ENZYME IMMOBILIZATION part II

COUPLING REACTIONS FOR ENZYME IMMOBILIZATION

A. Acylation Reactions
Carbonyl group:

Acyl group:

(strongly polarized)

O
RC

Compounds containing acyl group:

Acyl azide

Acylation reactions:

 The common mechanistic feature in acylation reactions is:

The attack of a nucleophile (in the case of proteins NH2, OH, or
SH groups) at an activated carbonyl group.
* Remarks:
(1) Nucleophiles are most effective in their unprotonated form (RNH2,
PhO, RS), i.e., at pH above their pKa values.
(2) High pH might cause irreversible denaturation of the protein as well as
fast hydrolysis of the reagent.
Acylation reactions are commonly carried out at pH 7.58.5 and
at 4C (to slow down hydrolysis of reagent).
Coupling of proteins via acylation reactions:
(1) Coupling of proteins to acyl azides

(2) Coupling of proteins to acid anhydrides

* Note: generation of free carboxyl groups

Enzyme located in an environment of excess negative charge.
(3) Coupling of proteins by activation of carboxyl group
(a) With carbodiimide

* pH 4.755 for the activation step.

* This is one of the most general methods for activating carboxyl
groups.
(b) With N-alkyl-5-phenylisoxazolium salt

* pH< 4.75 for the activation step.

(c) With N-ethoxycarbonyl--ethoxy-1,2-dihydroquinoline

B. Arylation and alkylation reactions

RX +RNH2 R-NH-R

Acylation versus alkylation:

Coupling of proteins:
(1) To halogen-substituted aromatic ring

(2) To polysaccharide supports (e.g., cellulose, agarose, and cross-linked

dextran)

 Remarks:
(1) Arylation and alkylation reactions are slower than acylation.
(2) Require an unprotonated nucleophile.
Amino groups are arylated at higher relative rates at alkaline pH
values (pH 8.59).
(3) Side reactions involve sulfhydryl groups cannot be eliminated.

C. The Cyanogen Bromide Method

It is a widely used method for immobilized enzyme.
Procedure:
(1) Activation of water insoluble polysaccharides

* Operated at high pH (1011.5).

(2) Coupling of enzymes

* Operated at mildly alkaline pH values.

* Structure III is most probably the major product.

D. Carbamylation and Thiocarbamylation Reactions

HOCOH

H2NCOH

H2NCNH2

O
Carbonic acid

O
Carbamic acid

O
Carbamide

HOC N
Cyanic acid

RN=C=O
Isocyanate

RN=C=S
Isothiocyanate

Coupling of protein:

* Isocyanates and isothiocyanates react with most protein nucleophiles.

Only the reaction with amino groups results in the formation of
stable products.
* Polymers containing isothiocyanate group have gained larger acceptance
than those containing isocyanate.
Due to the higher stability and the relative ease of preparation.

E. Amidination Reactions
Amidines:

NH
RCNH2

Procedure:
(1) Treat polymeric nitriles with alcohols and hydrogen chloride to obtain
imidoester functional groups

* Imines: compounds that contain a C=N bond

(2) Coupling of proteins

* Imidoesters are readily attacked by nucleophiles and react selectively

with - and -amino groups of proteins at pH 8.59.5 to form
amidines.
* Amidines are stable in neutral or acidic solutions, but hydrolyze slowly
at high pH.

F. Reactions with Polymeric Aldehydes

Polymers containing aldehyde groups:
(1) Synthetic polymers
(2) Oxidation of polysaccharides (with periodate or dimethyl sulfoxide)
Coupling of proteins:
Aldehydes react with amino groups on the protein.

Remarks:
(1) The reactions can be carried out under mild conditions.
(2) Sulfhydryl and imidazole groups may undergo similar reactions.
Could have deleterious effects on the activity of the bound enzyme.
(3) This method has limited applications.
* The bonds formed with the protein amino groups are reversible,
and the equilibrium is unfavorable particularly at low pH values.
* Many nucleophiles can reverse the equilibrium to generate the
free amine.

G. Reactions with Glutaraldehyde

Glutaraldehyde: OCHCH2CH2CH2CHO
Procedure:
(1) Treat polymer supports (containing primary amino groups) to yield
matrices containing the aldehyde function
(2) Coupling of proteins
Remarks:
(1) Proteins are bound irreversibly.
(2) The reaction can be carried out in aqueous solution within a rather wide
range of pH values (p9).
The rate of reaction increases with increasing pH.
(3) The nature of the reaction is not fully understood.
A probable mechanism:

H. Diazotization Reactions
A diazonium salt: ArNN+:X
Reaction of diazonium saltsazo coupling:

Coupling of proteins to polymeric diazonium salts:

The specificity of azo coupling is rather broad.

* The electrophilic aryldiazonium ion attacks mainly activated aromatic
rings, such as phenols (tyrosine) or imidazole (histidine), reacting at
pH 89.
* Amino groups (the -amino group of lysine) react under similar
conditions.
* The guanido group (arginine) and indole (tryptophan) also undergo
coupling, with a slower rate.

I. Thiol-Disulfide Interchange Reactions

Thiols: sulfur analogs of alcohols, containing sulfhydryl group SH.
They can be oxidized easily, two SH groups are converted into
disulfide links, SS.
Coupling of proteins:

The supportSSprotein bonds are stable under nonreducing

conditions; however, it can be reversed with low-molecular-weight
sulfhydryl reagent (Scheme 3).

J. Four-Component Condensation Reactions

Involve carboxylate, amine, aldehyde, and isocyanide.
Lead to the formation of an N-substituted amide.

Four-component condensation reactions can be carried out in an aqueous

medium at neutral pH and allow for considerable versatility and high
selectivity when applied to enzyme immobilization.
* Polymer: NH2
Additives in solution: CHO (acetaldehyde), NC
(cyclohexylisocyanide)
Coupling of proteins through COOH.
* Polymer: COOH
Additives in solution: CHO, NC
Coupling of proteins through NH2.
* Polymer: NC
Additives in solution: CHO (acetaldehyde), COOH (acetate)
Coupling of proteins through NH2.
* Polymer: NC
Additives in solution: CHO, NH2 (tris)
Coupling of proteins through COOH.
Tris, or Tris(hydroxymethyl) amino methane:

NH2
H O C H 2 C C H 2O H
C H 2O H

Remark: 4CC reactions can be used to advantage only when an enzyme is not

sensitive to aldehyde.

SUPPORT MATERIALS FOR ENZYME IMMOBILIZATION

Support materials:

Considerations of support materials:

(1) Influence on catalytic stability and kinetics
Microenvironment of the enzyme; shift of optimal pH and
temperature; diffusional resistance
(2) Capacity to bind protein
Affect the size of reactors.
(3) Surface charge and hydrophilicity
(4) Dimensional stability
Compaction in packed-bed reactors
(5) Chemical stability
Microbial attack; pH; temperature
(6) Ease of activation
(7) Interaction of the support with the analyte
(8) Cost, regenerability, and availability

SYNTHETIC POLYMERS FOR ENZYME IMMOBILIZATION

A. Polystyrene
It is the first synthetic polymer to be used for enzyme immobilization
(because of its availability and low price).
HNO3, H2SO4 (cat.)
Nitration

( CHCH2 )n

( CHCH2 )n

NO2
Fe, 30% HCl, heat

Na2CO3

Reduction

( CHCH2 )n

NH2
NaNO2, HCl
Diazotization

( CHCH2 )n

N2Cl

protein
azo coupling

* Remarks: the amount of bound protein is rather low because of the

hydrophobicity of the support.

B. Copolymers consisting of methacrylic acid

They contain carboxylic groups as the hydrophilic component.

Variation of the ratio of monomers gives carriers of different binding
capacities and hydrophilicities.
Increasing the content of hydrophilic groups increases the
hydrophilicity, but leads to a reduction in reactive groups.
The optimal ratio of hydrophilic to hydrophobic component is 3:1.

* Coupling of enzyme:
Arylation

Thiocarbamylation

C. Polyacrylamide
A hydrophilic, electrically neutral support.
Activation:
O
CNH2

H2NNH2 or HNO2

O
CN3

Coupling of enzyme (via acylation):

O

O
CN3

H2N-protein
pH 8.5, 0.05 M borate,
1 h at 0oC

D. Polyvinyl alcohol
A hydrophilic, electrically neutral support.

CNH-protein

E. Polyamides
The most important synthetic polyamides are nylons.
* Nylon-6,6 or -6,10: formed by the condensation of diamines with
dicarboxylic acids
Nylon-6, -11, or -12: self-condensed amino acids

* Physical forms of nylons commercially available: membrane, powder, tube,

and hollow fiber
They are mechanically strong and non-biodegradable.
* Nylon-6 and -6,6: nylons of shorter methylene chains
They are relatively hydrophilic, and are suitable for enzyme
immobilization.

* Enzyme immobilization by adsorption:

Enzymes are adsorbed on the surface of a nylon structure and then crosslinked with glutaraldehyde or bisimidates.
* Covalent immobilization of enzymes:
The polyamide backbone (peptide bonds) is chemically inert.
Reactive groups: terminal carboxyl and amine
The binding capacity is very poor.
* Approaches to increase the binding capacity of nylons:
(1) Controlled cleavage of peptide bonds to increase the number of amine
and carboxyl groups

In most cases, the cleavage is effected by mild acid hydrolysis.

The mechanical strength of the support might be impaired.

(2) Introduction of reactive centers via O-alkylation

(3) Introduction of reactive side chains via N-alkylation

F. Maleic Anhydride Copolymers

Copoly(ethylene-maleic anhydride):

Coupling of enzyme (via acylation):

 Enzyme located in an environment of excess negative charge.

Maleic anhydride copolymers could serve as starting materials for further
chemical modification.

The polyanionic enzyme conjugates exhibit improved stability at alkaline

pH values; conversely, the polycationic derivatives show higher stability
at acidic pH values.

INORGANIC SUPPORTS FOR ENZYME IMMOBILIZATION

Why inorganic supports?
(1) High mechanical strength
(2) Resistance to solvents and microbial attack
(3) Regenerability
(4) Structural stability over wide ranges of pH, pressure, and temperature

A. Controlled Pore Glass (CPG)

The most widely used fabricated support.
Preparation of controlled pore glass:
(1) Heat treatment (500700C) of borosilicate glass
Two phases formed: boric acid-rich phase (soluble in acids), and
silica-rich phase (insoluble in acids).
(2) Acid-leaching of the boric acid-rich phase
Result in porous glass with pore diameter of 3060 and 28% pore
volume
(3) Mild caustic treatment
Remove siliceous residue from pore interiors.
Enlarge the pore diameter (up to 3000 ); rather narrow pore
size distribution
* In most of the work on enzyme immobilization, CPG particles of 550
pore diameter and a surface area of around 40 m2/g are used.
* In general, controlled pore glass is most stable under acid conditions.
The silica continues to leach from the particles under alkaline
conditions.
CPG treated with a hydrophilic silane (e.g., aminopropyltriethoxysilane) shows improved durability.

-aminopropyltriethoxysilane:

OC2H5
C2H5OSiCH2CH2CH2NH2
OC2H5

Chemical modification of CPG:

Practice of the activation step:

* Weetall and Hersh (1969)
Clean the surface of the support.
Reflux the clean glass with a triethoxysilane in toluene for 12 to 36 h.
Wash the preparation and bake at 120C.
* Robinson et al. (1971)
Clean the surface of the support.
Place the clean glass in contact with a 1% solution (v/v) of silane in
acetone.
Evaporate the acetone.
Heat the glass at 120C for about 12 h.
Disadvantages of CPG in enzyme immobilization:

(1) Unstable during prolonged operation

(2) Severe diffusional resistance prevalent within the pores
(3) Relatively high cost

B. Porous Ceramics
Chemical modification: similar steps as in CPG
Advantages of using ceramics in enzyme immobilization:
(1) Non-expensive material
(2) Improved chemical and dynamic durability at elevated pH and
temperature.

C. Magnetic Ion Oxide (Magnetite, Fe2O3)

Why magnetite?
(1) Easy separation of the immobilized enzyme particles
(2) Can be used in magnetically stabilized fluidized-beds
Chemical modification:
(1) Silanized with -aminopropyltriethoxysilane
(2) Activated with glutaraldehyde; or
(2) Treated by phosgene (COCl2) to convert the amino groups to isocyanate
(N=C=O)
Phosgene (a highly poisonous gas):

* Phosgene undergoes the usual reactions of an acid chloride.

RNH2 + ClCCl
O

RNHCCl
O

RN=C=O

POLYSACCHARIDES AS SUPPORTS FOR ENZYME

IMMOBILIZATION
Polysaccharides commonly used as support materials:
(1) Cellulosecomposed of linear chains of 1,4-linked -D-glucose residues

(2) Starchcomposed of linear chains of 1,4-linked -D-glucose residues

(3) Dextrana linear water-soluble polysaccharide composed of 1,6-linked

-D-glucose residues; produced by microorganisms of the genus of
Leuconostoc

(4) Agaroseone of the components of agar; a complex mixture of

polysaccharide; extracted from red sea-water algae; composed of
alternating 1,3-linked -D-galactose and 1,4-linked 3,6-anhydro--Lgalactose residue

Cellulose is popular for enzyme immobilization, owing to:

(1) Comparatively low price
(2) Methods for chemical modification are well established.

 Functional derivatives of cellulose:

* Carboxymethyl (CM) cellulose (Structure I)

Hydrazine: H2NNH2
* DEAE-cellulose: Diethylaminoethyl ether of cellulose
CelluoseOCH2CH2N(C2H5)2
* p-Aminobenzyl ether of cellulose

Sephadex: commercially available dextran gels; prepared by cross-linking the

linear polysaccharide with epichlorohydrin
Epichlorohydrin:

O
CH2CHCH2Cl

ASSESSMENT OF ENZYME IMMOBILIZATION

Criteria for selection of technique for enzyme immobilization:
(1) Binding capacity of support materials
It is a function of charge density, functional groups, porosity, and
hydrophobicity of the support surface.
(2) Stability and retention of enzyme activity
It is dependent on functional groups on support material and
microenvironmental conditions.
Usually, immobilization results in a loss in enzyme activity and
stability.
Methods might be considered for determining the amount of bound protein:
(1) Measurement of the difference between the amount of protein put into the
reaction mixture and recovered in soluble state
Comment: only a rough estimate
The determination of a low protein concentration in a large
volume of wash solution cannot be accurate.

(2) Measurement of the difference between the activity input and activity
recovered in the soluble fraction
Comment: misleading
Enzyme activity may lose owing to inactivation under the
conditions of immobilization.
(3) Direct determination of the amount of protein bound to the carrier
(a) Elementary analysis
Comment: unsuitable as a general method, because
Carbon and nitrogen are common also as carrier constituents.
Sulfur content in proteins is very low.
(b) Amino acid analysis (after hydrolyzing the protein)
* Lysine, glutamic acid, aspartic acid, or tyrosine
They are involved in the formation of the covalent linkage
between enzyme and carrier.
Low yield obtained.
* Candidates for the calculatio of protein content: amino acids with
aliphatic side chains and not undergoing destruction during
hydrolysis.

* Alanine and leucine seem to be best suited, because

Valine and isoleucine require very long times of hydrolysis for

complete liberation as free amino acids.

The contents of alanine and leucine are moderately high in
most enzymes.
Causes of loss of enzyme activity after immobilization:
(1) Conformational change
(2) Steric hindrance

(3) Involvement of a reactive group in the active site in the binding to support
Can be avoided by using a reversible inhibitor during binding to
protect the active site.
(4) Denaturation or inactivation under the binding conditions
* In general, the more enzyme bound and the longer the time taken for
immobilization, the lower the retention of activity.

 At present, there seem to be few guidelines for the choice of immobilization

technique to obtain good activity retention.
An ideal support of universal applicability cannot be anticipated, owing
to the compositional and structural diversity of proteins.
The immobilization of an enzyme requires an empirical, essentially trial
and error approach.