Solvent enzyme activity

The discussion in the above Sect. 10.3 gives a clear indication that with a few exceptions in most ILs, enzymes remain active and perform better or comparable to conventional organic solvents. Enzyme activity is closely related to the structure and conformational change at the microenviromnent of the enzyme active site. As discussed before (Sect. 10.2) microenviromnents of the enzyme active site is affected by ILs, which has complicated solvation characteristics due to different interactions of large organic cations and anionic counterparts of ILs with the enzyme. So it is important to understand the stability of enzymes in ILs. 

Iborra and co-workers (Entry 8) examined the transesterification of N-acetyl-i-tyrosine ethyl ester in different ionic liquids and compared their stabilizing effect relative to that found with 1-propanol as solvent [36]. Despite the fact that the enzyme activity in the ionic liquids tested reached only 10 to 50 % of the value in 1-propanol, the increased stability resulted in higher final product concentrations. Fixed water contents were used in both studies. 

Entries 7, 8, and 10 describe so-called Idnetically controlled syntheses starting from activated substrates such as ethyl esters or lactose. In two reaction systems it was possible to demonstrate that ionic liquids can also be useful in a thermodynamically controlled synthesis starting with the single components (Entry 11) [39]. In both cases, as with the results presented in entry 6, the ionic liquids were used with addition of less than 1 % water, necessary to maintain the enzyme activity. The yields observed were similar or better than those obtained with conventional organic solvents. 

Further studies of Pseudomonas sp. lipase revealed a strong influence of the water content of the reaction medium (Entry 20) [48]. To be able to compare the enzyme activity and selectivity as a function of the water present in solvents of different polarities, it is necessary to use the water activity (a ) in these solvents. We used the... 

FIGURE 6.11. Comparison of the environment around the transition state of lysozyme in the enzyme-active site and in the reference solvent cage. 

Enzyme active sites, 136,148, 225. See also Protein active sites in carbonic anhydrase, 197-199 in chymotrypsin, 173 in lysozyme, 153, 157 nonpolar (hypothetical site), 211-214 SNase, 189-190,190 steric forces in, 155-158, 209-211, 225 in subtilisin, 173 viewed as super solvents, 227 Enzyme cofactors calcium ... 

Enzyme activity in organic solvents depends on parameters such as water activity, pH control, substrate-product solvation, enzyme form, and nature of the solvent. 

At the present time, "interest in reversed micelles is intense for several reasons. The rates of several types of reactions in apolar solvents are strongly enhanced by certain amphiphiles, and this "micellar catalysis" has been regarded as a model for enzyme activity (. Aside from such "biomimetic" features, rate enhancement by these surfactants may be important for applications in synthetic chemistry. Lastly, the aqueous "pools" solubilized within reversed micelles may be spectrally probed to provide structural information on the otherwise elusive state of water in small clusters. 

The effect of biphasic mixtures on the productivity includes the contributions of solvent partitioning on enzyme activity and stability. An important activity does not necessarily lead to increased productivity. We must then distinguish between the effect of the environment on activity and productivity. 

The active site of enzymes usually are located in clefts and crevices in the protein. This design effectively excludes bulk solvent (water), which would otherwise reduce the catalytic activity of the enzyme. In other words, the substrate molecule is desolvated upon binding, and shielded from bulk solvent in the enzyme active site. Solvation by water is replaced by specific interactions with the protein (Warshel et al., 1989). 

Enzymatic reactions are influenced by a variety of solution conditions that must be well controlled in HTS assays. Buffer components, pH, ionic strength, solvent polarity, viscosity, and temperature can all influence the initial velocity and the interactions of enzymes with substrate and inhibitor molecules. Space does not permit a comprehensive discussion of these factors, but a more detailed presentation can be found in the text by Copeland (2000). Here we simply make the recommendation that all of these solution conditions be optimized in the course of assay development. It is worth noting that there can be differences in optimal conditions for enzyme stability and enzyme activity. For example, the initial velocity may be greatest at 37°C and pH 5.0, but one may find that the enzyme denatures during the course of the assay time under these conditions. In situations like this one must experimentally determine the best compromise between reaction rate and protein stability. Again, a more detailed discussion of this issue, and methods for diagnosing enzyme denaturation during reaction can be found in Copeland (2000). 

The very high resolution for the ESR spectrum of cob(II)alamin in the enzyme system is undoubtedly due to the fact that all the coenzyme molecules are bound in an identical environment at the enzyme active site. This results in a homogeneous cobalt-benzimidazole geometry, because both identical binding sites, solvent, and solute molecules can no longer approach the Bia-molecule closely. In addition, the enzyme bound cob(II)alamin molecules are more isolated from one another and thus relaxation due to spin-spin interactions is less effective in broadening spectral lines. 

Persson, M., Costes, D., Wehtje, E. and Adlercreutz, P. (2002) Effects of solvent, water activity and temperature on lipase and hydroxynitrile lyase enantioselectivity. Enzyme and Microbial Technology, 30, 916-923. 

Dehal, S.S., Gagne, P.V., Crespi, C.L. and Parren, C.J. (2002) Effect of common organic solvent on human UGT enzyme activities. 11th North American ISSX Meeting, October 27-31, 2002, Orlando, Florida, USA, Abstract 370. 

Enzyme activity for the polymerization of lactones was improved by the immobilization on Celite [93]. Immobilized lipase PF adsorbed on a Celite showed much higher catalytic activity than that before the immobilization. The catalytic activity was further enhanced by the addition of a sugar or poly(ethylene glycol) in the immobilization. Surfactant-coated lipase efficiently polymerized the ring-opening polymerization of lactones in organic solvents [94]. 

Fontes tt al. [224,225 addressed the acid—base effects of the zeolites on enzymes in nonaqueous media by looking at how these materials affected the catalytic activity of cross-linked subtilisin microcrystals in supercritical fluids (C02, ethane) and in polar and nonpolar organic solvents (acetonitrile, hexane) at controlled water activity (aw). They were interested in how immobilization of subtilisin on zeolite could affected its ionization state and hence their catalytic performances. Transesterification activity of substilisin supported on NaA zeolite is improved up to 10-fold and 100-fold when performed under low aw values in supercritical-C02 and supercritical-ethane respectively. The increase is also observed when increasing the amount of zeolite due not only to a dehydrating effect but also to a cation exchange process between the surface proton of the enzyme and the sodium ions of the zeolite. The resulting basic form of the enzyme enhances the catalytic activity. In organic solvent the activity was even more enhanced than in sc-hexane, 10-fold and 20-fold for acetonitrile and hexane, respectively, probably due to a difference in the solubility of the acid byproduct. 

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