On the importance of the support material for enzymatic synthesis in organic media

Abstract

Enzymes were deposited on different porous support materials and these preparations were used to catalyze reactions in organic media. Reactions were carried out at specific water activities, achieved by equilibrating both the enzyme preparation and the substrate solution at the desired water activity before mixing them and thereby starting the reactions. The reaction rates obtained at the same water activity with different supports differed greatly, indicating a direct influence of the support on the enzyme. For horse liver alcohol dehydrogenase, Celite was the best support, and the reaction rate increased with increasing water activity. In the a-chymotrypsincatalyzed alcoholysis of N-acetyl-L-phenylalanine ethyl ester with 1-butanol, high rates were again obtained with Celite, but with this support only about one third of the ethyl ester was converted to butyl ester, the rest was hydrolyzed. With the polyamide support, Accurel PA6, alcoholysis was the dominating reaction, and by using a low water activity (0.33), hydrolysis was completely suppressed while still maintaining a high alcoholysis activity. Controlled pore glass (CPG), derivatized with either hexyl or glucosyl groups, had quite different properties as enzyme supports. For horse liver alcohol dehydrogenase, glucose-CPG was a much better support than hexyl-CPG, and in the a-chymotrypsin-catalyzed reactions, glucose-CPG favored hydrolysis, and hexyl-CPG alcoholysis, at water activities exceeding 0.8. The results are discussed considering the absorption of water on the enzymes, on the supports and the solubility of water in the reaction media; all these parameters were measured separately.The use of enzymes as catalysts in organic media is a new and promising field in biotechnology. Organic reaction media are favorable when hydrophobic compounds are to be converted and open up possibilities for reversal of hydrolytic reactions. Many promising results have been obtained for different synthetic applications, e. g. for interesterification of triglycerides, synthesis of peptides and resolution of racemic compounds [l, 21. However, in order to be able to choose reaction conditions for new conversions in a rational way, there is a need for basic studies concerning the mechanisms that govern the catalytic properties of enzymes in organic media.There are several methods for using enzymes in organic solvents. One practical approach, which is used in the present study, is to deposit the enzyme on a porous support material before adding it to the solvent. This type of preparation is easily removed after the conversion, which facilitates product isolation and enzyme re-use. Other approaches include the use of detergents to solubilize enzymes in reversed micelles [3] and the covalent modification of enzymes with, for example, poly(ethy1ene glycol) to make them soluble in organic media Enzymes. r-Chymotrypsin (EC 3.4.23 .I); horse liver alcohol dehydrogenase (EC 1.1.1.1).[4]. Although the water content in all these systems is low, water plays a crucial role in modulat...

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