1. IMMOBILISED ENZYMES

2. THE ECONOMIC ARGUMENT FOR IMMOBILISATION
Enzymes are expensive, they should be utilized in an efficient manner
As catalytic molecules, enzymes are not directly used up. After the reaction the enzymes cannot be economically recovered for re-use and are generally wasted
This enzyme residue remains to contaminate the product and its removal may involve extra purification costs
Simple and economic methods must be used
to separate the enzyme from the reaction product

3. This is known as IMMOBILISATION
Separation of enzyme and product using a two-phase system; * One phase containing the enzyme * The other phase containing the product
This is known as
IMMOBILISATION
The enzyme is imprisoned within its phase allowing its re-use or continuous use The separation prevents the enzyme from contaminating the product

4. METHODS OF IMMOBILISATION
Adsorption
Covalent binding
Entrapment
Membrane confinement

5. METHODS OF IMMOBILISATION

6. MATRICES FOR ENZYME IMMOBILISATION
Inert.
Physically strong and stable.
Should be cheap enough to discard.
Better if it could be regenerated after the useful lifetime of the immobilised enzyme.
The surface available to the enzyme.
More desirable properties:
* Ferromagnetism (e.g. magnetic iron oxide, enabling transfer of the biocatalyst by means of magnetic fields).
* Catalytic surface (e.g. manganese dioxide, which catalytically removes the inactivating hydrogen peroxide produced by most oxidases).
* Reductive surface environment (e.g. titania, for enzymes inactivated by oxidation).

7. ADSORPTION OF ENZYMES ONTO INSOLUBLE SUPPORTS
Mix the enzyme with a suitable adsorbent (under appropriate conditions of pH and ionic strength)
Incubate
Wash off loosely bound and unbound enzyme

8. EXAMPLES OF SUITABLE ADSORBENTS
Ion-exchange matrices
Porous carbon
Clays
Hydrous metal oxides
Glasses
Polymeric aromatic resins

9. PREPARATION OF IMMOBILISED INVERTASE BY ADSORPTION
   Support type   
% bound at
DEAE-Sephadex anion exchanger  
CM-Sephadex cation exchanger
pH 2.5   
0   
100
pH 4.7   
100    75
pH 7.0    34

10. specificity (kcat/Km)
EFFECT OF COVALENT ATTACHMENT TO A CHARGED MATRIX ON THE KINETIC CONSTANTS OF CHYMOTRYPSIN FOR N-ACETYL-L-TYROSINE ETHYL ESTER
   
Ionic strength (M)
kcat (s-1)
Km (mM
specificity (kcat/Km)
Free enzyme   
0.05   
184   
0.74   
249
1.00   
230   
0.55   
418
Enzyme attached to a negatively charged support   
300   
2.50   
120
280   
1.93   
145
Enzyme attached to a positively charged support   
 0.05   
119   
7.10    17
1.65   
5.82    28

11. COVALENT COUPLING TO INSOLUBLE MATRICES
Reactivity of protein side-chain nucleophiles is determined by their state of protonation (i.e. charged status) and roughly follows the relationship
-S- > -SH > -O- > -NH2 > -COO- > -OH >> -NH3+
where the charges may be estimated from a knowledge of the pKa values of the ionising groups and the pH of the solution.
Lysine residues are found to be the most generally useful groups for covalent bonding of enzymes to insoluble supports due to:
Their widespread surface exposure and high reactivity, especially in slightly alkaline solutions.
They also appear to be only very rarely involved in the active sites of enzymes.

12. Immobilising enzymes on Sepharose

13. Immobilising enzymes on Cellulose

14. Immobilising enzymes on Matrix with carboxylic acid moeity

15. Immobilising enzymes on PolyGlutaraldehyde

16. Immobilising enzymes on Glass

17. RELATIVE USEFULNESS OF CHARGED ENZYME RESIDUES FOR COVALENTCOUPLING
Content
Exposure
Reactivity
Stability of couple
Use
Aspartate    + ++
+   
Glutamate   
Arginine    -
±   
Histidine   
Lysine   
++   

18. RELATIVE USEFULNESS OF UNCHARGED ENZYME RESIDUES FOR COVALENTCOUPLING
Content
Exposure
Reactivity
Stability of couple
Use
Cysteine    - ++
Cystine    +
Methionine   
Serine   
Threonine   
Tyrosine   
Tryptophan

19. RELATIVE USEFULNESS OF OTHER MOEITY OF ENZYME FOR COVALENT COUPLING
Content
Exposure
Reactivity
Stability of couple
Use
C terminus    - ++ +
N terminus   
Carbohydrate
- ~ ++
Others   
 - ~ ++

20. Lysine residues are the most generally useful groups for covalent bonding of enzymes to insoluble supports due to their widespread surface exposure and high reactivity, especially in slightly alkaline solutions. They also appear to be only very rarely involved in the active sites of enzymes.

21. The most commonly used method for immobilizing enzymes on the research scale involves Sepharose (poly-{b-1,3-D-galactose-a-1,4-(3,6-anhydro)-L-galactose}), activated by Cyanogen bromide, because it is a commercially available beaded polymer which is highly hydrophilic and generally inert to microbiological attack

22. IMMOBILIZED ENZYME ACTIVITY DEPENDS ON CORRECT AND UNSTRAINED CONFORMATION

23. Immobilisation of an enzyme in the presence of saturating concentrations of substrate, product or a competitive inhibitor ensures that the active site remains unreacted during the covalent coupling and reduces the occurrence of binding in unproductive conformations

24. ENTRAPMENT OF ENZYMES WITHIN GELS OR FIBRES
Purely physical caging: Cellulose acetate
Involve covalent binding: Polyacrylamide gel

25. INVOLVE COVALENT BINDING
Lysine +
CH2=CH-CO-Cl (Acryloyl chloride)
CH2=CH-CO-NH-lysyl (Acryloyl amide) +
GEL
CH2=CH-CO-NH2 (Acrylamide) +
H2N-CO-CH=CH-CH=CH-CO-NH2 (Bisacrylamide)

26. CONFINEMENT OF ENZYMES INSIDE A MEMBRANE
Semipermeable membrane: Hollow fibre membrane
Membrane-bound droplets: Nylon-6,6
Liposome: Phospholipid

27. GENERALISED COMPARISON OF DIFFERENT ENZYME IMMOBILISATION TECHNIQUES
Characteristics
Adsorption
Covalent binding
Entrapment
Membrane confinement
Preparation
Simple
Difficult
Cost
Low
High
Moderate
Binding force
Variable
Strong
Weak 
Enzyme leakage
Yes No
Applicability
Wide
Selective
Very wide
Running Problems
Matrix effects
Large diffusional barriers
Microbial protection

28. Inhibition constant (ki, mM) Bound to polystyrene (hydrophobic)
EFFECT OF IMMOBILISATION USING A HYDROPHOBIC SUPPORT ON THE RELATIVE COMPETITIVE INHIBITION OF INVERTASE
   
Invertase
Inhibition constant (ki, mM)   
Soluble   
Bound to polystyrene (hydrophobic)
Aniline (hydrophobic)   
0.94   
0.39
Tris-(hydroxymethyl)-aminomethane (hydrophilic)   
0.45   
1.10
The Ki is reduced where both the inhibitor and support are hydrophobic.

29. Some of the more important industrial uses of immobilised enzymes
EC number
Product
Aminoacylase
L-Amino acids
Aspartate ammonia-lyase
L-Aspartic acid
Aspartate 4-decarboxylase
L-Alanine
Cyanidase
3.5.5.x
Formic acid (from waste cyanide)
Glucoamylase
D-Glucose
Glucose isomerase
High -fructose corn syrup
Histidine ammonia-lyase
Urocanic acid
Hydantoinasea
D- and L-amino acids
Invertase
Invert sugar
Lactase
Lactose-free milk and whey
Lipase
Cocoa butter substitutes
Nitrile hydratase
4.2.I.x
Acrylamide
Penicillin amidases
Penicillins
Raffinase
Raffinose-free solutions
Thermolysin
Aspartame
Some of the more important industrial uses of immobilised enzymes