摘要
分析预测海藻糖合酶的空间结构,并进行分子对接和序列比对,选择Lys490位点进行定点饱和突变。利用全质粒PCR方法构建饱和突变体库,并利用高效液相色谱法筛选优势突变。经过筛选获得优势突变体K490L、K490I,对比分析表明,突变体K490L、K490I的转化率较原始酶分别提高了18%和15%。突变体酶学性质分析表明,K490L、K490I的最适反应温度及最适pH与原始酶保持一致,皆为25℃和pH 8.0,但两种突变酶的耐热性及耐酸性较原始酶有明显增强。在pH 7.0环境下,突变体K490L、K490I放置60 min后,剩余酶活力均达80%以上,而原始酶低于68%。
This paper focus on improving trehalose synthase( Tre S) conversion rate,also meeting the needs of full scale commercialization using genetic modification method. First,the spatial structure of Tre S was predicted and analyzed. Second,the molecular docking and sequence has been aligned. As result,the Lys490 site was selected for site-saturation mutation. A full plasmid PCR method was used to construct the library of the saturated mutants.HPLC was used to screen dominant mutations. The results showed that the maltose conversion rate of the mutants K490 L and K490 I was 18% and 15% higher than the original enzyme,respectively. The optimum condition of the two mutants were the same as that of the original enzyme,which was 25 ℃ and pH 8. 0. However,the heat and acid resistance were significantly higher. At pH 7. 0 for 60 min,the residual enzyme activity of of the two mutants were over 80%,while the original enzyme was less than 68%.
引文
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