Μελέτη της δομής και της λειτουργίας υδρολυτικών ενζύμων σε συστήματα οργανωμένων νανοδομών

Abstract

Nanostructured systems are of considerable research interest in the field of enzyme biotechnology. The aim of this thesis is to study and understand the structure-function relationship of hydrolytic enzymes in nanostructured systems, leading to the rational design of novel nanobiocatalytic systems. The study focuses on two types of nanostructured systems: water in ionic liquid microemulsions and carbon-based nanomaterials. Water in ionic liquid microemulsions are novel water-nanodispersion systems which combine the advantages of both microemulsions and ionic liquids (IL). Such microemulsions can be formed using the ionic liquid [bmim]PF6 and the non-ionic surfactant Tween 20. Lipases entrapped in these IL microemulsions exhibited improved catalytic behaviour compared to other microheterogeneous systems, indicating the positive effect of the organized nanostructures on the catalytic behavior of enzymes. The composition of IL microemulsions affects significantly the catalytic characteristics and the structure of encapsulated lipases. Lipases in IL microemulsions adopt a rigid structure rich in β-sheets, which is responsible for the observed increase in thermostability. At the same time, lipases retain their α-helices, which are associated with their increased catalytic activity. A significant advantage of the use of IL microemulsions is the possibility of reuse of the entrapped enzymes, something that is not feasible in other microheterogeneous systems. Immobilization of lipases which are entrapped in IL microemulsions in gels prepared with (hydroxypropyl)methyl cellulose (HPMC) confers the improved catalytic features that the lipases exhibit in these microemulsions available to various non-conventional media. The immobilized lipase in IL microemulsion-based organogels led to increased synthetic activity, independently of the reaction medium. Moreover, the stability of immobilized lipases is increased compared with that exhibited by non-immobilized enzymes in an aqueous solution (up to 200 times) or IL microemulsions (up to 25 times). The increased stability of lipases in microemulsion-based organogels is due to the adoption of a more rigid structure, as in the case of IL microemulsions. Functionalized multi-walled carbon nanotubes (CNTs) and graphene oxide derivatives which bear chemical groups (such as carboxyl-, hydroxyl- and amino-groups) or aliphatic chains on their surface were used as immobilization carriers for lipases and esterases. Enzymes can be immobilized onto these nanomaterials with physical adsorption and covalent attachment, producing biomaterials that can be loaded with up to 1.65 mg enzyme per mg of nanomaterial. The immobilization yield and catalytic activity of enzymes is greatly affected by the geometry and the functional groups of the nanomaterials and the immobilization protocol. The experimental results suggest the functionalized CNTs as more appropriate immobilization carriers than the respective graphene oxide derivatives. Regarding the functional groups on the surface of the nanomaterials, carboxyl-groups seem to be recognized by the active site of hydrolytic enzymes, leading to reduced activity, while further modification of these nanomaterials with hexamethylenediamine have a positive effect on the catalytic activity of enzymes. The synthetic activity of immobilized hydrolases is up to 75 times greater than that of free enzymes. The covalent immobilization of enzymes producing materials with comparable biocatalytic activity to the physical absorption protocol and leads to significant stabilization of enzymes. The enzyme - nanomaterial interactions lead to structural changes of enzymes, particularly during non-covalent immobilization. The presence of nanomaterials in aqueous solution leads to reduction of enzyme’s α-helical content. This reduction has a negative impact on the catalytic activity of esterases and while for lipases it led to activation. This can be related to the interfacial activation of lipases. It seems that organized nanostructures (either microemulsions or nanomaterials) simulate in a better way the natural microenvironment of these enzymes compared with the aqueous solution, resulting in a structure of the enzyme observed in these systems which is closer to the active structure of lipases when they act in nature. These novel nanobiocatalytic systems can be used to develop biocatalytic processes, such as modification of bioactive natural compounds and the production of biodiesel. Conclusively, organized nanostructured systems significantly improve the catalytic activity of hydrolytic enzymes, leading to novel nanobiocatalytic systems with interesting properties. The systems developed in the present study are consistent with the requirements of 'green chemistry', as their structural components exhibit low toxicity and high biocompatibility. The results of this study demonstrate the significant benefits arising from the implementation of organized nanostructures to entrapment and immobilization of hydrolytic enzymes for the development of novel innovative biocatalytic systems, which constitute the basis for the development of numerous applications in the field of nanobiocatalysis and, in a more wide perspective, in the field of nanobiotechnology.