Amylopectin 3-amylase action

Figure 4-19 Schematic Representation of the Action of Starch-Degrading Enzymes. (A) Amylose and amylopectin, (B) action of a-amylase on amylose and amylopectin, (C) action of a debranching enzyme on amylose and amylopectin, (D) action of amyloglucosidase and debranching enzyme on amylose and amylopectin. Source Reprinted from H.S. Olsen, Enzymic Production of Glucose Syrups, in Handbook of Starch Hydrolysis Products and Their Derivatives, M.W. Kearsley and S.Z. Dziedzic, eds., p. 36, 1995, Aspen Publishers, Inc.

By use of a debranching enzyme, isoamylase or pullulanase, the a-D-(l — 6)-linkages in amylopectin are broken, thus opening up to beta-amylase action those parts of the amylopectin molecule that are otherwise resistant.14 In this way, syrups of very high maltose content can be made.19... 

Fig. 4.—Structures Around the Outermost Branch-points in the Limit Dextrins Remaining After Action of foeta-Amylase on Amylopectin and Glycogen. [Structure (b) is obtained uniquely by foeta-amylase action on phosphorylase limit-dextrins (see p. 315). For an explanation of the symbols, see footnote 49.]...

Amylase operates exclusively on nonreducing, terminal units in amylose or on the branches in amylopectin, to produce maltose directly, and its hydrolytic action on 4 — 1-a-D linkages is stopped by any branch points. Enzyme action is greatly impeded by secondary valence forces, as retro-gradation, for example, is accompanied by an increased resistance to j8-amylolysis.19 The enzyme can be crystallized relatively easily. The mode... 

The only example of this technique applied to the amylose component is that already described, of the action of Z-enzyme on the /3-limit dextrin. In the case of amylopectin, enzymic methods enable a distinction to be made between the proposed laminated and highly ramified structures (I and III, in Fig. 1, page 352). The method used by Peat and coworkers101 involves the successive action of /3-amylase and R-enzyme on waxy maize starch. /3-Amylolysis will degrade A-chains down to two or three units from the 6 —> 1-a-D interchain linkages. These latter linkages will protect the... 

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. 

The investigations carried out by Professor French and his students were based on sound experimental approaches and on intuitive theoretical considerations. The latter often resulted in new experiments for testing a hypothesis. On the basis of theoretical considerations, Professor French proposed a model for the structure of the amylopectin molecule, and the distribution of the linear chains in this molecule. This model was tested by utilizing enzymes that selectively cleave the linear chains, and the results substantiated the theoretical deductions. He proposed a theory on the nature and types of reactions occurring in the formation of the enzyme - starch complex during the hydrolysis of starch by amylases. In this theory, the idea of multiple attack per single encounter of enzyme with substrate was advanced. The theory has been supported by results from several types of experiments on the hydrolysis of starch with human salivary and porcine pancreatic amylases. The rates of formation of products, and the nature of the products of the action of amylase on starch, were determined at reaction conditions of unfavorable pH, elevated temperatures, and increased viscosity. The nature of the products was found to be dramatically affected by the conditions utilized for the enzymic hydrolysis, and could be accounted for by the theory of the multiple attack per single encounter of substrate and enzyme. 

Although all amylose molecules were once considered to be linear, many amylose molecules cannot be completely hydrolyzed by (3-amylase. With a concurrent or mixed action of pullulanase and beta-amylase, however, amylose can be completely hydrolyzed to maltose.150,151 These results rule out the theory that the incomplete hydrolysis of amylose by (3-amylase is a result of retrogradation, i.e. junction zone formation. It is now clear that the incomplete hydrolysis of an amylose preparation by (3-amylase is due to branching of some molecules. The (3-amylolysis limit of amylose varies from 72% to 95%152,153 compared with 55-61% for amylopectin. Amylose of most cereal starches, such as maize,154 rice,155,156 wheat157 and barley,158 give >80% (3-amylolysis... 

When the substrate was amylopectin (3-amylase limit dextrin, a different pattern of products was formed, namely Gl, G2 and G3, with no G6, G7 or higher sized dex-trins.14 Reaction with the (3-limit dextrin indicated that Gl, G2 and G3 are formed from the chains between the o-( 1 —6) branch linkages as the outer chains were removed by the action of (3-amylase (see Section 7.2 for a discussion of the action of (3-amylase). It further indicated that G6 and G7 from amylopectin were formed exclusively from longer unbranched outer chains. It also indicated that the number of glucosyl units between the branch linkages of amylopectin were sufficiently few that they could not yield the larger G6 and G7 products, but could give the smaller Gl, G2 and G3 products. 

The commercial importance of amylolytic enzymes is rapidly increasing. These enzymes catalyze the hydrolytic reactions of amylose (unbranched starch) and amylopectin (branched starch). Amylases, according to their difference in modes of action, can be divided into ... 

Because of the difficulties involved in separating the isoenzymes among themselves and from other enzymes such as amylases, it is not yet clear what the best acceptors are and what the products are for each isoenzyme, but some characterization of the enzymes has been done (e.g., Km for a-glucans such as amylopectin, different animal glycogens, and maltosaccha-rides). Characterization of the products has been minimal, but some progress has been made in our laboratory (see the next section, Mode of Action ). 

It should be noted that the proamylopectin in this still hypothetical pathway would be larger than the phytoglycogen found in the mutants lacking debranching activity. This is because proamylopectin would have a size comparable to amylopectin, while phytoglycogen, much smaller, may be the product of degradation of a proamylopectin unable to crystallize into amylopectin and may be so unprotected that it would be subject to the action of amylases. 

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