BT2252 - ETBT - UNIT 3 - Enzyme Immobilization.pdf

BT2252 - UNIT 3
ENZYME
IMMOBILIZATION
By
Er.A.Ganga., M.Tech., (Ph.D)
Assistant Professor
Department of Biotechnology
Kamaraj College of Engineering & Technology
ETBT
1

Applications of Enzyme
• Food Industry – (Pectinases – Fruit
juices, Jams, Jellies).
• Chemical Industry – Fine chemicals
(Amino acylase – L-aminoacids)
• Pharmaceutical Industry – (Penicillin G
amidase for the synthesis of Ampicillin)
• Cosmetic Industry – (Lipases - lightening
pigments, dyes, coumerin, benzyl
alcohol)
• Medical devices – (Urease immobilized
on artificial kidney machine removes
urea); Biosensors
05-04-2023 Er.A.Ganga/BT/4th Sem/ETBT/KCET 2
ETBT
2

Free Enzyme Vs Immobilized Enzyme
Characteristics Free enzyme Immobilized
enzyme
Price High Low
Efficient Low High
Activity Unstable Stable
Reusability &
recovery
Not possible Possible
Tolerance to
temperature & pH
Low High
Separate from
substrate/Product
Difficult Easy
Stability of the
product
Low High
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Topics
1. Physical and chemical techniques for enzyme immobilization
2. Kinetics of Immobilized enzymes – Factors affecting the kinetics of
immobilized enzymes.
3. Effects of internal and external diffusional limitations.
4. Determination of mathematical model for diffusional effects in
immobilized enzyme reaction.
Adsorption
Covalent binding
Matrix entrapment
Encapsulation
Cross-linking
Examples, Advantage &
Disadvantages
ETBT
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Enzyme Immobilization
Enzyme Immobilization
• Enzyme immobilization is defined
as
- arresting the movement of enzymes
or confinement of an enzyme(bio-
catalyst) in a distinct phase but allowing
it to exchange matter
(substrate/Product) with the later.
• Enzyme can be physically
entrapped or covalently bonded by
chemical means to an inert
insoluble matrix or carrier.
Need for enzyme immobilization
• Accelerates the chemical reaction
• Specificity of the enzyme can be
retained for a longer period.
• Enzymes can be reused.
• Products & Enzymes are easy to
separate.
• Promotes multi enzyme reactions.
• Protects enzyme from degradation and
deactivation.
• Product is stable.
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Support/Carrier/Matrix material
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Images of carrier molecules
SiO2 Silica Porous glass Bentonite clay
DEAE Cellulose
CM-Cellulose Polystyrene
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Characteristic of a solid support
• It should contain high surface area
• It should be inert.
• Physically strong and stable
• Cost effective
• Regenerable
• Reduction in product inhibition
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Enzyme Immobilization techniques
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Adsorption
• Involves the physical binding of enzyme on the surface of carrier matrix.
• Carrier may be organic or Inorganic.
• The process of adsorption involves weak interaction like vanderwaal or
hydrogen bonds.
• Carriers used: bentonite, silica, cellulose
• Ex: 1. Catalase (Peroxidases – for the conversion of H2O2, a toxic
compound produced as a byproduct of using O2 for respiration)
2. Invertase (Hydrolyses sucrose into glucose and fructose which
leads to the production of HFS (High fructose syrup))
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Adsorption
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Adsorption
Advantages
• Technologically simple
• Adaptable to many treatment
formats.
• Works at mild operation
condition and at wide range of
pH.
• This process does not produce
any by-product.
• Cost effective
Disadvantages
• Low stability of the immobilized
enzyme, leads to a fast washing
out of the enzyme carrier.
• Enzyme leakage due to
temperature, pH, ionic strength
of the solution
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12

Entrapment
• Enzymes are not attached directly to the support, rather they are
trapped inside the polymer matrix.
• Enzymes are held within suitable gels or fibres.
• It is done in such a way as to retain enzyme while allowing the
penetration of substrate.
• It can be classified into lattice and micro capsule types.
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Entrapment : Example – Lactose free enzyme
production
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Entrapment
Lattice type
• It involves entrapment of
enzymes within the interstitial
spaces of a cross-linked water
insoluble polymer.
• Synthetic Polymers used:
Polyacrylamide, PVA.
• Natural polymers: Starch
Capsule type
• It involves enclosing the
enzymes within semi-permeable
polymer membranes.
• Membranes used: Nitrocellulose
membrane, Nylon in the shape of
spheres.
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Entrapment
Advantages
• No chemical modification of the
enzyme.
• Relatively stable forms.
• Easy handling and re-usage
Disadvantages
• The enzyme might leak from
pores
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Covalent binding
• This technique involves the formation of covalent bond between the
functional groups present in water in-soluble carrier and the enzyme
molecules.
• Functional groups involved in bond formation : Amino group, Carboxyl
group, Sulfhydryl group, Hydroxyl group, Imidazole group, Phenolic groups,
Thiol group.
Advantages: No leakage of enzyme occurs even in the presence of substrate or
solution of high ionic strength.
Disadvantages: Covalent binding may alter the conformational structure and
active site of the enzyme, resulting major lose of activity and/or changes of the
substrate
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17

Cross linking
• Cross linking involves intermolecular cross linking of enzyme molecules in
the presence/absence of solid support.
• The method produces a 3-dimensional cross linked enzyme aggregate- CLEA
(insoluble in water) by means of a multifunctional reagent that links the
enzymes covalently to each other.
• Glutaraldehyde is a chemical reagent used as a cross-linker
Advantages: Very little desorption (Enzyme strongly bound); Higher stability
Disadvantages: Cross-linking may cause significant changes in the active site;
Not cost effective.
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Comparison between the methods
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Limitations of Enzyme Immobilization
• Cost of the carriers and immobilization process
• Changes in properties (selectivity)
• Mass transfer limitations. (Oil, water, O2, souluble sulphur dioxide,
phenolic components, gases/vapours, dissolved solutes, metal ions,
proteins)
• Problems with cofactors and regenerations
• Problems with multienzyme systems
• Enzyme activity loss during immobilization
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Conclusion
• Enzyme Immobilization is one of the most promising approaches for
exploiting enzyme based processes in biotransformation, diagnostics,
pharmaceuticals and food industries.
• Several hundreds of enzymes has been immobilized in a variety of
forms including penicillin G Acylase, lipases, proteases, invertases.
• We should focus our research on overcoming the current limitations to
expand the enzyme application horizon.
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21

Topics
Adsorption
Covalent binding
Matrix entrapment
Encapsulation
Cross-linking
Disadvantages
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22

Kinetics of Immobilized enzymes – Factors affecting
the kinetics of immobilized enzymes.
• Kinetic behavior of immobilized enzyme differs significantly from that of
the same enzyme in free solution.
• These changes may be due to conformational changes within the enzyme
structure due to immobilization procedure and due to the presence and
nature of immobilization support.
• Structure and stability of an immobilized enzyme plays a major role in
determining its kinetic behavior.
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23

Contd.
• If the immobilization process introduces any strain in the interior enzyme structure
then the enzyme is likely to be inactivated under unfavorable pH and temperature
resulting in denaturation. Enzyme gets strained when the process involves
single point of contact while immobilizing the enzyme.
• When there is a series of unstrained multipoint binding between the enzyme and
support, substantial stabilization may occur.
• Stability of an immobilized enzyme can be improved by derivatization of the
enzyme molecule.
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Example - Use of multipoint interaction between
enzyme & support for stabilization.
a. Activity of free underivatized chymotrypsin
b. Activity of chymotrypsin derivatized with acryloyl chloride (Acryloyl
chymotrypsin)
c. Activity of derivatized chymotrypsin entrapped in a polymer matrix
d. Activity of non-covalently entrapped chymotrypsin. (Degree of chymotrypsin
stability is dependent on the strength of the polymer used and so does the number of
non-covalent interactions
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Factors affecting the kinetics of immobilized
enzymes-Partitioning and Diffusion effect
There are two different ways in which a support polymer can affect the
microenvironment surrounding an immobilized enzyme.
 Partitioning effect: By nature of its physiochemical properties, the polymer
may either attract or repel substrate/inhibitors/products or other molecules
towards or away from its surface, by concentrating them or diluting them in the
immediate vicinity of the enzyme.
 Diffusion effect: The second way in which a polymer may affect the
microenvironment of the enzyme is by itself acting as a barrier to the free
diffusion of molecules to or from the enzyme.
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26

Effect of solute partition on the kinetics of
immobilized enzyme
Partitioning of charged molecules (eg. Substrates and products) occurs between the bulk solution and
the microenvironment; molecules of opposite charge to the immobilized enzyme surface being
partitioned into the microenvironment, whereas molecules possessing similar charge to the
immobilized enzyme surface are expelled, with equal effect, into bulk solution.
Solute partitioning may be quantified by the introduction of the electrostatic partition coefficient (L)
defined by A and is given as follows,
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Contd.
• Assuming Michaelis-Menten kinetics, the rate of reaction catalyzed by an
immobilized enzyme is given by equation 2, where the substrate concentration is the
concentration within the microenvironment.
• If the substrate is positively charged, then, equation 1 can be written as,
• Substituting [S] from equation 3 in equation 2, we get
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Contd.
• If the substrate is negatively charged,
• Substituting equation 6 in equation 5, we get the
following relationship.
• Km of an enzyme for the substrate is apparently
reduced if the [S] in the vicinity of enzyme’s active
site is higher than that measured in the bulk of the
solution.
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29

Effects of solute diffusion on the kinetics of
immobilized enzyme
• Diffusion limitations are constraints which are a function of physical size.
• If the pore diameter of the polymer matrix is smaller than that of the substrate molecule,
preventing the access to the enzyme, then reaction will not take place.
• When the immobilized enzyme and substrate are mixed, the substrate diffuses into the
particle and the enzyme begins to transform it at a rate depending on the substrate
concentration.
• Hence, a concentration gradient is rapidly set up through out the polymer matrix and a
steady state is reached in which the diffusion rate of the substrate up to a given point in the
interior of the polymer particle becomes equal to the rate of removal by the enzyme at that
point.
• The driving force for the net diffusive process is due to the concentration gradients, solutes
moving in the direction of higher to lower concentration.
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30

Types of diffusion effects
External diffusion
• This is due to the presence of a thin unstirred layer
of solvent surrounding the polymer particle; such a
layer is called Nernst layer.
• Solute diffuses through layer by a combination of
passive molecular diffusion and convection.
• Within certain limitation, the thickness of this layer
may be affected by the rate at which the bulk
solution surrounding the enzyme is stirred.
Internal diffusion
• This is due to the limitations of free diffusion of
solute within the polymer matrix imposed by the
nature of the matrix.
• Inside the polymer matrix, diffusion takes place only
by passive molecular diffusion and is unaffected by
stirring rate.
• The effects of internal diffusions are more apparent if
the enzyme is immobilized by entrapment within the
matrix of a polymer than it is immobilized on the
surface of such matrix.
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31

Topics
Adsorption
Covalent binding
Matrix entrapment
Encapsulation
Cross-linking
Disadvantages
ETBT
32

Topics
Adsorption
Covalent binding
Matrix entrapment
Encapsulation
Cross-linking
Disadvantages
ETBT
47

Applications of enzyme immobilization
Production of biodiesel
Textile Industry
Detergent Industry
Food Industry
Biomedical applications
Industrial production
Waste water management
05-04-2023 Er.A.Ganga/BT/4th Sem/ETBT/KCET
ETBT
48

Biomedical applications of Immobilized
enzyme
• Biosensors : Electronic devices that make use of an enzyme’s
specificity and the technique of enzyme immobilization.
• Eg. Glucose sensors
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Immobilized enzyme biosensors for various
applications
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Immobilized enzymes used for the
production of various antibiotics
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Immobilized enzyme for the conversion of
glucose syrup into fructose syrup
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Immobilized enzymes in food industry
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Immobilized enzyme for the production of
biodiesel
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Immobilized enzymes in biodiesel production
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Immobilized enzyme for bioremediation
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Acknowledgement
I would like to acknowledge and extend my
gratitude for the various web resources, research
articles for the references I have made on them
while preparing for the topic “Enzyme
Immobilization”
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Thank you
ETBT
58

BT2252 - ETBT - UNIT 3 - Enzyme Immobilization.pdf

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