Final Report Summary - CORESHELL (Marine nanobiotechnology: Manganese oxide-containing core-shell materials formed by proteins from marine organisms for biomedical and environmental applications) Core-shell materials are of enormous interest for many applications in nanotechnology and nanomedicine. Only recently, due to the achievements of the consortium, the generation of such nanoparticles by applying unique proteins from marine organisms has become possible. This IAPP was based on a long-term and very successful cooperation between the participating groups in Germany and Croatia. The consortium consisted of one partner from industry (NTM) and two partners from academia (RBI and UMC-Mainz). The aim of the project was to generate novel metal oxide nanocomposite materials, based on marine metal-oxide forming enzymes/proteins. Enzymatically active marine bacterial multicopper oxidase (MCO) and a sponge laccase, which catalyze the oxidation of Mn(II) to Mn(IV), should be immobilized on magnetic iron oxide nanoparticles to enzymatically fabricate core-shell materials. In addition, both enzymes should be applied in combination with silica or other metal oxide-forming proteins (silicateins) to generate nanoparticles containing multiple shells of various materials, which can be doped with fluorescent dyes and proteins during their formation at mild conditions. These core-shell nanoparticles should be used in drug delivery, removal of heavy metals from contaminated aqueous environments (remediation) and antifouling applications. This multidisciplinary project allowed an intense exchange of scientific expertise between the participating groups. The complementary skills and techniques available in the three partner institutions had strong synergetic effects. NTM is well experienced in biochemistry of biomineralization and recombinant protein expression. UMC-Mainz has international reputation and special knowledge in molecular biology of biosilicification of marine invertebrates (in particular sponges), gene technology, and nanoparticle synthesis/characterization. RBI is highly experienced in taxonomy, ecology and cell biology of marine organisms and marine chemistry. This resulted in an effective training especially of young researchers and postdoctoral fellows in both molecular biology and techniques used in biochemistry and nanotechnology, as well as in ecology. Highlights of this project were, among others, the isolation of the first poriferan laccase from the marine demosponge Suberites domuncula and the successful expression of the functionally active recombinant protein, as well as the isolation and characterization of the cDNAs encoding for ferritin cDNA and carbonic anhydrase II from the mussel Mytilus galloprovincialis. In addition, we succeeded to isolate and to characterize the ferritin cDNA and the cDNA encoding for the carbonic anhydrase II from the mussel M. galloprovincialis. The recombinant ferritin type 1 protein from S. domuncula has been prepared. The techniques for immobilization of the enzymes/proteins have been elaborated in order to generate ferromagnetic core-shell nanoparticles covered silicatein, laccase or ferritin. Core-shell nanoparticles with various metal oxide layers, including multilayered nanoparticles, have been prepared by the immobilized enzymes/proteins. We developed magnetic nanoparticles for scavenging toxic heavy metals or removal of manganese from seawater following a bio-inspired approach mimicking the formation of manganese nodules and polymetallic crusts in nature. A novel magnetic separator has been developed that allows the removal/recovery of the magnetic particles after loading with heavy metals. Magnetic iron oxide particles for antifouling applications have been developed. Nanoparticles with an enzymatically (via immobilized silicatein) synthesized titania coat were exposed to UV-A light to kill bacteria by photocatalytic reaction. The use of the magnetic separator allowed an easy collection of the magnetic particles at the end of the incubation period. We could demonstrate that the magnetic core-shell nanoparticles can be used for enrichment of manganese or removal of heavy metals from contaminated marine environments. The prototypes of the newly developed magnetic core-shell nanoparticles and separation devices have been successfully validated both in laboratory and field experiments. Several dissemination and communication activities and activities for the protection of knowledge have been performed. The results of this project have been published (40 publications) and presented during the 8 scientific conferences. In addition, the consortium members participated in public events in the frame of the initiative “Germany – Land of Ideas” (UMC-Mainz was one of the winners in the national competition 2011). Moreover, UMC-Mainz and NTM with their Center of Excellence “BIOTECmarin” participated as exhibitors in the German Pavilion at the Expo 2012, Yeosu, Korea (topic: “Deep-sea”). Three Summer school have been organized in 2012 (Zagreb, Croatia and 2014 and 2015 (Rovinj, Croatia), the first one together with the Marie Curie Initial Training Network BIOMINTEC and the last ones together with the EU FP7 Large-scale Integrating Project BlueGenics. Besides the events public presentations/lectures to non-experts have been given, among others, in the Multimedia Center in Rovinj in 2014. The SME (NTM) together with the university partner (UMC-Mainz) also demonstrated the results/products of the project in industrial fairs (CHINA HI-TECH FAIR 2014 in Shenzhen) and exhibitions (Medtech Rhineland-Palatinate 2014 and 2015). In summary, this project resulted in the development of several novel products that can be applied in different fields, from biomedicine to remediation of contaminated environments, and can be commercialized either alone or in combination. These products are:A. Recombinant proteins of commercial interest: (i) Laccase; (ii) Ferritin; and (iii) Carbonic anhydraseB. Core-shell nanoparticles: (i) Enzyme coated Fe2O3 nanoparticles (laccase, ferritin or carbonic anhydrase); (ii) MnO2-Fe2O3 nanoparticlesC. Special magnetic separatorD. Technologies based on use of core-shell nanoparticles: (i) Technology for photocatalytic killing of bacteria; (ii) Technology for removal of heavy metals from aquatic environment (remediation); (iii) Technology for enrichment of manganese: and (iv) Technology for drug delivery.In this project, we succeeded to introduce a variety of procedures for immobilization of enzymes/proteins on the surface of (magnetic) iron oxide nanoparticles. These enzymes can then be used for the generation of a (metal) oxide coat on the nanoparticles surfaces, e.g. silicatein: silica or titania layer; laccase: manganese oxide layer; ferritin: iron oxide layer; carbonic anhydrase: calcium carbonate layer. Even the fabrication of multi-layered core-shell particles has been demonstrated to be possible. The core-shell nanoparticles developed in the project are considered as highly innovative. The formation of metal oxide layers on these nanoparticles is controllable (enzyme mediated), allowing the fabrication of hierarchically ordered organic-inorganic composite materials. These products are novel and have multiple potential applications. Address of the project public website: http://coreshell.nanotecmarin.de/
