Sucrose isomerase (SI), catalysis sucrose to isomaltulose, has been wildly used in industrial production of isomaltulose. Here, rational design of Pantoea dispersa SI for improving its thermostability by predicting and substituting the unstable amino acid residues was studied using the computational-aided predictor FoldX. Through the mutation pool, two mutants of SI (V280L, S499F) displayed favorable characteristics on thermostability. The double mutant V280L/S499F were further constructed and showed prolonged half-life at 45 ℃, about 9-fold compared to the wild-type. Accordingly, the melting temperature of mutant V280L/S499F was improved to 54.2 ℃. To determine the recyclable ability of mutant V280L/S499F to bioconversion of isomaltulose, recombinant Corynebacterium glutamicum/pXMJ19/pdsiV280L/S499F was constructed and repeated batch conversion was performed in a 5 L bioreactor. The results shown that the maximum yield of isomaltulose by batch conversion reached to 451 g/L with a productivity of 45.1 g/L/h, and the conversion rate remained 83.2 ± 2.1% even after 15 repeated batches of biocatalysis. Structure-based molecule molding revealed that the interiors of mutant V280L/S499F was more tightly packed in ɑ-9 fold and a new hydrophobic network was formed in ɑ-17, which combined contributed to improving its thermostability. This work provides new reference for the sustainable production of isomaltulose.