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Enzymes - Definition, properties, classification and applications

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Content

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  • Definition
  • Properties of enzymes
  • Classification
  • Application of enzymes
  • Isolation of enzymes

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At the end of the session, student will be able to

  • Describe the properties of enzymes
  • Classify enzymes
  • Discuss the various applications of enzymes
  • Explain the different steps involved in isolation of enzymes

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Session Objectives

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Enzymes

  • Enzymes – soluble, colloidal organic catalysts - produced by living cells but are capable of acting independently of the cells
  • Accelerates and catalyses thousands of biochemical reactions in the

living cells

  • Functional unit of cell metabolism
  • Proteinaceous in nature

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Enzymes - Properties

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Enzymes

  • 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

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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

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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

Enzymes – Classification

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Enzymes – Classification �

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

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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

Enzymes contd.

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Applications:

Medicinal applications

Digestive disorders-

Papain (Papaya)

Pancreatin (animal

pancreas)

Deworming agents

Papain (Papaya)

Ficin (fig)

Anticancer agents

Asparginase

(E coli, guinea pig)

Inflammation

Bromelain (Pineapple)

Enzymes contd.

ß-Galactosidase (A. Oryzae)

Penicillinase (B. subtilis)

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Medicinal applications

Anti coagulant agents

Streptokinase (β-hemolytic

strepto cocci)

Urokinase ( Human urine)

Surface disinfectants

Trypsin (animal pancreas)

Diagnostic agents

Glucose isomerase (diabetes)

Upper Respiratory disorders

Chymotrypsin (Bovine

pancreas)

Enzymes contd.

SGOT, SGPT, ALP (Liver disorders)

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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

Enzymes contd.

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Industrial

applications

Textile industry

Amylase (Bacteria, fungi)

(Destarching)

Leather industry

Proteolytic enzymes(Bacteria,

and fungi)

(Bating)

Detergents

Alcalase

(Destaining)

Paper industry

Xylenase (Bacteria)

Cellulase (Bacteria)

(Bleaching of

pulp)

Organic compounds

Acetone

Enzymes contd.

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Source of Enzymes: Living cells

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1. Plant : Papain, Bromelain

2. Animal: Urokinase

3. Micro organism:

Advantages of micro organisms over other sources of enzymes

  • Growth is very fast - Grown on medium containing cheap raw materials
  • Genetic engineering and manipulations of microbial cell – possible – increase the yield of enzyme
  • Large quantity of enzymes are produced
  • Animal sacrifice can be minimized

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Enzymes - Production

Mitochondria, microsome, lysosome, ribosome

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Selection of microorganism:

  • Non-pathogenic
  • Should produce extra cellular enzymes
  • Fermentation time should be less
  • Must grow on medium with cheap raw material
  • Must give high yield of enzymes
  • Must be compatible with physical and chemical properties of medium
  • Strain must not produce byproducts – inhibit the growth of m/o

Aspergillus and Bacillus – industrial production

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Enzymes contd.

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Isolation of enzymes

1.Extraction Cell disintegration/ Cell disruption

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Removal of lipids

2.Preparation of crude enzyme Removal of nucleic acids

Centrifugation

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Addition of acid or base

3.Precipitation Salting out method

Addition of organic solvents

Addition of non-ionic polymers

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Dialysis

4.Purification Chromatographic techniques

Electrophoresis

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1.Extraction

  • Liberation of enzyme from the cells or cellular constituents
  • Break the cell wall or membrane – physical, chemical, mechanical
  • Extraction medium : Buffer solution, Temperature and pH : Optimum

Modification of extraction medium: To achieve enzyme with maximum activity buffers can be modified by adding

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EDTA (To remove heavy metals)

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Mercapto ethanol ( to prevent the breakage of

disulphide bond of enzymes containing cystein amino acid)

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Triton-X ( cell disruption)

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Isolation of enzymes contd.

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Plant tissues : By using hammer mill/chopper mill

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Animal tissues: Organs or in muscles

Defatted

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Minced - vertical cutter mixer

Frozen

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Colloidal mill

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Cell disintegration

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Disintegration of animal and plant tissues

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Summary

  • Enzymes are biocatalysts, high mol.wt, Proteinaceous and water soluble compounds
  • Activity is affected by temp, pH and heavy metals, specific in their

action

  • Classification according to IUMAB and site of action
  • Enzymes have medicinal, food and industrial applications
  • Main source is plant, animal and micro organisms
  • Isolation involves extraction, preparation of crude enzyme,

precipitation and purification