Enzymes immobilization in biochemical engineering

Enzymes immobilization in biochemical engineering

1. METHODS OF ENZYME AND IMMOBILIZED GROWTH

2. GROUP NO. 4 Zaineb Masood (1798) Malaika Ishtiaq (1799) Uswa Qamar (1795) Afefa Naeem (1772)

3. CONTENTS 01 03 02 03 04 Fundamentals of Enzyme and Cell Immobilization Kinetics of Immobilized Enzymes Methods of Cell Immobilization Advanced Topics and Industrial Applications

4. INTRODUCTION Enzyme Immobilization: It is the process of confining or localizing enzymes within or onto a solid support or matrix in a fixed space while retaining their catalytic activity. Essentially, it's about anchoring enzymes to a surface or within a structure DEFINITION:

5. INTRODUCTION DEFINITION: Cell immobilization: It is the process where whole cells are physically ,confined or localized within a specific matrix or support, allowing them to maintain their biological activity. This technique is used to retain cells for repeated or continuous use in bioprocesses.

6. IMPORTANCE For processing with isolated enzymes Retaining inside a reactor to avoid the loss and consequent replacement. In retaining the activity for longer period of time rather than the enzymes in solution. Increase catalytic efficiency for the multi-step conversion. To get the large throughout of the substrate. There are certainly some reasons to why the process of immobilization is desirable.

7. ADVANTAGES Simplified product separation and purification. Enzymes/cells are reusable, lowering operational costs. Enhanced stability against environmental factors (temperature, pH). Increased Stability: Easy Product Separation: Reusability Enables continuous processing for higher efficiency." Continuous Operation:

8. ADVANTAGES Better control of reaction parameter Improved Control: Reduced Contamination: Minimizes product contamination.

9. Industrial Biotechnology: Food Processing: Production of high-fructose corn syrup, dairy product processing, and flavor enhancement. Biofuel production: Enzyme therapy: Production of antibiotics, steroids, and other therapeutic compounds. Cellulosic ethanol production. Development of sensitive and specific biosensors for detecting glucose, cholesterol, and other analytes Using immobilized enzymes for targeted drug delivery or to correct metabolic deficiencies. Pharmaceuticals: Biosensors: APPLICATIONS:

10. TYPES OF IMMOBILIZATION 1. Adsorption: Factors and Applications Enzymes attach to support surfaces via weak forces. Surface Area of Adsorbent, PH of solution, temperature, ionic strength. Factors: Applications: Waste treatment, biosensors, some food processing.

11. TYPES OF IMMOBILIZATION 2. Covalent Binding: Strong Attachment Chemical bonds link enzyme/cell to support. Pros : It is Strong, stable immobilization. it also equires activation of support matrix. Cons: May reduce enzyme activity .

12. TYPES OF IMMOBILIZATION 3. Entrapment: Within a Matrix Enzyme/cells trapped in a gel or fiber. Gel: Alginate, polyacrylamide. Fiber: Synthetic or natural.

13. TYPESOF IMMOBILIZATION 4. Encapsulation: Enzymes or cells are enclosed within a semi-permeable membrane. maintain controlled reaction conditions. Provides a protective barrier against external factors.

14. TECHNIQUES OF IMMOBILIZATION: Adsorption: Physical vs. Ionic Electrostatic interactions, pH/ion-dependent. Physical: Weak van der Waals, hydrophobic interactions. Ionic: Stability, reversibility differences. Comparison:

15. TECHNIQUES OF IMMOBILIZATION: Adsorption: Support Material:i Silica gel: Activated carbon: Polymers: High surface area, versatile. Alumina: Good for ionic adsorption. High porosity, pollutant removal Varying properties for specific needs.

16. TECHNIQUES OF IMMOBILIZATION: Adsorption: Factors Surface area: pH: Temperature: Ionic strength: Higher = more binding. Affects enzyme/support charge. Impacts binding strength. Alters electrostatic interactions

17. TECHNIQUES OF IMMOBILIZATION: Adsorption: Applications Wastewater treatment: Biosensors: Chromatography: Food processing: Removal of pollutants. Enzyme immobilization for detection Enzyme-based separation. Some enzyme-catalyzed reactions.

18. TECHNIQUES OF IMMOBILIZATION: Adsorption: Pros & Cons Advantages: Simple, low cost, reversible. Disadvantages: Weak binding, leaching, environmental sensitivity.

19. TECHNIQUES OF IMMOBILIZATION: Covalent Binding: The Chemical Approach Covalent binding involves the formation of strong chemical bonds between the enzyme/cell and the support material. This method provides a very stable and durable immobilization. It requires the activation of the support matrix to create reactive groups that can form covalent bonds with the enzyme/cell.

20. TECHNIQUES OF IMMOBILIZATION: Covalent Binding: Support Matrices Common support matrices include agarose, cellulose, glass, and synthetic polymers. These materials possess or can be modified to possess functional groups for covalent attachment. The selection of the support depends on the enzyme/cell and the application requirements.

21. TECHNIQUES OF IMMOBILIZATION: Activation methods involve introducing reactive groups onto the support matrix. Covalent Binding: Activation Methods Common activating agents include glutaraldehyde, cyanogen bromide, and carbodiimides. The choice of activating agent depends on the support material and the enzyme/cell.

22. TECHNIQUES OF IMMOBILIZATION: Covalent Binding: Stability and Characteristics Strong binding allows for use in harsh conditions. Provides high stability, preventing enzyme/cell leaching. Can lead to some loss of enzyme activity due to chemical modification.

23. TECHNIQUES OF IMMOBILIZATION: Covalent Binding: Applications Used in applications requiring high stability, such as biosensors and industrial processes. Applied in the production of high-value pharmaceuticals and fine chemicals. Used in some diagnostic applications.

24. TECHNIQUES OF IMMOBILIZATION Entrapment: Confining Within a Matrix Entrapment involves physically trapping enzymes or cells within a porous matrix. This method relies on the matrix's pore size to prevent enzyme/cell leakage.

25. TECHNIQUES OF IMMOBILIZATION Entrapment: Fiber Matrices Fiber entrapment involves trapping enzymes or cells within synthetic or natural fibers. Fibers can provide a high surface area for enzyme/cell loading. This method offers good mechanical strength and stability.

26. TECHNIQUES OF IMMOBILIZATION Entrapment: Microencapsulation Microencapsulation involves enclosing enzymes or cells within small, spherical capsules. The capsule wall acts as a barrier, preventing enzyme/cell leakage. This method provides a protective microenvironment for the enzymes/cells.

27. TECHNIQUES OF IMMOBILIZATION Entrapment: Diffusional Limitations Entrapment can lead to diffusional limitations, affecting substrate and product transport. The pore size and matrix structure influence diffusion rates. Careful design of the matrix is crucial to minimize diffusion limitations.

28. Encapsulation: Membrane Confinement TECHNIQUES OF IMMOBILIZATION Encapsulation involves enclosing enzymes or cells within a semi-permeable membrane. Provides a protective barrier against external factors. This membrane allows substrate and product diffusion while retaining the biocatalyst.

29. Encapsulation: Membrane Materials TECHNIQUES OF IMMOBILIZATION Common membrane materials include polymers like cellulose acetate, alginate, and chitosan. Liposomes can also be used. The choice of material depends on the application and desired permeability.

30. TECHNIQUES OF IMMOBILIZATION Cross-linking: Chemical Bonding of Enzymes/Cells Cross-linking involves chemically linking enzymes or cells together using a multifunctional reagent. Glutaraldehyde is a common cross-linking agent. Forms a stable, three-dimensional network.

31. TECHNIQUES OF IMMOBILIZATION Cross-linking: Applications and Considerations Used in applications requiring high enzyme/cell loading and stability. Can lead to some loss of enzyme activity. Control of cross-linking is crucial to optimize performance.

32. DISCUSSION: Mass Transfer Limitations: Restricted substrate and product diffusion can affect reaction rates. Loss of Activity: Immobilization can alter enzyme conformation, reducing efficiency. Challenges in Immobilization Nano-Carriers : Use of nanoparticles for enhanced enzyme stability and efficiency. Biohybrid Systems: Combining synthetic and biological components for improved functionality. Emerging Trends

33. KINETICS OF IMMOBILIZED ENZYMES Immobilization alters enzyme-substrate interactions, often reducing catalytic efficiency. In case of immobilized enzymes reaction rates may be limited by mass transfer rather than intrinsic enzyme activity.

34. Mass Transfer Effects: Diffusion limitations Substrate must diffuse through the boundary layer to reach the enzyme. External Diffusion Limitations: Internal Diffusion Limitations: Substrate must penetrate the immobilization matrix, affecting enzyme efficiency.

35. VISION EFFECTIVENESS FACTOR THIELE MODULUS (Φ) Ratio of actual reaction rate to the rate without diffusion limitations. Indicates the efficiency of an immobilized enzyme system. Formula: η=Rate if no diffusion limitation existed /Actual reaction rate​ η = 1 → No diffusion limitations (ideal scenario). η < 1 → Presence of diffusion resistance reducing the reaction rate. Represents the relative importance of reaction rate to diffusion rate. Higher values indicate stronger diffusion limitations. Φ << 1 → Reaction rate is low compared to diffusion rate → minimal diffusion limitation. Φ >> 1 → Diffusion rate is low compared to reaction rate → strong diffusion limitation. MATHEMATICAL FORMUALTION Interpretation: Interpretation:

36. CASE STUDY: Kinetics of Immobilized Lactase in Dairy Processing Lactase (β-galactosidase) is an enzyme that hydrolyzes lactose into glucose and galactose. Introduction: In dairy processing, lactase is used to produce lactose-free milk for lactose-intolerant individuals. Immobilization of lactase enhances its operational stability, reusability, and efficiency in industrial applications.

37. CASE STUDY: Kinetics of Immobilized Lactase in Dairy Processing Covalent bonding to carriers like silica or cellulose Common Methods: immobilization of lactase Entrapment in alginate beads Adsorption on porous supports

38. Parameter Free Lactase Immobilized Lactase Vmax (Maximum rate) Higher; faster reaction in solution More stable under processing conditions Reusability Single-use More stable under processing conditions Stability (pH/temp) Sensitive to changes More stable under processing conditions CASE STUDY: Kinetics of Immobilized Lactase in Dairy Processing

39. KINETIC CHANGES IN IMMOBILIZED ENZYMES Km (Michaelis Constant): Substrate concentration at which the reaction rate is half of Vmax. Effect of Immobilization: Often increases due to mass transfer limitations (especially internal diffusion). The substrate takes longer to reach the enzyme’s active site

40. KINETIC CHANGES IN IMMOBILIZED ENZYMES Vmax (Maximum Velocity): The maximum rate at which the enzyme can convert substrate to product. Effect of Immobilization: Not all enzyme active sites may be equally accessible, and mass transfer resistance can limit substrate May decrease due to restricted enzyme mobility and slower substrate diffusion.

41. STRATEGIES TO MINIMIZE MASS TRANSFER LIMITATIONS Use of Highly Porous Carriers Increases the surface area and allows easier penetration of substrates into the immobilization matrix. Reduction of Particle Size: Smaller particles have shorter diffusion paths, allowing substrates to reach enzyme active sites faster. Optimization of Flow Conditions in Bioreactors Enhances external mass transfer by reducing the thickness of the boundary layer around enzyme particles.

42. IMPORTANCE OF CELL IMMOBILIZATION: 01 Enables continuous fermentation and biotransformation Immobilized cells can be retained in bioreactors for long-term operations, ideal for continuous processing. 02 Cell immobilization is a key technique in industrial biotechnology that involves restricting the movement of cells while maintaining their metabolic activity. Its importance lies in the following advantages: Protects cells from shear stress Immobilized systems reduce mechanical damage caused by stirring or fluid movement, especially in large-scale fermenters. 03 Facilitates prolonged use of biocatalysts Cells remain viable and functional over extended periods, enhancing productivity and reducing operating costs.

43. Cells are enclosed within a semi-permeable membrane (e.g., microcapsules), allowing exchange of nutrients and products. Techniques for Cell Immobilization a) Entrapment: Cells are physically enclosed within a porous gel matrix or fiber network. Alginate beads, Polyacrylamide gels Examples: Advantages: Simple and low-cost, Provides a protective environment b) Encapsulation Advantages: Better control over mass transfer, Prevents contamination

44. Techniques for Cell Immobilization: c) Surface Attachment (Adsorption or Covalent Bonding) Description: Cells are immobilized on solid supports via weak forces (adsorption) or strong chemical bonds (covalent linkage) Examples: Glass beads, Activated carbon Advantages: Minimal diffusion limitations, Easy to scale up

45. MATERIALS: Natural Polymers Examples: Alginate, Carrageenan, Agarose Common Polymers for Cell Immobilization: Advantages: Biocompatible and non-toxic, Suitable for live microbial cells Synthetic Polymers Examples: Polyvinyl alcohol (PVA), Polyurethane Advantages: Stronger mechanical properties, Greater durability in harsh condition

46. APPLICATIONS OF CELL IMMOBILIZATION: Industrial Fermentation Example: Immobilized yeast cells are used in the production of beer, wine, and ethanol. Benefits: Enables continuous fermentation, increasing productivity. Reduces cell washout and contamination. Enhances product consistency and quality. Wastewater Treatment Example: Use of immobilized microbial biofilms on carriers like activated carbon or plastic media. Benefits: Enhances the stability of microbial communities. Supports long-term treatment with minimal maintenance. Effective under variable environmental conditions.

47. DISCUSSION: Issue: Prolonged immobilization may reduce the viability and metabolic activity of cells over time. Cause: Accumulation of toxic by-products, Limited removal of waste compounds, Physical stress due to restricted space within the matrix. Consequence: Leads to decreased productivity and may require frequent re-immobilization or cell replacement . Cell Viability:

48. DISCUSSION: Issue: Immobilized systems can hinder the efficient transport of nutrients, oxygen, and substrates to cells. Cause: Dense gel matrices or thick biofilms, Poorly optimized carrier materials. Consequence: May result in uneven growth, local nutrient starvation, or low product formation. Nutrient Diffusion Limitations

49. Enzyme and Cell Co-Immobilization Intro Applications: Allows multi-step biochemical reactions within a single system. Increases reaction rates, minimizes intermediate loss, and improves product yields. Advantages: Co-immobilization involves the simultaneous immobilization of enzymes and living cells in the same matrix or on the same support. Bioconversion of starch to glucose and then to ethanol. Multi-enzyme systems for antibiotic synthesis or flavor compound production.

50. USE OF IMMOBILIZED SYSTEMS IN BIOREACTORS PACKED-BED REACTORS (PBR) FLUIDIZED-BED REACTORS (FBR) MEMBRANE REACTORS Immobilized particles are tightly packed into a column. Ideal for high-throughput, long-term operations. Common in pharmaceutical and organic acid production Particles are suspended by upward fluid flow. Offers better mass transfer and less clogging than PBR. Useful in wastewater treatment and high-viscosity processes. Combine immobilized catalysts with membrane separation. Allow selective removal of products and recycling of substrates. Applied in amino acid production, biohydrogen generation, and biosensor technologies. Immobilized cells and enzymes are integrated into various reactor designs for continuous and efficient production.

51. Mathematical Framework Modeling of Immobilized Systems: Kinetic models simulate the performance of immobilized biocatalysts in continuous systems. It incorporates: Tools: Diffusion limitations Reaction kinetics (e.g., modified Michaelis-Menten) Reactor design parameters Effectiveness Factor (η) and Thiele Modulus (Φ) are used to assess system efficiency.

52. CASE STUDY: INDUSTRIAL PRODUCTION OF AMINO ACIDS USING IMMOBILIZED CELLS Overview: Immobilized microbial cells are widely used in large-scale fermentation processes for the production of essential amino acids. Example Organism: Corynebacterium glutamicum or Escherichia coli (immobilized on alginate or other polymer matrices)

53. CASE STUDY: INDUSTRIAL PRODUCTION OF AMINO ACIDS USING IMMOBILIZED CELLS Common Amino Acids Produced: L-glutamic acid (flavor enhancer in foods) L-lysine (used in animal feed supplements) Advantages of Immobilization in This Context: Continuous Production: Enables long-term use of the same biomass, reducing downtime. Improved Yield and Productivity: High cell density and prolonged metabolic activity. Cost-Effective: Reduced need for cell regeneration and separation

54. Purpose: Monitoring blood glucose levels in diabetic patients. How It Works: Glucose oxidase (GOx) is immobilized on an electrode surface. Glucose in the sample reacts with the enzyme, generating an electrical signal. The signal is proportional to glucose concentration. USE OF IMMOBILIZED ENZYMES IN BIOSENSORS Immobilized enzymes play a crucial role in biosensing applications, especially in healthcare diagnostics. a) Glucose Oxidase-Based Biosensors

55. Purpose: Detection and monitoring of cholesterol levels in clinical diagnostics. How It Works: Enzymes like cholesterol oxidase are immobilized on transducers. Enzymatic reaction produces measurable signals (e.g., amperometric or optical). Significance: Fast, accurate, and non-invasive cholesterol measurement in point-of-care testing. USE OF IMMOBILIZED ENZYMES IN BIOSENSORS Immobilized enzymes play a crucial role in biosensing applications, especially in healthcare diagnostics. b) Cholesterol Biosensors

56. TECHNIQUES OF IMMOBILIZATION Smart Immobilization Materials Advanced carrier materials that respond to environmental stimuli such as pH, temperature, or substrate concentration. Functionality: Enable dynamic control of enzyme or cell activity. Improve stability and responsiveness of immobilized systems in fluctuating industrial environments. Examples: pH-sensitive hydrogels, Temperature-responsive polymers

57. TECHNIQUES OF IMMOBILIZATION 3D-Printed Biocatalysts: 3D printing technologies are being used to fabricate custom-designed structures for enzyme or cell immobilization. Advantages: Precision engineering of shape, porosity, and surface area. Enhanced mass transfer and reaction kinetics due to optimized geometry. Facilitates development of lab-on-a-chip systems or compact bioreactors.

58. Challenges in Scaling Up Immobilized Systems Optimization of Bioreactor Desig:n Issue: Bioreactor systems must be tailored to accommodate immobilized biocatalysts while ensuring uniform flow, minimal shear stress, and efficient mixing. Solution: Advanced modeling, computational fluid dynamics (CFD), and pilot-scale studies to develop scalable, efficient designs.

59. Challenges in Scaling Up Immobilized Systems Cost-Effectiveness of Immobilization Techniques Issue: High costs of carrier materials, enzyme purification, and immobilization procedures can limit commercial use. Solution: Use of inexpensive natural polymers (e.g., alginate, chitosan). Development of recyclable or multi-use matrices. Optimization of immobilization methods to reduce waste.

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