A recombinant xylose reductase enzyme with biotechnological potential for xylitol production.

Xylitol is a sugar alcohol widely used in the food industry due to its lower caloric value compared to sucrose. Industrial xylitol production relies primarily on chemical hydrogenation processes that require high energy input. Although biotechnological routes represent a more sustainable alternative, their industrial implementation is often limited by enzyme stability and cofactor costs. This study presents the design, expression, and biochemical analysis of recombinant xylose reductase from Neurospora crassa (rNcXR). The optimized gene was cloned into a salt-inducible T7-based expression system and expressed in Escherichia coli (E. coli) BL21-SI. The design incorporated an OmpT signal peptide to encourage secretion into the periplasm, and a 6×His tag at the C-terminus to simplify purification. The recombinant enzyme exhibited a specific activity of 20.87 U/mg, significantly exceeding values previously reported for both native and recombinant xylose reductases. Kinetic characterization revealed that the engineered rNcXR maintains a high substrate affinity while achieving a remarkably enhanced catalytic turnover compared to traditional fungal biocatalysts. Furthermore, experimental validation confirmed an optimized cofactor-binding pocket, supporting the structural enhancements predicted by molecular docking. The rNcXR displayed robust thermodynamic stability at an alkaline pH and maintained high catalytic efficiency at elevated temperatures up to 55 °C. These results demonstrate that the engineered rNcXR represents a resilient and high-performance biocatalyst with strong potential for continuous enzymatic xylitol production in industrial biorefineries.