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CHE1009-BIOCHEMICAL ENGINEERING
MODULE-II
LECTURE – 7

Dr.A.Babu Ponnusami
Associate Professor
SCHEME
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
 Effect of Mass-Transfer Resistance
 The immobilization of enzymes may introduce a new problem
which is absent in free soluble enzymes.
 It is the mass-transfer resistance due to the large particle size of
immobilized enzyme or due to the inclusion of enzymes in
polymeric matrix.
 The hypothetical path of a substrate from the liquid to the
reaction site in an immobilized enzyme, it can be divided into
several steps
 Hypothetical path

(1) Transfer from the bulk liquid to a relatively unmixed

liquid layer surrounding the immobilized enzyme;
(2) Diffusion through the relatively unmixed liquid layer;
and
(3) Diffusion from the surface of the particle to the active
site of the enzyme in an inert support.
Steps 1 and 2 are the external mass-transfer resistance.
Step 3 is the intraparticle mass transfer resistance.
 External Mass-Transfer Resistance

 If an enzyme is immobilized on the surface of an insoluble particle,

the path is only composed of the first and second steps, external
mass-transfer resistance.
 The rate of mass transfer is proportional to the driving force, the
concentration difference, as

Where CSb and CS are substrate concentration in the bulk of the

solution and at the immobilized enzyme surface, respectively.
The term kS is the mass-transfer coefficient (length/time) and
A is the surface area of one immobilized enzyme particle.
 During the enzymatic reaction of an immobilized enzyme, the rate
of substrate transfer is equal to that of substrate consumption.
 Therefore, if the enzyme reaction can be described by the
Michaelis-Menten equation,

 where a is the total surface area per unit volume of reaction

solution.
 This equation shows the relationship between the substrate
concentration in the bulk of the solution and that at the surface of
an immobilized enzyme.
Eq. (3.2) can be expressed in dimensionless form as:

where
 NDa is known as Damköhler number, which is the ratio of the
maximum reaction rate over the maximum mass-transfer rate.
 Depending upon the magnitude of NDa, Eq. (3.2) can be simplified, as
follows:
 1. If NDa < < 1, the mass-transfer rate is much greater than the reaction
rate and the overall reaction is controlled by the enzyme reaction,

 2. If NDa > > 1, the reaction rate is much greater than the mass-
transfer rate and the overall rate of reaction is controlled by the
rate of mass transfer that is a first-order reaction,
 To measure the extent which the reaction rate is lowered because
of resistance to mass transfer, we can define the effectiveness
factor of an immobilized enzyme, η, as

The rate that would be obtained with no mass-transfer resistance at

the interface is the same as Eq. (3.5) except that CS is replaced by
CSb. Therefore, the effectiveness factor is
 where the effectiveness factor is a function of xS and β. If xS is
equal to 1, the concentration at the surface CS is equal to the bulk
concentration CSb.
 Substituting 1 for xS in the preceding equation yields η = 1, which
indicates that there is no mass-transfer limitation.
 On the other hand, if xS approaches zero, η also approaches zero,
which is the case when the rate of mass transfer is very slow
compared to the reaction rate.
Problem:

 The Michaelis-Menten kinetic parameters for a soluble enzyme

for a certain substrate are found to be KM = 0.05 mol/L, rmax=10
mol/L min. After immobilizing this enzyme on the surface of
insoluble matrix by physical adsorption, it was found that the app
KM value was increased to 0.08 mol/L whereas the max rapp value
stayed the same as rmax. What is the effectiveness factor of the
immobilized enzyme when the substrate concentration is 1
mol/L?
Problem:

 The values of KM and rmax for an enzyme (21°C and pH = 7.1) are
0.004 kmol/m3 and 10 kmol/m3s, respectively. We immobilized
this enzyme by attaching it covalently to acrylamide-based
polymers that can be assumed to have spherical shape (diameter
= 1 mm). The effectiveness of the immobilized enzyme was
found to be 70 percent of the free enzyme when the concentration
of the substrate was 0.5 kmol/m3. The reaction was carried out in
a stirred reactor with an Immobilized Enzyme agitation speed of
50 rpm.
(a)Estimate the concentration of the substrate at the surface of the
immobilized enzyme.
(b) Estimate kS a.
 Internal Mass-Transfer Resistance

Assumptions are as follows:

1. The reaction occurs at every position within the immobilized
enzyme, and the kinetics of the reaction are of the same form as
observed for free enzyme.
2. Mass transfer through the immobilized enzyme occurs via
molecular diffusion.
3. There is no mass-transfer limitation at the outside surface of the
immobilized enzyme.
4. The immobilized enzyme is spherical.
The model developed by these assumptions is known as the
distributed model.
 For a steady-state condition, the change of substrate
concentration, dCS /dt, is equal to zero.
 After opening up the brackets and simplifying by eliminating all
terms containing dr2 or dr3, we obtain the second order
differential equation:

The above equation can be solved by substituting a suitable

expression for rS.
Let’s solve the equation first for the simple cases of zero-order
and first-order reactions, and for the Michaelis-Menten equation.
Zero-order Kinetics:

Let's assume that the rate of substrate consumption is constant (zero

order) with respect to substrate concentration as

Therefore, the effectiveness factor, the ratio of the actual reaction

rate to the rate if not slowed down by diffusion, is

First-order Kinetics:
If the rate of substrate consumption is a first order reaction with
respect to the substrate concentration,
Where ф is known as Thiele’s modulus, which is a measure of the
reaction rate relative to the diffusion rate.

When φ ≤ 0.1, the effectiveness factor is nearly equal to one, which is

the case when the rate of reaction is not slowed down by the
diffusion. On the other hand, when φ ≥ 0.1, the effectiveness factor is
inversely proportional to the Thiele’s modulus.
 Michaelis-Menten Kinetics:

Thiele's modulus (Φ) is defined slightly differently from the

first-order kinetics as
Diffusion effects in enzymes immobilized in a
porous matrix:

Relationship of
effectiveness factor
(η) with the size of
immobilized
enzyme particle and
enzyme loading
Problem:
An enzyme which hydrolyzes the cellobiose to glucose, β-
glucosidase is immobilized in a sodium alginate gel sphere (2.5 mm
in diameter). Assume that the zero-order reaction occurs at every
point within the sphere with k0 = 0.0795 mol/s m3, and cellobiose
moves through the sphere by molecular diffusion with DS = 0.6 ×
10-5 cm2/s (cellobiose in gel). Calculate the effectiveness factor of
the immobilized enzyme when the cellobiose concentration in bulk
solution is 10 mol/m3.