20-Feb-18

Immobilized Enzyme Systems

CHE F421 Biochemical Engineering

Semester-II, 2017-18
BITS Pilani (Rajasthan), India

Immobilized Enzyme Systems

Definition:
The restriction of enzyme mobility in a fixed space.
or
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.

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Immobilized Enzyme Systems

Advantages:
1. Reduce costs of operation compared to free enzyme
systems where additional separation and purification steps
are needed.
2. Some immobilization methods can increase enzyme activity.
3. A model system to study enzyme action in membrane-bound
enzymes that occur in the cell.
Disadvantages:
1. Many immobilized enzymes exhibit lower activity compared
to free enzymes.
2. More expensive to prepare than free enzymes.
3. Mass transfer limitations due to immobilization methods.

Methods of Enzyme Immobilization

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Matrix Entrapment of Enzymes

Matrix Entrapment
• 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 cannot diffuse out, but
smaller substrate and product molecules can.
Matrices for Entrapment: The matrix can be a particle, a
membrane, or a fiber.
Polymeric materials Solid material
• Ca-alginate • Agar * Activated carbon
• Polyacrylamide • Collagen * Porous ceramic

Membrane Entrapment
Membrane Materials
• Enzymes solution may be confined between thin semi-
permeable membranes. Membrane materials include;
• Nylon • Cellulose
• Polysulfone • Polyacrylate
Membrane Configurations
Hollow fiber configuration is a popular arrangement for
separating enzyme from substrate and product solution.

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Membrane Entrapment:
Diffusion Processes
• Semi-permeable membrane: Retains high-molecular
weight compounds (enzyme), small molecular weight
compounds (substrate or products) diffuses.

Membrane Entrapment:
Microencapsulation
• In this technique, microscopic hollow spheres are
formed.
• The spheres contain the enzymes, while the sphere is
enclosed within a porous membrane.
• The membrane can be polymeric or an enriched
interfacial phase formed around a microdrop.

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Enzyme Entrapment: Problems

• Enzyme leakage into solution.
– Can be overcome by reducing molecular wt. cutoff of
membranes or the pore size of solid matrices.
• Significant diffusional limitation.
– Can be eliminated by reducing the particle size of matrices
and/or capsules.
• Reduced enzyme activity and stability.
• Lack of control of micro-environmental conditions.
– Can be improved by changing process conditions

Surface Immobilization: Adsorption

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 since binding
forces are weak.
• Adsorption stability increases on cross-linking with
glutaraldehyde.
Solid Support Materials: may need surface pretreatment
(physically or chemically) for effective immobilization
Inorganic Organic
Alumina, Silica, Porous Glass, Cellulose, starch, activated
Ceramics, Diatomaceous Earth, carbon, Ion Exchange Resins
Clay or Bentonite.

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Surface Immobilization: Covalent Bonding

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.
• Support material functional groups are activated by using
chemical reagents such as cyanogen bromide,
carbodiimide, and glutaraldehyde.

Surface Immobilization
• The cross-linking of enzyme molecule with each other
using agents such as glutaraldehyde is another method of
enzyme immobilization.
– May cause significant changes in enzyme activity,
– May result in severe diffusion limitation.
• The most suitable support material and immobilization
method vary depending on the enzyme and particular
application.
• Criteria for selection of support material:
1. The binding capacity of the support material = f (charge
density, functional groups, porosity, and hydrophobicity)
2. Stability and retention of enzymatic activity.

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Surface Immobilization

Diffusional Limitations:
Immobilized Enzyme Systems
• Diffusional limitations are observed to various degrees in all
immobilized enzyme systems.
• Resistances vary depending on:
– Nature of support material (porous, nonporous)
– Hydrodynamic conditions
– Distribution of enzyme (inside or on surface)
• This occurs because substrate must diffuse from the bulk
solution up to the surface of the immobilized enzyme prior to
reaction.
• The rate of diffusion relative to enzyme reaction rate
determines whether limitations on intrinsic enzyme kinetics is
observed or not, and is characterized by Damkohler no. (Da).

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Diffusional Limitations:
Immobilized Enzyme Systems
• Damkohler Number:

where,
[Sb] is substrate conc. in bulk liquid (g/cm3);
kL is mass transfer coefficient (cm/s)

• If Da>>1, diffusion rate is limiting the observed rate

• If Da<<1, reaction rate is limiting.

Diffusional Effects on Surface-Bound

Enzymes on Non-porous Supports
Assumptions:
• Enzymes are bound and
evenly distributed.
• All enzyme molecules are
equally active.
• Substrate diffuses
through a thin film to
reach the reactive
surfaces.
• Protein structure and
intrinsic kinetic
parameters are unaltered.

Enzyme Loading (mg enzyme/cm2 )

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Diffusional Effects on Surface-Bound

Enzymes on Non-porous Supports
• At steady state the reaction rate is equal to the mass-
transfer rate:

where,
- is the maximum reaction rate per unit of
external surface area,
kL - is the liquid mass transfer coefficient.
• The equation can be solved graphically as shown:

Diffusional Effects on Surface-Bound Enzymes

on Non-porous Supports (cont.)

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Example
• Consider a system where a flat sheet of polymer coated with
enzyme is placed in a stirred beaker.
• The intrinsic maximum reaction rate (Vm) of the enzyme is 6 x 10-6
mol/s-mg enzyme.
• The amount of enzyme bound to the surface has been determined
to be 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
• (a) the bulk concentration of the substrate is 7 x 10-3 mol/l?
• (b) Sb =1 x 10-2 mol/l?

Diffusional Effects on Surface-Bound Enzymes

on Non-porous Supports (cont.)
• When the system is strongly mass transfer limited, [Ss] ≈0,
since the reaction is rapid compared to mass transfer, and

the system behaves as a pseudo first order.

• When the system is reaction limited (Da << 1), the reaction
rate is often expressed as,

Km,app is estimated experimentally as the value of [Sb], giving

one-half of the maximum reaction rate.

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Diffusional Effects in Enzymes Immobilized

in a Porous Matrix
• Substrate diffuses
through tortuous
pathway.
• Diffusion and reaction
are simultaneous in this
case.
• Assuming uniform =Sb;
distribution of enzyme negligible
in a spherical support film
particle and the kinetics
are expressed by M-M resistance
Kinetics.

Diffusional Effects in Enzymes Immobilized

in a Porous Matrix

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Diffusional Effects in Enzymes Immobilized

in a Porous Matrix

Diffusional Effects in Enzymes Immobilized

in a Porous Matrix (cont.)
• At steady state the diffusion rate is equal to reaction
rate:

(3.56)

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Diffusional Effects in Enzymes Immobilized

in a Porous Matrix (cont.)

Diffusional Effects in Enzymes Immobilized

in a Porous Matrix (cont.)

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Diffusional Effects in Enzymes Immobilized

in a Porous Matrix (cont.)

Diffusional Effects in Enzymes Immobilized

in a Porous Matrix (cont.)

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Diffusional Effects in Enzymes Immobilized

in a Porous Matrix (cont.)
• At steady state, the rate of reaction within matrix (rs)
is equal to the rate of diffusion through matrix
surface (Ns),

• Under diffusional limitations,

Diffusional Effects in Enzymes Immobilized

in a Porous Matrix (cont.)
• The effectiveness factor is defined as the ratio of the
reaction rate with diffusion limitation (or diffusion
rate) to the reaction rate with no diffusion limitation.
• The value of η is a measure of the extent of diffusion
limitation.
• For η < 1, the conversion is diffusion limited.
• For η ≈ 1, conversion is limited by reaction rate.

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Effectiveness Factor and

Thiele Modulus/Michaelis Constant
• Fig. 3.20 (Shuler) Theoretical relationship between η, Φ and β.

Diffusional Effects in
Enzymes Immobilized
in a Porous Matrix
(cont.)

• For a zero-order reaction rate (b  0), h  1 for a large

range of Thiele modulus values such as 1 < f < 100.
• For a first-order reaction rate (b ∞ ), h = (f, b) and h
is approximated to the following equation for high
values of f.

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Diffusional Effects in Enzymes

Immobilized in a Porous Matrix (cont.)
• Under diffusional limitations, the rate-constant Vm,app
and Km,app values are not true intrinsic rate constants,
but apparent values.
• To obtain true intrinsic rate constants the diffusion
resistances should be eliminated by:
– using small particle sizes,
– a high degree of turbulence around the particles, and
– high substrate concentrations.

Effectiveness Factor and

Particle Radius/Enzyme Loading
• Fig. 3.21. η as a function of enzyme loading and particle diameter.
As depicted in the
example in Fig.3.21,
Dp <= 10 µm and
enzyme loadings
of less than
10 mg/cm3 are
required for high
values of the
effectiveness factor
(η ≥ 0.8).

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Effectiveness Factor and

Particle Radius/Enzyme Loading
• In immobilized enzyme systems design:
– the main variables are Vm and R, since the Km, and De are
fixed.
• The particle size (R) should be as small as possible
within the constraints of:
– particle integrity,
– resistance to compression, and
– the nature of the particle recovery systems.

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“Typical” Production of Industrial Enzymes

Medicinal Uses of Enzymes

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Thanks

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