4-Nitrophenyl β-D-Xylopyranoside

4-Nitrophenyl β-D-Xylopyranoside (pNP-Xyl, CAS 2001-96-9) is a chromogenic aryl glycoside substrate essential for quantitative assays of β-xylosidase (EC 3.2.1.37) activity in hemicellulose degradation studies, biofuel research, and microbial enzymology. Hydrolysis by β-xylosidases cleaves the β-1,4-glycosidic linkage, releasing yellow 4-nitrophenolate (pNP, ε405 nm = 18,300 M⁻¹cm⁻¹ at pH 9-10) for sensitive spectrophotometric detection, enabling kinetic characterization of xylanolytic enzymes from fungi, bacteria, and rumen microbes. This substrate supports selective screening of GH3/GH39/ GH43 family hydrolases, inhibitor development for biomass saccharification, and differentiation of exo- vs. endo-xylanases when used in combination with pNP-xylobiosides, offering high specificity and stability for industrial biotechnology and plant cell wall analysis.

Appearance

• White to light yellow crystalline powder.
• Hygroscopic; soluble in water (5-10 mM), DMSO (>100 mM).

Source

• Koenigs-Knorr synthesis: β-D-xylopyranosyl bromide + 4-nitrophenol, Ag2CO3 catalysis, β-anomer selective crystallization.
• Enzymatic transxylosylation or commercial production (TCI, GBiosciences).

Molecular Weight and Structure

• C11H13NO7, 271.23 g/mol; mp 160°C; [α]D -85° ±5° (c=1, H2O).
• β-D-Xylp-(1→O)-C6H4-p-NO2; InChIKey ZKBWXYDDZTVQGQ-SKNVOPKUSA-N.

Sugar Specificity

• Highly selective for β-xylosidases (Km 0.1-0.8 mM); minimal α-xylosidase activity.
• Poor substrate for β-glucosidases (100x slower); GH3/GH39/GH43 family specific.

Biological Activity

• Hydrolysis: pNP-Xyl → D-xylose + pNP; optimal pH 4.5-6.0, 37-50°C.
• Vmax 100-500 µmol pNP/min/mg for microbial β-xylosidases; linear to 10 U/mL.

Purity and Microbial Contamination

• ≥98% HPLC purity; β-anomer >97%; free pNP <0.05%.
• Endotoxin-free; sterile lyophilized powder.

Identity and Quality Control

• 1H-NMR (J1,2 7.8 Hz β-anomer), HRMS (m/z 272.1 [M+H]+), optical rotation, TLC single spot.
• Functional assay: ΔA405 >0.2/min with β-xylosidase standard.

Shelf Life and Storage

• 3-5 years desiccated -20°C; light protected amber vials.
• Stock solutions stable 3 months -20°C; use fresh dilutions.

Application

• β-Xylosidase activity in biofuel enzyme cocktails; xylanase synergy studies.
• Rumen microbiology; plant pathology; GH family enzyme screening.

Key Characteristics

• Chromogenic (yellow pNP, ε405=18.3 mM⁻¹cm⁻¹); high sensitivity (0.01 U/mL detection).
• Thermostable; microplate compatible; distinguishes exo-xylanases.

Citation Links

• https://www.chemicalbook.com/ChemicalProductProperty_EN_CB8494683.htm
• https://www.benchchem.com/zh/product/b013790
• https://www.gbiosciences.com/Bioassays/Enzyme-Substrates/4-Nitrophenyl-beta-D-xylopyranoside
• https://pubmed.ncbi.nlm.nih.gov/17223215/
• https://www.bldpharm.com/products/2001-96-9.html
• https://www.smolecule.com/products/s8087581
• https://pubchem.ncbi.nlm.nih.gov/compound/91509
• https://www.sciencedirect.com/science/article/abs/pii/S0168165606010285
• https://www.tcichemicals.com/IN/en/p/N0620
• http://chemsynlab.com/en/product/2001-96-9.html