Immobilization of Alpha Amylase

Topics: Enzyme, Immobilized enzyme, Enzymes Pages: 6 (2041 words) Published: April 17, 2012
| 2012|
| Immobilisation of Alpha Amylase


A biocatalyst is termed immobilized, if its mobility has been restricted by chemical means. Immobilization of enzymes refers to techniques which represent variety of advantages over free enzyme catalysis including increased stability of enzyme, easy recovery of enzyme, easy separation of reactant and product, repeated or continuous use of a single batch of enzyme1 (Varavinit et al., 2002) which will ultimately save the enzyme, labor and overhead costs (Gerhartz, 1990). Immobilized enzymes have been widely used for many years in different industrial processes. Usually, immobilization of enzymes is carried out by three principle means, matrix assisted entrapment of enzyme, adsorption on a solid support, ionic or covalent binding (Swaisgood,1985; Zaborsky, 1973). Entrapment is taken as the most preferable method because it prevents excessive loss of enzyme activity after immobilization, increases enzyme stability in microenvironment of matrix, protects enzyme from microbial contamination (Cabral and Kennedy, 1993). Physical entrapment of á-amylase in calcium alginate beads has shown to a relatively easy, rapid and safe technique (Dey et al., 2003) in comparison with other immobilization methods. The method method of immobilization should be such that an enzyme faces as little conformational change as possible. The nature of the solid support or matrix plays an important role in retaining the actual confirmation and activity of enzyme in the processes that utilized immobilized biocatalysts. Thermostable á-amylase is one of the most important and widely used enzymes whose spectrum of application has widened in food, paper and detergent industries (Glazer et al., 1994, Nigam and Singh, 1995). These industries would find their boosted economy if á-amylase can be re-used which is possible by their immobilization. Therefore, the present study is attempted to immobilize á-amylase produced by Bacillus subtilis KIBGE-HAR by entrapment in calcium alginate beads. We also compared the kinetics of free and immobilized á-amylase in order to explore the benefits of immobilization of enzymes.

Because enzymes are biological catalysts that promote the rate of reactions but are not themselves consumed in the reactions in which they participate, they may be used repeatedly for as long as they remain active. However, in most of the industrial, analytical, and clinical processes, enzymes are mixed in a solution with substrates and cannot be economically recovered after the exhaustion of the substrates. This single use is obviously quite wasteful when the cost of enzymes is considered. Thus, there is an incentive to use enzymes in an immobilized or insolubilized form so that they may be retained in a biochemical reactor to catalyze further the subsequent feed. The use of an immobilized enzyme makes it economically feasible to operate an enzymatic process in a continuous mode. Numerous methods exist for enzyme immobilization, sometimes referred to as enzyme insolubilization. The overwhelming majority of the methods can be classified into four main categories: matrix entrapment, microencapsulation, adsorption, and covalent binding. Of these methods, matrix entrapment is the focus of this experiment. Many entrapment methods are used today, and all are based on the physical occlusion of enzyme molecules within a "caged" gel structure such that the diffusion of enzyme molecules to the surrounding medium is severely limited, if not rendered totally impossible. What creates the "wires" of the cage is the cross-linking of polymers. A highly cross-linked gel has a fine "wire mesh" structure and can more effectively hold smaller enzymes in its cages. The degree of cross-linking depends on the condition at which polymerization is carried out. Because there is a statistical variation in the mesh size, some of the enzyme molecules gradually diffuse toward the outer...
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