203. ENZYMETECHNOLOGY
UNIT I
1. Enzyme: Historical aspects, classification and nomenclature, EC number
2. Mechanism of enzyme action and properties of enzymes as catalysts
3. Sub‐cellular localization and organization of enzymes
4. Methods of enzyme assay: continuous and sampling techniques, coupled
enzyme assyas and expression of velocity of enzyme catalysed
reactions, activity, specific activity, turn over number and catalytic centre
activity
UNIT II
1. Enzyme purification techniques: Objectives and strategy, methods of
homogenization, methods of isolation , purification and crystallization
2. Criteria of purity and tabulation of purification data, stable storage of enzymes
3. Characterization of purified enzyme.
4. Coenzymes and cofactors.
.
UNIT III
1. Enzyme kinetics: Equilibrium and steady state theory, rate equation and
determination of Km and Vmax
2. Factors affecting rate of enzyme reaction: pH, temperatue and pressure
3. Enzyme inhibition : reversible and irreversible inhibition, their types, inhibitor
constant and its significance
4. Rapid reaction techniques
UNIT IV
1. Isoenzymes
2. Protein ‐ ligand binding: types, cooperativity, Hill and Scatchard plot.
Allosteric enzymes: Models of allostery, types and kinetics
3. Regulation of enzymes
4. Mechanism of action of dehydrogenases, kinases, metalloenzymes
UNIT V
1. Immobilization : Principle, kinetics of immobilised systems
2. Enzyme imobilisation ; Principle, method, activity, immobilized multienzyme
systems and applications
3. Effect of immobilization on : activity, kinetic parameters and stability
4. Applications of immobilized enzymes
Practicals:
1. Urease estimation by tritrimetric method
2. Urease estimation by colorimetric method
3. Acid phosphatase estimation
4. Alkaline phosphatase estimation
5. Determination of optimum time, optimum temperature & optimum pH
6. Determination of Km value
7. Acetylenline esterase/pseudocholinesterase estimation
8. Enzyme purification
Books
1. The nature of Enzymology by R.L. Foster
2. Enzymes by Dixon and Webb
3. Fundamentals of Enzymology by Price and Stevens
4. Enzyme Catalysis and Regulation by Hammes
5. Enzyme Reaction Mechanisms by Walsch
6. The Enzymes vol I and II by Boyer
7. Enzyme Structure and Mechanism by Alan Fersht
8. Enzyme Assays : A Practical Approach by Eisenthal and Danson
