Enzyme immobilization

By Saurabh Mandal

Enzyme immobilization

The term immobilized enzymes refers to enzymes physically confined or localized in a certain defined region of space with retention of their catalytic activities, and which can be used repeatedly and continuously. The major components of an immobilized enzyme system are the 1) enzyme 2) the matrix and 3) the mode of attachment The enzymes can be attached to the support by interactions ranging from reversible physical adsorption and ionic linkages to stable covalent bonds. The first industrial use of immobilized enzymes was reported in 1967 by Chibata and coworkers, who developed the immobilization of Aspergillus oryzae aminoacylase for the resolution of synthetic racemic D-L amino acids.

FACTOR THAT ARE IMPORTANT IMMOBILIZATION TECHNIQUE

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The characteristics of the matrix are of paramount importance in determining the performance of the immobilized enzyme system. Ideal support properties physical resistance to compression Hydrophilicity inertness toward enzymes ease of derivatization Biocompatibility resistance to microbial attack Availability at low cost

The physical characteristics of the matrices (such as mean particle diameter, swelling behavior, mechanical strength, and compression behavior) is the major importance for the performance of the immobilized systems and will determine the type of reactor used under technical conditions (i.e., stirred tank, fluidized, fixed beds). Typically pore size and particle size determine the total surface area and thus critically affect the capacity for binding of enzymes.

Methods Of Enzyme Immobilization

Adsorption: one of the simplest means of

immobilization that involves electrostatic forces such as vander waal forces, ionic bridges and hydrogen bonds, which are instrumental in establishing the reversible surface interactions between the support and the enzyme.

In this method :-

1. 2.

catalytic properties of the enzyme remain unaltered no change in the chemical nature of either the enzyme or the supporting matrix

3. 4.

allowing the regeneration of matrix reversible process

Moreover it serves as a rapid and economical means of immobilization A major limitation of this method lies in the possibility of the enzyme leaking from the support owing to changes in the reaction conditions.

Entrapment: the enzyme molecules are free in solution but their movement is restricted owing to the lattices in the gel The degree of porosity is set so as to prevent leakiness of the enzyme but allows unrestricted diffusion of the substrates as well as the products.
Entrapment may be achieved by a number of means which include the following: Temperature induced gelation: This is brought about by adding the enzyme to a 1-2 % preparation of agarose or gelatin kept at 45C, and then bringing down the temperature. Organic polymerization by chemical/photochemical reaction: Here, the enzyme is mixed with chemical monomers which are polymerized to form a cross linked network, trapping the enzyme.

1.

Cross linking: This technique involves joining of cells/enzymes to each other, giving rise to a three dimensional complex structure. This is achieved by the formation of covalent bonds, which in turn, is brought about by means of reagents like glutaraldehyde and toluene di-isocyanate. However, owing to the toxicity of these reagents, this method is not generally used when viable cells are involved.

2.

Covalent binding: This method involves the formation of covalent interactions between the functional groups present on the support surface and those present on the amino acid residues on the enzyme surface there are a number of reaction procedures.
to achieve this (e.g. formation of an isourea linkage or a diazo linkage, a peptide bond or an alkylation reaction)

Encapsulation: it is the enclosing of a droplet of solution-of enzyme in a semipermeable membrane capsule. The capsule is made up of cellulose nitrate and nylon. The method of encapsulation is cheap and simple but its effectiveness largely depends on the stability of enzyme although the catalyst is very effectively retained within the capsule. In this method a large quantity of enzyme is immobilized but the biggest disadvantage is that only small substrate molecule is utilized with the intact membrane. Chent (1977) has given the method of enzyme encapsulation.

 NONSPECIFIC ADSORPTION

The simplest immobilization method is nonspecific adsorption, which is mainly based on physical adsorption or ionic binding. In physical adsorption the enzymes are attached to the matrix through hydrogen bonding, van der Waals forces, or hydrophobic The nature of the forces involved in non-covalent immobilization results in a process can be reversed by changing the conditions that influence the strength of the interaction (e.g., pH, ionic strength, temperature, or polarity of the solvent). Such methods are therefore economically attractive, but may suffer from problems such as enzyme leakage from matrix when the interactions are relatively weak. IONIC BINDING- An obvious approach to the reversible immobilization of enzymes is to base the proteinligand interactions on principles used in chromatography. In ionic bonding the enzymes are bound through salt linkages. FORMATION OF DISULFIDE BONDS- These methods are unique because, even though a stable covalent bond is formed between matrix and enzyme, it can be broken by reaction with a suitable agent such as dithiothreitol (DTT) under mild conditions. Additionally, because the reactivity of the thiol groups can be modulated via pH alteration, the activity yield of the methods involving disulfide bond formation is usually highprovided that an appropriate thiol-reactive adsorbent with high specificity is used.

APPLICATION
DIARY INDUSTRY (immobilized

invertase systems and the production of lactose hydrolyzed milk by use of -galactosidase)

LEATHER INDUSTRY (Immobilized proteases offer a great deal of applications in this field)

FOOD INDUSTRY (conversion of glucose to high concentration fructose syrups by the immobised enzyme glucose isomerase) PHARMACEUTICAL INDUSTRY (the production of 6amino penicillanic acid or 7-amino deacetoxy cephalosporanic acid by means of immobilized systems)

They have been used as BIOSENSORS and BIOCHIP

Immobilized enzymes are used in several pathological and biochemical diagnosis purposes