Immobilization of a thermostable alpha-amylase by covalent binding to an alginate matrix increases high temperature usability.
Thermostable alpha-amylase was covalently bound to calcium alginate matrix to be used for starch hydrolysis at liquefaction temperature of 95 degrees C. 1-ethyl-3-(3-dimethylamino-propyl) carbodiimide hydrochloride (EDAC) was used as crosslinker. EDAC reacts with the carboxylate groups on the calcium alginate matrix and the amine groups of the enzyme. Ethylenediamine tetraacetic acid (EDTA) treatment was applied to increase the number of available carboxylate groups on the calcium alginate matrix for EDAC binding. After the immobilization was completed, the beads were treated with 0.1 M calcium chloride solution to reinstate the bead mechanical strength. Enzyme loading efficiency, activity, and reusability of the immobilized alpha-amylase were investigated. Covalently bound thermostable alpha-amylase to calcium alginate produced a total of 53 g of starch degradation/mg of bound protein after seven consecutive starch hydrolysis cycles of 10 min each at 95 degrees C in a stirred batch reactor. The free and covalently bound alpha-amylase had maximum activity at pH 5.5 and 6.0, respectively. The Michaelis-Menten constant (K(m)) of the immobilized enzyme (0.98 mg/mL) was 2.5 times greater than that of the free enzyme (0.40 mg/mL). The maximum reaction rate (V(max)) of immobilized and free enzyme were determined to be 10.4-mg starch degraded/mL min mg bound protein and 25.7-mg starch degraded/mL min mg protein, respectively. The high cumulative activity and seven successive reuses obtained at liquefaction temperature make the covalently bound thermostable alpha-amylase to calcium alginate matrix, a promising candidate for use in industrial starch hydrolysis process.
Food Agricultural and Biological Engineering Dept., The Ohio State University, 590 Woody Hayes Drive, Columbus, OH 43210, USA.
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D000464:Alginates / Q000737:chemistry*; D002234:Carbodiimides / Q000737:chemistry; D002621:Chemistry / Q000379:methods*; D003432:Cross-Linking Reagents / Q000737:chemistry; D004492:Edetic Acid / Q000737:chemistry; D004795:Enzyme Stability; D004800:Enzymes, Immobilized / Q000737:chemistry*; D020723:Glucuronic Acid / Q000737:chemistry; D006603:Hexuronic Acids / Q000737:chemistry; D006358:Hot Temperature; D007700:Kinetics; D011485:Protein Binding; D000516:alpha-Amylases / Q000737:chemistry*
D000464:Alginates; D002234:Carbodiimides; D003432:Cross-Linking Reagents; D004800:Enzymes, Immobilized; D006603:Hexuronic Acids; C074139:1-ethyl-3-(3-dimethylaminoethyl)carbodiimide; D020723:Glucuronic Acid; D004492:Edetic Acid; D000516:alpha-Amylases