1. Immobilization of Enzymes

2. 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

3. Introduction
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4. Introduction
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5. Introduction
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6. Introduction
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7. Introduction
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8. Need for Immobilization
• Accelerates the chemical reaction.
• Specificity & un-modified enzyme.
• Cost effective.
• Not difficult to separate.
• Attachment to polymers/matrix, causes re-use.
S

9. 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. S
• Potential in industrial & medicinal use.

10. 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).
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11. Methods of Immobilization
• Physical Methods • Chemical Methods
Adsorption Covalent Bonding
Entrapping Cross Linking
Membrane confinement Complexation & Chelation
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12. 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
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13. Carrier for Immobilized Enzymes
• Types of Carriers :
Naturally occuring
Structural proteins (Ex: ceratin, collagen)
Globular proteins (Ex: albumin)
Carbohydrates (Ex: dextran)
Synthetic organic
Ex: polyvinyls, epoxide,etc
Inorganic
Ex: glass, silica gel, bentonite, titania,etc. S

14. A • Non-specific binding like electrostatic or
D A
hydrophobic affinity binding to special ligand.
• Mostly explained in following terms:
S  Static pores
 Dynamic pores
O  Reactor Loading
R  Electro Deposition
P • NOTE: Adsorbent (mostly polymeric matrix)
T Ex: alumina, bentonite, CMC, Silica
gel, Titania, etc.
I
O
N

15. A
• Advantages:
D  Simple & Economical
S  Limited Loss of activity
 Can be Recycled, Regenerated & Reused (R3)
O
R • Disadvantages:
P  Relatively Low surface area for binding
 Exposure of enzyme to microbial attack.
T  Smaller particles cause high Pressure drop in
continuous packed bed reactor.
I  Yield are often low due to inactivation &
O desorption.
N

16. A
D
S
O
R
P
T
I
O
N

17. • Enzymes are held or entrapped within the
E suitable gels or fibres.
N • In a gel it may causes:
 Matrix polymerization or
T  Precipitation or
R  Coagulation
• Entrapment in calcium alginate is the most
A widely used for entrapment for :
P  Microbial
 Animal &
P  Plant enzymes/cells
I Ex: Glucose oxidase + Polyacrlamide (gel
entrapment)
N • NOTE: Adsorbent (mostly commonly used)
Ex: polyacrylamides, collagen, silica
G gel, alginates, etc.

18. E • Advantages:
N  No chemical modification
 Relatively stable forms.
T  Easy handling & reusage.
R • Disadvantages:
 May diffusion of substrate & product occurs.
A  Substrate accessibility may reduced due to
P free radical polymerization of gel.
 Enzyme in-activation.
P  Loss of enzyme content.
I
• NOTE: Sometimes covalent bonding may forms
N between the entrapped enzyme & the matrix.
G

19. E Enzyme + Sod.alginate
N Mixture is added dropwise
CaCl2 Solution
T
R Beads of Calcium alginates
A
P
P
I
N
G

20. Membrane
• Enzyme molecules (usually in aq. form) are
C confined within semi-permeable :
Reaction vessel
O o Partitioning into two chambers by a semi
N permeable membrane
F o One chamber contains the enzyme while
I the other have substrate & product.
Hollow fiber membrane
N o Entrapment in semi permeable fibres
E (cellulose, triacetate) or spheres
M (nylon, collodion).
E o In which, the enzyme will be in the
lumen/hollow space, while the
N fibres/spheres will be submerged in the
T substrate.

21. Membrane
Micro capsules
C o Enzymes are packed in microcapsules
formed by polymerization (like phase
O separation or chemical polymerization).
N Liposomes
F o Enzymes can be bounded in a concentric
I spheres of lipoidal membrane formed by
addition of phospholipid.
N • Advantages:
E  No enzyme leakage
M  No change in enzyme activity
E • Disadvantages:
N  Diffusional barrier to the substrate &
T product.
 Not cost effective.

22. Membrane
C
O
N
F
I
N
E
M
E
N
T

23. Membrane
C
O
N
F
I
N
E
M
E
N
T

24. Chemical
• Enzyme forms co-valent link with active group of
B the matrix (like terminal -NH2, -COOH,etc,).
• Support with groups like :
O
 -OH : support activation covalently by CNBr.
N
 -COOH : supports (like CMC) activation
D covalently by azide derivatives.
I  -NH2 : support activation covalently by
forming diazonium chlorides on treatment
N with NaNO2 + HCl.
NOTE: The functional group of enzyme which is
involved in the linkage, should not affect the active
G properties of the said enzyme.

25. Chemical
• Advantages:
B  Not affected by pH
O  Ionic Strength
N • Disadvantages:
 Active site may be modified
D  Not cost effective.
I NOTE: Adsorbent
Ex:
N Agarose, Cellulose, sepharose,
Polyacrlamide,etc.
G

26. Chemical
B
O
N
D
I
N
G

27. Cross
L • It involves cross linking of enzyme to a multi
functional reagent without use of any solid
I support.
N • Alternatively, chemical bridge of some other
molecule between & with the chemical support
K (i.e., reaction of enzyme with reagent bridge or
chemical bridge).
I
• Activated carriers are used.
N Ex : Sepharose by CNBr (most commonly
used)
Ex : Sepharose by ethyl chloroformate
G

28. Cross
• Advantages:
L • Strong linkage leads to low enzyme leakage
I while use.
• Higher stability (i.e., pH, ionic & substrate
concentration.
N
• Disadvantages:
K • Partially or wholly inactivation by active site
modification.
I • Not cost effetcive.
N
G

29. Cross
L
I
N
K
I
N
G

30. Uses of Immobilized Enzymes
• Biotransformation
• Secondary metabolite production
• Biosensors
• Enzyme-linked immunosorbent assays (ELISAs)
• Biological washing Powders
• Food Industry
• Seed Germination

31. Enzymes in Medicine
Glucose oxidase
Glucose Hydrogen peroxide
peroxidase
Dye: Blue---Green---Brown
Dye changes according to
amount of glucose
Enzyme-linked immunosorbent assays (ELISAs) S
detect antibodies to infections.

32. Enzymes in biological washing
Powders
• Proteases break down the coloured, insoluble proteins that
cause stains to smaller, colourless soluble polypeptides.
• Can wash at lower temperatures
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33. Enzymes in Food Industry
• 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. S

34. Enzymes in Seed Germination
starch
amylase
secreted
embryo plant
maltose
S

35. References
Pharmaceutical Biotechnology
By Dr. S.P. Vyas & Dr. V.K. Dixit
Enymes & its Immobilization Presentation
By Dr. S. Khanam
Internet Resources :
http://www.eplantscience.com
http://www.clickbiology.com
http://www.lsbu.ac.uk
http://www.tech-ceramics.co.uk

36. Thank You !!!
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