Engineering the GH1 β-glucosidase from Humicola insolens: Insights on the stimulation of activity by glucose and xylose

Abstract

The activity of the GH1 β-glucosidase from Humicola insolens (Bglhi) against p-nitrophenyl-β-D-glucopyranoside (pNP-Glc) and cellobiose is enhanced 2-fold by glucose and/or xylose. Kinetic and transglycosylation data showed that hydrolysis is preferred in the absence of monosaccharides. Stimulation involves allosteric interactions, increased transglycosylation and competition of the substrate and monosaccharides for the -1 glycone and the +1/+2 aglycone binding sites. Protein directed evolution has been used to generate 6 mutants of Bglhi with altered stimulation patterns. All mutants contain one of three substitutions (N235S, D237V or H307Y) clustered around the +1/+2 aglycone binding sites. Two mutants with the H307Y substitution preferentially followed the transglycosylation route in the absence of xylose or glucose. The strong stimulation of their pNP-glucosidase and cellobiase activities was accompanied by increased transglycosylation and higher monosaccharide tolerance. The D237V mutation favoured hydrolysis over transglycosylation and the pNP-glucosidase activity, but not the cellobiase activity, was stimulated by xylose. The substitution N235S abolished the preference for hydrolysis or transglycosylation; the cellobiase, but not the pNP-glucosidase activity of the mutants was strongly inhibited by xylose. Both the D237V and N235S mutations lowered tolerance to the monosaccharides. These results provide evidence that the fine modulation of the activity of Bglhi and mutants by glucose and/or xylose is regulated by the relative affinities of the glycone and aglycone binding sites for the substrate and the free monosaccharides.

Redefining Humicola sensu stricto and related genera in the Chaetomiaceae

“…The older names H. insolens and S. thermophilum are still frequently used in publications (e.g. Carniel et al., 2017 , Meleiro et al., 2017 , Meleiro et al., 2018 ), leading to confusion among researchers unaware that they are actually working with the organism (e.g. Meleiro et al., 2017 , Meleiro et al., 2018 ).…”

Progress in Ameliorating Beneficial Characteristics of Microbial Cellulases by Genetic Engineering Approaches for Cellulose Saccharification

“…Cel1A mutant of T. reesei also displayed enhanced pH stability and thermostability as well as 2.4-fold enhanced specific activity as compared to the wild-type Cel1A ( Guo et al, 2016 ). Two mutations D237V and N235S in bgl from Humicola insolens (bglhi) led to decline in tolerance to monosaccharides ( Meleiro et al, 2017 ). Following semi-rational engineering approach, Fang et al (2016) obtained bgl1A (bgl isolated from a marine microbial metagenomic library) variant A24S/F297Y with improved ethanol tolerance.…”

Microbial Beta Glucosidase Enzymes: Recent Advances in Biomass Conversation for Biofuels Application

“…The second step of deglycosylation involves the activity of the –COOH group acting as a base, which acted as a proton donor previously and now accepts the proton form a nucleophile such as water, monosaccharide, disaccharide, and monoterpenealcohol. It breaks the intermediate complex of the glucose–enzyme and sets the enzyme free with the production of a second product, and this completes the catalytic mechanism [149,150]. The catalytic mechanism of β-glucosidase of the family GH9 involves a single step, unlike the families GH1 and GH3 [141].…”