Title: CHARACTERIZATION AND DIRECTED EVOLUTION OF HEMICELLULOTIC ENZYMES USING NATURAL XYLOOLIGOACCHARIDE SUBSTRATES Authors
Submitted to: Enzyme Engineering Conference
Publication Type: Abstract Only
Publication Acceptance Date: May 5, 2005
Publication Date: September 9, 2005
Citation: Wagschal, K.C., Franqui Espiet, D., Lee, C.C., Robertson, G.H., Wong, D.W.S. 2005. Characterization of hemicellulotic degrading enzymes using natural substrates. Symposium on Biotechnology for Fuels and Chemicals, Denver, CO. Paper No. 1B-65. Abstract A10, Enzyme Engineering XVIII. Technical Abstract: There is much current interest in enzymes that degrade starch, cellulose and hemicellulose due to their increasing use in bio-bleaching and for the conversion of biomass to chemical feedstocks and fuel. Our group has been pursuing the discovery of enzymes that degrade biomass and their improvement through the use of directed evolution. One enzyme family of interest are the beta-xylosidases. We present here a new enzyme-coupled xylose assay,and an accompanying method for its use to characterize the kinetic parameters for the hydrolysis of natural xylooligosaccharide substrates. A Thermoanaerobacterium sp. beta-xylosidase was investigated, and it was found that at higher xylobiose concentrations the enzyme showed an increase in rate indicative of transglycosylation, while for xylotriose marked substrate inhibition was observed. At lower xylobiose concentraions, kcat was 2.7 sec-1, Km was 3.3 mM, and kcat/Km was 0.82 sec-1mM-1. Non-linear curve-fitting to a substrate inhibition model showed that for xylotriose the KI was 1.7 mM, kcat was 2.0, Km was 0.144 mM and kcat/Km was 14 sec-1mM-1. In addition, the assay appears to be amenable to conversion to both a solution and solid-phase high-throughput assay format for use in gene discovery and directed evolution studies.