Enzyme-coupled assay for beta-xylosidase hydrolysis of natural substrates
| Autor: | Dominic W. S. Wong, Kurt Wagschal, George H. Robertson, Diana Franqui-Espiet, Charles C. Lee |
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| Rok vydání: | 2005 |
| Předmět: |
Disaccharides
Applied Microbiology and Biotechnology Substrate Specificity chemistry.chemical_compound Hydrolysis Hydrolase Oxazines Xylobiose Escherichia coli Methods Glycosyl Benzothiazoles chemistry.chemical_classification ABTS Chromatography Xylose Ecology biology Substrate (chemistry) Hydrogen Peroxide Culture Media Kinetics Enzyme Xylosidases chemistry Chromogenic Compounds biology.protein Sulfonic Acids Thermoanaerobacterium Food Science Biotechnology Peroxidase |
| Zdroj: | Applied and environmental microbiology. 71(9) |
| ISSN: | 0099-2240 |
| Popis: | 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 β-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 . |
| Databáze: | OpenAIRE |
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