蓝状菌(Talaromyces leycettanus JCM12802)高温果胶甲酯酶PmeT在毕赤酵母中的高效表达及酶学性质

High-level expression and characterization of pectin methylesterase Pme T from Talaromyces leycettanus JCM12802 in Pichia pastoris

【目的】在毕赤酵母中高水平表达蓝状菌(Talaromyces leycettanus JCM12802)来源的高温果胶甲酯酶,并对其进行酶学性质研究,具有高催化效率的高温果胶甲酯酶有望能广泛应用于低甲氧基果胶的生产,优化生产工艺,提高转化率,降低生产成本。【方法】利用RT-PCR的方法,以蓝状菌(T.leycettanus JCM12802)总RNA为模板,克隆得到果胶甲酯酶基因(Pme T)的cDNA。将其插入表达载体p PIC9K,并转化毕赤酵母(Pichia pastoris)菌株GS115,高活性的阳性转化子进行高密度发酵研究。【结果】重组酵母的果胶甲酯酶表达水平达到428 U/m L,并进一步鉴定了重组果胶甲酯酶的酶学性质。该酶的最适反应温度为75°C,且在85°C以下具有较好的热稳定性。最适反应p H为4.0,在p H 2.0-7.0之间有较好的稳定性。【结论】用重组毕赤酵母可高效表达蓝状菌来源的高温果胶甲酯酶,为其今后在工业上的应用奠定了基础。

[Objective] We highly expressed the high temperature resistant pectin methylesterase from Talaromyces leycettanus JCM12802 in Pichia pastoris and studied its enzymatic properties. Pectin methylesterase with high catalytic efficiency at high temperature was expected to be widely used in the production of low-methoxyl pectin, for an optimal production process and conversion rate with reduced production cost. [Methods] We cloned the cDNA of pectin methylesterase gene （PmeT） from the total RNA of T. leycettanus JCM12802 as template by RT-PCR, which was inserted into the expression vector pPIC9K and transformed into P pastoris GS115. We cultivated the high activated positive transformant for high-density fermentation. [Results] The recombinant pectin methylesterase （r-PmeT） expression level reached 428 U/mL. We have identified the enzymatic properties of r-PmeT. The optimum reaction temperature of r-PmeT was 75℃, and its thermostability was below 85 ℃. The optimum pH was 4.0, and it was stable between pH 2.0 and 7.0. [Conclusion] The recombinant Pichia pastoris could express high level pectin methylesterase from T. leycettanus JCM12802, which shows excellent application potential in its future industrial.