Temperature proteinase activity

Because yams are stored in open systems at ambient temperatures (usually warm), tuber tissue was examined for proteinase activity at 40°C. Some tubers had high apparent polyphenol oxidase activity upon peeling of the tubers (tissue turned deep purple at the peeled surface) so that PYP was added to extracts to combine with polyphenolic compounds and protect the proteinase from reacting with these compounds. Earlier studies had shown some inhibition of alkaline proteinase activity by ferric ion (24) so that EDTA was also added to the extracts to chelate any free iron. Two alkaline pH optima were found, at 9.0 and 10.5. The alkaline proteinases of white potatoes (Solanum tuberosum) have pH optima between 8.6 and 9 (25) and those of Carilla chocola tubers have pH optima between 8.0 and 9.5 (26,27T, suggesting that alkaline... 

X SSC buffer 3MSodium chloride, 0.3Msodium citrate, pH at usted to 7 with sodium hydroxide. Sterilize by autodaving, store at room temperature. Proteinase K is obtained from Beckman, Somerset, hQ. Other sources may be acceptable. In using alternate sources, the occurrence of blue backgrounds between colonies, and oversize, blurry signals at colony sites after the final color development step indicates insufficient proteolytic activity. Prepare a solution of 200 pg/mL in IX SSC. 

Enzymatic synthesis of aliphatic polyesters was also achieved by the ringopening polymerization of cyclic diesters. Lactide was not enzymatically polymerized under mild reaction conditions however, poly(lacfic acid) with the molecular weight higher than 1 x 10" was formed using lipase BC as catalyst at higher temperatures (80-130°C). Protease (proteinase K) also induced the polymerization however, the catalytic activity was relatively low. 

Lactide was polymerized by lipase PC in bulk at high temperature (80-130°C) to produce poly(lactic acid) with Mw up to 2.7 x 105 [64, 65]. The molecular weight of the polymer from the D,L-isomer was higher than that from the d,d- and L,L-ones. Protease (proteinase K) also induced the polymerization of lactide, however, the catalytic activity was relatively low. 

Attempts have also been made to exploit the relatively high molecular activity of cod trypsin at low temperatures by incorporating the enzyme into herring "fermentations that proceed at 10°C. The preparation of brine-fermented round herring (matjes) is limited to certain seasons because of the balance of digestive enzymes in the fish at this time. Other studies have indicated that proteinases are important components in matje fermentation 41),... 

The presence of an indigenous proteinase in milk was suggested by Babcock and Russel in 1897 but because it occurs at a low concentration or has low activity in milk, it was felt until the 1960s that the proteinase in milk may be of microbial origin. Recent changes in the dairy industry, e.g. improved hygiene in milk production, extended storage of milk at a low temperature... 

The activity of this Proteinase K is optimal at 65°C, but the protein will rapidly denature at higher temperatures. One should take care that the incubation temperature does not exceed 6S°C. 

The keratin structure is destroyed through the use of proteolytic enzymes such as pronase and proteinase K. They are often used with chemical agents such as urea and thioglycolic to cleave the disulfide bonds and increase the dissolution rate of enzyme activity. The extracting procedures, using enzymatic digestion, last about 4-6 h and must be conducted at constant temperature and pH for providing maximum enzyme activity [155],... 

To isolate genomic DNA from E. coli, the cells are treated with lysozyme and then lysed by SDS in the presence of proteinase K. Proteinase K, which is active even in SDS solution, degrades proteins including nudeases. Cell debris, polysaccharides and unhydrolysed protein are removed by precipitation at room temperature with cetyltrimethylammonium bromide (CTAB). DNA is isolated from the supernatant by precipitation with alcohol. RNA can be removed from DNA preparations by incubation with DNase-free RNase. Further purification can be effected by a phenol/ chloroform/isoamyl alcohol (25 24 1) extraction, and/or by CsCl gradient centrifugation (see Sect. 4.3.4.2 ) to remove the remaining protein and RNA. 

Linked with the difference in sialic acid content is the finding that LH is inactivated more readily than FSH by proteolytic enzymes (Al). Reichert and Parlow showed that both FSH and LH preparations contained proteinase but could not demonstrate the relationship between the content of these proteolytic enzymes and the lability of the preparation (R7). Stockell Hartree found that one of her purified LH preparations, which had an activity five times that of NIH-LH-Sl when first prepared, lost half of its activity on storage at room temperature after drying in acetone and ether. However, other samples with this activity prepared by the same method were shown to be stable, and Stockell Hartree has suggested that the loss of activity in the one preparation was caused by failure to keep the substance properly desiccated (S27). 

Kaplan [66] investigated the persistence of VACV and determined inactivation curves at temperature ranges between 50°C-60°C. The results from this study demonstrated a rapid fall of infectivity followed by a complete inactivation at a much slower rate. VACV can be inactivated by pasteurization at 60°C in a variety of protein solutions [90], Approximately a 2 logi0 inactivation of VACV was observed after 1 h pasteurization in alphai-proteinase inhibitor solution within 3 h of exposure, no active VACV was detectable. Similarly, in human plasma protein solution, VACV was reduced by 4 logio within 30 min, and by 2 h vims activity was undetectable. 

For soluble and immobilized bromelain, temperature increase is accompanied by a decrease in residual enzyme activity. A more complex form of denaturation occurs with the immobilized enzyme, which may involve a two-phase process. Immobilization offers more resistance to denaturation at the higher temperature of 60°C where the second phase is prolonged by a factor of three [60]. Differential scanning calorimetry experiments showed that bromelain is an exceptional protease among the cysteine proteases, illustrated by the fact that its thermal denaturation is consistent with an irreversible two-state model [61]. Also, the far UV circular dichroism spectrum of bromelain differs from those of papain and chymopapain and therefore represents a third spectral class within the cysteine proteinase family [62],... 

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