INTRODUCTION:

Introduction to Enzyme Biotechnology:^1,2

Using enzymes to improve processes in the environmental, medical, and industrial domains is the main goal of enzyme biotechnology. Natural catalysts called enzymes effectively and sustainably speed up chemical reactions as they occur.

Key Features of Enzymes:

· Highly Specific: Enzymes are tailored to target specific reactions.

· Eco-Friendly: They minimize the need for chemicals.

· Reusability: Immobilized enzymes can be reused multiple times

Sources of Enzymes:

· Microbial – Bacteria (Bacillus subtilis), fungi (Aspergillus niger).

· Plant-Based – Papain (papaya), bromelain (pineapple).

· Animal-Based – Pepsin, trypsin.

Applications:

1. Pharmaceuticals: Enzymes are involved in drug production, like penicillin synthesis.

2. Food Industry: Used for lactose-free milk (lactase) and in baking (amylase).

3. Biofuel Production: Enzymes help convert biomass into ethanol and biodiesel.

4. Environmental Use: Waste treatment and pollution control.

5. Medical Diagnostics: Enzyme-based biosensors aid in disease detection

Enzyme Immobilization:^3,4

Introduction:

Enzyme immobilization is the process of physically or chemically attaching enzymes to a matrix or solid support to improve their stability, effectiveness, and reusability. This technique is frequently used to enhance enzymatic reactions in sectors like biotechnology, food, and pharmaceuticals.

Purpose of Enzyme Immobilization:

· Enhances enzyme stability under varying temperature and pH conditions.

· Allows enzyme reuse, lowering production costs.

· Improves process control in industrial settings.

· Prevents contamination of final products.

Types of Enzyme Immobilization

1. Adsorption Immobilization

2. Covalent bonding Immobilization

3. Entrapment Immobilization

4. Encapsulation Immobilization

Characteristics of Immobilized Enzymes:

· Higher Stability: Resistant to denaturation from heat or pH changes.

· Reusability: Can be reused multiple times.

· Controlled Reactions: Allows better regulation of enzyme activity.

· Reduced Contamination: Prevents mixing with the final product

Significance in Biotechnology:

· Pharmaceuticals: Drug synthesis, antibiotic production.

· Food Industry: Lactose-free milk, juice clarification.

· Biofuel Production: Enzymatic conversion of biomass.

· Waste Treatment: Biodegradation of pollutants.

Advantages of Enzyme Immobilization:

1. Increased Stability:

Better resistance to environmental changes.

2. Reusability:

Immobilized enzymes can be reused, reducing costs.

3. Enhanced Efficiency:

Stable enzyme activity ensures efficient industrial processes.

4. Reduced Contamination:

Less need for purification steps.

5. Easy Separation from Products:

Enzymes can be separated and reused easily.

6. Improved Process Control:

Allows better management of enzyme levels and reaction rates.

7. Eco-Friendly:

Reduces reliance on chemicals, making processes more sustainable.

8. Application in Continuous Processing:

Perfect for long-term industrial operations like drug manufacturing.

Applications of Enzyme Immobilization in Pharmaceuticals⁵

1. Drug Synthesis:

Immobilized enzymes assist in the production of antibiotics and other drugs.

· Example: Penicillin acylase for semi-synthetic penicillin’s

2. Enzyme-Based Drug Delivery:

Controlled release of therapeutic drugs using immobilized enzymes.

Example: L-asparaginase for leukaemia treatment.

3. Biocatalysts in API (Active Pharmaceutical Ingredient) Production:

Enhance the production of high-purity Active Pharmaceutical Ingredients.

Example: Lipases for chiral drug intermediates.

4. Biosensors for Disease Diagnosis:

Enzymes in biosensors detect diseases.

Example: Glucose oxidase for diabetes monitoring

5. Vaccine Production:

Enzyme immobilization helps in large-scale vaccine manufacturing.

Example: Immobilized enzymes help in the purification of biopharmaceuticals.

6. Enzyme Therapy:

Used in enzyme replacement therapies (ERT) for metabolic disorders.

Example: Immobilized lactase for treating lactose intolerance.

7. Prodrug Activation:

Some enzymes help convert prodrugs into active forms in the body.

Example: Cytochrome P450 in drug metabolism.

Methods of Enzyme Immobilization:

There are several methods used to immobilize enzymes to enhance their stability, reusability, and efficiency. Four main techniques include:

Adsorption Method of Enzyme Immobilization:⁶

Introduction:

This technique uses weak forces like hydrogen bonds, ionic interactions, or van der Waals forces to physically attach enzymes to a solid support.

Process of Adsorption:

Step-1 Preparation:

Select a solid carrier (e.g., glass beads, activated charcoal).

Step-2 Interaction:

Enzymes bind to the surface of the carrier.

Step-3 Washing:

Excess enzymes are washed off.

Advantages of Adsorption:

· Simple, low-cost, and effective for retaining enzyme activity.

· Mild conditions ensure enzyme activity is maintained.

· Reversible, allowing for enzyme reuse.

Disadvantages of Adsorption:

· Weak binding that may cause enzyme detachment under variable conditions.

· Limited stability due to enzyme leakage.

· Non-uniform enzyme distribution.

Applications:

· Penicillin production: Immobilized penicillin acylase for semi-synthetic penicillin.

· Biosensors: Immobilized glucose oxidase for diabetes testing.

· Drug Synthesis: Lipase for chiral drug production.

Covalent Bonding Method of Enzyme Immobilization:

Introduction:

This technique ensures long-term stability by attaching enzymes to a solid substrate using strong covalent bonds.

Process of Covalent Bonding:

Step-1 Selection of Carrier:

Use materials like silica, cellulose, or synthetic polymers.

Step-2 Activation of Carrier:

Modify the carrier surface using chemical reagents.

Step-3 Enzyme Attachment:

Enzymes bond with functional groups on the carrie.

Step-4 Washing and Stabilization:

Remove unbound enzymes and stabilize the immobilized ones:

Advantages of Covalent Bonding

· Strong binding and long-term stability.

· Enzymes can be reused multiple times.

· Resistant to environmental changes like pH and temperature

Disadvantages of Covalent Bonding:

· Complex, requiring chemical modifications that increase cost and time.

· Some enzyme activity may be lost during the process.

· Binding is irreversible, which limits flexibility.

Applications:

· Antibiotic production using penicillin acylase.

· Biosensors: Glucose oxidase for diabetes monitoring.

· Drug Synthesis: Lipases and proteases in pharmaceutical manufacturing.

Entrapment Method of Enzyme Immobilization:

Introduction:

This technique confines enzymes inside a porous gel or matrix, allowing substrates to flow through while the enzyme stays in its place.

Process of Entrapment:

Step -1 Selection of Matrix:

Use materials like calcium alginate, agar, or polyacrylamide.

Step -2 Mixing:

Enzymes are mixed with the matrix before solidification.

Formation:

The matrix solidifies, encapsulating the enzymes.

Advantages of Entrapment:

· Prevents Enzyme Leakage: Prevents enzyme leakage while preserving activity.

· Good Retention of Activity: The enzyme is not chemically modified, preserving its function.

· Continuous Use in Industrial Processes: Suitable for continuous industrial applications

Disadvantages of Entrapment:

· Diffusion Limitations: Diffusion limitations may slow reaction rates.

· Complex Preparation: Complex preparation conditions.

· Possible Enzyme Inactivation: Risk of enzyme inactivation during polymerization.

Applications in Pharmaceuticals:

· Antibiotic Production: Penicillin acylase immobilized in calcium alginate.

· Hormone Production: Entrapped enzymes for steroid transformations.

· Biosensors: Glucose oxidase entrapped for diabetes monitoring.

Encapsulation Method of Enzyme Immobilization:

Introduction:

Enzymes are protected by a semi-permeable membrane or microcapsule that permits substrate diffusion.

Process of Encapsulation:

Step-1 Selection of Encapsulation Material:

Polymers like alginate or chitosan are commonly used.

Step-2 Enzyme Suspension

The enzyme is suspended within the polymer solution.

Step-3 Membrane Formation

The polymer solidifies, forming microcapsules or vesicles.

Step-4 Washing and Stabilization

Excess unbound enzyme is removed, and the capsules are prepared for use.

Advantages of Encapsulation:

· Protects the Enzyme: Provides enzyme protection from environmental factors.

· Allows Controlled Diffusion: Allows controlled diffusion of substrates and products

· Reusable for Continuous Processing: Reusable for continuous industrial use.

Disadvantages of Encapsulation:

· Diffusion Limitations: Diffusion limitations can hinder reaction speed.

· High Cost and Complexity: High cost and complexity of membrane formation.

· Potential Enzyme Leakage: Risk of enzyme leakage if membranes are poorly formed

Applications in Pharmaceuticals:

· Drug Synthesis: Encapsulated penicillin acylase for antibiotic production.

· Enzyme-Based Drug Delivery: Controlled enzyme release for treatments.

· Biosensors: Encapsulated glucose oxidase for diabetes monitoring.

Plant Cell Immobilization⁷

Introduction:

This method helps with pharmacological, biotransformation, and environmental applications by immobilizing plant cells or tissues to increase their stability and reusability.

Methods of Plant Cell Immobilization:

1. Entrapment:

Plant cells are trapped in a matrix such as calcium alginate.

Example: Immobilization of Catharanthus roseus cells for anticancer drug production.

2. Encapsulation:

Cells are enclosed in a semi-permeable membrane.

Provides protection while allowing exchange of nutrients and metabolites.

3. Adsorption:

Plant cells are attached to a solid surface like glass beads

This method is simple but has weak cell attachment.

4. Covalent Bonding:

Cells are chemically bonded to solid carriers.

Provides strong attachment but may affect cell viability.

Applications of Immobilized Plant Cells:

· Secondary Metabolite Production: Production of pharmaceuticals like alkaloids and essential oils.

· Biotransformation: Modification of chemical structures, such as steroid transformation

· Environmental: Wastewater treatment and heavy metal removal.

· Food and Cosmetics Industry: Production of natural Flavors, Colores, and bioactive compounds.

Bacterial Cell Immobilization:

Introduction:

This technique increases stability and reusability in a variety of applications by containing bacterial cells on solid supports while preserving their biological activity.

Methods of Bacterial Cell Immobilization:

1. Entrapment:

Bacterial cells are embedded in a matrix like calcium alginate.

Example: E. coli for enzyme production

2. Encapsulation:

Bacteria are enclosed in a semi-permeable membrane.

Example: Lactic acid bacteria encapsulated for probiotic production.

3. Adsorption:

Bacteria are attached to surfaces like glass or activated carbon.

4. Covalent Bonding:

Bacteria are chemically bound to carriers.

5. Cross-Linking:

Bacterial cells are linked together using cross-linkers.

Applications of Immobilized Bacterial Cells:

· Antibiotic Production: Example: Streptomyces for penicillin.

· Vaccine Production: Using immobilized bacterial strains.

· Bioremediation: Wastewater treatment and oil spill cleanup.

· Fermentation: Used in food industries for dairy fermentation.

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Received on 22.02.2024 Revised on 05.08.2024

Accepted on 02.04.2025 Published on 10.05.2025

Available online from May 14, 2025

Res. J. Pharmacognosy and Phytochem. 2025; 17(2):168-172.

DOI: 10.52711/0975-4385.2025.00027

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