ABSTRACT
CALB, Candida antarctica lipase B; DCM, dichloromethane; DEE, diethoxyethane; DME, dimethoxyethane; DMF, dimethylforamide; DMSO, dimethyl sulphoxide; EEE, diethylene glycol diethyl ether; EG, ethylene glycol; MEA, 2-methoxyethyl acetate; PEG, polyethylene glycol; TCM, tetrachloromethane; THF, tetrahydrofuran; TMP, trimethyl phosphate. 16.2.1 Nearly Anhydrous Organic Solvent SystemsThe ability of enzymes to work in neat organic solvents was for long time taken with scepticism due to the assumption that enzymes are denatured in organic solvents. This prejudice, however, came from studying enzymes in mixtures of water and organic solvents, not in neat organic solvents containing less than 5% (v/v) of water (Griebenow and Klibanov, 1996). In contrary to aqueous-organic mixtures, enzymes are very rigid in the absence of water. As a consequence of protein rigidity, enzymes are much more stable in organic solvents than in water. The extreme thermostability of enzyme in 99% (v/v) organic medium was reported for the first time by Zaks and Klibanov (1984). The porcine pancreatic lipase was not only able to withstand heating at 100°C for many hours, but also exhibited a high catalytic activity at that temperature.The organic solvent systems containing little water represent the most widely used non-conventional media for enzymatic reactions. Among the reactions reported in nearly anhydrous organic solvent systems prevail those catalysed by hydrolases, particularly lipases and proteases. Hydrolases are primarily used for resolution processes, where one enantiomer of a racemic mixture is selectively modified to yield a separable derivative. In water, these enzymes catalyse the hydrolysis of esters to the corresponding alcohols and
acids, which obviously cannot occur in nearly anhydrous media. Addition of nucleophiles, such as alcohols, amines and thiols, leads to transesterification, aminolysis and thiotransesterification, respectively. Moreover, a reverse hydrolysis-the synthesis of esters from acids and alcohols-becomes thermodynamically favourable (Zaks and Klibanov, 1985). Several companies are currently using lipase-catalysed reactions in organic solvents for the production of useful intermediates (Table 16.3). For instance, BASF offers a broad range of enzymatically synthesized alcohols for manufacture of enantiopure drugs (Schmid et al., 2001). Table 16.3 Examples of processes involving lipase-catalysed reactions in organic media developed by several chemical and pharmaceu-tical companies Company Process Ref.BASF Synthesis of various enantiomerically pure alcohols, used as intermediates for synthesis of chemicals and pharmaceuticals, by
Despite high stability, enzymes generally exhibit lower activities in neat organic solvents than in aqueous reaction systems. The loss of biocatalytic activity has been ascribed to different reasons, including non-optimal hydration of the biocatalyst, restricted protein flexibility, suboptimal pH, diffusional limitations, unfavourable substrate desolvation, low stabilization of the enzyme-substrate intermediate and changes in the enzyme active site (Carrea and Riva, 2000; Klibanov, 1997; Toth et al., 2010).The main factor that has to be taken into account when performing biocatalysis in nearly anhydrous organic media is water activity. Even in neat organic solvent media, at least a few water molecules are required to remain bound to the enzyme. It became apparent, that fully dehydrated proteins are inactive. For instance, α-chymotrypsin and subtilisin need about 50 molecules of water per enzyme molecule to be catalytically active (Zaks and Klibanov, 1986). The more hydrophilic the solvent is, the more water has to be added to reach high activity, because hydrophilic solvents have a greater tendency to strip the essential water from the enzyme molecule (Klibanov, 2001). Water, acting as lubricant, allows enzymes to exhibit the conformational mobility required for optimal catalysis. In contrast, organic solvents lack water’s ability to create hydrogen bonds, and also have lower dielectric constants, leading to stronger intra-protein electrostatic interactions. The exception are hydrophilic solvents, such as glycerol, ethylene glycol or formamide, that are capable of forming multiple hydrogen bonds with enzyme molecules, thus partially mimic the water effects (Torres and Castro, 2004; Almarsson and Klibanov, 1996). Addition of small quantities of water or water-mimicking solvent to enzyme in anhydrous solvent can increase the enzyme activity by several
orders of magnitude. Thus, it is very important to control the amount of water in the reaction mixture and keep this parameter close to the optimal value. Another parameter that affects enzyme activity is pH. In many cases, an enzyme in neat organic solvents keeps the ionization state from the aqueous solution to which it was exposed before removal of water, (Xu and Klibanov, 1996; Klibanov, 1997; Zaks and Klibanov, 1985). This phenomenon is called pH memory. The enzymatic
activity can be therefore significantly enhanced if enzymes are lyophilized from solutions of the pH optimal for the catalysis. On the other hand, if the enzymatic reactions involve the formation or consumption of acidic or basic substances, the pH buffering capacity is needed. Triphenylacetic acid and its sodium salts are typical examples of pairs controlling pH in relatively polar solvents, while dendritic polybenzyl ether derivatives have been developed as the alternatives for more hydrophobic media (Dolman et al., 1997; Xu and Klibanov, 1996).Since enzymes are practically insoluble in most organic solvents, they are usually introduced into neat organic solvents as powders prepared by lyophilisation (Carrea and Riva, 2000; Torres and Castro, 2004). The protein denaturation, which may occur in the process of dehydration, is normally reversible upon rehydration in aqueous media. However, refolding in anhydrous organic solvents is not trivial due to the reduced structural mobility (Mattos and Ringe, 2001; Griebenow and Klibanov, 1995).
