Immobilized Enzyme Reactors

Recycle packed column reactor:

- allow the reactor to operate at high fluid velocities.
- a substrate that cannot be completely processed on a single pass
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.
 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
Sb
Enzyme

Low S concentration

DIFFUSION
DRIVING FORCE
 HIGH

Immobilized
Sb
Enzyme

REACTION

DIFFUSION
PRODUCT DRIVING FORCE
 HIGH

Immobilized
Sb
Enzyme

DIFFUSION
DRIVING FORCE
 HIGH

Immobilized
Sb
Enzyme
REACTION

PRODUCT
 Diffusional Limitation in
Immobilized Enzyme System
In immobilized enzyme system,
the overall production rate is determined
by
- liquid film mass transfer (external diffusion)
substrate, product
- intraparticle mass transfer (internal diffusion)
substrate, product in porous supports
- enzyme catalysis reaction
 Diffusional Limitation in
Immobilized Enzyme System
Diffusion effects in surface-bound enzymes
on nonporous support materials.
Ss
Sb
k2
E+S ES P E

Enzyme

Liquid Film Thickness, L

Ss: substrate concentration at 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.
Enzyme
-Substrate diffuses through a thin
liquid film surrounding the support Liquid Film Thickness, L
No intraparticle diffusion
surface to reach the reactive surface.
-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)

maximum rate of reaction Vm '

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/cm3)

 Diffusion effects in surface-bound enzymes
on nonporous support materials.

maximum rate of reaction Vm '

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/cm2-s):

J s k L ([Sb ] [S s ])
kL is the liquid mass transfer coefficient (cm/s).
The reaction rate is :
Vm '[S s ]
v d[S s ] / dt d[ P] / dt
K m [S s ]
Vm ' the maximum reaction rate per unit surface area.
(g/cm2-s)
Diffusion effects in surface-bound enzymes
on nonporous support materials.

According to the mass balance of substrate

at the surface :

Accumulation of substrate Ss =
substrate gain - substrate consumption

k2
E+S ES P E
Diffusion effects in surface-bound enzymes
on nonporous support materials.

According to the mass balance of substrate

at the surface :
Vm '[S s ]
d[S s ] / dt k L ([Sb [S s ])
K m [S s ]
At steady state, the reaction rate is equal to
the external diffusion rate:
Vm '[S s ]
k L ([Sb [S s ])
K m [S s ]
Diffusion effects in surface-bound enzymes
on nonporous support materials.

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

maximum rate of reaction Vm '

Da
maximum rate of diffusion k L [Sb ]
-Increase the bulk concentration of substrate.

-Increase the liquid film mass transfer coefficient kL.

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 (m2/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 ] Da << 1
v
K m, app [ Sb ]

K m,app can be determined experimentally.

 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.
 Diffusion Effects in Enzymes
Immobilized in a Porous Matrix
Assume:
-Enzyme is uniformly distributed in a
sphere support particle.
-There is not partitioning of the substrate
between the exterior and interior of the
support.
 Diffusion Effects in Enzymes
Immobilized in a Porous Matrix
At steady state, the intraparticle diffusion rate of
substrate equals to the reaction rate in a
spherical shell:
d[ S ] 2 d[ S ]
De 4r De 4r 2 v4r 2r
dr r r dr r
R

v is the reaction rate

per unit volume of support (mg/cm3-s).
De is the effective diffusivity (cm2/s). r r+r
 Diffusion Effects in Enzymes
Immobilized in a Porous Matrix
Dividing the two sides of the equation by 4r
yields
d[S ] 2 d[S ] 2
De r De r
r r r
dr dr vr 2
r
d d[S ] 2
When r 0
dr
(De
dr
r ) vr 2

Re-arrange this equation

d 2[S ] 2 d[S ]
De( r 2r ) vr 2
dr 2 dr
 Diffusion Effects in Enzymes
Immobilized in a Porous Matrix
Dividing the two sides of the equation by r2, yields,

d 2[ S ] 2 d[ S ] Vm" [S ]
De( )v v
dr 2 r dr K m [S ]

Then
d 2[S ] 2 d[S ] Vm" [S ]
De( )
dr 2 r dr K m [S ]
"
Vm is the maximum reaction rate per unit volume of support
(mg/cm3-s).
De is the effective diffusivity (cm2/s).
The above equation can be written in dimensionless form
by defining the following dimensionless variables:

[S ] r Km
S ,r ,
[S s ] R [S s ]

d 2 S 2 d S R 2Vm " S

2 r dr S s De S
dr
d2S 2 dS 2 S

dr
2 r dr S

Vm "
R =Thiele modules
S s De
 d2S 2 dS 2 S

dr
2 r dr S

With boundary conditions of

S 1, at r 1
d S / d r 0, at r 0
This differential equation can be solved numerically.
Refer to H. Fogler, Elements of Chemical Reaction Engineering
1999, p746 for analytical solution for first order reaction.
 At steady state, the rate of substrate consumption is equal to
the rate of substrate transfer through the external surface
of the support particle into the sphere.

2 d[S ]
rs N s 4R De
dr r R
Under diffusion limitations, the rate per unit volume is usually
expressed in terms of the effectiveness factor as follows:

"
Vm[S s ]
rs
K m [S s ]
 is the effectiveness factor.
reaction rate with intraparticle diffusion limitation

reaction rate withoutdiffusion limitation.

f (, )
Vm " Km
R
S s De [S s ]

1 the rate is diffusion limited.

1 the rate is reaction limited.

The relationship can be obtained empirically.

Analytical expression is available
for some special cases
 " Km
R
Vm
S s De [S s ]
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:

- Decrease the size of immobilized enzyme particle
- Increase the substrate concentration
- Increase the porosity or specific surface area of
the particle
 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 =

rate of substrate gain - substrate consumption rate
(production formation rate, or reaction rate)
At steady state,
Rate of substrate gain = substrate consumption 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 withoutdiffusion limitation.

f (, )
" Km
R
Vm
S s De [S s ]
1 the rate is diffusion limited.
1 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
Electrostatic and Steric Effects in Immobilized
Enzyme Systems

- The optimum pH for immobilized enzyme

system will shift from that of soluble free enzyme
Electrostatic effect

- The activity of enzyme toward a high-molecule-

weight substrate may be reduced.
Steric hindrance
 Excersize
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 KMapp. value was increased to
0.08 mol/L whereas the rmaxapp value stayed the
same as rmax. What is the effectiveness factor of
the immobilized enzyme when the substrate
concentration is 1 mol/L?
 Excersize 2
An enzyme which hydrolyzes the cellobiose to
glucose, -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 ko =
0.0795 mol/sm3, and cellobiose moves through
the sphere by molecular diffusion with Ds = 0.6
X 10-5 cm2 I s (cellobiose in gel). Calculate the
effectiveness factor of the immobilized enzyme
when the cellobiose concentration in bulk
solution is 10 mol/m3.
 Ex 3
The values of KM and rmax for an enzyme (21C 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
agitation speed of 50 rpm. (a) Estimate the concentration
of the substrate at the surface of the immobilized
enzyme, (b) Estimate ksa.