Beta Amylases

Hehre and coworkers showed that beta amylase from sweet potatoes, an inverting, a-specific exo-(l 4)-glucanase, catalyzes the hydrolysis of jS-maltosyl fluoride with complex kinetics which indicated the participation of two substrate molecules in the release of fluoride ion. Furthermore, the reaction was strongly accelerated by the addition of methyl ) -maltoside. Hydrolysis of a-maltosyl fluoride, on the other hand, obeyed Michaelis-Menten kinetics. The main product with both a- and yj-maltosyl fluoride was )S-maltose. The results with )3-maltosyl fluoride were interpreted by the assumption of a glycosylation reaction preceding hydrolysis by which a malto-tetraoside is formed by the replacement of fluoride ion by a second substrate molecule or added methyl -maltoside (see Scheme 5). 

Scheme 5.—Hydrolysis of a- and /J-Maltosyl Fluoride by Beta Amylase (R = o-d-G1uco-syl, R = H or a- Maltosyl).

A product manufactured in Japan as Alo mixture (Anomalously Linked Oligosaccharides) contains mainly a range of IMOS (glucose, isomaltose, and panose as principal constituents). It is produced from starch as substrate by the action of alpha-amylase, beta-amylase, and a transglucosidase. It is claimed to have favorable properties for application in the food industry.143... 

No attempt is made in this chapter to review in detail our present information concerning the beta amylases. However, because it is impossible to consider adequately the properties of the alpha amylases without reference to beta amylases, it seems desirable to describe briefly a few of the characteristic properties of the beta amylases. 

The data19 summarized in Figure 1 show that the extent of the hydrolysis of soluble potato starch by barley beta amylase reaches a limit which is independent of the concentration of the amylase. The data are typical of the action of beta amylases on unfractionated starches, when the hydrolyses are carried out at or near pH 4.5.1 3 6 19 20 Under these conditions, the hydrolysis of unfractionated starches usually ceases when 60 to 64% of the maltose theoretically obtainable from the substrate has been formed. The exact value of the limit obviously will depend upon the concentration of amylopectin in the starch and upon its structure. 

Blom and coworkers21 have reported that after pretreatment at pH 3.4 and under suitable conditions of action, beta amylase does not... 

The characteristic of beta amylases of reaching a limit in the hydrolysis of starches gives the investigator a simple criterion for ascertaining whether his amylase preparation has been freed from alpha amylase, which usually accompanies beta amylase in nature. If the hydrolysis of Lintner s soluble potato starch at pH 4.5 ceases when approximately 64%... 

If amylases are to be used as tools for the detailed study of the breakdown and structure of their substrates it is obviously important to separate them from other enzymes and from other naturally associated constituents which may influence the results. It is then equally important to study the properties of the purified amylase and to supply it with the chemical environment necessary to protect it from inactivation and to enable it to act efficiently. With beta amylases this ideal has often been approached. Beta amylases from several sources have been prepared by selective inactivation of other enzymes that accompany them in nature23 and highly active products have been obtained by extensive purification.20 24-26 Balls and his associates have recently reported the crystallization of beta amylase from sweet potato.27... 

The general status of the alpha amylases with respect to the above ideal is not so satisfactory. The difficulties involved in the purification of these amylases are increased because there are no simple criteria by which the investigator can make sure that the alpha amylase under consideration is not contaminated by beta amylase or by some other gluco-sidase.1 8,6 18 28 However, evidence is accumulating to indicate that these alpha amylases have important individual characteristics which will differentiate them from one another. Such characteristics should make these enzymes increasingly useful in the study of their substrates and thus amply repay continued efforts at their purification. 

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). 

In crude aqueous extracts of malted barley the alpha amylase is usually much more thermostable than the beta amylase. This difference in the stability of the two amylases in crude aqueous extracts was recog-... 

Examination of the ratios of the dextrinogenic to the saccharogenic activities of malted barley extracts before and after treatment shows that the results of the Ohlsson procedures23 are not always predictable.8 The concentration of the amylases in the extracts, and the kinds and concentrations of substances which accompany them, influence the results. The presence or the absence of calcium ions is an important factor. Calcium ions increase the inactivation of beta amylase of malted barley and protect the alpha amylase from inactivation at unfavorable temperatures and also at unfavorable hydrogen ion activities.28 With purification, both amylases become increasingly thermolabile and increasingly sensitive to unfavorable hydrogen ion activities.78... 

Very highly purified preparations of alpha amylase of malted barley give a value of approximately 4 to 1 for the ratio of their dextrinogenic to their saccharogenic activities when the measurements are made at 40° with Lintner s soluble potato starch.81 Under the same conditions approximately the same value is obtained with products precipitated by alcohol from malted barley extracts which had been treated to inactivate beta amylase.23 81 However, a constant value for these ratios is not proof that beta amylase is entirely absent. There is at present no satisfactory way of making certain that malted barley alpha amylase is not contaminated with traces of beta amylase. The crystallization of alpha amylase from malted barley has been reported since this manuscript was written.79... 

Free aldehyde groups do not appear to be essential to the action of malted barley alpha amylase. Myrback82,83 reports that, like beta amylase,1 3 this amylase also causes the hydrolysis of dextrins after their aldehyde groups have been oxidized to the corresponding acids. 

Table X 4 summarizes similar data for the hydrolysis by maltase-free malt alpha amylase of beta dextrins obtained from arrowroot starch by the action of beta amylase. The beta dextrins were precipitated with alcohol from the reaction mixture of arrowroot starch after it had reached a limit in the hydrolysis at 60% theoretical maltose. The beta dextrins were hydrolyzed extensively by malt alpha amylase. Glucose was liberated in very small amounts even in the later stages of the hydrolysis of these beta dextrins maltose was liberated in appreciable amounts and, at equivalent hydrolyses, appeared to be formed somewhat more rapidly from the beta dextrins (Table X) than from the untreated starch (Table IX). Upon hydrolysis with malt alpha amylase the molecular weights of the beta dextrins dropped appreciably but not as extensively as when arrowroot starch was hydrolyzed directly by malt alpha amylase.

Products Formed by Malted Barley Alpha Amylase from Beta Dextrine Formed from Arrowroot Starch by Beta Amylase (Data of Myrbiicku)... 

A Comparison of the Action of Purified Maltase-free Alpha Amylase from Malted Barley upon Corn Amylose, Corn Starch and upon Beta Dextrins Formed from Corn Starch by the Action of Beta Amylase (Data of Myrbdck and Thorsellu)... 

An interesting comparison is also given in Table XI86 of the action of malted barley alpha amylase upon corn starch, beta dextrins obtained from corn starch by the action of beta amylase, and upon corn amylose prepared according to the method of Meyer.6 90 These data show that, with equivalent amounts of enzyme, amylose was hydrolyzed much more rapidly and more extensively than unfractionated corn starch. Although the beta dextrins were hydrolyzed by this amylase, they were hydrolyzed much more slowly and less extensively than either of the other two substrates. These results are similar to those already considered for pancreatic amylase.41 64... 

Although the mechanism of the hydrolysis of starch by beta amylase has been well established, the hydrolysis of starch by the alpha amylases has proven much more complicated. The data already available show that alpha amylases from different sources hydrolyze starches very differently and that these differences are more marked in the early than in the late stages of the hydrolysis of starch. Unfortunately, sufficient strictly comparable data are not available at present to make possible clear cut statements as to the similarities and differences in the mode of action of the three amylases discussed here. However, it is evident that further work with these and other amylases will be amply repaid as it adds to our exact information and increases our understanding of these important catalysts and of the substrates upon which they act. 

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