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Enzymes by Catalytic Mechanism

Catalog Product Name EC No. CAS No. Source Price
DIA-489 Luciferase from Vibrio fischeri Vibrio fischeri Inquiry
DIA-490 Alkaline Phosphatase from Escherichia coli Escherichia coli Inquiry
DIA-491 Papain Papaya Inquiry
DIA-492 Phospholipase D Streptomyces sp. Inquiry
DIA-493 Pronase E Streptomyces griseus Inquiry
DIA-494 Alpha-Amylase (Thermostable) 9000-90-2, 9000-85-5 Bacillus sp. Inquiry
DIA-495 Alpha-Amylase from Aspergillus oryzae 9000-90-2 Aspergillus oryzae Inquiry
DIA-496 Alpha-Amylase from Bacillus licheniformis 9000-90-2, 9000-85-5 Bacillus licheniformis Inquiry
DIA-497 Alpha-Amylase from Bacilus subtilis Bacilus subtilis Inquiry

Enzymes are the biological catalysts that define the precision and efficiency of diagnostic assays. Their catalytic mechanisms determine substrate specificity, turnover rate, and stability—critical parameters for the reliability of in-vitro diagnostic (IVD) platforms.

Creative Enzymes provides a comprehensive portfolio of diagnostic-grade enzymes categorized by their catalytic mechanisms, enabling developers to select optimal candidates for colorimetric, fluorescent, chemiluminescent, and biosensor-based assays.

Understanding Catalytic Mechanisms in Diagnostic Enzymes

The classification of enzymes by catalytic mechanism is based on the International Union of Biochemistry and Molecular Biology (IUBMB) enzyme nomenclature, which organizes enzymes into six major classes according to the type of chemical reaction catalyzed. Each mechanism defines how the enzyme interacts with substrates, cofactors, and the diagnostic system's signal-generation pathway.

For diagnostic purposes, understanding these mechanisms ensures:

  • Accurate signal transduction and quantitation.
  • Compatibility with assay reagents and detection chemistry.
  • Optimization of kinetic parameters (Km, Vmax) and stability profiles.
  • Reduced interference from biological matrices such as serum, plasma, or urine.

Oxidoreductases

Catalytic Role:

Catalyze oxidation–reduction reactions by transferring electrons or hydrogen atoms between molecules.

Diagnostic Importance:

Oxidoreductases form the cornerstone of many colorimetric and electrochemical assays. For example, glucose oxidase (GOx) and cholesterol oxidase (ChOx) are routinely used to generate hydrogen peroxide, which reacts with chromogenic substrates in peroxidase-coupled systems.

Typical Examples:

  • Glucose oxidase (GOx): Used in blood glucose meters and biosensors.
  • Lactate dehydrogenase (LDH): Key biomarker in liver and cardiac panels.
  • Urate oxidase and xanthine oxidase: Used for uric acid and purine metabolism assays.
  • Glycerol-3-phosphate oxidase: Integral to triglyceride determination kits.

Key Features:

  • Cofactor dependence (e.g., FAD, NAD+, NADP+).
  • High substrate specificity with measurable redox activity.
  • Excellent compatibility with peroxidase-based chromogenic systems.

Transferases

Catalytic Role:

Transfer functional groups (such as phosphate, methyl, or amino groups) from one molecule to another.

Diagnostic Importance:

Transferases are essential in enzymatic assays for measuring metabolic intermediates and liver or cardiac function markers.

Representative Enzymes:

  • Alanine aminotransferase (ALT) and aspartate aminotransferase (AST): Indicators of hepatocellular injury.
  • Creatine kinase (CK): Key cardiac and muscular damage marker.
  • Hexokinase: Used in glucose quantification through ATP-dependent phosphorylation.

Assay Example:

In the hexokinase method for glucose, glucose is phosphorylated to glucose-6-phosphate, which is subsequently oxidized by glucose-6-phosphate dehydrogenase (G6PD), linking transferase and oxidoreductase reactions for optical detection.

Hydrolases

Catalytic Role:

Catalyze hydrolytic cleavage of chemical bonds, including esters, glycosides, and peptides.

Diagnostic Importance:

Hydrolases are indispensable for tests detecting metabolites, lipid profiles, or enzyme deficiencies.

Representative Enzymes:

  • Lipase and esterase: Used in triglyceride and cholesterol ester hydrolysis.
  • Alkaline phosphatase (ALP): Widely used in immunodiagnostic assays as a signal enzyme.
  • β-galactosidase and β-glucuronidase: Applied in reporter assays and conjugate detection.

Key Performance Indicators:

  • Robust activity at physiological pH (6.8–8.0).
  • Tolerance to surfactants and preservatives.
  • Stability during lyophilization for long-term storage.

Lyases

Catalytic Role:

Catalyze the addition or removal of groups to form or break double bonds without hydrolysis or oxidation.

Diagnostic Importance:

Lyases are valuable in specific metabolic assays where rapid equilibrium reactions are required.

Examples:

  • Pyruvate decarboxylase and aldolase: Applied in metabolic pathway evaluation.
  • Carbonic anhydrase: Used for CO₂ determination in blood gas analyzers.

Advantages for Diagnostics:

  • Rapid catalytic turnover.
  • Minimal interference from matrix components.
  • Compatibility with kinetic endpoint assays.

Isomerases

Catalytic Role:

Catalyze intramolecular rearrangements, converting one isomer into another.

Diagnostic Importance:

Isomerases enhance selectivity in metabolite conversion assays and improve the dynamic range of multi-enzyme systems.

Representative Enzymes:

  • Glucose-6-phosphate isomerase (GPI): Couples with hexokinase systems.
  • Triose phosphate isomerase (TPI): Used in coupled reactions with glycerol-3-phosphate dehydrogenase.

Diagnostic Advantage:

Their reversible reactions allow flexible integration into cascade assays for improved linearity and sensitivity.

Ligases (Synthetases)

Catalytic Role:

Catalyze the joining of two molecules using energy derived from ATP hydrolysis.

Diagnostic Importance:

Ligases are vital in molecular diagnostics, enabling DNA/RNA amplification and labeling processes.

Representative Enzymes:

  • DNA ligase: Seals nicks during PCR and next-generation sequencing (NGS) workflows.
  • Aminoacyl-tRNA synthetases: Investigated for enzyme-based biomarker assays.

Key Features:

  • High fidelity in nucleotide or peptide bond formation.
  • Temperature-resistant variants engineered for PCR and NGS kits.

Catalytic Mechanism–Driven Optimization

At Creative Enzymes, each enzyme's catalytic mechanism guides its design, engineering, and quality evaluation. Our optimization pipeline includes:

  • Molecular Engineering: Enhancing cofactor affinity, substrate recognition, and catalytic turnover.
  • Expression and Purification: Host-optimized systems (E. coli, yeast, mammalian) for high yield and purity.
  • Kinetic Profiling: Determination of Michaelis–Menten constants (Km, Vmax) under assay-relevant conditions.
  • Thermal and Chemical Stability Testing: Ensuring consistent performance across temperature and matrix variations.
  • Matrix Compatibility: Evaluating enzyme activity in serum, plasma, and urine to ensure diagnostic reproducibility.

This mechanism-based approach enables precise customization for diverse assay chemistries—from clinical biochemistry analyzers to point-of-care testing (POCT) devices.

Quality and Regulatory Alignment

All diagnostic-grade enzymes from Creative Enzymes are manufactured under ISO 9001 and ISO 13485 quality frameworks. Analytical validation follows IFCC and CLSI guidelines, ensuring:

  • Reproducibility (CV < 3 %).
  • Endotoxin level < 0.1 EU/mg (LAL tested).
  • Defined specific activity (U/mg) verified by spectrophotometric and chromatographic methods.
  • Batch-to-batch consistency validated by HPLC and electrophoretic profiling.

FAQs

  • Q1. Why is enzyme classification by catalytic mechanism important for diagnostic assay design?

    A1. Each catalytic mechanism determines how an enzyme interacts with substrates and detection reagents. Choosing the correct enzyme class ensures optimal reaction kinetics, minimizes cross-reactivity, and enhances assay stability.

  • Q2. Can Creative Enzymes modify catalytic properties to fit specific diagnostic formats?

    A2. Yes. Through site-directed mutagenesis and protein engineering, we can alter substrate affinity, cofactor usage, or thermal tolerance to match requirements for colorimetric, fluorescent, or biosensor systems.

  • Q3. What factors should be considered when selecting oxidoreductases for biosensors?

    A3. Key factors include electron transfer efficiency, oxygen dependence, mediator compatibility, and operational stability under continuous use.

  • Q4. How do hydrolases differ from transferases in clinical chemistry assays?

    A4. Hydrolases catalyze bond cleavage with water, typically generating measurable chromogenic or fluorogenic products, while transferases mediate group transfer reactions that often require coupled enzyme systems for detection.

  • Q5. Are these enzymes suitable for point-of-care testing (POCT)?

    A5. Absolutely. We provide lyophilization-compatible and thermostable enzyme formulations tailored for compact POCT devices, maintaining accuracy across variable ambient conditions.

  • Q6. What quality metrics define diagnostic-grade enzymes?

    A6. Key metrics include activity reproducibility (CV < 3 %), high purity (> 95 %), low endotoxin, and validated kinetic behavior under physiological pH and ionic strength conditions.

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