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. 2020 Apr 24;10(1):6998.
doi: 10.1038/s41598-020-63972-y.

A Highly Glucose Tolerant ß-Glucosidase From Malbranchea Pulchella (MpBg3) Enables Cellulose Saccharification

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A Highly Glucose Tolerant ß-Glucosidase From Malbranchea Pulchella (MpBg3) Enables Cellulose Saccharification

Lummy Maria Oliveira Monteiro et al. Sci Rep. .
Free PMC article

Abstract

β-glucosidases catalyze the hydrolysis β-1,4, β-1,3 and β-1,6 glucosidic linkages from non-reducing end of short chain oligosaccharides, alkyl and aryl β-D-glucosides and disaccharides. They catalyze the rate-limiting reaction in the conversion of cellobiose to glucose in the saccharification of cellulose for second-generation ethanol production, and due to this important role the search for glucose tolerant enzymes is of biochemical and biotechnological importance. In this study we characterize a family 3 glycosyl hydrolase (GH3) β-glucosidase (Bgl) produced by Malbranchea pulchella (MpBgl3) grown on cellobiose as the sole carbon source. Kinetic characterization revealed that the MpBgl3 was highly tolerant to glucose, which is in contrast to many Bgls that are completely inhibited by glucose. A 3D model of MpBgl3 was generated by molecular modeling and used for the evaluation of structural differences with a Bgl3 that is inhibited by glucose. Taken together, our results provide new clues to understand the glucose tolerance in GH3 β-glucosidases.

Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Purification, identification and deglycosylation effect of M. pulchella β-glucosidase (MpBgl3). (A) 10% SDS-PAGE stained with Coomassie Blue in (1) Dual Color Standards (BIO-RAD) molecular weight marker and (2) Crude extract produced by M. pulchella and (3) purified MpBgl3. (B) Zymogram in semi-denaturing conditions in gel 10% in (4) Dark band shows MpBgl3 activity. Effect of the deglycosylation on the MpBgl3 molecular weight. (C) Polyacrylamide gel electrophoresis 12% of the glycosylated and deglycosylated MpBgl3; (D) Determination of the molecular masses of glycosylated and deglycosylated MpBgl3 on SDS-PAGE. The band below the deglycosylated MpBgl3 is the PNGase F enzyme. Original photos of the gels can be found in Fig. S2.
Figure 2
Figure 2
Effect of temperature and pH, and temperature and pH stability on MpBgl3 activity. (A) Temperature effect showing optimum temperature at 50 °C; (B) pH effect showing as optimum pH 6.0; (C) Thermal inactivation in which 100% activity was measured at t = 0. (D) pH stability where 100% activity was measured at t = 0, immediately before the addition of different buffers.
Figure 3
Figure 3
Glucose effect on the MpBgl3 activity. The glucose concentrations tested were 0.05 M; 0.1 M; 0.25 M; 0.5 M and 1 M. As control, the activity was measured without glucose, and in this case the specific activity was 9.8 U/mg.
Figure 4
Figure 4
Influence of ions, EDTA and β-mercaptoethanol on MpBgl3 activity. The final concentration of each compound tested was 10 mM. The control sample was the activity measured in the absence of the compounds, represented in the graph as Bgl and therefore, equivalent to 9.8 U/mg. For this experiment the enzyme was previously dialyzed.
Figure 5
Figure 5
Far-UV circular dichroism spectrum of the purified MpBgl3.
Figure 6
Figure 6
Sequence alignment of GH3 family β-glucosidases. MpBgl3 (M. pulchella Bgl3), AnBgl3 (A. niger), AoBgl3 (A. oryzae Bgl3), AaBgl3 (A. aculeatus Bgl3). The secondary structures of the MpBgl3 model and AaBgl3 crystal structure are shown above and below the amino acid sequences, respectively. The residues conserved between the sequences are show in the blue boxes. The green numbers correspond to the Cys residues that participate in the disulfide bridge in MpBgl3. Residues that directly bind the glucose to the glycone-binding site are indicated by pink squares. Residues around the aglycone-binding site are indicated by green squares. The alignment was performed using MULTALIN and graphically displayed using ESPript.
Figure 7
Figure 7
Structural comparison of MpBgl3 and AaBgl3. The MpBgl3 and AaBgl3 structures are represented on the right (in gray) and on the left (in pink) sides, respectively. The glucose at the glycone and aglycone-binding sites are represented in green and yellow, respectively. (A) Representation of the residues involved in the glucose interaction at the glycone-binding site. (B) Depth depiction of both, the catalytic site of the glucose tolerant (MpBgl3) and glucose intolerant (AaBgl3) enzymes. Residues that differ between the two enzymes and that contribute to the difference in the shape of the aglycone-binding site are represented in cyan and blue for AaBgl3 and MpBgl3, respectively. (C) View of the active site entrance, illustrating the wider entrance to the substrate binding cleft for AaBgl3 (inhibited by glucose) and the narrower entrance for the MpBgl3 (glucose tolerant). (D) Electrostatic potential surface of MpBgl3 and AaBgl3 generated by Pymol program, highlighting the differences on electrostatic environment of the catalytic site entrance. The images were generated with Pymol program (W.L. Delano, The PyMol Molecular Graphics System, DeLano Scientific).

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