Metallo-β-lactamase, CAS No.9073-60-3

CAS: 9073-60-3 Cat. No.: M1508747 Número CE: 232-970-8 PubChem CID: 133358

GRADE & PURITY Bioactive, Recombinant, ActiBioPure™, High Performance, EnzymoPure™, Sterile, expressed in E.coli;≥2M units/EA

Accession #

P14488

Expression system

E. coli

Visão geral

Metallo-β-lactamase, also known as imipenem hydrolase, is a high-purity antibiotic-inactivating enzyme produced by genetic engineering technology. It can hydrolyze the β-lactam ring of carbapenem antibiotics, thereby abrogating their antimicrobial activity.

This product is a highly purified and highly active preparation obtained through purification processes. It is characterized by strong specificity, capable of hydrolyzing the β-lactam ring of antibiotics such as imipenem, meropenem, and ertapenem. It also exhibits hydrolytic activity against cefixime, cefoperazone, cefotaxime, ceftriaxone, and other cephalosporins.

Scope of Application

It can be used for the sterility test and microbial limit test of carbapenem antibiotic raw materials and their preparations, namely the sterility test for β-lactam antibiotics specified in Pharmacopoeia of the People's Republic of China (2020 Edition, Volume 4, Appendix: Sterility Test) and the microbial limit test specified in the corresponding appendix. It is also used as a reagent for the sterility test specified in USP-NF General Chapters 71 STERILITY TEST.

Usage Instructions

Sterility Test Method for Carbapenem Antibiotics (with reference to Pharmacopoeia of the People's Republic of China (2020 Edition, Appendix: Sterility Test)): Membrane Filtration Method or Direct Inoculation Method

Specifications

Product Name

Metallo-β-lactamase, CAS No.9073-60-3

Grau

ActiBioPure™, Bioactive, High Performance, Recombinant, Sterile, EnzymoPure™

Especificações e pureza

ActiBioPure™,Bioactive,High Performance,EnzymoPure™,Recombinant,sterile,expressed in E.coli;≥2M units/EA

Mecanismos bioquímicos e fisiológicos

Confers resistance to the different beta-lactams antibiotics (penicillin, cephalosporin and carbapenem) via the hydrolysis of the beta-lactam ring. Benzylpenicillin is a better substrate than cephalosporin C and ampicillin.

Número de acesso

P14488

CAS

9073-60-3

Número da Comissão das Enzimas

EC 3.5.2.6

Tipo de molécula

Enzyme

Product Line

Produtos biológicos

Armazenamento e envio

Concentração

expressed in E.coli;≥2M units/EA

Reconstituição

Reconstitute in sterile pre-chilled water.

Condições de armazenamento de armazenamento

Store at 2-8°C

Enviado em

Wet ice

Estabilidade e armazenamento

Store at 2-8℃ long term (24 months). Upon reconstitution, it is recommended to aliquot.

Definição da unidade

The amount of enzyme required to hydrolyze 1 unit of penicillin G in one hour at pH 7.0 and 37˚C.

Application Protocols

Only product-validated protocols can be listed as item-specific. For this SKU, no tested applications or recommended dilutions are provided in the Product Data.

• Item-specific protocols: Not specified for this item; refer to CoA/Spec Sheet.

General/literature example protocols (for planning; not product claims):

• Nitrocefin kinetic assay:
  • Prepare 50 mM HEPES, 150 mM NaCl, pH 7.5 with 50 µM ZnCl2.
  • Dilute enzyme to 0.5–5 nM; pre-equilibrate 10 min at 25°C.
  • Initiate with nitrocefin (20–200 µM). Monitor A482 over 60–180 s. Fit initial rates to Michaelis–Menten.
• Inhibitor IC50:
  • Pre-incubate enzyme with inhibitor (0–100 µM) for 5–15 min.
  • Add nitrocefin at KM-range concentration; record initial rates. Fit to four-parameter logistic to obtain IC50; perform Ki analysis as needed.
• Metal reconstitution:
  • Dialyze against chelator-containing buffer to prepare apo-enzyme, then titrate Zn2+ (0–200 µM) and assay to construct metal-activation curves.

Consult the CoA for any mandatory buffer components, storage additives, or specific activity units required for accurate assay setup.

Biological Roles

Metallo-β-lactamases (MBLs) are bacterial enzymes that confer the ability to hydrolyze β-lactam compounds by activating a water molecule at a zinc center, forming a nucleophile that attacks the β-lactam carbonyl (literature).

• Physiological context: Many Gram-negative bacteria express periplasmic MBLs; genes reside on chromosomes or mobile elements. Expression levels and metal homeostasis influence activity in vivo (literature).
• Cofactor biology: Cellular Zn2+ acquisition and trafficking (e.g., znu systems) modulate metalloenzyme maturation. Some subclasses can utilize alternative metals (Co2+, Mn2+) in vitro with altered kinetics (literature).
• Substrate scope: Broad across penicillins, cephalosporins, and carbapenems for B1/B3 classes; B2 enzymes often prefer carbapenems (literature). Detailed specificity is enzyme-variant dependent.
• Protein family: Belongs to the metallo-β-lactamase superfamily, which includes diverse hydrolases (RNA processing enzymes, glyoxalases), unified by a metal-dependent catalytic fold (literature).

This section provides general biochemical background to support experimental design. It does not imply any clinical or diagnostic application for this product.

Buffer Applications

While not a buffer reagent itself, metallo-β-lactamase performance depends critically on buffer composition (general/literature guidance).

• Common assay buffers (typical ranges; optimize for your enzyme variant):

  • HEPES: 25–50 mM, pH 7.0–8.0; good buffering near neutrality and minimal metal binding.
  • Tris: 25–50 mM, pH 7.5–8.5; convenient but can weakly complex metals—verify Zn2+ availability.
  • MOPS: 25–50 mM, pH 7.0–7.5; low metal interaction.
  • Ionic strength: 100–200 mM NaCl to enhance stability and reduce nonspecific adsorption.
  • Metal ions: Add ZnCl2 at micromolar levels (e.g., 10–100 µM) if reconstitution is required; avoid EDTA/EGTA unless intentionally inactivating the enzyme.
  • Additives: 0.01% Tween-20 to minimize surface loss; 10–20% glycerol for storage or low-temperature assays.

• Practical recipes (example, not item-specific):

  • HEPES 50 mM, NaCl 150 mM, pH 7.5, ZnCl2 50 µM; equilibrate enzyme ≥10 min before initiating with substrate (e.g., nitrocefin).

• pH range:

  • Activity commonly peaks between pH 6.5–8.5 (variant-dependent). Verify with a pH-rate profile for your preparation.

Refer to the product’s CoA for any mandatory excipients or stabilizers already present, and avoid buffer components that strip zinc.

Green Alternatives

Enzymes are often greener catalysts than harsh chemical reagents. For hydrolyzing β-lactams, metallo-β-lactamase offers a selective, aqueous, room-temperature route versus strong base or high-temperature conditions.

Comparison (general/literature):

• Process attributes:
  • Catalyst: Metallo-β-lactamase (biocatalyst) vs. NaOH/KOH or acid hydrolysis (stoichiometric chemicals).
  • Solvent: Water-based buffers vs. organic/strongly basic media.
  • Conditions: Ambient temperatures, neutral pH vs. elevated temperature/caustic conditions.
  • Selectivity: Enzyme shows β-lactam specificity; chemical hydrolysis may cause broader degradation.

Trade-offs:

• Metal dependency: Requires controlled Zn2+; chelator contamination can complicate workflows.
• Stability: Proteins may require cold-chain storage and are sensitive to denaturation.
• Cost: Enzymes can be more expensive per unit of conversion but reduce downstream neutralization and waste.

Green practice tips:

• Use aqueous buffers, recycle enzyme via immobilization when feasible, and operate at room temperature.
• Minimize organic cosolvent to what is strictly required for substrate/inhibitor solubility.

Note: These are general sustainability considerations; align with your process analytical targets and this item’s CoA for permissible buffers/additives.

Pharmaceutical Uses

For research use only. No therapeutic or diagnostic use is intended.

Relevant non-clinical, pre-formulation, and manufacturing research roles (general):

• Analytical reagent: Controlled degradation of β-lactam APIs/excipients to generate ring-opened reference materials for LC/UV/MS method development and stability-indicating assays.
• Bioassay system: Positive control for assays that measure β-lactam integrity or inhibitor potency during medicinal chemistry campaigns targeting MBLs.
• Process studies: Evaluate susceptibility of candidate β-lactam scaffolds to enzymatic hydrolysis under physiologically relevant conditions (pH 7–8, 25–37°C).
• Quality risk assessment: Stress-testing formulations containing β-lactam motifs to understand enzyme-mediated degradation pathways.

Item-specific pharmacopeial status, acceptable excipient lists, endotoxin levels, and GMP compliance are not specified for this item; refer to CoA/Spec Sheet if your workflow requires regulated-grade materials.

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.
  • Formulation/buffer: Not specified for this item; refer to CoA/Spec Sheet.
  • Concentration/activity units: Not specified for this item; refer to CoA/Spec Sheet.

• General/literature expectations for recombinant MBL proteins (informational; not item specifications):

  • Physical state: Typically provided as an aqueous protein solution or lyophilized cake/powder.
  • Solubility: Readily soluble in aqueous buffers; insoluble in organic solvents.
  • Stability: Enhanced by neutral pH (≈7–8), low ionic detergents (if any), cryoprotectants (e.g., 10–50% glycerol) and by avoiding metal chelators (EDTA) when activity is desired.
  • Isoelectric point (pI): Variant-dependent; many MBLs fall in the pI ≈ 5–7 range (literature), but sequence-specific values vary.
  • Cofactor dependence: Activity depends on bound Zn2+ (frequently di-zinc) or other divalent cations in certain subclasses.
  • Optimum temperature: Many mesophilic enzymes show robust activity at 25–37°C; thermostability varies by source (literature).
  • Optimum pH: Often near neutral to slightly alkaline (pH 6.5–8.5), substrate- and subclass-dependent (literature).

• Non-applicable small-molecule properties: Boiling point, melting point, logP, refractive index are not meaningful for proteins and are not provided.

Quality and Grades

• Item-specific grades (from Product Data):

  • Bioactive: Supplied in a form retaining enzymatic function under appropriate assay conditions.
  • Recombinant: Produced via heterologous expression, enabling consistent lot-to-lot identity compared to native isolates.
  • ActiBioPure™: Vendor designation indicating high activity retention and purity suitable for biochemical studies.
  • High Performance: Indicates performance-validated for demanding research applications (e.g., kinetics, inhibitor screening).
  • EnzymoPure™: Emphasizes enzyme purity and low contaminant protease/background activities.
  • Sterile: Manufactured/filtered to be free of detectable microbial contamination.

• What these grades imply in practice (general explanation):

  • Purity: Enzyme-focused purification to minimize host proteins and nucleic acids, supporting clean kinetic baselines.
  • Consistency: Recombinant production supports reproducible specific activity and buffer composition across lots.
  • Assay readiness: Sterility and bioactivity reduce the need for pre-use cleanup; compatible with cell-free biochemical assays.

• Not provided for this item:

  • Exact purity percentage, residual endotoxin, host-cell protein/DNA levels, stabilizers, and specific activity units are not specified for this item; refer to CoA/Spec Sheet.

Use the CoA to align grade specifications with your assay acceptance criteria (e.g., inhibitor IC50 profiling, mechanistic studies).

Reaction and Applications

Metallo-β-lactamases catalyze the hydrolytic opening of β-lactam rings through a metal-activated water/hydroxide, inactivating penams, cephems, carbapenems, and related β-lactam substrates (substrate scope varies by subclass; literature).

Key research applications (general/literature; expand according to your workflow):

• Enzyme kinetics: Measure kcat/KM with chromogenic substrates such as nitrocefin (Δλ ≈ 482 nm on hydrolysis) or fluorogenic β-lactams. Enables mechanism and variant comparison.
• Inhibitor discovery: Screen small-molecule chelators, zinc-site binders, and transition-state analogs; determine IC50/Ki using initial-rate or progress-curve analysis.
• Metal dependence: Probe mono- vs di-zinc states by apoprotein preparation (chelation) and controlled reconstitution with Zn2+ (or Co2+ as spectroscopic probe) to study active-site chemistry.
• Protein engineering: Mutagenesis of HxHxD and neighboring residues to dissect catalysis and substrate recognition.
• Biophysical characterization: Use ITC, DSF, CD, and metalloprotein spectroscopy (e.g., Co(II) d–d bands) to assess folding and metal occupancy.
• Analytical degradation: Controlled hydrolysis of β-lactam standards to generate ring-opened products for method development and mass-spectrometric identification.

Practical tips:

• Maintain defined Zn2+ levels; trace chelators in buffers can depress activity.
• Confirm linear initial rates by titrating enzyme concentration and ensuring ≤10% substrate consumption during measurement.
• Pre-equilibrate enzyme with buffer and metals before starting reactions; include appropriate blanks for spontaneous hydrolysis.

Reaction Conditions

General literature guidance for MBL-catalyzed hydrolysis (not item-specific; optimize empirically):

• Buffer: 25–50 mM HEPES or MOPS, pH 7.0–7.8; 100–200 mM NaCl.
• Metal ions: Ensure availability of Zn2+ (e.g., 10–100 µM ZnCl2) if enzyme requires reconstitution; avoid EDTA.
• Temperature: 25–37°C typical for assays; stability may allow broader ranges depending on variant.
• Enzyme loading: Commonly 0.1–100 nM for chromogenic substrates in cuvette assays; higher for poorly reactive substrates.
• Substrates: Nitrocefin (chromogenic), CENTA, β-lactam antibiotics (compound-dependent solubility; limit DMSO ≤1–2%).
• Readouts: UV–vis at 482 nm (nitrocefin), HPLC/LC–MS for non-chromogenic substrates.
• Kinetic regime: Keep substrate consumption ≤10% over measurement window; pre-warm and pre-equilibrate enzyme with buffer and metals.
• Quench: Add EDTA to chelate Zn2+ (inactivates) or acidify to pH <3 to stop the reaction for analytical sampling.

Expected performance (literature examples):

• Many MBLs show kcat in the 1–1000 s−1 range and KM from low µM to mM depending on substrate/class; your values will depend on the specific enzyme construct and metal state.

Note: The product’s exact activity units, optimal pH/temperature, and assay buffer are not specified for this item; consult the CoA for lot-specific recommendations.

Safety and Handling

• GHS classification and hazard statements (item-specific):

  • Signal word: Not specified for this item; refer to SDS.
  • H-statements/Pictograms: Not specified for this item; refer to SDS.

• General laboratory safety for recombinant proteins:

  • PPE: Lab coat, gloves, and eye protection when handling solutions or lyophilized material.
  • Engineering controls: Work in a clean area; use a biosafety cabinet if aerosol generation is possible during aliquoting.
  • Incompatibilities: Avoid strong oxidizers, extreme pH, and metal chelators (e.g., EDTA, EGTA) if enzymatic activity must be preserved. Avoid prolonged exposure to elevated temperatures.
  • Spill/cleanup: Wipe with absorbent material; decontaminate surfaces with detergent solution. For large spills, contain and collect for disposal as proteinaceous lab waste per institutional policy.
  • First aid (overview): If splashed in eyes or on skin, rinse with water for ≥15 minutes and remove contaminated clothing. If inhaled as aerosol, move to fresh air. If ingested, rinse mouth with water. Seek medical attention according to SDS guidance.
  • Special risks: This enzyme is catalytically active toward β-lactam rings; handle away from β-lactam-containing research materials when activity loss would be problematic. Proteinaceous materials may cause allergic sensitization in susceptible individuals.

Always consult the product-specific SDS for authoritative hazard, exposure, and disposal guidance. For research use only.

Solvent Selection

Not typically applicable to proteins as “solvent selection” in the small-molecule sense. For metallo-β-lactamase, the relevant medium is an aqueous buffer that maintains structure, metal occupancy, and activity.

• General guidance (literature/general):
  • Aqueous media: Use neutral to slightly alkaline buffers (e.g., HEPES, Tris) at ionic strength 50–200 mM.
  • Metal ions: Maintain required divalent cations (commonly Zn2+); avoid strong chelators (EDTA, EGTA) unless deliberately inactivating the enzyme.
  • Co-solvents: Keep organic cosolvents (DMSO, MeOH, acetonitrile) ≤1–5% v/v when required for substrate/inhibitor solubility; verify activity retention empirically.
  • Additives: Glycerol (10–20%) can help stability for storage and freeze–thaw; nonionic detergents (0.005–0.05% Tween-20) may reduce adsorption.

Please see Buffer Applications for more practical buffer systems tailored to assay types.

Storage and Reconstitution

• Item-specific from Product Data:

  • Storage conditions: Store at 2–8°C.
  • Shipping: Wet ice.

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

  • Exact formulation/buffer, presence of stabilizers (e.g., glycerol, salts), concentration, and reconstitution instructions (if lyophilized).

• General handling guidance for recombinant metallo-enzymes (informational; not item-specific):

  • If supplied lyophilized: Reconstitute with chilled, metal-compatible buffer (e.g., HEPES, MOPS) to the desired concentration; avoid EDTA/EGTA. Allow 5–10 min on ice for full dissolution.
  • If supplied in solution: Keep on ice during use; aliquot to avoid repeated freeze–thaw. For longer-term storage, some preparations tolerate −20°C or −80°C with 10–50% glycerol—confirm with CoA before freezing.
  • Protect from microbial contamination; the product is designated sterile, but maintain aseptic technique when opening and aliquoting.
  • Avoid prolonged exposure to room temperature and to air–water interfaces; use low-binding tubes.
  • For activity-critical work, pre-equilibrate with the working buffer and confirm metal occupancy by adding trace Zn2+ if recommended by the CoA.

Always follow the lot-specific CoA/SDS for definitive storage and reconstitution instructions. For research use only.

Structure and Identity

Brief: Metallo-β-lactamase (MBL) is an enzyme class (EC 3.5.2.-) that hydrolyzes β-lactam rings using a metal-activated water. This listing is for a recombinant, bioactive protein reagent.

• Item-specific identifiers from Product Data:

  • Name: Metallo-β-lactamase (recombinant)
  • CAS: 9073-60-3
  • PubChem CID: 133358 (class-level entry)
  • SMILES: Not specified for this item; refer to CoA/Spec Sheet.
  • InChIKey: Not specified for this item; refer to CoA/Spec Sheet.
  • Molecular formula: Not specified for this item; refer to CoA/Spec Sheet.
  • Molecular weight: Not specified for this item; refer to CoA/Spec Sheet.

• Structural features (literature/general):

  • Biopolymer type: Protein enzyme (typically 200–300 amino acids per domain; many exist as single-domain soluble periplasmic proteins).
  • Cofactor: Requires divalent metal ions, classically Zn2+ (mono- or di-zinc center depending on subclass); Co2+ can substitute in vitro for some variants (literature).
  • Active-site motifs: Conserved histidine-rich residues (e.g., HxHxD…H…H) coordinating metal ion(s) and activating water for nucleophilic attack (literature).
  • Subclasses: B1, B2, B3 MBLs, with differing metal usage and substrate scope (literature).
  • 2D/3D description: Compact αβ/ββ fold with a metallo-hydrolase active site pocket; no defined small-molecule SMILES applies because this is a polypeptide (literature).

Note: Exact amino acid sequence, isoform, tags, and calculated MW/pI are not provided in the Product Data and should be obtained from the item-specific CoA.

Synthetic Utility

As a biocatalyst, metallo-β-lactamase is a tool for chemo-selective transformation of β-lactam scaffolds in research settings (general/literature):

• Functional-group selectivity: Targets the strained four-membered β-lactam amide, opening to the corresponding amino acid/acid derivatives with high specificity compared to bulk amide hydrolysis.
• Chemo-compatibility: Operates in water at mild pH and temperature, preserving other sensitive groups.
• Analytical synthesis: Generates authentic hydrolysis products for standards, mechanistic probes, and isotope-label scrambling studies.
• Platform for inhibitor SAR: Enzyme provides a catalytic readout for structure–activity relationships on metal-binding pharmacophores and transition-state mimics.
• Immobilization potential: Can be coupled to solid supports for flow-through hydrolysis or scavenging of β-lactam impurities (requires empirical optimization to retain metal occupancy and activity).

Constraints and tips:

• Maintain Zn2+-loaded active sites; chelation or incorrect pH will depress conversion.
• If cosolvent is required for substrate solubility, validate activity retention and adjust ionic strength to stabilize the protein.
• Monitor conversions by UV-vis (chromogenic substrates), HPLC, or LC–MS; quench with chelators or acid to stop the reaction as needed.

Target Specificity

Only item-specific information may be listed here.

• Specific target/antigen, organism source, isoform, sequence tags, and subclass (B1/B2/B3) are not specified for this item; refer to CoA/Spec Sheet.

General (literature) note: Metallo-β-lactamases target the β-lactam core of susceptible substrates by coordinating and activating water at a Zn2+ center; fine specificity (penams, cephems, carbapenems) varies by enzyme subclass and sequence.