Semirational Engineering of Clostridium thermosuccinogenes l-Rhamnose Isomerase by Targeting Flexible Regions Adjacent to the Active Pocket for d-Allose Biosynthesis

d-Allose, one of important rare sugars, has attracted increasing attention due to its low caloric value and remarkable physiological activities. l-Rhamnose isomerase (L-RI) catalyzes the reversible conversion of d-allulose to d-allose. However, its industrial application is limited by inadequate catalytic efficiency. In this study, L-RI from Clostridium thermosuccinogenes was identified and systematically characterized. Using a semirational protein engineering strategy, loop residues adjacent to the catalytic core and potential active sites predicted by the HotSpot Wizard web server were targeted. The resultant triple mutant N137Q/D258R/A265I achieved a d-allose conversion rate of 21.8% and a productivity of 130.8 g·L–1·h–1 within 1 h using 600 g·L–1 d-allulose. Molecular dynamics (MD) simulations revealed that modulation of loop flexibility enhanced the enzyme turnover rate. By fine-tuning loop flexibility while maintaining the structural integrity of the enzyme, this engineered mutant reduces the risk of enzyme inactivation during prolonged high-temperature catalysis. Collectively, these results highlight its potential for industrial d-allose production and provide valuable insights for the modification of L-RIs and related enzymes.