Amylases action pattern

Fig. 6. —oZpha-Amylase Action Pattern Schematic Numbering of Bonds in an a-D-(1— 4)-Glucan. [Q, a-D-glucose residue , reducing D-glucose residue —, a-D-(l— 4) bond.]... 

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... 

Figure 9.72 Chromatograms of the action patterns of maltoheptaose after the indicated periods of incubation with a-amylase and a-glucosidase. Peaks 1, glucose 2, maltose 3, maltotriose 4, maltotetraose 5, maltopentaose (x) compound A 6, maltohexaose 7, maltoheptaose. (A) Pure maltoheptaose used for the assay. (B) Blank sample before the addition of substrate. (C-H) Chromatograms after 1, 5, 10,15, 20, and 30 minutes, respectively, of incubation. Chromatographic conditions column, 10 jum Nucleosil SA (250 mm X 4 mm) solvent, acetonitrile-water (72.527.5) flow rate, 0.7 mL/min temperature, 27°C detection, differential refractometer, full scale = 2 X 10-6 refractive index units. (From Haegel et aL, 1981.)...

The enzymic degradation and sjmthesis of starch has formed the basis of very extensive investigations, and it might be expected that the action of such enzymes is well understood. However, recent studies of detailed action-patterns have shown that some previous, widely accepted concepts require to be modified. Furthermore, in the case of the oZphc-amylases, order in the overwhelming mass of accumulated data is now becoming apparent. 

These hydrolytic enzymes—which take their name from the fact that the degradation products are sugars in the a-D configuration-are found in mammals, higher plants, fungi, bacteria, and Crustacea. It is now appreciated that both the properties and the detailed action pattern of an alpha-amylase depend on the source of the enzyme. 

Although early investigations indicated that a-amylolytic attack on amylose and smaller linear glucans was random (except for the linkage nearest each chain end) and that this mode of attack was independent of the source of the enzyme, it is now known that the detailed action-pattern of an alpha-amylase depends on the source of the enzyme. 

Quantitative studies have been used to confirm that the attack on amylose is not completely random. A comparison of the action patterns of alpha-amylases from higher plants and mammals shows that they are very different. At the achroic point of hydrolysis of amylose by plant enzymes, about 60% of the saccharides were larger than the... 

This enzyme, which yields jff-maltose as the initial product, has been crystallized from barley, wheat, sweet-potato, and soya-bean extracts. The properties of these enzymes are generally similar (for example, sulf-hydryl groups are essential for activity), but are not identical (for example, the pH optimum varies between 4 and 6). The action patterns of the various -amylases appear to be identical. 

The action pattern of /8-amylase on substrates of low molecular weight is, therefore, established, but that with amylose of DP 10 remains to be examined further. 

The action patterns of the a-amylases have been studied intensively during the past twenty years, but, since the various workers have used different substrates, enzymes of various activities and degrees of purity, incubation periods ranging from hours to months, and widely differing methods of analysis, it is not surprising that a great deal of often-contra-dictory information has been accumulated. The position has, fortunately, been clarified by Whelan and his coworkers and the major details of a-amylolysis are now established. 

Modified substrates may be used in testing the action patterns of purified enzymes. Thus, periodate-oxidized laminaran was used by Smith and coworkers to show the exo action of Basidiomycete (l- 3)-j8-n-glucanase. The oxidized-amylose substrate described earlier (see p. 266) has been used with alpha- and heta-amylases to illustrate how the method is completely general, and applicable to all classes of polysaccharides and poIysaccharidases. ° By use of oxidized pullulan, the endo nature of pullulanase action was confirmed. ... 

Figure 8. Action pattern of human salivary a-amylase on elsinan ( ) represents the sites cleaved by the enzyme G, a-D-glucopyranosyl residue...

Figure 9. Possible action pattern of Taka amylase, and release of tetra- and hep-tasaccharide ( ), site cleaved by Taka amylase (O), a-D-glucopyranosyl unit (-----------), (1 - 4)-D-glucosidic linkage (f), (1- 3)-D-glucosidic linkage.

A particular enzyme nomenclature problem which continues to arise, particularly in papers of clinical chemical origin, is the use of the term isoamylase for isoenzymes of a-amylase (EC 3.2.1.1) —a confusion with the true use of the term isoamylase (EC 3.2.1.68) an enzyme with an action pattern different from that of a-amylase. This gross misnomer stems from the sloppy use of the term amylase for a-amylase, which has been commented on before. In this report, the use of amylase by authors, has been taken by reporters as meaning a-amylase and not j3-amylase (EC 3.2.1.2). The use of the term isoamylase has been strictly reserved by reporters for isoamylase (EC 3.2.1.68). 

An amylase, previously detected in barley and described as a new barley amylase, has been further purified by immunoadsorption and ion-exchange chromatography. Analysis by isoelectric focusing and immunochemical techniques showed that the enzyme preparation did not contain the normal a-and 8-amylases usually found in barley and malt. The enzyme had a very low isoelectric point ca. pH 3.0) and was identified as an a-amylase on the basis of its action pattern on amylose. 

---