Immobilization of enzymes

Immobilization of enzymes


Ø  Definition

Ø  Advantages

Ø  Support or matrix materials

Ø  Methods of immobilization

Ø  Application of immobilized enzymes

Session Objectives

At the end of the session, student will be able to

Ø  Discuss advantages of immobilization

Ø   Discuss the support or matrix materials used

Ø  Explain the various methods of immobilization

Ø  Discuss the application of immobilized techniques


Ø  Enzymes – biocatalyst – promote the rate of reactions – but not themselves consumed in reactions

Ø  They may be used repeatedly – as long as they remain active

Ø  In most processes – enzymes are mixed in a solution with substrate – cannot be economically recovered after the reaction – generally get wasted

Ø  Hence enzymes are used – immobilized or insolubilised form – so that they may be retained in a biochemical reactor for further catalysis

Ø  Done by enzyme immobilization

Ø  Immobilization - imprisonment of cell or enzyme in a distinct support or matrix

Ø  The support or matrix on which the enzymes are immobilized allows the exchange of medium containing substrate

Ø  The practice of immobilization of cells is very old –

Ø  First immobilized enzyme -Nelson & Griffin -  adsorption of invertase on charcoal - 1916


Ø   Better stability

Ø   Increased functional efficiency of enzymes -  Better efficiency

Ø   Enzyme can be recovered and can be reused repeatedly

Ø  Enhanced reproducibility of the process

Ø  Less chance of contamination in products

Ø   Improved process control - ability to stop the reaction rapidly by removing the enzyme from  the reaction solution

Supports or Matrix used in immobilization technology

       The matrix or support immobilizes the enzyme by holding it permanently or temporarily for a brief period of time

       Wide variety of matrixes or carriers or supports available for immobilization

       Matrix -  cheap and easily available

       Their reaction with the components of the medium or with the enzyme should be minimum as possible

Supports or Matrix

       The matrixes or supports for immobilization of enzymes or whole cells are grouped into three major categories

(1)  Natural polymers

(2)  Synthetic polymers

(3)  Inorganic materials

Natural Polymers

(a) Alginate:

        Derived from the cell wall of some algae

        Calcium or magnesium alginate - commonly used matrix

       They are inert and have good water holding capacity.

(b) Chitosan and chitin:

       Polysaccharides - cell wall of fungi - exoskeleton of Arthropods

        The various functional groups in enzymes can bind to the – OH group of chitin and can form covalent bonds  

(c) Collagen:

       Main structural protein – animal connective tissue

       Proteinaceous support -  good porosity and water holding capacity

       The side chains of the amino acids in the collagen + of enzyme can form covalent bonds to permanently hold the enzyme to the support

(d) Carrageenan:

       Sulfated polysaccharide - some red algae

       Good gelling properties together with its high protein holding capacity makes it good support for immobilizing enzymes

(e) Cellulose:

       Most abundant polymer of nature

       Cheapest support available as carrier of enzymes

       The hydroxyl group - monomer units (glucose) can form covalent bonds with that of the amino acids of enzyme

(f) Starch:

        A natural polymer of amylose and amylo-pectin

        It has good water holding capacity

(g) Pectin:

       Structural polysaccharide -  plants - primary cell wall -  they also acts as the inter-cellular cementing material in plant tissues

       Gelling agent with good water holding capacity

Synthetic polymers

       Ion exchange resins or polymers -  insoluble supports with porous surface

        Their porous surface can trap and hold the enzymes or whole cells


      Diethylaminoethyl cellulose (DEAE cellulose)

      Polyvinyl chloride (PVC)

      UV activated Polyethylene glycol (PEG)

Inorganic Materials

(a) Zeolites:  

     They are microporous, alumino silicate minerals with good adsorbing properties and extensively used for immobilizing enzymes and whole cells

(b) Ceramics:

     They are nonmetallic solids consisting of metal and nonmetal atoms held in ionic and covalent bonds

(c) Diatomaceous earth:

       Siliceous sedimentary rocks - formed - fossilized accumulations of the cell wall of diatoms

        Celite - trade name - diatomaceous earth

       It is a good adsorbent and are resistant to high pH and temperature

(d) Silica

(e) Glass

(f) Activated carbon


Types of Immobilization

Based on support or matrix and types of bonds involved

         Adsorption method

         Covalent binding method

         Cross linking method

         Entrapment method


Adsorption Method of Immobilization

       Oldest and simplest method of enzyme  immobilization

       Nelson & Griffin -  charcoal to adsorb invertase for the first time in 1916

        Enzyme is adsorbed to external surface of the support

       No permanent bond formation between carrier and the enzyme in adsorption method

The weak bonds (low energy bonds) involved are mainly:

(a) Ionic interaction

(b) Hydrogen bonds

(c) Van der Waal forces

       For significant surface bonding the carrier particle size must be small (500 Å to 1 mm diameter)

       The greatest advantage of adsorption method is that there will not be “pore diffusion limitations” since enzymes are immobilized externally on the support or the carrier

Adsorbents :Alumina, Amberlite CG, Bentonite, CMC, Calcium phosphate , Porous carbon, Silica gel, clay, glass, collagen, cellulose, titania etc

Adsorbent pack


Water-jacketed column


Preconditioning solution


Enzyme solution circulated at desired temp for several hours


Enzyme solution drained the column wash with water


Immobilized enzyme column


Ø   Simple process, economical (cheapest)

Ø   Limited loss of activity

Ø   Immobilized enzyme and matrix can be recycled, regenerated


Ø   Desorption of enzymes from carrier

Ø   Enzyme is exposed and can be prone to proteolytic and microbial attack

Covalent binding Method of Immobilization

       Formation of covalent between – chemical groups of carrier and enzymes

Chemical groups – Carrier

       Amino groups

       Imino groups

       Hydroxyl groups

       Carboxyl groups

       Thiol groups

       Imidazole groups

Chemical groups – enzyme

       Alpha carboxyl group at ‘C’ terminal of enzyme

       Alpha amino group at ‘N’ terminal of enzyme

       β and γ carboxyl groups of Aspartate and Glutamate

       Phenol ring of Tyrosine

       Thiol group of Cysteine

       Hydroxyl groups of Serine and Threonine

       Imidazole group of Histidine

       Indole ring of Tryptophan

       Carriers or supports commonly used for covalent bonding are:

 Carbohydrates: Eg. Cellulose, cellulose, Agarose

 Synthetic agents: Eg. Polyacrylamide

 Protein carriers: Collagen, Gelatin

 Amino group bearing carriers:  Eg. Amino benzyl cellulose

 Inorganic carriers: Porous glass, silica

 Cyanogen bromide (CNBr) - agarose and CNBr Sepharose


Ø  No enzyme leakage or desorption problem

Ø   Higher stability

Ø  A variety of support with different functional groups available

Ø  Strong linkage


Ø  Enzyme may be partially or wholly inactivated by active site modification - overcome through immobilization in the presence of  a competitive inhibitor

Ø  Chemical modification of enzyme – loss of functional conformation

Cross linking or Copolymerization method of Immobilization


                   â Cross link

Bi or multifunctional reagent

 Eg: Glutaraldehyde, diazobenzidine, N, N hexamethyl di iso cyanate, dimethyl derivative (adipimate, suberimate) etc

Basic approaches

Cross- linking of enzyme with glutaraldehyde

Adsorption of enzyme onto surface followed by cross-linking

Eg. Cross linking trypsin – adsorbed to colloidal silica particles


        Not suitable for macromolecules

        Hard to regenerate the immobilized enzyme for reuse

        Cross linking may denature or structurally modify the enzyme – loss  of catalytic activity

Entrapment method of Immobilization


                                  â Physically entrapped

Porous matrix

(Water soluble polymers)

(Calcium alginate, PAA, starch, Agar, Cellulose triacetate)

(Covalent or non-covalent bond)


Pore size is adjusted to prevent loss of enzyme - Done – adjusting the concentration of polymer used


Ø   Enzyme activity is not damaged

Ø   Fast method of immobilization

Ø  Cheap (low cost matrixes available)

Ø  Easy to practice at small scale

Ø  Mild conditions are required

Ø  Less chance of conformational changes in enzyme

Ø  Minimize enzyme leaching


Ø  Loss of enzyme activity due to free radicals produced during polymerization

Ø   Chance of microbial contamination

Applications of Immobilized Enzymes

Ø  Various immobilized enzymes are frequently in use in several large industrial processes

Ø  Some of the immobilized enzymes are utilized in drug manufacture and drug analysis

Drug Manufacture:

Ø  Production of antibiotics

Ø  Production of amino acids

Ø  Other medicinal compounds

i. Production of antibiotics


Ø  Production of semi-synthetic 6 – amino penicillinic acid (6-APA) is an important intermediate for the production of semi-synthetic penicillins like ampicillin, amoxycilin 

Ø  This intermediate is derived from penicillin G or penicillin V by acylation with enzymes penicillin amidase

Ø  Immobilized penicillin amidase is now being used in place of native or soluble enzyme

Immobilized enzyme (source)

Method of immobilization




amidase (E.coli)

Trapping into cellulose triacetate fibres or covalent bonding to CN Br-activated sephadex

Penicillin G



amidase (E.coli)

Absorption on Bentonite or covalent bonding to amberite XDA-7 with glutaraldeyde

Penicillin V



amidase (E.coli)

Trapping into cellulose triacetate fibres




amidase (E.coli)

Trapping into cellulose triacetate fibres

D-phenyl glycine methyl ester


Other antibiotics:


Immobilized enzyme or cells

Cephalosporin derivates

Cephalosporin amidase


Cells of Bacillus species

Tylosin and nikkomycin

Cells of Streptomyces species

iii. Production of amino acids

Ø    Amino acids are in great demand for their nutritional and medical applications

Ø   Chemical synthesis produces only racemic mixtures.

Ø   d-isomer of the racemic mixture generally devoid of nutritional value    

     Thus, it is desirable to obtain l-amino acids

Ø    Immobilized enzymes are helpful to get these physiologically active isomers

Ø    For example, immobilized amino acylase can be used for the production of l-amino acids

iv. Other medicinal compounds

Ø  A few notable examples are given below, which are produced by immobilized enzymes


Immobilized enzyme

Used  in




Vitamin. B12

Propioni bacterium species

As. Vitamin

High fructose corn syrup

Glucose isomerase

Commercial application


Bacillus substilis cells



Ø     Enzymes are biocatalysts, high m.w, Proteinaceous and water soluble compounds

Ø     Activity  is affected by temp, pH and heavy metals, specific in their action

Ø     Classification according to IUMAB and site of action

Ø     Enzymes have medicinal, food and industrial applications

Ø     Main source is plant, animal and micro organisms

Ø     Isolation involves extraction, preparation of crude enzyme, precipitation and purification

Ø  Imprisonment of enzyme or cell in / on a distinct phase that allows exchange with but it is separated from bulk phase

Ø    Stable, economical and reusable

Ø    Adsorption method

Ø    Imprisonment of enzyme or cell in / on a distinct phase that allows exchange with but it is separated from bulk phase

Ø    Stable, economical and reusable

Ø    Covalent binding, cross linking, entrapment and microencapsulation method

Ø  Immobilized enzymes are utilized in drug manufacture  and drug  analysis

Ø  Production of antibiotics, steroids, amino acids and other medicinal compounds


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