Interfacial activation-based molecular bioimprinting of lipolytic enzymes.

Abstract

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...

Enantioselective recognition mechanism of secondary alcohol by surfactant-coated lipases in nonaqueous media

“…It was suggested that the two conformers, "closed" poor active and "open" active forms, should be taken into account when we employ lipases as a biocatalyst, especially in a water-restricted environment where lack of interfacial activation is observed (Lowrier et al, 1996). In this regard, Mingarro et al reported a novel concept for the activation of lipolytic enzymes, interfacial activation-based molecular bioimprinting (IAMI), for use in nonaqueous media (Mingarro et al, 1995(Mingarro et al, , 1996. Their strategy is based on the molecular imprinting technique of other enzymes (Russell and Klibanov, 1988): the combination of the creation of the activated form of enzymes in aqueous media and the retention of the imprinted conformation in nonaqueous media.…”

Immobilized Aspergillus niger Lipase with SiO2 Nanoparticles in Sol-Gel Materials

“…A similar effect may occur with the presence of hydrophobic alkyl groups in the gel. Lipophilic interactions between the hydrophobic parts of the gel and lipase during the sol-gel process can fix the encapsulated enzyme in a more active conformation, and enhance the observed activity [24,25]. However, the gelatin behavior was poor with a large excess of MTMS due to the lack of the network-forming ability of TMOS.…”

Induction of catalytic activity in proteins by lyophilization in the presence of a transition state analogue

“…It can be best illustrated by (bio)molecular imprinting, an approach which enables the introduction of new binding sites into protein molecules (Braco et al, 1990;Dabulis and Klibanov, 1992) and the alteration of an enzyme's catalytic properties such as activity (Mingarro et al, 1995), substrate specificity (Johnsson et al, 1995;O'Rich, and Dordick, 1997) or enantioselectivity (Stahl et al, 1990(Stahl et al, , 1991. However, if ligands can be employed to alter protein conformations in organic solvents in a predictable manner, it should be possible to use this approach for the induction of ''de novo'' catalytic activity in existing proteins via imprinting with transition state analogues (TSA) (Lerner et al, 1991).…”