Syllabus
Various techniques used for immoblized enzyme, Chemical
modifications.
Application of immobilized enzyme in biotechnology
Kinectics of immobilized enzyme,
Kinectics of inhibition of immobilized enzyme.
Mass transfer effects on enzyme kinetics both in free and
immobilized enzyme system
Immobilized Enzyme Systems
Enzyme Immobilization:
To restrict enzyme mobility in a fixed space.
Immobilized Enzyme Systems
Enzyme Immobilization:
- Easy separation from reaction mixture, providing the
ability to control reaction times and minimize the
enzymes lost in the product.
- Re-use of enzymes for many reaction cycles, lowering the
total production cost of enzyme mediated reactions.
- Ability of enzymes to provide pure products.
- Possible provision of a better environment for enzyme
activity
- Diffusional limitation
Methods of Enzyme Immobilization
• Three major Methods of Enzyme
Immobilization are :
- Entrapment
- Surface Immobilization
- Cross-linking
These are further broadly divided as in next
slide
Classification of Immobilization
Methods for Enzymes
Selecting an Immobilization Technique
•It is well recognized that no one method
can be regarded as the universal method for
all applications or all enzymes. Consider,
– widely different chemical characteristics of
enzymes
– different properties of substrates and products
– range of potential processes employed
Immobilization by Entrapment
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.
•Gel entrapment places the enzyme within the interstitial
•spaces of crosslinked, water-insoluble polymer gels.
•Polyacrylamide gels:
•Polysaccharides: The solubility of alginate and kCarrageenan varies with the cation, allowing these soluble
polymers to be crosslinked upon the addition of CaCl2 and
KCl, respectively.
•Variations of pore size result in enzyme leakage, even after
washing. The effect of initiator used in polyacrylamide gels
can be problematic.
cont……….
Immobilization by Entrapment in microcapsule
•Microencapsulation encloses enzymes within spherical,
•semi-permeable membranes of 1-100 mm diameter.
•Urethane prepolymers, when mixed with an aqueous
•enzyme solution crosslink via urea bonds to generate membranes of varying
hydrophilicity.
•Alternatively, photo-crosslinkable resins can be gelled by UV-irradiation.
•Advantage of Entrapment
– Enzymes are immobilized without a chemical or structural modification. A very
general technique.
•Disadvantage of Entrapment
– High molecular weight substrates have limited diffusivity, and cannot be treated with
entrapped enzymes.
Entrapment
- Matrix Entrapment
- Membrane Entrapment
(microencapsulation)
Matrix Materials used in Entrapment :
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
Challenges in Entrapment Method
- 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.
Immobilization by Carrier Binding
or Surface Immobilization
•Attachment of an enzyme to an insoluble carrier creates an active surface catalyst.
Modes of surface attachment classify carrier methods into physical adsorption, ionic
binding and covalent binding.
•Physical Adsorption: Enzymes can be bound to carriers
•by physical interaction such as hydrogen bonding and/or
• van der Waal’s forces.
–
–
–
the enzyme structure is unmodified
carriers include chitosan, acrylamide polymers and silica-alumina
binding strength is usually weak and affected by temperature and the concentration of
reactants.
•Ionic Binding: Stronger enzyme-carrier binding is obtained with solid supports containing
ion-exchange residues.
–
–
cellulose, glass-fibre paper, polystyrene sulfonate
pH and ionic strength effects can be significant
Surface immobilization
According to the binding mode of the enzyme, this
method can be further sub-classified into:
- Physical Adsorption: Van der Waals
Carriers: silica, carbon nanotube, cellulose, etc.
Easily desorbed, simple and cheap,
enzyme activity unaffected.
- Ionic Binding: ionic bonds
Similar to physical adsorption.
Carriers: polysaccharides and synthetic polymers
having ion-exchange centers.
•Covalent attachment of soluble enzymes to an insoluble support is the most
common immobilization technique.
– Amino acid residues not involved in the active site can be used fix the enzyme to
a solid carrier
•Advantages:
•1. Minimal enzyme leaching from the support results
• in stable productivity
•2. Surface placement permits enzyme contact with
• large substrates
•Disadvantages:
•1. Partial modification of residues that constitute the active site
decreases
activity
•2. Immobilization conditions can be difficult to optimize (often done
• in the presence of a competitive inhibitor)
- Covalent Binding: covalent bonds
Carriers: polymers contain amino, carboxyl,
sulfhydryl, hydroxyl, or phenolic groups.
- Loss of enzyme activity
- Strong binding of enzymes
Most Convenient Residues for
Covalent Binding
•Amino acid residues with polar and reactive functional groups are best for
covalent binding, given that they are most often found on the surface of the
enzyme.
•The data shown in next slide is the most convenient residues for binding in
descending order.
•The average percent composition of proteins (reactive residues only) is shown,
along with the number of potential binding reactions in which the amino acids
partake.
•Abundance(%)Reactions
CH2
+
NH3
4
Lysine (Lys)
Cysteine (Cys)
CH2 SH
CH2
OH
HN
N
Histidine (His)
CH2 CH2 C O
3
CH2
27
•
3.4
31
•
3.4
16
•
2.2
13
•
4.8
4
•
4.8
4
•
3.8
6
•
1.2
7
Aspartic Acid (Asp)
O
CH2
7.0
Tyrosine (Tyr)
CH2
O
CH2 C O
•
Glutamic Acid (Glu)
NH C NH2 Arginine (Arg)
+NH2
Tryptophan (Trp)
N
H
Covalent Attachment Techniques
•Cyanogen bromide activates supports with vicinal hydroxyl groups (polysaccharides, glass
beads) to yield reactive imidocarbonate derivatives:
•Diazonium derivatives of supports having aromatic amino groups are activated for enzyme
immobilization:
•Under the action of condensing agents (Woodward’s reagent K), carboxyl or amino groups
of supports and amino acid residues can be condensed to yield peptide linkages.
•Other methods include diazo coupling, alkylation, etc.
Immobilization by Crosslinking
•Bi- or multi-functional compounds serve as reagents for intermolecular
crosslinking of enzymes,
•creating insoluble aggregates that are effective heterogeneous catalysts.
•Reagents commonly have two identical functional groups which react with
specific amino acid residues.
•Common reagents include glutaraldehyde, carbodimide and diisocyanates,
•Involvement of the active site in crosslinking can lead to great reductions in
activity, and the gelatinous nature of the product can complicate processing.
Cross-linking is to cross link enzyme
molecules with each other using agents
such as glutaraldehyde.
Features: similar to covalent binding.
Several methods are combined.
Immobilized Enzymes
• Advantages
• Retention in reactor
• Separation from reaction
components is facilitated
• Usable in a wide range of
reactor configurations
• High catalytic loadings
• Enhanced stability toward
T, pH, solvent, etc.
• Modified selectivities
• Disadvantages
• Mass-transfer limitations
• Loss of activity upon
immobilization
• Impractical for solid
substrates
Application of ImmobilizedEnzymes
1-High-fructose corn syrups (HFCS)
2-GLUCOSE ISOMERASE
a Treatment with activated carbon.
3-Use of immobilised raffinase
4-Use of immobilised Invertase
5-Production of amino acids
6- Use of immobilised lactase
7- Production of antibiotics
Effect of Immobilization on Operational
Stability
•Given that activity of enzymes is dictated by structure and
conformation, the environmental change resulting from immobilization
affects not only maximum activity, but the stability of the enzyme
preparation.
– The factors that inactivate enzymes are not systematically understood,
and depend on the intrinsic nature of the enzyme, the method of
immobilization, and the reaction conditions employed.
– In general, immobilized enzyme preparations demonstrate better
stability
Note that the immobilized preparation is ften more stable than the soluble
enzyme and displays a period during which no enzyme activity appears to be
lost.
immobilized
enzymes
free (soluble)
enzymes
Effects of Immobilization on Enzyme
Stability and Use
•Design of enzymatic processes requires knowledge of:
– reactant and product selectivity
– thermodynamic equilibria that may limit product yield
– reaction rate as a function of process conditions ([Enzyme], [substrate(s)],
[Inhibitors], temperature, pH, …)
•Two design issues that we have not considered are:
– enzyme stability
– efficiency losses associated with the use of homogeneous (soluble)
catalysts
•Immobilization of an enzyme allows
•it to be retained in a continuous reactor,
•but its initial activity and its stability
•directly influence its usefulness
•in industrial applications.
Effects of Enzyme
Immobilization on Activity
Enzyme Stability
•Although enzyme storage stability is important, it is the operational
stability of an enzyme that governs its reactor performance.
– Operation stability is a complex function of temperature, pH,
[substrate] and the presence of destabilizing agents.
•Generally, the rate of free enzyme deactivation is first order with a
deactivation constant, kd:
d[E]T
 k d [E]T
dt
•Integrating this expression yields the concentration of active
enzyme as a function of time:
[E]T  [E]T,o ek dt
Yields of the concentration of active enzyme
as a function of time:
6.0
[Enzyme] *1E6 M
5.0
No decay
4.0
kd = 6E-6 s-1
3.0
kd = 3E-5 s-1
2.0
1.0
0.0
0
20
40
60
Time (hours)
80
100
Effect of Thermolysin Instability on APM
Production
•Recall the rate expression developed for APM synthesis by thermolysin:
d[ZAPM] k 2 [E]T [ZLAsp][LPM]

dt
K1  [ZLAsp]
•If thermolysin deactivation were adequately described as a first order process,
the observed reaction rate would have an explicit time dependence, as shown
below:
d[ZAPM] k 2 [ZLAsp][LPM]

[E]T,o ek dt
dt
K1  [ZLAsp]
•where [E]T,o represents the initial enzyme concentration and kd is the
deactivation rate constant.
•The conversion versus time profile for aspartame synthesis by a batch process
can be developed from this expression by integration.
Effect of Thermolysin Instability on APM
Production
•The evolution of [L-Asp] and conversion
with time for a batch process is shown
below.
– Depending on the relative rates of reaction
and enzyme deactivation, the ultimate
conversion can be strongly affected
APM Synthesis by Thermolysin
APM Synthesis by Thermolysin
Batch Process at 40C
Batch Process at 40C
0.90
0.018
0.80
0.016
kd = 3E-5 s-1
[L-Asp]: M
0.014
0.012
0.010
kd = 6E-6 s-1
0.008
0.006
kd = 0 s-1
0.004
L-Asp Conversion
0.020
0.70
0.50
0.40
0.30
0.000
0.00
40
60
Time (hours)
80
[LPM]o 0.0182 M
[LAsp]o 0.0182 M
k2
2.65 M-1s-1 K1
0.0103 M-1s-1
[E]o
4.85E-06 M
100
kd = 3E-5 s-1
0.20
0.10
20
kd = 6E-6 s-1
0.60
0.002
0
kd = 0 s-1
0
20
40
60
Time (hours)
80
[LPM]o 0.0182 M
[LAsp]o 0.0182 M
k2
2.65 M-1s-1 K1
0.0103 M-1s-1
[E]o
4.85E-06 M
100
Industrial Enzymatic Synthesis of Aspartame
•The unique regio and stereoselectivity afforded by enzymes has been exploited on an
industrial scale Aspartame production.
Ph
CO2 H
•The process employs a protease,
•thermolysin, to catalyze the
•condensation of the modified Asp
•and Phe).
H2N
X
N
H
H
CO2H
Amine-protected (X)
L-aspartic acid
(Z-L-Asp)
H
H
CO2 Me
O
•The forward reaction is written as:
CO2H
NH
-L-aspartyl-L-phenylanaline methyl ester
-aspartame (APM)]
Ph
+
CO2H
H2N
H
CO2 Me
Methyl ester of
L-phenylanaline
(L-PM)
thermolysin
X
N
HH
NH
H
Ph
+
CO2 Me
O
(APM)
•Note however, that the synthesis reaction is equilibrium limited by the reverse
(hydrolysis) reaction for which proteases are known. Furthermore, the equilibrium
strongly favours hydrolysis.
OH2
Factors Affecting Immobilize
Enzyme Kinetics
• pH effects
- on enzymes
- enzymes have ionic groups on their active sites.
- Variation of pH changes the ionic form of the active sites.
- pH changes the three-Dimensional structure of enzymes.
- on substrate
- some substrates contain ionic groups
- pH affects the ionic form of substrate
affects the affinity of the substrate to the enzyme.
• Effect of Temperature
- on the rate of enzyme catalyzed reaction
d[P]
v
 k [ES]
2
dt
k2=A*exp(-Ea/R*T)
T
k2
v
- enzyme denaturation
d[ E ]

 kd [ E]
Denaturation rate:
T
dt
kd=Ad*exp(-Ea/R*T)
Where kd: enzyme denaturation rate constant;
Ea: deactivation energy
Kinetics of immobilized enzyme
External Mass Transfer
External Mass Transfer
The governing expression is the Nernst equation:
*
N  k (S  S )
s
s o
ks = Mass transfer coefficient (cm/sec). This is determined
from well-established, empirical correlations;
S* = Substrate concentration at the solid-liquid interface;
So = Substrate concentration in the bulk solution.
At steady state, the enzymatic reaction rate cannot exceed the rate of substrate
diffusion to the enzyme. This can be written as follows:
v
A
catalyst
 v' 
V'
K
 k (S  S * )
s o
 (S * )
max
m
(S * )
For external mass transfer, we must evaluate the catalytic activity
normalized to the surface area of the catalyst (Acatalyst). The “prime”
notation indicates this.
Simpler to use dimensionless variables:
S*
*
x 
S
o
v
;
K
S
m
o
Da 
;
V'
max
k (S )
s o
Here, Da represents the Damköhler Number
Da = (Maximum Reaction Rate)/(Maximum Flux through the Diffusion
Layer)
If Da << 1, then the reaction rate is much less than the rate of diffusion
and we are in the kinetically-limited regime.
If Da >> 1, then the reaction rate is much greater than the rate of
diffusion and we are in the diffusionally-limited regime.
We can rewrite as follows:
v' 
x*
V'
max
x*  v
;
x*
1  x*

*
Da
x v
Thus, we can express the observed rate (v’) in terms of the
dimensionless substrate concentration (x*) at the catalyst surface.
If no diffusional limitations (i.e., S* = So, and x* = 1). Then:
v'
S*  S

o
V'
(S ) V '
max o  max
K  (S )
1 v
m
o
Let’s divide by x*:
v' 
V'

1  

 (1  v) x* 
max



*
1

v


x  v 
v 


x* 
max
V'
External Effectiveness Factor
We define this as the Effectiveness Factor, E, where the E stands for
external.
 (1  v) x* 

E  
 x*  v 
The effectiveness factor requires that you know b (=1/v) and Da.
This is often difficult as you need to know the intrinsic kinetics of the
immobilized enzyme (e.g., V’max and Km). Use the Observable Damkohler Number.
v'
Da 
k (S )
s o
Intraparticle Mass Transfer

p
D
D
H
eff
s 
where
H  (1   ) 2 (1  2.1044  2.089 3  0.948 5 )
Assume: Immobilized enzyme is uniformly distributed (e.g.,homogeneously loaded);
Transport of solute is described by Fick’s law;
Isothermal reaction at constant pH;
Negligible electrostatic effects.
Derivation of Key Expressions
S
i   N  v[ S ]
s
i
t
where



  ex
 ey
 ez
x
y
z
For a spherical particle of radius R, and at steady-state;
d 2S
dS
v[ S ]
V
[S ]
i 2 i 
i 
max i
rdr D
K  [ S ]D
dr 2
eff
m
i eff
S
0
t
,
then;
In terms of the same dimensionless numbers as for external diffusion;

2 Vmax
R

x
K
D
d 2 x 2 dx R 2 v[ S i ]
m
eff



 
1  bx
d r 2 r d r Deff [ S o ]
Hence, the concentration profile of substrate in the porous particle will
depend on the size of the particle, effective diffusivity, and the intrinsic
kinetic parameters. All three factors can be combined into a single
dimensionless parameter known as the Thiele Modulus, .
R

3
V
max
K D
m eff
For first-order reactions (e.g., when [Si] << Km, we can have an
analytical solution to the intraparticle effectiveness factor:
 
i
1 1
1

  tanh 3 3 
Far more useful is the Observable Modulus:
v
R
obs

 
D [S ]  3 
eff o

i   0 .3
1
and

2
i 3

1

Enzyme Reactors,
• Different Types of Enzyme Reactors,
• Heterogenous reaction system, transient
analysis of Enzyme Reactors
• Process design and operational strategies
of immobilized Enzyme Reactors
The most common definition for immobilized
enzymes is that proposed by Katchalski-Katzir
in the 1960s:
-“Enzymes physically confined or
localized in a certain defined region of space
with retention of their catalytic activities, which
can be used repeatedly and continuously.”
According to this definition, three types of
immobilized enzymes can be distinguished:
1. Heterogenization of the soluble enzyme by coupling
to an insoluble support by adsorption or covalent
binding, by cross-linking of the enzyme or entrapment
in a lattice or in microcapsules such as alginate
Beads
2. Retention of the enzyme by means of ultrafiltration
Membranes
3. Use of whole cells for biotransformations using their
enzyme apparatus
Enzyme Reactors
Membrane reactors have been used quite recently as have
applications of whole cell processes.
Retention of the cells within the reactor may be achieved
by membrane separation or by the same immobilization
methods that are used for isolated enzymes (34).
In principle, the cell itself can be regarded as a form of
native immobilization of enzymes.
\Biosensors are a very special form of carrier-fixed
biocatalysts.
The major goal behind immobilization is the recovery of the
biocatalysts,
separation from products and reactants, and
subsequent reuse
-in batch or continuous processes.
Subsequent reuse in batch or continuous processes is especially
important for a reduction of the catalyst costs.
Enzymes immobilized on a support often show enhanced stability
when compared with the soluble form.
When considering the use of soluble or
carrier-fixed enzymes, the following
topics we have to be addressed:
1. Additional costs for support and chemicals performing
the immobilization have to be balanced against the
increase of stability.
2. Loss of activity during the immobilization step.
3. When the catalyst is immobilized only by adsorption or
entrapment without covalent attachment, its leakage
from the carrier support has to be examined and
compared with the overall deactivation rate.
4. Mass transfer limitations for enzymes on a support may
cause problems when adjusting of the pH is necessary
during the reaction.
5. With soluble enzymes, higher volumetric activities at
high catalyst concentrations are possible, enabling
conversion of poor substrates at reasonable rates.
6. Whereas membrane reactors can be easily sterilized
before use, this is not possible for reactors with carrierfixed enzymes.
To prevent microbial contamination, these processes are
quite often operated at higher temperatures.
7. When enzymes are to be used together with
organic solvents to increase reactant or product
solubility or to alter their kinetics, it may become
necessary to immobilize them on a support
The support will at the same time act as a water
pool to maintain the enzymatic activity.
In such systems, water-insoluble organic
solvents have less effect on the enzyme stability
than water-soluble solvents.
Process design and operational strategies
of immoblized enzyme reactors
• The final decision for a certain reactor
design should be based on an
• optimization process covering all relevant
factors contributing
• to the overall costs, including investment,
catalyst
• consumption, or productivity.
Comparison of Processes Using Soluble
or Carrier-Fixed Enzymes
Reactors for Immobilized
Enzymes
• The methods for the heterogenisation (or
localization) of enzymes
•
– by coupling them to insoluble supports or
– by entrapment.
• The types of reactors used for immobilized
enzymes are summarized in Figure given
bellow.
Reactors for immobilized enzymes. (a–c)
(a) Batch reactors with complete backmixing;
(b) Stirred-tank reactor;
(c) Fixed-bed reactor;
Fluidized-bed reactor. (d–f) are
Continuously operated reactors
with complete back mixing.
(g–h) are the Continuously operated
reactors with plug-flow behavior.
(i) Reactor with the enzyme immobilized in or
on a membrane that may at the same time
separate two phases such as water and
organic solvent.
(j) reactor with physically separated enzyme
and organic solvent in order to prevent
denaturation of the protein
The principles developed for general heterogeneous
catalysis in synthetic chemistry are valid, resulting
in well-known reactor configurations.
Differences between enzyme catalysis and other
systems result from the nature of the biocatalyst
and reaction medium.
For example, soft particles containing the
biocatalyst, such as alginate beads, may limit the
pressure drop in fixed-bed reactors.
The decision as to specific reactor design will
be based on a careful analysis of the kinetic
properties of the reaction system.
For example, if the enzyme shows a strong
substrate-surplus inhibition, a continuously
operated reactor with complete backmixing
working at high conversion is
advantageaous.
• A reaction with strong product inhibition may utilize a
batch reactor or a plug flow reactor to achieve higher
volume and catalyst specific productivities.
• An extractive bioreactor may be used if substrates and
products show different solubilities.
• By using this reactor configuration, the destabilizing
effect of organic solvents may also be overcome,
because the enzyme is separated from the organic
phase, which is used to extract the insoluble product .
• The aqueous phase containing the enzyme will be
saturated until the maximum solubility of with the
substrate is reached.
• Reactions using biocatalysts are normally performed in
aqueous solution at temperatures between 10 and 80 C
and at ambient pressure.
• Due to the inhibition of some enzymes by heavy metals,
the materials of construction must not release these
elements.
• Reactors are operated under conditions that prevent
microbial contamination.
• The reactor itself as well as the substrate may be sterilized
prior to reaction by using chemical agents (ethanol,
formaldehyde, ethylenoxide, Velcorin) or steam.
• Ultraviolet rays may be used to sterilize
the immobilized enzyme on its support .
• Alternatively, the immobilization may be
performed under sterile conditions.
• Antibacterial agents may be added to the
reaction mixture to prevent microbial
growth while the reactor is running.
• In some cases, the reactants may act as
sterilants or inhibitors of microbial growth, such
as ketones or alcohols.
• At higher concentrations (more than 500
mmol/L), solutions may become autosterile
because of osmotic pressure effects.
• Ndustrial processes are often performed at
elevated temperatures, above 55 C, reducing
the danger of microbial contamination.
• For a constant product quality and reproducibility
of downstream processing, the reactor should
be operated at constant conversion.
• To overcome the deactivation per unit of time
that shows all biocatalysts as a result of
denaturation processes, either the residence
time has to be increased or fresh enzyme has to
be supplied.
• The latter is especially easy for soluble
enzymes. For carrier-fixed enzymes,
a
combination of both methods is used, as
discussed later.
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.
• The immobilization of enzymes onto particulate carriers
that may be packed into a column (the ‘‘packed-bed’’
reactor), such as a typical HPLC column, facilitates
repetitive use of the enzyme and also allows the
automation of enzymatic assays.
• Open-tubular reactors have also been constructed by
covalently immobilizing an enzyme onto the inner wall of a
nylon or polyethylene tube.
• Immobilized enzyme reactors are used in conjunction with
a pump, to force a buffer, or mobile phase, through the
reactor at a steady rate, an injector located between the
pump and the reactor to allow the introduction of substrate
solutions, and a detector located close to the column exit.
• The mobile phase contains all required cosubstrates and activators
required for the enzymatic reaction, but does not contain the analyte
substrate.
• A typical packed-bed system may use a 25-cm long reactor with a 5mm inner-diameter, packed with the carrier-enzyme solid phase at
high pressures.
• Flow rates of 0.5–2 mL/min and sample injection volumes of 10–100
mL are common.
• Detection involves the same principles used in homogeneous
enzymatic assays, and flow-through optical absorbance and
fluorescence detectors, and amperometric and potentiometric
electrochemical detectors may be employed, with detector volumes
of the order of tens of microliters being standard.
• Enzyme reactor systems may be of the continuous flow
or the stopped-flow variety.
• Continuous flow systems are further categorized as open
or closed systems.
• The open system, shown in Figure , continuously pumps
fresh buffer through the injector, reactor and detector,
ultimately into a waste reservoir for discarding.
• This arrangement is preferred for the testing of enzyme
reactors, since unreacted substrate, cofactors and the
products of the enzymatic reactions will not be
reexposed to the column.
Diagram of an open enzyme reactor system
• Closed systems may be employed when
buffer recycling is possible, that is when
the buffer contains high concentrations of
all necessary cosubstrates, when
complete consumption of injected
substrate occurs within the reactor, and
when products of the enzymatic reaction
do not inhibit the immobilized enzyme.
• A closed system for immobilized oxidase
enzymes is shown in Figure below.
Diagram of a closed enzyme
reactor system.
• Both open and closed continuous flow
systems rely on the fixed time, or endpoint
method for the determination of substrate
concentrations.
• At a fixed and constant flow rate, the
injected volume of substrate will spend a
fixed time on the column, and this time is
related to the volume of the column (that
volume not occupied by stationary phase)
and the mobile-phase flow rate.
• Indicator reactions that are chemical in
nature may be introduced either into the
mobile phase or at the end of the column
by the method of postcolumn reagent
addition.
• Postcolumn addition of reagents dilutes
the column eluent, so that, when possible,
the addition of indicator reagents to the
mobile phase is preferable.
• The conditions under which chemical indicator
reactions are used often necessitates the use of
postcolumn addition, however.
• Figure given below shows an experimental
setup for urea assays using an immobilized
urease reactor.30 The postcolumn addition of
sodium hydroxide allows the NHþ4 produced by
the reactor to be detected as NH3 at an
ammonia gas-sensing electrode placed in a flow
cell.
Enzyme reactor system for urea based on immobilized
urease and potentiometric detection.
• Stopped-flow enzyme reactor systems have
been designed for automated kinetic assays.
• A diagram of a stopped-flow reactor that uses a
postcolumn chemical indicator reaction is shown
in Figure below.
• In this system, the flow rate of themobile phase
through the reactor dictates the residence time
of the analyte on the column.
Stopped-flow enzyme reactor with
absorbance detection
THEORETICAL TREATMENT OF PACKED-BED
ENZYME REACTORS
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Packed-bed enzyme reactors, those employing enzymes immobilized
onto a particulate phase that is subsequently packed into a column, may
be characterized by their column capacity, C, and the degree of reaction
P.
The parameter C is defined by the equation.
where k is the decomposition rate constant for the enzyme–substrate
complex (either k2 or kcat),
Et is the total number of moles of enzyme immobilized, and the value of
β is a constant for a given reactor, and is equal to the ratio of reactor
void volume to total reactor volume (i.e., β is always less then unity).
The degree of reaction, P, varies between zero (no product formed) and
unity (complete conversion of substrate).
An equation equivalent to the Michaelis–Menten equation has been
derived for immobilized enzymes in packed-bed reactor systems, and
is given in Eq.
where Q is the volume flow rate of the mobile phase. In general, this
equation predicts that for a given column capacity, the degree of
reaction, P, is inversely related to the mobile-phase flow rate, Q. That
is, the faster the analyte plug flows through the reactor, the less likely
will be its complete conversion into product.