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首页> 外文期刊>The protein journal >Cloning, expression and characterization of a trehalose synthase gene from rhodococcus opacus
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Cloning, expression and characterization of a trehalose synthase gene from rhodococcus opacus

机译：不透明红球菌海藻糖合酶基因的克隆，表达与鉴定

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摘要

Trehalose is a unique disaccharide capable of protecting proteins against environmental stress. A novel trehalose synthase (TreS) gene from Rhodococcus opacus was cloned and expressed in Escherichia coli Top10 and BL21 (DE3) pLysS, respectively. The recombinant TreS showed a molecular mass of 79 kDa. Thin layer chromatography (TLC) result suggested that this enzyme had the ability to catalyze the mutual conversion of maltose and trehalose. Moreover, high-performance liquid chromatography (HPLC) result suggested that glucose appeared as a byproduct with a conversion rate of 12 %. The purified recombinant enzyme had an optimum temperature of 25 C and pH optimum around 7.0. Kinetic analysis revealed that the K m for trehalose was around 98 mM, which was a little higher than that of maltose. The preferred substrate of TreS was maltose according to the analysis of k cat/K m. Both 1 and 10 mM of Hg2+, Cu2+ and Al3+ could inhibit the TreS activity, while only 1 mM of Ca2+ and Mn2+ could increase its activity. Five amino acid residues, Asp244, Glu 286, Asp354, His147 and His353, were shown to be conserved in R. opacus TreS, which were also important for α-amylase family enzyme catalysis.
机译：海藻糖是一种独特的二糖，能够保护蛋白质抵抗环境压力。克隆了一个来自Rhodococcus opacus的新型海藻糖合酶（TreS）基因，并分别在大肠杆菌Top10和BL21（DE3）pLysS中表达。重组TreS的分子量为79kDa。薄层色谱（TLC）结果表明该酶具有催化麦芽糖和海藻糖相互转化的能力。此外，高效液相色谱（HPLC）结果表明，葡萄糖作为副产物出现，转化率为12％。纯化的重组酶的最适温度为25℃，最适pH约为7.0。动力学分析表明，海藻糖的K m约为98 mM，略高于麦芽糖。根据k cat / K m的分析，TreS的优选底物是麦芽糖。 1和10 mM的Hg2 +，Cu2 +和Al3 +均可抑制TreS活性，而只有1 mM的Ca2 +和Mn2 +可以提高其TreS活性。五个氨基酸残基，Asp244，Glu 286，Asp354，His147和His353被证明在不透明芽孢杆菌TreS中是保守的，这对于α-淀粉酶家族酶催化也很重要。

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