Immobilized Biocatalysts

Are enzymes, cells or organelles (or

combinations of these) which are in a
state that permits their reuse

Enzymes modified to a water insoluble

form

• Soluble enzymes retained within reactors

by a non-permeable ultrafiltration
membrane

• Soluble enzymes with their mobility

restricted by attachment to another
molecule.
 Why Immobilize?
Facilitates process control
(optimization of product yield and
quality)

Easier Enzyme/Product Recovery

(Immobilized Enzymes do not mix
with products)

More Stable Enzymes

Make continuous processes possible

Industrial Application Limited By:
Comparatively low cost of
soluble enzymes

Traditional attitudes

The investment needed for

introducing new equipment to
already implanted processes

The nature and cost of the

immobilizing support and the
immobilizing process (including
losses of activity)

The performance of the system

Characterizing Immobilized Enzymes
ENZYME
Biochemical Properties: mass, cofactors/
coenzymes, functional groups, purity, source,
availability
Kinetics: activity, pH- and T-profile,
inhibitors, stability, solvent tolerance
CARRIER
Chemical Properties: composition, functional
groups, swelling, matrix and pore size, stability
Mechanical Properties: mean wet particle diameter,
abrasion, compression behaviour, sedimentation
velocity, flow resistance
IMMOBILIZED ENZYME
Method: binding, yield, intrinsic kinetics
Mass Transfer: external and internal diffusion,
effectiveness, partitioning
Stability: operational, storage, handling
Performance: productivity, consumption

Immobilized Enzymes: Methods and Applications, W.Tisher and F.Wedekind, Topics Curr. Chem.
(1999) 200, 95-126
 Synthesized and chemically modified
acrylate- and styrene-based polymers

Synthetic polymers provide

several advantages, e.g.
inertness to micro-organism
attack, higher chemical
resistance, and the option to
use complex buffer components.
 Ideal Carrier/
Support Specifications
Cheap

Inert

Physically strong and stable

Increases enzyme specificity

Reduces product inhibition

Shifts the pH optimum to the desired value

Discourages microbial growth and nonspecific adsorption

Possess other properties useful for
particular purposes
(a) ferromagnetism: enables biocatalyst
transfer by means of magnetic fields
(b) catalytic surface: removes
inactivating hydrogen peroxide
produced by oxidases
(c) reductive surface environment: for
enzymes inactivated by oxidation

High porosity; Pore size distribution

must not hinder free diffusion of:
(a) enzyme into the carrier
(b) substrates to the enzyme
(c) products out of the carrier
 Immobilized Enzyme

Maximum Binding Capacity(surface density of binding

sites; volumetric surface area sterically
available to the enzyme)

Actual Capacity (no. of potential coupling sites in the

enzyme molecules; electrostatic charge distribution;
surface polarity or hydrophobic- hydrophilic balance)

Expressed Activity and Apparent Kinetics

(affected by nature of support)
 Immobilization Methods
Immobilized Enzymes

Attachment to Encapsulation/
Crosslinking
(Prefab)Carriers Inclusion

Covalent Direct Membrane

Binding Crosslinking Devices
Ionic
Binding Co-Crosslinking Microcapsules
Adsorptive
Binding Crystallization Liposomes/
+ Crosslinking Reversed
Metal
Micelles
Binding Copolymerization
(Bio)affinity After Chemical Organic
Binding Modification Solvents
Immobilization
Methods
 E
E
E
E E Gel
E
lattice
E Covalent E
E
E binding
E E E
E E
E Support E
E Binding Entrapment E
E
E E Fiber
E
Models of
E
E +- +
Immobilization E
-+ E -
E
E +- +
+ E
- -
+ E - E
E -
-
+ - + Crosslinking
- +
- +
E -
E Micro
Van der
+
-+
- +
E E E capsule
+
-
-
E
Waals -
+ + +
-
E
-
E E
binding -
+
-
+
E
-
+
E E
-+
E -
-
+ E
E
Ionic E
binding E
 Adsorption On Carriers

Simple way of immobilizing with a wide

range of applicability

Capable of high enzyme loading (1 g/g)

Can be accomplished by:

(a) Hydrogen Bonding
(b) Hydrophobic Interaction
(c) Ionic Forces

Materials for adsorption: silica gel,

(hydrous) metal oxides, glass, organic
polymers, porous carbon, clay, etc.
 Adsorptive Binding

MIXING Mixing enzyme with

suitable adsorbent

React at appropriate pH, ionic

INCUBATION
strength, and time

WASHING Wash off loosely bound and

unbound enzyme

Immobilized Enzyme in a
PRODUCT
directly usable form

Adsorbed enzyme applied in aqueous medium can be cross-linked to

minimize enzyme leakage but this eliminates the possibility for re-
using of carrier (acceptable when enzyme and carrier are inexpensive)
 Preparation of Immobilized
Invertase by Adsorption*

Support Type
% bound at DEAE-Sephadex CM-Sephadex
anion cation
exchanger exchanger
pH 2.5 0 100

pH 4.7 100 75

pH 7.0 100 34
*Woodward, 1985
 Covalent Binding
Small amounts of enzymes are immobilized
(~0.02 g/g to 0.3 g/g)

Very strong binding (less enzyme leakage)

Relative usefulness of various groups

dependent on their availability and
reactivity

Reactivity of protein side-chain nucleophiles

determined by their state of
protonation (pKA and pH dependent)

Often involve lysine residues (especially in

slightly alkaline solutions)
Relative Usefulness of Enzyme Residues
for Covalent Coupling
Stability
Residue Content Exposure Reactivity Use
of couple
Aspartate + ++ + + +
Arginine + ++ - ± -
Cysteine - ± ++ - -
Cystine + - ± ± -
Glutamate + ++ + + +
Histidine ± ++ + + +
Lysine ++ ++ ++ ++ ++
Methionine - - ± - -
Serine ++ + ± + ±
Threonine ++ ± ± + ±
Tryptophan - - - ± -
Tyrosine + - + ± +
C terminus - ++ + + +
N terminus - ++ ++ ++ +
Carbohydrate - ~ ++ ++ + + ±
Others - ~ ++ - - - ~ ++ -
 Covalent Coupling Reactions
O
OH
OH
+ NH2 ENZYME C
CN O
O
HN ENZYME

1. Coupling to CNBr-activated Polysaccharide

Simple, mild, successful method

Hydroxyl groups combine with CNBr to give the

reactive cyclic imido-carbonate. This reacts
with primary amino groups (i.e. mainly lysine
residues) on the enzyme under mildly basic
conditions (pH 9 - 11.5)
 Covalent Coupling Reactions
O
O NH2 H
O Si +
N H
O H
O
O
O N + NH2 ENZYME
O Si N H
O H
H
H
O N ENZYME
O Si N N
O H
H

2. Coupling to Dexolan DAP with butanedial/ glutaraldehyde

 Covalent Coupling Reactions

Glutaraldehyde is a bifunctional reagent which may be

used to cross-link enzymes or link them to supports

Useful for producing immobilised enzyme membranes

(biosensors) by cross-linking the enzyme plus a non-
catalytic diluent protein within a porous sheet (e.g.
lens tissue paper or nylon net fabric)

Use of trialkoxysilanes allows even such apparently

inert materials as glass to be coupled to enzymes
 Covalent Coupling Reactions
O O
+ NH2 ENZYME
O

3. Coupling to Eupergit C HO N ENZYME

H

4. Ethyl chloroformate

Produce similar intermediates as coupling with CNBr

without inherent toxicity

5. Carbodiimides

Very useful bifunctional reagents (allow the coupling of

amines to carboxylic acids)
Careful control of the reaction conditions and choice of
carbodiimide allow a great degree of selectivity in this
reaction.
The effect of
covalent
coupling on
the expressed
activity of an
immobilised
enzyme.

(a) Immobilised enzyme (E) with its active site unchanged

and ready to accept the substrate molecule (S), as shown in
(b). (c) Enzyme bound in a non-productive mode due to the
inaccessibility of the active site. (d) Distortion of the
active site produces an inactive immobilised enzyme.
 Crosslinking

In itself is both expensive and

insufficient (best used in conjunction
with one of the other methods)

It is used much more widely as a means of

stablilizing adsorbed enzymes (preventing
leakage from polyacrylamide gels)

ε-amino groups of the protein’s lysine

residues react with glutaraldehyde forming
a simple Schiffs base (Branner-Jørgensen)
 CLECS

Cross-linked Enzyme Crystals

Are insoluble and have high

operational stability in
aqueous systems as well as
in organic solvents
 Crosslinking
PROTEIN
(CH2)4 (CH2)4 (CH2)4
N NH2 N
Glutaraldehyde
CH CHO CH
(CH2)3 (CH2)3 (CH2)3
CH CH CH

N N N
(CH2)4 (CH2)4 (CH2)4

PROTEIN
 Crosslinking Whole Cells
FERMENTATION,
Enzymes are made available and at the
CENTRIFUGATION, CELL
same time contained in cell debris
DISPRUPTION

GLUTARALDEHYDE Proteins (enzymes/cells) are linked to

CROSS-LINKING each other through lysine residues

DILUTION AND Cationic flocculant is added to separate

FLOCCULATION cells from the water phase

FILTRATION AND Crosslinked aggregate is separated and

EXTRUSION extruded

DRYING AND Dried particles are screened to obtain a

SEIVING product of uniform size

IMMOBILIZED CELLS
 Entrapment of Enzymes
Often done in gels or fibres (with
polyacrylamide, calcium alginate, gelatin)

A convenient method for use in processes

involving low molecular weight substrates
and products

Entrapped amounts may be in excess of 1 g/g

May be a purely physical caging or involve

covalent binding

The method of choice for the immobilisation

of microbial, animal and plant cells, where
calcium alginate is widely used
 Entrapment Media
POLYACRYLAMIDE

widely used for entrapment of enzyme

non-ionic (enzyme properties and diffusion

characteristics are minimally affected)

dimethylaminopropionitrile, the
polymerization initiator, is highly toxic

requirement to purge the monomer solution

with nitrogen is also troublesome, although
not totally crippling
 Entrapment Media

CALCIUM ALGINATE

as widely used as polyacrylamide

gelation of calcium alginate occurs

by calcium ion crosslinking (meaning
beads can be formed in extremely
mild conditions, which ensure that
enzyme activity yields of over 80%
can be routinely achieved)

calcium ions can be easily exchanged

for sodium ions and displaced by
other ions (a property that is both
advantageous and disadvantageous)
 Entrapment Media
GELATIN

requires only simple equipment

reagents are relatively inexpensive and

nontoxic

retention of enzymatic activities is

typically 25-50% of the original free
enzyme

mass transfer resistance is relatively low

compared to other entrapment methods

rate of enzyme loss due to leakage is high

 Recombinant Microorganisms Immobilized
as Whole-Cell Biocatalysts

Entrapment of wild-type or
recombinant cells in gluten gel
matrices

Application of a single or
multi-step enzymatic activity

Gluten is advantageous for use as a new material for enzyme and

cell immobilization because it is biodegradable, cheaper and
readily available
 Enzyme Entrapment: GEL

REACTION WITH ACRYLOYL

CHLORIDE Surface lysine residues
form acryloyl amides with
CH2=CH-CO-Cl

REACTION WITH Copolymerization and

ACRYLAMIDE & crosslinking to form a gel
BISACRYLAMIDE w/CH2=CH-CO-NH2 and H2N-CO-CH=CH-
CH=CH-CO-NH2

ENZYME TRAPPED IN
GEL
 Enzyme Entrapment: FIBER

EMULSIFICATION Enzyme emulsified with

cellulose acetate in methylene
chloride

EXTRUSION Emulsion extruded through

a spinneret into a
solution of aqueous
precipitant

ENZYME TRAPPED IN
FIBRE
Membrane confinement of enzymes

Costly but very easy to use for a wide

variety of enzymes

May involve no (or few) additional

research and development costs
associated with other immobilization
methods

Achieved through a number of quite

different methods (depending on the
semi-permeable nature of the membrane)
 Membrane Confinement
DISSOLUTION Enzyme is dissolved in an aqueous solution of
1,6-diaminohexane

DISPERSION
Solution is dispersed in hexanedioic acid and
chloroform mixture. A thin polymeric (Nylon)
shell is formed around the aqueous droplets
which traps the enzyme

WASHING Droplets are washed free of non-confined

enzyme and transferred back to aqueous
solution

MICRO-
CAPSULES
Generalised comparison of different
enzyme immobilisation techniques

Covalent Membrane
Characteristics Adsorption Entrapment
binding confinement
Preparation Simple Difficult Difficult Simple
Cost Low High Moderate High
Binding force Variable Strong Weak Strong
Enzyme leakage Yes No Yes No
Applicability Wide Selective Wide Very wide
Running
High Low High High
Problems
Matrix effects Yes Yes Yes No
Large diffusional
No No Yes Yes
barriers
Microbial
No No Yes Yes
protection