Submitted to: Applied Biochemistry and Biotechnology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/14/2008
Publication Date: 5/16/2008
Citation: Wong, D., Chan, V.J., Batt Throne, S.B. 2008. Cloning and characterization of a novel exo-a-5-L-arabinanase gene and the enzyme. Applied Biochemistry and Biotechnology. 79(6):941-949.
Interpretive Summary: Pectins are complex polymers of carbohydrates present in plant cell walls, accounting for about one-third of all primary cell wall macromolecules. The chemical composition varies depending on the origin of the pectin. Arabinose, a five-carbon sugar, is a major component existing as a polymer. The breakdown of polymeric arabinan requires several groups of enzymes, and may contribute to the unfolding of the plant cell wall structure. This report describes the isolation and cloning of a novel gene, resulting in the production of an enzyme that could degrade linear arabinan into arabinose. These results will enable the development of enzyme systems for further optimization of biomass degradation in the conversion to biofuel and bioproducts.
Technical Abstract: A novel exo-α-L-arabinanase gene (arn3) was isolated, cloned, and expressed in E. coli. The recombinant enzyme (ARN3) had a pH optimum of 6.0-7.0 and a pH 3.0-7.0 stability range. The temperature optimum was 50oC with stability < 50oC. The enzyme showed considerable loss of activity by the addition of 0.4 mM Ag+ (70%), Cu++ (77%), and Zn++ (58%). ARN3 cleaved CM-arabinan, debranched arabinan, and linear arabinan at a decreasing rate, and is inactive on sugar beet arabinan, wheat arabinoxylan, and p-nitrophenyl-α-L-arabinofuranoside. The enzyme hydrolyzed debranched arabinan and synthetic arabino-oligosaccharides entirely to arabinose. The apparent Km and Vmax values were determined to be 6.2±0.3 mg/ml and 0.86±0.01 mg/ml/min, respectively (37oC, pH 7.0). Multiple sequence alignment and homology modeling revealed unique short sequences of amino acids extending the loop involved in partial blocking of one end of the substrate-binding site on the surface of the molecule.