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

Influence of GA3 and NAA on certain carbohydrate fractions in corms of saffron crocus (Crocus sativus L.) during development

Application of gibberellic acid (GA3) and napthaleneacetic acid (NAA) to corms of saffron crocus (Crocus sativus L.) markedly influenced degradation of reserve carbohydrates in the corm tissues. GA3 stimulated the breakdown of starch and promoted the accumulation of soluble sugars, especially sucrose. Although NAA treatment stimulated starch breakdown for the first 14 days after the treatment, the effect was less pronounced as compared to that of GA3. NAA treatment promoted the accumulation of reducing sugars but suppressed accumulation of non reducing sugars in the corm tissues. Corms treated with GA3 showed an increased accumulation of total pentoses and total ketoses. In NAA treated corms, accumulation of total pentoses was stimulated but that of total ketoses was suppressed. The effects of GA3 and NAA on carbohydrate changes in the corm tissues are discussed in the light of their effects on bud development

Evolutionary divergence in Chenopodium and validation of SNPs in chloroplast rbcL and matk genes by allele-specific PCR for development of Chenopodium quinoa-specific markers

The genus Chenopodium comprises about 150 species, of which Chenopodium quinoa and C. album are important for their nutritional value. Evaluation of variation in qualitative morphological traits of plants and SNPs in chloroplast rbcL and matK gene sequences in 19 accessions representing C. quinoa and C. album indicated that the accessions IC-411824 and IC-411825, which have white seeds, belong to C. quinoa rather than C. album. This observation was also supported by a time tree that indicated IC-411824 and IC-411825 to be a sister clade to accessions of C. quinoa with an estimated age of 1.2 Mya. Whereas multiple alignments of rbcL gene sequences from the 19 accessions revealed 1.26% parsimony-informative sites with 0.68% interspecific sequence diversity, alignment of nucleotide sequences of amplicons representing the matK gene revealed 4.97% parsimony-informative sites and 2.81% interspecific sequence diversity. Validation of SNPs in the cp rbcL and matK regions of 36 accessions belonging to C. quinoa and C. album was performed by allele-specific PCR with primers carrying a single base change at the 3′ end. We report the first C. quinoa-specific SNP-based primer, R1RQ-AFR, designed from rbcL sequences, that could differentiate quinoa from 64 genera including 13 species of the genus Chenopodium. With an estimated age of 10.5–4.1 million years (Myr), the Himalayan chenopods are evolutionarily younger than the Andean chenopods. The results establish the paraphyletic origin of the genus Chenopodium

Amino acid sequence of the basic subunit of 13S globulin of buckwheat

A 26 kDa basic subunit of 13S globulin has been purified from grains of common buckwheat (Fagopyrum esculentum Moench). The amino acid composition of the protein closely matches the W.H.O. recommended values for a nutritionally balanced protein. The sequence of 17 N-terminal amino acid residues of the protein revealed 73.3 and 66.7% homology with soya bean glycinin and pea legumin, respectively

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

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

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

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