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%.