1. Pharmaceutical Biotechnology PHR 403
Chapter 3: Enzyme Technology

2. Learning objectives: In this chapter, students will learn about-
Enzymes
commercial uses of enzyme
immobilized enzyme technology and reactors
application of immobilized enzymes in manufacturing and preparation of different biopharmaceuticals.
Other uses of enzyme technology

3. Sources Lipases Tripsin Rennets Papain Proteases Amylase
Animal
Lipases
Tripsin
Rennets
Plant
Papain
Proteases
Amylase
Soyabean lipoxygenase
Microbial
Extracellular enzyme
Cellulase
Plymethylgalacturonase
Polygalacturonase
Pectimethylesterase
Intracellular enzyme
Invertase
Uric oxidase
Asparaginase

4. Comparsion Animal/ Plant Vs. Microbial enzyme
Microbial enzymes have 2 advantages over animal and plant enzymes-
Firstly:
Economical, can be produced on large scale, within the limited space and time.
Easy to extract and purify
Secondly:
They are capable of producing large variety of enzyme
Can grow in wide range of environment.
Show genetic flexibility
Short generation times

5. Historical perspective
1913
Enzyme containing detergent was developed
But it was less stable
1965
New stable enzyme protease was introduced
1970
First commercial process was used to produce fructose from glucose by isomerization
Present
2000 enzymes have been isolated
1000 enzymes are recommended for various applications.
50 microbial enzymes has industrial applications.

6. Properties of enzyme Presence of species specificity
Macromolecule differ in different species
Phylogenetic variations gave rise to variation in different macromolecules
Example: Protease of 2 closely related microorganisms may be related in some properties but differs in many other properties.
Variations in activity and ability
Most microbial enzymes are extracellular
Extracellular enzyme are influenced by temperature, pH etc.
Optimum environment for extracellular enzyme correlates with the optimum growth environment for the bacteria. Bacillus coagulans (Thermophillic) has different amylase activity than Bacillus licheniformis (Mesophillic)
Intracellular enzymes are not that much influenced by environment
Optimum temperature and pH

7. Properties of enzyme Substrate specificity Activation and inhibition
The stability of the enzyme is also increase by many factors, such as:
High concentration of respective enzyme
Presence of their substrate
Presence of ion. eg.Ca2+
Reduced amount of water content
Substrate specificity
Activation and inhibition
Same enzymes isolated from diferent sources show different responses. i.e., β- galactosidase from fungal origin does not require Cobalt, where the same enzyme from bacterial origin needs that.

8. Methods of enzyme production
Isolation of microorganism, strain development and preparation of inoculums
Medium formulation and preparation
Sterilization of medium, maintenance of culture and fluid filtrations
Purification of enzyme

9. Isolation of microorganism, strain development, and preparation of inoculum
Aim:
Production of enzyme in high amount
Completion of fermentation process in short time
Maximum utilization of culture medium in low cost.

10. Medium formulation and preparation
Carbohydrate sources: Molases, Barley, Corn, wheat and starch hydrolase
Protein: Meals of Soybean, Cotton seed, peanut and whey, corn steep liquor and yeast hydrolysate.

11. Sterilization Continuous sterilization
Surface culture Vs Submerged culture
Anti-foaming agents. Eg: Oils

12. Purification Preparation of concentrated solution
Clarification of concentrated enzyme
Addition of preservatives
Precipitation of enzyme
Drying
Packaging

13. Enzyme immobilization technology
Enzymes can increase the rate of reaction many folds.
But, majority of the enzyme are fairly unstable
Industrial application is often hampered by a lack of long-term operational stability and the technically challenging recovery process and reuse of the enzyme.
In order to make enzyme utilization in biotechnological processes more favourable, different methods for cost reduction have been put into practice and, immobilization is one of them

14. Immobilized enzyme
The term ‘immobilized enzymes’ refers to ‘enzymes physically confined or localized in a certain defined region of space.
with retention of their catalytic activities
which can be used repeatedly and continuously
substantially simplifies the manipulation with the biocatalyst and the control of the reaction process while enhancing the stability of the enzyme under both storage and operational conditions.

15. What is enzyme immobilization?
The term “immobilized enzymes” refers to “enzymes physically confined or localized in a certain defined region of space with retention of their catalytic activities, and which can be used repeatedly and continuously.”
It is a process of confining the enzyme molecules to a solid support over which a substrate is passed and converted to products.
The major components of an immobilized enzyme system are-
enzyme,
matrix, and
the mode of attachment.

16. Continued…
The enzymes can be attached to the support by interactions ranging from-
reversible physical adsorption and ionic linkages to
stable covalent bonds.
However, as a consequence of enzyme immobilization, some properties such as catalytic activity or thermal stability become altered.

17. Continued…
Besides the application in industrial processes, the immobilization techniques are the basis for making a number of biotechnological products with applications in-
diagnostics,
bioaffinity chromatography, and
biosensors.

18. Choice of Support
The characteristics of the matrix are of paramount importance in determining the performance of the immobilized enzyme system.
Ideal support properties include-
physical resistance to compression, hydrophilicity,
inertness toward enzymes,
ease of derivatization,
biocompatibility,
resistance to microbial attack, and
availability at low cost

19. Classification of Support
According to their chemical composition, supports can be classified as-
inorganic and
organic.
The organic supports can be subdivided into-
natural and
synthetic polymers

20. Classification of Support
Organic
Natural polymers:
• Polysaccharides: cellulose, dextrans, agar, agarose, chitin, alginate
• Proteins: collagen, albumin
• Carbon
Synthetic polymers:
• Polystyrene
• Other polymers: polyacrylate polymethacrylates, polyacrylamide, polyamides, vinyl, and allyl-polymers

21. Continued… Inorganic Natural minerals: bentonite, silica
Processed materials: glass (nonporous and controlled pore), metals, controlled pore metal oxides.

22. Immobilization criteria
There are a number of requirements to achieve a successful immobilization:
The biological component must retain substantial biological activity after attachment
It must have a long-term stability
The sensitivity of the enzyme must be preserved after attachment
Overloading can block or inactivate the active site of the immobilized biomaterial, therefore it must be avoided

23. Methods of enzyme immobilization
Irreversible enzyme Immobilization
Covalent bond formation
Entrapment
Reversible enzyme Immobilization
Adsorption
Non-specific adsorption
Ionic binding
Hydrophobic adsorption
Affinity binding
Chelation or metal binding
Formation of disulfide bond

24. Methods of enzyme immobilization
Alternatively it can be classified as-
Immobilization ‘inside’ a support
Immobilization ‘on’ a support
a. Entrapment
b. Microencapsulation
2. Immobilization ‘on’ a support
a. Adsorption
b. Covalent bonding
c. Cross linking

25. Advantages of immobilized enzyme
Reuse
Continuous use
Less labour intensive
Saving in capital cost
Minimum reaction time
Less chance of contamination of products
More stability
Improved process control and
High enzyme : substrate ratio.

26. covalent attachment/cross-linking
1. Adsorption
Schematics of the three most common enzyme immobilization techniques: (A) physical adsorption, (B) entrapment and (C)
covalent attachment/cross-linking

27. 1. Adsorption
Enzyme is immbilized by bonding to either the external or internal surface of a career or support such as
Mineral support (Aluminium oxide, Clay)
Organic support ( Starch)
Modified sapharose and ion exchange resins.
Bonds with low energy is involved (ionic, Hydrogen, van der waals forces)
For external immobilization the carrier particle must be very small (500 Å- 1mm)

28. 1. Adsorption Processes of immobilization using adsorption-
Static process: Enzyme is immobilized on the carrier simply by allowing the solution containing the enzyme to contact the carrier without stirring
The dynamic batch process: Carrier is placed into the enzyme solution and mixed by stirring or agitation
The reactor loading process: Carrier is placed into the reactor that will be subsequently employed for processing. The enzyme solution is transferred to the reactor and adsorption occurs in a dynamic environment.
The electrodeposition process: Carrier is placed proximal to one of the electrodes in enzyme bath, the current put on, enzyme migrates to the carrier and deposited on the surface.

29. 1. Adsorption Advantages:
Easy and cheap
Cause little or no conformational change in the enzyme or destruction of its active centre
Disadvantage: The adsorbed enzyme may leak from the carrier during use due to weak binding force between the enzyme and the carrier
Use:
Vinegar production: Adsorption of Acetobacter sp. onto the Wood chips
Manufacture of malted beverage

30. Covalent bonding
Formed between the chemical groups of the enzyme and the chemical groups of the support
Can be used under varieties of conditions like under a broad range of pH, ionic strength and other variable conditions .
Involves 2 steps- attachment of coupling agent and activation of groups
Covalent attachment may be directed to a specific group (amine, hydroxyl, tyrosil etc). Sulfohydril group is least involve.

31. Methods of covalent bonding
Diazonization (Support- amino group and tyrosil/ histidyl group of the enzyme
Formation of peptide bond
Group activation (Cyanogen bromide to a support containing glycol group i.e. cellulose)
Polyfunctional reagents (Glutaraldehyde forms bond between amino group of the support and amino group of the enzyme)

32. A problem to consider in Covalent bonding
Enzyme may undergo conformtional change when undergoes reactions at the active site
Problems can be overcome through immobilization in the presence of substrate or competitive inhibitors
Common activated polymers are Cellulose or polyacrylamides

33. Entrapment
Enzymes can be physically entrapped inside a matrix of a water soluble polymers (Cellulose, triacetate, agar, gelatin, carrageenan, alginate etc).
Pore size should be adjusted to prevent the loss of enzyme (agar and carrageenan have larger pore size (<10 µ)

34. Methods of enzyme entrapment
Inclusion on gels (enzyme entrapped in gels)
Inclusion fibres (Enzyme entrapped in fibre format)
Inclusion in microcapsules (Enzyme entrapped in microcapsules formed monomer mixtures such as polyamine chloride)

35. Cross linking or Copolymerization
Covalent bonding between groups via polyfunctional reagents such as glutaraldehyde.
Disadvantage: They can denature enzyme,
Advantage: Cheap and simple but not much used with pure proteins with high intrinsic activity.

36. Encapsulation