Action of Malt a-Amylase

This enzyme is prepared most simply by heating malt extracts to a temperature at which the /3-amylase is inactivated. The temperature is not the same for all malt extracts. Impurities in the extracts seem to have a more or less protective action on the enzymes. In most cases, heating at 70° for about fifteen minutes will be suitable. Other methods of separating the enzymes are reviewed by Bamann and Myrback.  

It is not easy to show that a preparation of malt a-amylase is enzy-mically homogeneous and free from /3-amylase and maltase. As will be shown later, the enzyme liberates D-glucose as a primary product from starch. Thus the appearance of D-glucose is not evidence of the presence of maltase. But the absence of this enzyme is most easily demonstrated 

In contrast to the /S-amylase, the malt a-amylase (and other liquefying amylases) very rapidly lower the viscosity of starch pastes. When the relative viscosity has dropped to half the original value, only about 0.1% of the D-glucosidic linkages in the starch have been hydrolyzed. The malt a-amylase is a typical liquefying enzyme. 

The a-amylases rapidly alter the starch in such a way that the reaction mixture is no longer colored by iodine. With malt a-amylase and potato starch this is the case when about 7% of the D-glucosidic linkages are hydrolyzed, that is, when the apparent yield of maltose is 15-20%. (The experiments were carried out with a preparation of soluble starch.) 

When examining curves of this type, one involuntarily inquires if both phases of the reaction are caused by the same enzyme or if the second phase is not an action of traces of /3-amylase. But if malt extracts are heated to temperatures causing inactivation of the /3-amylase, a variation of time and temperature does not alter the relation between the velocities of the two phases nor the relation between these velocities and the time necessary to change the starch so that it is not colored by iodine. These relations also are not altered if the a-amylase is partly removed from the solutions by adsorption on bentonite, activated carbon, aluminum 

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Hopkins and coworkers have compared the action of malt a-amylase with that of bacterial amylase (from B. suhtilis). The bacterial enzyme closely resembled the malt enzyme in respect to the kinetics of the reaction and produced much the same products. 

Acid hydrolysis Action of 0-amylase Action of malt a-amylase... 

Later, MacGregor and MacGregor16 reported that barley malt a-amylase had an action pattern that was similar to that of B. amyloliquefaciens a-amylase, forming... 

The course of the optical rotation during the action of the malt a-amylase shows that the enzyme, as pointed out by Ohlsson, liberates the hydrolysis products in the a-configuration. A simple calculation shows that this is the case not only with the fermentable sugar but with the dextrins as well. 

In order to understand why the malt a-amylase acts so slowly on a normal hexaose (Fraction PDXII) we have tried to apply the Michaelis-Menten theory and have determined the affinity constants of the enzyme-substrate compounds. If the concentration of starch and dextrin are expressed as moles of maltose per liter we find the affinity constant, K, between malt a-amylase and starch to be about 200 and for /3-amylase about 170. The affinity constant a-amylase to a-dextrin is so small that no values could be obtained. The constant certainly is smaller than 12. Thus, it is clear that the slow action of the malt a-amylase on the dextrin is due to a very low affinity of the enzyme to the substrate. In other words, the enzyme can readily attach itself to a long chain, but only with difficulty to chains shorter than a certain value. In the dextrinization period of the action of the enzyme on starch, a mixture of a-dextrins is produced with an average chain length of about 7 units. It can be assumed, therefore, that the enzyme readily attaches itself to chains that contain more than about 8 units provided that the chains are normal and contain no branchings or other anomalies. When anomalies occur these... 

In an experiment with 8.5 g. of amylose and malt a-amylase, the enzyme action was stopped when the degree of hydrolysis was 22.4%, that is, at the end of the dextrinization phase. Fermentable sugar was removed by fermentation (17%) and the non-fermentable a-dextrins were fractionated with alcohol. Six fractions were obtained (Table XI) having chain lengths from 4 to 10 units. 

In order to explain the action of the malt a-amylase, the assumption had been made formerly (Freudenberg, Meyer) that the enzyme has the capacity of attacking all D-glucosidic linkages in a chain, except the two terminal ones, with about the same probability. If this be the case, the distribution of the products at a certain degree a of hydrolysis can be calculated approximately according to the formula... 

We will discuss first whether there is an absolutely definite limit of action for all amylases. In the case of the action of /5-amylase on starch and on a-dextrins this question seems to be settled, but in the case of the malt a-amylase the answer is less certain. But certainly the action of the malt amylase practically stops at a certain limit. There is, however, almost always a very slow further action. It is possible that this slow saccharification of the limit dextrins is due not to the amylases but to other carbohydrases which have no action on starch but which are capable of attacking products with short chains. Under all circumstances it must be kept in mind that when in an experiment the saccharification for practical purposes has stopped and the limit dextrins have been isolated, this does not necessarily mean that the limit dextrins will not be further attacked by the enzyme used. But the velocity of this action is certainly very small compared with the velocity of the action on starch. Thus, it must be admitted that experiments involving the isolation of the limit dextrins after the action of a certain amylase on starch are in most cases not strictly reproducible. TJie total yield and chain length distribution of limit dextrins may vary, but their general character is not affected. If a limit dextrin produced by a certain amylase is treated with the same enzyme for a very long time, it is very often transformed to another limit dextrin of lower molecular weight with concomi-... 

Arrow-root starch (500 g.) in 8 liters of water containing 1 g. of sodium chloride was hydrolyzed at pH 6.5 with 50 ml. of saliva. After twenty-four hours, 50% of maltose had been formed. The reaction mixture was kept under toluene for thirty-two days. The reducing power then corresponded to 89% maltose. Fermentation experiments, however, showed that 63% maltose and 14% D-glucose had been formed, the latter by the secondary action of maltase. As mentioned above, the salivary amylase does not form D-glucose as a primary product but differs in this respect from malt a-amylase. The pancreatic enzyme is... 

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