Probing the role of residues lining the active site in the generation of glucose-tolerant variants of a fungal GH1 enzyme

Abstract The hydrolytic breakdown of cellobiose into glucose, catalysed by β-glucosidases, is the last and rate-limiting step in cellulose saccharification for producing fermentable glucose in the bioethanol industry. This limitation arises because β-glucosidase activity is inhibited by factors such as temperature, pH, and glucose accumulation in reactors. Enzyme inactivation leads to the buildup of cello-oligosaccharides, which, in turn, inhibit upstream cellulases. Therefore, glucose-tolerant β-glucosidases are preferred for the formulation of industrial cellulase cocktails. In this study, we have recombinantly expressed, purified, and biochemically characterised a β-glucosidase from the cellulolytic fungus Fusarium odoratissimum (FoBgl-WT). FoBgl-WT exhibits optimal cellobiose hydrolysis over a broad pH range (4.5-7.5), an important and industrially desirable property for its application in bioreactors. However, the glucose tolerance of FoBgl-WT was ∼0.56 M. Structure-based analyses were carried out to map the residues lining the active site of FoBgl, and their roles in stabilising the product glucose (or even the substrate, cellobiose) were elucidated through a series of site-specific mutations, followed by biochemical characterisation of the resulting FoBgl mutants. Among all the mutants generated, FoBgl-K256I-Y325F exhibits >2.5-fold greater glucose tolerance (∼1.4 M) than FoBgl-WT. Further, we have observed that the FoBgl-K256W and FoBgl-K256I mutants exhibit improved kinetic properties, such as catalytic efficiencies. The structure-based rational engineering efforts improve glucose tolerance and the kinetic properties of FoBgl mutants, making it a useful and promising candidate enzyme for industrial cellulase cocktails. Competing Interest Statement The authors have declared no competing interest.