Inactivation protease

The presence of proteases is a threat for protein stability. A simple strategy for protection against proteolytic degradation is based on the fast handling of the sample and use of low temperatures. One additional precaution is the addition of protease inhibitors, especially during disruption and extraction steps. In some cases, there is a need for a combination of inhibitors. Sometimes, pH adjustment is effective in inactivating proteases without affecting the stability of the protein product. 

Other protease inhibitors (e.g., PHMB) as well as inactivating proteases, can also potentially react with amino acid residues of the protein being isolated. Table 3-1 lists the commonly used protease inhibitors together with their targets and recommended concentrations. 

Since incubation of the protease with a large excess of DFP could nonspecifically phosphorylate tyrosine residues (10), the control and inactivated protease samples were analyzed by ES mass spectrometry (8). The ES mass spectrum of a fully active control sample of CMV protease is shown in Figure 1, where a series of multiply charged ions (from +12 to +34) provided an average Mf value of 27,910 (calculated Mr=27,909). The deconvoluted spectrum (Fig. 1, inset), shows that only 30% of the protease was full length (observed... 

A protease species with 2 DIP groups incorporated is also apparent in samples incubated with 1.26, 1.63 and 2.07 mM DI, although even in the sample incubated with 2.07 mM DFP, the diphosphorylated components represent less than 5% of the inactivated protease. Based on Figure 3, the... 

Diisopropylfluorophosphate (DIFP) irreversibly inactivates proteases such as trypsin and chymotrypsin by reacting with ... 

Inactivate protease prior to digestion with restriction enzymes. For this purpose, add phenylmethylsulfonylfluoride (PMSF, a covalent protease inhibitor, usually stored as a 100 mM stock solution(17.4 mg/ml in isopropanol) at -20°C) to TE (at 25°C) just before use to a final concentration of 80 pg/ml. PMSF solutions have a short effective half-life in water and should be replaced twice with a fresh solution (each incubation 1 h). Moreover, PMSF is not stable in alkaline conditions or at higher temperatures. This dangerous chemical (handled in the hood) can be inactivated by rendering the used solution alkaline and leaving at room temperature. 

The amidase from Klebsiella oxytoca is robust, stable, and does not require cofactors. It can be heated to 70 °C for 10 min without loss of activity, and therefore, heat treatment of the biomass used in the biotransformation was used to stabilize the enzyme, presumably by inactivating proteases. 

Significantly different from the inactivated protease-treated ARS-2 group. (p<0.05). 

More than 97% of the available soybean meal is used for feed where extensive heat treatment is necessary to maximize feed conversion efficiency by livestock. Toasting inactivates protease inhibitors (especially trypsin and chymotrypsin inhibitors) and the enzyme urease, and improves protein digestibility. None of these objectives can be obtained without protein being denatured and loss in water solubility however, depending on the method used, meals with great differences in protein solubilities or dispersibilities can be produced. The optimum amount of heat treatment in toasting soybean meal is still debated among animal nutritionists. 

These harmful effects suggest a need to find ways to inactivate the inhibitors before soybeans are consumed. Although heat is often used to inactivate protease inhibitors, such inactivation is often incomplete. High temperature may also destroy lysine and sulfur amino acids and thus damage protein nutritional quality. 

Collagen Gives strnctnral support. Functions as signaling molecnle. Inactivates proteases. Protects exogenously added growth factors. Demidova-Rice et al. (2012)... 

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