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Immobilization of Enzymes
Introduction
• Immobilization of enzymes can be defined as the confinement of an enzyme (bio-catalyst) in a distinct phase, separated from the bulk phase but allowing it to exchange with the latter.
• Bulk Phase consists of a substrate, an effecter or inhibitor.
• Immobilized enzyme is either physically entrapped or covalently bonded by chemical means to an inert insoluble matrix or carrier.
• In other words, it involves the restrictive localization of enzymes.
• Matrix is generally a high molecular weight polymer.
Ex : cellulose, polyacrlamide, alginate, etc. S
Introduction
S
EnzymeIn
Solution Form
Substrate/Bulk
Phase/Inhibitor
Introduction
SSubstrate/Bulk
Phase/Inhibitor
EnzymeIn
Solution Form
Introduction
S
EnzymeIn
Solution Form
EnzymeIn
Encapsulated Form
Substrate/Bulk
Phase/Inhibitor
Introduction
S
EnzymeIn
Solution Form
EnzymeIn
Encapsulated Form
Substrate/Bulk
Phase/Inhibitor
Introduction
S
EnzymeIn
Solution Form
EnzymeIn
Encapsulated Form
Substrate/Bulk
Phase/Inhibitor
Need for Immobilization
S
• Accelerates the chemical reaction.
• Specificity & un-modified enzyme.
• Cost effective.
• Not difficult to separate.
• Attachment to polymers/matrix, causes re-use.
Advantages of Immobilized Enzymes
• Recovered at the end of the reaction thereby can be reused.• Economy of the reaction is improved.• Easy separation of enzyme from the products occurs.• Stability of immoblilised enzyme increases.• Enhanced enzyme properties.• Efficiency of the catalytic reaction is better in a few cases.• Better control of reaction can be achieved.• Catalytic process can be operated continuously.• Multi enzyme reaction possible.• Potential in industrial & medicinal use. S
Methods of Immobilization
• Parameters for Method Selection :- Overall catalytic activity. Effectiveness of the catalytic utilization. Deactivation & Regeneration characteristics. Cost effective. Intended application of immobilized enzyme. Toxicity of immobilized enzyme. Waste disposal (of immobilization process).
S
Methods of Immobilization
• Physical Methods
AdsorptionEntrappingMembrane confinement
• Chemical Methods
Covalent BondingCross LinkingComplexation & Chelation
S
Carrier for Immobilized Enzymes
• Ideal Characteristics of the Carrier:- Low Cost & of optimum quality Inertness Physical Strength Stability Regenerability Enhancement of enzyme specificity Reduction of product inhibition
S
• Types of Carriers :Naturally occuring
Structural proteins (Ex: ceratin, collagen)Globular proteins (Ex: albumin)Carbohydrates (Ex: dextran)
Synthetic organicEx: polyvinyls, epoxide,etc
InorganicEx: glass, silica gel, bentonite, titania,etc.
S
Carrier for Immobilized Enzymes
A• Non-specific binding like electrostatic or
hydrophobic affinity binding to special ligand.• Mostly explained in following terms:
Static pores Dynamic pores Reactor Loading Electro Deposition
• NOTE: Adsorbent (mostly polymeric matrix)Ex: alumina, bentonite, CMC, Silica gel,
Titania, etc.
DSORPTION
A
ADSORPTION
• Advantages: Simple & Economical Limited Loss of activity Can be Recycled, Regenerated & Reused (R3)
• Disadvantages: Relatively Low surface area for binding Exposure of enzyme to microbial attack. Smaller particles cause high Pressure drop in
continuous packed bed reactor. Yield are often low due to inactivation &
desorption.
E
E
E
E
ADSORPTION
MATRIX
ENTRAPPING
• Enzymes are held or entrapped within the suitable gels or fibres.
• In a gel it may causes: Matrix polymerization or Precipitation or Coagulation
• Entrapment in calcium alginate is the most widely used for entrapment for :
Microbial Animal & Plant enzymes/cells
Ex: Glucose oxidase + Polyacrlamide (gel entrapment)
• NOTE: Adsorbent (mostly commonly used)Ex: polyacrylamides, collagen, silica gel,
alginates, etc.
ENTRAPPING
• Advantages: No chemical modification Relatively stable forms. Easy handling & reusage.
• Disadvantages: May diffusion of substrate & product occurs. Substrate accessibility may reduced due to
free radical polymerization of gel. Enzyme in-activation. Loss of enzyme content.
• NOTE: Sometimes covalent bonding may forms between the entrapped enzyme & the matrix.
ENTRAPPING
Enzyme + Sod.alginate Mixture is added dropwise
CaCl2 Solution
Beads of Calcium alginates
CONFINEMENT
Membrane• Enzyme molecules (usually in aq. form) are
confined within semi-permeable :Reaction vesselo Partitioning into two chambers by a semi
permeable membraneo One chamber contains the enzyme while
the other have substrate & product.Hollow fiber membraneo Entrapment in semi permeable fibres
(cellulose, triacetate) or spheres (nylon, collodion).
o In which, the enzyme will be in the lumen/hollow space, while the fibres/spheres will be submerged in the substrate.
CONFINEMENT
MembraneMicro capsuleso Enzymes are packed in microcapsules
formed by polymerization (like phase separation or chemical polymerization).
Liposomeso Enzymes can be bounded in a concentric
spheres of lipoidal membrane formed by addition of phospholipid.
• Advantages: No enzyme leakage No change in enzyme activity
• Disadvantages: Diffusional barrier to the substrate &
product. Not cost effective.
CONFINEMENT
Membrane
E
EE
E E
E E
CONFINEMENT
Membrane
E
EE
E E
E E
BO
N
D
I
N
G
Chemical• Enzyme forms co-valent link with active group of
the matrix (like terminal -NH2, -COOH,etc,).
• Support with groups like :
-OH : support activation covalently by CNBr.
-COOH : supports (like CMC) activation covalently by azide derivatives.
-NH2 : support activation covalently by forming diazonium chlorides on treatment with NaNO2 + HCl.
NOTE: The functional group of enzyme which is involved in the linkage, should not affect the active properties of the said enzyme.
BO
N
D
I
N
G
Chemical• Advantages:
Not affected by pH Ionic Strength
• Disadvantages: Active site may be modified Not cost effective.
NOTE: Adsorbent
Ex: Agarose, Cellulose, sepharose, Polyacrlamide,etc.
BO
N
D
I
N
G
Chemical
E
E
E
E
E
E
LI
N
K
I
N
G
Cross• It involves cross linking of enzyme to a multi
functional reagent without use of any solid
support.
• Alternatively, chemical bridge of some other
molecule between & with the chemical support
(i.e., reaction of enzyme with reagent bridge or
chemical bridge).
• Activated carriers are used.Ex : Sepharose by CNBr (most commonly
used)Ex : Sepharose by ethyl chloroformate
LI
N
K
I
N
G
Cross• Advantages:• Strong linkage leads to low enzyme leakage
while use.• Higher stability (i.e., pH, ionic & substrate
concentration.
• Disadvantages:• Partially or wholly inactivation by active site
modification.• Not cost effetcive.
LI
N
K
I
N
G
Cross
E
E
E
E
E
E
E
E
E
E
E
E
Uses of Immobilized Enzymes
• Biotransformation• Secondary metabolite production• Biosensors• Enzyme-linked immunosorbent assays (ELISAs)• Biological washing Powders• Food Industry• Seed Germination
Glucose oxidaseGlucose Hydrogen peroxide
peroxidase
Dye: Blue---Green---Brown
Dye changes according to amount of glucose
Enzyme-linked immunosorbent assays (ELISAs) detect antibodies to infections.
Enzymes in Medicine
S
• Proteases break down the coloured, insoluble proteins that
cause stains to smaller, colourless soluble polypeptides.
• Can wash at lower temperatures
Enzymes in biological washing Powders
S
• Pectinase break down substances in apple cell walls and enable greater juice extraction.
Lactase breaks down lactose in milk into glucose and galactose. This makes milk drinkable for lactose intolerant people.
Enzymes in Food Industry
S
starch
embryo plant
amylasesecreted
maltose
absorbed
Enzymes in Seed Germination
S
References
Pharmaceutical BiotechnologyBy Dr. S.P. Vyas & Dr. V.K. Dixit
Enymes & its Immobilization PresentationBy Dr. S. Khanam
Internet Resources :http://www.eplantscience.comhttp://www.clickbiology.comhttp://www.lsbu.ac.ukhttp://www.tech-ceramics.co.uk
Thank You !!!
S