Cloning and characterization of a novel exo-α-1,5-L-arabinanase gene and the enzyme

A novel exo-α-1,5-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 50°C with a stability less than or equal to 45°C. The recombinant ARN3 cleaved carboxymethyl (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−1 min−1, respectively (pH 7.0, 37°C, CM-arabinan). 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.