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Applied and Environmental Microbiology, September 2005, p. 5318-5323, Vol. 71, No. 9
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.9.5318-5323.2005

Enzyme-Coupled Assay for ß-Xylosidase Hydrolysis of Natural Substrates

Kurt Wagschal,* Diana Franqui-Espiet, Charles C. Lee, George H. Robertson, and Dominic W. S. Wong

USDA Agricultural Research Service, Western Regional Research Center, 800 Buchanan Street, Albany, California 94710

Received 14 December 2004/ Accepted 31 March 2005

We describe here a new enzyme-coupled assay for the quantitation of D-xylose using readily available enzymes that allows kinetic evaluation of hemicellulolytic enzymes using natural xylooligosaccharide substrates. Hydrogen peroxide is generated as an intermediary analyte, which allows flexibility in the choice of the chromophore or fluorophore used as the final reporter. Thus, we present D-xylose quantitation results for solution-phase assays performed with both the fluorescent reporter resorufin, generated from N-acetyl-3,7-dihydroxyphenoxazine (Amplex Red), and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS), whose corresponding radical cation has an absorbance maximum at ~400 nm. We also describe a useful solid-phase variation of the assay performed with the peroxidase substrate 3,3'-diaminobenzidine tetrahydrochloride, which produces an insoluble brown precipitate. In addition, kinetic parameters for hydrolysis of the natural substrates xylobiose and xylotriose were obtained using this assay for a glycosyl hydrolase family 39 ß-xylosidase from Thermoanaerobacterium sp. strain JW/SL YS485 (Swiss-Prot accession no. O30360). At higher xylobiose substrate concentrations the enzyme showed an increase in the rate indicative of transglycosylation, while for xylotriose marked substrate inhibition was observed. At lower xylobiose concentrations kcat was 2.7 ± 0.4 s–1, Km was 3.3 ± 0.7 mM, and kcat/Km was 0.82 ± 0.21 mM–1 · s–1. Nonlinear curve fitting to a substrate inhibition model showed that for xylotriose Ki was 1.7 ± 0.1 mM, kcat was 2.0 ± 0.1 s–1, Km was 0.144 ± 0.011 mM, and kcat/Km was 14 ± 1.3 mM–1 · s–1.

* Corresponding author. Mailing address: USDA Agricultural Research Service, Western Regional Research Center, 800 Buchanan Street, Albany, CA 94710. Phone: (510) 559-6453. Fax: (510) 559-5940. E-mail: kwagschal{at}pw.usda.gov.

Applied and Environmental Microbiology, September 2005, p. 5318-5323, Vol. 71, No. 9
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.9.5318-5323.2005




This article has been cited by other articles:

  • Fan, Z., Wagschal, K., Chen, W., Montross, M. D., Lee, C. C., Yuan, L. (2009). Multimeric Hemicellulases Facilitate Biomass Conversion. Appl. Environ. Microbiol. 75: 1754-1757 [Abstract] [Full Text]  


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