Download - Immobilized ennzymes.pptx
-
7/27/2019 Immobilized ennzymes.pptx
1/39
IMMOBILIZEDENZYMES
Module 2
-
7/27/2019 Immobilized ennzymes.pptx
2/39
Introduction
Restriction of enzyme mobility
Advantages
1. Enzyme reutilization
2. Elimination of enzyme recovery and
purification
3. Improve enzyme activity
-
7/27/2019 Immobilized ennzymes.pptx
3/39
Immobilization techniques
Two major types
1. Entrapment
2. Surface immobilization
-
7/27/2019 Immobilized ennzymes.pptx
4/39
Entrapment
Physical enclosure of enzyme
1. Matrix entrapment
2. Membrane entrapment
3. Microencapsulation
-
7/27/2019 Immobilized ennzymes.pptx
5/39
Matrix entrapment
Physical enclosure of enzyme by using
matrices
Matrices are usually polymeric material
Polymer materials like Ca-alginate, agar,
collagen, polyacrylamide and k-carrageein
Solid matrices also used
E.g. activated carbon, porous ceramic anddiatomaceous earth
The matrix can be a particle, a membrane or a
fiber
-
7/27/2019 Immobilized ennzymes.pptx
6/39
-
7/27/2019 Immobilized ennzymes.pptx
7/39
Membrane entrapment
Semi permeable membrane is used
Membrane of nylon, cellulose, polysulfone and
polyacrylate
Hollow fiber arrangement is commonly used
Semi permeable membrane retains high
molecular weight compounds (enzymes) while
allows low molecular weight compounds(substrate and product) to access enzyme
-
7/27/2019 Immobilized ennzymes.pptx
8/39
Microencapsulation
Form of membrane entrapment
Microscopic hollow spheres are formed
Spheres contain the enzyme solution
Sphere is enclosed in porous membrane
Membrane can be polymeric or an enriched
interfacial phase formed around a micro drop
-
7/27/2019 Immobilized ennzymes.pptx
9/39
Disadvantages of entrapment
1. Enzyme leakage into solution
2. Diffusional limitation
3. Reduced enzyme activity and stability
4. Lack of control of micro environmental
conditions
-
7/27/2019 Immobilized ennzymes.pptx
10/39
Surface immobilization
Two types
1. Adsorption
2. Covalent binding
-
7/27/2019 Immobilized ennzymes.pptx
11/39
Adsorption
Attachment of enzymes on the surface of
supporting particle
Weak physical force of attachments
Van der Waals or dispersion forces
Active site of enzymes are unaffected
Adsorption is stabilized by cross-linking with
glutaraldehyde
Supporting materials are alumina, silica, porous
glass, ceramics, cellulose (CMC, DEAE
cellulose), starch etc
-
7/27/2019 Immobilized ennzymes.pptx
12/39
Covalent binding
Retention of enzymes on support surface by
covalent bond
Enzyme molecule bind to support material via
certain functional group
Functional groups are amino, carboxyl, hydroxyl
and sulfhydryl
Functional group on support material are usuallyactivated by using cyanogen bromide,
carbodiimide and glutaraldehyde
-
7/27/2019 Immobilized ennzymes.pptx
13/39
Cross-linking of enzyme
Done using glutaraldehyde, 2,2-disulfonic acid
Done in different ways
1. Enzyme can cross-linked with glutaraldehyde to
form an insoluble aggregate
2. Adsorbed enzyme is cross-linked
3. Impregnation of porous support with enzyme
solution
-
7/27/2019 Immobilized ennzymes.pptx
14/39
Two major criteria for support selection
1. Binding capacity of support material
Function of charge density, functional groups,
porosity and hydrophobicity of support
surface
2. Stability and retention of enzymatic activity
Function of functional group and micro-environmental conditions
-
7/27/2019 Immobilized ennzymes.pptx
15/39
Diffusional limitations in
immobilized enzyme systems
-
7/27/2019 Immobilized ennzymes.pptx
16/39
Diffusion resistance depends on nature of
supporting material, hydrodynamic conditions
surrounding the support material
Damkohler number determines diffusion resistancehave significant effect on the rate of enzymatic
reaction rate
Sb is substrate concentration in bulk liquid (g/cc) and kL
is mass transfer coefficient (cm/s)
-
7/27/2019 Immobilized ennzymes.pptx
17/39
Damkohler no of CSTR
For non-ideal reactor
-
7/27/2019 Immobilized ennzymes.pptx
18/39
If Da>>1 diffusion rate is limiting
If Da
-
7/27/2019 Immobilized ennzymes.pptx
19/39
us on e ec s n sur ace- ounenzymes on nonporous support
materialsAssumptions
1. Enzymes are bound and evenly distributed on
the surface
2. All enzyme molecules are equally active
3. Substrate diffuses through a thin liquid film
surrounding the support surface
4. Immobilization has not altered protein structureand intrinsic kinetic parameters (Vm, Km)
-
7/27/2019 Immobilized ennzymes.pptx
20/39
At steady state
Quadratic equation w.r.t Ss
When Da>>1 system is mass transfer limited
Ss =0 and system behaves pseudo first order
-
7/27/2019 Immobilized ennzymes.pptx
21/39
If Da
-
7/27/2019 Immobilized ennzymes.pptx
22/39
Diffusion effects in enzymes immobilized
in a porous matrix
Substrate diffuses through tortuous pathway
React with enzyme immobilized on the pore
surface
Diffusion and reaction are simultaneous in thiscase
Tortuousity:- Ratio of actual distance substrate
travelled to minimum distance between thepoints
-
7/27/2019 Immobilized ennzymes.pptx
23/39
Assumptions
1. Enzyme is uniformly distributed in a spherical
support particle
2. Reaction kinetics are expressed by Michaelis-Menten kinetics
3. No partition of the substrate between the
exterior and interior of the support
-
7/27/2019 Immobilized ennzymes.pptx
24/39
At steady state:-
With boundary conditions [S] = [Ss] at r = R
and d[S]/dr =0 at r = 0
De is the effective diffusivity of substrate within
the porous matrix
-
7/27/2019 Immobilized ennzymes.pptx
25/39
Previous equation can be written in
dimensionless form
-
7/27/2019 Immobilized ennzymes.pptx
26/39
Where
With boundary conditions at and
at
-
7/27/2019 Immobilized ennzymes.pptx
27/39
The rate of substrate consumption is equal to
the rate of substrate transfer through external
surface of the support particle at steady state
into sphere
Under diffusion limitation, the rate per unit
volume is expressed as
-
7/27/2019 Immobilized ennzymes.pptx
28/39
Effectiveness factor, Ratio of the reaction
rate with diffusion limitation to the reaction rate
with no diffusion limitation
Value ofis a measure of the extend ofdiffusion limitation
When
-
7/27/2019 Immobilized ennzymes.pptx
29/39
-
7/27/2019 Immobilized ennzymes.pptx
30/39
To eliminate diffusion resistance use small
particle sizes, a high degree of turbulence
around particle and high substrate
concentration Main variable in designing immobilized
enzyme systems are Vm and R
Km and De are fixed Particle size should be small as possible
-
7/27/2019 Immobilized ennzymes.pptx
31/39
Higher enzyme content higher enzyme activity
per unit of reactor volume
But lower effectiveness factor
For higher (0.8)
1. Enzyme loading should be less than
10mg/cm
2. Particle size
-
7/27/2019 Immobilized ennzymes.pptx
32/39
Electrostatic and steric effects in
Immobilized enzyme systems
Immobilized enzyme in charged matrix
experience shift in pH
Bulk pH of immobilized will shift to that of
soluble enzyme Charged matrix will repel or attract substrate,
product, H+ ions depending on type and
quantity of surface charged Shift in pH activity profile is given by
-
7/27/2019 Immobilized ennzymes.pptx
33/39
F is Faraday constant (96500 coulomb/eq.g)
is electrostatic potential
pHi and pHe are internal and external pH values
z is charge on the substrate R is gas constant
Intrinsic activity of enzyme is altered
Alteration of apparent kinetics due change in pH
Enzyme activity towards high molecular weightsubstrate is reduced by immobilization
Due to steric hinderance by the support
-
7/27/2019 Immobilized ennzymes.pptx
34/39
Immobilization results in increase in thermal
stability
Thermal stability due to the presence of
thermal diffusion barriers and constraints onprotein folding
L l d ti f
-
7/27/2019 Immobilized ennzymes.pptx
35/39
Large scale production of
enzymes
Eg:- proteases (subtilism, rennet), hydroleases
(pectinase, lipase, lactase), isomerases
(glucose isomerase) and oxidases (glucose
oxidase) Steps in production of enzymes
1. Overproducing strains of certain organisms
2. Separation and purification of an enzymefrom organism by cell disruption, removal of
cell debris and nucleic acid
3. Precipitation of protein
-
7/27/2019 Immobilized ennzymes.pptx
36/39
Uses of enzymes
-
7/27/2019 Immobilized ennzymes.pptx
37/39
-
7/27/2019 Immobilized ennzymes.pptx
38/39
-
7/27/2019 Immobilized ennzymes.pptx
39/39