A Novel α-L-Arabinofuranosidase of Family 43 Glycoside Hydrolase (Ct43Araf) from Clostridium thermocellum

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.

Evaluation of Avena sativa Derived Arabinoxylans as Native Biological Response Modifiers in Broilers

“…Chemically AXs are two pentose sugars; α-Larabinofuranose that is attached to the beta-(1,4) linked xylans backbone (Ahmed et al, 2013). Pharmacological activities of cereals derived polysaccharides especially AXs decreased the glucose level in diabetes patients and increased the fecal output (Lo et al, 2005).…”

Novel Trifunctional Xylanolytic Enzyme Axy43A from Paenibacillus curdlanolyticus Strain B-6 Exhibiting Endo-Xylanase, β- d -Xylosidase, and Arabinoxylan Arabinofuranohydrolase Activities

“…Endo-xylanases hydrolyze the backbone of xylan to produce xylooligosaccharides (XOSs) in an endo-acting manner; however, their activities are frequently hampered by the arabinose substitutions (4). ␣-L-Arabinofuranosidases (AFs) include various types of enzymes that release arabinose from arabinose-containing substrates, e.g., p-nitrophenyl-␣-L-arabinofuranoside (pNPAf), arabinoxylo-oligosaccharides (AXOSs), arabinoxylans, linear and branched arabinans, and arabinogalactans (1,5), while arabinoxylan arabinofuranohydrolases (AXHs) are atypical AFs that prefer to release arabinose from the nonreducing terminal xylose of arabinoxylans (6) and AXOSs (7), including pNPAf (7). Based on the mode of action, AXHs are categorized into two groups.…”

Crystallization and preliminary X-ray crystallographic analysis of a novel α-L-arabinofuranosidase (CtGH43) fromClostridium thermocellumATCC 27405

“…The truncated 903 bp gene encoding the N-terminus of CtGH43 (family 43 glycoside hydrolase from Clostridium thermocellum) is part of a full-length family 43 glycoside hydrolase (Ct43Araf) which also contains two tandem family 6 carbohydrate-binding modules, CtCBM6A (405 bp) and CtCBM6B (402 bp), at the C-terminus (Ahmed et al, 2013). CtGH43 was expressed, biochemically characterized and shown to be predominantly an -l-arabinofuranosidase (Ahmed et al, 2013).…”

“…CtGH43 was expressed, biochemically characterized and shown to be predominantly an -l-arabinofuranosidase (Ahmed et al, 2013). The truncated CtGH43 shows a unique ability to catalyze both p-nitrophenol--l-arabinofuranoside (pNPAf) and pnitrophenol--l-arabinopyranoside (pNPAp), although the catalytic efficiency was greater with pNPAf (Ahmed et al, 2013).…”

“…CtGH43 was expressed, biochemically characterized and shown to be predominantly an -l-arabinofuranosidase (Ahmed et al, 2013). The truncated CtGH43 shows a unique ability to catalyze both p-nitrophenol--l-arabinofuranoside (pNPAf) and pnitrophenol--l-arabinopyranoside (pNPAp), although the catalytic efficiency was greater with pNPAf (Ahmed et al, 2013). Phylogenetic analysis of CtGH43 from C. thermocellum shows a close evolutionary relationship with GH43s linked to family 6 carbohydrate-binding modules (CBM6s) from C. cellulolyticum, C. papyrosolvens, C. cellulyticum and Acetovibrio cellulolyticus .…”