Organic solvent, effect, enzymatic

Finally, in the sense that the imposition of conformational restrictions or specific solvent effects on an organic molecule are forms of strain, non-covalent catalysis by the cycloamyloses may provide a simple model for the investigation of strain and distortion effects in enzymatic reactions. 

Metal ion-catalyzed hydrolytic processes have been studied for a long time, and many interesting systems have been explored which give valuable information about catalysis. However, with very few exceptions the catalysis afforded by these systems in water is disappointing when compared with enzymatic systems where a metal ion cofactor activates a substrate and a nucleophilic or basic group in an acyl or phos-phoryl transfer process. It has been noted that bulk water may not be a good medium to approximate the medium inside the active site of an enzyme where it is now known that the effective dielectric constants resemble those of organic solvents rather than water. 

Tween 85 is used extensively for RME [84]. Russell and coworkers [234] used Tween 85/isopropanol microemulsions in hexane to solubilize proteins and not only showed >80% solubilization of cytochrome C at optimum conditions, but also proved that Tween 85 does not have a detrimental effect on the structure, function, and stability of subtilisin and cytochrome C. There are other reports available on the extraction and purification of proteins using Tween 85-RMs and also on the stability of protein activity in these systems [234]. It has also been shown that Tween 85-RMs can solubilize larger amounts of protein and water than AOT. Tween 85 has an HLB of 11, which indicates that it is soluble in organic solvents. In addition, it is biodegradable and can be successfully used as an additive in fertihzers [235,236]. Pfammatter et al. [35] have demonstrated that RMs made of Tween 85 and Span 80 can be successfully used for the solubilization and growth of whole cells. Recently, Hossain et al. [84] showed an enhanced enzymatic activity of Chromobacterium viscosum Hpase in AOT/Tween 85 mixed reverse micellar systems when compared to that in classical AOT-RMs. This is due to the modification of the interface in AOT-RMs caused by the co-adsorption of Tween 85, and increased availability of the oHve oil molecules (substrate) to the enzyme. 

The proportions of water and organic solvent can be varied from pure water to almost pure organic solvent. In order to retain enzymatic activity there seems to be a need for a little water. However, this minimal amount of water is sometimes considerably less than a monolayer of water around the enzyme molecules. The rest of the medium can be an organic solvent. The effects of water on biocatalysis in non-conventional media are treated below. 

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. 

Based on a suggestion by Odell and Earlam [119] that crown ethers and cryptands can cause proteins to dissolve in methanol, Broos and coworkers [120] investigated the effects of crown ethers on the enzymatic activity of a-chymotrypsin in the transesterification reaction of N-acetyl-L-phenylalanine ethyl ester with n-propanol in organic solvents. They observed a 30-fold rate acceleration when 18-crown-6 was used in octane. At that time, it was proposed that the water- and cation-complexing... 

The application of enzymatic acylation for the resolution of racemic alcohols in organic solvent has shown to be an effective method to rapidly synthesize chiral alcohols. The racemic alcohols are treated with the lipase and acylating agent one enantiomer remains unconverted whereas the second enantiomer is esterified and easily separated by distillation (Scheme 7.2). Vinyl acetate or isopropenyl acetate are typical acylating agents, as the generated vinyl alcohol tautomerizes rapidly... 

Enzymatic enantioselectivity in organic solvents can be markedly enhanced by temporarily enlarging the substrate via salt formation (Ke, 1999). In addition to its size, the stereochemistry of the counterion can greatly affect the enantioselectivity enhancement (Shin, 2000). In the Pseudomonas cepacia lipase-catalyzed propanolysis of phenylalanine methyl ester (Phe-OMe) in anhydrous acetonitrile, the E value of 5.8 doubled when the Phe-OMe/(S)-mandelate salt was used as a substrate instead of the free ester, and rose sevenfold with (K)-maridelic acid as a Briansted-Lewis acid. Similar effects were observed with other bulky, but not with petite, counterions. The greatest enhancement was afforded by 10-camphorsulfonic acid the E value increased to 18 2 for a salt with its K-enanliomer and jumped to 53 4 for the S. These effects, also observed in other solvents, were explained by means of structure-based molecular modeling of the lipase-bound transition states of the substrate enantiomers and their diastereomeric salts. 

A. C. Ross, G. Bell, and P. J. Halling, Organic solvent functional group effect on enzyme inactivation by the interfacial mechanism, J. Mol. Cat. B Enzymatic 2000, 8, 183-192. 

Horseradish peroxidase is an excellent candidate with which to elucidate enzymatic kinetics in organic solvents. It is an active enzyme with turnover numbers exceeding 320 s 1 in organic media (XI) and hence is susceptible to diffusional limitations which must be overcome. Peroxidase also catalyzes mechanistically identical reactions in aqueous and organic media. Therefore, direct kinetic comparisons between aqueous and organic reactions can be made and the effects of the organic solvent on reactivity and substrate specificity can be directly compared to aqueous-based catalysis. 

One intriguing aspect of enzymatic catalysis in low water environments (this includes 85% dioxane where the water activity is substantially less than unity) is the effect of reaction pH. In aqueous solutions, the ionogenic functional groups of an enzyme respond to the pH of the solution. Definitive pH optima exist for all enzymes. In organic solvents, however, the lack of water as a bulk solvent makes pH an unmeasurable variable although there will be a small concentration of protons in the vicinity of... 

The different catalytic responses of peroxidase in dioxane and methanol versus acetone are intriguing. It is clear that the effects of water-miscible solvents on enzymatic catalysis are not equivalent and for the first time quantitative kinetic data have been obtained which highlight this. However, the cause of this effect remains unresolved. We are continuing and expanding this kinetic study to include other solvents, both water-miscible and immiscible, and other phenols. This future study will enable rational and quantitative approaches for peroxidase-catalyzed phenolic polymerizations to be based on optimal solvent and phenol choices. From a more fundamental standpoint, this work has shown that enzymes may be more active in organic media than in water as long as optimal conditions are employed. There is no reason to believe peroxidase is unique in this respect. 

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