STUDIES ON STABILIZATION OF AMYLASE BY COVALENT COUPLING TO SOLUBLE POLYSACCHARIDES

In order to enhance thermal and proteolytic stability of amylase produced from a Bacillus sp., glycosylation was performed employing two chemical routes, one involving cyanogen bromide activation and the other sodium metaperiodate oxidation. Glycosylated enzyme obtained via the cyanogen bromide activation method was found to show better stability compared to the other route. There was little or no change in the optimum temperature and pH for the maximal activity of the enzyme after glycosylation. The pH stability of the glycosylated enzyme remained almost the same as that of the native enzyme, but it showed enhancement in its thermal stability, resistance to urea denaturation, and proteolytic digestion. At 100-degrees-C, the conjugated enzyme retained 50% of its total activities after 20 min incubation in the absence of Ca2+ and substrate. In the presence of substrate and Ca2+, the stability was much higher. Native enzyme lost all of its activities within 10 min of incubation at 100-degrees-C. The action of proteolytic enzymes was much slower on the conjugated enzyme compared to the native enzyme. There was no change in the affinity for the substrate after glycosylation. Glycosylation of other commercially available amylases was also performed and enhancement in thermal stability was observed. The cause(s) of carbohydrate-induced protein stabilization was also looked into. Amylase was conjugated with dextran, acetylated dextran, and methylated dextran. The results showed that amylase could be stabilized by conjugation with modified dextran, but higher stability was achieved by conjugating the protein with hydrophilic polysaccharides, acetylated dextran, than by hydrophobic polysaccharide, methylated dextran. The results are discussed in relation to the possible elucidation of carbohydrate-induced protein stabilization.