Fig 1.
Catalytic reaction mechanism of the retaining β-glycosidases.
After the formation of the glucosyl-enzyme intermediate (step 1), the entry of a water molecule leads to hydrolysis (step 2) and the entry of a sugar leads to transglycosylation (step 3).
Table 1.
Characterization of the selected mutants.
Fig 2.
Positions of the mutations inserted in Bglhi in each mutant.
The 3-D structures of (a) N235S, (b) A141T/N235S, (c) D237V/P389H/E395G/K475R, (d) D237V, (e) N89YH307Y and (f) H307Y were modelled using the crystal structure of Bglhi (PDB code: 4MDO) as template with the MODELLER 9.10 software. The amino acid substitutions identified by random mutagenesis and functional selection are indicated as yellow spheres in Fig 2. The catalytic residues (E166 and E377), the glycone and the aglycone-binding sites are shown as red, green and blue spheres, respectively. The inset shows a ribbon representation of the +1/+2 aglycone binding region of the wild-type Bglhi (in green) showing the catalytic residues (E166 and E377), together with the mutants N235S (in magenta), D237V (in light blue) and H307Y (in yellow). The highly conserved positions of the main chain atoms resulted in the mottled appearance of the main chain after superposition of the different structures.
Fig 3.
Effect of increasing concentrations of pNP-Glc, glucose and xylose on the pNP-glucosidase activity.
(a) Bglhi, (b) N89Y/H307Y, (c) H307Y, (d) D237V/P389H/E395G/K475R, (e) D237V, (f) A141T/N235S, (g) N235S. (A) Effect of increasing concentrations of pNP-Glc on the pNP-glucosidase activity in the absence (black lines and dots) or in the presence of glucose (red lines and dots) or xylose (blue lines and dots) at fixed concentrations equal to MCmax. (B) Effect of increasing concentrations of glucose (red lines and dots) or xylose (blue lines and dots) on the pNP-glucosidase activity of each mutant enzyme at 90–95% saturating concentrations of pNP-Glc (2 mM for Bglhi, N89Y/H307Y and H307Y; 1.5 mM for D237V/P389H/E395G/K475R, A141T/N235S and N235S; 3 mM for D237V). All experiments were repeated three times using three separate pure enzyme preparations. Each point represents the mean of duplicate assays ± SD (error bars are not evident, as they lie within the area of the symbol).
Table 2.
Kinetic parameters for the stimulation of the pNP-glucosidase activity of Bglhi and mutants against pNP-Glc in the absence or presence of fixed concentrations of glucose or xylose.
Table 3.
Glucose- and xylose-stimulation of the pNP-glucosidase activity of Bglhi and mutants.
Fig 4.
Effect of increasing concentrations of cellobiose and xylose on the cellobiase activity.
(a) Bglhi, (b) N89Y/H307Y, (c) H307Y, (d) D237V/P389H/E395G/K475R, (e) D237V, (f) A141T/N235S, (g) N235S. (A) Effect of increasing concentrations of cellobiose on the cellobiase activity, in the absence (black lines and dots) or in the presence of fixed concentrations of xylose (blue lines and dots). The fixed concentrations of xylose were equal to MCmax (Bglhi, N89Y/H307Y and H307Y) or MT (D237V/P389H/E395G/K475R, D237V, A141T/N235S and N235S). (B and C) Effect of increasing concentrations of xylose on the cellobiase activity of each enzyme at 90–95% saturating concentrations of cellobiose (5 mM for Bglhi, 40 mM for N89Y/H307Y, 70 mM for H307Y and D237V, 10 mM for D237V/P389H/E395G/K475R, 2 mM for A141T/N235S and 1 mM for N235S). The red asterisks (*) in Fig C indicate the MT concentrations. The experiments were repeated three times using three separate pure enzyme preparations. Each point represents the mean of duplicate assays ± SD (note that the error bars are not visible, as they lie within the area of the symbol).
Table 4.
Kinetic parameters for the stimulation of the cellobiase activity of Bglhi and mutants by cellobiose in the presence or absence of fixed concentrations of xylose.
Table 5.
Rates of pNP- and glucose liberation and percentages of hydrolysis (%H) and transglycosylation (%T) of pNP-Glc by Bglhi and mutants.
Fig 5.
Time-course analysis of the reaction products formed by Bglhi and mutants against cellobiose.
(a) Bglhi, (b) N89Y/H307Y, (c) H307Y, (d) D237V/P389H/E395G/K475R, (e) D237V, (f) A141T/N235S, (g) N235S. The reactions were performed at 90–95% saturating concentrations of cellobiose (see legend to Fig 4) in the absence (indicated as “control”) or presence of glucose (indicated as “Plus G1”) or xylose (indicated as “Plus X1”). The final concentrations of each monosaccharide were equal to MCmax (Bglhi, N89Y/H307Y and H307Y) or MT (D237V/P389H/E395G/K475R, D237V, A141T/N235S and N235S). The reaction times were 0 (lanes t0), 5 min (lanes t1), 10 min (lanes t2) and 24 h (lanes t3). Standards: G1, glucose; G2, cellobiose; G3, cellotriose; G4, cellotetraose; Ge, gentibiose; X1, xylose; X2, xylobiose; SG2, equimolar mixture of sophorose and cellobiose. The volumes of each aliquot applied to the TLC plates were adjusted aiming the best visualization of the products.
Fig 6.
Tandem mass spectrometry analysis of glucopyranosyl-xylose (A), cellotriose (B) and cellotetraose (C). The sodium adducts of glucopyranosyl-xylose (m/z 335), cellotriose (m/z 527) and cellotetraose (m/z 689) were analyzed by MS/MS and the proposed interpretation of mass spectra are indicated on the structures.