Genetic and biochemical characterization of an α-l-arabinofuranosidase isolated from a compost starter mixture
Enzymes that are involved in the breakdown of arabinoxylan biomass are becoming more important as the need to harness renewable energy sources becomes necessary. A gene encoding an α-l-arabinofuranosidase not previously described (1581 bp) was isolated from a culture seeded with a compost starter mixed bacterial population. Sequence analysis of the putative catalytic domain determined that the enzyme, termed deAFc, is a glycoside hydrolase family 43 member. The gene was cloned into Escherichia coli with a C-terminal His-tag and its recombinant product expressed and purified. deAFc appeared to be monomeric under the gel-permeation chromatography conditions employed, and kinetic analysis using several artificial glycoside substrates revealed Km values between 0.251 and 0.960 mM and kcat values between 0.13 and 1.22 s−1. The purified enzyme was stable up to 45 °C, had an activity temperature optimum of 47 °C, and a pH profile that was essentially invariant between pH 5 and 8.5. deAFc was observed to release xylose only when incubated with synthetic xylopyranoside substrates, while release of arabinose was observed from arabinoxylan and branched arabinan as well as from synthetic chromophore or fluorophore-tagged α-l-arabinofuranoside substrates.
Journal: Enzyme and Microbial Technology - Volume 40, Issue 4, 5 March 2007, Pages 747–753