Covalent Bonding

2.2. Covalent Bonding

In this particular instance the ‘enzyme molecules’ in question are duly attached to the carrier matrix by the formation of covalent bonds. Consequently, the actual strength of bondage happens to be quite strong ; and hence, there is absolutely no loss of enzyme during usage. The formation of colvalent bond usually takes place particularly with the side chains of amino acids present in the enzyme ; how- ever, their actual strength of reactivity being exclusively linked to the status of ‘charge’ present in them as given below :

—S – > — SH > — O – > — NH > — COO – > — OH >> — NH + 2 3 Thus, the various functional moieties mostly present in enzyme which actively take part in the

formation of the numerous viable chemical bonds are : sulphide, sulphhydril, oxide, amino, carboxyl, hydroxyl, ammonium, imino, amide, methylthiol, guanidyl, imidazole, and phenol ring.

The covalent bonding of an enzyme may be accomplished either by activating the polymer with

a reactive moiety (i.e., copolymerization with ethylene, anhydride of maleic acid) or by effectively em- ploying the bifunctional reagent to serve as a bridge between the two entities : enzyme and polymer, whereby 3D-network may be obtained by cross-linking with low molecular weight bifunctional agent(s). In doing so, the enzyme invariably may get inactivated because the reactions normally engage a functional moiety strategically located at the ‘active site’ of the enzyme. Thus, the overall net effect being the substantial loss of enzymatic activity. Importantly, such an overwhelmingly loss in the enzymatic activity may be overcome by judiciously carrying out the ‘enzyme immobilization’ either in the presence of a competitive inhibitor or an enzyme substrate.

COVALENT BONDING

EM

EM = ENZYME MOLECULE

CROSS LINKING

Fig. 5.2. Covalent Bonding and Cross Linking of Enzyme Immobilization.

Fig. 5.2 Illustrates the covalent bonding and cross linking explicitely. The activated polymers that are solely employed are : hydrogels duly incorporated with a host of

typical chemical entities, such as : azide group ; diazo group ; carbodiimide group. The various enzymes immobilized by covalent bonding using typical carrier matrix and binding reaction are summarized below :

S.No. Enzymes

Carrier Matrix Used

Binding Reaction Involved

1 α -Amylase

DEAE-Cellulose*

Direct coupling

2 Amyloglucosidase

—do—

Cyanuric chloride

4 Glucose Isomerase

—do—

—do—

5 Glucose Oxidase

Porous Glass

Carbodiimide Activation

ENZYME IMMOBILIZATION

Advantages of Covalent Bonding : The various important advantages of the covalent bonding in enzyme immobilization are as enumerated below :

(1) Adsorption of enzymes to the carrier matrices is quite easy and convenient, and hence used extensively.

(2) Covalent bonding attachment is not reversed by pH, ionic strength or substrate. (3) Relatively broader spectrum of bonding reactions, and of matrices with functional moieties

capable of either having covalent bondage or prone to be activated to yield such groups renders this method into a highly acceptable one.

(4) Support with Functional groups : Various typical examples using support with functional groups are :

CHOH CH O C ≡ N CH  O () a + BrCN

C = NH CHOH

[Very R ea ctiv e]

[R ea ctiv e]

E  NH 2 + Enzyme  N H (p H 8 .9 ) 2

+ EN NH ZYM

CH O C NH ENZYME   CH O C NH ENZYME   CH OH 

CH OH 

IM M O B IL IZ E D E N Z Y M E A C T IVAT IO N O F S E P H A R O S E

() b CHOH + 2

+ ENZYME  NH 2

T R IA Z IN E A C T IVAT IO N OF SUPPORT

Fig. 5.3. Immobilization of Enzymes ; Using Supports with – OH Moieties that are Activated by Covalent Bonding with : (a) Cyanogen Bromide ; and (b) Triazine.

PHARMACEUTICAL BIOTECHNOLOGY

(a) 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 as illus- trated in Fig. 5.3(a) and (b). The reaction with the enzyme protein in each instance

involves the –NH 2 moiety of lysine.

(b) With – COOH Groups : Carboxymethyl cellulose (CMC) may be activated either via acyl-isourea formation or azide derivative formation as given in Fig. 5.4(a) and (b) be- low. The reaction essentially involves the participation of amino (– NH 2 ) moiety present in lysine ; besides, a host of other amino acids viz., cysteine, serine, tyrosine — are also made use of in the covalent bonding phenomenon.

Fig. 5.4. Immobilization of Enzymes using CMC Supports Having — COOH with — NH 2

Group or with Hydrazine (NH 2 –NH 2 ) Group via Covalent Bondage Involving :

ENZYME IMMOBILIZATION

(c) With – NH 2 Group : The amino functional moiety containing support material may be converted easily to the corresponding diazonium chloride salt by suitably treating with

a mixture of sodium nitrite (NaNO 2 ) and diluted hydrochloric acid (HCl) between 0-5°C (diazotization). The enzyme protein gets hooked up with the resulting diazotized deriva- tive thereby establishing an appropriate azo-linkage involving the corresponding the tyrosine residue of enzyme protein as illustrated in Fig. 5.5(a). In certain specific instances one may make use of glutaraldehyde (i.e., an aliphatic aldehyde) to

strategically activate the support material essentially having the —NH 2 functional group. Thus, the reaction predominantly involved as Schiff’s base formation between two entities, namely : (i) amino moiety of the support material ; and (ii) amino moiety of one of the amino acids present in the protein, as depicted in Fig. 5.5(b).

Interestingly, it has been observed that the exact number of bonds existing between the support material and the corresponding enzyme molecule in the course of the very covalent bondage is dis- tinctly variable, such as : papain molecule* has 17 covalent bonds ; and subtilopeptidase immobilized duly on semialdehyde starch analogue possesses 8 covalent bonds.

* Papain — a proteolytic enzyme, is usually bound adequately to porous glass which is linked via three azo bondages per enzyme molecule.

PHARMACEUTICAL BIOTECHNOLOGY

Fig. 5.5. Immobilization of Enzymes using Supports with Specific —NH 2 Moiety Involving

(a) Formation of Diazonium Chloride ; (b) Activation with Glutaraldehyde.

Loss in Enzyme Activity : Immobilization by covalent bonding may ultimately lead to a certain extent of loss in enzyme activity due to the involvement of the specific active site in the process of immobilization. Another school of thought suggests that immobilization of the enzyme in a particular orientation affords either a distortion of the active site or renders it more or loss unavailable. However, the ensuing loss in enzyme activity may be minimised to a considerable extent by adopting the following methods, such as :

(a) Immobilization of the enzyme in the presence of its substrate at a saturated concentration only, and

(b) Incorporation of a ‘competitive inhibitor’ (i.e., it gets bound to the active site*). The above possible effects of enzyme mobilization exclusively by the help of covalent binding

upon the enzyme activity may be illustrated explicitely in Fig. 5.6.

CORRECT ORIENTATION AND E CONFORMATION [ACTIVE SITE AVAILABLE

-] ENZYME ACTIVE

E SITE INACCESSIBLE TO SUBSTRATE INCORRECT ORIENTATION [ACTIVE

-] ENZYME INACTIVE

INCORRECT CONFORMATION E [ACTIVE SITE DISTORTED -

ENZYME INACTIVE ]

E = Enzyme M = Matrix

Fig. 5.6. Enzyme Immobilization : Probable Effects by Covalent Bonding upon the Enzyme Activity. * In such a critical situation, the prevailing active site is duly preoccupied and held in the correct conforma-

ENZYME IMMOBILIZATION