组脂肪酶最适温度为35℃,在0℃仍可保持66%的相对酶活性,对热敏感。结论:实现了低温脂肪酶在毕赤酵母的成功表达,为进一步研究低温酶催化机制和工业化应用打下基础。更多还原

【Abstract】 Objective: Lipase is one of primary enzymes applied in industry in the world.To clone and express a cold-adapted lipase gene from Geotrichum candidum ch-3 by gene engineering.Methods: A cold-adapted lipase gene lip was amplified with PCR from the genomic DNA of G.candidum ch3,and it was then linked into pBluescriptⅡSK(+) vector.After it was confirmed by sequencing analysis,it was correctly inserted into the expression vector pPIC9α of Pichia pastoris.Then the recombinant pPIC9α plasmid with target gene was induced and expressed in P.pastoris.Results: The lip gene was expressed successfully by using P.pastoris as host strain and the activity of it was 37U/mL.SDS-PAGE analysis showed that the molecular weight of the lipase protein was about 62kDa,larger than the theoretical value.This result indicated that the expression protein of lip gene might be glycosylated.The optimal temperature for recombinant lipase was 35℃.At 0℃,the enzyme still remained 66% of its activity.It was thermoliable. Conclusion: The successful expression of cold-adapted lipase in recombinant P.pastoris was realized,and it will be the foundation for the study on catalytic reaction mechanism of cold-adapted lipase and its industrialized application.更多还原