Computer-Aided Rational Design Enhances 2-O-α-Glucosyl Glycerol Biosynthesis by Sucrose Phosphorylase in...

Industrial biosynthesis of value-added glycosides in high-viscosity concentrated substrates still faces unprecedented challenges. Herein, manifold computational approaches are combined to rationally engineer the thermal tolerance of sucrose phosphorylase (SPase) for strengthening the performance in high-viscosity system. Through rational combination of ΔΔG, B-factor, and conservative amino acids, mutant V70I emerged as a superior mutant with an elevated optimal temperature, enhanced thermal tolerance, and augmented catalytic activity among 120 candidates. In a super high-viscosity system (267.8 mPa·s), mutant V70I demonstrated remarkable industrial potential as a biocatalyst, achieving 423 g/L 2-O-α-glucosyl glycerol (2-O-α-GG) production from 1.8 M sucrose and 2.16 M glycerol (a 208 g/L increase over the wild type) at 45 °C. This study highlights the industrial merits of rational integrated computational approaches, enabling efficient enzyme engineering for superior biocatalysis in high-viscosity systems.