Purification and characterization of a highly thermostable alpha-L-Arabinofuranosidase from Geobacillus caldoxylolyticus TK4.
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.
Canakci
Sabriye
S
Department of Biology, Faculty of Arts and Sciences, Karadeniz Technical University, 61080, Trabzon, Turkey. sabriye@ktu.edu.tr
Belduz
Ali Osman
AO
Saha
Badal C
BC
Yasar
Ahmet
A
Ayaz
Faik Ahmet
FA
Yayli
Nurettin
N
D001406:Bacillaceae / Q000201:enzymology*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D006358:Hot Temperature; D007700:Kinetics; D008970:Molecular Weight; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity
D001426:Bacterial Proteins; D011994:Recombinant Proteins; D006026:Glycoside Hydrolases; C019713:alpha-N-arabinofuranosidase