Endo-Beta-Galactosidase - Specific Activity ＞140 U/mg;Activity ＞14 U/ml , CAS No.55072-01-0

CAS: 55072-01-0 Cat. No.: E489844

GRADE & PURITY Specific Activity ＞140 U/mg;Activity ＞14 U/ml

Storage

Store at 2-8°C

Shipped In

Wet ice

Übersicht

Endo-beta-galactosidase cleaves internal ß(1-4) galactose linkages in unbranched, repeating poly-N-acetyllactosamine structures.

Product Description

Endo-Beta-Galactosidase cleaves internal β(1-4) galactose linkages in unbranched, repeating poly-N-acetyllactosamine structures. Sulfated structures such as keratan sulfate are also cleaved. Branching and/or fucosylation of the substrate may decrease or eliminate cleavage.

Endo-Beta-Galactosidase is useful for identifying and removing poly-N-acetyllactosamine structures on many biologically important glycoconjugates.

Source Recombinant from Bacteroides fragilis

Specificity Cleaves internal ß(1-4) galactose linkages in unbranched, repeating poly-N-acetyllactosamine [GlcNAc-ß(1-3)Gal-ß(1-4)]n structures are the preferred substrate. Sulfated structures such as keratan sulfate are also cleaved. Branching and/or fucosylation of the substrate may decrease or eliminate cleavage. Sulfation of C-6 on galactose will block cleavage. Oligosaccharidesof the neo-lacto group are cleaved at greatly educed rates depending on the deviation from the preferred substrate. For example, Gal-ß(1-3)GlcNAc-ß(1-3)Gal-ß(1-4)Glc is cleaved at 5×10-5 the rate of keratan sulfate. Specificity is similar to the Escherichia freundii enzyme. Specificity is similar to the Escherichia freundii enzyme except that it is limited to cleaving N-acetyllactosamine extensions on tetraantennary structures of erythropoietin.

Purity Endo-Beta-Galactosidase is tested for contaminating protease as follows: 10 μg of denatured BSA is incubated for 24 hours at 37°C with 2 μL of enzyme. SDS-PAGE analysis of the treated BSA shows no evidence of degradation.

EC 3.2.1.103

CAS 55072-01-0

Specific Activity >140 U/mg

Activity >14 U/ml

Molecular weight ~32,000 daltons

Optimum pH 5.8

Specific Activity

One unit of Endo-Beta-Galactosidase is defined as the amount that will liberate one µmole of reducing sugar per minute at 37˚C and pH 5.8 from bovine corneal keratan sulfate.

Contents

60 µl aliquot of enzyme (0.9 U) in 20 mM tris-HCl, pH 7.5

1 vial reaction buffer- 250mM Sodium phosphate, pH 5.8

Suggested usage

For glycoproteins:

1. Add up to 100 µg of glycoprotein to a tube.

2. Add 4 ul 5X buffer and water to 19 µl.

3. Add 1 µl enzyme.

4. Incubate at 37˚C for 2 hrs.

Procedure for oligosaccharides:

Same as above except incubate from several hours to several days depending on the substrate. Add bovine serum albumen to 2 mg/ml to stabilize the protein during extended incubations.

Storage

Stability Stable at least 24 months when stored properly. Several days exposure to ambient temperature will not reduce activity. Active for at least 5 days under reaction conditions.

The production strain of E. coli has been extensively tested and does not produce any detectable glycosidases.

Specifications

Spezifikationen & Reinheit

Specific Activity ＞140 U/mg;Activity ＞14 U/ml

Storage

Store at 2-8°C

Verschickt in

Wet ice

Dieses Produkt erfordert Kühlkettenversand. Grundversand und andere Economy-Optionen sind nicht verfügbar.

Application Protocols

No item-specific tested applications or unit definitions are provided for SKU E489844; consult the CoA/Spec Sheet. The protocol below is a general, literature-based starting point for glycoprotein digestion.

General protocol: Trimming poly-LacNAc on a glycoprotein

1. Prepare buffers and samples

• Buffer: 50 mM sodium acetate, pH 5.8, 100 mM NaCl, 5% glycerol.
• Desalt the glycoprotein into buffer; optional: pretreat with sialidase to remove terminal sialic acids.

2. Set up reactions

• Combine substrate (e.g., 10–100 µg) with endo-β-galactosidase at an initial ratio of 1–10 mU per µg substrate (adjust per supplier unit definition).
• Final volume 20–100 µL; avoid >10% organic solvent or strong detergents.

3. Incubate

• 30–37 °C for 1–16 h with gentle mixing. For membrane proteins, include 0.01–0.05% nonionic detergent if necessary and validated.

4. Monitor progress

• Analyze aliquots by SDS-PAGE with lectin blotting (e.g., ECL/galectin probes) or release/label glycans for HILIC–FLD/LC–MS.

5. Quench and clean up

• Heat inactivate enzyme (if compatible) or perform buffer exchange/ultrafiltration.

6. Controls

• Include no-enzyme and heat-inactivated enzyme controls to confirm specificity.

Adjust pH, temperature, and enzyme load based on your substrate and observed kinetics. For quantitative workflows, calibrate using a known glycoprotein standard.

Biological Roles

General biological context (literature; not item-specific)

• Functional class: Glycoside hydrolase that cleaves internal β-D-galactosidic linkages within poly-N-acetyllactosamine chains and keratan sulfate–like motifs.
• Natural occurrence: Homologous activities are found in bacteria (catabolism of host or environmental glycans), fungi, and some animal tissues (glycan remodeling/turnover in lysosomes or extracellular matrix).
• Pathway relevance: Modulates the length and presentation of poly-LacNAc on N- and O-glycans, which in turn affects lectin (e.g., galectin) binding, cell–cell adhesion, and signaling cascades in development and immune recognition.
• Substrate selectivity: Prefers internal β-1,4-linked galactose within repeating Galβ1-4GlcNAc units; activity can be impeded by sialylation, fucosylation, or sulfation proximal to the cleavage site.
• Structural consequences: Cleavage shortens glycan chains, altering glycoprotein physicochemical properties (hydrodynamic radius, charge distribution when paired with other enzymes) and potentially receptor–ligand affinity in model systems.

Research implications

• Tool enzyme for dissecting glycan-dependent processes in glycobiology, such as selectin-mediated adhesion, galectin binding, and modulation of ECM components rich in keratan sulfate.
• Complements exoglycosidases (β-galactosidase, β-hexosaminidase) and endoglycosidases (PNGase F, Endo H) in constructing targeted digestion schemes to map glycome architecture.

Note: This product is for research use only. No clinical or diagnostic claims are made.

Buffer Applications

While Endo-β-galactosidase is not itself a buffer component, its performance strongly depends on buffer choice. The following guidance is general/literature-based and should be optimized per lot.

Common buffer systems for activity assays

• Sodium acetate (pH 5.0–6.0): Frequently supports robust activity for enzymes acting on keratan sulfate–like or poly-LacNAc substrates.
• MES/MOPS (pH 6.0–7.0): Useful when slightly higher pH is desired for protein compatibility.
• HEPES or phosphate (pH 6.8–7.2): For workflows that must remain near neutral pH (e.g., to preserve protein structure), acknowledging that some enzyme variants may show reduced rates.

Practical buffer tips

• Ionic strength: 50–150 mM total salt often balances stability and substrate binding; fine-tune to your substrate.
• Additives: 5–10% glycerol can stabilize the enzyme during prolonged incubations; avoid chelators or detergents unless validated for your preparation.
• Preservatives: Sodium azide (0.02–0.05%) can prevent microbial growth but may inhibit certain enzymes; verify compatibility.

Example starting recipe (general; not item-specific)

• 50 mM sodium acetate, pH 5.8; 100 mM NaCl; 5% glycerol. Incubate substrate with enzyme at 37 °C; monitor progress and adjust conditions as needed.

Always consult the CoA/Spec Sheet for any manufacturer-recommended buffers or incompatibilities for this specific item.

Green Alternatives

Enzymatic deglycosylation with Endo-β-galactosidase is itself a greener alternative to harsh chemical methods for glycan cleavage.

Comparison of approaches (general literature)

• Enzyme-catalyzed hydrolysis:
  • Pros: Aqueous buffers; high chemo-, regio-, and stereoselectivity; mild temperature (25–37 °C); minimal byproducts; amenable to reuse/immobilization.
  • Cons: Narrow substrate scope depending on enzyme source; sensitive to denaturants and extremes of pH; cost and need for cold-chain logistics.
• Chemical hydrolysis (acidic or basic):
  • Pros: Inexpensive reagents; broad, nonselective cleavage.
  • Cons: Harsh conditions (strong acid/base, elevated temperatures); degradation of sensitive glycan/protein structures; racemization/peeling reactions; hazardous waste.

Greener practice tips (general)

• Use aqueous buffers and avoid organic cosolvents unless necessary for substrate solubility.
• Employ minimal effective enzyme loadings and recycle enzyme via immobilization or ultrafiltration when feasible.
• Optimize pH/temperature to reduce reaction time and energy consumption.
• Choose benign buffers (e.g., acetate, HEPES) and minimize azide or heavy-metal preservatives when compatible with your workflow.

Note: No item-specific green certifications or metrics are provided; consult the CoA/Spec Sheet for any stated stabilizers or preservatives that may influence EHS assessments.

Pharmaceutical Uses

This product is supplied for research use only. No medical or clinical use is intended or implied.

General roles in pharmaceutical R&D/manufacturing (literature; not item-specific)

• Analytical characterization: Employed in biopharmaceutical development to simplify complex glycan structures on candidate biologics for LC-MS or HPLC profiling during process development and comparability studies.
• Glycoengineering research: Used in research settings to remodel glycan termini or truncate poly-LacNAc extensions prior to further enzymatic steps, facilitating the production of analytical standards or model materials.
• Raw material considerations: When used in regulated environments, documentation typically includes CoA with activity definition, concentration, buffer composition, bioburden/endotoxin, and adventitious agent testing appropriate to a research-grade reagent.

Formulation/excipient status

• There is no pharmacopeial monograph specific to endo-β-galactosidase for use as an excipient, and it is not typically incorporated directly into finished dosage forms.

Compatibility and residues

• Residual enzyme in process samples is generally removed by ultrafiltration, chromatography, or heat inactivation. Verification by activity assays or peptide MS is recommended when required.

For any application that interfaces with GMP processes, qualify the enzyme in-house and consult your quality unit; refer to the CoA/Spec Sheet for lot-specific details.

Physical Properties

Item-specific properties

• Appearance: Not specified for this item; refer to CoA/Spec Sheet.
• Molecular weight: Not specified for this item; refer to CoA/Spec Sheet.
• pH of supplied formulation: Not specified for this item; refer to CoA/Spec Sheet.
• Extinction coefficient, specific activity, and unit definition: Not specified for this item; refer to CoA/Spec Sheet.

General/literature characteristics of endo-β-galactosidases (for context; not specifications)

• Physical state: Typically supplied as an aqueous solution or lyophilized powder containing stabilizers (e.g., salts, buffers, protein stabilizers).
• Solubility: Readily soluble in aqueous buffers; activity generally diminished in high concentrations of organic solvents or strong denaturants (literature).
• Optimal pH (typical): Many endo-β-galactosidases show maximal activity in mildly acidic to neutral pH ranges, approximately pH 5.0–7.0, depending on source (literature).
• Temperature profile (typical): Active at ambient to physiological temperatures (25–37 °C); thermal denaturation occurs at elevated temperatures; stability often enhanced by glycerol and salts (literature).
• Isoelectric point and exact hydrodynamic dimensions vary by source and formulation (literature).

Note: Classical small-molecule properties (boiling point, melting point, refractive index, logP, pKa) are not applicable to proteins. For lot-specific data (concentration, buffer composition, stabilizers, activity units), consult the CoA/Spec Sheet.

Quality and Grades

Item-specific grade/purity

• Grade/Purity: Not specified for this item; refer to CoA/Spec Sheet.

How to interpret enzyme quality (general guidance)

• Activity units: Enzyme quality is best defined by specific activity (e.g., units per mg protein) and by a stated unit definition (substrate, conditions, and readout). Absent item-specific values, confirm these on the CoA.
• Purity descriptors: For proteins, purity is often given by SDS-PAGE band patterns, densitometry (%), host-cell protein/DNA, and endotoxin levels, rather than traditional chemical assay purity.
• Formulation: Stabilizers (e.g., glycerol, BSA, salts) can enhance shelf life but may affect downstream assays (MS, electrophoresis). Check the CoA for excipients if compatibility with your workflow is critical.
• Lot-to-lot consistency: Enzyme preparations can vary in activity with storage and shipping; always calibrate by running a control digestion to verify performance with your substrate.
• Contaminants to monitor: Proteases, nucleases, and trace metals may compromise sensitive substrates. Use protease-free and nuclease-free consumables where required and verify with supplier specifications.
• Documentation: For regulated workflows, request a detailed Specification Sheet (activity, concentration, buffer, stabilizers, bioburden/endotoxin, and recommended storage) and retain CoA for traceability.

Note: In the absence of explicit grade terminology (e.g., “recombinant, ≥95% by SDS-PAGE”), treat this item as a research-use enzyme and qualify it empirically for critical applications.

Reaction and Applications

Scope (literature/general knowledge)

• Catalytic function: Hydrolyzes internal β-D-galactosidic linkages within repeating poly-N-acetyllactosamine [Gal(β1→4)GlcNAc] and related sequences on N- and O-glycans, glycolipids, and proteoglycans. Some preparations also act on keratan sulfate–like structures, depending on enzyme source.

Analytical applications

• Glycomics: Trimming poly-LacNAc extensions to map branching and chain length on glycoproteins by LC-MS, CE, or HPLC.
• Glycoprotein characterization: Sequential digestion with endo-β-galactosidase followed by exoglycosidases to resolve isomeric glycans and confirm antennary structures.
• Histology/glycobiology: Modifying cell-surface glycans to probe lectin binding and cell–cell interaction dependence on poly-LacNAc.

Preparative and bioprocess uses

• Glycoengineering: Tailoring glycan profiles on recombinant proteins prior to further enzymatic remodeling (e.g., to improve homogeneity for analytics; research use only).
• Oligosaccharide production: Generating defined fragments from poly-LacNAc polymers for standards or binding studies.

Practical tips (general)

• Substrate accessibility is critical; denaturation or mild detergent can expose glycans on heavily glycosylated proteins—balance with potential enzyme inhibition.
• Pre-treatment with sialidase (neuraminidase) and fucosidase can increase susceptibility by removing capping groups that sterically hinder endo-β-galactosidase.
• Use enzyme-to-substrate ratios titrated by activity (units) rather than mass; maintain consistent buffer pH and ionic strength.
• Verify progress by monitoring release of oligosaccharides (HILIC–MS) or changes in glycoprotein migration (SDS-PAGE/Western with lectin probes).

Reaction Conditions

Item-specific recommended conditions are not provided; refer to the CoA/Spec Sheet. The following are general, literature-based starting points for endo-β-galactosidases and should be optimized experimentally.

Typical starting conditions (general)

• Buffer: 25–50 mM sodium acetate (pH 5.5–6.0) or MES (pH 6.0–6.5). Include 50–150 mM NaCl if needed for stability.
• Temperature: 30–37 °C.
• Enzyme-to-substrate: Begin with 1–10 mU per µg glycoprotein or 0.01–0.1 U per mg glycan polymer; titrate based on activity units defined by the supplier.
• Additives: 5–10% glycerol for stability; avoid strong detergents and >10–20% organic cosolvents unless validated.
• Time: 1–16 h depending on substrate accessibility and enzyme load.

Substrate preparation (general)

• Remove sialic acids and terminal fucose if they hinder access to internal β-gal linkages (e.g., with sialidase and fucosidase pretreatments).
• Denaturation: For tightly folded glycoproteins, mild denaturation (e.g., 0.1% nonionic detergent or brief heat) can increase accessibility but may impact enzyme activity—optimize empirically.

Monitoring and endpoints

• Assay release of oligosaccharides by HILIC–FLD/MS or changes in lectin binding/mobility shifts on SDS-PAGE.
• Quench by heating (e.g., 65–95 °C for 10 min, as compatible) or by buffer exchange.

These conditions are illustrative and not specifications for SKU E489844.

Safety and Handling

Item-specific hazard information

• Signal word: Not specified for this item; refer to SDS.
• Hazard statements (H-codes): Not specified for this item; refer to SDS.
• GHS classification/pictograms: Not specified for this item; refer to SDS.

General safety guidance for protein enzymes (literature/best practice)

• Potential hazards: Protein enzymes may cause allergic sensitization upon inhalation or skin contact; avoid aerosolization and contact with skin/eyes. Assume low acute toxicity but handle as a protein allergen.
• PPE: Lab coat, nitrile gloves, safety glasses or face shield as needed. Use a certified biosafety cabinet when preparing aerosols or handling powders.
• Engineering controls: Work in well-ventilated areas; avoid generating dust or sprays. Use secondary containment when transporting open containers.
• Incompatibilities: Avoid strong oxidizers, extreme pH, and organic solvents that denature proteins. Sodium azide and heavy metals can inhibit or inactivate enzymes.
• First aid (overview; refer to SDS):
  • Inhalation: Move to fresh air; seek medical attention if symptoms persist.
  • Skin contact: Wash with soap and water; remove contaminated clothing.
  • Eye contact: Rinse cautiously with water for several minutes; remove contact lenses if present and easy to do.
  • Ingestion: Rinse mouth; do not induce vomiting; seek medical advice.
• Spill response: Absorb aqueous spills with inert material; clean with water and mild detergent; avoid generating aerosols.
• Waste: Dispose according to institutional and local regulations; decontaminate with appropriate disinfectants or heat where compatible.

Always consult the product SDS for authoritative safety information.

Solvent Selection

Endo-β-galactosidase is a protein biocatalyst and is not used as a solvent. The relevant consideration is buffer and additive compatibility for preserving activity.

General buffer compatibility (literature/general guidance)

• Preferred media: Aqueous buffers such as sodium acetate, MES, MOPS, HEPES, or phosphate, typically in the pH 5.0–7.0 range depending on enzyme source.
• Ionic strength: Moderate salt (e.g., 50–150 mM NaCl) can stabilize some enzymes; excessive ionic strength can reduce activity or substrate binding.
• Organic cosolvents: Many enzymes lose activity in >10–20% v/v organic solvents; minimize DMSO/MeOH/ACN unless validated for your substrate and enzyme lot.
• Detergents: Nonionic surfactants (0.01–0.1% Tween-20 or Triton X-100) can sometimes aid solubilization of membrane glycoproteins but may inhibit activity; titrate empirically.
• Metal ions: Heavy metals can inhibit; include EDTA if compatible with your substrate and if the enzyme does not require divalent cations (most endo-β-galactosidases do not, literature).

When to choose which buffer

• Acidic substrates or keratan sulfate–rich matrices: Sodium acetate or MES near pH ~5.5–6.0 is often effective.
• Neutral glycan analyses or protein-compatible workflows: HEPES or phosphate near pH 6.8–7.2 may preserve protein integrity while maintaining activity.

Always confirm optimal conditions for your specific source/lot using small-scale tests; consult the CoA/Spec Sheet for any item-specific recommendations.

Storage and Reconstitution

Item-specific storage/shipping

• Storage Conditions: Store at 2–8 °C (per Product Data).
• Shipped in: Wet ice (per Product Data).

Item-specific details not provided

• Formulation (buffer, stabilizers), concentration, and reconstitution instructions: Not specified for this item; refer to CoA/Spec Sheet.

General guidance (literature/best practice; apply only if consistent with CoA)

• Short-term storage: Keep at 2–8 °C. Minimize freeze–thaw cycles; aliquot upon first use if the enzyme is concentrated and stable at 4 °C for limited periods.
• Long-term storage: Many enzymes benefit from freezing at –20 °C or –80 °C in 10–50% glycerol; only apply if the CoA permits freezing and lists compatible stabilizers.
• Reconstitution of lyophilized enzyme: If supplied as a powder, reconstitute in the recommended buffer to the stated concentration. Gently mix without vigorous vortexing to avoid foaming/denaturation. Allow to fully dissolve on ice.
• Working solutions: Prepare fresh in activity buffer; keep on ice during setup. Include stabilizers (e.g., 5–10% glycerol) if compatible.
• Handling: Avoid repeated warming; return promptly to cold storage after use. Use low-protein-binding tips and tubes for dilute solutions.

Always follow the CoA/Spec Sheet for lot-specific instructions and the SDS for safe handling during storage and disposal.

Structure and Identity

• Item name: Endo-β-Galactosidase (enzyme; glycoside hydrolase)
• CAS: 55072-01-0
• SKU: E489844
• Category: Protein (research enzyme)

Item-specific identifiers

• Molecular formula: Not specified for this item; refer to CoA/Spec Sheet.
• Molecular weight: Not specified for this item; refer to CoA/Spec Sheet. (Proteins are polypeptides with heterogeneous mass depending on source and formulation.)
• SMILES: Not applicable to proteins.
• InChIKey: Not applicable to proteins.

General structural description (literature/general knowledge)

• Macromolecule type: Protein enzyme composed of amino acids folded into one or more domains typical of glycoside hydrolases.
• Functional architecture: Contains a catalytic domain characteristic of β-galactoside–cleaving enzymes; often accompanied by carbohydrate-binding modules that enhance affinity for complex glycans (literature).
• Active site features: Conserved acidic residues (e.g., glutamate/aspartate) commonly serve as catalytic acid/base and nucleophile in retaining or inverting mechanisms of glycoside hydrolysis (literature).
• Substrate recognition: Binds internal β-linked D-galactose residues within poly-N-acetyllactosamine or keratan sulfate–like motifs on glycoconjugates (literature).

Two-dimensional depiction in words (general)

• A globular protein surface with a cleft-like active site that accommodates repeating Gal(β1→4)GlcNAc (LacNAc) units; subsites flanking the scissile β-galactosidic bond position and orient the glycosidic oxygen for acid/base catalysis (literature).

Synthetic Utility

Role as a biocatalyst (literature/general)

• Selective C–O bond cleavage: Catalyzes hydrolysis of internal β-D-galactosidic linkages within poly-LacNAc sequences, enabling site-specific depolymerization of complex glycans under mild, aqueous conditions.
• Chemo- and regioselectivity: Targets internal bonds without indiscriminately removing terminal monosaccharides, complementing exoglycosidases in stepwise glycan editing.

Applications in synthesis and analysis

• Oligosaccharide synthesis: Controlled partial digestion generates defined oligosaccharide fragments that can be isolated and used as standards or ligands in binding studies.
• Glycan remodeling: Prepares glycoproteins with tailored glycan length prior to further enzymatic elaboration (sialyltransferases, fucosyltransferases) or deglycosylation (PNGase F/Endo H) for structural studies.
• Protecting-group-free strategies: Enzymatic editing avoids chemical protecting-group manipulations typical of carbohydrate synthesis.

Operational guidance

• Substrate preparation: De-sialylation and de-fucosylation may be required to expose susceptible internal β-gal linkages.
• Reaction monitoring: Track by HILIC–MS, MALDI-TOF, or fluorophore-labeled glycan assays. Adjust enzyme load and time to tune fragment distribution.
• Workup: Quench by heat inactivation (if compatible) and purify products via size-exclusion, SPE (graphitized carbon), or HILIC.

Note: Specific activity, unit definitions, and optimal conditions for this catalog item are not provided here; consult the CoA/Spec Sheet and perform pilot reactions to calibrate performance.

Target Specificity

This section typically applies to antibodies or affinity reagents. Endo-β-galactosidase is an enzyme, not a binding antibody, so antigen/epitope specificity, clone, isotype, and species reactivity do not apply.

General substrate specificity (literature; provided for context only)

• Recognizes internal β-D-galactosidic linkages within poly-N-acetyllactosamine [repeating Gal(β1→4)GlcNAc] and related keratan sulfate–like structures.
• Cleavage is often impeded by terminal capping groups (e.g., sialylation, fucosylation) adjacent to the scissile bond and by certain sulfate patterns.

For validated, lot-specific substrate scope and any known inhibitors/requirements, consult the CoA/Spec Sheet.