MOESM1 of Lignocellulose binding of a Cel5A-RtCBM11 chimera with enhanced β-glucanase activity monitored by electron paramagnetic resonance

Additional file 1: Figure S1. Smoothed far ultraviolet circular dichroism spectra of the BsCel5A-CBM3 (black line) and BsCel5A-CBM11 (red line). The insert presents the percentage of secondary structure elements in both proteins as estimated by deconvolution using the ContinII software [33]. See the relevant section in “Materials and Methods” for further experimental details

Time-course analysis of the reaction products formed by Bglhi and mutants against cellobiose.

<p>(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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188254#pone.0188254.g004" target="_blank">Fig 4</a>) 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 MC_max (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.</p

Glucose- and xylose-stimulation of the <i>p</i>NP-glucosidase activity of Bglhi and mutants.

<p>Glucose- and xylose-stimulation of the <i>p</i>NP-glucosidase activity of Bglhi and mutants.</p

Catalytic reaction mechanism of the retaining β-glycosidases.

<p>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).</p

Rates of <i>p</i>NP^- and glucose liberation and percentages of hydrolysis (%H) and transglycosylation (%T) of <i>p</i>NP-Glc by Bglhi and mutants.

<p>Rates of <i>p</i>NP^- and glucose liberation and percentages of hydrolysis (%H) and transglycosylation (%T) of <i>p</i>NP-Glc by Bglhi and mutants.</p

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.

<p>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.</p

Kinetic parameters for the stimulation of the <i>p</i>NP-glucosidase activity of Bglhi and mutants against <i>p</i>NP-Glc in the absence or presence of fixed concentrations of glucose or xylose.

<p>Kinetic parameters for the stimulation of the <i>p</i>NP-glucosidase activity of Bglhi and mutants against <i>p</i>NP-Glc in the absence or presence of fixed concentrations of glucose or xylose.</p

Engineering the GH1 β-glucosidase from <i>Humicola insolens</i>: Insights on the stimulation of activity by glucose and xylose - Fig 6

<p><b>Tandem mass spectrometry analysis of glucopyranosyl-xylose (A), cellotriose (B) and cellotetraose (C).</b> The sodium adducts of glucopyranosyl-xylose (<i>m/z</i> 335), cellotriose (<i>m/z</i> 527) and cellotetraose (<i>m/z</i> 689) were analyzed by MS/MS and the proposed interpretation of mass spectra are indicated on the structures.</p

Effect of increasing concentrations of cellobiose and xylose on the cellobiase activity.

<p>(a) Bglhi, (b) N89Y/H307Y, (c) H307Y, (d) D237V/P389H/E395G/K475R, (e) D237V, (f) A141T/N235S, (g) N235S. <b>(A)</b> 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 MC_max (Bglhi, N89Y/H307Y and H307Y) or MT (D237V/P389H/E395G/K475R, D237V, A141T/N235S and N235S). <b>(B and C)</b> 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).</p

Characterization of the selected mutants.

<p>Characterization of the selected mutants.</p