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Addis Ababa University

Addis Ababa Institute of Technology


School of Chemical & Bio Engineering

Course Title: ChEg 4190: Biochemical Engineering

Credit hour: Lect. 2hr / Tut. 3hr

Instructor: Lemessa E. Room E-107,

E-mail: leme3388@gmail.com

lemessa.etana@aait.edu.et

A/Year: Semester II, 2014


1
Chapter 4: Immobilized Enzymes
1. Introduction
2. Immobilization Technique
3. Diffusion Limitation in Immobilized
Enzyme System
1. Introduction to Immobilized Enzymes systems
Immobilization - Definition
The containment of enzyme solution within a
confined space for the purpose of retaining and
re-using enzyme in processing equipment.
There are many advantages that accompany
immobilized enzymes and many methods for
immobilization.
any immobilized enzyme, by definition, must
comprise two essential functions, namely the
non-catalytic functions (NCF) that are designed to aid
separation (e.g. isolation of catalysts from the
application environment, reuse of the catalysts and
control of the process) and the
catalytic functions (CF) that are designed to convert
the target compounds (or substrates) within the time
and space desired
Immobilized Enzyme Systems
Advantages
1. Reduce costs of operation compared to free enzyme
systems where additional separation and purification steps
are needed.
2. A model system to study enzyme action in membrane-
bound enzymes that occur in the cell.
3. Some immobilization methods can increase enzyme
activity.
Improvement-by-immobilization is focused mainly on utilization of available
immobilization techniques to alter (or improve) enzyme performance, to
suit the desired application. Thus, the native enzyme might be not suitable
for a desired process, because of its poor performance such as lower
activity, or stability or selectivity. (three catalytic characteristics)
genetically engineered enzymes performance can be
further improved by immobilization techniques and many
examples have revealed that enzyme-immobilization
techniques are indeed an indispensable complimentary
tool in enzyme engineering, due to its potential for:
combination of immobilization and improvement,
modulation of enzyme performance by selecting appropriate
method of immobilization, and
combination of different immobilization methods.
Benefits from use of the immobilized enzymes rather than the soluble
counterparts, for instance as:
reusable heterogeneous biocatalysts, with the aim of reducing
production costs by efficient recycling and control of the process,
stable and reusable devices for analytical and medical applications
selective adsorbents for purification of proteins and enzymes
fundamental tools for solid-phase protein chemistry and
effective micro devices for controlled release of protein drugs
Disadvantages
1.Many immobilized enzymes exhibit lower
activity compared to free enzymes.
Enzymes may be stressed
The shape of the active site may be changed
2.More expensive to prepare than free
enzymes.
3.Mass transfer limitations due to
immobilization methods.
Immobilization criteria
There are a number of requirements to achieve a
successful immobilization:
The biological component must retain
substantial biological activity after attachment
It must have a long-term stability
The sensitivity of the enzyme must be
preserved after attachment
Overloading can block or inactivate the active
site of the immobilized biomaterial, therefore,
must be avoided
Properties of support material
The form, shape, density, porosity, pore size distribution,
operational stability and particle size distribution of the supporting
matrix will influence the result
The ideal support is cheap, inert, physically strong and stable
Ideally, it should:
increase the enzyme specificity (kcat/Km)
shift the pH optimum to the desired value for the process
discourage microbial growth and non-specific adsorption
Some matrices may possess other properties which are useful for
particular purposes such as
ferromagnetism (e.g. magnetic iron oxide, enabling transfer of
the biocatalyst by means of magnetic fields)
a catalytic surface (e.g. manganese dioxide, which
catalytically removes the inactivating hydrogen peroxide
produced by most oxidases)
2. Immobilization Technique
Immobilization methods

a) adsorption
b) covalent binding
c) entrapment
d) encapsulation
1. Entrapment

1.1 Matrix Entrapment of Enzymes

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
1.2. Membrane Entrapment
Enzymes solution may be confined between
thin semi-permeable membranes.
Membrane materials include;
Nylon
Cellulose
Polysulfone
Polyacrylate
Membrane Entrapment: Diffusion Processes
2. Surface Immobilization
2.1. Adsorption: Attachment of enzymes to
stationary solids by weak physical forces (van der
Waals or dispersion forces).
Active site is normally unaffected and nearly full
activity is observed. Desorption of enzymes is a
common problem.
Solid Support Materials:

Alumina Diatomaceous Earth


Silica Clay
Porous Glass Cellulose Materials
Ceramics Activated Carbon
Ion Exchange Resin
Starch
2.2. Covalent Bonding: The retention of enzyme on support
surfaces by covalent bonding between functional groups
on the enzyme and those on the support surface.

Functional Groups on Enzymes:

Amino (protein-NH2)

Carboxyl (protein-COOH)

Hydroxyl (protein-OH)

Sulfhydryl (Protein-SH)
Active site of enzyme must not participate in
covalent bonding. Enzyme inhibitors are added to
enzyme solution during covalent bonding treatment.
3. Diffusion Limitation in Immobilized Enzyme
System

external diffusion the transport


of substrates towards the surface,
and products away
internal diffusion the transport of
the substrates and products,
within the pores of immobilised
enzyme particles
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
In immobilized enzyme systems, the overall
production rate is determined by
- liquid film mass transfer (external diffusion)
- intraparticle mass transfer (internal diffusion)
substrate, product in porous supports
- enzyme catalysis reaction
3.1. Diffusion Effects in Surface-bound Enzymes
on Nonporous Support Materials

Ss
Sb
k2
E+S ES 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.
Ss
Assume: 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 Thickness, L


liquid film surrounding the support 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.
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 ]
is the maximum reaction rate per unit of
Vm ' 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)


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.
The external diffusion rate Js (g/cm2-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 [S s ]
Vm ' the maximum reaction rate per unit surface area.
(g/cm2-s)
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 ([ S b ] [ 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
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.


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.
When the system is strongly reaction limited,
[Sb] [Ss]
the overall reaction rate is equal to the rate:

Vm '[ S b ]
v Da << 1
K m, app [ S b ]

where Vm'
K m ,app K m 1
k L ([ Sb ] K m )
Km,app is increased. It is a function of mixing speed and Sb.
Thank you!
Questions??????
Next week
3.2 Diffusion Effects in
Enzymes Immobilized
in a Porous Matrix
3.2 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
Assume:
- Enzyme is uniformly distributed in a
spherical support particle.
- The reaction kinetics follows Michaelis-
Menten 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 [Ss ]
is effectiveness factor.
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 the rate is .
1 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