الفهرس 
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Abstract
The study of green oxidation technologies increases the attention in enzyme research. In this perspective, the development of oxidation technologies based on enzymes is assembled by laccase enzymes. Laccases belong to the multicopper oxidases superfamily forming a phylogenetically divergent subgroup of so-called ”blue oxidases”, which contain four copper atoms per enzyme molecule, arranged into three metallocentres between three structural domains that are widely applied in the biotechnical industry due to their ability to catalyse the oxidation of various aromatic compounds. Laccases are relatively easy to be produced heterologously in industrial host organisms. In this thesis, the sequences of seventeen laccase genes of Coprinopsis cinerea had been analysed using different bioinformatic software tools to obtain a complete theoretical image of the nature, structure, and function of these laccases. Moreover, the three-dimensional structures of all laccases were predicted by homology modelling. In order to produce different C. cinerea laccases, seventeen pYSK laccase plasmids under control of the Agaricus bisporus gpdII-promoter, were transformed into E. coli competent cells XL1-Blue. Subsequently, these vectors containing each laccase gene were then transformed into Coprinopsis cinerea strain FA2222 for heterologous laccase expression. Positive laccase reactions of transformants were observed on solid agar plate supplemented with 0.5 mM ABTS [2,2’-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid)] or 0.2 mM DMP (2,6-dimethoxyphenol). Two to eight transformants for each laccase had been tested using YMG liquid media supplemented with 0.1mM CuSO4. Afterwards, the clones with the highest laccase activity in YMG media were chosen to be optimized in a modified Kjalke medium with the addition of 0.1 mM CuSO4. Laccase activities within cultures were determined with ABTS until days 8 – 11 of cultivation. Various laccase activity had been rapidly increased to the maximum peak between day 7 and 9 with 2200, 35200, 269.6, 10008, and 434 mU/ml for laccases Lcc7, Lcc5, Lcc2, Lcc6, and Lcc11, respectively. Several C. cinerea laccases in culture supernatants were purified using Äkta FPLC (Fast Protein Liquid chromatography) and separated in native SDS-PAGE (Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis). Furthermore, in this study, C. cinerea Lcc5, in conjunction with Vanillyl alcohol, had been used to manufacture Medium-Density Fiberboard (MDF) in different thicknesses (2 and 8 mm). Vanillyl alcohol was chosen based on the oxygen consumption test and the hydroxyl group content measurements of Lcc5 on wood fibres in conjunction with five different mediators: 4-hydroxybenzoic acid, 1-hydroxy benzotriazole, Guaiacol, 2,6-dimethoxy-phenol and Vanillyl alcohol. The physical and mechanical properties of MDF had been measured to evaluate the effect of different laccase-mediator systems on the board properties. MDF was assessed for the mechanical (Modulus of Rupture (MOR) and Internal Bonding (IB)) and physical (Thickness Swelling (TS)) properties according to European Standard (EN 622-5). For thin MDF (2 mm), only MOR and TS were measured, not IB. Mechanical properties revealed the highest MOR values of 55.10 and 38 N/mm2 for 2 and 8 mm thick MDF, respectively, and IB of 0.61 N/mm2 for 8mm boards using the treatment of Lcc5 + Van alc dissolved in McIlvaine buffer, pH 6.0. For the physical properties, without using a hydrophobic agent, all treatments’ thickness swelling exceeded the European standard values. Another vital application of C. cinerea Lcc5 is the biotransformation of lignin into valuable products. In this study, C. cinerea Lcc5 had been used to bio-transform calcium lignosulphonate under mild reaction conditions. The reactivity of Lcc5 from C. cinerea with Ca-lignosulphonate had been determined. Oxygen consumption test and measurements of hydroxyl groups from different treatments on Ca-lignosulphonate, either with solely Lcc5 or in conjunction with different mediators; HBA, Van alc, HBT, Gu and DMP. Moreover, the determination of fungal growth and colour change on lignin-containing solid agar media had been achieved using different C. cinerea laccase strains inoculated on Minimal media containing 1% lignin. Lcc5 alone in the presence of lignin consumed more oxygen as compared to all other treatments with lignin. In the case of Lcc5 with different mediators in the presence of lignin, the oxygen consumed during the reaction is much less than Lcc5 alone with lignin. Lcc5, in conjunction with Van alc on lignin, had a higher amount of hydroxyl group during the incubation time till 24 hours. All lignin treatments with Laccase Mediator Systems (LMS) have demonstrated lower hydroxyl groups’ content during the reaction time following LMS (Van alc). Thus, LMS (Van alc) reaction was the best among all laccase or LMSs reactions with lignin during the experiment time. Moreover, all laccases’ cultures are capable of growing on lignin-containing agar media with different growth rates. The change in colour of lignin dissolved in media from light brown to dark brown had been observed. For a more accessible overview, the following list of experimental tasks summarises the main aims of this thesis : 1. Bioinformatics analysis and structure prediction of C. cinerea laccases. 2. Recombinant overexpression and purification of some C. cinerea laccases. 3. Using the characterised Lcc5 in some biotechnological Applications : a. Medium Density Fiberboards Production in Pilot Scale. b. Enzymatic Transformation of Lignin into Renewable Chemicals.