IMMOBILIZED ENZYMES AND ITS APPLICATION

2. Presented by
V. SELVAM
09-607-016

3. OUTLINE
• Definition Of Immobilized Enzyme
• Discovery of Enzyme Immobilization
• Uses of Immobilized Enzyme
• Preparation Techniques
• Form of Immobilized Enzyme
• Application Of Immobilized Enzyme

4. Definition
• An enzyme that is physically
attached to a solid support
over which a substrate is
passed and converted to
product.
• "immobilized" means “unable
to move or stationary”

5. Discovery of Enzyme Immobilization
• In 1916, Nelson and Griffin discovered that invertase
“exhibited the same activity when absorbed on a solid
(charcoal or aluminium hydroxide)”.
• Invertase catalyzes the hydrolysis (breakdown)
of sucrose (table sugar) to fructose and glucose

6. Why Immobilized Enzyme ???
• Increased Resistance
to changes in pH and
temperature.
• Easily Separated
• Highly Reusable
• Easy to Handle

7. Preparation Techniques Of Immobilized Enzyme
Adsorption
Carrier-
Binding
Cross-Linking Entrapping
Lattice-Type
Microcapsule-
Type

8. Adsorption
• Enzyme is attached to the external or internal
surface of a carrier material or support
• e.g. mineral supports like aluminum oxide or
clay; organic support (e.g. clay) or modified
sepharose and ion exchange resins

9. Procedures for immobilization by
adsorption
Static process
The dynamic batch process
The reactor loading process
The electrodeposition process

10. Demerits
• Active site of the immobilized enzyme may
be blocked by the matrix-Reducing the
activity of the enzyme.
• Slowest among those listed before

11. Carrier-Binding
• Binding of enzymes to water-insoluble
carriers
• A covalent bond is established between the
functional groups in the carrier and the
functional groups in the amino acid residues
of the enzyme, like
OH, NH2, and COOH.

12. Contd……
• Amount of enzyme bound to the carrier & the
activity after immobilization depend on the
nature of the carrier
Selection of the carrier depends on
• Particle size
• Surface area
• Molar ratio of hydrophilic to hydrophobic
groups
• Chemical composition

13. Contd…
Commonly used carriers
Polysaccharide derivatives such as
- cellulose,
- dextran,
- agarose, and
- polyacrylamide gel.,etc

14. The possible effects
of enzyme
immobilization by co
valent binding on
enzyme with carrier
material

15. Cross-Linking
• Intermolecular cross-linking of the protein,
either to other protein molecules or to functional
groups on an insoluble support matrix
• Relatively low enzymatic activity
• Most common reagent
- glutaraldehyde
(Zaborsky 1973; Cao 2005b; Guisan 2006)

16. Entrapping
• Physically entrapped within a matrix of water-
soluble polymer
• may or may not be covalent bond formation
• covalent bonding -treated with a reagent
• E.g. Acryloyl chloride is used to prepare lysine
residues for binding by forming acryloyl amides.
These are then co-polymerized and cross-linked
With acrylamide and bis acrylamide to form a gel
containing entrapped enzyme

17. TYPES OF ENTRAPMENT
Lattice Type : It
involves entrapping
enzymes within
interstitial spaces of
cross linked water
insoluble polymer.
Microcapsule Type
The entrapping involves
enclosing the enzymes within
semi permeable polymer
membrane.

18. Form of Immobilized Enzyme

19. APPLICATION OF IMMOBILIZED
ENZYMES
• Immobilized biocatalysts in dairy industry
• Immobilized enzymes in pharmaceutical industry
• Immobilized enzymes in High fructose syrup
Production
• Immobilized fumarase for the production of malic
acid
• Immobilized enzyme assay for detection of Heroin

20. Immobilized biocatalysts in
dairy industry
• Lactose is a disaccharide that occurs naturally in
both human and cow's milk
• lactose intolerants -incapable of digesting lactose
• disaccharide lactose via hydrolysis into its
monosaccharide components, glucose and galactose
• b –galactosidase
• hydrolysis also enhances the sweetness and
solubility of the sugars

21. Contd….
• First company-Centrale del Latte of Milan,
Italy
• Neutral lactase from yeast entrapped in
synthetic fibres

22. Immobilized enzymes in
Pharmaceutical industry
• Production of 6-aminopenicillanic acid (6-APA) by
the deacylation of the side chain in either penicillin
G or V, using penicillin acylase (penicillin amidase)
• More than 50% of 6-APA
• Partially purified amidase from E.coli trapped into
cellulose triacetate fibres can be used for production
of 6-APA from Penicillin G

23. Immobilized enzymes in High
fructose syrup Production
• conversion of glucose syrups to high fructose
syrups by the enzyme glucose isomerase
• less expensive than sucrose
• fructose is twice as sweet as sucrose
• commercial preparations use either the
adsorption or the cross-linking technique

24. Immobilized fumarase for the
production of malic acid
• Tanabe Seiyaku and Kyowa Hakko- Japan,
• use of immobilized nonviable cells of
Brevibacterium ammoniagenes or B. flavus as
a source of fumarase.
• Malic acid is becoming food acidulant in
competition with citric acid.

25. Immobilized enzyme assay
• Detecting Heroin and Its Metabolites
• use of heroin is illegal
• need to detect and identify heroin and its
derivative

29. Reference Articles

30. Reference Books

31. Be HAPPY…………….