A novel a-L-Arabinofuranosidase of Family 43 Glycoside Hydrolase (Ct43Araf ) from Clostridium thermocellum

Articles in International JournalsThe 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 a-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 50uC. Ct43Araf and CtGH43 showed maximum activity with rye arabinoxylan 4.7 Umg21 and 5.0 Umg21, 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 a-L-arabinofuranosidase from C. thermocellum having the capacity to degrade both pnitrophenol- a-L-arabinofuranoside and p-nitrophenol-a-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% b-sheets, 49% random coils but only 3% a-helices

A novel multienzyme complex from a newly isolated facultative anaerobic bacterium, Paenibacillus sp. TW1

A multienzyme complex from newly isolated Paenibacillus sp. TW1 was purified from pellet-bound enzyme preparations by elution with 0.25% sucrose and 1.0% triethylamine (TEA), ultrafiltration and Sephacryl S-400 gel filtration chromatography. The purified multienzyme complex showed a single protein band on non-denaturing polyacrylamide gel electrophoresis (native-PAGE). The high molecular mass of the purified multienzyme complex was approximately 1,950 kDa. The complex consisted of xylanase and cellulase activities as the major and minor enzyme subunits, respectively. The complex appeared as at least 18 protein bands on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and as 15 xylanases and 6 cellulases on zymograms. The purified multienzyme complex contained xylanase, α-L-arabinofuranosidase, carboxymethyl cellulase (CMCase), avicelase and cellobiohydrolase. The complex could effectively hydrolyze corn hulls, corncobs and sugarcane bagasse. These results indicate that the multienzyme complex that is produced by this bacterium is a large, novel xylanolytic-cellulolytic enzyme complex

Characterization of Paenibacillus curdlanolyticus B-6 Xyn10D, a Xylanase That Contains a Family 3 Carbohydrate-Binding Module ▿ †

Paenibacillus curdlanolyticus B-6 Xyn10D is a xylanase containing a family 3 carbohydrate-binding module (CBM3). Biochemical analyses using recombinant proteins derived from Xyn10D suggested that the CBM3 polypeptide has an affinity for cellulose and xylan and that CBM3 in Xyn10D is important for hydrolysis of insoluble arabinoxylan and natural biomass