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

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

摘要:

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。

Abstract:

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%.