PRACTICAL WORK Extraction, Analysis and Identification of Biomolecules

Objective:
This practical module provides hands-on experience in isolating, analyzing, and identifying biomolecules from natural sources. It integrates theoretical knowledge with laboratory techniques essential for microbial biotechnology.

1. Key Biomolecules Studied

  • Secondary Metabolites: Antibiotics (e.g., penicillin), antimicrobial peptides, and toxins.

  • Fungal Metabolites: Antifungals and mycotoxins with medical and industrial relevance.

2. Core Techniques Covered

  • Extraction Methods:

    • Liquid-Liquid Extraction: Separation using immiscible solvents.

    • Solid-Phase Extraction: Adsorption onto solid supports for purification.

    • Supercritical Fluid Extraction: Eco-friendly method using supercritical CO₂.

  • Chromatography:

    • HPLC/GC: High-resolution separation of complex mixtures.

    • Column Chromatography: Purification using solvent gradients.

  • Spectrometry:

    • Mass Spectrometry (MS): Identification via mass/charge ratio (m/z) and fragmentation patterns.

    • Nuclear Magnetic Resonance (NMR): Structural elucidation of molecules.

  • Electrophoresis:

    • Agarose Gel Electrophoresis: DNA/RNA analysis.

    • SDS-PAGE: Protein separation by size.

3. Practical Sessions (TP) Highlights

  • TP1: Antibiotic Susceptibility Testing
    Goal: Evaluate antibiotic efficacy using disc diffusion assays.
    Techniques: Zone of inhibition measurement, resistance mechanism analysis.

  • TP2: Bacterial Resistance Identification
    Goal: Detect resistance genes via PCR and electrophoresis.
    Techniques: DNA extraction, PCR amplification, gel electrophoresis.

  • TP3: Enzyme Kinetics (Beta-Galactosidase)
    Goal: Measure enzymatic activity using ONPG hydrolysis.
    Techniques: Spectrophotometry, Michaelis-Menten kinetics.

  • TP4: Biomolecule Extraction & Purification
    Goal: Isolate compounds from plant matrices (e.g., tea leaves).
    Techniques: Solvent extraction, column chromatography, rotavaporation.

  • TP5: Mass Spectrometry Analysis
    Goal: Identify purified biomolecules using MS/MS.
    Techniques: Sample preparation, ionization (ESI/EI), spectral interpretation.

4. Theoretical Concepts Applied

  • Biomolecule Properties: Solubility, polarity, and chemical interactions.

  • Kinetics: Enzyme activity measurements and inhibition mechanisms.

  • Genetic Mechanisms: Horizontal gene transfer, plasmid-borne resistance.

  • Analytical Interpretation: Spectral data analysis, database matching.

5. Applications and Valorization

  • Medical: Antibiotic development, diagnostic tools.

  • Industrial: Bio-pesticides, enzyme production, waste valorization.

  • Research: Metabolite profiling, structural biology.

Conclusion:
This module equips students with critical skills in biomolecule handling, from extraction to advanced characterization. It bridges theory and practice, preparing them for challenges in biotech research, pharmaceuticals, and industrial applications.