IMMOBILISED ENZYMES

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

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

METHODS OF IMMOBILISATION Adsorption Covalent binding Entrapment Membrane confinement

METHODS OF IMMOBILISATION

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).

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

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

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

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

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.

Immobilising enzymes on Sepharose

Immobilising enzymes on Cellulose

Immobilising enzymes on Matrix with carboxylic acid moeity

Immobilising enzymes on PolyGlutaraldehyde

Immobilising enzymes on Glass

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

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

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

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.

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

IMMOBILIZED ENZYME ACTIVITY DEPENDS ON CORRECT AND UNSTRAINED CONFORMATION

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

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

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)

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

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

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.

Some of the more important industrial uses of immobilised enzymes EC number Product Aminoacylase 3.5.1.14 L-Amino acids Aspartate ammonia-lyase 4.3.1.1 L-Aspartic acid Aspartate 4-decarboxylase 4.1.1.12 L-Alanine Cyanidase 3.5.5.x Formic acid (from waste cyanide) Glucoamylase 3.2.1.3 D-Glucose Glucose isomerase 5.3.1.5 High -fructose corn syrup Histidine ammonia-lyase 4.3.1.3 Urocanic acid Hydantoinasea 3.5.2.2 D- and L-amino acids Invertase 3.2.1.26 Invert sugar Lactase 3.2.1.23 Lactose-free milk and whey Lipase 3.1.1.3 Cocoa butter substitutes Nitrile hydratase 4.2.I.x Acrylamide Penicillin amidases 3.5.1.11 Penicillins Raffinase 3.2.1.22 Raffinose-free solutions Thermolysin 3.2.24.4 Aspartame Some of the more important industrial uses of immobilised enzymes