Migration of reactive trace compounds from Novozym ® 435 into organic solvents and ionic liquids

Abstract

The commercial form of immobilized Candida antarctica lipase B (CALB), known as Novozym ® 435, is a catalyst routinely used in enzymatic reactions. However, we observed a number of compounds migrated from this enzyme preparation into organic solvents and ionic liquids (ILs). These compounds were further identified by GC–MS analysis as a mixture of 17 substances, including 5 major components: glycerol, benzoic acid, 2-hydroxyethyl benzoate, 2-hydroxyethyl sorbate, and sorbic acid. The importance of this discovery is that all five major compounds are reactive in the presence of CALB, especially the last four compounds are potential acyl donors in enzymatic (trans)esterification reactions. We then quantified the migration of these acyl donors into various aqueous solutions, organic solvents and ILs, and observed that the migration into polar organic solvents and ILs was rapid (10–30 min). We also measured the reactivities of these acyl donors with 1-propanol in Novozym ® 435-catalyzed (trans)esterifications. Our data suggest that the initial reaction rates of sorbic acid and sorbate ester were very fast; although the initial rates of benzoic acid and benzoate were much slower, their conversions into propyl benzoate were significant within 24 h period. The presence of these compounds in Novozym ® 435 may not have considerable impact on fast reactions involving high substrate concentrations, however, it is important to realize that these trace compounds may affect the enzyme activity, and may cause confusion during the analysis of enzymatic reactions.