【摘要】 利用前期克隆得到的红色亚栖热菌CBS-01(Meiothermus ruber CBS-01)的海藻糖合酶基因(TreS),构建天然蛋白异源宿主表达载体并在大肠杆菌中获得较高的表达量.经纯化后测定该酶在pH 6.5,反应温度为50℃时活性最高.酶学性质的研究表明,TreS的最适底物为麦芽糖,其对麦芽糖的转化效率约是海藻糖的2.5倍;该酶在30～60℃、pH 3.5～9.0范围内都保留较高的酶活;2 mmol/L的一价阳离子Na~+、K~+对TreS有激活作用;低温有利于反应向海藻糖方向进行,并能减少葡萄糖副产物的生成;20℃时,几乎没有葡萄糖生成,麦芽糖至海藻糖的转化率可达65%.为工业化大规模生产海藻糖奠定了理论基础.更多还原

【Abstract】 The previously cloned thermostable trehalose synthase gene（TreS）was used to construct a native protein heterogenous expression plasmid and transformed into E.coli.Overexpression of this gene was achieved and the enzyme after isolation and purification showed the highest activity at pH 6.5 and 50℃.Kinetic analysis showed that the enzyme had a 2.5-fold higher catalytic efficiency(k_cat/K_m)for maltose than for trehalose,which indicated that the maltose is the preferred substrate.The maximum conversion rate of maltose into trehalose by the enzyme was increase at lower temperatures,and reached 65%at 20℃.The enzyme could maintain very high activity（above 90%）at pH 3.5～8.5 and 60°for 5 h.Enzyme activity was activated by low concentration of Na⁺ and K⁺.These results provided that the stable TreS was suitable for the industrial production of trehalose.更多还原