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Abstract
The study describes a comparative analysis of biochemical, structural and functional properties of two recombinant derivatives from Clostridium thermocellum ATCC 27405 belonging to family 43 glycoside hydrolase. The family 43 glycoside hydrolase encoding α-L-arabinofuranosidase (Ct43Araf) displayed an N-terminal catalytic module CtGH43 (903 bp) followed by two carbohydrate binding modules CtCBM6A (405 bp) and CtCBM6B (402 bp) towards the C-terminal. Ct43Araf and its truncated derivative CtGH43 were cloned in pET-vectors, expressed in Escherichia coli and functionally characterized. The recombinant proteins displayed molecular sizes of 63 kDa (Ct43Araf) and 34 kDa (CtGH43) on SDS-PAGE analysis. Ct43Araf and CtGH43 showed optimal enzyme activities at pH 5.7 and 5.4 and the optimal temperature for both was 50°C. Ct43Araf and CtGH43 showed maximum activity with rye arabinoxylan 4.7 Umg−1 and 5.0 Umg−1, respectively, which increased by more than 2-fold in presence of Ca2+ and Mg2+ salts. This indicated that the presence of CBMs (CtCBM6A and CtCBM6B) did not have any effect on the enzyme activity. The thin layer chromatography and high pressure anion exchange chromatography analysis of Ct43Araf hydrolysed arabinoxylans (rye and wheat) and oat spelt xylan confirmed the release of L-arabinose. This is the first report of α-L-arabinofuranosidase from C. thermocellum having the capacity to degrade both p-nitrophenol-α-L-arabinofuranoside and p-nitrophenol-α-L-arabinopyranoside. The protein melting curves of Ct43Araf and CtGH43 demonstrated that CtGH43 and CBMs melt independently. The presence of Ca2+ ions imparted thermal stability to both the enzymes. The circular dichroism analysis of CtGH43 showed 48% β-sheets, 49% random coils but only 3% α-helices.
Highlights
Plant cell wall is mainly composed of complex structural polysaccharides like cellulose and hemicellulose [1,2]
Rye arabinoxylans contain arabinose and xylose in the A/X ratio of 0.49–0.82 and ferulate residues attached to arabinose as esters at its O-5 position [4] but in wheat arabinoxylans the arabinose to xylose ratios [A/X] varies from 0.47 to 0.58 [5]
The digested fragment of Ct43Araf was cloned into NheI-XhoI digested pET-21a(+) expression-vector, while PCR amplified and digested DNA of CtGH43 was cloned earlier in pET-28a(+) vector earlier [21], resulting in respective recombinant plasmids
Summary
Plant cell wall is mainly composed of complex structural polysaccharides like cellulose and hemicellulose [1,2]. The heteropolymers of pentoses like D-xylose, L-arabinose and hexoses viz. D-mannose, D-glucose and D-galactose constitutes the hemicellulose. Xylans are hetero-polysaccharides with 1,4-linked-bD-xylopyranose backbone chains containing arabinose, glucuronic acid, or its 4-O-methyl ether, acetic, ferulic, and p-coumaric acids side chains depending mainly on the source of xylans [3]. Rye arabinoxylans contain arabinose and xylose in the A/X ratio of 0.49–0.82 and ferulate residues attached to arabinose as esters at its O-5 position [4] but in wheat arabinoxylans the arabinose to xylose ratios [A/X] varies from 0.47 to 0.58 [5]. The cereal arabinoxylans are composed majorly of a backbone of 1,4linked-b-D-xylopyranosyl residues substituted with single aarabinofuranosyl substituents attached to the O-2, O-3 or to both O-2,3 of the xylose residues [6,7]. It has been documented that aL-arabinofuranosyl and to a lesser extent a-L-arabinopyranosyl side chains are attached to the b-D-galactopyranose main chain by
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