Enzymes
Definition,
properties, classification and applications
Content
Ø Definition
Ø Properties of enzymes
Ø Classification
Ø Application of enzymes
Ø Isolation of enzymes
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
Enzymes – Properties
•
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 – co enzyme 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
Enzymes – Classification
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 method 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:
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 strepto cocci)
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)
Chacolate 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 cell – possible – increase the yield of enzyme
Ø Large quantity of enzymes are produced
Ø Animal sacrifice can be minimized
Selection of microorganism:
Ø Non-pathogenic
Ø Should produce extra cellular enzymes
Ø Fermentation time should be less
Ø Must grow on medium with cheap raw material
Ø Must give high yield of enzymes
Ø Must be compatible with physical and chemical
properties of 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 enzyme 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 disulphide bond of enzymes containing
cystein amino acid)
Triton-X
(cell disruption)
Disintegration of animal and plant tissues
Plant
tissues: By using hammer mill/chopper mill
Animal
tissues: Organs or in 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 micro
organisms
Ø Isolation involves extraction, preparation
of crude enzyme, precipitation and purification