Catalytic enzymes stability

Selected entries from Methods in Enzymology [vol, page(s)] Theory, 63, 340-352 measurement, 63, 365 cryosolvent [catalytic effect, 63, 344-346 choice, 63, 341-343 dielectric constant, 63, 354 electrolyte solubility, 63, 355, 356 enzyme stability, 63, 344 pH measurements, 63, 357, 358 preparation, 63, 358-361 viscosity effects, 63, 358] intermediate detection, 63, 349, 350 mixing techniques, 63, 361, 362 rapid reaction techniques, 63, 367-369 temperature control, 63, 363-367 temperature effect on catalysis, 63, 348, 349 temperature effect on enzyme structure, 63, 348. 

Efforts should be made to stabilize an enzyme s activity. Certain agents (such as glycerol, ammonium ions, boric acid, polyethylene glycol, and even talcum powder or bentonite clay) have proven widely to be effective enzyme stabilizers. For multisubstrate enzymes, inclusion of one particular substrate with the enzyme (in the absence of other substrates or cofactors) often stabilizes an enzyme s catalytic activity. Such a substrate may also assist in unlocking the enzyme from a particularly inactive conformational form. In addition to substrates, other ligands and effectors (including reaction products. 

In vitro enzymatic polymerizations have the potential for processes that are more regio-selective and stereoselective, proceed under more moderate conditions, and are more benign toward the environment than the traditional chemical processes. However, little of this potential has been realized. A major problem is that the reaction rates are slow compared to non-enzymatic processes. Enzymatic polymerizations are limited to moderate temperatures (often no higher than 50-75°C) because enzymes are denaturated and deactivated at higher temperatures. Also, the effective concentrations of enzymes in many systems are low because the enzymes are not soluble. Research efforts to address these factors include enzyme immobilization to increase enzyme stability and activity, solubilization of enzymes by association with a surfactant or covalent bonding with an appropriate compound, and genetic engineering of enzymes to tailor their catalytic activity to specific applications. 

Industrial exploitation of biocatalysts requires that they be rendered more stable to withstand harsh operating conditions (high temperatures and other conditions that favom the loss of catalytic activity), hence increasing biocatalyst life. The following sections discuss the mechanisms of enzyme stability and stabilization. 

Ever since it was discovered that enzymes can be catalytically active in neat organic solvents, the question of how to select the correct solvent for a specified enzymatic conversion has been of crucial importance. The solvent can influence an enzymatic reaction both by direct interaction with the enzyme and by influencing the solvation of the substrates and products in the reaction medium. An example of direct interaction between solvent and enzyme is when the solvent acts as an inhibitor of the enzyme. In other cases the solvent causes conformational changes in the enzyme, thereby changing its catalytic properties. The solvent can also influence the amount of water bound to the enzyme, but this effect can largely be avoided by the use of fixed water activity as described above. Direct interaction between solvent and enzyme can influence enzyme stability as well as activity. 

In most experimental studies of the influence of organic solvents on enzyme stability, the remaining catalytic activity after exposure to different solvents has been measured. In such a study it was found that the remaining activity of a... 

The primary purpose of iimnobilization is to stabilize the enzyme. Stabilization typically reduces the activity of the enzyme. Part of the reason is stearic, but the reduction of activity could also occur because the site of attachment of the enzyme and substratum is also the portion of the enzyme responsible for its catalytic activity. 

Molded Dry Chemistry. In general, most enzymes are very fragile and sensitive to pH. solvent, and elevated lemperaiurts. The catalytic activity of most enzymes i> reduced dramatically ils the temperature is increased, Typi cal properties of diagnostic enzymes are given in Table 1. t he presence of ionic salts and other chemicals can considerably influence enzyme stability. To keep or sustain enzymatic activity, the redox centers must remain intact. The bulk of the enzyme, polymeric in composition, is an insulaior. thus. altering ii does not reduce the enzyme s catalytic activity, li... 

As discussed earlier, the enzymic reaction catalyzed by glutamine synthetase requires the presence of divalent metal ions. Extensive work has been conducted on the binding of Mn2+ to the enzyme isolated from E. coli (82, 109-112). Three types of sites, each with different affinities for Mn2+, exist per dodecamer n, (12 sites, 1 per subunit) of high affinity, responsible for inducing a change from a relaxed metal ion free protein to a conformationally tightened catalytically active protein n2 (12 sites) of moderate affinity, involved in active site activation via a metal-ATP complex and n3 (48 sites) of low affinity unnecessary for catalysis, but perhaps involved in overall enzyme stability. The state of adenylylation and pH value alter the metal ion specificity and affinities. 

Enzyme Stability. Loss of enzyme-catalytic activity may be caused by physical denaturation, eg, high temperature, drying/freezing, etc or by chemical denaturation, eg, acidic or alkaline hydrolysis, proteolysis, oxidation, denaturants such as surfactants or solvents, etc. pH has a strong influence on enzyme stability, and must be adjusted to a range suitable for the particular enzyme. If the enzyme is not sufficiendy stable in aqueous solution, it can be stabilized by certain additives a comprehensive treatment with additional examples is available (27). 

Finally, self-assembled monolayers (SAMs) on gold electrodes constitute electrochemical interfaces of supramolecular structures that efficiently connect catalytic reactions, substrate and product diffusion and heterogeneous electron transfer step when enzymes are immobilised on them. Resulting enzyme-SAM electrodes have demonstrated to exhibit good performance and long-term enzyme stability. 

The possibility of having membrane systems also as tools for a better design of chemical transformation is today becoming attractive and realistic. Catalytic membranes and membrane reactors are the subject of significant research efforts at both academic and industrial levels. For biological applications, synthetic membranes provide an ideal support to catalyst immobilization due to their biomimic capacity enzymes are retained in the reaction side, do not pollute the products and can be continuously reused. The catalytic action of enzymes is extremely efficient, selective and highly stereospecific if compared with chemical catalysts moreover, immobilization procedures have been proven to enhance the enzyme stability. In addition, membrane bioreactors are particularly attractive in terms of eco-compatibility, because they do not require additives, are able to operate at moderate temperature and pressure, and reduce the formation of by-products. 

Abstract This chapter discusses the potential usefulness of ionic liquids with respect to biocatalysis by illustrating the stability and activity of enzymes in ionic liquids in the presence or absence of water. Ionic liquids are a class of coulombic fluids composed of organic cations like alkyl-substituted imidazolium, pyrrolidin-ium, and tetraalkylammonium ions and anions such as halides, tetrafluoroborates, hexafluorophosphates, tosylates, etc. The possibility of tunable solvent properties by alternation of cations and anions has made ionic liquids attractive to study biocatalysis which warrants an understanding of enzyme stability and activity in ionic liquids. This chapter systematically outlines the recent studies on the stability of enzymes and their reactivity toward a wide range of catalytic reactions. A careful approach has been taken toward analysis of relationship between stabil-ity/activity of enzymes versus chaotropic/kosmotropic nature of cations and anions of ionic liquids. 

The catalytic properties, pH-activity profile and enzyme stability can be enhanced in the immobilized form. 

Water levels also have important general effects on enzyme behavior. If too little water is present, the catalytic activity of most enzymes falls dramatically. On the other hand, reduction in water levels often leads to an increase in enzyme stability. A decline in catalytic activity at high water levels is also commonly observed, with several possible explanations ... 

In 1993, a thermostable aminoacylase from Bacillus stearothermophilus was characterized by Sakanyan et al.l51. The enzyme hydrolyzes N-acyl derivatives of aromatic amino acids preferentially and even has some dipeptidase activity. Its optimal reaction temperature is 70 °C after incubation for 15 min, 90% of the original activity was retained. The authors write that the similarity of the B. stearothermophilus enzyme sequence with that of other enzymes such as aminoacylase I, acetylornithine deacetylase and carboxypeptidase G2 suggests a common origin. The aminoacylase from B. stearothermophilus is well characterized the gene has been completely sequenced1511, cloned into E. coli and overexpressed[51> 671 and studied for catalytic and stability properties16 1 the intrinsic one Zn2+ ion per subunit seems to have a predominantly structural role and activity can be restored to the apo-enzyme by Co2+ and particularly by Cd2+ (3-fold activity ) but not by Zn2+. 

One striking observation about the reactions catalyzed by RTA and pertussis toxin is that the enzymes appear to use opposite catalytic strategies. The analogous non-enzymatic reactions, hydrolysis of adenosine 5 -monophosphate (AMP) and NAD, both have highly dissociative AnD mechanisms with oxocarbenium ionlike transition states. In the ADP-ribosylation of protein Gid by pertussis toxin, the AnDn mechanism is more synchronous, with more nucleophile participation and lower oxocarbenium ion character than the non-enzymatic reaction. " In contrast, the RTA-catalyzed depurination of RNA" and DNA substrates proceeds through stepwise Dj,j An mechanisms where the enzyme stabilizes an oxocarbenium ion to the point that it becomes a discrete intermediate. This is illustrated with a hypothetical free energy surface for each reaction (Fig. 14). 

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