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Immobilized Enzymes

Immobilized Enzymes

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Published by NITIN

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Categories:Types, Research, Science
Published by: NITIN on May 16, 2010
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05/27/2013

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What is an Immobilized Enzyme?
 The term
"immobilized"
means unable to move or stationary. And that is exactly whatan immobilized enzyme is
: an enzyme that is physically attached to a solid supportover which a substrate is passed and converted to product.
It is important to understand the changes in physical and chemical properties whichan enzyme would be expected to undergo upon immobilization (sometimes referred to asinsolubilization). There are a number of factors that affect the rate of theenzyme's catalytic activities. Changes have been observed in thestabilityof enzymes and in their  kinetic propertiesdue to the micro environment and the product's characteristics.
Benefits of Immobilizing an Enzyme
 There are a number of advantages to attaching enzymes to a solid support and a fewof the major reasons are listed below:
Multiple or repetitive use of a single batch of enzymes.
The ability to stop the reaction rapidly by removing the enzyme from the reactionsolution (or vice versa).
Enzymes are usually stabilized by bounding.
Product is not contaminated with the enzyme (especially useful in the food and pharmaceutical industries).
Analytical purposes - long 1/2-life, predictable decay rates, elimination of reagent preparation, etc.
Methods of Immobilization
:
: the binding of enzymes to water-insoluble carriers.
: intermolecular cross-linking of enzymes by bi-functional or multi-functional reagents.
: incorporating enzymes into the lattices of a semi-permeable gel or enclosing the enzymes in a semi-permeable polymer membrane
1. Carrier-Binding
 
The carrier-binding
 
method is the oldest immobilization technique for enzymes. In thismethod, the amount of enzyme bound to the carrier and the activity after immobilizationdepend on the nature of the carrier. The following picture shows how the enzyme is bound to the carrier:The selection of the carrier depends on the nature of the enzyme itself, as well asthe:
Particle size
Surface area
Molar ratio of hydrophilic to hydrophobic groups
Chemical compositionIn general, an increase in the ratio of hydrophilic groups and in the concentration of  bound enzymes, results in a higher activity of the immobilized enzymes. Some of themost commonly used carriers for enzyme immobilization are polysaccharide derivativessuch as cellulose, dextran, agarose, and polyacrylamide gel.According to the binding mode of the enzyme, the carrier-binding method can befurther sub-classified into:
i.
 
ii.
 
iii.
 
i. Physical Adsorption Mode
 
This method for the immobilization of an enzyme is based on the physicaladsorption of enzyme protein on the surface of water-insoluble carriers. Hence, themethod causes little or no conformational change of the enzyme or destruction of itsactive center. If a suitable carrier is found, this method can be both simple and cheap.However, it has the disadvantage that the adsorbed enzyme may leak from the carrier during use due to a weak binding force between the enzyme and the carrier. The earliestexample of enzyme immobilization using this method is the adsorption of beta-D-fructo-furanosidase onto aluminum hydroxide. The processes available for physical adsorptionof enzymes are:
Static Procedure
 
Electro-deposition
 
Reactor Loading Process
 
Mixing or Shaking Bath Loading
 Of the four techniques, the most frequently used in the lab is
Mixing-Bath Loading.
For commercial purposes the preferred method is
Reactor Loading .
 A major advantage of adsorption as a general method of immobilizing enzymes isthat usually no reagents and only a minimum of activation steps are required. Adsorptiontends to be less disruptive to the enzymatic protein than chemical means of attachment because the binding is mainly by hydrogen bonds, multiple salt linkages, and Van der Waal's forces. In this respect, the method bears the greatest similarity to the situationfound in natural biological membranes and has been used to model such systems.Because of the weak bonds involved, desorption of the protein resulting fromchanges in temperature, pH, ionic strength or even the mere presence of substrate, isoften observed. Another disadvantage is non-specific, further adsorption of other proteinsor other substances as the immobilized enzyme is used. This may alter the properties of the immobilized enzyme or, if the substance adsorbed is a substrate for the enzyme, therate will probably decrease depending on the surface mobility of enzyme and substrate.
ii. Ionic Binding Mode
The ionic binding
 
method relies on the ionic binding of the enzyme protein to water-insoluble carriers containing ion-exchange residues.
Polysaccharides
and
synthetic polymers
 
having ion-exchange centers are usuallyused as carriers. The binding of an enzyme to the carrier is easily carried out, and theconditions are much milder than those needed for the covalent binding method. Hence,the ionic binding method causes little changes in the conformation and the active site of 

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