Action of 0-Amylase

Obviously, more work on this subject is necessary, but the problem is complicated by the fact that some authors have reported that the conversion of amylose to maltose by the action of /3-amylase is incomplete. Limits of from 70 to 97 % have been recorded. Apparently, variations depend on the actual D. P. of the amylose and on the concentration of enzyme. Meyer and coworkers19-214 maintain that these lower limits may be due to retro-gradation phenomena, which is not unreasonable in view of the fact that... 

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... 

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. 

A third mechanism, proposed by French,involves multiple attack, in which the enzyme remains associated with a given substrate molecule long enough to remove several maltose residues before attacking another amylose molecule. With amylose of DP 44, sweet-potato /3-amylase removed about four maltose residues per effective encounter. i The multiple-attack mechanism is, in fact, intermediate between the single-chain and multi-chain patterns. In agreement with this view, Whelan and Bailey found that the action of /3-amylase on maltosaccharides of DP 6 and 7 and on amylose of DP 49 was intermediate between single-chain and multichain, but varied with the pH and temperature of the experiments. 

I = cross-linked -> = action of a-amylase > = action of 3-amylase Scheme 3... 

The /3-amylases in the absence of the a-amylases are incapable of degrading whole starches completely. The hydrolysis proceeds rapidly until about 50 to 55 % of the theoretical amount of maltose is produced and then very slowly until a limit of about 61 to 68% is reached (101), The solution is still viscous and the residue, called a /3-amylase limit dextrin, is unfer-mentable. The limit dextrin arises from the inability of /3-amylase to act beyond a branch point in the randomly branched amylopectin molecule and may be envisaged as a pruned amylopectin structure. In the case of potato starch, the /3-limit dextrin includes all the associated phosphate. The limit dextrin contains one end group for every 10 to 12 D-glucose residues (102), in contrast to one in every 25 or 30 residues for the original amylopectin. The initial attack of /3-amylase on amylopectin is about 20 times as fast as on amylose (103), Maltose in amounts of 53 to 62 % of the theoretical have been reported from the action of /3-amylases on amylopec-tins separated from various starches (104). When the /3-limit dextrin is cleaved by acid hydrolysis or by the action of a-amylase, the structure is opened and new chain ends are made available which can be further acted upon by i3-amylase. 

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. 

Since the action of a-amylase is random some reducing sugars will accumulate during conversion. The quantity of sugars depends on the amount and purity of the enzyme used. The sugar content in the converted paste should not exceed 3%. The product composition can be analyzed by titration and chromatographic separation. 

The mechanism of the action of beta-amylase involves interaction of the starch with imidazole and carboxyl groups of the enzyme, resulting in a glycosyl ester intermediate (fi form of the D-glucose residue) that is hydrolyzed by addition of water to the carboxyl group, thus releasing the maltose in the /3-anomeric configuration. 

A study of the action of beta-amylase on enzymically synthesized, branched oligosaccharides, containing 4 to 7 D-glucose residues, showed that the rate of hydrolysis is considerably lessened as the enzyme approaches the branch-points, and action stops 2 or 3 D-glucose units away from the branch-point. ... 

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... 

The Schardinger dextrins have also been reported " to lie stable to alpha-type amylases. However, in a study of the action of salivary amylase, French and coworkers " found that while the a-dextrin is essentially completely resistant, the /3-dextrin is attacked very slowly indeed and the 7-dextrin is attacked about 1 % as rapidly as is starch. Here it is clear that the ring size exerts an effect possibly the smaller rings have greater rigidity and hence cannot adapt their shape to that inquired by the enzyme. 

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