Temperature 3-amylase action

The effect of temperature on the kinetics of alpha-amylase action has been studied, and, from the variation of reaction velocity, activation energies have been determined for several enzymes, as shown in Table V. The variation of with temperature has also been studied, and, from such measurements, the heat of formation of the enzyme—substrate complex has been calculated to be —3.4 Kcal. per mole for sorghum alpha-amylase and —7 Kcal. per mole for hog-... 

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. 

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.)...

This enzyme is prepared most simply by heating malt extracts to a temperature at which the /3-amylase is inactivated. The temperature is not the same for all malt extracts. Impurities in the extracts seem to have a more or less protective action on the enzymes. In most cases, heating at 70° for about fifteen minutes will be suitable. Other methods of separating the enzymes are reviewed by Bamann and Myrback. ... 

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... 

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. 

The manufacture of beer as now practiced is, in fact, much too costly. Malting is an operation which involves considerable losses in nutritive materials. The catalysts which are developed during this part of the manufacture, although very expensive, are far from performing all the necessary work. The amylase of the malt has two functions the one, liquefaction the other, saccharification. Now, these two effects are exerted at differait temperatures, and the saccharification carmot be conducted in the desired way without neglecting the liquefaction. Consequently, undissolved starch always remains in the husks. On the other hand, the proteolytic enzymes are abundant but of a quality not suited to the work desired. They can induce an advanced hydrolysis, but exert only a poor dissolving action. 

Studies of the hydrolyses of cyclohexa-, cyclohepta-, and cyclo-octa-amyloses by Taka-amylase A (one of the a-amylases from Aspergillus oryzae) indicated that each cycloamylose binds to the same active site of the enzyme. Since there is little difference in the respective AG, Af/, and AS values for enzymic hydrolysis of these cycloamyloses, their binding modes appear to be similar. The extent of multiple attack on the cycloamyloses was not affected by temperature. A 4-phenylazobenzoyl derivative of Taka-amylase A has been used to investigate the active site of the enzyme. A. oryzae a-amylase has a synergistic effect on the action of the glucoamylase from A. awamori var. kawachi ... 

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