Surface-charged β-glucosidase synergizes cellulase for cellulose affinity in ionic liquid pretreated biomass in situ saccharification†

Protein surface charge engineering has promising applications for a deeper understanding of the adsorption and action mechanisms between enzymes and substrates. The engineering modification of β-glucosidase (BGL) is conducive to greatly reducing the negative feedback inhibition in biomass resource utilization and improving the utilization efficiency. Herein, the mutants BGL-14 and BGL-1, which had the largest difference in surface ζ-potential (−12.5 mV and −4.6 mV) obtained by rational design in previous studies, were selected to explore their adsorption behaviors on cellulose and lignin using an adsorption isotherm model. The results showed that more negative charges on the enzyme surface facilitated adsorptive contact with substrates while repelling lignin to reduce competitive adsorption, and that the monolayer coverage might be the main mechanism for cellulose/lignin adsorption of enzyme. Secondly, commercial cellulases were added to the modified BGLs to form a “cocktail” of enzymes (BGL-C) to synergistically participate in the in situ saccharification of biomass pretreated with ionic liquids (ILs). The BGL-14-C synergistic hydrolysis system presented a strong conversion ability in low concentrations of ILs, and its catalytic biomass production of reducing sugars at 5% (v/v) 1-ethyl-3-ethyl-imidazolium diethylphosphate ([EEIM]DEP) reached a yield of 1.24 g L⁻¹, which was 130% and 136% of that of WT-C and BGL-1-C, respectively. BGL-1-C, on the other hand, exhibited higher thermal stability, with the catalytic rate remaining at 51% when the temperature was increased from 50 °C to 70 °C in 1-ethyl-3-methyl-imidazolium diethylphosphate ([EMIM]DEP). Our study delves into the role of enzyme surface charge in reducing unproductive adsorption and increasing the probability of contact with the substrate, providing a theoretical basis for understanding the role of surface charge in mixed systems.