来源于Pseudomonas putidaP06的海藻糖合酶能以麦芽糖为底物，一步转化生成海藻糖，但其麦芽糖转化率相对较低。通过分子改造提高海藻糖合酶的转化率，使其满足产业化需要，是一种非常有效的方法。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%。

The trehalose synthase fromPseudomonas putidaP06 can convert maltose into trehalose in one step, but the conversion rate of maltose to trehalose is lower. In order to improve the conversion rate and meet the needs of industrialization, molecular modification of trehalose synthase is a very effective method. Analysis and predict the spatial structure of trehalose synthase (TreS), molecular docking and sequence alignment, the Lys490 site of trehalose synthase was selected for site-saturation mutation. The full - length PCR method was used to construct the library of saturated mutants, and the dominant mutations were screened by high performance liquid chromatography (HPLC). The results showed that the conversion rate of maltose from the mutants K490L and K490I was 18% and 15% higher than the original enzyme, respectively. And the optimum reaction temperature and optimum pH of K490L and K490Iwere consistent with the original enzyme, 25oC and pH8.0, the heat resistance and acid resistance of two mutant enzymes were obviously higher than the original enzyme. The mutants K490L and K490Iwas respectively setted at pH7.0 for 60 min, the residual enzyme activity was more than 80%, while the residual enzyme activity of original enzyme was less than 68%.