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.

Inducible lysine decarboxylases (LDCs) are essential in various cellular processes of microorganisms and plants, especially under acid stress, which induces expression of genes encoding LDCs. In this study, a novel Serratia marcesenes LDC (SmcadA) was successfully expressed in E. coli and structural modeling showed the protein as a decamer with a molecular weight of approximately 75 kDa. Further structural analysis of the protein network interactions showed that specific N-terminal and C-terminal residues with significant node size-degree correlation and high color intensity possessed high values of betweenness, suggesting they played a key role in protein function and activity. Molecular dynamics simulations further revealed that hydrogen bonds were vital interactions for stabilization of cofactor PLP and formation of intermediates, hence improved biocatalysis. Furthermore, mutations coffered structural changes of the protein residues and PLP hence altered the type of interacting residues with the ligand, causing changes in conformation of PLP. Moreover, temperature also induced changes in orientation of cofactor PLP and amino acid residues. This work therefore demonstrates the role of protein-ligand interactions in altering cofactor and binding site residue conformations thus contributing to improved biocatalysis.