Enzyme deactivation protein denaturation

When the rate of an enzyme catalyzed reaction is studied as a function of temperature, it is found that the rate passes through a maximum. The existence of an optimum temperature can be explained by considering the effect of temperature on the catalytic reaction itself and on the enzyme denaturation reaction. In the low temperature range (around room temperature) there is little denaturation, and increasing the temperature increases the rate of the catalytic reaction in the usual manner. As the temperature rises, deactivation arising from protein denaturation becomes more and more important, so the observed overall rate eventually will begin to fall off. At temperatures in excess of 50 to 60 °C, most enzymes are completely denatured, and the observed rates are essentially zero. 

Although the interest of scientists in peroxidase enzymes has increased tremendously during the past decades, the application of these enzymes as biocatalysts in industrial processes is still negligible. Often the low activity and the fragile nature of these enzymes make their use challenging and sometimes results in poor productivities. Different aspects including heme deactivation (Chap. 12), redox potential modulation (Chap. 4), protein denaturation, and substrate availability have to be dealt with. 

The definition of a more efficient enzymatic system could be based on the separation of the catalytic cycle of the enzyme and the degradation step by the Mn3+ reactive species in MnP systems. The Mn3+-chelates present several advantages in their use as oxidants. They are more tolerant to protein denaturing conditions such as extremes of temperature, pH, oxidants, organic solvents, detergents, and proteases, and they are smaller than proteins therefore, they can penetrate microporous barriers inaccessible to proteins. The optimization of the production of the Mn3+-chelate will have to be compatible with the minimal consumption and deactivation of the enzyme. 

Enzymes in the presence of water can attack and break apart proteins and biological body waste products and dirt. Why is it advised not to boil the washing The enzyme is a protein and can be deactivated or denatured if heated too strongly. 

In spite of a long-time paradigm that enzymes can be active only in their natural aqueous media and other solvents cause deactivation and denaturation of proteins, at present a growing number of investigations are devoted to enzymatic reactions in organic solvents (Klibanov, 2001 Ke et al., 1996 Koskinen and Klibanov, 1996 and references therein). Such enzymes as a-chymotrypsin, subtilisin ribonuclease, pancreatuc lipase, and horse radish peroxidase have been found to be markedly active in organic solvents (alcohols, amines, tiols,anhydrous alkanes, acetonitril, dichloromethane, methyl acetate, etc.). 

Other mechanisms for enzyme denaturation in the presence of surfactants have also been proposed. One hypothesis is that the high charge densities of ionic surfactants increase the probability of them binding strongly to protein sites. This causes conformational changes of the enzyme which subsequently leads to further enzyme deactivation [99,103],... 

In enzymatic reactions, SCFs have shown their potential as better alternatives to conventional organic solvents, due to easy product recovery, lower heating requirements, and side reactions. The first attanpt to use SCFs in enzymatic reactions dates to 1985, when an alkaUne phosphatase was used, followed by p-cresol and p-chloro-phonol oxidation with polyphenol oxidase (Hammond et al., 1985). The fluids used in enzymatic reactions are those that have low critical temperatures to avoid the thermal denaturation of the enzyme. Therefore, CO2, ethane, and fluoroform have been the commonly used. Superaitical water, however, cannot be used due to a high critical temperature Q1AA°C), which deactivates the enzyme. On the other hand, it is well known that enzymes, and proteins in general, are not affected by high pressure (Lanza et al., 2004 Prasanth and Abraham, 2009). 

Reaction temperature is well known for its effect on enzyme activity. Most lipases are active in the temperature range 40-75 °C. Within this temperature range, higher temperature usually increases the reaction rate. Nevertheless, lipases are deactivated faster at higher reaction temperature due to protein denaturation. Cheong and co-workers (2007) found that 1,3-specific lipase from RML was quite stable at 65 °C and could be reused in at least ten continuous batches of enzymatic partial hydrolysis (120h) to produce DAG without significant loss of catalytic activity. 

Fig. 9.54 Deactivation of enzymes by protein unfolding. (From https //upload.wikimedia.orgfyirikipedia/ commons/ /I d/Process of Denaturation.svg).

Effect of Temperature and pH. The temperature dependence of enzymes often follows the rule that a 10°C increase in temperature doubles the activity. However, this is only tme as long as the enzyme is not deactivated by the thermal denaturation characteristic for enzymes and other proteins. The three-dimensional stmcture of an enzyme molecule, which is vital for the activity of the molecule, is governed by many forces and interactions such as hydrogen bonding, hydrophobic interactions, and van der Waals forces. At low temperatures the molecule is constrained by these forces as the temperature increases, the thermal motion of the various regions of the enzyme increases until finally the molecule is no longer able to maintain its stmcture or its activity. Most enzymes have temperature optima between 40 and 60°C. However, thermostable enzymes exist with optima near 100°C. 

Most ultrasonic experiments are carried out in temperature controlled systems to ensure that isothermal conditions are maintained. Even a small general increase in microbial temperature can influence both the active and passive transport systems of the cell membrane/wall and this in turn may lead to an increased uptake of compounds. If the temperature is not controlled then sonication could result in a large temperature increase which will lead to the denaturation (deactivation) of enzymes, proteins and other cellular components present within the microorganism [7]. 

Another protein in soybeans that is destroyed by extrusion is the trypsin inhibitor, which is produced in the pancreas. Without the action of trypsin, the animal cannot use protein, as it is trypsin that splits or hydrolyzes the protein molecule. Other less important enzyme inhibitors that are denatured by the extruder relate to fats and the carbohydrate fraction of a diet. As the heat needed to deactivate enzymes is less than that needed to prepare oilseeds for oil extraction, the effect on the amino acids is much less severe, thus making them more available to the animal or higher in digestibility. 

Although very interesting biotranformations have been reported in supercritical carbon dioxide, this solvent has been found to affect enzyme activity adversely. CO can react reversibly with free amino groups (lysine residues, specifically) on the surface of the protein to form carbamates, leading to low activity enzyme. [21]. Furthermore, carbon dioxide dissolves in water at molar concentrations at moderate pressures (<100 bar) and rapidly forms H COj. This can create some problems in biocatalytic reactions because many enzymes are denatured (unfolded and/or deactivated) at low pH. Enzymes can also be denatured by pressurization/depressuriza-tion cycles. For all of them, it is necessary to develop new enzyme stabilization strategies. 

Deactivation generally refers to a change in the physical structure of the enzyme, often caused by an increase in temperature. Some of the amino acids in a protein chain are hydrophobic. Others are hydrophilic. Proteins in solution fold into elaborate but characteristic shapes to increase like-to-like interactions within the polymer and between the polymer and the solvent. The folded state is the native or natural state and is the state in which enzymes have their catalytic activity. At high temperatures, random thermal forces disrupt the folded chain and destroy the catalytic sites. Very high temperatures will cause coagulation or other structural and chemical changes. This leads to irreversible deactivation, and the proteins are denatured. Mammalian... 

Comparison with Free Lipase and Novozyme To evaluate the catalyst performance, free lipase and a commercial immobilized lipase (Novozyme) were also tested. The results of the activity per gram of protein are plotted in Figure 11.10. The specific activity of immobilized lipase is lower than that of the free enzyme. It is known that the residual activity of an enzyme usually decreases significantly. A decrease in the rate observed can usually be ascribed to conformational changes, steric effects, or denaturation. For the monolithic biocatalysts, the immobilized activity was found to be 30 to 35%, and for Novozyme around 80% in the first run. Deactivation of the commercial sample in consecutive runs is probably due to the instability observed for the support matrix under reaction conditions... 

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