Learn about Immobilized Enzyme systems

S
 IMMOBILIZED ENZYME SYSTEMS

IMMOBILIZED :enzyme mobility gets restricted in a fixed space

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Advantages of immobilized enzymes:
- 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

Disadvantages of immobilized enzymes:

- Problem in diffusional mass transfer
- Enzyme leakage into solution
- Reduced enzyme activity and stability
- Lack of controls on micro environmental conditions
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Methods of immobilization

1) Entrapment Immobilization

2) Surface Immobilization

3) Cross-linking

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1) Entrapment Immobilization

It is the physical enclosure of enzymes in a small space.

- Matrix entrapment (matrices used are polysaccharides,
proteins, polymeric materials, activated carbon, porous
ceramic and so on)
- Membrane entrapment (microcapsulation or trapped
between thin, semi-permeable membranes)

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Advantage is enzyme is retained.

Disadvantages are
- substrate need to diffuse into enzyme and
product need to diffuse out
- reduced enzyme activity and enzyme stability owing
to the lack of control of micro environmental
conditions

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2) Surface Immobilization

- Physical adsorption (Carriers are silica,

carbon nanotube, cellulose, and so on; easily
desorbed; simple and cheap; enzyme activity
unaffected )
- Ionic binding (Carriers are polysaccharides
and synthetic polymers having ion-exchange
centers)
- Covalent binding (Carriers are polymers
containing amino, carboxyl, hydroxyl, or
phenolic groups; loss of enzyme activity;
strong binding of enzymes)

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Methods of immobilization

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3) Cross linking

is to cross link enzyme molecules with each other

using agents such as glutaraldehyde.

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Comparison between the methods
Characteristics Adsorption Covalent Entrapment Membrane
coupling 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 problems High Low High High
Matrix effects Yes Yes Yes No

Large diffusional No No Yes Yes

barriers

Microbial protection No No Yes Yes

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Immobilized enzyme reactor (example)

- Allow the reactor to

operate at high fluid
velocities

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 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

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- An immobilized enzyme
tends to decompose upon
physical stirring.

- The batch system is

generally suitable for the
production of rather small
amounts of chemicals.

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 Diffusion Limitations in Immobilized ENZYME SYSTEMS

Mass transfer resistance is present

- due to the large particle size of the immobilized enzymes
- due to the inclusion of enzymes in polymeric matrix

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 Diffusion Limitations in Immobilized ENZYME SYSTEMS

Mass transfer resistance are divided into the following:

- External mass transfer resistance (step 1 & 2)
(during transfer of substrate from the bulk liquid
to the relatively unmixed liquid film surrounding
the immobilized enzyme and during diffusion
through the relatively unmixed liquid film)
- Intra-particle mass transfer resistance (step 3)
(during diffusion from the surface of the particle
to the active site of the enzyme in an inert support)

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External mass-transfer resistance:
Surface bound Enzyme Immobilize on nonporous support
Ss
Ss
Assumption: SbSb
- Enzymes are bound and 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 structure is unaltered
- M-M kinetic parameters (Vm, Km) are
unaltered. Enzyme
Enzyme

Liquid Film
Liquid filmThickness,
thickness,L L
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External mass-transfer resistance:
Diffusional mass transfer across
Ss
the liquid film: Ss
SbSb
JS = kL (Sb Ss)

kL liquid mass transfer

coefficient (cm/s)
Sb substrate concentration in
the bulk solution (mol/cm3)
Ss substrate concentration at
the immobilized enzyme Enzyme
Enzyme
surface (mol/cm3)

Liquid Film
Liquid filmThickness,
thickness,L L
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 External mass-transfer resistance:
At steady state (rate of S transfer
=rate of S consumption by enzymatic Ss
Ss
reaction), the reaction rate is equal to Sb
the external diffusion rate:

Js = v
Vm Ss
JS = kL (Sb Ss) =
Km + Ss

Vm maximum reaction rate per

unit of external surface area
(e.g. mol/cm2.s) Enzyme
Enzyme
Km is the M-M kinetic constant
(e.g. mol/cm3) Liquid Film
Liquid filmThickness,
thickness,L L
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External mass-transfer resistance:
Vm [Ss]
JS = kL ([Sb] [Ss]) =
Km + [Ss]
Dimensionless form of the above equation,

1 [Ss] [Ss]
=
Da 1 + [Ss]

where
[Ss]= x = [Ss] / [Sb]

Da = Vm / (kL [Sb] ) is the Damkhler number

= [Sb] / Km is the dimensionless substrate

concentration
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Damkhler number (Da)

Maximum rate of reaction Vm

Da = =
Maximum rate of external diffusion kL [Sb]

If Da >> 1, rate of diffusion is very slow and therefore the

system is strongly MT limited
JS = kL ([Sb])=v

If Da << 1, rate of reaction is very slow and therefore the

system is strongly reaction kinetic limited
Vm [Sb]
v =
Km + [Sb]
If Da = 1, rates of diffusion and reaction are comparable.
Diffusion and reaction occurs simultaneously
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Internal mass transfer resistance:
Enzyme Immobilize in Porous Matrix
Assumption:
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.
= Sb
3. There is no mass-transfer limitation at the
outside surface of the immobilized enzyme.

4. The enzyme is immobilized on spherical

surfaces

The model developed by these assumptions is known as the

distributed model
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Internal mass transfer resistance:

general mass transfer equation in spherical system

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Internal mass transfer resistance:
Foe one dimension and steady-state condition equation 14. 50
becomes:

Substrate consumption can be expressed by the Michaelis-Menten

equation

By substituting rs and changing it to dimensionless form, we

obtain
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Where X=Ss/Sb =r/R
=Sb/Km

(which is dimensionless and combine particle dia, diffusivity and kinetic parameters)

A. Can be solved numerically with some boundary conditions

B. C. 1: at =1,
x=1
B. C. 2: at =0,
dx/d =0
B. Can be solved analytically also with some boundary conditions 24
 Effectiveness factor ()

Under internal diffusion limitations, the rate per unit volume is

expressed in terms of the effectiveness factor as follows:

Vm [ S b ]
rs =
K m [S b ]

reaction rate with intraparticle diffusion limitation or actual rxn rate

=
reaction rate without diffusion limitation.

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Effectiveness factor ()
reaction rate with intraparticle diffusion limitation or actual rxn rate
=
reaction rate without diffusion limitation.

Vm [SS] x
Km + [SS] 1+x
= =
Vm [Sb]
Km + [Sb] 1+
Effectiveness factor is a function of and x
Da, : measure the extent of limiting mechanism

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For maximum conversion rates, particle
size should be small (Dp 10 mm) and
enzyme loading should be optimized.
As depicted in the example in Fig. 3.21,
Dp 10 mm and enzyme loadings of
less than 10 mg/cm3 are required for
high values of the effectiveness factor
( 0.8).

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Example 3.4 in Shuler & Kargi:
Consider a system where a flat sheet of polymer coated with
enzyme is placed in a stirred beaker. The intrinsic maximum
reaction rate of the enzyme is 6 x 10-6 mols/s.mg enzyme. The
amount of enzyme bound to the surface has been determined
to be maximum 1 x 10-4 mg enzyme/cm2 of support. In
solution, the value of Km has been determined to be 2 x 10-3
mol/l. The mass-transfer coefficient can be estimated from
standard correlations for stirred vessels. We assume in this
case a very poorly mixed system where kL = 4.3 x 10-5 cm/s.

What is the reaction rate, when the bulk concentration of the

substrate ([Sb]) is
(a) 7 x 10-3 mol/l and
(b) 1 x 10-2 mol/l?
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 Solution

Data provided:
Vm = 6 x 10-6 x 1 x 10-4 mols/s.cm2
= 6 x 10-10 mols/s.cm2
Km = 2 x 10-3 mol/l = 2 x 10-6 mol/cm3
kL = 4.3 x 10-5 cm/s
Sb = 7 x 10-3 mol/l OR 1 x 10-2 mol/l
= 7 x 10-6 mol/cm3 OR 1 x 10-5 mol/cm3
Equation to be solved:
Vm [Ss]
JS = kL ([Sb] [Ss]) =
Km + [Ss]

where Ss should be solved for, which can then be used to

calculate JS.
Use solver 29
Solution to Example 3.4 in Shuler & Kargi:

6.E-10 J_S for part (a)

J_S for part (b)
J_S or r_S (mol/s.cm )
2

5.E-10 r_S
4.E-10
3.E-10
2.E-10
1.E-10
0.E+00
0 0.002 0.004 0.006 0.008 0.01
C_Ss (mol/l) 30
Example 3.5
D. Thornton and co-workers studied the hydrolysis of sucrose at pH = 4.5 and
25C using crude invertase obtained from baker's yeast in free and immobilized
form. The following initial velocity data were obtained with 408 units of crude
enzyme (1 unit = quantity of enzyme hydrolyzing 1 mol of sucrose/min when
incubated with 0.29 M sucrose in a buffer at pH 4.5 and 25C).

a. Determine the Km and Vm for this reaction using both free and immobilized
enzyme.
b. Do the data indicate any diffusion limitations in the immobilized enzyme 31
preparation?
Solutions

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 Solutions
From a double-reciprocal plot of 1lV
versus 1/S for free enzyme

-1/Km = -20 and Km = 0.05 M.

1/Vm = 2 and Vm = 0.5 mmol/l. min

From a double-reciprocal
plot of 1lV versus 1/S for the immobilized
enzyme,

-1/Km = -20 and Km = 0.05 M.

1/Vm = 3 and Vm = 0.33 mmol/l. min
Km measure the amount of substrate
required for effective catalysis to occur. If
Km values same (for free and immobilized
enz) means Ss=Sb and there is no diffusion
limitation. 33
Problem 3.14

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Notes

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Transport & Kinetic Processes in Catalytic Reactions

external diffusion of A

bulk gas phase

Reactant A

hydrodynamic
boundary
layer

porous catalyst particle

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Courtesy of Prof. David Rockstraw, New Mexico State University.
Transport & Kinetic Processes in Catalytic Reactions

external diffusion of A

internal diffusion of A

bulk gas phase

hydrodynamic
boundary
layer

porous catalyst particle

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Courtesy of Prof. David Rockstraw, New Mexico State University.
Transport & Kinetic Processes in Catalytic Reactions

external diffusion of A

internal diffusion of A

adsorption of A
A + S AS bulk gas phase

hydrodynamic
boundary
layer

porous catalyst particle

S catalyst adsorption site 38

Courtesy of Prof. David Rockstraw, New Mexico State University.
Transport & Kinetic Processes in Catalytic Reactions

external diffusion of A

internal diffusion of A reaction of A to B

AS BS
adsorption of A
A + S AS bulk gas phase

hydrodynamic
boundary
layer

porous catalyst particle

39
Courtesy of Prof. David Rockstraw, New Mexico State University.
Transport & Kinetic Processes in Catalytic Reactions

external diffusion of A

internal diffusion of A reaction of A to B

AS BS
adsorption of A desorption of B
A + S AS bulk gas phase BS B + S

hydrodynamic
boundary
layer

porous catalyst particle

40
Courtesy of Prof. David Rockstraw, New Mexico State University.
Transport & Kinetic Processes in Catalytic Reactions

external diffusion of A

internal diffusion of A reaction of A to B internal diffusion of B

AS BS
adsorption of A desorption of B
A + S AS bulk gas phase BS B + S

hydrodynamic
boundary
layer

porous catalyst particle

41
Courtesy of Prof. David Rockstraw, New Mexico State University.
Transport & Kinetic Processes in Catalytic Reactions

external diffusion of A external diffusion of B

internal diffusion of A reaction of A to B internal diffusion of B

AS BS
adsorption of A desorption of B
A + S AS bulk gas phase BS B + S

hydrodynamic
boundary
layer

porous catalyst particle

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Courtesy of Prof. David Rockstraw, New Mexico State University.
Active site

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 At small particle diameters, k is large, and at its maximum value, but P is
also large, resulting in a low rate of rxn & conversion.
At large particle diameters, the P is small, but so is the overall rate constant,
kr , and the rate of rxn, resulting in low X.
Thus, we see a low X at both large and small particle diameters with an
optimum in between.

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How mass transfer occurs?

Mass transfer across an interface between a gas

and a liquid or between two liquid phases.

Such interfaces exist in absorption, distillation,

extraction, and stripping.

Need to develop theoretical models to describe mass

transfer between a fluid and such an interface.
Film Theory
A simple theoretical model for mass transfer
to or from a fluid-phase boundary was
suggested in 1904 by Nernst.

He postulated that the entire resistance to

mass transfer in a given phase is in a thin,
stagnant region of that phase at the
interface, called a film.
Internal mass transfer resistance:
The material balance for the spherical shell with thickness dr

Ds=Deff

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