Engineering Sucrose Isomerase by Truncation and Directed Evolution for Thermostability and Efficient Isomaltulose Production

Isomaltulose, a functional reducing sugar with promising applications, is biosynthesized from sucrose using sucrose isomerase (SIase). Nonetheless, the practical application of SIase has been hindered by its instability and reduced activity. In this study, we modified <i>Pantoea dispersa</i> SIase (PdSIase) by truncating certain flexible regions, resulting in PdSIaseΔ32, which exhibited a 2.29-fold increase in activity. Additionally, a high-throughput screening platform was designed to facilitate the directed evolution of PdSIaseΔ32, leading to the identification of PdSIaseΔ32-V300D/D330T, which demonstrated a 1.7-fold improvement in half-life at 45 °C. Structural modeling and molecular dynamics simulations revealed that the enhanced thermostability stemmed from the formation of new hydrogen bonds between the chains. Importantly, the bioconversion of 800 g/L sucrose with PdSIaseΔ32-V300D/D330T resulted in the production of 791.2 g/L isomaltulose, achieving a conversion rate of 98.9%. Our findings provide a fast screening tool and a theoretical foundation for advancing SIases, thereby facilitating the large-scale biosynthesis of isomaltulose.