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    13 Isolation of Enzyme

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    Rizki fitriana dewi
    This document discusses the process of isolating an enzyme from its source. It begins by outlining the basic steps: 1) cell disruption to break open the cells, 2) centrifugation to separate cell debris, and 3) decantation to separate and collect the enzyme. It then provides details on important considerations for isolation like the source of the enzyme (plants, animals, microbes), its location within or outside the cell, and factors that impact its stability. The document discusses various sources of enzymes and includes tables listing examples. It also outlines methods for microbial fermentation, cell disruption techniques, common buffers used, and isolation methods that are appropriate for different protein sources.

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    13 Isolation of Enzyme

    Uploaded by

    Rizki fitriana dewi
    34 views22 pages
    This document discusses the process of isolating an enzyme from its source. It begins by outlining the basic steps: 1) cell disruption to break open the cells, 2) centrifugation to separate cell debris, and 3) decantation to separate and collect the enzyme. It then provides details on important considerations for isolation like the source of the enzyme (plants, animals, microbes), its location within or outside the cell, and factors that impact its stability. The document discusses various sources of enzymes and includes tables listing examples. It also outlines methods for microbial fermentation, cell disruption techniques, common buffers used, and isolation methods that are appropriate for different protein sources.

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    This document discusses the process of isolating an enzyme from its source. It begins by outlining the basic steps: 1) cell disruption to break open the cells, 2) centrifugation to separate cell debris, and 3) decantation to separate and collect the enzyme. It then provides details on important considerations for isolation like the source of the enzyme (plants, animals, microbes), its location within or outside the cell, and factors that impact its stability. The document discusses various sources of enzymes and includes tables listing examples. It also outlines methods for microbial fermentation, cell disruption techniques, common buffers used, and isolation methods that are appropriate for different protein sources.

    Copyright:

    © All Rights Reserved
    Download as PDF, TXT or read online from Scribd
    Download as pdf or txt
    34 views22 pages

    13 Isolation of Enzyme

    Uploaded by

    Rizki fitriana dewi
    This document discusses the process of isolating an enzyme from its source. It begins by outlining the basic steps: 1) cell disruption to break open the cells, 2) centrifugation to separate cell debris, and 3) decantation to separate and collect the enzyme. It then provides details on important considerations for isolation like the source of the enzyme (plants, animals, microbes), its location within or outside the cell, and factors that impact its stability. The document discusses various sources of enzymes and includes tables listing examples. It also outlines methods for microbial fermentation, cell disruption techniques, common buffers used, and isolation methods that are appropriate for different protein sources.

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    Isolation of an Enzyme

    Deva Krisna Kadarani MSi


    Chemistry Department of FST
    UIN MAULANA MALIK IBRAHIM MALANG
    Scopes

    • Enzymes source characteristics (bacteria, yeast,


    plant, etc)
    • Buffer solution
    • Isolation of enzymes
    Basic step of isolation an enzyme

    1) Cell disruption
    2) Centrifugation
    3) Decantation
    Important things on isolation of enzyme

    • Objective
    • Lab scale : high purity, high cost production
    • Industrial scale : medium purity, low cost production
    and high yields
    • Source: plant, animal, microbes
    • Location: intracellular and extracellular
    • Stability of enzymes
    Source of enzymes

    Microorganism
    Plants (4%) Animal (8%) (>80%)
    (Papain, bromelain) (Rennet) (Yeast, Fungi and
    Bacteria)
    Plants as sources of enzymes
    Industrial
    Enzymes No (EC) Sources Extra/Intra
    use
    Actinidin 3.4.22.14 Kiwi fruit E Food

    -Amylase 3.2.1.1 Malted barley E Brewing

    -Amylase 3.2.1.2 Pancreas E Food

    Bromelain 3.4.22.4 Pineapple latex E Brewing

    -Glucanase 3.2.1.6 Malted barley E Brewing

    Ficin 3.4.22.3 Fig latex E Food

    Lipoxygenase 1.13.11.12 Soybeans I Food

    Papain 3.4.22.2 Pawpaw latex E Meat


    Animal as sources of enzymes

    Industrial
    Enzymes No (EC) Sources Extra/Intra
    use
    Catalase 1.11.1.6 Liver I Food

    Chymotrypsin 3.4.21.1 Pancreas E Leather

    Lipase 3.1.1.3 Pancreas E Food

    Rennet 3.4.23.4 Abomasum E Cheese

    Trypsin 3.4.21.4 Pancreas E Leather


    Yeast as sources of enzymes

    Enzymes No (EC) Sources Extra/Intra Industrial use

    Invertase 3.2.1.26 Saccharomyces I/E Confectionery

    Lactase 3.2.1.23 Kluyveromyces I/E Dairy

    Lipase 3.1.1.3 Candida E Food

    Raffinase 3.2.1.22 Saccharomyces I Food


    Fungi as sources of enzymes

    Enzymes No (EC) Sources Extra/Intra Industrial use

    Aminoacylase 3.5.1.14 Aspergillus I Pharmeceutical

    -Amylase 3.2.1.1 Aspergillus E Starch

    Catalase 1.11.1.6 Aspergillus I Food

    Cellulase 3.2.1.4 Trichoderma E waste

    Rennet 3.4.23.6 Mucor miehei E Cheese

    Protease 3.4.23.6 Aspergillus E Baking

    Pectinase 3.2.1.15 Aspergillus E Drinks


    Bacteria as sources of enzymes

    Enzymes No (EC) Sources Extra/Intra Industrial use

    Asparaginase 3.5.1.1 E. coli I Health

    -Amylase 3.2.1.1 Bacillus E Starch

    -Amylase 3.2.1.2 Bacillus E Starch


    Glcose
    3.5.1.5 Bacillus I Fructose syrup
    isomerase
    Penicillin
    3.5.1.11 Bacillus I Pharmeceutical
    amidase

    Protease 3.4.21.14 Bacillus E Detergent

    Pullulanase 3.2.1.41 Klebsiella E Starch


    Why microbe as best source of enzyme ?
    • Low-cost production
    • Easy and quick to extracted and purified
    • Predictable and controlable products
    • Able to used genetical manipulation
    • Less toxic
    General flowchart of Isolation and purification
    Animal Plant Microorganisms

    Grinding Fermentation
    Extracellular Intracellular
    Extraction enzyme enzyme

    Filtration Disruption

    Concentration Purification Pure Enzyme


    Type of microbial enzyme

    • Intracellular enzyme • Extracellular enzyme


    • Inside cell, precisely on membrane • Outside cell, diffused into
    cell environment
    • Unstable in outer cell environment • No cell wall degradation
    • Isolation depend on buffer and • Using centrifugation
    temperature (supernatant), filtration and
    • Endogen enzymes concentration
    • Exogen enzymes
    Production of microbial enzyme

    • Submerged Fermentation • Surface Fermentation


    • Produced enzyme by microbes on • Produced enzyme on surface of
    liquid medium solid medium
    • High yields • Medium yields
    • Aeration is essential
    Industrial Fermentors
    (submerged fermentation – liquid medium)
    Growth Curve

    discontinous Semi continous


    Disruption cell method
    • Physical • Chemical
    • Freeze thawing : N2 liquid H2O • Organic solvent : ethyl
    • Osmotic pressure acetate, toluene
    • Mechanic • Detergent : SDS, Triton X-100
    • Solid shear : plant, bacteria with sand tween
    • Liquid shear : plant and animal tissue
    by using blender
    • Enzymatic
    • High pressure disruption : animal cell • Cellulase : plant cell
    (1000 psi) and bacteria (1000-2000 • Lysozyme : bacteria
    psi) • Citinase : fungi
    • Ultrasonication
    Isolation method
    Isolation method Protein or enzyme source
    Cell lysis (osmotic shock) Intracellular protein (low yields but
    reduced protease release)
    Enzymatic digestion Intracellular protein (for lab scale using
    lysozyme, 37oC, 15 min, combined with
    mechanical disruption
    Grinding with abrasive (sand) Plant tissue, bacteria
    Ultrasonication Intracellular protein
    French press Plant tissue, bacteria
    Fractional precipitation Extracellular protein
    Buffer Advantage Disadvantage
    Phosphate Low cost Weak at pH 8-11
    Compatible into : Inhibit enzymes : dehydrogenase, kinase
    • Gel permeation and DO NOT SUITABLE in anion-exchange
    cation-exchange chromatography
    chromatography
    • Cross-linking reagents
    TRIS Low cost Poor buffer below pH 7
    Suitable for gel permeation Passes through biological membrane
    and anion exchange Sensitive to temperature
    chromatography
    Borate Low cost Form complexes with oligosaccharides,
    mono-
    Citrate Low cost Binds to some protein and metal complex
    Carbonate Low cost Limited solubility
    Effective buffer criteria
    • Water-soluble
    • Does not permeate biological membranes
    • pKa between 6 and 8
    • Minimal effect on biochemical reaction
    • Chemically stable
    • Does not absorb UV light
    • Easy to purify
    Assignment
    • Create a group (about 3-4 people) then make a presentation about
    isolation and characterization of an enzyme.
    • Please send me via google classroom assignment by 3 may 2021,
    23:59 pm
    • If you have any que about this, do not hesitate to ask me personally
    or in our group chat
    Thank You

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