Queennie Anne B.

Casauran
10-5484
Enzyme Immobilization
Enzyme immobilization is defined as a process of confining the enzyme molecules to a solid
support over which a substrate is passed and converted to products. Most enzymes are globular
protein and are soluble in water in which it is very difficult or impractical to separate the enzyme for reuse
in a chemical processes. Immobilized enzymes where developed in order to treat that difficulty.
Immobilized enzymes are enzymes that are physically confined or localized in certain defined
region of space wherein its enzymatic activities are being retained, and are used repeatedly and
continuously. Immobilized enzymes can be easily separated from the reaction solution and can be easily
retained in a reactor. Furthermore, immobilized enzyme may show selectively altered chemical or
physical properties and it may act out the realistic natural environment where the enzyme came from
which is the cell. Immobilized enzymes are often used rather than the soluble enzymes because
immobilized enzymes is protected from degradation and deactivation, can be re-used in which it lowers
the total production cost of mediated reactions, ability to stop the reaction rapidly by removing the enzyme
from the reaction stability, enhanced stability, easy separation of the enzyme from the product, and the
product is not contaminated with the enzyme.
The major components of an immobilized enzyme system are the enzyme, the matrix, and the
mode of attachment or support. Immobilized enzymes can be attached to the support by interactions
ranging from reversible physical adsorption and ionic linkages to stable covalent bonds. The
characteristics of the matrix are of importance in determining the performance of the immobilized enzyme.
Meanwhile, supports are classified as inorganic and organic according to their chemical composition as
presented in Table 1.
Table 1. Classification of Supports
INORGANIC
Natural Polymers
Polysaccharides: cellulose, dextrance,
agar, agarose, chitin, alginate
Proteins: collagen, albumin
Carbon
Natural Minersls: bentonite, silica

Processed Materials: glass(nonporous and
controlled pore), metals, contolled pore metal
oxides
Synthethic Polymers
Polystyrene
Others: polyacrylate polymethacrylates,
polyacrylamide, polyamides, vinyl and
allyly-polymers
Source: Viera and Brena, 2001
Immobilization techniques is classified into two methods, the chemical method (covalent bond
formation dependent) and physical method (non-covalent bond formation dependent), Fig. 1.

Figure 1. Immobilization Techniques

Physical Methods for Immobilization
Adsorption. This method is the simplest way to immobilize enzymes wherein the enzyme is
adsorbed physically on a surface-active adsorbent by contacting an aqueous solution of enzyme with an
adsorbent. It involves the weak interactions like Van der Waals or hydrogen bonds. The support in this
method may be organic or inorganic. The advantages of adsorption technique are as follows; (1) the
procedure of immobilization is simple, (2) It is possible to separate and purify the enzymes while being
immobilized, (3) the enzymes are not usually deactivated by adsorption, and (4) the adsorption is a
reversible process. However, adsorption also have several disadvantages; (1) the bonding strength is
weak, (2) the state of immobilization is very sensitive to pH, ionic strength, and temperature, and (3) the
amount of enzymes loaded on a unit amount of support is usually low.

Immobilization
Techniques
Physical
Methods
Adsorption
Entrapment
Encapsulation
Chemical
Methods
Covalent
Binding
Cross Linking
Entrapment. Enzymes or cells are not directly attached to the support surface but simply trapped
inside the polymer matrix. This method has a major advantage in the fact that there is no chemical
modification of the enzyme , therefore, the intrinsic properties of an enzyme are not altered. There are
different approaches to entrap enzymes such as gel or fiber entrapping and minro-encapsulation. The
practical use of these methods is limited by mass transfer limitations through membranes or gels.

Encapsulation. Enzymes can be immobilized within semi permeable membrane microcapsules.
This can be done by the interfacial polymerization technique. Organic solvent containing one component
of copolymer with surfactant is agitated in a vessel and aqueous enzyme solution id introduced. The
polymer membrane is formed at the liquid-liquid interface while the aqueous phase is dispersed as small
droplets. The immobilized enzyme produced by this technique provides an extremely large surface area.

Chemical Methods for Immobilization.
Covalent binding. The covalent attachment of enzyme molecules to water-insoluble,
functionalized supports are the most widely used method for immobilizing enzymes. 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. However, covalent binding may alter the conformational
structure and active center of the enzymes resulting in major loss of activity and/or changes in the
substrate.

Cross linking. Cross linking involves intermolecular cross linking enzymes molecules in the
presence/ absence of solid support. This method produces a 3-dimensional crossed-linked enzyme
aggregate by means of a multifunctional reagent that links covalently to the enzyme molecules.

Table 2 shows the comparison between the immobilization methods
CHARACTERISTICS ADSORPTION
COVALENT
BINDING
ENTRAPMENT ENCAPSULATION
Preparation simple difficult difficult simple
Cost low high moderate high
Binding Force variable strong weak strong
Enzyme Leakage yes no yes no
Applicability wide selective wide very wide
Running Problems high low high high
Matrix Effects yes yes yes no
Large diffusional barriers no no yes yes
Microbial protection no no yes yes