双孢蘑菇来源非特异性过氧合酶在毕赤酵母中的表达及生化表征

Expression and Biochemical Characteristics of Unspecific Peroxygenase Derived from Agaricus bisporus var.bisporus in Pichia pastoris

本研究以来源于(Agaricus bisporus var. bisporus,Abvb)的非特异性过氧合酶AbvbUPO为目标蛋白,实现了其在毕赤酵母GS115中的异源分泌表达。Westernblot检测结果表明重组AbvbUPO分子量为35ku。酶生化表征研究发现,非特异性过氧合酶AbvbUPO的最适反应温度和pH分别为35℃和3.0。通过过氧化氢耐受性实验,发现2 mM以上的过氧化氢会导致AbvbUPO钝化。为保持AbvbUPO在反应过程中的活力,原位生成过氧化氢的AbvbUPO酶级联催化反应被应用于其催化能力的鉴定。以乙基苯为底物的AbvbUPO酶级联催化反应,经过4h的反应后,产物β-苯乙醇的得率可达14.40%。上述研究表明,AbvbUPO是一种不耐受高温和高浓度过氧化氢的中温酶,但在原位生成过氧化氢的级联反应中AbvbUPO可保持良好的催化性能。本研究为非特异性过氧合酶的异源表达和生物催化应用提供了一个良好的借鉴。

In this study,an unspecific peroxygenase AbvbUPO derived from Agaricusbisporus var.bisporus was used as the target protein to achieve heterologous expression in Pichia pastoris GS115.Western blot results showed that the molecular mass of AbvbUPO was 35 ku.Biochemical characterization of the enzyme revealed that the optimal reaction temperature and pH of the unspecific peroxygenase AbvbUPO were 35℃and 3.0,respectively.Hydrogen peroxide tolerance experiments revealed that hydrogen peroxide above 2.0 mM deactivated AbvbUPO.In order to maintain the activity of AbvbUPO in the reaction process,the insitu production of H2O2 occurring in the AbvbUPO enzyme cascade catalytic reaction was used for the identification of its catalytic ability.After the 4 h AbvbUPO enzyme cascade catalytic reaction with ethylbenzene as the substrate,the yield of the productβ-phenylethanol reached 14.40%.The above research results show that AbvbUPO was a mesophilic enzyme that could not tolerate high temperature and high concentrations of H2O2,but can maintain good catalytic performance in the cascade reaction of insitu H2O2 production.This study provides a reference for the heterologous expression and biocatalytic application of unspecific peroxygenase.