Thermostable Recombinant β‑(1→4)-Mannanase from C. thermocellum: Biochemical Characterization and Manno-Oligosaccharides Production

Functional attributes of a thermostable β-(1→4)-mannanase were investigated from Clostridium thermocellum ATCC 27405. Its sequence comparison the exhibited highest similarity with Man26B of C. thermocellum F1. The full length <i>Ct</i>Manf and truncated <i>Ct</i>ManT were cloned in the pET28a­(+) vector and expressed in E. coli BL21­(DE3) cells, exhibiting 53 kDa and 38 kDa proteins, respectively. On the basis of the substrate specificity and hydrolyzed product profile, <i>Ct</i>Manf and <i>Ct</i>ManT were classified as β-(1→4)-mannanase. A 1.5 fold higher activity of both enzymes was observed by Ca²⁺ and Mg²⁺ salts. Plausible mannanase activity of <i>Ct</i>Manf was revealed by the classical hydrolysis pattern of carob galactomannan and the release of manno-oligosaccharides. Notably highest protein concentrations of <i>Ct</i>Manf and <i>Ct</i>ManT were achieved in tryptone yeast extract (TY) medium, as compared with other defined media. Both <i>Ct</i>Manf and <i>Ct</i>ManT displayed stability at 60 and 50 °C, respectively, and Ca²⁺ ions imparted higher thermostability, resisting their melting up to 100 °C

Association constants (<i>K</i>_a) and free energy of binding of <i>Ct</i>CBM35 from affinity electrophoresis and relative fluorescence intensities.

<p><i>AE: Affinity Electrophoresis.</i></p

Qualitative binding of <i>Ct</i>CBM35 with insoluble mannan (A) using 12% SDS-PAGE.

<p>Lane 1: High range unstained molecular weight marker (200 kDa - 10 kDa), lane 2: Purified <i>Ct</i>CBM35, lane 3: unbound <i>Ct</i>CBM35, lane 4: bound <i>Ct</i>CBM35, lane 5: Bovine serum albumin (BSA) as control, lane 6: unbound BSA, lane 7: bound BSA. (B) Adsorption of <i>Ct</i>CBM35 to insoluble mannan. The main panel shows the equilibrium adsorption isotherm ([B] versus [F]) for <i>Ct</i>CBM35. Adsorption assay was done at 4°C, as described under methods section. Initial protein concentrations of <i>Ct</i>CBM35 were 0.2–19 µM. In the small panel showing a linear regression plot of 1/[B] versus 1/[F] concentrations to derive the association constant (<i>K</i>_a). (C) Scatchard plot of [B]/[F] vs [B]. The curved line was fitted to data points for <i>Ct</i>CBM35 by least square regression analysis. (D) a semi-logarithmic plot ([B] vs log [F]) for adsorption data of <i>Ct</i>CBM35. In both the plots the standard errors in two dimensions are indicated by vertical and horizontal bars.</p

Binding parameters of <i>Ct</i>CBM35 on binding with insoluble mannan derived from adsorption isotherm analysis.

*<p><i>values are mean ± SD (n = 3).</i></p

Denaturing SDS-PAGE (12%) of recombinant <i>Ct</i>CBM35 purified by IMAC.

<p>Denaturing SDS-PAGE (12%) of recombinant <i>Ct</i>CBM35 purified by IMAC.</p

Affinity electrophoresis of <i>Ct</i>CBM35 using 7.5% native PAGE in presence of varying concentrations of (A) carob galactomannan (B) konjac glucomannan (C) 10 mM Ca²⁺ incorporated with carob galactomannan (D) 10 mM Ca²⁺ incorporated with konjac glucomannan (E) A non linear regression plot of inverse relative migration of <i>Ct</i>CBM35 (1/r) against polysaccharide concentration (%, w v⁻¹), (•) carob galactomannan (in red), (▴) konjac glucomannan (in green) and (•) in presence of 10 mM Ca²⁺ ion with carob galactomannan (in light blue), (▴) in presence of 10 mM Ca²⁺ ion with konjac glucomannan (in dark blue).

<p>Affinity electrophoresis of <i>Ct</i>CBM35 using 7.5% native PAGE in presence of varying concentrations of (A) carob galactomannan (B) konjac glucomannan (C) 10 mM Ca²⁺ incorporated with carob galactomannan (D) 10 mM Ca²⁺ incorporated with konjac glucomannan (E) A non linear regression plot of inverse relative migration of <i>Ct</i>CBM35 (1/r) against polysaccharide concentration (%, w v⁻¹), (•) carob galactomannan (in red), (▴) konjac glucomannan (in green) and (•) in presence of 10 mM Ca²⁺ ion with carob galactomannan (in light blue), (▴) in presence of 10 mM Ca²⁺ ion with konjac glucomannan (in dark blue).</p

Protein melting curve of <i>Ct</i>CBM35 (—) in absence of 10 mM Ca²⁺ ion, (— —) in presence of 10 mM Ca²⁺ ion.

<p>Protein melting curve of <i>Ct</i>CBM35 (—) in absence of 10 mM Ca²⁺ ion, (— —) in presence of 10 mM Ca²⁺ ion.</p

Dynamic light scattering of <i>Ct</i>CBM35 in conjugation with 0.1% (w/v) (A) carob galactomannan, (B) konjac glucomannan and (C) 10 mM Ca²⁺ ion.

<p>Dynamic light scattering of <i>Ct</i>CBM35 in conjugation with 0.1% (w/v) (A) carob galactomannan, (B) konjac glucomannan and (C) 10 mM Ca²⁺ ion.</p

Tryptoptophan fluorescence emission spectrum of <i>Ct</i>CBM35 in presence of (A) carob galactomannan (%, w/v), represented in lines: (red) without polysaccharide, (in green) 0.01, (light blue) 0.04, (dark green) 0.06, (dark red) 0.08. (B) konjac glucomannan (red) without polysaccharide, (yellow) 0.01, (deep blue) 0.04, (light blue) 0.06, (dark green) 0.08, (dark red) 0.1.

<p>(C) Hill plot of log [(F_o−F)/F] vs log [carob galactomannan] (D) Hill plot of vs log [konjac glucomannan] used to derive association constant (<i>K</i>_a).</p

Amino acid residues of <i>Ct</i>CBM35 in the modeled structure (A) without Ca²⁺ ion (B) with Ca²⁺ ion (C) superimposed structure of both (A) and (B) showing the Ca²⁺ ion binding pocket to compare the altered positions of the amino acid residues in absence and presence of Ca²⁺ ion.

<p>Amino acid residues of <i>Ct</i>CBM35 in the modeled structure (A) without Ca²⁺ ion (B) with Ca²⁺ ion (C) superimposed structure of both (A) and (B) showing the Ca²⁺ ion binding pocket to compare the altered positions of the amino acid residues in absence and presence of Ca²⁺ ion.</p