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IMMOBILIZED
BIOCATALYST
Properties of Immobilized
Enzymes
The properties of immobilized enzyme preparations
are governed by the properties of both the
enzyme and the carrier material.
Due to immobilization, the properties of enzymes
will be altered such as catalytic activity with
respect to the support matrix.
The change in the enzyme properties in the
immobilized enzyme is due to the enzyme and
the substrate reacts in the microenvironment
which is different from the enzyme substrate
reaction in the bulk solution environment.
Properties of Immobilized
Enzymes
As far as manufacturing costs are concerned the yield of
immobilized enzyme activity is mostly determined by the
immobilization method and the amount of soluble enzyme
used.
Under process conditions, the resulting activity may be further
reduced by mass transfer effects, lead to lowered efficiency.
More precisely, the yield of enzyme activity after immobilization
depends not only on losses caused by the binding procedure but may
be further reduced as a result of diminished availability of enzyme
molecules within pores or from slowly diffusing substrate molecules.
Support selection
Selection criteria differ among one another, depending on the
biocatalyst of interest, but there are still few basic features that must
be considered.
Material used as a carrier should have chemical, physical and
biological stability during processing, as well as in the reaction
conditions; sufficient mechanical strength, especially for its
utilization in reactors and industry; should be nontoxic both for
the immobilized cell/bioparticle, as well for the product; also
should have adequate function groups for binding biocatalyst
and high loading capacity.
Profitability of the material application and its processing costs
always have to be taken upon consideration.
Other criteria, such as physical characteristics (porosity, swelling,
compression, material and mean particle behavior), as well as
possibility for microbial growth, biodegradability, solubility, are
more application specific.
Carrier materials can be divided into those of inorganic and organic
origin
type
and
strength
of
non-covalent
proteinmatrix
Regenerability:
This property is of interest in case of expensive carrier materials.
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
Enzyme
Low S concentration
R
E
M SF
L
I
F AN
TR
Sb
E
C
N
E
R
FE
F
N
DI
O
TI
N
C
IO
A
T
R
A
R
TT
T
A
N
E
IC
C
R
N
T
C
O
E
C
L
E
DIFFUSION
DRIVING FORCE
HIGH
Immobilized
Enzyme
REACTION
PRODUCT
R
E
M SF
L
I
F AN
TR
Sb
E
C
N
E
R
FE
F
N
DI
O
TI
N
C
IO
A
T
R
A
R
TT
T
A
N
E
IC
C
R
N
T
C
O
E
C
L
E
DIFFUSION
DRIVING FORCE
HIGH
Immobilized
Enzyme
E
L
IC
T
R
A
P R
A FE
R
T NS
N
I
A
R
T
R
E
M SF
L
I
F AN
TR
Sb
DIFFUSION
DRIVING FORCE
HIGH
Immobilized
Enzyme
R
E
M SF
L
I
F AN
TR
Sb
REACTION
E
L
IC
T
R
A
P R
A FE
R
T
IN ANS
TR
PRODUCT
Diffusional Limitation in
Immobilized Enzyme Systems
In immobilized enzyme systems, the overall production rate is
determined by
- liquid film mass transfer (external diffusion)
The transport of substrates towards the surface, and products away)
- intraparticle mass transfer (internal diffusion)
The transport of the substrates and products, within the pores of
immobilised enzyme particles
- enzyme catalysis reaction
Diffusional Limitation in
Immobilized Enzyme System
Diffusion Effects in Surface-bound Enzymes
on Nonporous Support Materials
Ss
E+S
ES
Sb
k2
P E
Enzyme
Liquid Film Thickness, L
Ss
Sb
Enzyme
Liquid Film Thickness, L
No intraparticle diffusion
Vm '
maximum rate of reaction
Da
kL
[ Sb ]
Vm '
maximum rate of reaction
Da
Js
(g/cm 2-s):
J s k L ([ Sb ] [ S s ])
kL
Vm '[ S s ]
v
K m [Ss ]
'
Vm '[ S s ]
J s k L ([ Sb ] [ S s ])
K m [S s ]
J s k L ([ Sb ] [ S s ])
v k L [ Sb ]
Da>>1
Vm '
maximum rate of reaction
Da
-Increase
D2 / 3
1
/
2
AB
U
k L 0.6
1
/
6
d p1 / 2
Vm '[ Sb ]
v
K m, app [ Sb ]
Da << 1
where
K m ,app
Vm'
Km 1
k L ([ Sb ] K m )
Vm" [ S s ]
rs
K m [S s ]
is
.
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.
1
1
the rate is
the rate is
boundary film
resistances
minimized by
Rbulk
decreasing particle
size (increase
surface area/volume
ratio)
increasing [R]bulk
improved mixing,
agitation
increasing porosity
optimizing
distribution of
enzyme/cells
Vmax [R]
v
K m [R]
Summary of Diffusion
Effects
Summary of Diffusion
Effects
Summary of Diffusion
Effects
f ( , )
R
1
1
"
Vm
S s De
Km
[S s ]
Kinetics of immobilized
enzyme
- on enzymes
- enzymes have ionic groups on their active sites.
- Variation of pH changes the ionic form
sites.
of the active
Effect of Temperature
- on the rate of enzyme catalyzed reaction
d [ P]
v
k [ ES ]
2
dt
k2=A*exp(-Ea/R*T)
T
k2
- enzyme denaturation
T
d[E]
kd [E]
Denaturation rate:
dt
kd=Ad*exp(-Ea/R*T)
Where kd: enzyme denaturation rate
constant; Ea: deactivation energy
Enzyme Stability
The stability of an immobilized enzyme is highly dependent on
many factors, including:
the properties of its interaction with the carrier
the binding position and the number of the bonds
the freedom of the conformation change in the matrix
the microenvironment in which the enzyme molecule is
located
the chemical and physical structure of the carrier
the properties of the spacer (for example, charged or neutral,
hydrophilic or hydrophobic, size, length) linking the enzyme
molecules to the carrier
the conditions under which the enzyme molecules were
immobilized
Enzyme Stability
Although enzyme storage stability is important, it is the
d[E]T
k d [E]T
dt
[E]T [E]T,o e k dt
Note that the immobilized preparation is often more stable than the
soluble
enzyme and displays a period during which no enzyme activity
appears to be lost.
immobilized
enzymes
free (soluble)
enzymes
Effects of Immobilization on
Enzyme Stability and Use
enzyme stability
efficiency losses associated with the use of homogeneous
(soluble) catalysts