- By Trupti Barbuddhe
What Is 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.
What Is An Immobilized Enzyme?
An immobilized enzyme is one whose movement in space has
been restricted either completely or to a small limited region.
Why Immobilize Enzymes?
• Protection from degradation and deactivation.
• Re-use of enzymes for many reaction cycles, lowering the
total production cost of enzyme mediated reactions.
• Ability to stop the reaction rapidly by removing the enzyme
from the reaction solution.
• Enhanced stability.
• Easy separation of the enzyme from the product.
• Product is not contaminated with the enzyme.
An Ideal Carrier Matrices For Enzyme Immobilization Inert.
• Physically strong and stable.
• Cost effective.
• Regenerable.
• Reduction in product inhibition.
METHODS FOR ENZYME IMMOBILIZATION
Physical Methods For Immobilization
ADSORPTION
• Involves the physical binding of the enzyme on the surface
of carrier matrix.
• Carrier may be organic or inorganic.
• The process of adsorption involves the weak interactions
like Vander Waal or hydrogen bonds.
• Carriers: - silica, bentonite, cellulose, etc.e.g. catalase &
invertase
ADVANTAGES
• Simple and economical
• Limited loss of activity
• Can be Recycled,Regenerated & Reused.
DISADVANTAGES
• Relatively low surface area for binding.
• Exposure of enzyme to microbial attack.
• Yield are often low due to inactivation and desorption.
Entrapment
• In entrapment, the enzymes or cells are not directly attached to the support surface,
but simply trapped inside the polymer matrix.
• Enzymes are held or entrapped within the suitable gels or fibres.
• It is done in such a way as to retain protein while allowing penetration of substrate.
• It can be classified into lattice and micro capsule types.
• Inclusion in gels: Poly acrylamide gel, Poly vinyl alcohol gels. Inclusion in fibers:
Cellulose and Poly -acryl amide gels.
• Inclusion in micro capsules: Polyamine. Polybasic - acid chloride monomers.
Lattice-Type Entrapment
• Entrapment involves entrapping enzymes within the interstitial
spaces of a cross-linked water-insoluble polymer.
• Some synthetic polymers such as polyarylamide,
polyvinylalcohol, etc... and natural polymer (starch) have been
used to immobilize enzymes using this technique.
Microcapsule Type Entrapmet/ Encapsulation/Membrane
Confinement
It involves enclosing the enzymes within semi -permeable polymer
membranes e.g. semi permeable collodion or nylon membranes in the
shape of spheres.
ADVANTAGES
• No chemicalmodification.
• Relatively stableforms.
• Easy handling and re-usage.
DISADVANTAGES
• The enzyme may leak from the pores.
Covalent Binding
• Based on the binding of enzymes and water-insoluble carriers by
covalent bonds
• The functional groups that may take part in this binding are Amino
group, Carboxyl group, Sulfhydryl group, Hydroxyl group, Imidazole
group, Phenolic group, Thiol group, etc*
• Disadvantages: covalent binding may alter the conformational
structure and active center of the enzyme, resulting in major loss of
activity and/or changes of the substrate
• Advantages the binding force between enzyme and carrier is so
strong that no leakage of the enzymes occurs, even in the presence
of substrate or solution of high ionic strength.
Cross Linking
• Cross linking involves intermolecular cross linking of enzyme
molecules in the presence/absence of solid support.
• The method produces a 3 dimensional cross linked enzyme
aggregate (insoluble in water) by means of a multifunctional
reagent that links covalently to the enzyme molecules.
Advantages of cross linking:-
1. Very little desorption(enzyme strongly bound)
2. Higher stability (i.e. ph, ionic & substrate concentration)
Disadvantages of cross linking:-
1. Cross linking may cause significant changes in the active site.
2. Not cost effective.
Comparison Between The Methods
Limitations Of Enzyme Immobilization
• Cost of carriers and immobilization.
• Changes in properties (selectivity).
• Mass transfer limitations.
• Problems with cofactor and regeneration.
• Problems with multienzymes systems.
• Activity loss during immobilization.
Immobilized Enzymes Reactors
Bioreactor:- device, usually a vessel, used to direct the
activity of a biological catalyst transformation to achieve
a desired chemical
Fermenter:- Type of bioreactor in which the biocatalyst is
a living cell.
Types of Reactors
Batch
• No flow of material in or out of reactor
• Changes with time
Fed- Batch
• Either an inflow or an outflow of material but not both
• Changes with time
Continuous
• Flow in and out of reactor
• Continuous Stirred Tank Reactor (CSTR)
• Plug Flow Reactor (PFR)
• Steady State Operation
Types of an Enzyme Reactor
1.Batch reactor
• Stirred tank batch reactor [STBR]
• Plug flow batch reactor [PFBR]
• Packed-bed batch reactor[PBBR]
• Fluidised-bed batch reactor [FBBR]
2.Continous reactor
• Continous Stirred tank reactor [CSTR]
• Continous Plug flow reactor [CPFR]
Batch Reactor
• Common when soluble enzymes are used.
• Fitted with fixed baffles that improve the stirring
efficiency.
• Entire product is removed.
• Enzymes & Substrate molecule have identical
residence time.
Continuous reactors
• Substrate added continuously removed
simultaneously.& product
• Certain advantages over batch reactors.
• Control over the product formation convenient
operation of the system & easy automation of
the entire process.
• 2 types - Continuous stirred tank reactor (CSTR)
& Plug flow reactor (PFR).
• Choice of continuous reactor is based on the
Kinetic considerations.
• CSTR is ideal for good product formation
Examples of Immobilized enzymes
• Immobilized glucosidase & glucose isomerase are used
in production of fructose from starch.
• Immobilized L-Aminoacylase which resolves a mixture
of D- and L-amino acids.
• Immobilization of Microbial cells.
