Enzyme Immobilization

• Difficulties of using free enzyme:

– Practical: Contamination of the products with residual
enzyme
– Economic: Use of enzyme for a single reactor pass. Hence,
part of the overall potential enzymatic activity is lost.
Enzyme immobilization:
• A technique where freedom movement of the enzyme is
restricted and localized to an inert support or carrier.
• If the enzyme is immobilized, it becomes an independent
phase within the reaction system:
– Easily retained in the reactor
– Extend its useful active life
– Prevent contamination of the products
Advantages of immobilization:
• Immobilized enzyme can be reused easily (the ability to use
the immobilized enzyme repeatedly is actually the factor that
determines its effectiveness).
• Higher reaction optimum temperature (immobilization
provides a more rigid external backbone for the enzyme
molecule, allowing it to maintain its activity at higher
temperatures than if it is in free form).
• Enzyme is dispersed over a large surface, which results in an
enhanced catalytic performance, especially in organic media.
Disadvantages of immobilization:
• Additional external and internal mass transfer resistances for
both substrates and products.
• By-products adsorption on the surface of the immobilized
enzymes, which results in a loss in activity (solvents are
usually used to dissolve the by-product, which clogs the active
sites of the immobilized enzyme)
Immobilization Techniques:
Physical adsorption:

• Advantages:
– Easy
– Does not require expensive and toxic chemicals
– Retain the activity
– Regeneration feasibility
• Disadvantages:
– Subject to diffusion (internal and external) limitation
– By product deposition
– Poor adsorption results in its leaching off the support
surface
It is possible to strengthen
the attachment between
the water-soluble enzyme
and the water-insoluble
surfaces by using
multifunctional agents that
are bifunctional in nature
and have low molecular
weight, such as
glutaraldehyde
Physical adsorption:
• Enzyme adsorption can be described by the Langmuir isotherm:
a ads, max K ads a free
a ads 
1  K ads a free
 Δh ads 
K ads  β exp   aads,max =  (1 +  T)
 RT 

Where,
aads: adsorbed lipase activity
afree: lipase activity present in the supernatant solution at
equilibrium
Dhads: enthalpy change of adsorption
,  and b are contestants (can be experimentally determined)
Physical adsorption:
• In porous structure, increasing the temperature results in an
increase in the equilibrium amount of enzyme adsorbed (unlike
the general behavior of physical adsorption)

• This is a result of the increase in the diffusion of lipase into the

micropores due to expansion of the pores and the reduction of
the solution viscosity at higher temperatures.
Entrapment within a polymer matrix
• Much more stable than physical adsorption
• Unlike the covalent bonding this method uses a relatively simple
procedure.
• Offers a good compromise between stability of the
heterogeneous biocatalyst and activity loss, and hence this
technique has received considerable attention in recent
Sol–gel process
• Aqueous solution of the enzyme is mixed with alkoxysilanes as
inorganic-organic matrix precursor.
• Sol-gel material is then obtained by hydrolysis and condensation
of the precursor
• Amorphous silica matrix that entraps the enzyme is then
produced.
• Lipase entrapped in sol-gel has been used for biodiesel
production and was easily recovered from reaction media.
• However, under the same operating conditions, it was found that
immobilized lipase on ceramic beads were more efficient, mainly
due to diffusion limitations.
Special Case (Lipase immobilization)
Preparing immobilized lipases in porous structures (used to
enhance the interfacial area) has several problems especially in
almost anhydrous media:
– Lipases are only active at the oil water-interface
– When inside a porous structure, lipase molecules become
inaccessible to external surfaces, which prevent their activation.
– It is therefore proposed to use hydrophobic support that
resembles the surface of drops of the natural substrates to
immobilize lipase on.
– In this case, the adsorbed lipases are in open form, with the
active sites accessible for substrate and the immobilized enzyme
exhibits enhanced activity