Enzyme Activation in Nonaqueous Media

Indeed, this is the basis behind many of the methods used to activate enzymes for use in organic solvents, as we will describe below. 

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L. Dai and A. M. Klibanov, Striking activation of oxidative enzymes suspended in nonaqueous media, Proc. Natl. Acad. Sd. 1999, USA 96, 9475-9478. 

Dai L and Klibanov AM. Striking Activation of Oxidase Enzymes Suspended in Nonaqueous Media. Proceedings-National Academy of Sciences USA Physical Sciences 1999 96 9475-9478. 

Akbar U, Aschenbrenner CD, Harper MR et al. (2007) Direct solubilization of enzyme aggregates with enhanced activity in nonaqueous media. Biotechnol Bioeng 96(6) 1030-1039 Albetghina L (2000) Protein engineering in industrial biotechnology. CRC Press, Boca Raton 376 pp. ISBN 9057024128... 

Lee CM, Bagdasarian M, Zeikus JG (1990) Characterization of Thermoanaerobacter glucose iso-merase in relation to saccharidase synthesis and development of single-step process for sweetener production. Appl Environ Microbiol 56 2895-2901 Lee MY, Dordick JS (2002) Enzyme activation for nonaqueous media. Curr Opin Biotechnol... 

The transformations described thus far were catalyzed by enzymes in their traditional hydrolytic mode. More recent developments in the area of enzymatic catalysis in nonaqueous media (11,16,33—35) have significantly broadened the repertoire of hydrolytic enzymes. The acyl—enzyme intermediate formed in the first step of the reaction via acylation of the enzyme s active site nucleophile can be deacylated in the absence of water by a number of... 

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. 

Despite these advantages, native enzymes almost universally exhibit very low activities in organic solvents-often 4-5 orders of magnitude lower than in aqueous solutions. This loss in catalytic activity may be attributed to several factors, including a decrease in the polarity of the enzyme s microenvironment, the loss of critical water residues from the enzyme s surface, the decreased conformational mobility of the enzyme s structure, ground-state stabilization of hydrophobic substrates, and deactivation during the preparation of the biocatalyst for use in nonaqueous media,... 

A particularly interesting, and extremely simple, method of activating enzymes for use in nonaqueous media is lyophilization in the presence of nonbuffer salts. In the process, a great deal about excipient-enzyme-water interactions has been learned along with an appreciation of how enzymes adjust to novel microenvironments while retaining their intrinsic catalytic properties. 

Yang L, Dordick JS, and Garde S. Hydration of Enzyme in Nonaqueous Media is Consistent with Solvent Dependence of its Activity. BiophysJ2004 87 812— 821. 

Immobilization, dehned as the physical confinement or localization of an enzyme into a specihc micro-environment, has been a very common approach to prepare enzymes for aqueous as well as nonaqueous applications. For nonaqueous enzymol-ogy, immobilization improves storage and thermal stability, facilitates enzyme recovery, and enhances enzyme dispersion. In addition, immobilized enzymes are readily incorporated in packed bed bioreactors, allowing for continuous operation of reactions. Moreover, lyophilized enzyme powders often aggregate and attach to reactor walls, particularly when the water activity is moderately high. The major disadvantage of immobilization is low activity, induced by pore diffusion mass transfer limitations and by alteration of protein stmcture. For enzymes in nonaqueous media, the following broad categories of immobilization exist ... 

Triantafyllou, A. O., Wehtje, E. Adlercreutz, P., and Mattiasson, B., How do additives affect enzyme activity and stability in nonaqueous media , Biotechnol. Bioeng., 54, 67-76, 1997. 

Almarsson, O. and Klibanov, A. M., Remarkable activation of enzymes in nonaqueous media by denaturing organic cosolvents, Biotechnol. Bioeng., 49, 87-92, 1996. 

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