Title: GENETIC AND BIOCHEMICAL CHARACTERIZATION OF A NOVEL BACTERIAL A-L-ARABINOFURANOSIDASE Authors
Submitted to: Enzyme and Microbial Technology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: June 14, 2006
Publication Date: March 5, 2007
Citation: Wagschal, K.C., Franqui Espiet, D., Lee, C.C., Accinelli, R., Robertson, G.H., Wong, D. 2007. Genetic and biochemical characterization of a novel bacterial a-l-arabinofuranosidase. Enzyme and Microbial Technology. (40):747-753. Interpretive Summary: Hemicellulose is a major component of the woody part of plants, and represents a significant renewable source of chemical energy. However, it would first be desirable to break the hemicellulose down into simpler components, that could be converted by other processes to a more useable form of the energy, e.g. bioethanol. To do so enzymatically requires a suite of different enzymes, a critical one of which is termed an arabinofuranosidase. We describe here the isolation and biochemical characterization of a novel arabinofuranosidase that may find use in the breakdown of hemicellulose.
Technical Abstract: Enzymes that are involved in the breakdown arabinoxylan biomass are becoming more important as the need to harness renewable energy sources becomes necessary. A gene encoding a novel a-L-arabinofuranosidase termed deAFc (1581 bp) was isolated from a culture seeded with a compost starter mixed bacterium population. Sequence analysis of the putative catalytic domain determined that the enzyme is a glycoside hydrolase family 43 member. The gene was cloned into E. coli with a C-terminal His-tag and its recombinant product expressed and purified. The enzyme appeared to be monomeric under the gel-permeation chromatography conditions employed. The enzyme had Km values between 0.251 mM and 0.960 mM, and kcat values between 0.13 sec-1 and 1.22 sec –1 for a series of artificial glycoside substrates. The purified enzyme was stable up to 45 °C, had an activity temperature optimum of 47 oC, and a pH profile that was essentially invariant between pH 5 and pH 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 a-L-arabinofuranoside substrates.