203 : ENZYME TECHNOLOGY
UNIT I
1. Enzyme : Historical aspects, classification and nomenclature, EC number.
2. Mechanism of enzyme catalysis and action
3. Sub cellular localization and rganization of enzymes
4. Methods of enzyme assay: continuous and sampling techniques, coupled enzyme assay
and methods and significance of enzyme turnover number; specific activity
UNIT II
1. Enzyme purification techniques : objectives and strategy; methods of
homogenization; method 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, Cofactors and Isoenzymes
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, temperature and pressure
3. Enzyme inhibition : reversible and irreversible inhibition, their type,
inhibitor constant and its significance
4. Rapid reaction techniques
UNIT IV
1. Protein- ligand binding : types, cooperativity, Hill and Scatchard plot, Allosteric
enzymes : Models of allostery, types and kinetics
2. Regulation of enzymes
3. Mechanism of action of Chymotrypsin; Ribonuclease; Lysozyme; Metallo-enzymes
4. Degradation of enzymes
UNIT V
1. Enzyme immobilization; techniques; experimental procedures and effect of
immobilization on kinetic parameters
2. Principle and Industrial application of immobilized systems
3. Enzymes in Medical diagnosis and enzyme therapy
4. Enzymes during aging
Practical Exercises :
1. Protein estimation methods: Lowry, Bradford and Spectrophotometeric.
2. Urease estimation in plant tissues
3. Assay of Acid phosphatase in plant seeds
4. Assay of Alkaline phosphatase in Kidney and Liver
5. Determination of optimum pH, temperature & time
6. Determination of Km value of alkaline phosphatase
7. Acetylcholinesterase estimation in Rat /Goat Brain
8. Enzyme purification: Ammonium sulphate precipitation, Ion exchange
chromatography , molecular sieve chromatography.
9. Checking of purity of enzyme by PAGE
10.Molecular weight determination of enzyme by Gel Filtration
11. Immobilization of HRP (Horse reddish peroxidase).
12. Kinetic properties of Immobilised HRP
13.Sub-cellular fractionation of rat liver and marker enzyme assays.
Reference 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
9 Enzyme Biotechnology by G. Tripathi
10 Practical Biochemistry by Plummer.
11 Practical Biochemistry by Sawhney and R. Singh
Note: All text books are of latest editions.
