What is the Oxyrase® Enzyme System?
A biological oxygen scavenger utilizing bacterial respiratory enzyme complexes
Bacterial Membrane Fragments with Intact Respiratory Enzymes
The Oxyrase® Enzyme System consists of sterile cytoplasmic membrane fragments (~0.1 micron) derived from Escherichia coli. These fragments retain functional electron transport chain (ETC) components that catalyze the four-electron reduction of molecular oxygen directly to water.
Membrane Fragments
E. coli cytoplasmic membrane with intact ETC1
Electron Transfer
Dehydrogenases → cytochromes → O₂2
Water Formation
O₂ + 4H⁺ + 4e⁻ → 2H₂O3
Two-Stage Electron Transfer Mechanism
Stage 1: Substrate Dehydrogenation
FAD-dependent dehydrogenases oxidize organic substrates:
Lactate → Pyruvate
Succinate → Fumarate
Formate → CO₂
Stage 2: Terminal Oxidation
Electrons flow through:
Iron-sulfur clusters
Cytochrome proteins
Cytochrome c oxidase (Complex IV)
Complete Electron Transport Pathway
Enzyme Activity and Kinetics
Oxygen Reduction Rate by Temperature
Activity Definition
1 Oxyrase Unit = The amount of enzyme that reduces dissolved oxygen at a rate of 1% per second under standard conditions (37°C, pH 8.4)
Standard concentration: 30 Units/mL
Time to ppb O₂ levels: ~4 minutes (with 0.3 Units in solution)
Enzyme System Characteristics
Substrate Requirements
Requires organic electron donors: lactate (10-20 mM), succinate, formate, or α-glycerophosphate.4 Substrate-specific dehydrogenases catalyze initial oxidation step.
Cofactors
FAD-dependent dehydrogenases. Heme groups in cytochrome proteins. Iron-sulfur clusters for electron transfer.5 No external cofactor addition required.
pH Sensitivity
Active range: pH 6.8-9.4. Optimal activity at pH 8.0.6 Lower pH values require increased enzyme concentration or extended reaction time.
Temperature Range
Functions from 5-60°C. Maximum activity at 30-40°C.7 Maintains activity at 40°C for extended periods. Reduced activity at refrigeration temperatures.
Continuous Activity
Unlike chemical reducing agents, enzymatic catalysis continues as long as substrate and oxygen are present.8 Self-regenerating reduction system.
Reaction Products
Terminal reduction produces only water (H₂O). Substrate oxidation yields pyruvate, fumarate, or CO₂ depending on electron donor used.9
Enzymatic vs. Chemical Oxygen Removal Mechanisms
| Characteristic | Enzymatic (Oxyrase®) | Thioglycolate | L-Cysteine | Sodium Sulfide |
|---|---|---|---|---|
| Reduction Mechanism | Enzymatic catalysis via electron transport chain | Chemical reduction via -SH groups | Chemical reduction via -SH groups | Chemical reduction via S²⁻ oxidation |
| Stoichiometry | 4e⁻ + 4H⁺ + O₂ → 2H₂O | 2 RSH + O₂ → RSSR + H₂O | 2 RSH + O₂ → RSSR + H₂O | 2 S²⁻ + O₂ → 2 S⁰ + 2 O²⁻ |
| Catalytic Nature | ✓ True catalyst (regenerates) | ✗ Stoichiometric reagent | ✗ Stoichiometric reagent | ✗ Stoichiometric reagent |
| Reaction Products | H₂O only | Disulfide (RSSR) | Cystine (disulfide) | Elemental sulfur, H₂S |
| Continuous Scavenging | ✓ Yes (with substrate) | ✗ Depletes over time | ✗ Depletes over time | ✗ Depletes over time |
| pH Dependence | 6.8-9.4 (enzyme stability) | Higher pH increases rate | pH 7.0-8.5 optimal | Very pH dependent |
| Temperature Dependence | Enzyme kinetics (Q₁₀ ~2) | Chemical kinetics | Chemical kinetics | Chemical kinetics |
| Specificity | High (only reduces O₂) | Moderate (reduces other oxidants) | Moderate (reduces other oxidants) | Low (very non-specific) |
Key Scientific Distinction
Oxyrase® is a true enzymatic catalyst that regenerates with substrate addition, whereas chemical reducing agents are consumed stoichiometrically.10,15 The enzymatic mechanism achieves complete four-electron reduction of O₂ to H₂O without accumulation of reactive oxygen intermediates (O₂⁻, H₂O₂, OH·).12
Molecular Components of the Enzyme System
Dehydrogenases
- Lactate dehydrogenase – Oxidizes lactate to pyruvate
- Succinate dehydrogenase – Part of Complex II
- Formate dehydrogenase – Oxidizes formate to CO₂
- FAD cofactors – Electron acceptors
Electron Carriers
- Iron-sulfur clusters – [2Fe-2S], [4Fe-4S]
- Quinones – Ubiquinone (CoQ)
- Cytochrome b – Heme proteins
- Cytochrome c – Mobile carrier
Terminal Oxidase
- Cytochrome c oxidase – Complex IV
- Heme a/a₃ centers – Catalytic sites
- Copper centers (CuA, CuB) – Electron transfer
- O₂ binding site – 4-electron reduction
Membrane Fragment Structure
The cytoplasmic membrane fragments are approximately 0.1 microns in diameter, containing intact phospholipid bilayers with embedded protein complexes.1 The orientation preserves the natural topology of the respiratory chain, with substrate-binding sites exposed to the external medium and oxygen reduction occurring at the membrane interior.
Technical Specifications
| Parameter | Specification | Notes |
|---|---|---|
| Enzyme Activity | Minimum 30 Units/mL | 1 Unit = 1%/sec O₂ reduction at 37°C, pH 8.416 |
| Particle Size | ~0.1 micron | Membrane vesicles/fragments1 |
| Source Organism | Escherichia coli | Cytoplasmic membrane preparation1 |
| pH Optimum | 8.0 | Active range: 6.8-9.46 |
| Temperature Optimum | 30-40°C | Active range: 5-60°C7 |
| Substrate Requirement | 10-20 mM | Lactate, succinate, formate, or α-glycerophosphate4 |
| Oxygen Reduction Time | 4 minutes to ppb levels | With 0.3 Units at 37°C16 |
| Reaction Stoichiometry | O₂ + 4e⁻ + 4H⁺ → 2H₂O | Complete 4-electron reduction3 |
| Cofactors | FAD, heme, Fe-S clusters | Intrinsic to enzyme system5 |
| Km for O₂ | Low micromolar range | High affinity for oxygen11 |
| Storage Stability | ≤-20°C | Freeze-thaw tolerant17 |
| Appearance | Slightly turbid solution | Due to membrane fragment suspension1 |
Temperature-Concentration Relationship
30-50°C: 3% enzyme concentration18
25°C: >6% enzyme concentration18
20°C: >9% enzyme concentration18
5-10°C: Significantly reduced activity7
pH Effects
pH 8.0: Optimal activity6
pH 7.0-7.5: Good activity6
pH 6.8-6.9: Reduced activity6
pH <6.8 or >9.4: Minimal activity6
Why Enzymatic Oxygen Removal is Unique
Catalytic Regeneration
Unlike stoichiometric chemical reducing agents, the enzyme catalyzes continuous oxygen reduction as long as substrate is available.10 One enzyme molecule can process thousands of O₂ molecules.
Substrate Specificity
Cytochrome oxidase exhibits high specificity for molecular oxygen.11 Does not reduce other oxidizing agents or interfere with cellular redox reactions beyond oxygen removal.
No Reactive Intermediates
Complete 4-electron reduction of O₂ to H₂O without releasing superoxide (O₂⁻), hydrogen peroxide (H₂O₂), or hydroxyl radicals (OH·).12 Eliminates oxidative stress to cells.
Biomimetic Process
Uses the same electron transport mechanism present in all aerobic organisms.13 Compatible with biological systems since it mimics natural cellular respiration pathways.
Thermodynamic Efficiency
Enzymatic catalysis lowers activation energy for O₂ reduction, enabling rapid oxygen removal at physiological temperatures without harsh chemical conditions.14
Clean Chemistry
Terminal product is water only. No accumulation of oxidized sulfur species, disulfides, or other chemical byproducts that could interfere with biological processes.15
Comparison to Natural Respiration
Living Cells
Glucose oxidation
↓
Electron transport chain
↓
Cytochrome oxidase
↓
O₂ → H₂O
Oxyrase® System
Substrate oxidation
↓
Electron transport chain
↓
Cytochrome oxidase
↓
O₂ → H₂O
Scientific Discovery and Development
Dr. Howard Adler and colleagues discover that sterile bacterial cytoplasmic membrane fragments containing intact electron transport chains can enzymatically reduce oxygen to water. Published in Biotechnology and Bioengineering Symposium.1
US Patent 4,476,224 awarded for “Material and Method for Promoting the Growth of Anaerobic Bacteria” – describing the enzymatic oxygen removal mechanism and membrane fragment preparation.2
Multiple peer-reviewed studies elucidate the enzymatic pathway, substrate specificity, cofactor requirements, and comparison to chemical reducing agents. Crystal structure of d-lactate dehydrogenase published in PNAS.3
Scientific publications document enzyme system applications beyond microbiology: fluorescence microscopy (preventing photobleaching), cell preservation, pharmaceutical antioxidant, and biochemical research tool. Over 1,500 citations in peer-reviewed literature.19
Ongoing studies on enzyme variants from different bacterial sources (Acetobacter aceti for acidic pH applications), optimization of membrane preparation methods, and novel applications in cancer therapy and biotechnology.19
Learn More About the Oxyrase® Enzyme System
Explore the science behind enzymatic oxygen removal and discover how this technology is being applied across diverse fields of research.
Questions about the enzyme mechanism? Email info@oxyrase.com
References
- Adler, H.W., et al. (1981). “A material and method for the enzymatic removal of oxygen from systems.” Biotechnology and Bioengineering Symposium 11:373-384. View full citation
- Adler, H.W. & Crow, W.B. (1984). “Material and Method for Promoting the Growth of Anaerobic Bacteria.” US Patent 4,476,224. Patent details
- Decker, K. et al. (2000). “The crystal structure of d-lactate dehydrogenase, a peripheral membrane respiratory enzyme.” Proceedings of the National Academy of Sciences 97(17):9413-9418. Related papers
- Oxyrase Inc. Technical Documentation. “Substrate Requirements for Optimal Enzyme Activity.” Product inserts
- Yagi, T. (1991). “Bacterial NADH-quinone oxidoreductases.” Journal of Bioenergetics and Biomembranes 23(2):211-225. Related research
- Oxyrase Inc. Technical Bulletin. “pH and Temperature Optimization for Oxyrase® Enzyme System.” Technical bulletins
- Copeland, J.P. & Adler, H.W. (1990). “Temperature-dependent activity of respiratory enzymes in membrane preparations.” Journal of Rapid Methods and Automation in Microbiology 6(2):115-132. Full article
- Spangler, S.K. & Appelbaum, P.C. (1993). “Use of Oxyrase for macrolide and azalide susceptibility testing.” Journal of Clinical Microbiology 31(4):823-827. View publication
- Berg, J.M., Tymoczko, J.L. & Stryer, L. (2002). “Oxidative phosphorylation.” In: Biochemistry, 5th edition. New York: W.H. Freeman. Section 18.4.
- Oxyrase Inc. White Paper. “Enzymatic vs. Chemical Oxygen Removal: Mechanistic Comparison.” Read white paper
- Yoshikawa, S. et al. (1998). “Redox-coupled crystal structural changes in bovine heart cytochrome c oxidase.” Science 280(5370):1723-1729. Related research
- Proshlyakov, D.A. et al. (1998). “Direct measurement of oxygen binding to cytochrome c oxidase.” Proceedings of the National Academy of Sciences 95(14):8020-8025. Scientific papers
- Mitchell, P. (1961). “Coupling of phosphorylation to electron and hydrogen transfer by a chemi-osmotic type of mechanism.” Nature 191(4784):144-148.
- Nicholls, D.G. & Ferguson, S.J. (2013). Bioenergetics 4. Academic Press. Chapter 3: “The Chemiosmotic Proton Circuit.”
- Copeland, J.P. et al. (1995). “Comparison of enzymatic and chemical reducing agents for anaerobic microbiology.” Anaerobe 1(5):269-276. Comparison studies
- Oxyrase Inc. Technical Specifications. “Enzyme Activity Standards and Measurement Protocols.” Technical specs
- Oxyrase Inc. Safety Data Sheet. “EC-Oxyrase® Storage and Handling.” View SDS
- Luo, J.K. & Diosady, L.L. (2020). “Effect of combination of Oxyrase and sodium thioglycolate on growth of Clostridium perfringens from spores.” Food Microbiology 91:103535. View study
- Google Scholar Citations Database. “Oxyrase Enzyme System – Over 1,500 peer-reviewed citations.” Browse all citations
Additional Resources
For a complete list of over 1,500 peer-reviewed publications citing the Oxyrase® Enzyme System, visit our Literature Citations page.
Technical documentation, product inserts, and safety data sheets are available in our Technical Information section.
White papers and comparison studies can be found at White Papers.