Effect of Immobilization Method on Activity of Alpha-Amylase
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Date
2007-06
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The Ohio State University
Abstract
In this study, alpha-amylase is immobilized by surface binding or by physical entrapment using calcium alginate gel beads. The objectives are to compare the effects of immobilization method and the bead size on the activity of immobilized alpha-amylase.
Process yield per unit of enzyme is improved by immobilization. Physical entrapment of enzymes in calcium alginate beads has shown to be a relatively easy, rapid and safe technique. Entrapment requires that the immobilized enzyme should be a large enough molecule to be kept inside the gel matrix but the substrate and product should be small enough to pass through the pores of the gel. Starch molecules are very large, often reaching a molecular weight of 80 million Daltons. It is expected that starch hydrolysis reaction could occur more effectively if enzyme bound to surface. Therefore, a comparison of different methods of immobilization of α-amylase to calcium alginate beads would provide useful information on the efficiency of the hydrolysis of starch into smaller sugars.
For surface adsorption, calcium alginate beads are prepared by dripping a sodium alginate solution through needles of varying gauge into a calcium chloride solution. The beads are then exposed to an alpha-amylase solution. For entrapment, a sodium alginate solution containing alpha-amylase is dripped into a calcium chloride solution to produce beads containing immobilized enzyme. For both immobilization methods, three bead sizes (2.04, 2.26, and 2.74 mm diameter) are prepared. Starch degradation is monitored as a function of time by a starch-iodine colorimetric assay.
The initial velocities for entrapped enzyme are 0.8857, 0.7261, and 0.5595 mg starch degraded / (g bead * min) and for surface bound enzyme are 1.7792, 1.4485 and 1.1491 mg starch degraded / (g bead * min) for small, medium, and large beads, respectively.
Surface adsorption requires less enzyme than the entrapment to achieve the same catalytic activity. The results reveal an increase of enzyme activity with decreasing bead diameter indicating the larger surface/mass ratio allows a greater amount of immobilized enzyme to have access to substrate. The surface immobilized enzyme yields higher activity per gram beads than the enzyme immobilized by entrapment because enzyme entrapped within the alginate gel-matrix is less accessible to high molecular weight starch. Thus, the combination of surface immobilization and bead size optimization should offer more efficient immobilization of alpha-amylase which leads to starch more cost-effective starch liquefaction process.
Advisor: Gonul Kaletunc
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Keywords
alpha-amylase, enzyme immobilization, alginate, entrapment, adsorption