Chapter 3: Enzymes

Prof. Zakaria Al-Qodah

Department of Chemical Engineering

School of Engineering

1st Semester (2024-2025)

Presentation Outline: Lectures 5-8
Immobilized Enzyme Systems

Methods of Enzyme Immobilization

Diffusional Limitations:

Effectiveness of Immobilized Enzymes

Overview of Industrial and Medicinal Enzymes

 Biocatalyst Immobilization - Definition
The containment of enzyme solution within a confined space with
retention of its catalytic activity for the purposes of:

Increasing enzyme concentration

Retaining and re-using enzyme in processing equipment.

The term "immobilization" is applicable also for cells, either

microbial, plant or animal cells and even for cell organelles.

The first discovery of this process was by Nelson and Griffin 1961,
when they find that invertase is immobilized (adsorbed) on charcoal
and able to hydrolyze sucrose.

There are many advantages that accompany immobilized enzymes

and many methods for immobilization.
 Immobilized Enzyme Systems

Advantages
Reduce costs of operation compared to free enzyme systems
where additional separation and purification steps are needed.

Some immobilization methods can increase enzyme activity.

A model system to study enzyme action in membrane-bound

enzymes that occur in the cell.

These facts suggest that energy saving, resources saving, low

pollution process and low waste products could be achieved by
using immobilization
 Immobilized Enzyme Systems

Disadvantages
Many immobilized enzymes exhibit lower activity compared to
free enzymes.

More expensive to prepare than free enzymes.

Mass transfer limitations due to immobilization methods.

Some immobilization methods could cause inhibition

Biocatalyst detachment attrition could be noticed

Methods of Enzyme Immobilization

Major immobilization methods.

 Matrix Entrapment of Enzymes

Entrapment involves entrapping enzymes within the

spaces of a cross-linked water-insoluble polymer
The enzyme solution is mixed with a polymeric fluid that solidifies
into various forms, depending on application (usually small beads).

The polymeric material is semi-permeable.

 Large molecular weight enzymes can not

diffuse out

 but smaller substrate and product molecules

can.
 Matrices for Entrapment

Ca-alginate

 Agar

Polyacrylamide

 Collagen

k-carrageenin
Steps for enzyme and cell immobilization
Selection of the suitable enzyme or cell

2- Selection of the suitable carrier.

3- Selection of the suitable immobilization technique.

Selection of the suitable enzyme or cell to perform a

required reaction is carried out through several screening
researches;

 while selection of the suitable carrier and immobilization

technique depends on the type of the biocatalyst.
Steps of enzyme immobilization using Ca-alginate
 Dissolve 9 g of Na-alginate in 300 ml DW.

 Stir until all sodium alginate is completely dissolved. The final

solution contains 3% alginate by weight.

 Thoroughly suspend about 1 g of the enzyme in the alginate

solution. Let air bubbles escape.

 Drip the enzyme-alginate mixture from a burette into 1000 ml of

crosslinking solution.

 The crosslinking solution is prepared by adding an additional

0.05M of CaCl2 solution with agitation with magnetic stirrer.

 Gel formation can be achieved at room temperature

Steps of enzyme immobilization using Ca-alginate
Steps of enzyme immobilization using Ca-alginate
Carrier-binding methods
This method is based on binding of the biocatalyst with a non-
soluble carrier by a covalent bond, ionic bond, physical
adsorption or bio-specific binding.

Several materials can be used such as:

 Polysaccharides: cellulose, dextran and agarose

derivatives.

 Proteins: gelatin, albumin.

 Synthetic polymers: Polystyrene derivatives, ion exchange

resins, polyurethane.

 Inorganic materials: glass, sand, ceramic and magnetite.

Support Bonding
Figure1: Steps of CNBr enzyme immobilization method

Figure 2: The spacer effect in enzyme immobilization.

Advantages and disadvantages of the covalent binding
method
Advantages:
The enzyme doesn't leak from the carrier.
2- The enzyme can be easily interacts with the substrate because it
is in the surface of the carrier.
3- The enzyme stability is often increased due to its strong binding
with the carrier.

Disadvantages:
 The yield activity of the enzyme is low due to using of toxic
materials during immobilization.
 The optimal immobilization conditions are difficult to be find.
 Renewable of the carrier and recovery of the enzyme is impossible.
Physical adsorption

This method depends on the physical interaction between

the carrier and the biocatalyst (enzyme or cells).

These forces such as hydrogen bonding, Van der Waals

force and hydrophobic interaction.

But the enzyme binding is weaker than the covalent or

ionic bonding methods.

So, it can be affected by the environmental conditions such

as temperature and solute concentration.

Example of surface carriers is activated carbon, silica,

alumina, clay, etc.
Effect of Immobilization Methods on the
Retention of Enzymatic Activity of Aminoacylase
Comparison between surface and entrapment
methods
 Surface adsorption only one external mass transfer
resistance

 Passive: No adverse effects

 Week binding: Detachment is possible

 Entrapment Two mass transfer resistances

 Active and could have negative effects

 Restricted in the pores: no leaking

 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 I L
F AN
M SFER Sb
Enzyme TR

Low S concentration C E
EN
E R
F F
DI O N
N T I
T IO A C
N T R A
A T TRDIFFUSION
C E IC
N
C O LEC
T R
DRIVING FORCE
E
 HIGH

Immobilized I L
F AN
M SFER Sb
Enzyme TR

REACTION C E
EN
E R
F F
DI O N
N T I
T IO A C
N T R A
A T TRDIFFUSION
C E IC
PRODUCT
N
C O LEC
T R
DRIVING FORCE
E
 HIGH

Immobilized I L
F AN
M SFER Sb
Enzyme TR
LE
TIC
AR
- P R
RA FE
NT NS
I A
TR
DIFFUSION
DRIVING FORCE
 HIGH

Immobilized I L
F AN
M SFER Sb
Enzyme TR
REACTION E
L
T IC
AR
A-P ER
TR SF
IN AN
TR 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
Ss: substrate concentration at surface;
Liquid Film Thickness, L
Sb: substrate concentration in bulk solution.
 Diffusion effects in surface-bound enzymes
on nonporous support materials.
Assume:
Enzyme are evenly distributed on Ss
Sb
the surface of a nonporous support
material.
All enzyme molecules are equally
active.
Substrate diffuses through a thin
Enzyme
liquid film surrounding the support
surface to reach the reactive surface. 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:
Damköhler 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/cm 2-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 k L.

The liquid film mass transfer coefficient k L:
 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)
ν 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 4r  De 4r 2 v 4r 2 r
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+Δr
Diffusion Effects in Enzymes
Immobilized in a Porous Matrix
Dividing the two sides of the equation by 4r
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 ( De r ) vr 2
dr dr
Re-arrange this equation

d 2[S ] 2 d[S ] 2
De( r  2r ) vr
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
 
2 r dr S 
dr
Vm "
 R =Thiele modules
S s De
 d2S 2 dS 2 S
 
2 r dr S  
dr

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 4R 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 without diffusion limitation.
  f ( ,  )
" K
 R
Vm
 m
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
 Vm " Km
 R 
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 without diffusion limitation.

  f ( ,  )

Vm " Km
 R 
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