Biocatalysis water

In order to broaden the field of biocatalysis in ionic liquids, other enzyme classes have also been screened. Of special interest are oxidoreductases for the enan-tioselective reduction of prochiral ketones [40]. Formate dehydrogenase from Candida boidinii was found to be stable and active in mixtures of [MMIM][MeS04] with buffer (Entry 12) [41]. So far, however, we have not been able to find an alcohol dehydrogenase that is active in the presence of ionic liquids in order to make use of another advantage of ionic liquids that they increase the solubility of hydrophobic compounds in aqueous systems. On addition of 40 % v/v of [MMIM][MeS04] to water, for example, the solubility of acetophenone is increased from 20 mmol to 200 mmol L ... 

To carry out the enzymatic amidation of carboxylic acids, normally two strategies are considered the use of ionic liquids or the removal of water from the reaction media at high temperature or reduced pressure. For instance, one of the first examples of the use of ionic liquids in biocatalysis has been the preparation of octanamide from octanoic acid as starting material and ammonia in the presence of CALB (Scheme 7.3) [11]. 

The role of biocatalysis in two-phase systems has many parallels with the subject we have covered under extractive reactions. It appears that a two-phase system was originally considered for transformations of water insoluble substances like steroids. Now, a series of treatises are available which teach us that the maximum value of the apparent equilibrium constant for a second-order reaction in a two-phase system can exceed the equilibrium... 

However, the transfer of this technology from laboratory to industrial scale requires advances in the engineering of biocatalysis environment, particularly when one or more components are poorly water soluble [5-8]. 

The present review deals with the same topic as the articles cited above, but modified with different parameters influencing biocatalysis and reactant partition in water-organic two-liquid phase bioreactors. Interactions between these phenomena are also discussed. 

The protein-containing colloidal solutions of water-in-organic solvents are optically transparent. Hence, absorption spectroscopy, circular dichroism spectroscopy and fluorescence spectroscopy are found to be convenient for studying biocatalysis [53]. The reversed micelles are interesting models for studying bioconversion, since the majority of the enzymes in vivo act inside or on the surface of biological membranes. 

Biocatalysis localization in the biphasic medium depends on physicochemical properties of the reactants. When all the chemical species involved in the reaction are hydro-phobic, catalysis occurs at the liquid-liquid interface. However, when the substrate is hydrophobic (initially dissolved in the apolar phase) and the product is hydrophilic (remains in the aqueous phase), the reaction occurs in the aqueous phase [25]. The majority of biphasic systems use sparingly water-soluble substrates and yield hydrophobic products therefore, the aqueous phase serves as a biocatalyst container [34,35] [Fig. 2(a)]. Nevertheless, in some systems, one of the reactants (substrate or product) can be soluble in the aqueous phase [23,36-38] (Fig. 2(b), (c)). 

A common characteristic of metabolic pathways is that the product of one enzyme in sequence is the substrate for the next enzyme and so forth. In vivo, biocatalysis takes place in compartmentalized cellular structure as highly organized particle and membrane systems. This allows control of enzyme-catalyzed reactions. Several multienzyme systems have been studied by many researchers. They consist essentially of membrane- [104] and matrix- [105,106] bound enzymes or coupled enzymes in low water media [107]. 

Another environmental issue is the use of organic solvents. The use of chlorinated hydrocarbons, for example, has been severely curtailed. In fact, so many of the solvents favored by organic chemists are now on the black list that the whole question of solvents requires rethinking. The best solvent is no solvent, and if a solvent (diluent) is needed, then water has a lot to recommend it. This provides a golden opportunity for biocatalysis, since the replacement of classic chemical methods in organic solvents by enzymatic procedures in water at ambient temperature and pressure can provide substantial environmental and economic benefits. Similarly, there is a marked trend toward the application of organometal-lic catalysis in aqueous biphasic systems and other nonconventional media, such as fluorous biphasic, supercritical carbon dioxide and ionic liquids. ... 

Biocatalysis has traditionally been performed in aqueous environments, but this is of limited value for the vast majority of nonpolar reactants used in chemical synthesis. For a long time it was assumed that all organic solvents act as denaturants, primarily based on the flawed extrapolation of data obtained from the exposure of aqueous solutions of enzyme to a few water-miscible solvents, such as alcohols and acetone, to that of all organic sol vents. 

A major cause of suboptimal activity in organic solvent results from the removal of essential water during enzyme dehydration. All enzymes require some water in order to retain activity through the provision of conformational flexibihty. Particularly in the case of lipases, the amount of water can be so low that it appears that none is required. For example, following the development of suitable techniques to analyse low water concentrations, it has been reported that the lipase from Rhizomucor miehei retains 30 % of its optimum activity with as little as two or three water molecules per molecule of enzyme.Owing to the apparent absence of water in some exceptional cases, the term biocatalysis in anhydrous solvent is commonly used, although in the vast majority of cases a monolayer of water is required for optimal activity (although this is often stUl well below its solubility limit in water-immiscible solvent). ... 

Because enzymes are insoluble in organic solvent, mass-transfer limitations apply as with any heterogeneous catalyst. Water-soluble enzymes (which represent the majority of enzymes currently used in biocatalysis) have hydrophilic surfaces and so tend to form aggregates or stick to reaction vessel walls rather than form the fine dispersions that are required for optimum efficiency. This can be overcome by enzyme immobilization, as discussed in Section 1.5. 

An ionic liquid can be used as a pure solvent or as a co-solvent. An enzyme-ionic liquid system can be operated in a single phase or in multiple phases. Although most research has focused on enzymatic catalysis in ionic liquids, application to whole cell systems has also been reported (272). Besides searches for an alternative non-volatile and polar media with reduced water and orgamc solvents for biocatalysis, significant attention has been paid to the dispersion of enzymes and microorganisms in ionic liquids so that repeated use of the expensive biocatalysts can be realized. Another incentive for biocatalysis in ionic liquid media is to take advantage of the tunability of the solvent properties of the ionic liquids to achieve improved catalytic performance. Because biocatalysts are applied predominantly at lower temperatures (occasionally exceeding 100°C), thermal stability limitations of ionic liquids are typically not a concern. Instead, the solvent properties are most critical to the performance of biocatalysts. 

Promising developments of ionic liquids for biocatalysis reflect their enhanced thermal and operational stabilities, sometimes combined with high regio- or enantioselectivities. Ionic liquids are particularly attractive media for certain biotransformations of highly polar substrates, which cannot be performed in water owing to equilibrium limitations 297). 

The scope and limitations of biocatalysis in non-conventional media are described. First, different kinds of non-conventional reaction media, such as organic solvents, supercritical fluids, gaseous media and solvent-free systems, are treated. Second, enzyme preparations suitable for use in these media are described. In several cases the enzyme is present as a solid phase but there are methods to solubilise enzymes in non-conventional media, as well. Third, important reaction parameters for biocatalysis in non-conventional media are discussed. The water content is of large importance in all non-conventional systems. The effects of the reaction medinm on enzyme activity, stabihty and on reaction yield are described. Finally, a few applications are briefly presented. 

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. 

Semenov, A N., Khmelnitsky, Y.L., Berezin, I.V. and Martinek, K. (1987) Water-organic solvent systems as media for biocatalytic reactions the potential for shifting chemical equilibria towards higher yield of end products. Biocatalysis, 1, 3-8. 

Wehtje, E., Svensson, I., Adlercreutz, P. and Mattiasson, B. (1993) Continuous control of water activity during biocatalysis in organic media. Biotechnol. Techniques, 7, 873-878. 

During biocatalysis in organic media, the small amounts of water present are associated with various components of the system dissolved in the solvent, bound... 

Table 1.4 Solvent descriptors of organic solvents commonly used for biocatalysis. Sw/o (solubility of water in solvent, wt%) So/w (solubility of solvent in water, wt%) and e (dielectric constant) values from [78], log P (P = partition coefficient between octanol and water), ET (empirical polarity parameter by Reichardt-Dimroth) and HS (Hildebrand solubility parameter, )l, cm J, ) from [79].

Natural polyols have been used as substrates for the so-called combinatorial biocatalysis , a proposed approach to drug discovery [33]. For instance, complementary enzymatic regioselectivity was applied to produce a combinatorial library of 167 distinct selectively acylated derivatives of the flavonoid bergenin (11) on a robotic workstation [34]. Another lead compound, the antitumoral paclitaxel (12, a molecule with very low water solubility) has been similarly derivatized, initially exploiting the selectivity of the protease thermolysin for its side-chain C-2 OH. 

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