Immobilization of enzymes

Immobilization
of enzymes

Content

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

Immobilization

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

  Advantages:

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

       Example:

     
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

(g)Charcoal

Types of Immobilization

Based on support or
matrix and types of bonds involved

         Adsorption method

         Covalent binding method

         Cross linking method

         Entrapment method

         Microencapsulation

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

â

Water-jacketed column

â

Preconditioning
solution

â

Enzyme solution
circulated at desired temp for several hours

â

Enzyme solution
drained the column wash with water

â

Immobilized enzyme
column

Advantages:

Ø   Simple process, economical (cheapest)

Ø   Limited loss of activity

Ø   Immobilized enzyme and matrix can be recycled,
regenerated

Disadvantages:

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

Advantages:

Ø  No
enzyme leakage or desorption problem

Ø   Higher stability

Ø  A
variety of support with different functional groups available

Ø  Strong
linkage

Disadvantages:

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

Enzymes

                   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

Disasdv: 

        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

Enzymes

                                  â Physically entrapped

Porous matrix

(Water soluble
polymers)

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

(Covalent or
non-covalent bond)

â

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

Advantages:

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

Disadvantages:

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

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

Precursor

Product

Penicillin

amidase (E.coli)

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

Penicillin G

6-APA

Penicillin

amidase (E.coli)

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

Penicillin V

6-APA

Penicillin

amidase (E.coli)

Trapping into cellulose triacetate fibres

6-APA

Ampicillin

Penicillin

amidase (E.coli)

Trapping into cellulose triacetate fibres

D-phenyl glycine methyl ester

Amoxycillin

Other antibiotics:

Antibiotics

Immobilized enzyme or cells

Cephalosporin derivates

Cephalosporin amidase

Bacitracin

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

Compounds

Immobilized enzyme

Used  in

Dopamine

B-Tyrosinase

Parkinsonism

Vitamin. B12

Propioni bacterium species

As. Vitamin

High fructose corn syrup

Glucose isomerase

Commercial application

Proinsulin

Bacillus substilis cells

Diabetes

Summary

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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