INTRODUCTION TO IMMOBILIZED ENZYME KINETICS

The kinetics of immobilized enzyme reactions are influenced by a variety of factors:

• Mass transfer resistances
• Possible reactions between support and the enzyme.
• Micro-environmental conditions.

Effect of Mass transfer resistances on Immobilized Enzyme Kinetics: (Mass transfer- Catalytic Reaction coupling)
Mass transfer resistances play a very important role in determining the immobilized enzyme kinetics.
The Substrate has to cross the film above the solid support and then diffuse through the matrix to the enzyme molecule embedded in the matrix. So, the enzyme will have to encounter two mass transfer resistances :

1. External Mass transfer Resistance encountered while passing through the film.
2. Internal Mass transfer Resistance encountered while diffusing through the matrix.

Possible reactions between support and the enzyme
A proper choice has to be made while selecting a support for the immobilization of the enzyme. The chemical nature of the support material and any possible reactions between the support and the enzyme should be taken into account. The reaction between support and the enzyme causes chemical modifications within the enzyme. These are called conformational effects.

Micro-environmental conditions
Micro-environmental conditions like pH, temperature and the ionic environment with in the immobilized enzyme particle influence the immobilized enzyme kinetics.

Thus immobilization may alter the intrinsic properties of the enzyme. Some times entirely different rate constants and entirely different equations should be used to characterize the immobilized enzyme kinetics.

ENZYME ENGINEERING UNIT V 4 JNTU BIOTECHNOLOGY

I presented the material for the Unit V under the following headings.

• Enzyme Immobilization; Advantages and Disadvantages
• Methods of Enzyme Immobilization
• Physical Methods of Enzyme Immobilization
• Chemical methods of Enzyme Immobilization
• Cross Linking for Enzyme Immobilization
• Criteria for selecting support for Enzyme Immobilization
• Comparison of Enzyme Immobilization Techniques

You can follow the above links to access the material

COMPARISION of ENZYME IMMOBILIZATION TECHNIQUES

You can refer to the table 4.11 in Bailey text book. But here i have a modified form of it that will be more easy fro you to memorize and even with a mention of IMTR and EMTR.

CRITERIA for SELECTING SUPPORT for IMMOBILIZATION of ENZYMES

The criteria that should be taken into account for selecting a support for enzyme immobilization are:

• Surface properties of the support.
• Interactions between support and enzyme.
• Interactions between support and reaction mixture.
• Physical and Mechanical properties of the support.

Surface properties of the support
A variety of support materials are used for immobilization of enzymes.

Activated charcoal, silica, alumina, clay, glass etc., are used as support materials for adsorption.
Cellulose, carboxymethyl cellulose, dextrose, agarose are some of the important supports used in covalent immobilization.
The choice of the support depends on the surface properties of the support material.
The surface should have the capacity to adsorb the enzyme or have functional groups essential for attachment of enzymes.
If the support doesn't have the functional groups essential for attachment of enzymes, they should be modified/activated.
In many covalent immobilization processes the first step is the surface modification or activation. Two commonly used for surface modification or activation are silanisation and attachment of flexible spacer arm moeties .

Interactions between support and the enzyme
The support binds the enzyme through certain functional groups.
If the support binds to the residues with in the active site and neat the active site then the enzyme activity will be reduced.
Attachment of support near active site of the enzyme renders it unavailable for the catalytic process.

Interactions between support and reaction mixture
The support will interact with the reaction mixture also.
For example:
A charged support causes the concentration of the opposite charged ions near its surface. This affects the pH of the local environment around the enzyme.
Similarly the hydrophobicity or hydrophilicity of the support will influence the local concentrations of the solutes and solvents according to their hydrophobicity or hydrophilicity.
Thus the interaction between the support and the reaction mixture creates a local environment around the enzyme that differs from the bulk solution.

Physical and mechanical properties of the support
The physical properties of the support that are significant are:

• Porosity
• Mechanical strength
• Compressibility
• Swelling properties.

Porosity of the material determines the amount of enzyme that can be immobilized and the amount of substrate that would be available to the enzymes immobilized on the internal surfaces of the support.
High mechanical strength is requires if the immobilized catalysis are to be used in agitated reactors. High compressibility is undesirable if the enzymes are to be used for large scale packed column applications.

CROSS LINKING for ENZYME IMMOBILIZATION

Enzymes can be immobilized through cross linking using various bi- or multi- functional reagents.
The most widely used methods uses Glutaraldehyde to introduce cross links between the amino groups of the enzyme molecules.
Glutaraldehyde is a bifunctional reagent and hence cross linked and enzyme molecules. Multifunctional reagents can cross link many enzyme molecules togather.
Other reagents used for cross-linking include:

• Bisbiazo-benzidine.
• Cyanuric chloride.
• Hexa methyl-Di-isocyanate

Particles of cross-linked enzyme alone are gelatinous and lack mechanical strength. So they are first adsorbed onto a support/ carrier and then we cross link the enzymes.

Example:
Cross linking of Lipase
Sodium alginate solution is prepared by heating with a Bunsen burner and poured into a petriplate.
Later enzyme solution of lipase is poured onto the solidified algiante so that the enzyme gets adsorbed onto the solid.
Then glutaraldehyde solution is spread on the support containing adsorbed enzyme for cross-linking.

Advantages:

• The enzyme is strongly bound to the solid surface.
• The enzyme activity is high relative to the adsorption process.
• Substrate specificity of the enzyme can be changed.

Disadvantages:

• Preperation is difficult.
• Regeneration of enzyme is incompressible.
• General applicability is low.

CHEMICAL METHODS OF IMMOBILIZATION

Covalent bonding and ionic bonding are used to bind the enzyme to the support and thus immobilize the enzyme.

Covalent Bonding
The enzyme is made to covalently bind to the support. Surface modification (of the support) is usually the first step in enzyme immobilization by covalent bonding.

Cellulose, Carboxy methyl cellulose, Dextrose, Agarose are some of the important supports in covalent attachment. The choice of the support depends on the surface properties.
The support should have functional groups that facilitate the attachment/covalent bonding of the enzyme to the support.
If the support doesn't have the functional groups essential for attachment of enzymes, they should be modified/activated.
In many covalent immobilization processes the first step is surface modification or activation. Two common methods of surface modification or surface activation are :

• Silanisation
• Attachment of flexible spacer arm moeties.

Silanisation
An organofunctional silane group is coated onto the support. such coatings are then derivatized to aldehyde groups using glutaraldehyde to which enzymes are attached.

Attachment of flexible spacer arm moeties
Flexible spacer arm moeties such as n-propyl amine are attached to the support. They offer more flexibility and the enzyme attached to them have their structure better protected.

Advantages

• Enzyme activity is high.
• Substrate specifity of the enzyme is changeable.
• The strong covalent attachments hold the enzyme tightly and thus contribute to the stability by preventing the deactivation of the enzyme.

Disadvantages

• Covalent interaction between substrate and enzyme disturb the native enzyme structure.
• Regeneration of enzyme is impossible.
• Preperation is difficult.
• Cost is high.

Ionic Bonding
The enzyme is made to bind to the support through ionic bonding.

DEAE sephadex, DEAE cellulose, DOllen-50, Carboxymethyl Cellulose and amberlite are the supports usually used.

Advantages

• Low cost.
• Regeneration is possible.
• Preperation is easy.
• Overall enzyme activity is high.

Disadvantages

• Not fit for industrial use.
• Ionic interactions between substrate and enzyme disturbs the enzyme activity.
• Effectiveness gradually decreases.
• Substrate specificity is unchangeable.

ENZYME ENGINEERING Question Papers (2007,Supple)

SET :1

1. State the importance of enzymes in medical diagnosis.
2. How enzymes are preliminarily purified from a crude enzyme extract?
3. Give an account of various factors that affect enzyme catalyzed reaction.
4. Derive the general rate equation of Alberty for multi-substrate enzyme catalyzed reaction.
5. Explain the kinetics of competitive inhibition. Add a note on its importance.
6. Write the merits and demerits of enzyme immobilization techniques.
7. Give an account of different types of immobilized enzyme reactors.
8. What are the different types of transducer used in biosensors?

SET :2

1. State the importance of coenzymes and cofactors in enzyme catalysis.
2. Describe the procedures of enzyme isolation from natural sources.
3. Distinguish between the lock & key and Induced Fit models for binding of a substrate to an enzyme.
4. Discuss about the Eadie-Hofstee and Hanes plot and state their edge over the LB plot.
5. How Koshland-Nemethy-Filmer (KNF) model account for allosteric regulation.
6. Discuss the important criteria and prerequisite for selecting support for enzyme immobilization. [16]
7. Enumerate the kinetics of immobilized enzymes.
8. Enumerate the application of enzymes in analysis.

SET :3

1. What are enzymes? Justify its role as biocatalysts.
2. Discuss the methods employed for isolating enzymes soluble in cytoplasm and subcellular organelles from animal, Plant and microbial sources.
3. Distinguish between the lock & key and Induced Fit models for binding of a substrate to an enzyme.
4. Discuss the kinetics for reversible reactions.
5. How Koshland-Nemethy-Filmer (KNF) model account for allosteric regulation.
6. Give an account of analytical application of immobilized enzymes.
7. Formulate and calculate dimensionless groups and effectiveness factor.
8. Enumerate the application of enzymes in analysis.

SET :4

1. Discuss enzyme classification in terms of four-digit classification number?
2. Describe the procedures of enzyme isolation from natural sources.
3. Explain the energetics of enzyme-substrate complex formation.
4. Derive Michaelis-Menten equation. State the importance of MM constant.
5. Explain various types of enzyme inhibition with relevant examples.
6. Discuss the important criteria and prerequisite for selecting support for enzyme immobilization.
7. Give an account of different types of immobilized enzyme reactors.
8. Enumerate the application of enzymes in analysis.

ENZYME ENGINEERING Question Papers (2006,Supple)

SET : 1

1. Explain the enzymw classification with examples for each class.

Explain the terms apoenzyme, holoenzyme, coenzyme and prosthetic group.

2. Explain the different methods available for enzyme purification.

How do you characterize an enzyme?

3. Discuss the application of enzymes in food, pharma and medical fields.

How are enzymatic assays developed? Discuss with an example.

4. What are the different types of inhibition.

Discuss briefly the kinetics of single substrate reactions and multiple substrate reactions.

5. Explain the following :

a) Allosteric regulation

b) Deactivation enzymes

c) Energitics of enzyme substrate complex formation.

d) Factors affecting enzyme activity

6. What are the different techniques used for enzyme immobilization? Comment on the advantages and disadvantages of each of these techniques.

7. Write short notes on :

a) Applications of immobilized enzymes

b) Uses of enzymes in different areas.

8. Comment on any two of the following :

a) Mass transfer effects in immobilized enzymes.

b) Biosensors

c) Applications of enzyme electrodes.

SET : 2

1. Discuss the six main classes of enzymes.

Comment on the applications of enzymes in pharmaceutical insustries.

2. Define the terms :

a) Turnover number

b) Michaelis menten constant.

Discuss the different types of enzyme inhibition.

3. What are the different immobilization techniques that can be used for immobilization?

Comment on the different applications of immobilized enzymes.

4. Write notes on :

a) Fluidized – bed reactors

b) Packed bed reactors.

5. What are biosensors? Discuss their applications in different areas.

6. Derive the michaelis menten equation.

Discuss allosteric enzymes and the kinetics of allosteric enzymes.

7. Write a note on the development of enzyme assays.

Discuss the different methods involved in purification of enzymes.

8. Write notes on :

a) Mechanism of enzyme action.

b) Applications of enzymes in analysis.

SET :3

1. What are enzymes? How are they classified? Explain.

How are enzymes extracted from various sources? What precautions are required to be taken while isolating the enzymes?

2. Explain what you understand by the following :

a) Turnover number

b) Michelis menten constant

c) Prosthetic group

d) Allosteric enzymes

3. Discuss the uses of enzymes in different areas.

What do you understand by the terms competitive and noncompetitive inhibition? Explain.

4. Write short notes :

a) End product inhibition

b) Significance of end product inhibition in biological systems.

c) Applications of immobilized enzymes.

d) Activation energy.

5. Derive the michaelis menten equation.

How do the kinetics differ in the case of reactions catalyzed by allosteric enzymes.

6. Discuss the various physical and chemical techniques used for enzyme immobilization.

7. Write notes on :

a) Kinetics of immobilized enzyme reactors.

b) Immobilized enzyme reactors.

8. Discuss the principle behind design of enzyme electrodes and their applications as biosensors in different areas.

SET :4

1. Comment on the applications of enzymes in different areas.

How can you obtain crude enzyme extracts from different sources? Discuss with examples?

2. Derive the Michaelis – Mente equation.

Discuss different types of enzyme inhibition with kinetic models.

3. Explain the following :

a) Turnover number

b) Deactivation of enzymes.

c) Allosteric enzymes.

d) Allosteric regulation.

4. Discuss the physical techniques available for enzyme immobilization.

Comment on the advantages and disadvantages of enzyme immobilization.

5. Discuss the design of immobilized enzyme reactors.

Discuss the mass transfer in immobilized enzyme reactors.

6. Discuss the methods involved in enzyme purification.

7. Write short notes :

a) Development of enzyme assays.

b) Characterization of enzymes

8. Design of enzyme biosensors.

Application of biosensors in different areas.