Enzyme immobilization

Introduction
Enzymes are the most powerful catalysts:
High catalytic activity
Rate enhancements of 107-1017
High turnover number

High selectivity
Stereo-, chemo-, and regioselectivity

Mild reaction conditions

Near-ambient temperature and pH

Environmentally Friendly
Biodegradable
Easy to produce

Industrial Applications of Enzymes

Production Scale
[tpy]

Product

Enzyme

>1 000 000

High-fructose corn
syrup

Glucose isomerase

>100 000

Lactose-free milk

lactase

> 10 000

Acrylamide

nitrilase

> 1000

6-aminopenillanic
acid

Penicillin amidase

> 100

Ampicillin

Penicillin amidase

The catalytic power of enzymes allows for the production of many products on an

industrial scale. However, general enzyme stability has limited enzymatic catalyst in
industrial preparations.

Enzyme Stability
Denaturant Type

Target

End Product

Temperature

Hydrogen bonds

Highly disordered structure

Aggregates

Acids

Buried uncharged group

(histidine, peptide bonds)

Random coil

Alkali

Buried uncharged groups

(tyrosine, cysteine)

Random coil

Salts

Polar and non-polar groups

Highly disordered structure

Solvents

Non-polar groups

Highly disordered peptide with large helical

regions

Surfactants

Hydrophobic domains

Disordered with large helical regions

Denaturants disrupt the secondary structure of the enzymes resulting in catalytically

inactive enzymes
P. V. Iyer et al., Process
Biochemistry 43 (2008)
1019-1032

Overcoming Enzyme Stability Issues

Immobilization

Immobilization provides a fast method for stabilizing enzymes by restricting the enzymes
structural mobility
R. A. Sheldon, Adv. Synth.
Catal. 2007, 349, 1289-1307
P. Lopez-Serrano et al.,
Biotechnology Letters 24:

Historical Background:
( 1823 vinegar production, sludge, attachment to equipment )
50s 60s : immobilization of enzymes
( 1916 Nelson Griffin: invertase ads.on charcoal
1948 Sunmer: jack bean urease)
1950 1970: intensive investigations on immobilized enzymes and other
proteins
( e.g.antigens -> affinity chromatography )
1969 first industrial appt.of immobilized enzyme
Optical resolution of DL aminoacids with immobilized amino acylase (
Chibata et al. )
Since 1960 investigations on immobilized cells
Industrial applications of immobilized microbial cells:
1973
L aspartic acid -Escherichia coli (aspartase )
1974 L - malic acid Brevibacterium ammoniagenes
( fumarase)
1982
L alanin Pseudomonas dacunhae ( L-aspartate decarboxylase )

Immobilized Enzyme Systems

Enzyme Immobilization:
To restrict enzyme mobility in a fixed space.

Immobilization
Immobilized biocatalysts can be made from
killed cells, cell fragments, purified enzymes,
and respiring cells
Definition: Enzymes or cells which are
physically confined to a defined region in
space while retaining their catalytic activity
and have the ability to be repeatedly and
continuously used

Advantages of Immobilization

Higher dilution rates can be used in a CSTR.

Easier product recovery.
Ability of enzymes to provide pure products.
Possible provision of a better environment for enzyme activity
Lowered viscosity.
Easier enzyme recovery. providing the ability to control reaction
times and minimize the enzymes lost in the product.
Enzyme reuse is economically advantageous
(reduction in waste and catalyst production), for many reaction
cycles, lowering the total production cost of enzyme mediated
reactions
Stabilization of the enzyme: Immobilized enzymes typically have
greater thermal and operational stability than the soluble form of
the enzyme

Disadvantages of Immobilization
Mass transfer is inhibited
Reduced activity of enzymes
Bursting of immobilization medium due
to cell growth
Cells/enzymes can leak out of
immobilization medium

Enzyme Immobilization
There are two approaches for immobilizing
enzymes:
Carrier-bound immobilization
Carrier-free immobilization

P. Lopez-Serrano et al.,
Biotechnology Letters 24:
1379-1383, 2002

11

Immobilization criteria

There are a number of requirements to achieve a successful

immobilization:
The biological component must retain substantial biological activity
after attachment
It must have a long-term stability
The sensitivity of the enzyme must be preserved after attachment
Overloading can block or inactivate the active site of the immobilized
biomaterial, therefore, must be avoided

Immobilized Enzyme Systems

Methods of Enzyme Immobilization:
- Entrapment

- Surface Immobilization
- Cross-linking

Immobilization methods
a)
b)
c)
d)

adsorption
covalent binding
entrapment
encapsulation

Immobilization Techniques
Figure 1. Classification of enzyme immobilization methods

Properties of support material

The form, shape, density, porosity, pore size distribution, operational

stability and particle size distribution of the supporting matrix will
influence the result
The ideal support is cheap, inert, physically strong and stable
Ideally, it should:
increase the enzyme specificity (kcat/Km)
shift the pH optimum to the desired value for the process
discourage microbial growth and non-specific adsorption

Some matrices may possess other properties which are useful for
particular purposes such as
ferromagnetism (e.g. magnetic iron oxide, enabling transfer of the biocatalyst
by means of magnetic fields)
a catalytic surface (e.g. manganese dioxide, which catalytically removes the
inactivating hydrogen peroxide produced by most oxidases)

Table 2. Chemical classification of matrixes used for enzyme

immobilization

Natural polymers

Minerals

Polysaccharides

Attapulgite clays

Synthetic
polymers

Cellulose

Bentonite

Polystyrene

Starch

Kieselghur

Dextran

Pumice stone

Synthetic
materials
Nonporous glass

Polyacrylates and poly-

Controlled pore
glass

Agar and agarose

methacrylates

Controlled pore
metal

Alginate

Polyacrylamide

Carrageenan
Chitin and chitosan

Hydroxyalkyl
methacrylates

Proteins

Glycidyl methacrylates

Collagen

Maleic anhydride
polymers

Gelatin
Albumin
Silk
Carbon materials
(activated carbon)

Vinyl and allyl

polymers

Polyamides

oxides
Metals

 Carrier-Binding
The oldest immobilization technique for enzymes
Some of the most commonly used carriers for enzyme
immobilization are polysaccharide derivatives such as
cellulose, dextran, agarose, and polyacrylamide gel.
The selection of the carrier depends on the nature
of the enzyme itself, as well as the:
- Particle size
- Surface area
- Molar ratio of hydrophilic to hydrophobic groups
- Chemical composition
the carrier-binding method can be further sub-classified into:
Physical Adsorption
Covalent Binding
Ionic Binding

1-1 : Physical Adsorption

Of enzyme protein on the surface of waterinsoluble carriers.

Advantages : no reagents and only a minimum of

activation steps are required

Disadvantages : the adsorbed enzyme may leak

from the carrier during use due to a weak
binding force between the enzyme and the
carrier. Moreover, the adsorption is nonspecific, further adsorption of other proteins
or other substances

Immobilization by Adsorption
Binding forces are ionic, hydrophobic,
hydrogen bonds, or Van der Waals
interactions
Binding is simple (stir together in a
beaker) but is reversible. Substrate
addition can cause desorption.

Typical Adsorbents

Cellulose
Polystyrene resins
Kaolinite
Glass
Alumina
Silica gel

Adsorption and Ionic binding

Simplest immobilization method

Mix the enzyme and support in suitable conditions

First immobilized enzyme model: invertase on the activated charcoal

(Nelson and Griffin, 1916)
Forces are weak so leakage is generally a problem
Supports such as alluminium hydroxide are often utilized
With a suitable charged matrix, ionic interactions may also be promoted
This technique is technically undemanding and economically attractive
Regeneration is easy
Best known industrial example: amino acylase immobilized on DEAESephadex in the production of amino acids

Adsorption
simple adsorption of
cell/enzyme onto support
(carrier) with adsorptive
properties
anion exchange resins (DEAE
cellulose, Sephadex)
cation exchange resins
(carboxymethylcellulose)

1-2 : Covalent Binding

Based on the binding of enzymes and water-insoluble carriers by
covalent bonds
The functional groups that may take part in this binding are Amino
group,
Carboxyl
group,
Sulfhydryl
group,
Hydroxyl
group, Imidazole group, Phenolic group, Thiol group, Threonine
group,Indole group

Disadvantages : covalent binding may alter the conformational

structure and active center of the enzyme, resulting in major loss
of activity and/or changes of the substrate

Advantages : the binding force between enzyme and carrier is so

strong that no leakage of the enzymes occurs, even in the
presence of substrate or solution of high ionic strength.

Covalent Bonding
Most extensively used method of
immobilization due to high bond
strength
Three elements- structural polymer,
enzyme molecule and bridge molecule
Activity of an immobilized enzyme is
a strong function of the
hydrophilicity of the structural
polymer

Covalent Bonding To Support

Bridging molecule needs to be small, have two
reactive groups, and not bond at the active site

Copolymerization of Enzymes
Copolymerization is performed with
multifunctional bridge molecules to yield
and insoluble product
Usually an inert protein is also included

Chemistry of Covalent Immobilization

CLECs - Glutaraldehyde
A proposed mechanism for the
reaction between glutaraldehyde and
proteins

Cross-linking by glutaraldehyde could occur through schiff base formation but the mechanism is still not fully elucidated.
Thus, the optimal concentration of glutaraldehyde for cross-linking is determined empirically.

1-3 : Ionic Binding

Of the enzyme protein to water-insoluble
carriers containing ion-exchange residues

Polysaccharides and synthetic polymers having

ion-exchange centers are usually used as
carriers

Advantages : the enzyme to carrier linkages is

much stronger for ionic binding

Disadvantages : the binding forces between

enzyme proteins and carriers are weaker than

those in covalent binding

Cross-Linking
Either to other protein molecules or to functional
groups on an insoluble support matrix
It is used mostly as a means of stabilizing
adsorbed enzymes and also for preventing leakage
from polyacrylamide gels
The most common reagent used for
cross-linking is glutaraldehyde

Disadvantages : Cross-linking reactions are

carried out under relatively severe conditions.

These harsh conditions can change the
conformation of active center of the enzyme; and
so may lead to significant loss of activity.

Gel-fibre entrapment and encapsulation

Entrapment

Enzymes may be entrapped within the matrix of a polymeric gel

Incubate the enzyme together with the gel monomers
Promote gel polymerization
Polyacrylamide and polymethacrylamide gels are examples
Gel pore size is a crucial factor

Encapsulation

Encapsulation involves entrapping the enzymes within a semipermeable

membrane such as cellulose nitrate and nylon-based membranes

Immobilized Enzyme Systems

Entrapment
- Matrix Entrapment

- Membrane Entrapment
(microencapsulation)

Entrapping Enzymes
Based on the localization of an enzyme within the lattice
of a polymer matrix or membrane
It can be classified into lattice and micro capsule types.
This method differs from the covalent binding and cross
linking in that the enzyme itself does not bind to the gel
matrix or membrane. This results in a wide applicability

Disadvantages : The conditions used in the chemical

polymerization reaction are relatively severe and result

in the loss of enzyme activity.

Immobilized Enzyme Systems

Matrix Materials:
Organics: polysaccharides, proteins, carbon, vinyl and allyl
polymers, and polyamides. e.g. Ca-alginate, agar,
K-carrageenin, collagen

Immobilization procedures:
Enzyme + polymer solution polymerization
extrusion/shape the particles

Inorganics: activated carbon, porous ceramic.

Shapes: particle, membrane, fiber

Immobilized Enzyme Systems

Entrapment
Challenges:
- enzyme leakage into solution
- diffusional limitation

- reduced enzyme activity and stability

- lack of control micro-environmental
conditions.
It could be improved by modifying matrix or
membrane.

Immobilized Enzyme Systems

Entrapment Immobilization is based on the
localization of an enzyme within the lattice of a
polymer matrix or membrane.
- retain enzyme
- allow the penetration of substrate.
It can be classified into matrix and micro
capsule types.

Alginate Matrix
Binding of Ca2+ to G

Eggbox model for

Ca2+ binding

Structure of the
Alginate-Ca2+ Matrix

Cell entrapment protocol

- external gelation
M

Ca++

Dropwise addition of
alginate/cells into CaCl2
gelation bath

DNA entrapment protocol

- emulsification/internal gelation
Ca++

alginate droplet
containing DNA,
microcrystalline CaCO3
alginate in oil
emulsion

6.5

7.5

CaCO3

Membrane coating
polyanion core
(alginate)

polycation
membrane
chitosan
poly-L-lysine
co-guanidine

oil recycle
canola oil: 40oC

KCl

carrageenan: 40oC

5oC
static mixer

separator
settler

yeast
40oC

static mixer
carrageenan beads
to bioreactor

Immobilized yeast
technology

Kenics static mixer to

encapsulate brewing
yeast

Continuous brewing

gas out
beer out
bead disengagement
section

draft tube
temperature
control jacket
medium in
sparger - air in

Labatt continuous
airlift reactor

Microencapsulation
spherical ultrathin semipermeable membrane
enclosing cell/enzyme
suspension/solution
interfacial polymerization
reaction (nylon)
NH2(CH2)6NH2 + ClCO(CH2)8COCl
NH2(CH2)6NH-CO(CH2)8CONH(CH2)6NH-CO(CH2)8CO-

nylon 6-10 polyamide

HCl

Microencapsulation protocol
- interfacial polymerization
oil soluble
cross-linker

M
chitosan

cells/chitosan
in oil emulsion

Encapsulation of lobster carotenoids as

natural food pigment

Membranes

Membrane Microencapsulation
Membrane polymerized around aqueous
enzyme solution in colloidal suspension
(particle sizes on the order of 100-10 m)
mixing

Organic solvent

Aqueous enzyme solution

Add polymer

Microencapsulation
Advantages
High surface to volume ratio
Thin membrane
Relatively benign attachment
method
Problems
More easily damaged than gel beads

Other Membrane Immobilization

Methods- HFM
Hollow fiber reactors use micro- or ultrafiltration membranes to retain high MW
enzymes, but pass low MW compounds
Substrate and products
outside the membrane

Substrate diffuses
through membrane

Product diffuses out

of the membrane
Enzymes or
cells

Close-up view of fiber

Hollow Fiber Reactor

Enzymes or cells in

Substrate in

Product out

Two Types of Cartridges

Comparison of immobilization techniques

adsorption and gel entrapment
simple, gentle and efficient
enzyme/cells often released (leaky); solved by cross-linking
gas buildup may be problem

microencapsulation

size exclusion (eg. antibodies)

only small substrates can be used
may lead to inactivation

covalent attachment and cross-linking

strong attachment
laborious and expensive
often leads to significant inactivation

Kinetic Properties

There is usually a decrease in specific activity of an enzyme upon

insolubilization: denaturation caused by the coupling process
Microenvironment after immobilization may be drastically different from
that existing in free solution: the physical and chemical character of the
support matrix, or interactions of the matrix with substrates or products
involved in the enzymatic reaction
The Michaelis constant may decrease by more than one order of magnitude
when substrate of opposite charge to the carrier matrix

The diffusion of substrate can limit the rate of the enzyme reaction: the
thickness of the diffusion film determines the concentration of substrate
in the vicinity of the enzyme and hence the rate of reaction
The effect of the molecular weight of the substrate can also be large. This
may be an advantage in some cases, since the immobilized enzymes may
be protected from attack by large inhibitor molecules

Effects of solute diffusion on the kinetics of

immobilized enzymes

external diffusion the transport of

substrates towards the surface, and
products away
internal diffusion the transport of
the substrates and products, within
the pores of immobilised enzyme
particles

Reaction kinetics or mass transfer control

diffusional resistances
minimized by
decreasing particle size
(increase surface
area/volume ratio)
increasing [R]bulk
improved mixing,
agitation
increasing porosity
optimizing distribution
of enzyme/cells

boundary film

Rbulk

Vmax [R]
v
K m [R]

CH3CHOHCOOH + O2

CH3COOH + CO2 + H2O

L-lactate-oxygen 2-oxidoreductase

Kinetics of immobilized enzymes

Partitioning effect
The solution lying within a few molecular diameters (10 nm)
from the surface of an immobilized enzyme will be influenced
by both the charge and hydrophobicity of the surface
The Km of an enzyme for a substrate is apparently reduced if [S]
in the vicinity of the enzyme's active site is higher than that
measured in the bulk of the solution

Kinetics of immobilized enzymes

A high concentration of ionising groups may cause a partitioning of gases

away from the microenvironment with consequent effects on their apparent
kinetic parameters
It is also a useful method for protecting oxygen-labile enzymes by 'salting out'
the oxygen from the vicinity of the enzyme
Partition of hydrogen ions The pH of the microenvironment may differ
considerably from the pH of the bulk solution

Enzyme immobilised on charged supports:

free enzyme
enzyme bound to a (+)ly charged
support; a bulk pH of 5 is needed to
produce a pH of 7 within the
microenvironment
enzyme bound to a (-)ly charged
support; a pH of 7 within the
microenvironment is produced by a
bulk pH of 9

Kinetics of immobilized enzymes

If the surface is predominantly hydrophobic
Hydrophobic molecules will partition into the microenvironment of
the enzyme and hydrophilic molecules will be partitioned out into the
bathing solution
Partition will affect the apparent kinetic constants of the enzyme

E.g. the reduction in the Km of immobilised alcohol

dehydrogenase for butanol
If the support is polyacrylamide, the Km is 0.1 mM but if a more
hydrophobic copolymer is used as the support, the Km is reduced to
0.025 mM

Kinetics of immobilized enzymes

A similar effect may be seen in the case of competitive inhibitors

Invertase
Ki, mM

free

Bound to PS
(hydrophobic)

Aniline (hydrophobic)

0.94

0.39

Tris-(hydroxymethyl)aminomethane
(hydrophilic)

0.45

1.20

Kinetics of immobilized enzymes

Enzymatic depolymerisation (including hydrolysis) of macromolecules may

be affected by diffusional control
Large molecules diffuse fairly slowly. After reaction, the cleaved fragments
normally retain their ability to act as substrates for the enzyme
They are likely to be cleaved several times while they are in the vicinity of
the immobilised enzyme
This causes a significant difference in the molecular weight profiles of the
fragments produced by the use of free and immobilised enzymes
Immobilized enzyme produces a small quantity of well-hydrolysed low
molecular weight product with the majority of the substrate molecules
unchanged

Kinetics of immobilized enzymes

Co-immobilization of the necessary enzymes for the pathway results in a

rapid conversion through the pathway due to the localized high
concentrations of the intermediates
The reduction in the apparent lag phase is most noticeable when there
are more enzymes in the pathway

It is least pronounced where the

flux through the pathway is
controlled by the first step
Mixture of free enzymes
Co-immobilized enzymes

Application of immobilized enzymes

Bioreactors
Large scale production or conversion of various compounds

Tannase from Aspergillus oryzae to

hydrolyze tea tannins
tannins represent 25% of extractables
in tea leaves
cause creaming (turbidity) on cooling
desire tea to be clear and bright
tannase controlled hydrolysis of tannins,
retaining flavour
encapsulated tannase remained stable
for 1 month
3 successive batch cycles during
48 h processing

Application of immobilized enzymes

Biosensors
An analytical device which can detect and quantify specific analytes in complex
samples

Sample
Solution

Biological
Detection Transducer
Element
Signal
Processor
Readout
Signal

Immobilized Enzyme Reactors

Recycle packed column reactor:

- allow the reactor to operate at high fluid velocities.

Fluidized Bed Reactor:

- a high viscosity substrate solution
- a gaseous substrate or product in a continuous reaction system
- care must be taken to avoid the destruction and
decomposition of immobilized enzymes

- An immobilized enzyme tends to decompose

upon physical stirring.
- The batch system is generally suitable for the production
of rather small amounts of chemicals.