Enzyme-coupled assay for beta-xylosidase hydrolysis of natural substrates.
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 approximately 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 beta-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 k(cat) was 2.7 +/- 0.4 s(-1), K(m) was 3.3 +/- 0.7 mM, and k(cat)/K(m) was 0.82 +/- 0.21 mM(-1) . s(-1). Nonlinear curve fitting to a substrate inhibition model showed that for xylotriose K(i) was 1.7 +/- 0.1 mM, k(cat) was 2.0 +/- 0.1 s(-1), K(m) was 0.144 +/- 0.011 mM, and k(cat)/K(m) was 14 +/- 1.3 mM(-1) . s(-1).
Wagschal
Kurt
K
Agricultural Research Service, U.S. Dept. of Agriculture, Western Regional Research Center, 800 Buchanan Street, Albany, CA 94710, USA. kwagschal@pw.usda.gov
Franqui-Espiet
Diana
D
Lee
Charles C
CC
Robertson
George H
GH
Wong
Dominic W S
DW
D052160:Benzothiazoles; D002863:Chromogenic Compounds / Q000378:metabolism*; D003470:Culture Media; D004187:Disaccharides / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D006861:Hydrogen Peroxide / Q000378:metabolism; D006868:Hydrolysis; D007700:Kinetics; D010078:Oxazines / Q000378:metabolism*; D013379:Substrate Specificity; D013451:Sulfonic Acids / Q000378:metabolism*; D046950:Thermoanaerobacterium / Q000201:enzymology* / Q000235:genetics; D014994:Xylose / Q000378:metabolism*; D014995:Xylosidases / Q000378:metabolism*
D052160:Benzothiazoles; D002863:Chromogenic Compounds; D003470:Culture Media; D004187:Disaccharides; D010078:Oxazines; D013451:Sulfonic Acids; C002502:2,2'-azino-di-(3-ethylbenzothiazoline)-6-sulfonic acid; C014180:resorufin; D014994:Xylose; D006861:Hydrogen Peroxide; D014995:Xylosidases; C026579:exo-1,4-beta-D-xylosidase; C004173:xylobiose