Purification and characterization of a highly thermostable α-l-Arabinofuranosidase from Geobacillus caldoxylolyticus TK4

Abstract

The gene encoding an alpha-L: -arabinofuranosidase from Geobacillus caldoxylolyticus TK4, AbfATK4, was isolated, cloned, and sequenced. The deduced protein had a molecular mass of about 58 kDa, and analysis of its amino acid sequence revealed significant homology and conservation of different catalytic residues with alpha-L: -arabinofuranosidases belonging to family 51 of the glycoside hydrolases. A histidine tag was introduced at the N-terminal end of AbfATK4, and the recombinant protein was expressed in Escherichia coli BL21, under control of isopropyl-beta-D-thiogalactopyranoside-inducible T7 promoter. The enzyme was purified by nickel affinity chromatography. The molecular mass of the native protein, as determined by gel filtration, was about 236 kDa, suggesting a homotetrameric structure. AbfATK4 was active at a broad pH range (pH 5.0-10.0) and at a broad temperature range (40-85 degrees C), and it had an optimum pH of 6.0 and an optimum temperature of 75-80 degrees C. The enzyme was more thermostable than previously described arabinofuranosidases and did not lose any activity after 48 h incubation at 70 degrees C. The protein exhibited a high level of activity with p-nitrophenyl-alpha-L: -arabinofuranoside, with apparent K (m) and V (max) values of 0.17 mM and 588.2 U/mg, respectively. AbfATK4 also exhibited a low level of activity with p-nitrophenyl-beta-D: -xylopyranoside, with apparent K (m) and V (max) values of 1.57 mM and 151.5 U/mg, respectively. AbfATK4 released L: -arabinose only from arabinan and arabinooligosaccharides. No endoarabinanase activity was detected. These findings suggest that AbfATK4 is an exo-acting enzyme.

Enzymatic Mechanism for Arabinan Degradation and Transport in the Thermophilic Bacterium Caldanaerobius polysaccharolyticus

“…In addition, the enzymes function with maximum activity at high temperatures, up to 75°C for CpAbf51A. Comparable data (with pH 5.0 to 7.0 and temperatures of 60°C to 75°C) were also reported for arabinofuranosidases and arabinanases from other thermophilic bacteria (10,(39)(40)(41)(42)(43)(44)(45)(46)(47)(48)(49)(50). However, higher optimum temperatures from 80°C to 90°C in the same pH range were reported for enzymes from Caldicellulosiruptor saccharolyticus (46,51), Sulfolobus solfataricus (52), Clostridium thermocellum (53), and Thermotoga maritima (54).…”

Two distinct arabinofuranosidases contribute to arabino-oligosaccharide degradation in Bacillus subtilis

“…The primary structure of B. subtilis AbfA is very similar to that of the characterized AbfA from Geobacillus stearothermophilus T-6 [71 % identity (Gilead & Shoham, 1995)], AbfATK4 from Geobacillus caldoxylolyticus TK4 [70 % identity (Canakci et al, 2007)] and Araf51 from Clostridium thermocellum [63 % identity (Taylor et al, 2006)], and AF activity was reported for the B. subtilis abfA gene product (Wipat et al, 1996). The amino acid sequence of Abf2 displays high identity to characterized AFs from Bacillus pumilus, ArfA [65 % identity (Degrassi et al, 2003)], Thermobacillus xylanilyticus, AbfD3 [64 % identity (Debeche et al, 2000)], Clostridium cellulovorans, ArfA [60 % identity (Kosugi et al, 2002)] and Clostridium stercorarium ArfB [56 % identity (Zverlov et al, 1998)].…”

A new recombinant phosphotriesterase homology protein from Geobacillus caldoxylosilyticus TK4: An extremely thermo- and pH-stable esterase

“…G. caldoxylosilyticus TK4 has been deposited in the National Collections of Industrial Food and Marine Bacteria under the number 14283 [13]. E. coli JM101 and E. coli BL21(DE3)pLysS were used as the host for cloning and gene expression, respectively.…”