Engineering Glucose-Tolerant Variants by Probing Residues Lining the Active Site of a Fungal β-Glucosidase.

The hydrolytic breakdown of cellobiose into glucose, catalyzed 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 characterized 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 stabilizing the product glucose (or even the substrate, cellobiose) were elucidated through a series of site-specific mutations, followed by biochemical characterization of the resulting FoBgl mutants. Among all the mutants generated, FoBgl-K256I and FoBgl-K256W exhibit ~2-2.5-fold enhancement in glucose tolerance. Further, the FoBgl-K256W variant displayed a two-fold improvement in catalytic efficiency compared to FoBgl-WT. Double mutants of FoBgl-WT targeting the +2 subsite of glucose binding further enhanced the glucose tolerance levels; however, the catalytic efficiencies decreased considerably. 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.