Deciphering ligand specificity of a Clostridium thermocellum family 35 carbohydrate binding module (CtCBM35) for Gluco- and Galacto- Substituted mannans and Its calcium induced stability

Articles in International JournalsThis study investigated the role of CBM35 from Clostridium thermocellum (CtCBM35) in polysaccharide recognition. CtCBM35 was cloned into pET28a (+) vector with an engineered His6 tag and expressed in Escherichia coli BL21 (DE3) cells. A homogenous 15 kDa protein was purified by immobilized metal ion chromatography (IMAC). Ligand binding analysis of CtCBM35 was carried out by affinity electrophoresis using various soluble ligands. CtCBM35 showed a manno-configured ligand specific binding displaying significant association with konjac glucomannan (Ka = 14.3×104 M−1), carob galactomannan (Ka = 12.4×104 M−1) and negligible association (Ka = 12 µM−1) with insoluble mannan. Binding of CtCBM35 with polysaccharides which was calcium dependent exhibited two fold higher association in presence of 10 mM Ca2+ ion with konjac glucomannan (Ka = 41×104 M−1) and carob galactomannan (Ka = 30×104 M−1). The polysaccharide binding was further investigated by fluorescence spectrophotometric studies. On binding with carob galactomannan and konjac glucomannan the conformation of CtCBM35 changed significantly with regular 21 nm peak shifts towards lower quantum yield. The degree of association (Ka) with konjac glucomannan and carob galactomannan, 14.3×104 M−1 and 11.4×104 M−1, respectively, corroborated the findings from affinity electrophoresis. The association of CtCBM35with konjac glucomannan led to higher free energy of binding (ΔG) −25 kJ mole−1 as compared to carob galactomannan (ΔG) −22 kJ mole−1. On binding CtCBM35 with konjac glucomannan and carob galactomannan the hydrodynamic radius (RH) as analysed by dynamic light scattering (DLS) study, increased to 8 nm and 6 nm, respectively, from 4.25 nm in absence of ligand. The presence of 10 mM Ca2+ ions imparted stiffer orientation of CtCBM35 particles with increased RH of 4.52 nm. Due to such stiffer orientation CtCBM35 became more thermostable and its melting temperature was shifted to 70°C from initial 50°C

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

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

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

A Simplified Multiplex PCR Assay for Simultaneous Detection of Six Viruses Infecting Diverse Chilli Species in India and Its Application in Field Diagnosis

Chilli is infected by at least 65 viruses globally, with a mixed infection of multiple viruses leading to severe losses being a common occurrence. A simple diagnostic procedure that can identify multiple viruses at once is required to track their spread, initiate management measures and manage them using virus-free planting supplies. The present study, for the first time, reports a simplified and robust multiplex PCR (mPCR) assay for the simultaneous detection of five RNA viruses, capsicum chlorosis orthotospovirus (CaCV), chilli veinal mottle virus (ChiVMV), large cardamom chirke virus (LCCV), cucumber mosaic virus (CMV), and pepper mild mottle virus (PMMoV), and a DNA virus, chilli leaf curl virus (ChiLCV) infecting chilli. The developed mPCR employed six pairs of primer from the conserved coat protein (CP) region of the respective viruses. Different parameters viz., primer concentration (150–450 nM) and annealing temperature (50 °C), were optimized in order to achieve specific and sensitive amplification of the target viruses in a single reaction tube. The detection limit of the mPCR assay was 5.00 pg/µL to simultaneously detect all the target viruses in a single reaction, indicating a sufficient sensitivity of the developed assay. The developed assay showed high specificity and showed no cross-amplification. The multiplex PCR assay was validated using field samples collected across Northeast India. Interestingly, out of 61 samples collected across the northeastern states, only 22 samples (36%) were positive for single virus infection while 33 samples (54%) were positive for three or more viruses tested in mPCR, showing the widespread occurrence of mixed infection under field conditions. To the best of our knowledge, this is the first report on the development and field validation of the mPCR assay for six chilli viruses and will have application in routine virus indexing and virus management

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

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

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

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