Enzyme-coupled assay for beta-xylosidase hydrolysis of natural substrates

Abstract

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).

FIG. 1.

Solid-phase assay of E. coli BL21(DE3) transformed with vector only or with the β-xylosidase gene (β-xyl). Bacterial colonies were grown and expressed in the presence of 1 mM IPTG at 30°C overnight on a Nytran SPC membrane placed on LB_carb. Cell lysis and detection were performed as described in the text by incubating the membrane overnight at room temperature using DAB as the peroxidase substrate. The image was obtained using an AlphaImager imaging system (Alpha Innotech Corp., San Leandro, CA).

FIG. 2.

d-Xylose standard curves generated using ABTS as the chromophore or Amplex Red as the fluorophore. The error bars are generally about the size of the symbols. mOD/min 420 nm, 10⁻³ unit of optical density at 420 nm/minute; rfu/sec Ex/Em 550/590 nm, relative fluorescence units/second at an excitation wavelength of 550 nm and an emission wavelength of 590 nm.

FIG. 3.

Reaction conditions were 45°C, 50 mM phosphate (pH 6.0), and 0.1% BSA. (A) Nonlinear regression fitting to the Michaelis-Menten equation of hydrolysis rate versus xylobiose concentration (only 0.104 mM to 2.08 mM shown) using 0.018 μM enzyme. (Inset) Lineweaver-Burk reciprocal plot of the data. (B) Nonlinear regression fitting to the Michaelis-Menten equation with substrate inhibition of hydrolysis rate versus xylotriose concentration using 0.0027 μM enzyme. R² = 0.993.