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

【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.