天蓝色链霉菌海藻糖合酶的异源表达、活性分析及重组菌全细胞转化合成海藻糖的条件优化

从天蓝色链霉菌Streptomyces coelicolor克隆得到海藻糖合酶基因 (ScTreS)，在大肠杆菌Escherichia coli BL21(DE3) 中进行了异源表达，通过 Ni-NTA 亲和柱对表达产物进行分离纯化得到纯酶，经 SDS-PAGE 测定其分子量约为62.3 kDa。研究其酶学性质发现该酶最适温度35 ℃；最适pH 7.0，对酸性条件比较敏感。通过同源建模和序列比对分析，对该基因进行定点突变。突变酶K246A比酶活比野生酶提高了1.43倍，突变酶A165T相对提高了1.39倍，海藻糖转化率分别提高了14%和10%。利用突变体重组菌K246A进行全细胞转化优化海藻糖的合成条件并放大进行5 L罐发酵，结果表明：在麦芽糖浓度300 g/L、初始反应温度和pH分别为35 ℃和7.0的条件下，转化率最高达到71.3%，产量为213.93 g/L；当底物浓度增加到700 g/L时，海藻糖产量仍可达到465.98 g/L。

Heterologous expression of Streptomyces coelicolor trehalose synthase and whole-cell biocatalyst production of trehalose in Escherichia coli

The trehalose synthase (ScTreS) gene from Streptomyces coelicolor was successfully cloned and heterologously expressed in Escherichia coli BL21(DE3). The protein purified by Ni-NTA affinity column showed an apparent molecular weight (MW) of 62.3 kDa analyzed by SDS-PAGE. The optimum temperature of the enzyme was 35 °C and the optimum pH was 7.0; the enzyme was sensitive to acidic conditions. By homologous modeling and sequence alignment, the enzyme was modified by site-directed mutagenesis. The relative activities of the mutant enzymes K246A and A165T were 1.43 and 1.39 times that of the wild type, an increased conversion rate of 14% and 10% respectively. To optimize the synthesis conditions of trehalose, the mutant strain K246A was cultivated in a 5-L fermentor and used for whole-cell transformation. The results showed that with the substrate maltose concentration of 300 g/L at 35 °C and pH 7.0, the highest conversion rate reached 71.3%, and the yield of trehalose was 213.93 g/L. However, when maltose concentration was increased to 700 g/L, the yield of trehalose can reach 465.98 g/L with a conversion rate of 66%.