Sweet potato 3-amylase

Using the pure anomers of maltose in the same way, we found that crystalline hog pancreatic a-amylase causes the very rapid synthesis of maltotetraose from a-maltose but not from /3-maltose whereas, crystalline sweet potato /3-amylase causes the very rapid synthesis of the same compound, specifically from /3-maltose. Configurational inversion again marks this latter condensation as glycosyl-hydrogen interchange, or glycosyl transfer. 

Figure 2.5 Logarithmic scale comparison of k,d and kuncat (= (rnon) for some representative reactions at 25 °C. The length of each vertical bar represents the rate enhancement. (Wolfenden, 2001). ADC arginine decarboxylase ODC orotidine 5 -phosphate decarboxylase STN staphylococcal nuclease GLU sweet potato /3-amylase FUM fumarase MAN mandelate racemase PEP carboxypeptodase B CDA E. coli cytidine deaminase KSI ketosteroid isomerase CMU chorismate mutase CAN carbonic anhydrase.

Initially, the action of maltose (3-amylase on amylose gave complete conversion into maltose,235 and amylose was considered to be a completely linear a-(l—>4) glucan. However, when highly purified crystalline sweet potato (3-amylase was used, the... 

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. 

Figure 7. Loss of activity on heat treatment (60°C) of sweet potato /3-amylase (%) and its conjugate with dextran (O) (31)...

Maltosylamine has proved to be an effective specific inhibitor of sweet potato /3-amylase.This result extends the observation that 1-aminoglycosides are specific inhibitors of glycoside hydrolases, and also demonstrates that enzymes acting with inversion, as well as those acting with retention, of anomeric configuration can be inhibited by glycosylamines. /3-Maltosylamine, which acted as a reversible inhibitor, appeared to be directed to the active-site... 

Collado, L.S. and Corke, H. 1999. Accurate estimation of sweet potato amylase activity by flour viscosity analysis. J. Agric. Food Chem. 47, 832-835. 

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). 

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... 

Maltose (3-amylases are primarily found in plants and have been isolated from sweet potatoes,27 soybeans,28 barley29 and wheat.30 Maltose (3-amylases are also elaborated by bacteria, e.g. by Bacillus polymyxa,31 B. megaterium,32 B. cereus33 and Pseudomonas sp. BQ6.34 These (3-amylases all produce (3-maltose and a high molecular weight (3-limit dextrin. The limit dextrins result when the enzyme reaches an a-(l—>-6) branch linkage, which it cannot pass. Approximately half of an amylopectin molecule is converted to (3-maltose the remaining half is the (3-limit dextrin. 

The amino acid compositions of the beta-amylase from sweet potato, soya bean, wheat, and malted sorghum have been determined, and are shown in Table XIII. These results may suggest that the beta-amylase from these different sources differs in structure. This is, perhaps, not surprising, but it should be noted that the sorghum amylase contained 9% of pentose, and the important, sulfiir-containing amino acid cysteine was not reported and the soya-bean enzyme still contained traces of aipba-amylase and phosphatase. The sequence of amino acids, or the three-dimensional structure of any... 

Table XIV shows the results of the few reports of the molecular size of beta-amylase. No crystallized enzyme from cereals or beans has yet been examined by physical techniques. There is also a problem regarding the important, sweet-potato enzyme the only full report in the literature has been contradicted, and it has been suggested, without experimental details, that subunits of beta-amylase exist. In addition, in the case of cereal fcefo-amylases, where there is the complication of free and bound enzyme, the wheat and malted-barley enzyme has been reported to contain...

It has been reported that a yellow substance is liberated from crystalline, sweet-potato beta-amylase when the enzyme is subjected to gel filtration on Sephadex at pH 8.8 in the presence of pyrophosphate buffer. The chromophoric material can also be obtained by treating the enzyme with acetic acid, and can readily be reattached. 

Measurements of the molecular activity have indicated values of 250,000 for sweet-potato befo-amylase, and 11,800—14,200 for wheat befc-amylase. ... 

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. 

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