Immobilization of enzymes

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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

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

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)

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