1. Applied Enzymes Catalysis-4
Dr. A.K.M. Shafiqul Islam
School of Bioprocess Engineering

2. Enzyme immobilization
Four types of immobilization
Adsorption Method
Covalent bonding
Entrapment
Encapsulation

3. Covalent bonding With – OH Group :
Supports of this type may be activated specifically for the covalent bonding by subjecting it to treatment with either cyanogen bromide or triazine. The reaction with the enzyme protein in each instance involves the –NH2 group of lysine.

4. Covalent bonding
Using Supports with – OH group that are Activated by Covalent Bonding with Cyanogen Bromide.

5. Covalent bonding
Using Supports with – OH group that are Activated by Covalent Bonding with Triazine.

6. With – COOH Groups :
Carboxymethyl cellulose (CMC) may be activated either via acyl-isourea formation or azide derivative formation.
The reaction involves the participation of amino (– NH2) moiety present in lysine, cysteine, serine, tyrosine — are also made use of in the covalent bonding phenomenon.

7. Immobilization of Enzymes using CMC Supports Having — COOH with — NH2 Group or with Hydrazine (NH2–NH2) Group via Covalent Bondage Involving Acyl Urea .

8. Immobilization of Enzymes using CMC Supports Having — COOH with — NH2 Group or with Hydrazine (NH2–NH2) Group via Covalent Bondage Involving Azide Derivative.

9. With – NH2 Group :
The amino functional group containing support material may be converted easily to the corresponding diazonium chloride salt by suitably treating with a mixture of sodium nitrite (NaNO2) and diluted hydrochloric acid (HCl) between 0-5°C (diazotization).

10. Immobilization of Enzymes using Supports with Specific —NH2 group Involving Formation of Diazonium Chloride

11. Immobilization of Enzymes using Supports with Specific —NH2 group Involving Activation with Glutaraldehyde.

12. Entrapment
Entrapment refers to the phenomenon whereby the enzyme molecules are either held or entrapped within the appropriate fibres or gels.
This entrapment may or may not necessarily be accomplished via covalent bonding existing between the enzyme entities (molecules) and the carrier matrix.
In a situation when the covalent bonding is needed, the enzyme molecules essentially required to be treated with synthetic reagents e.g., acryloyl chloride, cellulose acetate etc.

13. Entrapmentn Method
The various steps involved in ‘entrapment’ are as stated below:
The enzyme(s) may be dissolved in a solution of the polymer’s precursors.
Polymers may be selected from a variety of materials
natural gels (e.g., cellulose triacetate, alginate, agar, gelatin) ;
synthetic gels e.g., polyacrylamide gels.
In order to check and prevent the possible leakage of the low molecular weight enzymes from the body of the gel, the average pore size of the gel must be maintained as large as possible.
Two important aspects in ‘entrapment’ process, namely:
excessive diffusion limitation, and
variability of pore size

14. Entrapmentn Method
Polymer entrapment
liposome entrapment

15. Entrapment
Example
Lysine residues may be prepared by employing acryloyl chloride resulting into the formation of the corresponding acryloyl amides. The acryloyl amides are first copolymerized, and secondly cross-linked with either acrylamide and bisacrylamide to give rise to the formation of the desired ‘gel’ which comprises of the ‘entraped enzyme’ that may be further exploited in the form of a thin film on a solid support or as small beads.

16. Entrapment
Example
Cellulose acetate fibres also used to entrapment of enzymes.
Enzyme and cellulose acetate is blended together to obtain an ‘emulsion’ in an organic solvent, methylene chloride. The resulting emulsion is subjected to the process of ‘extrusion’ to obtain fibres into a solution of an aqueous precipitant.
Calcium alginate is the material used for the entrapment of microbial, plant cells, and animal cells.

17. Encapsulation
Encapsulation or microencapsulation or membrane confinement is another effective approach of enzyme immobilization.
In this method the enzyme molecules, invariably taken up in an aqueous medium, may be strategically confined within a semipermeable membrane that ideally permits an almost absolute ‘free movement’ of the enzymes in either direction to the products and substrates but fails to allow their migration and Escape.

18. Encapsulation

19. Encapsulation There are two enzyme entrapping methods:
1. Phase Separation:
Membranes are usually made by adopting the process of phase-separation, that essentially bears a close resemblance to homogenization of water in oil.
In this particular instance one phase is obviously not miscible with the other but eventually gives rise to a droplet with the other phase upon adequate mixing. Thus, ultimately the ‘enzyme’ gets entrapped right within this droplet,

20. Encapsulation
2. Chemcical Polymerization: The chemical polymerization aids in the preparation of the specific water-insoluble membrane, and thus the enzyme in question gets duly entrapped during this on-going phenomenon of polymerization.

21. Encapsulation 2. Chemcical Polymerization:
Examples: Two type examples are as follows:
Semipermeable collodion or nylon membranes in the shape of spheres (round beads) are invariably utilized for the microencapsulation of an enzyme. These materials are also available commercially.
Fibres of celluclose triacetate may also be employed for the entrapment of enzymes within this synthetic material. However, these fibres may be either woven into a suitable fabric or packed into the columns carefully.

22. I hope you would not mind to have a small test today.
What is meant by enzyme specificity? Describe lock-and-key hypothesis for enzyme specificity?
What is Lineweaver-Burk plot? How can it be used to calculate Michaelis-Menten constant?
Define and discuss competitive and noncompetitive inhibitor.