Enzymes in nonaqueous media

Perhaps the biggest impact on the practical utilization of enzymes has been the development of nonaqueous enzymology (11,16,33,35). The use of enzymes in nonaqueous media gready expands the scope of suitable transformations, simplifies thek use, and enhances stabiUty. It also provides an easy means of regulation of the substrate specificity and regio- and enantioselectivity of enzymes by changing the reaction medium. 

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. 

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. 

Examples of Reactions Catalyzed by Enzymes in Nonaqueous Media... 

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 ... 

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

However, the above does not answer the main question how can one employ isolated enzymes for the preparation of surfactants In fact, the answer is simple Use hydrolytic enzymes in nonaqueous media. Indeed, many hydrolytic enzymes, such as lipases, proteases, and glycosidases, available in large quantities, are very robust and inexpensive, and do not require any cofactors to manifest their catalytic activity. As any other catalyst, enzymes cannot influence the equilibrium of a chemical reaction and therefore the removal of water from the reaction medium forces them to work in reverse, i.e., to synthesize a chemical bond rather than to break it. Consequently, there is a principal difference between microbial and enzymatic synthesis of surfactants regarding the type of enzymes involved and the reaction medium. The former is a biosynthetic process catalyzed by living microorganisms and as such dependent solely on their viability, whereas the latter is an organic synthesis whereby enzymes are used as substitutes for chemical catalysts. The two approaches are complementary not only in terms of the production methods but because the surfactant structures amenable to both methodologies are quite different. 

In the last few years the use of enzymes in nonaqueous media, especially in SFCs has received much attention. As described in Sec. II, SCFs offer some advantages of organic solvents, such as dissolving properties for hydrophobic compounds, ease of enzyme recovery, separation of products and unreacted substrates, and the possibility of performing reactions that are thermodynamically unfavorable in water. One of the best advantages of SCFs is their excellent transport properties and adjustable solvent power, due to their low viscosity and high diffusivity. 

Other immobilization methods are based on chemical and physical binding to soHd supports, eg, polysaccharides, polymers, glass, and other chemically and physically stable materials, which are usually modified with functional groups such as amine, carboxy, epoxy, phenyl, or alkane to enable covalent coupling to amino acid side chains on the enzyme surface. These supports may be macroporous, with pore diameters in the range 30—300 nm, to facihtate accommodation of enzyme within a support particle. Ionic and nonionic adsorption to macroporous supports is a gentle, simple, and often efficient method. Use of powdered enzyme, or enzyme precipitated on inert supports, may be adequate for use in nonaqueous media. Entrapment in polysaccharide/polymer gels is used for both cells and isolated enzymes. 

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... 

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... 

Ease of immobilization, e.g., via simple adsorption onto nonporous surfaces enzymes cannot desorb from these surfaces in nonaqueous media... 

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. 

Peptide condensation -in nonaqueous media [ENZYMES IN ORGANIC SYNTHESIS] (Vol 9)... 

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

Popp JL, Kirk TK, Dordick JS (1991) Incorporation of p-cresol into lignins via peroxidase-catalysed copolymerization in nonaqueous media. Enzyme Microb Technol 13 964—968... 

---