Activating Enzymes for Use in Organic Solvents

Potential Advantages of Employing Enzymes in Organic Media [1] 

Thermodynamic equilibria favor synthesis over hydrolysis 

Suppression of water-dependent side reactions (e.g., hydrolysis of acid anhydrides and halides, polymerization of quinones) 

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

Ease of product recovery from low-boiling, high-vapor-pressure solvents 

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

Activating Enzymes for Use in Organic Solvents Table 3.1 Methods for enzyme activation for use in non-aqueous media. 

Crown ethers are heterocyclic chemical compounds that, in their simplest form, are cyclic oligomers of ethylene oxide. The essential repeating unit of any simple crown ether is ethyleneoxy, i.e., —CH2CH20—, which repeats twice in dioxane and six times in 18-crown-6. Crown ethers can activate enzymes for use in organic solvents through two methods (a) direct addition of 18-crown-6 to the reaction solvent [93], or (b) co-lyophilization of the enzyme with 18-crown-6, the latter being the most effective [94, 95]. 

Eppler et al. [103] viewed these results as having a potential relationship to salt-activated enzyme preparations, particularly in relation to the mobility of enzyme-bound water. Specifically, the authors examined both water mobility [as measured by T2-derived correlation times, (tc)D20] and NaF-activated enzyme activity and observed a linear relationship. This suggests that the salt-activated enzymes contain a more mobile water population than salt-free enzymes, which facilitates a more aqueous-like local environment and dramatically increases enzyme activity through increased flexibility. Therefore, enzyme activation appears to correlate with the properties of enzyme-associated water. Once again, the physicochemical properties of water dictate enzyme structure, function, and dynamics. Hence, salt activation has proven to be a useful technique in activating enzymes for use in organic solvents and has provided a quantitative tool to better understand the role of water in enzymatic catalysis in dehydrated media. 

The simplest way to prepare a biocatalyst for use in organic solvents and, at the same time, to adjust key parameters, such as pH, is its lyophilization or precipitation from aqueous solutions. These preparations, however, can undergo substrate diffusion limitations or prevent enzyme-substrate interaction because of protein-protein stacking. Enzyme lyophilization in the presence of lyoprotectants (polyethylene glycol, various sugars), ligands, and salts have often yielded preparations that are markedly more active than those obtained in the absence of additives [19]. Besides that, the addition of these ligands can also affect enzyme selectivity as follows. 

Purified MeHNL was crystallized by the sitting-drop vapor-diffusion method. The 10-20 mm bipyramidal crystals formed were cross-linked with glutaraldehyde and used as biocatalyst for the synthesis of optically active cyanohydrins. The cross-linked crystals were more stable than Celite-immobilized enzymes when incubated in organic solvents, especially in polar solvents. After six consecutive batch reactions in dibutyl ether, the remaining activity of the cross-linked crystals was more than 70 times higher than for the immobilized enzymes. Nevertheless, the specific activity of the cross-linked crystals per milligram protein was reduced compared with the activity of Celite-immobilized enzymes [53],... 

It was reported that PEGylated lipase entrapped in PVA cryogel could be conveniently used in organic solvent biocatalysis [279], This method for enzyme immobilization is more convenient in comparison to other types of immobilization that take advantage of enzyme covalent linkage to insoluble matrix, since the chemical step which is time consuming and harmful to enzyme activity is avoided. The application of this catalytic system to the hydrolysis of acetoxycoumarins demonstrated the feasibility of proposed method in the hydrolysis products of pharmaceutical interest and to obtain regioselective enrichment of one of the two monodeacetylated derivatives. 

Figure 13. Preparation of immobilized enzymes with different solubilities in aqueous solutions and organic solvents. Procedure A mixture of an enzyme (3 mg) and the polymer (10 mg) was incubated at pH 7.5 for 20 min. Ammonium phosphate (0.1 M, pH 7, 1 mL) was then added to react with the remaining active ester. After 20 min, the solution was ready for use, or lyophilization to give the immobilized enzyme as a powder to be used for reaction in organic solvents. Each gram of the polymer contains approximately 0.7 mmol of the active ester.

It is worth noting that the enzyme can be withdrawn and recycled by using supercritical CO2. The success of the polymerizations carried out in organic solvents stems directly from the sustained activity of several lipases in organic solvents. In this respect, it must be noted that water has a manifold influence on the course of the polymerization. On the one hand, water can initiate the polymerization. On the other hand, a minimum amount of water has to be bound to the surface of the enzyme to maintain its conformational flexibility, which is essential for its catalytic activity [94]. Lipase-mediated polymerization cannot therefore be achieved in strictly anhydrous conditions. 

Lipase has been used in organic solvents to produce useful compounds. For example, Zark and Klibanov (8) reported wide applications of enzymes to esterification in preparing optically active alcohols and acids. Inada et al (9) synthesized polyethylene glycol-modified lipase, which was soluble in organic solvent and active for ester formation. These data reveal that lipases are very useful enzymes for the catalysis different types of reactions with rather wide substrate specificities. In this study, it was found that moditied lipase could also synthesize esters and various lipids in organic solvents. Chemically moditied lipases can help to solve today s problems in esteritication and hopefully make broader use of enzymatic reactions that are attractive to the industry. 

It can be noted that the way in which the enzyme is prepared in the dry form for catalysis in organic solvent is responsible for striking differences (up to two orders of magnitude) in the enzyme-specific activity. Furthermore, it is worth mentioning that the transesterification activity of lipase from B. cepacia entrapped in sol gel (sol gel-AK-lipase BC) was 83% of the activity in water measured using tributyrin as a substrate [6]. Analogously, in the case of CALB lyophilized with methoxypoly(ethylene glycol) (CALB -i- PEG) the activity was 51% of the activity in water in the hydrolysis of vinyl acetate [7]. It is important to note that, for both... 

Crude Solid. The simplest way to use enzymes in organic solvents is to suspend a precipitate or a lyophilisate. The enzyme does not need to be of high purity, but some care should be taken during the preparation. In aqueous solution, the enzyme has an optimal pH, dictated by the ionization state under which the amino acids involved in the catalysis must be to allow activity. The solid enzyme must be in the same ionization state when used in organic solvents (15). For this purpose, it is important to precipitate the enzyme or lyophilize it from a solution buffered at this pH. This applies to the other forms of solid enzyme preparations. The other important point is the drying of the preparation. It has been observed that the secondary structure of proteins can be affected by lyophilization (16). This can be avoided by the use of lyoprotectants such as sorbitol (17) or salts such as KCl (18). 

The specificity of enzyme reactions can be altered by varying the solvent system. For example, the addition of water-miscible organic co-solvents may improve the selectivity of hydrolase enzymes. Medium engineering is also important for synthetic reactions performed in pure organic solvents. In such cases, the selectivity of the reaction may depend on the organic solvent used. In non-aqueous solvents, hydrolytic enzymes catalyse the reverse reaction, ie the synthesis of esters and amides. The problem here is the low activity (catalytic power) of many hydrolases in organic solvents, and the unpredictable effects of the amount of water and type of solvent on the rate and selectivity. 

Aqueous solutions are not suitable solvents for esterifications and transesterifications, and these reactions are carried out in organic solvents of low polarity [9-12]. However, enzymes are surrounded by a hydration shell or bound water that is required for the retention of structure and catalytic activity [13]. Polar hydrophilic solvents such as DMF, DMSO, acetone, and alcohols (log P<0, where P is the partition coefficient between octanol and water) are incompatible and lead to rapid denaturation. Common solvents for esterifications and transesterifications include alkanes (hexane/log P=3.5), aromatics (toluene/2.5, benzene/2), haloalkanes (CHCI3/2, CH2CI2/I.4), and ethers (diisopropyl ether/1.9, terf-butylmethyl ether/ 0.94, diethyl ether/0.85). Exceptionally stable enzymes such as Candida antarctica lipase B (CAL-B) have been used in more polar solvents (tetrahydrofuran/0.49, acetonitrile/—0.33). Room-temperature ionic liquids [14—17] and supercritical fluids [18] are also good media for a wide range of biotransformations. 

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