Feed Research Institute, Chinese Academy of Agricultural Sciences, No. 12 Main Street South, Zhongguancun, Beijing, 100081, China.
Protein J. 2013 Mar;32(3):223-9. doi: 10.1007/s10930-013-9476-3.
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 Hg(2+), Cu(2+) and Al(3+) could inhibit the TreS activity, while only 1 mM of Ca(2+) and Mn(2+) could increase its activity. Five amino acid residues, Asp(244), Glu(286), Asp(354), His(147) and His(353), were shown to be conserved in R. opacus TreS, which were also important for α-amylase family enzyme catalysis.
海藻糖是一种独特的二糖，能够保护蛋白质免受环境压力的影响。我们从红球菌属（Rhodococcus opacus）中克隆并表达了一种新型海藻糖合酶（TreS）基因，分别在大肠杆菌 Top10 和 BL21（DE3）pLysS 中表达。重组 TreS 的分子量为 79 kDa。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°C，最适 pH 值约为 7.0。动力学分析表明，TreS 对海藻糖的 K m 值约为 98 mM，略高于麦芽糖。根据 k cat/K m 的分析，TreS 的首选底物是麦芽糖。1 和 10 mM 的 Hg(2+)、Cu(2+)和 Al(3+)均可抑制 TreS 活性，而只有 1 mM 的 Ca(2+)和 Mn(2+)可提高其活性。在红球菌属 TreS 中，有 5 个氨基酸残基（Asp244、Glu286、Asp354、His147 和 His353）保守，这些残基对α-淀粉酶家族酶的催化也很重要。
