X‑β‑Man

X‑β‑Man (CAS 129787‑67‑3) is a synthetic β‑mannosidase‑specific chromogenic substrate composed of a 5‑bromo‑4‑chloro‑3‑indoxyl aglycone linked via a β‑glycosidic bond to D‑mannose. When cleaved by β‑mannosidase or β‑mannosidase‑containing enzymes, the indoxyl moiety is released and undergoes rapid oxidation in air to form an intensely colored, water‑insoluble indigo‑like precipitate, enabling visual or spectrophotometric detection of enzyme activity. This substrate is commonly used in microbiology to screen for or enumerate mannose‑utilizing bacteria or yeast, as well as in histochemistry and recombinant‑enzyme assays where spatial localization or high‑sensitivity readouts are required. Compared with other X‑type substrates, X‑β‑Man offers good selectivity for β‑mannosidases and is compatible with a range of buffers and culture‑medium components.

Appearance

• Off‑white to pale yellow crystalline powder or lyophilized solid.
• Fine, free‑flowing texture suitable for dissolution in aqueous or mixed aqueous‑organic solvents.

Source / synthesis

• Synthetically produced by coupling 5‑bromo‑4‑chloro‑3‑indoxyl with β‑D‑mannose under glycosylation conditions.
• Commercially manufactured by specialty carbohydrate and enzyme‑substrate suppliers as a research‑use‑only product.

Molecular weight and structure

• Molecular formula: C14H15BrClNO6\text{C}_{14}\text{H}_{15}\text{BrClNO}_{6}C14​H15​BrClNO6​.
• Molecular weight: 408.63 g/mol.
• Structure: β‑D‑mannopyranoside of 5‑bromo‑4‑chloro‑3‑indolyl with a β‑glycosidic linkage to the 3‑position of the indole ring.

Sugar specificity

• Specifically hydrolyzed by β‑mannosidases (EC 3.2.1.25) and closely related β‑mannan‑ or β‑mannoside‑cleaving enzymes.
• Shows minimal cross‑reactivity with other β‑glycosidases such as β‑galactosidase or β‑glucosidase when used under optimized conditions.

Biological activity

• Acts as a chromogenic reporter for β‑mannosidase‑mediated mannobiose and mannooligosaccharide hydrolysis.
• Hydrolysis yields 5‑bromo‑4‑chloro‑3‑indoxyl, which dimerizes and oxidizes to a blue–violet indigo derivative, providing a sensitive, low‑background signal for both solution‑based and solid‑medium assays.

Purity and microbial contamination

• Typically supplied at ≥95–98% purity by HPLC or equivalent analytical methods.
• Certificates of analysis for research‑grade material usually confirm low levels of residual solvents, acceptable heavy‑metal content, and absence of significant microbial contamination when stored and handled aseptically.

Identity and quality control

• Identity confirmed by NMR, HPLC, MS, and elemental analysis, with batch‑specific certificates of analysis provided by major suppliers.
• Quality‑control parameters include appearance, solubility, UV/Vis spectral data, and assay performance in a defined β‑mannosidase activity system.

Shelf life and storage

• Shelf life is typically 12–24 months when stored as a dry powder at –20 °C in a tightly sealed container, protected from moisture and light.
• Aqueous working solutions should be prepared immediately before use or stored frozen for short periods to minimize degradation and oxidation of the indoxyl moiety.

Application

• Screening and enumeration of β‑mannosidase‑producing microorganisms on selective agar media.
• Enzyme‑activity assays for recombinant or purified β‑mannosidases in solution or immobilized formats.
• Histochemical localization of β‑mannosidase activity in tissue sections or whole cells.

Key characteristics

• High sensitivity and low background due to formation of an insoluble blue–violet dye upon β‑mannosidase hydrolysis.
• Defined β‑mannopyranoside linkage providing selectivity for β‑mannosidases over other β‑glycosidases.
• Crystalline, stable powder with known molecular weight and formula suitable for quantitative assays.

citations 

• ChemicalBook – 5‑bromo‑4‑chloro‑3‑indoxyl β‑D‑mannopyranoside: https://amp.chemicalbook.com/ProductChemicalPropertiesCB0494814_EN.htm
• Biosynth – Enzyme substrates (X‑substrates, including X‑β‑Man): https://www.biosynth.com/category/enzyme-substrates/view-all?page=4
• Biosynth – X‑substrates (blue–green) overview page: https://www.biosynth.com/research-products/x-substrates-blue-green
• Cymit Quimica – 5‑Bromo‑4‑Chloro‑3‑Indolyl β‑D‑mannopyranoside (X‑Man): https://cymitquimica.com/cas/129787-67-3/
• Geneseq / 金赛途 – X‑β‑Man (CAS 129787‑67‑3) product page: http://www.geneseqtools.com/cn/product03_cid_212_pid_496.html
• Shenzhen Hepattack Bioscience – X‑substrates including X‑β‑Man: https://www.hepattack.com/en/product02_page_3.html
• Sigma‑Aldrich – related X‑substrate (α‑D‑galactopyranoside) page: https://www.sigmaaldrich.com/AT/en/search/5-bromo-4-chloro-3-indoxyl-%CE%B1-d-galactopyranoside
• Level / Carbosynth catalog (enzyme substrates and X‑substrates): http://www.level.com.tw/i/assets/upload/files/Agency/CBR/Catalog/Enzyme%20Substrate%20and%20Detergent%20Catalogue.pdf
• BiDePharm – related enzyme‑substrate portal (for purity/QC context): https://www.bidepharm.com/products/129541-41-9.html
• Sigma‑Aldrich – X‑α‑Man (5‑bromo‑4‑chloro‑3‑indolyl α‑D‑mannopyranoside) reference page: https://www.sigmaaldrich.com/DE/de/search/5-bromo-4-chloro-3-indoxyl-%CE%B1-d-galactopyranoside