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IMMOBILIZED ENZYME SYSTEMS
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Advantages of immobilized enzymes:
- Easy separation from reaction mixture, providing the ability to control
reaction times and minimize the enzymes lost in the product
1) Entrapment Immobilization
2) Surface Immobilization
3) Cross-linking
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1) Entrapment Immobilization
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Advantage is enzyme is retained.
Disadvantages are
- substrate need to diffuse into enzyme and
product need to diffuse out
- reduced enzyme activity and enzyme stability owing
to the lack of control of micro environmental
conditions
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2) Surface Immobilization
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Methods of immobilization
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3) Cross linking
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Comparison between the methods
Characteristics Adsorption Covalent Entrapment Membrane
coupling confinement
Preparation Simple Difficult Difficult Simple
Cost Low High Moderate High
Binding force Variable Strong Weak Strong
Enzyme leakage Yes No Yes No
Applicability Wide Selective Wide Very wide
Running problems High Low High High
Matrix effects Yes Yes Yes No
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Fluidized bed reactor
- A high viscosity substrate
solution
- A gaseous substrate or
product in a continuous
reaction system
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- An immobilized enzyme
tends to decompose upon
physical stirring.
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Diffusion Limitations in Immobilized ENZYME SYSTEMS
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Diffusion Limitations in Immobilized ENZYME SYSTEMS
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External mass-transfer resistance:
Surface bound Enzyme Immobilize on nonporous support
Ss
Ss
Assumption: SbSb
- Enzymes are bound and evenly
distributed on the surface of a nonporous
support material.
- All enzyme molecules are equally active.
- Substrate diffuses through a thin liquid
film surrounding the support surface to
reach the reactive surface.
- Enzyme structure is unaltered
- M-M kinetic parameters (Vm, Km) are
unaltered. Enzyme
Enzyme
Liquid Film
Liquid filmThickness,
thickness,L L
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External mass-transfer resistance:
Diffusional mass transfer across
Ss
the liquid film: Ss
SbSb
JS = kL (Sb Ss)
Liquid Film
Liquid filmThickness,
thickness,L L
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External mass-transfer resistance:
At steady state (rate of S transfer
=rate of S consumption by enzymatic Ss
Ss
reaction), the reaction rate is equal to Sb
the external diffusion rate:
Js = v
Vm Ss
JS = kL (Sb Ss) =
Km + Ss
1 [Ss] [Ss]
=
Da 1 + [Ss]
where
[Ss]= x = [Ss] / [Sb]
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Internal mass transfer resistance:
Foe one dimension and steady-state condition equation 14. 50
becomes:
(which is dimensionless and combine particle dia, diffusivity and kinetic parameters)
Vm [ S b ]
rs =
K m [S b ]
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Effectiveness factor ()
reaction rate with intraparticle diffusion limitation or actual rxn rate
=
reaction rate without diffusion limitation.
Vm [SS] x
Km + [SS] 1+x
= =
Vm [Sb]
Km + [Sb] 1+
Effectiveness factor is a function of and x
Da, : measure the extent of limiting mechanism
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For maximum conversion rates, particle
size should be small (Dp 10 mm) and
enzyme loading should be optimized.
As depicted in the example in Fig. 3.21,
Dp 10 mm and enzyme loadings of
less than 10 mg/cm3 are required for
high values of the effectiveness factor
( 0.8).
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Example 3.4 in Shuler & Kargi:
Consider a system where a flat sheet of polymer coated with
enzyme is placed in a stirred beaker. The intrinsic maximum
reaction rate of the enzyme is 6 x 10-6 mols/s.mg enzyme. The
amount of enzyme bound to the surface has been determined
to be maximum 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.
Data provided:
Vm = 6 x 10-6 x 1 x 10-4 mols/s.cm2
= 6 x 10-10 mols/s.cm2
Km = 2 x 10-3 mol/l = 2 x 10-6 mol/cm3
kL = 4.3 x 10-5 cm/s
Sb = 7 x 10-3 mol/l OR 1 x 10-2 mol/l
= 7 x 10-6 mol/cm3 OR 1 x 10-5 mol/cm3
Equation to be solved:
Vm [Ss]
JS = kL ([Sb] [Ss]) =
Km + [Ss]
5.E-10 r_S
4.E-10
3.E-10
2.E-10
1.E-10
0.E+00
0 0.002 0.004 0.006 0.008 0.01
C_Ss (mol/l) 30
Example 3.5
D. Thornton and co-workers studied the hydrolysis of sucrose at pH = 4.5 and
25C using crude invertase obtained from baker's yeast in free and immobilized
form. The following initial velocity data were obtained with 408 units of crude
enzyme (1 unit = quantity of enzyme hydrolyzing 1 mol of sucrose/min when
incubated with 0.29 M sucrose in a buffer at pH 4.5 and 25C).
a. Determine the Km and Vm for this reaction using both free and immobilized
enzyme.
b. Do the data indicate any diffusion limitations in the immobilized enzyme 31
preparation?
Solutions
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Solutions
From a double-reciprocal plot of 1lV
versus 1/S for free enzyme
From a double-reciprocal
plot of 1lV versus 1/S for the immobilized
enzyme,
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Notes
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Transport & Kinetic Processes in Catalytic Reactions
external diffusion of A
hydrodynamic
boundary
layer
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Courtesy of Prof. David Rockstraw, New Mexico State University.
Transport & Kinetic Processes in Catalytic Reactions
external diffusion of A
internal diffusion of A
hydrodynamic
boundary
layer
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Courtesy of Prof. David Rockstraw, New Mexico State University.
Transport & Kinetic Processes in Catalytic Reactions
external diffusion of A
internal diffusion of A
adsorption of A
A + S AS bulk gas phase
hydrodynamic
boundary
layer
external diffusion of A
hydrodynamic
boundary
layer
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Courtesy of Prof. David Rockstraw, New Mexico State University.
Transport & Kinetic Processes in Catalytic Reactions
external diffusion of A
hydrodynamic
boundary
layer
40
Courtesy of Prof. David Rockstraw, New Mexico State University.
Transport & Kinetic Processes in Catalytic Reactions
external diffusion of A
hydrodynamic
boundary
layer
41
Courtesy of Prof. David Rockstraw, New Mexico State University.
Transport & Kinetic Processes in Catalytic Reactions
hydrodynamic
boundary
layer
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Courtesy of Prof. David Rockstraw, New Mexico State University.
Active site
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At small particle diameters, k is large, and at its maximum value, but P is
also large, resulting in a low rate of rxn & conversion.
At large particle diameters, the P is small, but so is the overall rate constant,
kr , and the rate of rxn, resulting in low X.
Thus, we see a low X at both large and small particle diameters with an
optimum in between.
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How mass transfer occurs?
Ds=Deff
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