Interfacial activation-based molecular (bio)-imprinting (IAMI) has been developed to rationally improve the performance of lipolytic enzymes in nonaqueous environments. The strategy combinedly exploits (i) the known dramatic enhancement of the protein conformational rigidity in a water-restricted milieu and (ii) the reported conformational changes associated with the activation of these enzymes at lipid-water interfaces, which basically involves an increased substrate accessibility to the active site and/or an induction of a more competent catalytic machinery. Six model enzymes have been assayed in several model reactions in nonaqueous media. The results, rationalized in light of the present biochemical and structural knowledge, show that the IAMI approach represents a straightforward, versatile method to generate manageable, activated (kinetically trapped) forms of lipolytic enzymes, providing under optimal conditions nonaqueous rate enhancements of up to two orders of magnitude. It is also shown that imprintability of lipolytic enzymes depends not only on the nature of the enzyme but also on the "quality" of the interface used as the template.Nonaqueous enzymology has emerged in the last decade as a promising research field (for reviews, see, for example, refs. 1-3), not only because of a large number of widely recognized advantages (2) but especially as a result of the realization that enzymes placed in nearly anhydrous environments can exhibit exciting features, such as an increased conformational rigidity, which confers on the protein greatly enhanced (thermo)stability (4), or a profoundly altered substrate specificity (5). In particular, one interesting facet of the exploitation of the drastically lowered flexibility of a protein in very low-water media is the so-called ligand-induced enzyme "memory" (or simply ligand memory) (6), presumably based on inducing, upon ligand binding, an enzyme conformational change that (after freeze-drying of the solution) will be preserved in the lyophilized sample when exposed to anhydrous solvents. This strategy, also referred to as molecular (bio)imprinting (7,8), has been extended to nonenzymic proteins and other macromolecules (9, 10) and shares a conceptual analogy with the previously known molecular imprinting of polymers (for a review, see ref. 11). In the case of enzyme (bio)imprinting, only scarce efforts have been reported so far, which have focused on a very few related proteases and made use of competitive inhibitors (amino acid derivatives) as print molecules (6-8). Although these preliminary results seem indeed encouraging in terms of an activity enhancement or even an apparent (stereo)selectivity alteration in the organic milieu, the nature of the presumptive conformational changes induced has not been characterized, and the molecular reasons for the experimental observations are still open to debate (12).Paradoxically, in this context, no effort aimed at assessing the possibility of imprinting lipolytic enzymes has been reThe publication costs of th...