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TOPIC 4

IMMOBILIZED
BIOCATALYST

Properties and Characterization


of Immobilized Biocatalysts

Properties of Immobilized
Enzymes
The properties of immobilized enzyme preparations
are governed by the properties of both the
enzyme and the carrier material.
Due to immobilization, the properties of enzymes
will be altered such as catalytic activity with
respect to the support matrix.
The change in the enzyme properties in the
immobilized enzyme is due to the enzyme and
the substrate reacts in the microenvironment
which is different from the enzyme substrate
reaction in the bulk solution environment.

Properties of Immobilized
Enzymes
As far as manufacturing costs are concerned the yield of
immobilized enzyme activity is mostly determined by the
immobilization method and the amount of soluble enzyme
used.
Under process conditions, the resulting activity may be further
reduced by mass transfer effects, lead to lowered efficiency.
More precisely, the yield of enzyme activity after immobilization
depends not only on losses caused by the binding procedure but may
be further reduced as a result of diminished availability of enzyme
molecules within pores or from slowly diffusing substrate molecules.

Support selection
Selection criteria differ among one another, depending on the
biocatalyst of interest, but there are still few basic features that must
be considered.
Material used as a carrier should have chemical, physical and
biological stability during processing, as well as in the reaction
conditions; sufficient mechanical strength, especially for its
utilization in reactors and industry; should be nontoxic both for
the immobilized cell/bioparticle, as well for the product; also
should have adequate function groups for binding biocatalyst
and high loading capacity.
Profitability of the material application and its processing costs
always have to be taken upon consideration.
Other criteria, such as physical characteristics (porosity, swelling,
compression, material and mean particle behavior), as well as
possibility for microbial growth, biodegradability, solubility, are
more application specific.
Carrier materials can be divided into those of inorganic and organic
origin

Characteristics of the Carrier


Functional groups:
The type of activation, presence, distribution and density of functional
groups determines the activity yields of an immobilization reaction, and
stability and operational stability of the carrier-fixed enzyme.
Most immobilization procedures proceed via a nucleophilic attack of
amino groups on activated carrier functional groups.
This is in many cases the appropriate way to couple an enzyme on a
carrier surface, because it avoids the enzyme getting into contact with
highly reactive organic compounds of low molecular weight.

Characteristics of the Carrier


Permeability and surface area:
In most cases a large surface area (>100m2 g1) and high porosity are
desirable, so that enzyme and substrate can easily penetrate.
A pore size of >30 nm seems to make the internal surface accessible for
immobilization of most enzymes.

Hydrophilicity/hydrophobicity of the carrier matrix:


It influences
interaction.

type

and

strength

of

non-covalent

proteinmatrix

In addition, it can influence the adsorption, distribution and availability


of the substrate and product.

Characteristics of the Carrier


Insolubility:
This is essential, not only for prevention of enzyme loss, but also to
prevent contamination of the product by dissolved matrix and
enzyme.
Mechanical stability/rigidity:
These properties are dependent on the type of reactor. If used in a
stirred tank reactor, the support should be stable against sheer forces
to minimize abrasion. Production of fines (particles below 10050
mm) can lead to plugging of sieve plates and filters.

Characteristics of the Carrier


Form and size of support:
The particle size will have an influence on filtration times from stirred
tank reactors in repeated batch mode. Furthermore, this factor is
important for the performance in column reactors regarding back
pressure and flow rates, which of course are correlated. For this
purpose a size of spherical particles in the range of 150300 mm is
preferred.
Resistance to microbial attack:
During long term usage the support has to be stable against
microbial degradation.

Regenerability:
This property is of interest in case of expensive carrier materials.

Mass Transfer Effects


Identify the parameters which affect the
mass
transfer
of
immobilized
enzymes/cells. Explain.

Mass Transfer Effects


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

A reduced reaction rate may result from external diffusional

restrictions on the surface of carrier materials.


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

Effects of Enzyme Immobilization on


Activity

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
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

Diffusional Limitation in
Immobilized Enzyme System
Immobilized enzyme system normally

includes
- insoluble immobilized enzyme
- soluble substrate, or product
They are heterogeneous systems

Substrate

HIGH

Immobilized
Enzyme
Low S concentration

R
E
M SF
L
I
F AN
TR

Sb

E
C
N
E
R

FE
F
N
DI
O
TI
N
C
IO
A
T
R
A
R
TT
T
A
N
E
IC
C
R
N
T
C
O
E
C
L
E

DIFFUSION
DRIVING FORCE

HIGH

Immobilized
Enzyme
REACTION

PRODUCT

R
E
M SF
L
I
F AN
TR

Sb

E
C
N
E
R

FE
F
N
DI
O
TI
N
C
IO
A
T
R
A
R
TT
T
A
N
E
IC
C
R
N
T
C
O
E
C
L
E

DIFFUSION
DRIVING FORCE

HIGH

Immobilized
Enzyme
E
L
IC
T
R
A
P R
A FE
R
T NS
N
I
A
R
T

R
E
M SF
L
I
F AN
TR

Sb

DIFFUSION
DRIVING FORCE

HIGH

Immobilized
Enzyme

R
E
M SF
L
I
F AN
TR

Sb

REACTION

E
L
IC
T
R
A
P R
A FE
R
T
IN ANS
TR

PRODUCT

Diffusional Limitation in
Immobilized Enzyme Systems
In immobilized enzyme systems, the overall production rate is
determined by
- liquid film mass transfer (external diffusion)
The transport of substrates towards the surface, and products away)
- intraparticle mass transfer (internal diffusion)
The transport of the substrates and products, within the pores of
immobilised enzyme particles
- enzyme catalysis reaction

Diffusional Limitation in
Immobilized Enzyme System
Diffusion Effects in Surface-bound Enzymes
on Nonporous Support Materials
Ss

E+S

ES

Sb

k2
P E

Assume the enzyme catalyzed


reaction rate follows Michaelis-Menten
type kinetics.

Enzyme
Liquid Film Thickness, L

Ss: substrate concentration at the surface;


Sb: substrate concentration in bulk solution.

Diffusion Effects in Surface-bound Enzymes


on Nonporous Support Materials
Assume:

Ss

Sb

-Enzyme are evenly distributed on the


surface of a nonporous support
material.
-All enzyme molecules are equally
active.
-Substrate diffuses through a thin
liquid film surrounding the support
surface to reach the reactive surface.

Enzyme
Liquid Film Thickness, L
No intraparticle diffusion

-The process of immobilization has not altered the enzyme


structure and the intrinsic parameters (Vm, Km) are unaltered.

Diffusion Effects in Surface-bound Enzymes


on Nonporous Support Materials
To determine the significant effect of external diffusion
resistance on the rate of enzyme catalytic reaction rate:
Damkhler numbers (Da)

Vm '
maximum rate of reaction
Da

maximum rate of diffusion k L [ Sb ]


Vm '

is the maximum reaction rate per unit of


external surface area (e.g. g/cm2-s)

kL

is the liquid mass transfer coefficient (cm/s)

[ Sb ]

Is the substrate concentration in bulk solution (g/cm 3)

Diffusion Effects in Surface-bound Enzymes


on Nonporous Support Materials

Vm '
maximum rate of reaction
Da

maximum rate of external diffusion k L [ Sb ]


When Da >> 1, the external diffusion rate is limiting;
Da << 1, the reaction rate is limiting;
Da 1, the external diffusion and reaction
resistances are comparable.

Diffusion Effects in Surface-bound Enzymes


on Nonporous Support Materials
The external diffusion rate

Js

(g/cm 2-s):

J s k L ([ Sb ] [ S s ])
kL

is the liquid mass transfer coefficient (cm/s).

If the product formation rate is :

Vm '[ S s ]
v
K m [Ss ]
'

Vm ' the maximum reaction rate per unit surface area.


(g/cm2-s)

Diffusion Effects in Surface-bound Enzymes


on Nonporous Support Materials
k2
E+S
ES P E
At steady state, the reaction rate is equal to
the external diffusion rate:

Vm '[ S s ]
J s k L ([ Sb ] [ S s ])
K m [S s ]

With the equation and known Sb, KL, Vm or Km,


to determine numerically or graphically:
- The substrate concentration at the surface.
- The reaction rate.

J s k L ([ Sb ] [ S s ])

Graphical solution for reaction rate per unit of surface area


for enzyme immobilized on a non-porous support

Diffusion Effects in Surface-bound Enzymes


on Nonporous Support Materials
When the system is strongly external diffusion
(liquid film mass-transfer) limited, [Ss]0,
the overall reaction rate is equal to the rate:

v k L [ Sb ]

Da>>1

The system behaves as pseudo first order.


The rate is a linear function of bulk substrate concentration.

Diffusion Effects in Surface-bound Enzymes


on Nonporous Support Materials
To increase the overall reaction rate
with external diffusion limitation

Vm '
maximum rate of reaction
Da

maximum rate of diffusion k L [ Sb ]


-Increase

-Increase

The liquid film mass transfer coefficient kL:

D2 / 3
1
/
2

AB
U
k L 0.6

1
/
6

d p1 / 2

(H. Fogler, Elements of Chemical Reaction Engineering 1999, p705)

DAB is mass diffusivity of the substrate in the liquid phase,


a function of temperature and pressure (m 2/s)
is the kinematic viscosity (m2/s), a function of temperature.
U is the free-system liquid velocity
(velocity of the fluid flowing past the particle) (m/s).
dp is the size of immobilized enzyme particle (m).
At specific T and P, increasing U and decreasing dp increase
the liquid film mass transfer coefficient and
the external diffusion rate.

Diffusion Effects in Surface-bound Enzymes


on Nonporous Support Materials
When the system is strongly reaction limited,
[Sb] [Ss]
the overall reaction rate is equal to the rate:

Vm '[ Sb ]
v
K m, app [ Sb ]

Da << 1

where

K m ,app

Vm'
Km 1

k L ([ Sb ] K m )

Km,app is increased. It is a function of mixing speed and S b.

Diffusion Effects in Enzymes


Immobilized in a Porous Matrix
- Substrate diffuses through the tortuous
pathway within the porous support to reach
the enzyme.
- Substrate reacts with enzyme on the pore
surface.
Ex. Spherical support particles
Sr

Diffusion Effects in Enzymes


Immobilized in a Porous Matrix
Assume:
- Enzyme is uniformly distributed in a
spherical support particle.
- The reaction kinetics follows MichaelisMenten kinetics.
- There is no external diffusion limitation.

Under internal diffusion limitations, the rate per unit volume is


expressed in terms of the effectiveness factor as follows:

Vm" [ S s ]
rs
K m [S s ]

is
.
Vm" is the maximum velocity per volume of the support.
K m is the M-M constant.
[ S s ] is the substrate concentration on the surface of the support.

reaction rate with intraparticle diffusion limitation

reaction rate without diffusion limitation.

1
1

the rate is

the rate is

Relationship of effectiveness factor with the size of


immobilized enzyme particle and enzyme loading

At specific conditions (T, P) for a fixed system,


To increase the intra-particle mass transfer rate:
-

the size of immobilized enzyme particle

the porosity or specific surface area of


the particle

Reaction kinetics or mass transfer


control
diffusional

boundary film

resistances
minimized by

Rbulk

decreasing particle

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

Vmax [R]
v
K m [R]

Electrostatic and Steric Effects in


Immobilized Enzyme Systems
The optimum pH for immobilized enzyme

system will shift from that of soluble free


enzyme
The charged matrix may repel or attract
substrate, product, cofactors or H+
Electrostatic effect
The activity of enzyme toward a high-

molecule-weight substrate may be


reduced.
Steric hindrance

Summary of Diffusion
Effects

- Determine the support to be non-porous or


porous.
- Identify the substrate determining the
reaction rate.
- Conduct mass balance of the substrate of
interest.
Accumulation of substrate of interest =
At steady
state, gain - substrate
rate
of substrate
Rate of substrate
gain = substrate
consumption
rate (production
formation rate,
consumption
rate
or
reaction rate)

Summary of Diffusion
Effects

In surface-bound enzymes on nonporous


support materials.
Consider external diffusion rate (liquid film mass
transfer rate)
At steady state, the reaction rate per unit
surface area is equal to the rate of net substrate
gain in regard to the external diffusion.
With the equation and known Sb, KL, Vm or Km,
to determine graphically or numerically:
- The substrate concentration at the surface.
- The reaction rate.

Summary of Diffusion
Effects

In surface-bound enzymes on porous


support materials.
Consider intraparticle diffusion rate.
At steady state, the reaction rate per unit
volume is equal to the rate of net substrate
gain in regard to the intraparticle diffusion.

is the effectiveness factor.

reaction rate with intraparticle diffusion limitation

reaction rate without diffusion limitation.

f ( , )
R

1
1

"
Vm
S s De

Km

[S s ]

the rate is diffusion limited.


the rate is reaction limited.

At specific conditions (T, P) for a fixed system,


To increase the intra-particle mass transfer
rate:
- Decrease the size of immobilized enzyme
particle
- Increase the substrate concentration
- Increase the porosity or specific surface
area of the particle

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


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 wellhydrolysed 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

Kinetics of immobilized
enzyme

Factors Affecting Immobilize


Enzyme Kinetics
pH effects

- on enzymes
- enzymes have ionic groups on their active sites.
- Variation of pH changes the ionic form
sites.

of the active

- 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

- enzyme denaturation
T

d[E]

kd [E]
Denaturation rate:
dt

kd=Ad*exp(-Ea/R*T)
Where kd: enzyme denaturation rate
constant; Ea: deactivation energy

Enzyme Stability
The stability of an immobilized enzyme is highly dependent on
many factors, including:
the properties of its interaction with the carrier
the binding position and the number of the bonds
the freedom of the conformation change in the matrix
the microenvironment in which the enzyme molecule is
located
the chemical and physical structure of the carrier
the properties of the spacer (for example, charged or neutral,
hydrophilic or hydrophobic, size, length) linking the enzyme
molecules to the carrier
the conditions under which the enzyme molecules were
immobilized

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

Note that the immobilized preparation is often 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.

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