Aspergillus oryzae溶解多糖单加氧酶基因的克隆，异源表达及酶学特性分析

发布时间：2018-01-10 10:36

  本文关键词：Aspergillus oryzae溶解多糖单加氧酶基因的克隆，异源表达及酶学特性分析　出处：《云南大学》2016年硕士论文　论文类型：学位论文

  更多相关文章： 溶解多糖单加氧酶 米曲霉 毕赤酵母 异源表达 酶活

【摘要】：溶解多糖单加氧酶(Lytic polysaccharide monooxygenases, LPMOs)是一类能够促进生物质如几丁质和纤维素等降解获得可溶性寡糖的氧化酶。本研究根据己报道的米曲霉LPMOs基因序列(GenBank:BAE61530.1)设计特异性引物,PCR扩增得到长度为1266 bp的溶解多糖单加氧酶基因lpmo,将该基因克隆到T载体上进行测序验证,测序结果和己知序列相似度100%。基因lpmo和载体pET32a、pPIC9K用相同的限制性内切酶进行双酶切、转化到大肠杆菌DH5a中,挑取转化子进行酶切和测序验证,获得了重组表达载体pET32a-l-pmo(ZH-1)和pPIC9K-lpmo(ZH-2).重组载体pET32a-lpmo和pPIC9K-lpmo分别通过化学转化法和电转法导入大肠杆菌BL21和毕赤酵母GS115中。重组毕赤酵母GS115/pPIC9K-lpmo经甲醇诱导5天后,其酶活较高。重组大肠杆菌BL21/pET32a-lpmo I在IPTG浓度0.2-1mg/mL时诱导表达16h后表达量均为600 mg/L,其最适表达温度为16-28℃。表达的蛋白用Ni亲和层析柱纯化出相对较单一重组蛋白,经过SDS-PAGE电泳检测结果表明,纯化后的重组蛋白的分子量大小约为68 kDa。根据酶学性质研究表明,纯化后的LPMOs对底物壳聚糖的最适反应温度为50℃；超过50℃,显色反应表明,酶活随温度升高逐渐降低；低于50℃,酶活随温度降低而随之变化。其最适反应的pH值约为6.0,当pH大于6.0时酶活逐渐降低,说明LPMOs在弱酸条件下活性较高。金属离子对重组LPMOs酶活也有一定程度的影响,低浓度Cu2+、Al3+、Fe3+金属离子对重组LPMOs酶活具有促进作用,高浓度Cu2+、Al3+、Fe3+对酶活具有抑制作用；Zn2+对酶活无影响。本实验研究表明,通过构建异源表达载体,提高LPMOs表达量。LPMOs在50℃和pH 6.0条件下酶活相对较高。为今后纤维素降解工业生产乙醇提供一定技术支持。
[Abstract]:Lytic polysaccharide monooxygenases. LPMOs is a kind of oxidase that can promote the degradation of biomass such as chitin and cellulose to obtain soluble oligosaccharides. The LPMOs gene sequence of Aspergillus oryzae has been reported in this study. GenBank: BAE61530.1). A 1266bp LPM gene was amplified by PCR and cloned into T vector for sequencing. Lpmo and pET32a pPIC9K were digested with the same restriction endonuclease and transformed into Escherichia coli DH5a. The transformants were digested and sequenced. Recombinant expression vectors pET32a-l-pmoguanZH-1) and pPIC9K-lpmo-ZH-2) were obtained. The recombinant vectors pET32a-lpmo and pPIC9K-lpmo were introduced into Escherichia coli BL21 and Pichia pastoris GS115 by chemical transformation and electrotransposition respectively. The recombinant Pichia pastoris G. S115 / pPIC9K-lpmo was induced by methanol for 5 days. The recombinant Escherichia coli BL21/pET32a-lpmo I was induced to express at 0.2-1 mg / mL IPTG for 16 h and the expression level was 600 mg/L. The optimal expression temperature was 16-28 鈩，

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