ARS | Publication request: Functional cloning of an endo-alpha-1,5-L-arabinanase gene from a metagenomic library

Submitted to: Protein and Peptide Letters
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: December 1, 2008
Publication Date: July 16, 2009
Citation: Wong, D., Chan, V.J., Mccormack, A.A. 2009. Functional cloning of an endo-alpha-1,5-L-arabinanase gene from a metagenomic library. Protein and Peptide Letters. 16:1411-1435.

Interpretive Summary: Arabinose, a five-carbon sugar, is abundantly found as building blocks of various complex polymers of carbohydrates present in plant cell wall. These arabinose-containing polymers belong to the hemicellulose fraction accounting for a major portion of the cell wall biomass. The breakdown of polymeric arabinans and the utilization of the five-carbon sugars are achieved by microbes that produced several specific enzymes. This report describes the isolation and cloning of a novel gene of microbial source, and the production of the enzyme that could cleave arabinans internally to trimeric fragments. This novel enzyme could form part of a larger enzyme system for the complete breakdown of biomass in the conversion to biofuel and bioproducts.

Technical Abstract: A novel endo-alpha-L-arabinanase gene (arn2) was isolated, and expressed in E. coli in active form. The recombinant enzyme (ARN2) had optimum activity at pH 6.0 and 45o-50oC with stability between pH 5.0-8.0 and at temperatures up to 45oC . The recombinant ARN2 catalyzed internal cleavage of alpha-1,5 glycosidic bonds of CM-arabinan, debranched arabinan, linear arabinan, and sugar beet (native) arabinan at rates of decreasing order, and was inactive on wheat arabinoxylan and p-nitrophenyl-alpha-L-arabinofuranoside. Kinetic analysis showed that branching in the arabinan did not significantly affect the apparent Km values, and the difference in the reaction rates was mostly due to the chemical step after substrate binding. The enzyme hydrolyzed arabino-oligosaccharides of DP>=6 to smaller oligomers and mostly arabinotriose. Natural and modified arabinans were cleaved to oligomers of various chain lengths, which were progressively hydrolyzed to yield arabinotriose. The pattern of degradation revealed an endo-acting mechanism with arabinotriose as the end product.