ENZYME IMMOBILIZATION part II

COUPLING REACTIONS FOR ENZYME IMMOBILIZATION

A. Acylation Reactions

Carbonyl group:(strongly polarized)

Acyl group:RC

O

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, orSH 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 asfast hydrolysis of the reagent.

Acylation reactions are commonly carried out at pH 7.58.5 andat 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 carboxylgroups.

(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 +R’NH2 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-linkeddextran)

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 pHvalues (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

O

H2NCOH

O

H2NCNH2

OCarbonic acid Carbamic acid Carbamide

HOC NCyanic acid

RN=C=OIsocyanate

RN=C=SIsothiocyanate

Coupling of protein:

* Isocyanates and isothiocyanates react with most protein nucleophiles.

Only the reaction with amino groups results in the formation ofstable products.

* Polymers containing isothiocyanate group have gained larger acceptancethan those containing isocyanate.

Due to the higher stability and the relative ease of preparation.

E. Amidination Reactions

Amidines:RCNH2

NH

Procedure:

(1) Treat polymeric nitriles with alcohols and hydrogen chloride to obtainimidoester functional groups

* Imines: compounds that contain a C=N bond

(2) Coupling of proteins

* Imidoesters are readily attacked by nucleophiles and react selectivelywith - and -amino groups of proteins at pH 8.59.5 to formamidines.

* Amidines are stable in neutral or acidic solutions, but hydrolyze slowlyat 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 thefree amine.

G. Reactions with Glutaraldehyde

Glutaraldehyde: OCHCH2CH2CH2CHO

Procedure:

(1) Treat polymer supports (containing primary amino groups) to yieldmatrices 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 widerange 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 salts—azo coupling:

Coupling of proteins to polymeric diazonium salts:

The specificity of azo coupling is rather broad.

* The electrophilic aryldiazonium ion attacks mainly activated aromaticrings, such as phenols (tyrosine) or imidazole (histidine), reacting atpH 89.

* Amino groups (the -amino group of lysine) react under similarconditions.

* The guanido group (arginine) and indole (tryptophan) also undergocoupling, 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 intodisulfide links, SS.

Coupling of proteins:

The supportSSprotein bonds are stable under nonreducingconditions; however, it can be reversed with low-molecular-weightsulfhydryl 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 aqueousmedium at neutral pH and allow for considerable versatility and highselectivity 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:

H O C H 2 C C H 2O H

N H 2

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 andtemperature; 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).

( CHCH2 )nHNO3, H2SO4 (cat.)

Nitration( CHCH2 )n

NO2

Fe, 30% HCl, heat Na2CO3

Reduction

NH2

( CHCH2 )n

NaNO2, HCl

Diazotization( CHCH2 )n

N2Cl

proteinazo coupling

* Remarks: the amount of bound protein is rather low because of thehydrophobicity 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 bindingcapacities and hydrophilicities.

Increasing the content of hydrophilic groups increases thehydrophilicity, 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:

CNH2

OH2NNH2 or HNO2

O

CN3

Coupling of enzyme (via acylation):

CN3

OH2N-protein

pH 8.5, 0.05 M borate,1 h at 0oC

CNH-protein

O

D. Polyvinyl alcohol

A hydrophilic, electrically neutral support.

E. Polyamides

The most important synthetic polyamides are nylons.

* Nylon-6,6 or -6,10: formed by the condensation of diamines withdicarboxylic 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 enzymeimmobilization.

* Enzyme immobilization by adsorption:

“Enzymes are adsorbed on the surface of a nylon structure and then cross-linked 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 amineand 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 furtherchemical modification.

The polyanionic enzyme conjugates exhibit improved stability at alkalinepH values; conversely, the polycationic derivatives show higher stabilityat 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 Å and 28% porevolume

(3) Mild caustic treatment

Remove siliceous residue from pore interiors.

Enlarge the pore diameter (up to 3000 Å); rather narrow poresize distribution

* In most of the work on enzyme immobilization, CPG particles of 550 Åpore 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 alkalineconditions.

CPG treated with a hydrophilic silane (e.g., -aminopropyltriethoxysilane) shows improved durability.

-aminopropyltriethoxysilane:C2H5OSiCH2CH2CH2NH2

OC2H5

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 inacetone.

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 andtemperature.

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 RNHCCl RN=C=O

O O

POLYSACCHARIDES AS SUPPORTS FOR ENZYMEIMMOBILIZATION

Polysaccharides commonly used as support materials:

(1) Cellulose—composed of linear chains of 1,4-linked -D-glucose residues

(2) Starch—composed of linear chains of 1,4-linked -D-glucose residues

(3) Dextran—a linear water-soluble polysaccharide composed of 1,6-linked-D-glucose residues; produced by microorganisms of the genus ofLeuconostoc

(4) Agarose—one of the components of agar; a complex mixture ofpolysaccharide; extracted from red sea-water algae; composed ofalternating 1,3-linked -D-galactose and 1,4-linked 3,6-anhydro--L-galactose 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 thelinear polysaccharide with epichlorohydrin

Epichlorohydrin:CH2CHCH2Cl

O

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, andhydrophobicity of the support surface.

(2) Stability and retention of enzyme activity

It is dependent on functional groups on support material andmicroenvironmental conditions.

Usually, immobilization results in a loss in enzyme activity andstability.

Methods might be considered for determining the amount of bound protein:

(1) Measurement of the difference between the amount of protein put into thereaction mixture and recovered in soluble state

Comment: only a rough estimate

The determination of a low protein concentration in a largevolume of wash solution cannot be accurate.

(2) Measurement of the difference between the activity input and activityrecovered in the soluble fraction

Comment: misleading

Enzyme activity may lose owing to inactivation under theconditions 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 linkagebetween enzyme and carrier.

Low yield obtained.

* Candidates for the calculatio of protein content: amino acids withaliphatic side chains and not undergoing destruction duringhydrolysis.

* 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 inmost 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 toprotect the active site.

(4) Denaturation or inactivation under the binding conditions

* In general, the more enzyme bound and the longer the time taken forimmobilization, the lower the retention of activity.

At present, there seem to be few guidelines for the choice of immobilizationtechnique to obtain good activity retention.

An ideal support of universal applicability cannot be anticipated, owingto the compositional and structural diversity of proteins.

The immobilization of an enzyme requires an empirical, essentially trialand error approach.