Tsuneo Yamane 1 Introduction

Although it has not been explicitly recognized in the literature, biotransformations in organic media can be classified as either solvent-based or solvent-free reactions. In the former systems, one or more substrates are dissolved in an inert organic solvent that does not participate in the reaction in any respect, but to provide an environment for the enzyme to exert its action on the dissolved substrate(s). In a solvent-free system, no other compounds but substrate(s) and enzyme are present in a reactor. In principle, one substrate can be used in a large excess over another and, if so, it may also act as a solvent for other reactants. Examples of such "neat" biotransformations can be found even in the early literature (1-3).

One of the big attractions of enzymatic solvent-free synthesis is potentially very high volumetric productivity. This, however, does not apply to all reactions, and in many instances, it may actually take longer to achieve the desired degree of substrate conversion in the absence of added solvent. In this case, volumetric productivity in the reactor [(mass of product formed)(reactor volume)-1 h-1] should be calculated for both solvent-based and solvent-free systems using the same volume of the reaction mixture and the same amount of the enzyme in order to make an economically justified choice between the two. Similarly, there is no risk of solvent-induced inactivation of the biocatalyst in a solvent-free system, but the overall loss of enzyme activity can still be significant if the reaction time is too long. It should be added that the avoidance of organic solvents is particularly advantageous to the food industry where stringent regulations related to the use of organic solvents are in force. Also, no fireproof and explosion-proof equipment/procedures are necessary for the solvent-free processing and the environment at the factory is less hazardous to the health of the workers.

From: Methods in Biotechnology, Vol. 15: Enzymes in Nonaqueous Solvents: Methods and Protocols Edited by: E. N. Vulfson, P. J. Halling, and H. L. Holland © Humana Press Inc., Totowa, NJ

As enzymatic reactions in organic solvents, solvent-free systems can be classified in several groups depending on particular features of the biotransformation in question (Table 1). Thus, the reaction can be carried out with either free or immobilized enzymes with one or more substrates that are either used in nearly stoichiometric quantities or one is present in large excess. Further, solvent-free biotransformations can be implemented in a monophasic reaction mixture as well as in biphasic liquid-liquid and even biphasic liquid-solid systems. The latter, "solid-phase glycerolysis of fats," was extensively studied in the author's laboratory for many years (4-9).

Currently, a number of biotransformations of low- to mid-value symmetrical fats and oils (e.g., 1,3-distearoyl-2-oleoyl-glycerol, 1,3-dioleoyl-2-palmitoyl-glycerol and 1,3-dibehenoyl-2-oleoyl-glycerol) are being produced in solvent-free systems. Similar solvent-free transesterifications are used for the manufacture of low-volume high-value food additives and pharmaceuticals, including simple alkyl and terpenyl esters such as ethyl isovalerate, heptyl oleate, geranyl acetate, and citronellyl acetate (flavors and fragrances), and glycerides enriched in polyunsaturated fatty acids (PUFAs) for biomedical applications (10,11). Esters of C2-C8 alcohols (e.g., isopropyl myristate, isopropyl palmitate, and 2-ethylhexyl palmitate are currently produced commercially for applications in cosmetics and personal care products [see the review article by Vulfson et al. (12)]. Structured lipids containing mono-, di-, or tri-PUFAs are also expected to be manufactured in a solvent-free enzymatic process (13,14), as triglycerides enriched with ds-5,8,11,14,17-eicosahexaenoic acid (EPA) or ds-4,7,10,13,16,19-docosapentaenoic acid (DHA) have numerous health benefits and are better adsorbed in the intestine (15).

In addition to "conventional" solvent-free systems, where substrates and products are completely miscible liquids at the reaction temperature, two other more sophisticated approaches to increasing mutual miscibility or solubility of substrate(s) in the reaction mixture have been developed (see review by Vulfson et al. [12]). In these systems, one of the substrates is a solid at ambient temperature and is only sparingly soluble in another substrate (e.g., a sugar and fatty acids). To overcome the problem of solubility, a prior "hydrophobization" of the sugar moiety either by 1-0-alkylation (ethyl-, n- and ¿so-propyl or butyl-) or by acetonization (2,3-isopropylidene sugar, sugar acetal) is carried out and the product is esterifiied with molten fatty acids in a solvent-free process (16-20). Alternatively Vulfson and coworkers used molten substrate(s), the crystallization of which under the reaction conditions was deterred for sufficient time to complete the biotransformation.

The enzyme performance in solvent-free substrate mixtures (as well as in conventional solvent-based biotransformation) is crucially dependent on the water content (or thermodynamic water activity, aw) of the medium. Thus, aw

Table 1

Classification of Solvent-Free Biotransformations

Number of phases:

Monophasic (homogeneous, liquid) Biphasic (heterogeneous, liquid) Biphasic (heterogeneous, solid) Number of substrates: One substrate Two substrates:

Extended systems:

Molten substrate/eutectic mixtures Substrates derivatized to increase mutual miscibility Enzyme

Free (suspended enzyme powder) Immobilized (on or within solid support)

in the system must be carefully controlled, as some water is essential for the maintenance of high catalytic activity of the bicatalyst (see 21-23) and other contributions to this volume). However, an excess of water in the system is undesirable, as it leads to decreased yields of product resulting from hydrolytic side reactions and/or an unfavorable position of equilibrium. Thus, in esterification and transesterification reactions, the removal of water and volatile alcohols, respectively, is necessary to shift the equilibrium in favor of the product formation. Strategies to achieve this include application of vacuum, bubbling of dry inert gas, and the use of selective adsorbents such as silica gel, molecular sieves, and so forth. Other methods to increase the product yield in reactions where (side) products are not volatile are available too: fractional crystallization (wintering) of saturated fatty acid liberated from acidolysis (11) and tautomerization of liberated vinyl alcohol when using vinyl esters of carboxylic acids as substrates for transesterifications (24).

In the following sections, lipase-catalyzed solvent-free biotransformations of lipids are exemplified by two reactions. One is transesterification between EPA ethyl ester (EPAEE) and tricaprylin to yield 1(3)-EPA-2,3(1,2)-dicaprylin (25). This is a typical monophasic solvent-free system:

Nearly stoichiometric quantities Excess amount of one substrate

The other is the biphasic solvent-free esterification of glycerol with free fatty acid (FFA) to give to 1,3-diacylglycerol (26):



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