Enzymes
Definition, properties, classification, applications & Isolation
Session Objectives
At the end of the session, student will be able to
Ø Describe the properties of enzymes
Ø Classify enzymes
Ø Discuss the various applications of enzymes
Ø Explain the different steps involved in isolation of enzymes
Enzymes
Ø Enzymes – soluble, colloidal organic catalysts – produced by living cells but are capable of acting independently of the cells
Ø Accelerates and catalyses thousands of biochemical reactions in the living cells
Ø Functional unit of cell metabolism
Ø Proteinaceous in nature
Properties of Enzymes
• Proteinaceous and High molecular weight – 12,000 – 1 million
• Colloidal in nature
• Soluble – water, buffer and dilute alcohol
• Insoluble – Conc. Alcohol, acetone and other organic solvent
• Highly selective and specific – action
• Required in small quantities relative to the substrate concentration
• 35˚ C – 40˚ C
• Temp above 65˚ C or 0˚ C – loss of catalytic activity – due to the disruption of polypeptide chains
• Hence stored – low temp – moisture free container
• Enzymes – require – a specific, heat stable, low molecular weight organic molecule – Coenzyme
• Some enzymes – coenzyme and one more metal ions – activity
• Does not change the final equilibrium position of reactions – only the rate of attainment of equilibrium – increased
Non-protein part – coenzyme or metal (Prosthetic group) + Protein part (Apoenzyme) = Enzymes
Structure of enzyme
• Co enzymes – Coenzyme A, FAD, NAD
• Co factors – Glucose – 6-phosphate – Mg, Arginase – Mn
• Protein – Linear chain of amino acid residues joined – peptide bond
• Catalytic activity depends – L-amino acid sequence and peptide bond constituting the protein molecule
• Localized folding of primary structure – secondary structure
• Overall folding – molecule – tertiary structure
• Agglomeration of several folded chains – Quaternary structure
Classification of Enzymes
IUB classification:
1. Oxido reductases – catalyzing oxidation and reduction between two substrates
S reduced + S’ oxidized = S oxidized + S’ reduced
Eg. Alcohol dehydrogenase, lactate dehydrogenase
2. Transferase – catalyzing – transfer of S, C, N, P other than H
S – G + S’ = S’- G +S
Eg. Hexokinase, acetyl transferase
3. Hydrolases – catalyzing – hydrolysis of ester, ether, peptides, glycosyl, C-C, C-halide bonds
Acylcholine + H2O = Choline + acid
Eg. Urease
4. Lyases – Catalyses removal of groups from substrates – other than hydrolysis leaving double bond – acts on C-C, C-O, C-N, C-S bond
Eg. Aldolase, fumerase
5. Isomerase – catalyses – interconversion – optical, geometric or positional isomers —- Eg. Alanine isomerase
6. Ligases – an enzyme that can catalyze the joining of two large molecules by forming a new chemical bond, usually with accompanying hydrolysis of chemical group on one of the larger molecules. Eg. Pyruvate carboxylase, glutamine synthetase
Other methods of classification
1. Extracellular enzymes or exoenzymes – secreted outside the cell
Eg. Cellulose, polyglucturonase
2. Intracellular enzymes or endoenzymes – secreted within the cell
Eg. Invertase, asparaginase
Applications of Enzyme:
Medicinal applications
Digestive disorders-
Papain (Papaya)
Pancreatin (animal pancreas)
ß-Galactosidase (A. Oryzae)
Penicillinase (B. subtilis)
Deworming agents
Papain (Papaya)
Ficin (fig)
Anticancer agents
Asparginase (E coli, guinea pig)
Inflammation
Bromelain (Pineapple)
Anti-coagulant agents
Streptokinase (β-hemolytic streptococci)
Urokinase (Human urine)
Surface disinfectants
Trypsin (animal pancreas)
Diagnostic agents
Glucose isomerase (diabetes)
SGOT, SGPT, ALP (Liver disorders)
Upper Respiratory disorders
Chymotrypsin (Bovine pancreas)
Food industrial applications
Tenderization of meat
Papain (Papaya)
Bromelain (Pineapple)
Ice cream industry
Lactase (prevention of lactose crystals)
Beverage industry
Invertase (yeast)
Chocolate industry
Invertase (yeast)
Juice & wine processing
Pectinase
Industrial applications
Textile industry (Destarching)
Amylase (Bacteria, fungi)
Leather industry (Bating)
Proteolytic enzymes (Bacteria, and fungi)
Detergents (Destaining)
Alcalase
Paper industry (Bleaching of pulp)
Xylenase (Bacteria)
Cellulase (Bacteria)
Organic compounds
Acetone
Enzymes – Production
Source of Enzymes: Living cells
1. Plant:
Papain, Bromelain
2. Animal: Urokinase
3. Microorganism:
Advantages of microorganisms over other sources of enzymes
Ø Growth is very fast – Grown on medium containing cheap raw materials
Ø Genetic engineering and manipulations of microbial cells – possible – increase the yield of enzyme
Ø Large quantities of enzymes are produced
Ø Animal sacrifice can be minimized
Selection of microorganisms:
Ø Non-pathogenic
Ø Should produce extracellular enzymes
Ø Fermentation time should be less
Ø Must grow on medium with cheap raw material
Ø Must give a high yield of enzymes
Ø Must be compatible with physical and chemical properties of the medium
Ø Strain must not produce byproducts – inhibit the growth of m/o Aspergillus and Bacillus – industrial production
Isolation of enzymes
1. Extraction
Cell disintegration/ Cell disruption
Removal of lipids
2. Preparation of crude enzyme
Removal of nucleic acids
Centrifugation
Addition of acid or base
3. Precipitation
Salting out method
Addition of organic solvents
Addition of non-ionic polymers
Dialysis
4. Purification
Chromatographic techniques
Electrophoresis
1. Extraction
Ø Liberation of enzymes from the cells or cellular constituents
Ø Break the cell wall or membrane – physical, chemical, mechanical
Ø Extraction medium: Buffer solution, Temperature, and pH : Optimum
Modification of extraction medium: To achieve enzyme with maximum activity buffers can be modified by adding
EDTA (To remove heavy metals)
Mercapto ethanol (to prevent the breakage of disulfide bond of enzymes containing cysteine amino acid)
Triton-X (cell disruption)
Disintegration of animal and plant tissues
Plant tissues: By using a hammer mill/chopper mill
Animal tissues: Organs or muscles
Defatted
â
Minced – vertical cutter mixer
â
Frozen
Colloidal mill
â
Cell disintegration
Summary
Ø Enzymes are biocatalysts, high mol.wt, Proteinaceous and water-soluble compounds
Ø Activity is affected by temp, pH, and heavy metals, specific in their action
Ø Classification according to IUMAB and site of action
Ø Enzymes have medicinal, food, and industrial applications
Ø Main source is plant, animal, and microorganisms
Ø Isolation involves extraction, preparation of crude enzyme, precipitation, and purification
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