Immobilization of whole cells has been defined as the physical confinement or localization of intact cells to a certain defined region of space with preservation of some, or most, catalytic activity.1 Some of the advantages of whole-cell immobilization in comparison with enzyme immobilization are the higher stability and enzyme activity, multivariate enzyme applications, and the lower cost.
On the other hand, disadvantages of using whole cell immobilization in comparison with enzyme immobilization are linked to the increased diffusion barriers caused by the much larger sizes of cells in comparison with enzymes.
There are five different types of techniques for cell immobilization:
- Covalent bonding
it is one of the simplest & oldest methods of cell immobilization. For the first time in 1916 Nelson & Griffin used charcoal to adsorb invertase. This method is based on physical adsorption of enzyme protein on the surface of water insoluble carriers.2
Enzymes can be adsorbed physically on a surface active adsorbent by contacting an aqueous solution of an enzyme with an adsorbent. Commonly employed adsorbents are ammonia-exchange resins, carbon, cation exchange resins, calcium carbonate, celluloses, clays, collagen etc.
In this method there is no permanent bond formation between carrier & enzyme as only weak bonds stabilize the enzymes to support the carrier. The weak bonds involved are (a) Hydrogen bonds, (b) Van der Waal forces, (c) Ionic interaction.
The cell immobilization by adsorption method can be done by the following ways:
a. Reactor loading process: in this method the carrier is placed into the reactor and the enzyme solution is transferred to the reactor with agitation of the whole content in the reactor.
b. Electrode-position process: in this process the carrier is placed in the vicinity of one of the electrode in an enzyme bath & electric current is applied resulting in migration of enzyme towards the carrier which thus results in positioning of enzyme on the surface of the carrier.
c. Static process: in this technique the cell is immobilized by allowing it to be in contact with the carrier without agitation.
d. Dynamic Process: this process involves admixing of enzyme with the carrier under constant agitation using mechanical shaker.
2. Covalent Bonding:
This is the most widely used method for immobilization. This method involves formation of covalent bonds between the chemical groups in enzyme & the chemical groups on the support or carrier. Chemical groups in the carrier that can form covalent bonds with support are hydroxyl groups, amino groups, carboxyl groups, methylthiol groups and phenol ring.
The cell immobilization by covalent bonding method can be done by the following ways:
a. Peptide bond: involves bonding between amino or carboxyl groups of the support & enzyme.
b. Poly-functional reagents: this method involves use of a bi-functional or multi-functional reagent which forms covalent bond between amino group of the support & amino group of the enzyme.
c. Diazoation: this method involves bonding between amino group of support & histidyl group of enzyme.
In this method the cells are not directly attached to the support surface. The cells are simply trapped inside the polymer matrix. Entrapment is carried out by mixing the biocatalyst into a monomer solution followed by polymerization initiated by a change in temperature or by a chemical reaction.3
Here the cells are physically entrapped inside a porous matrix. The matrix used will be a water soluble polymer. Pore size of matrix is adjusted with the concentration of the polymer used. for example commonly used matrixes for entrapment are (a) Agar, (b) Gelatin, (c) Alginate, (d) Polyacrylamide gels etc.
In this method enzymes are directly linked by covalent bonds between various groups of enzymes through multifunctional reagents. In this method there is no matrix or support involved. Commonly used agents are Gluteraldehyde, Diazonium salt. This technique is generally cheap & simple and used in commercial preparations and industrial applications.
In this method immobilization is done by enclosing the enzymes in a membrane capsule. The capsule is made up of semi-permeable membrane like nylon. In this method the effectiveness depends upon the stability of enzymes inside the capsule.4 It is a cheap & simple method. This method is advantageous because in this method large quantity of enzymes can be immobilized by encapsulation.
Thus the cell immobilization is done by some of the above given methods.
- Berillo D, Kirsebom H, Mattiasson B. Cryostructured and Crosslinked Viable Cells Forming Monoliths Suitable for Bioreactor Applications. 2013;57(5):339.
- Karel, Steven. The immobilization of whole cells: Engineering Principles. Chemical Engineering Science. 2016: 1321-1354.
- Gray CJ, Weissenborn MJ, Eyers CE, Flitsch SL. Enzymatic reactions on immobilised substrates. 2013:42(15):6378.
- Engelmark C, Kadow M, Wikmark Y, Rothstein M. A general protein purification and immobilization method on controlled porosity glass: biocatalytic applications. 2014;50(65):9134.
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