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🧪 Enzyme Analysis Techniques – Overview
Enzyme analysis techniques are essential for studying enzyme activity, kinetics, specificity, inhibition, and stability. These methods are widely used in biotechnology, food science, pharmaceuticals, diagnostics, and clinical biochemistry.
🔍 1. Enzyme Activity Assays
✅ Colorimetric Assays
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Measure color change due to enzyme action.
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Example: Amylase breaking down starch (iodine test), peroxidase with guaiacol.
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Spectrophotometer used to measure absorbance.
✅ Fluorometric Assays
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Use fluorescent substrates; very sensitive.
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Common in diagnostic and clinical assays (e.g., protease activity).
✅ Turbidimetric Assays
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Measure reduction in turbidity (cloudiness) due to enzymatic action (e.g., lysozyme breaking bacterial cell walls).
✅ Titrimetric Assays
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Involve titration to measure product or substrate concentrations.
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Example: Lipase activity via free fatty acid titration.
🧬 2. Enzyme Kinetics
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Michaelis-Menten kinetics: Determines Km (substrate affinity) and Vmax (max activity).
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Tools: Lineweaver-Burk, Eadie-Hofstee plots.
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Helps in understanding catalytic efficiency and comparing enzyme variants.
🚫 3. Enzyme Inhibition Studies
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Types: Competitive, Non-competitive, Uncompetitive
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Measure how inhibitors affect enzyme activity.
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Important for drug discovery and therapeutic enzyme design.
🧊 4. Enzyme Stability Tests
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Temperature and pH stability profiles.
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Thermal inactivation curves (e.g., T₅₀ or half-life).
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Storage and shelf-life testing for industrial enzymes.
🧪 5. Protein Estimation & Enzyme Purity
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Bradford Assay, Lowry Method, or BCA Assay for total protein.
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SDS-PAGE to check enzyme purity.
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Specific activity = (Enzyme activity per mg of total protein)
🧰 6. Modern & Instrument-Based Techniques
| Technique | Purpose | Example |
|---|---|---|
| Spectrophotometry | Absorbance-based activity | NADH oxidation (340 nm) |
| Fluorometry | Low-concentration detection | Protease or phosphatase assays |
| HPLC/UPLC | Separation of substrates/products | Enzyme kinetics |
| Mass Spectrometry (MS) | Enzyme identification & modification | Proteomics |
| Isothermal Titration Calorimetry (ITC) | Binding & thermodynamics | Enzyme-inhibitor interactions |
| Surface Plasmon Resonance (SPR) | Real-time binding kinetics | Drug-enzyme studies |
📦 Applications
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Clinical Diagnostics: Liver enzymes (ALT, AST), amylase, lipase
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Food Industry: Protease, amylase, lactase assays
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Pharmaceuticals: Enzyme inhibitors and drug development
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Environmental: Enzyme biosensors for pollutant detection
🎓 Learn & Practice
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Use model enzymes like catalase, peroxidase, urease, amylase
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Perform lab-based activity assays + calculations of enzyme units, specific activity
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Ideal for B.Sc./M.Sc. project work, internships, or lab-based job roles
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