Mechanistic Investigation in Ultrasound-Assisted (Alkaline) Delignification of <i>Parthenium hysterophorus</i> Biomass

Delignification of biomass is a primary step in biomass pretreatment in fermentation based synthesis of alcoholic biofuels. This paper attempts to give mechanistic insight into ultrasound-assisted delignification of biomass. <i>Parthenium hysterophorus</i> (carrot grass) has been used as the model biomass. The approach of study is to couple simulations of cavitation bubble dynamics to the experiments on delignification. Best values of delignification parameters with ultrasound have been identified as temperature = 303 K, NaOH concentration = 1.5% w/v, and biomass concentration = 2% w/v. Characterization of delignified biomass has been carried out using FTIR spectroscopy and XRD and FESEM techniques. Both physical and chemical effects of transient cavitation contribute to delignification. The physical effect of shock waves leads to depolymerization of lignin matrix through homolytic cleavage of phenyl ether α–O–4 and β–O–4 bonds. The chemical effect of radical generation causes hydroxylation/oxidation of the aromatic moieties and side chain elimination. Due to these peculiar mechanisms, ultrasound treatment gives effective delignification at ambient temperature and with lesser requirement of delignifying agents. Cavitation also causes decrystallization of cellulose due to partial depolymerization. Kinetic analysis of delignification at best values of parameters has revealed 2-fold enhancement with ultrasound as compared to mechanically agitated treatment

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

Structure modeling and functional analysis of recombinant dextransucrase from <i>Weissella confusa</i> Cab3 expressed in <i>Lactococcus lactis</i>

<p>The dextransucrase gene from <i>Weissella confusa</i> Cab3, having an open reading frame of 4.2 kb coding for 1,402 amino acids, was amplified, cloned, and expressed in <i>Lactococcus lactis</i>. The recombinant dextransucrase, <i>Wc</i>Cab3-rDSR was expressed as extracellular enzyme in M17 medium with a specific activity of 1.5 U/mg which after purification by PEG-400 fractionation gave 6.1 U/mg resulting in 4-fold purification. <i>Wc</i>Cab3-rDSR was expressed as soluble and homogeneous protein of molecular mass, approximately, 180 kDa as analyzed by SDS-PAGE. It displayed maximum enzyme activity at 35°C at pH 5.0 in 50 mM sodium acetate buffer. <i>Wc</i>Cab3-rDSR gave <i>K</i>_m of 6.2 mM and <i>V</i>_m of 6.3 µmol/min/mg. The characterization of dextran synthesized by <i>Wc</i>Cab3-rDSR by Fourier transform infrared and nuclear magnetic resonance spectroscopic analyses revealed the structural similarities with the dextran produced by the native dextransucrase. The modeled structure of <i>Wc</i>Cab3-rDSR using the crystal structures of dextransucrase from <i>Lactobacillus reuteri</i> (protein data bank, PDB id: 3HZ3) and <i>Streptococcus mutans</i> (PDB id: 3AIB) as templates depicted the presence of different domains such as A, B, C, IV, and V. The domains A and B are circularly permuted in nature having (β/α)₈ triose phosphate isomerase-barrel fold making the catalytic core of <i>Wc</i>Cab3-rDSR. The structure superposition and multiple sequence alignment analyses of <i>Wc</i>Cab3-rDSR with available structures of enzymes from family 70 GH suggested that the amino acid residue Asp510 acts as a nucleophile, Glu548 acts as a catalytic acid/base, whereas Asp621 acts as a transition-state stabilizer and these residues are found to be conserved within the family.</p

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

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

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

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

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

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