THE INFLUENCE OF THE MODE OF ENZYME PREPARATION ON ENZYMATIC ENANTIOSELECTIVITY IN ORGANIC-SOLVENTS AND ITS TEMPERATURE-DEPENDENCE

The enantioselectivities of subtilisin Carlsberg and Rhizo-mucor miehe i lipase in organic solvents are found to strongly depend on the metho d by which the enzymes are prepared. For the transesterification betwe en sec-phenethyl alcohol and vinyl butyrate in dioxane at 7 degrees C, the enantioselectivity of subtilisin precipitated with isopropanol is more than twice that of the enzyme prepared by lyophilization from aq ueous buffer. Furthermore, the temperature dependence of the enantiose lectivity is influenced by the mode of enzyme preparation. For example , in the aforementioned process the enantioselectivities of subtilisin lyophilized from aqueous buffer and crosslinked subtilisin crystals i ncrease when the temperature is raised from 7 to 45 degrees C. In cont rast, the enantioselectivities decrease with temperature for the enzym e precipitated from aqueous solution with acetone or isopropanol and f or the enzymatic hydrolysis in water. The temperature dependence of th e enantioselectivity of subtilisin lyophilized from buffer is markedly affected by the solvent: In acetonitrile and nitromethane the enzyme is more enantioselective at higher temperatures, while negligible temp erature effects have been found in tetrahydrofuran and pyridine. Lyoph ilized lipase exhibits striking temperature dependencies of its enanti oselectivity in dioxane, acetonitrile, and nitromethane, while showing almost none in pyridine, triethylamine, and tetrahydrofuran. The resu lts underscore the importance of the mode of enzyme recovery on enanti oselectivity and its temperature dependence in enzymatic reactions in organic solvents (in contrast to those in water). (C) 1996 John Wiley & Sons, Inc.