title: 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 Ca(2+) and Mg(2+) 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 Ca(2+) 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.
authors.affiliation: Department of Biotechnology, Indian Institute of Technology Guwahati, Guwahati, Assam, India.
authors.forename: Shadab
authors.initials: S
authors.lastname: Ahmed
authors.forename: Ana Sofia
authors.initials: AS
authors.lastname: Luis
authors.forename: Joana L A
authors.initials: JL
authors.lastname: Bras
authors.forename: Arabinda
authors.initials: A
authors.lastname: Ghosh
authors.forename: Saurabh
authors.initials: S
authors.lastname: Gautam
authors.forename: Munishwar N
authors.initials: MN
authors.lastname: Gupta
authors.forename: Carlos M G A
authors.initials: CM
authors.lastname: Fontes
authors.forename: Arun
authors.initials: A
authors.lastname: Goyal
mesh_terms: D003001:Cloning, Molecular; D048013:Acetivibrio thermocellus / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D011994:Recombinant Proteins; D006026:Glycoside Hydrolases; C019713:alpha-N-arabinofuranosidase
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