Submitted to: Applied and Environmental Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: March 31, 2005
Publication Date: September 1, 2005
Citation: Wagschal, K.C., Franqui Espiet, D., Lee, C.C., Robertson, G.H., Wong, D. 2005. Enzyme-coupled assay for beta-xylosidase hydrolysis of natural substrates. Applied and Environmental Microbiology. 71:5318-5323. Interpretive Summary: The United States produces nearly 3 billion gallons of fuel ethanol from grain every year. This may sound significant, but it represents just a fraction of a percent of the energy needed for gasoline alone. This work is part of a larger effort to expand the biomass sources used to make ethanol to include crop residues. Hemicellulose is a major chemical constituent of crop residues, and this work will help define new options for biorefining processes to reduce the chemical and energy costs associated with ethanol production, such that the use of crop residues is economically feasible. The project strategy starts with the identification of enzymes that are able to degrade hemicellulose to fermentable sugars, which can then be readily converted to ethanol or other chemical feedstocks. The enzymes are then engineered to tailor their activities to the desired process parameters using a multi-disciplinary protein modification tool known as directed evolution. The work described in this paper involves the development of an assay system that measures the relative efficiency, with respect to hemicellulose degradation, of a family of enzymes such that the best one can be selected for further improvement in an iterative process.
Technical Abstract: We are currently developing enzyme systems that convert hemicellulose to monosaccharides for use as a chemical feedstock and for the production of bioethanol. One approach we are using is the directed evolution of enzymes involved in hemicellulose biodegradation. In order to select for improved mutants using this strategy, an in-vitro screen emulating the final commercial-scale bioreactor process conditions was desired, thus requiring a selection assay that monitors the depolymerization of natural xylan or xylooligosaccharide substrates to xylose monosaccharides. We describe here a new enzyme-coupled assay for the quantitation of xylose using readily available enzymes that allows the kinetic evaluation of hemicellulolytic enzymes degrading natural xylooligosaccharide substrates. Hydrogen peroxide is generated as an intermediary analyte, thereby allowing flexibility in the choice of chromophore or fluorophore used as the final reporter. Kinetic parameters were obtained for a b-xylosidase from a Thermoanaerobacterium sp. strain JW/SL YS485 (SwissProt O30360) using the natural substrates xylobiose and xylotriose. Interestingly, at higher substrate concentrations xylobiose showed an increase in rate indicative of transglycosylation, while for xylotriose marked substrate inhibition was observed. At lower xylobiose concentrations kcat was 2.9 ± 0.3 sec-1and the Km was 1.7 ± 0.3 mM. Non-linear curve fitting to a substrate inhibition model showed that for xylotriose the Ki was 1.1 mM, kcat was 6.2 ± 0.5 sec-1 and Km was 0.5 ± 0.1 mM.