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The biocatalyst activity is influenced by a number of factors, such as affinity and substratum concentration, temperature, pH-medium, the presence of activators and inhibitors. Experts identify four types of elements that determine the catalytic spree of an enzyme. First, we need a chemical apparatus in a dynamic center, capable of deforming or polarizing bonds of a substratum, which makes the latter more reactive. Secondly, a fixation core is needed that immobilizes substrata in the correct position to other reaction groups participating in chemical transformation.
Thirdly, it is not superfluous to determine the right orientation to medium, thanks to which each stage passes with minimal vibrational motion around its links. Finally, a method for fixing substratum should help to reduce activation punch of the biocatalyst complex in a transition. All these components to some extent affect the ferment fabric, so they cannot be considered in isolation.
An elementary act of catalysis occurs spontaneously in an aggressive center of ferment when a configuration is attained between reacting groups of the substratum and biocatalyst located at distances on the order of lengths of chemical ties.
At the stage of interaction, while the combination is formed and resulting product is cleaved in biocatalyst, conformational changes take place. Such external agents as tem, ionic strength of the solution, viscosity, may impact these relaxation stages. However, a direct act of catalysis in the established dynamic configuration no longer requires thermal energy.
The study of how does pH affect enzyme activity is significant not only from a theoretical but purely practical aspect.
When choosing an activity unit, you need to know the best conditions for its procedure, as well as the effects of various elements on it. Similar preliminary analysis is usually needed for successful purification of the biocatalyst since this process must be quantitatively monitored by systematic determinations of preparation spree at all steps.
Exploration of pH effect on enzymes and stability of catalase is regarded as essential components impressing storage parameters of the product. The fact is that the spree of catalysts frequently increases simultaneously with a decrease in their stability. In the cell, thanks to the regulatory system, after breakdown, new portions of ferment immediately appear in place of the destroyed ones. Thus, the system remains stable and effective due to the fact that any factor that destroys biocatalysts could be neutralized by cellular biosynthesis.
As a material for examination, potato disks were applied. The catalase preparation of potato cells was obtained by mechanical grinding in a laboratory mill and homogenization of object with phosphate-citrate buffer (pH 7.4). The first stage of purification is the precipitation of the biocatalyst preparation with 96.5% ethyl alcohol at a tem of 2-4 ° C.
The precipitate was separated in a refrigerated centrifuge at 5000 g and dried under vacuum. The ferment was released from ballast proteins by fractionating ammonium sulfate. The residue precipitated when ammonium sulfate was saturated with 60-80%. It was dissolved in a minimal number of buffers. From the ferment solution, low-molecular-weight impurities were removed by gel filtration on Sephadex G-25. An enzyme preparation was achieved with an 80-fold purification degree and a specific thread of 545.2 units for 1 mg of protein.
Catalase is ferment from a group of oxidoreductases formed by an oxidation-reduction reaction, during which water and oxygen are separated from two molecules of hydrogen peroxide. When storing and processing potatoes, catalase performs a negative function. Its action leads to the liberation of oxygen, intensifying oxidative processes in the product. As a result of this chain reaction leading to the formation of hydroperoxides and other deeper elements of oxidation and lipid decomposition, an unpleasant odor and a sharp rancid taste in the product arise.
Each enzyme has its own pH-optimum. The reply of a medium has a significant effect on biocatalyst potency. For the manifestation of their optimal action, there is often a narrow pH measurement range. In some cases, the pH shift per unit reduces thread by 80%. Therefore, in experimental conditions of working with ferment, it is quite essential to sustain optimal pH for catalase at a constant level.
For example, pepsin of gastric juice, which catalyzes the hydrolysis of proteins, has a pH optimum at pH = 2. At pH = 3, it loses half own activity, and at pH 4 the spree of pepsin is not measured. The pH optimum of amylase that ferments the cleavage of starch is 7.0. And in this case, a change in pH per unit leads to a decrease in thread by 50%, and – by 2 units there is a complete loss of activity.
Relatively rare cases occur when the pH optimum of enzyme activity is in a strongly acidic or alkaline medium. This is typical for such ferments as pepsin and arginase (catalyzes the cleavage of arginine). Most proteins at extreme pH values are unstable. The optimal pH level is a significant characteristic of the biocatalyst, but not as exclusive to ferment alone.
The fact is that a lot of enzymes have similar pH-optimum values. The mechanism of effect of pH on enzyme activity is that a change in this index leads to a change in the degree of ionization of amino- (NH3) and carbon (COO-) groups in the ferment molecule. As a result, the protein molecule undergoes conformational rearrangements, which affects relationship between a biocatalyst and the substrata.
Results and Conclusions
Temperature and pH are the two most essential factors must be taken into account when catalase is inactivated for the long-term preservation of potatoes, as well as for the preparation. The ferment potency was defined in the pH range 4-9 at the optimum perature and expressed as a percentage of the maximum. The catalase optimum pH is concentrated in the range of 7.3-7.8.
At an optimal pH value, the impact of perature on fermenting thread was determined, so temperature balance composed 37 ± 2 ° C. The study of thermal and pH-stability of the biocatalyst is of considerable interest, since these indices may be important criteria in the choice of storage conditions and food processing regimes products. In the analysis of kinetics of inactivation of catalase, after a certain time interval, samples were taken from the incubation medium and the residual activity of biocatalyst was defined.
Using data on the dynamics, we calculated rates of inactivation. The ferment exhibited the greatest stability at a pH of 8.0 and a perature of 20 ° C. We counted change in the entropy ΔS ≠ of the transient state of the biocatalyst from an active form to the inactivated one. It follows from the received evidence that a high concentration of H + ions leads to an intensive transition of the protein globule of the biocatalyst to a chaotic coil at low temperatures; apparently, this is due to aggressive destruction of electrostatic ties. The reverse situation is noted at pH 8.0. At high temperatures, there is an intensive demolition, which is explained by the annihilation of hydrophobic interactions of nonpolar regions of molecules.
Thus, on the basis of the study, it could be deduced that inactivation of catalase at low temperatures is dominated by H + ions, and at high ones, by thermal energy. The biocatalyst has low acid and thermal stability, which makes it possible to use these features to suppress ferment activity during storage and selection of processing parameters.
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