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ARS Home » Research » Publications at this Location » Publication #202792
Title: PURIFICATION AND CHARACTERIZATION OF A HIGHLY THERMOSTABLE ALPHA-L-ARABINOFURANOSIDASE FROM GEOBACILLUS CALDOXYLOLYTICUS TK4

Author
item CANAKCI, SABRIYE - TURKEY
item BELDUZ, ALI - TURKEY
item Saha, Badal
item YASAR, AHMET - TURKEY
item AYAZ, FAIK - TURKEY
item YAYLI, NURETTIN - TURKEY

Submitted to: Applied Microbiology and Biotechnology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/8/2007
Publication Date: 4/1/2007
Citation: Canakci, S., Belduz, A.O., Saha, B.C., Yasar, A., Ayaz, F.A., Yayli, N. 2007. Purification and characterization of a highly thermostable alpha-L-arabinofuranosidase from Geobacillus caldoxylolyticus TK4. Applied Microbiology and Biotechnology. 75:813-820.

Interpretive Summary: Various agricultural residues and processing by-products such as corn stover, corn fiber, wheat straw, and rice hulls can serve as abundant feedstocks for production of fuel ethanol. These feedstocks generally contain 20-35% hemicellulose, which needs to be broken down to sugars. These sugars are then fermented to ethanol. Arabinose residues are widely distributed in hemicellulose as side chains. These side chains, however, restrict the enzymatic breakdown of hemicellulose. The enzyme, arabinofuranosidase, releases arabinose from the side chains. In this research, the gene encoding the enzyme from a bacterium was isolated, cloned, and sequenced. The cloned enzyme was purified and characterized. It was found to be highly thermostable. The enzyme has potential to be used in the conversion of hemicellulose to sugars.

Technical 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 IPTG 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 deg C), and it had an optimum pH of 6.0 and an optimum temperature of 75-80 deg C. The enzyme was highly thermostable than previously described arabinofuranosidases and did not lose any activity after 48 hours incubation at 70 deg C. The protein exhibited a high level of activity with p-nitrophenyl alpha-L-arabinofuranoside, with apparent Km and Vmax 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 Km and Vmax 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.