Abstract: Sucrose isomerase(SIase)is a kind of important industrial enzyme, which can produce non-carcinogenic isomaltulose by intramolecular transglycosylation of sucrose. Isomaltlose is a new type of sweetener that can replace sucrose. It has the characteristics of low calorie, low glycemic index (GI), non-cariogenic and anti-obesity, which can meet the requirements of functional sweetener to a large extent. In order to obtain a high-quality strain with a high SIase activity, the Erwinia rhubarb YS-1 was used as the initial strain. The strain was irradiated for 50 s 300 W power, at 28 ℃, irradiation distance of 2 mm and working gas flow of 12.0 L/min by atmospheric room temperature plasma technology(ARTP). By measuring the SIase activity and detecting the flocculation effect of the bacteria, the target strain was screened. In order to further improve the enzyme activity of the mutant strain SIase, Box-Behnken response surface test was used to optimize the fermentation conditions of the target strain on the basis of single factor test. The results showed that a total of 380 mutant strains were obtained after multi-round and multi-batch mutation, a mutant strain DXL-13(14.75 U/mL) whose SIase activity was 10.01 times higher than that of the original strain YS-1(1.34 U/mL) was obtained. In addition, the viscosity of the bacterial fermentation broth decreased, and the bacterial flocculation rate reached 85.62%. The decrease of the viscosity of bacterial fermentation broth indicated that the cells were more easily combined with the flocculant, which was beneficial to the separation of bacteria and product in industry. The quadratic regression equations of sucrose mass concentration, Na₂HPO₄·12H₂O mass concentration and fermentation time were established by response surface methodology. The optimal culture condition of mutant DXL-13 was 76 g/L sucrose, the mass concentration of Na₂HPO₄·12H₂O was 2.7 g/L and the fermentation time was 12.5 h. The results showed that the mass concentration of Na₂HPO₄·12H₂O had a significant effect on the SIase activity (P< 0.05), and the order of influence of each factor was: Na₂HPO₄·12H₂O mass concentration> fermentation time> sucrose mass concentration. There was an interaction between sucrose mass concentration and fermentation time. Under these conditions, the maximum activity of SIase reached 41.14 U/mL, which was 29.70 times higher than that of the original strain.