Engineering Laccases: In Search for Novel Catalysts

Laccases (p-diphenol oxidase, EC 1.10.3.2) are blue multicopper oxidases that catalyze the reduction of dioxygen to water, with a concomitant oxidation of small organic substrates. Since the description at the end of the nineteenth century of a factor catalyzing the rapid hardening of the latex of the Japanese lacquer trees (Rhus sp.) exposed to air laccases from different origins (plants, fungi bacteria) have been continuously discovered and extensively studied. Nowadays, molecular evolution and other powerful protein modification techniques offer possibilities to develop tailored laccases for a wide array of applications including drug synthesis, biosensors or biofuel cells. Here, we give an overview on strategies and results of our laboratory in the design of new biocatalysts based on laccases

Laccase from Aspergillus niger: A novel tool to graft multifunctional materials of interests and their characterization

In the present study, we propose a green route to prepare poly(3-hydroxybutyrate) [(P(3HB)] grafted ethyl cellulose (EC) based green composites with novel characteristics through laccase-assisted grafting. P(3HB) was used as a side chain whereas, EC as a backbone material under an ambient processing conditions. A novel laccase obtained from Aspergillus niger through its heterologous expression in Saccharomyces cerevisiae was used as a green catalyst for grafting purposes without the use of additional initiator and/or cross-linking agents. Subsequently, the resulting P(3HB)-g-EC composites were characterized using a range of analytical and imagining techniques. Fourier transform infrared spectroscopy (FT-IR) spectra showed an increase in the hydrogen-bonding type interactions between the side chains of P(3HB) and backbone material of EC. Evidently, X-ray diffraction (XRD) analysis revealed a decrease in the crystallinity of the P(3HB)-g-EC composites as compared to the pristine individual polymers. A homogeneous P(3HB) distribution was also achieved in case of the graft composite prepared in the presence of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) as a mediator along with laccase as compared to the composite prepared using pure laccase alone. A substantial improvement in the thermal and mechanical characteristics was observed for grafted composites up to the different extent as compared to the pristine counterparts. The hydrophobic/hydrophilic properties of the grafted composites were better than those of the pristine counterparts

Probing the Surface of a Laccase for Clues towards the Design of Chemo-Enzymatic Catalysts

Systems featuring a multi-copper oxidase associated with transition-metal complexes can be used to perform oxidation reactions in mild conditions. Here, a strategy is presented for achieving a controlled orientation of a ruthenium–polypyridyl graft at the surface of a fungal laccase. Laccase variants are engineered with unique surface-accessible lysine residues. Distinct ruthenium–polypyridyl-modified laccases are obtained by the reductive alkylation of lysine residues precisely located relative to the T1 copper centre of the enzyme. In none of these hybrids does the presence of the graft compromise the catalytic efficiency of the enzyme on the substrate 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid). Furthermore, the efficiency of the hybrids in olefin oxidation coupled to the light-driven reduction of O2 is highly dependent on the location of the graft at the enzyme surface. Simulated RuII–CuII electron coupling values and distances fit well the observed reactivity and could be used to guide future hybrid designs.L.S. was the recipient of a MinistHre de l’Education Nationale fellowship. This study was supported by grants from the Agence Nationale de la Recherche (ANR-09-BLANC-0176 and ANR-15-CE07-0021-01) and from the Ministerio de EconomÍa, Industria y Competitividad (CTQ2016-79138-R). We thank Elise Courvoisier-Dezord from the Plateforme AVB (AMU): Analyse et Valorisation de la Biodiversit8 and Yolande Charmasson for help in the production of the recombinant enzymes, as well as Pascal Mansuelle and R8gine Lebrun from the Plateforme Prot8omique (CNRSAMU) for help in acquiring mass spectrometry data.Peer ReviewedPostprint (published version

Towards robust alkane oxidation catalysts: electronic variations in non-heme iron(II) complexes and their effect in catalytic alkane oxidation

A series of non-heme iron(II) bis(triflate) complexes containing linear and tripodal tetradentate ligands has been prepared. Electron withdrawing and electron donating substituents in the para position of the pyridine ligands as well as the effect of pyrazine versus pyridine and sulfur or oxygen donors instead of nitrogen donors have been investigated. The electronic effects induced by these substituents influence the strength of the ligand field. UV-vis spectroscopy and magnetic susceptibility studies have been used to quantify these effects and VT 1H and 19F NMR spectroscopy as well as X-ray diffraction have been used to elucidate structural and geometrical aspects of these complexes. The catalytic properties of the iron(II) complexes as catalysts for the oxidation of cyclohexane with hydrogen peroxide have been evaluated. In the strongly oxidising environment required to oxidise alkanes, catalyst stability determines the overall catalytic efficiency of a given catalyst, which can be related to the ligand field strength and the basicity of the ligand and its propensity to undergo oxidation

Catalyst stability determines the catalytic activity of non-heme iron catalysts in the oxidation of alkanes

A series of iron(II) bis(triflate) complexes [Fe(L)(OTf)2] containing linear tetradentate bis(pyridylmethyl)diamine ligands with a range of ligand backbones has been prepared. The backbone of the ligand series has been varied from a two-carbon linkage [ethylene (1), 4,5-dichlorophenylene (2) and cyclohexyl (3)] to a three-carbon [propyl (4)) and a four-carbon linkage (butyl (5)]. The coordination geometries of these complexes have been investigated in the solid state by X-ray crystallography and in solution by 1H and 19F NMR spectroscopy. Due to the labile nature of high-spin iron(II) complexes in solution, dynamic equilibria of complexes with different coordination geometries (cis-α, cis-β and trans) are observed with ligands 2–5. In these cases, the geometry observed in the solid state does not necessarily represent the only or even the major geometry present in solution. The ligand field strength in the various complexes has been investigated by variable temperature magnetic moment measurements and UV-vis spectroscopy. The strongest ligand field is observed with the most rigid ligands 1 and 2, which generate complexes [Fe(L)(OTf)2] with a cis-α coordination geometry and the corresponding complexes [Fe(L)(CH3CN)2]2+ display spin crossover behaviour. The catalytic properties of the complexes for the oxidation of cyclohexane, using hydrogen peroxide as the oxidant, have been investigated. An increased flexibility in the ligand results in a weaker ligand field, which increases the lability of the complexes. The activity and selectivity of the catalysts appear to be related to the strength of the ligand field and the stability of the catalyst in the oxidising environmen

The Rapid Exchange of Zinc (2+) Enables Trace Levels to Profoundly Influence Amyloid-beta Misfolding and Dominates Assembly Outcomes in Cu (2+)/Zn (2+) Mixtures

This work was supported by the Biotechnology and Biological Sciences Research Council Project GrantBB/M023877/1 and Quota Studentship