特异腐质霉内切葡聚糖酶Ⅱ基因在毕赤酵母中的表达及酶学性质  

Expression of Humicola insolens (NC3) endo-β-glucanaseⅡ gene in Pichia pastoris and anylysis of enzymic properties

作　　者：郑海英[1] 黄平[1] 蔡少丽[1] 柯崇榕[1] 林国滟[1] 黄建忠[1] 

机构地区：[1]福建师范大学工业微生物教育部工程研究中心生命科学学院福建省现代发酵技术工程研究中心,福建福州350108

出　　处：《微生物学通报》2012年第2期145-153,共9页Microbiology China

基　　金：基金福建省发改委产业化关键技术项目(闽发改投资[2009]958号)

摘　　要：【目的】在毕赤酵母中表达特异腐质霉Humicola insolens的中性内切葡聚糖酶Ⅱ,并对其性质加以研究。【方法】利用RT-PCR的方法,以特异腐质霉(Humicola insolens)NC3总RNA为模板,克隆到中性内切葡聚糖酶Ⅱ基因(egⅡ)的cDNA。将其插入表达载体pPIC9K,重组质粒经线性化后电击转化毕赤酵母(Pichia pastoris)菌株GS115。【结果】SDS-PAGE和酶活的检测结果均表明:egⅡ基因在毕赤酵母中成功表达。重组酶的部分酶学性质研究表明,该酶的最适反应温度为70°C,且在65°C以下具有较好的热稳定性。最适反应pH为6.5,在pH 6.0?7.0之间有较好的稳定性。【结论】用重组毕赤酵母可高效表达外源中性内切葡聚糖酶,为其今后在工业应用奠定了基础。[Objective] The purpose was to clone and express endo-1,4-β-glucanase gene of Humicola insolens in Pichia pastoris expression system.[Methods] An endo-1,4-β-glucanaseⅡ（egⅡ） cDNA gene was isolated from the fungus Humicola insolens NC3 by RT-PCR.Subsequently,we cloned the egⅡgene into expression vector pPIC9K,and then transformed into Pichia pastoris GS115.[Results] SDS-PAGE and CMC enzyme activity analysis demon-strated that recombinant EGⅡ protein was successfully expressed after induction in shake flasks.The endo-1,4-β-glucanase exhibited maximum activity at 70 °C and pH 6.5,and was stable between pH 6.0 and 7.0 and below 65 °C.[Conclusion] P.pastoris expression system is an efficient way of production of endo-1,4-β-glucanase.The recombinant endo-1,4-β-glucanase could be a candidate for industrial applications.

关 键 词：中性内切葡聚糖酶Ⅱ 特异腐质霉 毕赤酵母 

分 类 号：Q78[生物学—分子生物学]