Submitted to: Applied Microbiology and Biotechnology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/17/2008
Publication Date: 8/5/2008
Citation: Canakci, S., Kacagan, M., Inan, K., Belduz, A.O., Saha, B.C. 2008. Cloning, Purification, and Characterization of a Thermostable Alpha-L-arabinofuranosidase from Anoxybacillus kestanbolensis AC26Sari. Applied Microbiology and Biotechnology. 81(1):61-68. Interpretive Summary: Various agricultural residues (corn stover, wheat straw) and processing by-products (corn fiber, 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 break down 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 thermostable and active on hemicellulosis substrates. It has potential to be used in the conversion of hemicellulose to sugars.
Technical Abstract: The gene, AbfAC26Sari, encoding an alpha-L-arabinofuranosidase from Anoxybacillus kestanbolensis AC26Sari, was isolated, cloned, sequenced, and characterizated. On the basis of amino acid sequence similarities, this 57 kDa enzyme could be assigned to family 51 of the glycosyl hydrolase classification system. Characterization of the purified recombinant alpha-L-arabinofuranosidase produced in Escherichia coli BL21 revealed that it is active at a broad pH range (pH 4.5 to 9.0) and at a broad temperature range (45-85 deg C), and it has an optimum pH of 5.5 and an optimum temperature of 65 deg C. Kinetic experiment at 65 deg C with p-nitrophenyl alpha-L-arabinofuranoside as a substrate gave Vmax and Km values of 1019 U/mg and 0.139 mM, respectively. The enzyme had no apparent requirement of metal ions for activity, and its activity was strongly inhibited by 1 mM Cu**2+ and Hg**2+. The recombinant arabinofuranosidase released L-arabinose from arabinan, arabinoxylan, oat spelt xylan, arabinobiose, arabinotriose, arabinotetraose, and arabinopentaose. Endoarabinanase activity was not detected. These findings suggest that AbfAC26Sari is an exo-acting enzyme.