Enhancing the Catalytic Efficiency of <scp>l</scp>-Rhamnose Isomerase for <scp>d</scp>-Allose Synthesis through Simultaneous Engineering of Noncatalytic and Catalytic Pockets

d-Allose is a crucial rare sugar that holds significant application value in the food, pharmaceutical, and healthcare industries. The most prevalent method for the production of d-allose is its conversion from d-allulose, which is catalyzed by l-rhamnose isomerase (l-RI). However, this enzyme demonstrates relatively low catalytic efficiency toward d-allulose. To enhance the catalytic efficiency of l-RI derived from <i>Clostridium stercorarium</i>, key amino acids in the noncatalytic pockets were mutated to improve the probability of d-allulose entering the catalytic pocket and to increase the local concentration of the substrate in the active region. Simultaneously, the impact of steric hindrance on the lid around the catalytic pocket was reduced by mutating the amino acid His99. Notably, the catalytic efficiency of the combined mutant E53D/A142G/E273D/H99A toward d-allulose was increased by 170% compared to that of the wild-type enzyme. Moreover, <i>Bacillus subtilis</i> 168 whole cells expressing this l-RI variant achieved a 36.5% conversion rate of d-allose from 100 g/L d-allulose within 90 min. This study presents a highly efficient biocatalyst with the potential for industrial production of d-allose.