Aspergillus oryzae, amylase

Reducing power of the reaction products at the hydrolysis of maltose, maltotriose, maltotetraose, maltopentaose and maltohexaose catalyzed by Aspergillus oryzae -amylase at Pjj 5.2 and 370C. Enzyme concentration 1.62x10 6 m. 

Fischer, brilliant results were achieved, and in succession the a-amylases of pig pancreas, of Bacillus subtilis, of human saliva, of human pancreas, and of Aspergillus oryzae, and the /3-amylase of malt, were successfully crystallized. Important biological deductions were gained from this study whereas the amylases of human pancreas and saliva cannot be distinguished from one another, amylases from pig pancreas and from human pancreas are different. These differences are manifested in molecular weight, crystalline forms, electrophoretic mobility, and influence of the pH on the activity however, all the amylases have the same specific biochemical action. The identity of the enzymes seems to be dependent on the species and not on the organ. Interest in biologically active proteins led Meyer to a study of the protein hormones, a field in which he was very active at the time of his death. 

While alpha amylases from many sources are known, the present discussion will deal mainly with the alpha amylase of malted barley, pancreatic amylase and the amylase of Aspergillus oryzae. These... 

Even after extensive purification, the amylase of Aspergillus oryzae is relatively stable in aqueous solutions held at ordinary room temperature. Its lability increases with increasing temperatures and becomes very rapid between 50° and 60°. This loss of activity may be retarded by the presence of substrate and by the presence of calcium ions.4070... 

The amylase of Aspergillus oryzae is most active in slightly acid solutions. When reacting in the presence of 0.01 M acetate at 40° it is most active71 at pH 5.0. 

The amylase of Aspergillus oryzae causes a very rapid decrease in the viscosity of its substrates and a very rapid disappearance from its reaction mixtures of products which give color with iodine. When examined under favorable conditions71 at 40° with Lintner s soluble potato starch, the achroic point was reached with highly purified maltase-free amylase when approximately 12% of the glucose linkages of the substrate had been ruptured. 

When measured at 40° with Lintner s soluble potato starch, the ratio of the dextrinogenic to the saccharogenic activities is approximately 6 to 1 for the amylase of Aspergillus oryzae. This value is given by both crude and purified preparations of the amylase if the measurements are carried out under comparable conditions. This constancy in the ratio of these two activities has led to the conclusion that, like pancreatic amylase, the amylase of Aspergillus oryzae is not accompanied in nature by beta amylase. Reacting mixtures of the amylase of Aspergillus oryzae and starch exhibit alpha mutarotation.72... 

The data show that the extent of the hydrolysis of starch by the amylase of Aspergillus oryzae depends within wide limits upon the concentration of amylase used. Like those for pancreatic amylase already discussed (Figure 2), these hydrolysis curves show a change from a rapid to a slow phase of the reaction and tend to flatten at higher values as the concentration of amylase is increased. Again, with different concentrations of the amylase of Aspergillus oryzae there is no evidence of a common limit such as is observed with different concentrations of beta amylase (Figure 1). 

A Comparison of the Hydrolysis of Soluble Potato Starch by Purified Maltase-free Pancreatic Amylase or Amylase of Aspergillus oryzae (Extent of Hydrolysis as Percent Theoretical Maltose)... 

Taken as a whole, the results indicate that the amylase of Aspergillus oryzae causes the rapid random hydrolysis both of the straight and of the branched chain components of starch and that it hydrolyzes very slowly products with average molecular weights of penta- and tetra-saccharides. 

Studies of the rate of the hydrolysis of dextrins isolated from a reaction mixture after the extensive hydrolysis of starch by maltase-free malted barley alpha amylase, led Myrback11 to conclude that the flattening of the reaction curves with this amylase is not due to equilibrium between the amylase and the products of the hydrolysis. As indicated above, similar conclusions have been reached for pancreatic amylase and for the amylase of Aspergillus oryzae.41,7a... 

Enz5mies Proteases Amylases Cellulases Various Bacilli, e.g. Bacillus licheniformis Bacillus subtilis, Aspergillus oryzae Trichoderma viride, Penicillium pinophilum... 

The most important features are, first, that some of the a-amylases— i.e., those in digests Numbers 4, 5, 6, and 7 have catalyzed glycosylations from a-maltosyl fluoride at one-tenth to one-one hundredth the enzyme concentrations required for action on a-D-glucosyl fluoride. The Aspergillus oryzae a-amylase, which had the weakest synthetic action on a-D-glucosyl fluoride of the enzymes tested at 100 ftg/ml, has produced under conditions of this experiment an extended series of maltosaccharides at 1 fig/ml. 

An even more serious disadvantage of this technique is that it often impairs the biological activity of the modified protein. The activities of hen s egg lysozyme (EC 3.2.1.17) and alpha amylase from Aspergillus oryzae (EC 3.2.1.1) were lessened by diazo coupling of glycosides or aniline.12 Whether the decrease in activity was due to the modification of critical residues, or to the introduction of aromatic structures, is not yet clear however, enzymes subjected to the diazocoupling conditions in the absence of the diazonium salts retained their activity, implying that the reaction conditions themselves were not responsible for the loss of activity. 

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