Amylase inactivation

Enzyme—Heat—Enzyme Process. The enzyme—heat—enzyme (EHE) process was the first industrial enzymatic Hquefaction procedure developed and utilizes a B. subtilis, also referred to as B. amjloliquefaciens, a-amylase for hydrolysis. The enzyme can be used at temperatures up to about 90°C before a significant loss in activity occurs. After an initial hydrolysis step a high temperature heat treatment step is needed to solubilize residual starch present as a fatty acid/amylose complex. The heat treatment inactivates the a-amylase, thus a second addition of enzyme is required to complete the reaction. 

After 30 hours, the maximum and critical fermentation is underway and the pH must remain above 4.0 for optimal fermentation. However, accompanying bacterial contamination from various sources such as yeast contamination, improper cleaning procedures, slow yeast growth, or excessive temperatures can result in a pH below 4.0. The remaining amylase enzymes, referred to as secondary conversion agents, are inactivated and can no longer convert the dextrins to maltose. Under these circumstances, the fermentor pH continues to drop because of acid production of the bacteria, and the pH can drop to as low as 3.0. The obvious result is a low ethanol yield and quaUty deterioration. 

Fig. 24. Kinetics of add inactivation of a-amylase (Bac. subtilis) in solution (/, 2) and immobilized on Biocarb (3) 1) pH 2 2) and 3) pH 4. A/A0 is the value of relative enzymatic activity (compared to the initial activity A0 before inactivation), substrate — starch...

Fig. 25. Reactivation of inactivated of 1) a-amylase at different pH values of the solution, 2) oc-amylase immobilized in Biocarb...

The magnitude of inhibition of polygalacturonase was found to be dependent on preincubation of inhibitor with the enzyme. Similar observations have been reported for other enzyme inhibitors (Shivaraj and Pattabiraman, 1980 Sharma and Pattabiraman, 1980 Padmanabhan and Shrasti, 1990). However, preincubation of the inhibitor with substrate did not show any effect on inhibitor activity. In contrast, Shivaraj and Pattabiraman (1980) and Buonocore et al. (1977), have observed inactivation of amylase inhibitor activity on pretreatment with starch. 

As mentioned above, certain metal ions may be necessary for activity or stability. Thus calcium is needed for bacterial a-amylase. Magnesium or cobalt is needed with glucose isomerase. Calcium stabilises the starch-liquifying bacterial a-amylases but inactivates the glucose isomerase that may be used subsequently. Many enzymes contain an additional non-... 

The reason for the non-response of the Hagberg Falling Number to fungal a-amylase is that it is inactivated at 75°C rather than the 87°C of cereal a-amylase. It turns out that fungal a-amylase preparations improve loaf volumes considerably. Most of this effect is produced by a lipoxygenase enzyme that is also present. 

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... 

Pancreatic amylase is very labile and sensitive to its chemical environment. Its lability is accelerated by purification and by such factors as dilution of its aqueous solutions, dialysis of its aqueous solutions against water, unfavorable hydrogen ion activities and unfavorable temperatures.29-31, 36,33 The loss of amylase activity in solutions of pancreatic amylase increases with increasing temperature and is very rapid between 50° and 60°. The inactivation of pancreatic amylase in aqueous solution may be retarded by the addition of certain anions, of which the chloride ion is outstanding 37-39 by the addition of certain cations, of which... 

It is important to note that no evidence of maltase activity was found in the amylase preparations even when the highest concentrations used in these comparisons were allowed to react for twenty-four hours with one per cent maltose under the conditions for the hydrolysis of starch. Similarly, no evidence was obtained for the presence of any other contaminating or extraneous carbohydrases in the amylase preparations. Partial inactivation of the amylase under a number of different conditions failed to give any evidence of selective inactivation such as might be expected if more than one enzyme were present. The substrates used for measuring the activity of the partially inactivated amylase were starch and starch hydrolyzates that had already been extensively (58) J. Blom, Agnete Bak and B. Braae, Z. physiol. Chem., 250, 104 (1937). 

Similarly, the addition of substrate to hydrolysis mixtures that had reached stages of very slow rates of change with dialysis resulted in extensive hydrolysis of the added substrate. Comparisons showed that the new substrate was hydrolyzed to practically the same extent in the same time as the original substrate.41 These findings show not only that active amylase was present but that no appreciable inactivation of the amylase had taken place in the dialyzing hydrolysis mixtures when the... 

On the other hand, marked inactivation of the amylase (86 to 87 %) occurred when solutions of the same purified pancreatic amylase preparations were dialyzed under the same conditions but in the absence of substrate.41 These results give experimental evidence for the suggestion often advanced that the amylase unites with its substrate, in this case with the larger less readily dialyzable products of the hydrolysis of starch, and thus is protected from appreciable inactivation due to dialysis. 

On the other hand, in crude aqueous extracts of malted barley the alpha amylase is much more sensitive to high hydrogen ion activities than the beta amylase. This difference between the two amylases also was utilized by Ohlsson28 to inactivate the alpha amylase and to prepare beta amylase relatively free from alpha amylase. 

Examination of the ratios of the dextrinogenic to the saccharogenic activities of malted barley extracts before and after treatment shows that the results of the Ohlsson procedures23 are not always predictable.8 The concentration of the amylases in the extracts, and the kinds and concentrations of substances which accompany them, influence the results. The presence or the absence of calcium ions is an important factor. Calcium ions increase the inactivation of beta amylase of malted barley and protect the alpha amylase from inactivation at unfavorable temperatures and also at unfavorable hydrogen ion activities.28 With purification, both amylases become increasingly thermolabile and increasingly sensitive to unfavorable hydrogen ion activities.78... 

Very highly purified preparations of alpha amylase of malted barley give a value of approximately 4 to 1 for the ratio of their dextrinogenic to their saccharogenic activities when the measurements are made at 40° with Lintner s soluble potato starch.81 Under the same conditions approximately the same value is obtained with products precipitated by alcohol from malted barley extracts which had been treated to inactivate beta amylase.23 81 However, a constant value for these ratios is not proof that beta amylase is entirely absent. There is at present no satisfactory way of making certain that malted barley alpha amylase is not contaminated with traces of beta amylase. The crystallization of alpha amylase from malted barley has been reported since this manuscript was written.79... 

The alpha amylase of malted barley, the amylase of Aspergillus oryeae and pancreatic amylase all are thermolabile proteins that rapidly lose their amylase activities upon exposure to unfavorable temperatures, to unfavorable hydrogen ion activities, or to other unfavorable chemical environments. The loss of amylase activity in aqueous solutions increases with increasing temperatures and is exceedingly rapid for each of these amylases at 50°. The inactivation of each of these amylases at unfavorable temperatures or at unfavorable hydrogen ion activities may be retarded by the presence of suitable concentrations of calcium ions. 

The hydrolysis curves for the three alpha amylases considered in this review are all similar in shape. They show a rapid hydrolysis in the early stages with a slow secondary stage of hydrolysis which cannot be explained as due either to inactivation of the enzymes or to the influence of products formed during the hydrolysis. The extent of the hydrolysis and the position of the break in the hydrolysis curve depend upon the concentration of enzyme the break in the hydrolysis curve does not appear to be a fixed point characteristic of any one enzyme. With each of the alpha amylases discussed here the slowing down of the hydrolysis appears to be due largely to the replacement of the original substrate by products for which the amylase has relatively low affinity. 

The purification of some enzymes inactivates them because substances essential for their activity but not classed as a prosthetic group are removed. These are frequently inorganic ions which are not explicit participants in the reaction. Anionic activation seems to be non-specific and different anions are often effective. Amylase (EC 3.2.1.1), for example, is activated by a variety of anions, notably chloride. Cationic activation is more specific, e.g. magnesium is particularly important in reactions involving ATP and ADP as substrates. In cationic activation it seems very likely that the cation binds initially to the substrate rather than to the enzyme. 

Cellulases can also be eliminated fiom a mixture with xylanases by selective thermal inactivation. Cellulases are more thermolabile than xylanases in tiie cdlulolytic systems of the fungus Y-94 (79), T. harzianum 20), and Tkermoascus aurantiacus (77), but not in the Trickoderma reesei system (Biely, P. and Vrsanska, M., Slovak Academy of Sciences, Bratislava, unpublished results). Since cellulase thoixud inactivation causes a significant loss of xylanase also, a more convenient way to eliminate cellulase activity is by selective chemical or biological inhibition or inactivation. There appear, however, to be no reports on the existence of natural inhibitors that would be specific for cellulases. Such inhibitors of amylases and pectinases are known to occur in plants (27). 

Dekker et al. [170] studied the extraction process of a-amylase in a TOMAC/isooctane reverse micellar system in terms of the distribution coefficients, mass transfer coefficient, inactivation rate constants, phase ratio, and residence time during the forward and backward extractions. They derived different equations for the concentration of active enzyme in all phases as a function of time. It was also shown that the inactivation took place predominantly in the first aqueous phase due to complex formation between enzyme and surfactant. In order to minimize the extent of enzyme inactivation, the steady state enzyme concentration should be kept as low as possible in the first aqueous phase. This can be achieved by a high mass transfer rate and a high distribution coefficient of the enzyme between reverse micellar and aqueous phases. The effect of mass transfer coefficient during forward extraction on the recovery of a-amylase was simulated for two values of the distribution coefficient. These model predictions were verified experimentally by changing the distribution coefficient (by adding... 

Tomazic, S.J. and Khbanov, A.M. (1988) Mechanisms of irreversible thermal inactivation of Bacillus -amylases. J. Biol. Chem., 263, 3086-3091. 

The percentage decreases in the activities of a-amylase incubated at 70, 80, and 85 °C are given in Table 3.1. Calculate the inactivation constants of a-amylase at each temperature. 

Table I (104) shows the yields of products from maltose to malto-pentaose recovered from digests of 0.2 M crystalline a-D-glucosyl fluoride with crystalline a-amylase preparations from six different biological sources. The digests were incubated at 30 °C for 10 minutes, heat inactivated, and chromatographed for product isolation and analysis.

Two types of enzymes in milk are important those useful as an index ol heat treatment and those responsible tor bad flavors. Phosphatase is destroyed by the heat treatments used to pasteurize milk hence its inactivation is an indication of adequate pasteurization. Lipase catalyzes the hydrolysis of milk fat which produces rancid flavors. It must be inactivated by pasteurization or more severe heat treatment to safeguard the product against off-flavor development Other enzymes reported to have been found in milk include catalase, peroxidase, protease, diastase, amylase, oleinase. reductase, aldehydrase. and lactase. 

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