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.
Highlights
We have recently described the catalytic properties of the Bglhi, which in addition to high glucose and thermal tolerance was shown to have high catalytic efficiency for cellobiose hydrolysis
Using the Bglhi in a combined protein engineering, kinetic and transglycosylation study, we have investigated the mechanisms of glucose/xylose stimulation of the cellobiase activity of a GH1 β-glucosidase, which has yielded novel insights as to the stimulation of pnitrophenolate ion (pNP-)glucosidase activity
Our results suggest that allosteric interactions, increased transglycosylation and competition of the monosaccharides glucose and xylose with the substrate at the -1 glycone and the +1/+2 aglycone binding sites all play a role in the stimulation mechanism
Summary
The construction of the pET28_bglhi plasmid containing the Bglhi has been previously described [15], and random mutagenesis of the bglhi coding sequence was performed by Engineering the GH1 β-glucosidase from Humicola insolens error-prone PCR (epPCR) using the pET28_bglhi plasmid as template. The determination of the rates of pNP- and free glucose release from pNP-Glc allows the estimation of the probabilities of the hydrolysis and the transglycosylation reactions catalyzed by a given mutant enzyme under particular experimental conditions. Reactions were performed under various conditions as follows: i) in the presence of xylose or glucose at MCmax (100 mM for Bglhi, 200 mM for the N89Y/ H307Y mutant and 500 mM for H307Y); ii) in the presence of xylose or glucose at the maximal concentration that did not apparently inhibit the enzymatic activity (maximum tolerance, MT), corresponding to 30 mM for D237V/P389H/E395G/K475R, 300 mM for D237V, 50 mM for A141T/N235S and 30 mM for N235S. Data acquisition and processing were performed using the MassLynx V4.1 (Waters Corporation)
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