Nonaqueous biocatalysis

In the last decade, biocatalysis in nonaqueous media, using hydrolases, has been widely used for organic chemists. The possibilities that these biocatalysts offer for the preparation of different types of organic compounds, depending upon the nucleophile... 

For some recent reviews on the use of enzymes in nonconventional media, see (a) Dreyer, S., Lembrecht, J., Schumacher, J. and Kragl, U., Enzyme catalysis in nonaqueous media past, present, and future in biocatalysis in the pharmaceutical and biotechnology industries, 2007, CRC Press, pp. 791-827 . (b) Torres, S. and Castro, G.R., Non-aqueous biocatalysis in homogeneous solvent systems. Food Technol. BiotechnoL, 2004, 42, 271-277 (c) Carrea, G. and Riva, S., Properties and synthetic applications of enzymes in organic solvent. Angew. Chem. Int. Ed., 2000, 39, 2226-2254. 

Vofi H,Miethe P (1992) Enzymes entrapped in liquid crystals a novel approach for bio-catalysis in nonaqueous media. In Tramper J, Vermae MH, Beet HH, Stockar UV (eds) Biocatalysis in non-conventional media, progress in biotechnology. Elsevier, London 8 739... 

Cyclodextrins (sometimes called cycloamyloses) make up a family of cyclic oligosaccharides composed of 5 or more a-D-glucopyranoside units linked 1 —> 4, as in amylose (a fragment of starch), and have been used as activating macrocyclic excipients in nonaqueous biocatalysis. Griebenow et al. observed that lyophilization of methyl-[3-cyclodextrin with subtilisin Carlsberg in a 6 1 weight ratio resulted in a 164-fold rate enhancement in THF for the transesterification rate of sec-phenethyl alcohol with vinyl butyrate (Scheme 3.3) [98]. 

Yang, Z. and Pan, W. (2005). Ionic liquids green solvents for nonaqueous biocatalysis. Enzyme Microb. Tech., 37, 19-28. 

One of the greatest hurdles for the application of biocatalysis is the need to operate processes under conditions that can differ dramatically from those in which the enzymes evolved. Many techniques are used in order to preserve catalytic activity and minimize the costs associated with the biocatalyst. In cases where the cost of the biocatalyst is a concern, an enzyme might be immobilized and used in a packed column or a fluidized bed reactor so as to enable reuse. Here also the enzyme must be stable for extended periods and may even be used under nonaqueous conditions and elevated temperatures. Recombinant technology has revolutionized the applications of biocat-... 

Adjustable miscibility with organic compounds (may be hydrophobic or hydrophilic) Unique ability to dissolve polar substrates in a nonaqueous environment (potential for biocatalysis see Section 5.2.2.5) Good solvents for mulfiphasic catalysis May provide a solution to product separation from catalyst/solvent May influence reaction selectivity by selective extraction of reaction intermediates... 

Enzyme immobilisation on mesoporous silica has also proved attractive for biocatalysis applications, particularly in nonaqueous solvents, since immobilisation of enzymes results in increased mechanical stability and potential for catalyst recyclingMany factors, such as relative size of nanopore/enzyme, nanopore volume, surface characteristics of support/enzyme influence enzyme loading and activity Enzymes... 

Scheme 2.6 Combinatorial biocatalysis of glycosidase and lipase in nonaqueous media for constructing a three-dimensional array of glycoconjugates...

Biocatalysis in organic solvents has unique advantages compared to traditional aqueous enzymology/fermentation. Often times in nonaqueous media enzymes exhibit properties drastically different from those displayed in aqueous buffers. These novel properties are given in Table 4.3. In addition to those mentioned in Table 4.3, the solubility of hydrophobic substrates and/or products increases in organic solvents, which diminishes diffusional barriers for bioconversions, and thus speeds up the reactions and improves the potential for direct applications in industrial chemical processes. Once organic solvent becomes a reaction medium, there cannot be contamination, which thus precludes release of proteolytic enzymes by microbes and favors the direct application of the process in an industrial setting. Most proteins (enzymes) inherently function in an aqueous environment, and hence their behavior in nonaqueous solvents is completely different due to the loss in the three-dimensional structure. Thus, only polar solvents... 

Biocatalysis in nonaqueous systems has proven itself as a powerful tool. HIP allows significant improvement in enzyme activity in nonaqueous systems. A combination of directed evolution and rational enzyme design is likely to result in many more exciting developments in the near future. 

Nonaqueous IL microemulsions were also used as catalysts to improve reaction efficiency. Gayet et al. established an IL-in-oil microemulsion system with benzylpyridinium bis(trifluoromethanesulfonyl)imide ([BnPyrJNTfj), TX-lOO, and toluene, in which the Matsuda-Heck reaction between methoxybenzene diazotate and 2,3-dihydrofuran took place [46]. The reaction yield in this IL-in-oil microemulsion was twice as high as that in neat ILs. The results provided a basis for designing a nonaqueous IL microemulsion microreactor and also showed that nonaqueous IL microemulsion might have good prospects of applications in biocatalysis and nanomaterial synthesis. 

Regarding the role of water, it has been well known that a certain amoimt of water is required for biocatalysis in nonaqueous environment, but different enzymes require different amounts of water for their optimal catalytic performance. It is the amoimt of water associated with the enzyme, rather than the total water content in the reaction system, that is the key determinant of the enzyme properties (e.g., activity, stability, and specificity) [32], and thermodynamic water activity (aj has been generally considered as a parameter to quantify the hydration level on the enzyme [33]. 

The use of nonaqueous reaction media also offers a number of attractive options for increasing the productivity of enzymatic syntheses. In particular, it has been conclusively shown that enzyme-catalyzed reactions proceed readily in nearly stoichiometric mixtures of substrates in the absence of bulk solvents, e.g., enzymatic solvent-free processing [7] and biocatalysis in... 

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