Surface charge engineering of β-glucosidase using rational design improves catalytic capacity and ionic liquid tolerance

Abstract

β-Glucosidases are susceptible to external factors affecting catalytic efficiency in biomass resource utilization. In this study, a total of 14 positively-oriented and negatively-oriented surface-charged β-glucosidase mutants were constructed by rational design and surface charge engineering, having a gradient in ζ-potential from −4.6 mV to −12.7 mV. The negatively-oriented charged mutant BGL-14 (K283E/K332E) with the lowest ζ-potential (-12.7 mV) exhibited a 14 % increased substrate conversion after 2 h in 6 % (v/v) 1-ethyl-3-ethyl-imidazolium diethylphosphate ([EEIM]DEP) compared to wild-type. The inactivation kinetics indicate that the final plateau activity of positively-oriented charged BGL-1 (D9K/D239N/D273N/E291R) with the highest surface ζ-potential (-4.6 mV) was 75.29 % in 6 % (v/v) [EEIM]DEP, which was 5.23-fold higher than that of the wild-type. Furthermore, the presence of [EEIM]DEP considerably promoted the solubility and enzymatic activity of β-glucosidase, such that the catalytic capacity of BGL-14 was increased by 42 % in 6 % (v/v) [EEIM]DEP compared to 1-ethyl-3-methyl-imidazolium diethylphosphate ([EMIM]DEP). Changes in β-glucosidase charge, as well as structure caused by mutations, have a crucial role in IL tolerance, and the effect of charged residue substitution on IL appears to be complex, enzyme-specific, and highly dependent on the IL ion assemblies. In addition to the electrostatic repulsion of IL ions, other factors may be an extra molecular basis for enhancing tolerance/resistance to charge engineering in the IL environment.