Potato enzyme, inactivation

In the authors experience, the pigments of a wide variety of plant materials have been stable in the aqueous acetone extract. However, problems have been encountered with pigment degradation for certain potato cultivars with high polyphenol oxidase activity. This problem can be circumvented by putting the aqueous acetone extract (uncapped) in a boiling water bath for 5 min. After heating, the volume of acetone lost by evaporation should be replenished. This enzyme inactivation step has been found unnecessary for most materials. 

Table II gives the results of residual trypsin inhibitor levels for the various soymilk preparations. The 90 and 120 sec microwave treatments were the most effective in inactivating the trypsin inhibitor complex while hot water treated and unheated samples showed the highest levels. It is not surprising to find that microwave processing is more efficient than hot water in suppressing trypsin inhibitor considering the rapid penetration of food material by microwaves and the susceptibility of protein action to small heat induced changes in tertiary structure. Hence, Collins and McCarty (12) found microwaves produced a more rapid destruction of endogenous potato enzymes (polyphenol oxidase and peroxidase) than hot water heating.

The sweet potato enzyme could be inactivated by o-phenanthroline and 2,2 -bipyridine the activity could be restored by the addition of Zn2+ and partially by added Mn2+ or Co2+ (69). The amino acid composition revealed an usually large content of tyrosine. Later experiments were able to quantitate the amount of Mn present at two Mn per mole enzyme (70). Additionally, the native enzyme was shown to be EPR inactive however, treatment of the enzyme with acid led to the appearance of a six-line pattern typical of aqueous Mn2+ coincident with the loss of the purple color, further evidence for the association of Mn with the 555-nm absorbance band. 

Presently, the vast majority of information on the Mn site in these acid phosphatases comes from the enzyme from sweet potato tubers. This 110-kDa enzyme is identical to the previously reported sweet potato enzyme likewise, a 55-kDa subunit was found (78). However, the enzyme possesses only one Mn per enzyme molecule. At 293 and 77 K, no EPR signal could be detected for the native enzyme. Inactivation of the enzyme by heat treatment or the addition of acid results in the appearance of a six-line EPR pattern due to aquated Mn(II). As in the case of Mn SODs, this was taken as evidence for Mn(III) in the native... 

Lipase and alkaline phosphatase in milk are ther-molabile (Fig. 2.37), whereas acid phosphatase is relatively stable. Therefore, alkaline phosphatase is used to distinguish raw from pasteurized milk because its activity is easier to determine than that of lipase. Of all the enzymes in the potato tuber (Fig. 2.38), peroxidase is the last one to be thermally inactivated. Such inactivation patterns are often found among enzymes in vegetables. In such cases, peroxidase is a suitable indicator for controlling the total inactivation of all the enzymes e. g., in assessing the adequacy of a blanching process. However, newer developments aim to limit the enzyme inactivation to... 

Figure 6. Maceration of potato tuber tissue pre-incubated for 2 h with 0,05 U/ml of PL3 enzyme activity. Con = Control incubated with the inactivated enzyme.

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... 

Freshly cut oranges or their juices may be exposed in an open glass for several hours without appreciable loss of I he vitamin because of the protective effect of the acids present and the practical absence of enzymes that catalyze its destruction. In potatoes, when baked or boiled, there is a slight loss of the vitamin, blit if they are whipped lip with air while hot, as in the production of mashed potatoes, a large fraction of the initial vitamin content usually will be lost. In freezing foods, it is common practice to dip them in boiling water or to treat them briefly with steam to inactivate enzymes, after which they arc frozen and stored at very low temperatures. In this state, the vitamin is reasonably stable. Vuamin C degradation in dehydrated food systems is described shortly. 

Similar Mn-containing enzymes were subsequently isolated from other plant sources spinach leaves (71), rice plant cultured cells (72), soybeans (73-75), and the tubers of the sweet potato Kintoki (76-81) (Table III). Sweet potatoes have recently been reported to possess two different acid phosphatases which were immunologically distinct but which have similar molecular weights and metal content (106). Interestingly, sulfhydryl reagents have been shown to inactivate the soybean enzyme (75). 

Both potato tuber and potato leaf ADP-Glc PPases are plas-tidic the leaf enzyme is in the chloroplast, and the tuber enzyme is in the amyloplast (74). The ferredoxin-thioredoxin system is located in the chloroplast and thus, with photosynthesis, reduced thioredoxin is formed and activated within the leaf ADP-Glc PPase. At night, oxidized thioredoxin is formed it oxidizes and inactivates the ADP-Glc PPase. This activation/inactivation process during the light/dark cycle allows a fine tuning and dynamic regulation of starch synthesis in the chloroplasts. Thioredoxin isoforms are present in many different subcellular locations of plant tissues cytosol, mitochondria, chloroplasts, and even nuclei (75) and are also present in amyloplasts (76). 

The following experiments illustrate that when studying the involvement of phospholipase in the host-pathogen interaction, the total contribution of enzyme of host origin may be considerably higher than previously realized. Rodionov and Zakharova (32) recently reported very high rates of autolytic hydrolysis of membrane lipids in homogenates of potato leaves (26-37% of the phospholipids were hydrolyzed after 2 h at 0-1 ). Our laboratory recently confirmed this observation and proceeded to study sosie of the properties of the lipolytic acyl hydrolase activity in potato leaves (6). Lipolytic acyl hydrolase activity is apparently inactivated by polyphenol oxidase or its toxic quinone products. 

Still more recent work by the same authors has suggested an alternative possibility. It has been generally assumed that L-ascorbic acid has no effect on the polyphenolase system other than its effect as a reducing agent for the o-quinone formed by the oxidation of the phenols. It has now been shown that ascorbic acid itself has an inhibitory action on the polyphenolase enzyme. When polyphenolase prepared from potato was treated with ascorbic acid under anaerobic conditions, and the ascorbic acid subsequently removed by dialysis, the activity of the enzyme was very considerably reduced. The enzyme after such treatment could not be reactivated by the addition of cupric salts and appeared to bo irreversibly inactivated. It was also shown that neither dehydroascorbic acid nor the further oxidation products of dehydroascorbic acid were responsible for this result. There is at present no explanation of the mechanism of this inhibitory action of ascorbic acid, but it is quite clear that, if these results are confirmed, other explanations are possible of why these enzymes do not exert their full potential effect in vivo. 

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