3

Chapter -3Immobilization of Enzymes

Motivation

•Consider two different modes of a continuous process involving enzymatic reactions as shown below

S,E

S,E,P S,P

S

(a) (b)

Immobilized Enzyme

Advantages of method (b)

•Reusability of enzyme•Reduced separation costs• Some enzymes are more stable if they are immobilized. (Many native

enzymes are “immobilized” in the cell.)

Definitions

•Enzyme Immobilization • Enzyme immobilization may be defined as a process of confining the

enzyme molecules to a solid support over which a substrate is passed and converted to products.

• Immobilized Enzyme• An immobilized enzyme is one whose movement in space has been restricted

either completely or to a small limited region.

Ideal Characteristics of Supports

• Inert.

•Physically strong and stable.

• Cost effective.

• Possibility to regenerate.

•Reduction in product inhibition.

Immobilization Techniques

Immobilization TechniquesPhysical methods

Adsorption

Entrapment

Encapsulation

Chemical methodsSupport

Copolymerization

Cross Linking

Entrapment EncapsulationAdsorption

Copolymerization Cross LinkingSupport

Rapid Reaction on the surface

Effect of Mass-Transfer Resistance

•Due to the large particle size of immobilized enzyme or due to inclusion of enzyme in polymeric matrix.

External Mass Transfer Resistance

•Enzymes are immobilized on surface of uncharged, nonporous flat plate.•Entire surface is uniformly accessible to substrate in adjacent fluid.•At steady state product and substrate do not accumulate at the

surface and rate of mass transfer equals rate of reaction

Dimensionless form

Concentration profiles

Distance from the surface

Conc

entr

atio

n

Distance from the surface

Conc

entr

atio

n

Reaction Limited Mass Transfer Limited

Rate of Reaction Vs Concentration

Concentration

rate

of r

eact

ion

rate

of r

eact

ion

Reaction LimitedMass Transfer Limited

Concentration

Effectiveness Factor

•

Example

•

Internal Mass Transfer Resistance

•Enzymes are immobilized to a porous support having large internal surface areas.

Internal Mass Transfer Resistance

•

rdr

In – Out = Accumulation – Generation

• First-order Kinetics

Zero-order Kinetics

Concentration Profiles

Effectiveness Factor

•

Effectiveness FactorNo resistance to

pore diffusionStrong pore

diffusion effects

•

Effective Diffusion Coefficient

•

[E. M. Renkin, J. Gen. Physiol. 38 (1954) 225-243]

Effective Diffusivities in Biological gels

•The gel structure increases the path length for diffusion, and asa result decreases the diffusion rate.•One of the most reliable techniques is the thin-disk method

• A diffusion cell with two compartments divided by a thin gel. • Each compartment contains a well-stirred solution with different solute

concentrations.

•Effective diffusivity can be calculated from the mass flux verses timemeasurement

Simultaneous External & Internal Mass Transfer Resistance

•

P.3.3