O'Donnell, A.; Abbas, G.

{beta}-glucosidase (BglB) from Paenibacillus polymyxa was mutated (G23S, Rosetta/Foldit numbering; G26S, conventional numbering) to assess structural and functional changes. Foldit modeling and prior Design 2 Data (D2D) database results led us to hypothesize that this mutation would increase substrate binding affinity and catalytic efficiency, with a moderate reduc-tion in thermal stability. The mutant protein was expressed, purified, and analyzed using kinetics and thermal stability as-says. Relative to the wild-type (WT), G23S exhibited a similar binding affinity (similar Km), an approximately 2-fold in-crease in turnover number (kcat) and catalytic efficiency (kcat/Km), an almost 14-fold increase in maximum reaction velocity (Vmax) and a slight decrease in thermostability (T50). The results largely support the hypothesis, indicating that changes in residue 23 can enhance catalytic power while minimally compromising stability.