Amylose acetate enzymic hydrolysis

Cellulose differs from amylose principally in the stereochemistry of the acetal linkages, which are a in amylose but P in cellulose. a-Amylase is specific for al 4 bonds and is not able to hydrolyse pi 4 bonds. An alternative enzyme, termed cellulase, is required. Animals do not possess cellulase enzymes, and thus cannot digest wood and vegetable fibres that are predominantly composed of cellulose. Ruminants, such as cattle, are equipped to carry out cellulose hydrolysis, though this is dependent upon cellulase-producing bacteria in their digestive tracts. 

Both endoamylases (a-amylases) and exoamylases (glucoamylases) are produced by microorganisms, and these enzymes are also utilized by plants and mammals for the biodegradation of starch. The endoamylases generally hydrolyze only the main chain acetal bonds in either amylose or amylopectin and are not active on the branch points of the latter, but many of the exoamylases can cleave either main chain or branch bonds. Some exoamylases, however, cannot catalyze the hydrolysis of the 1,4-glycosidic bonds on units containing a 1,6-gIycosidic branch point. 

Two methods were used one is the iodine method that was used to determine dextrinization or the ratio of hydrolysis of the starch, and the other is the phenolphifaalein method lhat was used to determine CD formation. Starch-dextrinizing activity was determined in accordance with Fuwa (19) and Pongasawasdi and Yagisawa (20) with slight modifications. The reaction mixture containing 100 (iL of diluted enzyme aliquot and 300 pL of 0.5% soluble starch prepared in 0.1 M acetate buffer, pH 5.5, was incubated at 55 °C for 10 min. The enzyme reaction was stopped by the addition of 4.0 mL of 0.2 M HCl solution. Then, 0.5 mL of iodine solution (0.3 g/L I2 and 3.0 g/L KI) was added to form an amylose-iodine complex with residual amylose. The final volume was adjusted to 10 mL with distilled water. The absorbance of the blue color of the amylose-iodine complex was measured by spectrophotometer at 700 nm, and a decrease in absorbance was verified, when compared to a control tube with heat-inactivated enzyme. One unit of enzyme activity was defined as the quantity of enzyme that reduces the blue color of the starch-iodine complex by 10% per minute. 

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