Enzymes

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


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