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The aim of this study was to immobilize the redox enzyme laccase over TiO2-SiO2-Au thin film coated ITO glass substrates in order to prepare electrochemically active surfaces for biosensor applications. Colloidal TiO2-SiO2-Au solution was synthesized by sol-gel route and thin film was deposited onto the substrates by dipcoating method. The cysteamine was utilized as a linker for immobilization of enzyme covalently through gold active sites. Preliminary studies were conducted by using invertase as model enzyme and Pyrex glasses as substrates. The effect of immobilization parameters such as immobilization temperature, concentration of enzyme deposition solution, immobilization time for laccase were examined. Leakage studies were conducted and storage stability of immobilized laccase was determined. Highest laccase activity was achieved when immobilization was performed with 50 &micro / g/ml solution at 4&deg / C for 2 hours. Laccase activity decreased after 4 hours of impregnation in enzyme solution. Laccase leakage was observed in the first usage of substrates and 55% activity decrease was determined in the subsequent use which might be attributed to the presence of uncovalently adsorbed enzyme on the fresh samples. In air and in buffer storage stabilities were also tested. It was found that the activity of samples almost vanished after 6 days regardless of storage conditions. Both enzymes had more activity on ITO substrate.

The work presented in this thesis describes the occurrence and properties of two multicopper oxidases derived from lichens. Despite numerous data on laccases and tyrosinases in fungi and flowering plants, this is the first report of the occurrence of these enzymes in lichenized ascomycetes. Extracellular laccase and tyrosinase activity was measured in 50 species of lichens from different taxonomic groupings and contrasting habitats. Out of 27 species tested from suborder Peltigerineae, all displayed laccase and tyrosinase activity that correlated to each other, while activity was absent in species tested from other lichen groups. Identification of the enzymes as laccases and tyrosinases was confirmed by the ability of lichen thalli or leachates to readily metabolize substrates such as 2,2’-azino(bis-3-ethylbenzthiazoline-6-sulfonate) (ABTS), syringaldazine and o-tolidine in case of laccase and L-dihydroxyphenylalanine (L-DOPA), Ltyrosine and epinephrine in case of tyrosinase in the absence of hydrogen peroxide. The activities of both enzymes were highly sensitive to cyanide and azide, and tyrosinase activity was sensitive to hexylresorcinol. Laccase activity had typical pH and temperature optima and an absorption spectrum with a peak at 614 nm. Tyrosinases could be activated by sodium dodecyl sulphate (SDS) and had typical tyrosinase molecular masses of approx. 60 kDa. The diversity of laccase isoforms in 20 lichen species from suborder Peltigerineae was investigated. The molecular masses of the active forms of most laccases varied between 135 and 190 kDa, although some lichens within the family Peltigeraceae had laccases with higher masses, typically varying from 200 to over 350 kDa. Most species contained one oligomeric laccase isoform. Desiccation and wounding stimulated laccase activity, while only wounding stimulated tyrosinase activity. The ability of laccases to decolorize dye is a classic attribute of laccases, and one with biotechnological potential. The ability of eight lichen species to decolourize different types of dyes was therefore tested. Interestingly, results showed that not only species belonging to suborder Peltigerineae but also species from other lichen group effectively decolourised dyes after 48 h suggesting that other oxidases appear to have ability to decolorize. Hopefully, our work could contribute to the better knowledge and application of lichen multicopper oxidases. / Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2012.

Polyphenol oxidases are enzymes that catalyze the oxidation of certain phenolic substrates to quinones in the presence of molecular oxygen. Polyphenol oxidases are widely used in several applications. In food industry, they are used for enhancement of flavor in coffee, tea and cocoa production, and determination of food quality. In medicine, they have several uses in treatments of Parkinson&rsquo / s disease, phenlyketonurea and leukemia. In wastewater treatment, they are used for the removal of phenolic pollutants from wastewaters. In pharmaceutical industry, differentiation of morphine from codeine is possible by means of polyphenol oxidase immobilized electrodes. In this study, a thermophilic fungus, Thermomyces lanuginosus was evaluated in terms of poyphenol oxidase production. The effect of different nutrient sources, inducers and fermentation parameters on enzyme production were investigated and maximum PPO activity of 97 U/ml was observed in bioreactor experiments at 50&deg / C, 400 rpm and pH 8.0 in a fermentation medium containing 1.4% yeast extract, 0.3% MgSO4, 1% KH2PO4, 0.003% CuSO4, 0.032% gallic acid. Type of polyphenol oxidase produced by Thermomyces lanuginosus was determined as laccase. For biochemical characterization studies, the enzyme was enriched by electrophoresis. Temperature and pH optima for the enzyme were determined as 60&deg / C and 8.0, respectively. Enzyme retained 67% activity after 1 h incubation at 80&deg / C and retained 87% of its activity after 1 hour of incubation at pH 9.0 at room temperature. The enzyme obeys Michealis-Menten kinetics with Km and Vmax values being 5 mg /ml catechol and 38 U/ml, respectively. Molecular weight of the enzyme was determined as 29 kDa and isoelectric point of enzyme was found to be approximately 6.0.

In this study, the aim was to find an economical plant source for polyphenol oxidase (PPO) production as an alternative to mushroom and possible application areas by characterization and immobilization of the PPOs. For this purpose, tissues of various plants of no commercial value were screened for their PPO activities. Mulberry leaf tissues showed the highest PPO activity against 4-methyl catechol which was comparable to that of mushroom. Average Km and Vmax values of free mulberry leaf PPOs were found as 7 mM and 218 U/ml, respectively. Mulberry leaf PPOs were immobilized in a polypyrole matrix and the Km and Vmax values of immobilized PPOs were calculated as 35 mM and 3 U/ml, respectively. Mulberry leaf PPO was the most active at 45&deg / C and pH 7. By using electrophoretic analysis, laccase and catechol oxidase type activities of PPOs and in addition, peroxidase activity were detected. Molecular weights of laccase, peroxidase and catechol oxidase were found to be about 62, 64 and 62-64 kDa, with pI values of 8.0-8.5, 4.5 and 10, sequentially.

ABSTRACT UTILIZATION OF SCYTALIDIUM THERMOPHILUM PHENOL OXIDASE IN BIOORGANIC SYNTHESES Kaptan, Yelda M.S., Department of Biotechnology Supervisor: Prof. Dr. Z&uuml / mr&uuml / t B. &Ouml / gel Co-supervisor: Prof Dr. Ufuk Bakir September 2004, 90 pages In this study, the ultimate aim was to utilize phenol oxidases of Scytalidium thermophilum in bioorganic syntheses. For this purpose, studies were conducted towards enhancing the production of phenol oxidases by Scytalidium thermophilum, developing a suitable method for laccase activity assays, analyzing the effects of organic solvents on phenol oxidase activity and analysis of the biotransformation of a number of organic substrates by phenol oxidases of Scytalidium thermophilum. In order to enhance the production of phenol oxidases, induction experiments were carried out with gallic acid, syringaldazine and chlorogenic acid. Gallic acid was found as the most effective inducer for phenol oxidase production. Inductive effect of edible mushroom Agaricus bisporus was also assayed, however, the phenolic compounds released by mushroom did not represent any induction for phenol oxidase activity of Scytalidium thermophilum. Different substrates were tested and catechol was determined as the most suitable substrate rather than syringaldazine and ABTS. Molar extinction coefficient (e) of catechol was calculated as 3450 M-1 cm-1 and 3700 M-1 cm-1 by using &ldquo / substrate blank&rdquo / and &ldquo / enzyme blank&rdquo / respectively at 420 nm. Kinetic parameters, Km and Vmax for the enzymatic reactions in which catechol was used as substrate were calculated as 52.03 mM and 0.253 U/ml respectively from Lineweaver-Burk plot and as 41.25 mM and 0.2055 U/ml from Hanes-Woolf plot. Effect of some organic solvents on phenol oxidases of Scytalidium thermophilum was assayed and DMSO was found as an appropriate solvent for the organic substrates. Phenol oxidase containing culture supernatant could oxidize benzoin, hydrobenzoin and benzoyl benzoin.

L'objectif de ce projet est de développer une biocathode enzymatique en utilisant une laccase qui catalyse la réduction de dioxygène en eau. La laccase de T. Versicolor produite au laboratoire a été choisie sur la base de sa bonne stabilité et son potentiel redox élevé. Cette laccase a été immobilisée par liaison covalente (EDC/NHS) ou par adsorption sur la surface d'une électrode de carbone fonctionnalisée. L'activité de laccase en présence d'ABTS et le courant de réduction d'O2 ont été évalués. Le transfert direct d'électrons (TED) a eu lieu entre l'électrode et la laccase immobilisée. Pour fonctionnaliser les surfaces des électrodes de carbone deux approches ont été étudiées. Dans une première approche, les surfaces ont été électrochimiquement fonctionnalisées par réduction de sel de diazonium pour générer des groupements amines ou carboxyliques. La laccase a été ensuite immobilisée par une liaison covalente sur des surfaces fonctionnalisées par des groupements carboxyliques et le courant a atteint une densité de 25,1±6,1 µA∙cm-2. L'oxydation de cette laccase a permis d'atteindre une densité de courant de 166,8±21,4 µA∙cm-2. Une autre stratégie de fonctionnalisation des électrodes qui consiste en traitement des surfaces par plasma a été étudiée. Le procédé plasma est innovant, simple et rapide. Différentes paramètres du plasma ont été étudiés. Selon le type de plasma (air, O2 et N2), des groupements carboxyliques, carbonyles et amines/amides ont été générés. La réduction d'O2 a également été effectuée par TED. Une densité de courant de l'ordre de 108 µA∙cm-2, a été obtenue pour la laccase immobilisée d'une façon covalente sur des surfaces traitées par plasma N2.

Stuttgart, Univ., Diss., 2008.

Thesis (Ph. D.)--Chemistry and Biochemistry, Georgia Institute of Technology, 2009. / Committee Chair: Ragauskas, Arthur; Committee Member: Bunz, Uwe; Committee Member: Cairney, John; Committee Member: Collard, David; Committee Member: Singh, Preet. Part of the SMARTech Electronic Thesis and Dissertation Collection.