Effects on enzyme stability

Dixon and Webb provided a useful list of various causes for the shape of the temperature effect seen in enzyme-catalyzed reactions (1) effect on enzyme stability (2) effect on the actual velocity of the reaction (especially on kcat) (3) effect on affinity(ies) of the substrate(s)... 

As one might expect, water has a dramatic effect on enzyme stability in SCFs. Lozano et al. found that the half-life time of a-chymotrypsin decreased exponentially in SCCO2 with increasing water content from 0 to 15 wt% [28]. Kashe et al. found that a-chymotrypsin, trypsin and penicillin amidase partially unfolded during pressure reduction in humid SCCO2. They suggested that... 

The common approach to maintain minimal polarization is to operate at high shear rates [103] however, this can be harmful to biocatalysts. To decrease gel-layer formation in the surface of the membrane, Hakoda and co-workers [178] applied an electric field of 50 and 100 V to a ceramic membrane module used in the lipolysis of triolein in a reversed micellar system. These authors reported a slight increase in the filtration flux (about 15%), without deleterious effects on enzyme stability for an operation time length of 12 h. The electrokinetic phenomena leading to the observations occurred even in apolar media, since small amounts of water or surfactant were present in such media [178]. 

Despite their positive effects on enzyme stability, excessive use of organic solvents decreases the yield, which is mainly due to the dilution of the substrates (Ji et al., 2006). Samukawa et al. (2000) tested the use of different solvents on Lipozyme TL activity, and it was found that the yield increased along with the hydrophobic-ity of the solvent. In contrast, hydrophilic solvents are much less efficient (Doukyu and Ogino, 2010 Klibanov, 1997). For example, the use of acetone (log P = -0.23) showed a less than 20% yield compared to 80% when n-hexane was used. The negative effect of hydrophobicity is due to the solvent s interaction with the essential water layer surrounding the lipase molecule (Iso et al., 2001), which results in unfavorable conformational changes in the enzyme structure and reduces activity. 

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. 

High pressure has proven to be a useful tool in biochemistry for the study of a number of cell-mediated processes, the most important being the effect on gene expression. The pressure effect on the stability of enzymes and biopolymers is a topic of general interest that may generate a number of possible applications in the area of food science. Biochemistry and biophysics continue to attract many new research groups, especially in the field of protein chemistry. Pressure may be a tool to obtain unique textures and provide biochemical products with new properties. 

Some drugs can also increase the amount of active CYP enzyme via effects on protein stabilization or the transcriptional apparatus. This process is called induction. 

Sadana and collaborators (Sadana, Raju and Shahin, 1989) have proposed an empirical stability index (SI) for enzyme deactivation, which makes more quantitative the effect of different variables on enzyme stability. 

Chemical modification of proteins has been extensively studied over the years to identify which amino acids are involved in catalysis. Much less work has been carried out on its influence on enzyme stability. Chemical modification of proteins may yield stabilization, destabilization or no effect at all. Martinek and Berezin (1978) reported the dependence of the thermostability of chymotrypsin on the degree of alkylation of its amino groups up to 30% alkylation the stability rose slightly at 90% substitution stability increased markedly, with a maximum (110-fold) at 95% stability fell to nearly initial values when 100% amino groups were modified. (With these modifications, the optimum pH of the errzyme can change and one must therefore be cautious in comparing two different... 

Other post-translational modifications of proteins (e.g., phosphorylation) are extremely important mechanisms of regulation of enzyme activity. Very tittle work has been done on the effects of such modifications on enzyme stabilization. 

The preceding discussion dealt with membrane-bearing systems. In the case of acellular systems, i.e. with crude cell extracts or purifled enzymes, the validity of logKow as a criterion for biocompatibility is questionable. As a result, other parameters, such as interfacial tension, have been suggested to predict the effect of solvents on enzyme stability [30]. 

Below, solvent effects on activity and stability of enzymes will be discussed, while solvent effects on enzyme selectivity is a large topic which is treated in a separate chapter. Solvent effects on equilibria are treated in Section 1.4. 

Erom the results presented in Figure 6.4 it is clear that the loss of enzyme activity over time is dramatic when either ClAA or AA concentrations exceed 100 mM, resulting in half-life times in the range of 5-7 h at industrially relevant concentrahons. The first aldol condensation product also rapidly inactivates DERA at concentrahons above 100 mM. Although the final product 1 seems to have a less pronounced effect on the stability of the enzymes. Figure 6.4 indicates that the achvity in the presence of the compound is low. 

Activity and stability are often comparable to values in aqueous media. Many substrates which cannot be made to react in water or in pure organic solvents such as hexane owing to lack of solubility can be brought to reaction in microemulsions. Whereas enzyme structure and mechanism do not seem to change upon transition from water to the microemulsion phase (Bommarius, 1995), partitioning effects often are very important. Besides an enhanced or diminished concentration of substrates in the vidnity of microemulsion droplets and thus of enzyme molecules, the effective pH values in the water pool of the droplets can be shifted in the presence of charged surfactants. Frequently, observed acceleration or deceleration effects on enzyme reactions can be explained with such partitioning effects (Jobe, 1989). 

Deglycosylation had no marked effect on the stability of the acid carboxypeptidase. Both native and deglycosylated enzymes were stable up to 50°C and active in the presence of 4 M urea after 24 h. The molecular structures of both enzymes were not impaired by pepsin at pH 2.5 (Figure 25). These two enzymes had similarly great resistance to peptic digestion after 24h. 

Srivastava PK, Sharma VK, Kalonia DS, Grant DF (2004) Polymorphisms in human soluble epoxide hydrolase effects on enzyme activity, enzyme stability, and quaternary structure. Arch Biochem Biophys 427 164-169... 

---