Enhanced Thermostability of an l -Rhamnose Isomerase for d -Allose Synthesis by Computation-Based Rational Redesign of Flexible Regions

d -Allose is a low-calorie rare sugar with great application potential in the food and pharmaceutical industries. The production of d -allose has been accomplished using l -rhamnose isomerase (L-RI), but concomitantly increasing the enzyme’s stability and activity remains challenging. Here, we rationally engineered an L-RI from Clostridium stercorarium to enhance its stability by comprehensive computation-aided redesign of its flexible regions, which were successively identified using molecular dynamics simulations. The resulting combinatorial mutant M2-4 exhibited a 5.7-fold increased half-life at 75 °C while also exhibiting improved catalytic efficiency. Especially, by combining structure modeling and multiple sequence alignment, we identified an α0 region that was universal in the L-RI family and likely acted as a “helix-breaker”. Truncating this region is crucial for improving the thermostability of related enzymes. Our work provides a significantly stable biocatalyst with potential for the industrial production of d -allose.