Neste trabalho relata-se pela primeira vez a determinação de adenosina por um biossensor baseado em tirosinase. O biossensor foi desenvolvido mediante a modificação de um eletrodo de carbono impresso (SPE) com nanopartículas de ouro (AuNPs), tirosinase (Tyr) e Nafion, denominado biossensor Nafion/Tyr/AuNPs/SPE. As AuNPs sintetizadas possuem diâmetro médio de 15,0 ± 1,1 nm e sua função é melhorar a via de condução de elétrons entre a enzima e o eletrodo. Utilizou-se o aprisionamento com filme Nafion® para evitar a lixiviação enzimática da superfície do eletrodo. A tirosinase imobilizada apresentou boa atividade frente ao substrato catecol. Verificou-se que a adenosina atua como um inibidor do tipo não-competitivo. O biossensor é estável durante pelo menos 45 dias. Além disso, foi realizada a eletro-oxidação da adenosina para sua determinação. O biossensor apresenta sensibilidade superior em comparação com SPE, AuNPs/SPE e Nafion/AuNPs/SPE. As curvas de calibração revelaram duas faixas lineares para as concentrações de adenosina, de 1,0 × 10-5 mol L-1 até 5,0 × 10-5 mol L-1 e entre 6,0 × 10-5 mol L-1 e 1,2 × 10-4 mol L -1. O limite de detecção (3 × (desvio padrão + média dos brancos)/coeficiente angular da curva) foi de 7,0 × 10-7 mol L-1. / In this work we report for the first time the determination of adenosine by a biosensor based on tyrosinase. The biosensor was developed by modifying a screen-printed carbon electrode (SPE) with gold nanoparticles (AuNPs), tyrosinase (Tyr) and Nafion, denoted as Nafion/Tyr/AuNPs/SPE biosensor. The synthesized AuNPs have a mean diameter of 15.0 ± 1.1 nm and their function is to improve the electron conduction pathway between the enzyme and the electrode. The entrapment with Nafion® film was selected to prevent the enzyme lixiviation from the electrode surface. Immobilized tyrosinase showed good activity with the catechol substrate. It was found that adenosine acts as a non-competitive type inhibitor. The biosensor is stable for at least 45 days. In addition, the electro-oxidation of adenosine was performed for its determination. The biosensor has superior sensitivity compared to SPE, AuNPs/SPE and Nafion/AuNPs/SPE. Calibration curves revealed two linear ranges for adenosine concentrations of 1,010-5 mol L-1 up to 5,010-5 mol L-1 and from 6,010-5 mol L-1 to 1,210-4 mol L-1. The detection limit (3 × (standard deviation + mean of blanks)/slope of the curve) was 7,010-7 mol L-1.

>Magister Scientiae - MSc / Tyrosinase is an enzyme widely distributed in the biosphere and is found in many species of bacteria, fungi, animals, and plants; it is associated with melanin production. Even though it possesses many beneficial properties such as photoprotection, but overproduction causes undesirable effects such melasma, solar lentigines etc. Therefore, tyrosinase enzyme inhibitors are of far-ranging importance in cosmetics, medicinal products, and food industries. This study is aimed to test anti-tyrosinase activity in 37 plants from 20 families using mushroom tyrosinase inhibition method; each plant was extracted with methanol. The results showed that 17 plant extracts, exerted a considerable level of in vitro tyrosinase inhibition comparable to positive controls of kojic acid in the same solvent systems when evaluated spectrophotometrically. Among plant extracts, those that showed an inhibition rate >50 % at 50 μg/ml and ˃60 % at 200 μg/ml were A. karroo (Hayne.), A. afra Jacq. Ex Willd, C. geifolia (L.), E. racemosa (L.), H. petiolare Hilliard & B.L.Burt, M. quercifolia (L.), M. communis (L.), P. rigida (Wikstr.), P. ecklonii (Benth.), P. ericoides (L.), S. Africanacaerulea (L.), S. Africana-lutea (L.), S. antarcticus (Willd.), S. lucida (L.) F.A.Barkley, S. hamilifolius (L.), S. furcellata R.Br and T riparia which exhibited great anti-tyrosinase activity.

Le travail de cette thèse a consisté à développer des microbiocapteurs de type ISFET (Ion Sensitive Field Effect Transistor) et conductimétrique en utilisant l'enzyme tyrosinase pour la détection de pesticides dans l'eau. Ces deux types de capteurs ont été mis au point à l'Institut International de Formation de Science en Matériaux (ITIMS), Viet Nam. En particulier, les étapes de diffusion de dopants ont été réalisées par la technique Spin On Glass (SOG). La biofonctionnalisation de ces capteurs a été réalisée par immobilisation de l'enzyme tyrosinase avec de la bovine sérum albumine (BSA) en présence de vapeur de glutaraldéhyde. Le temps de contact avec le glutaraldéhyde, la charge en enzyme, le pH, la nature du substrat, le temps pour chaque mesure... ont été optimisé afin d'obtenir les meilleurs caractéristiques analytiques possibles. Ces capteurs nous ont permis de détecter en phase aqueuse des dérivés phénoliques ainsi que des pesticides de type diuron et atrazine. La limite de détection est de 2,15 ppb pour l'atrazine et 2,33 ppb pour le diuron, le temps de réponse est de 1 à 5 minutes, l'activité enzymatique reste de 90% après 23 jours dans un tampon à 4°C et la dérive est de 5%.

The first study reports the development of a reusable, single-step system for the detection of specific substrates using oxidase-functionalized Fe3O4 nanoparticles (NPs) as a bienzyme system and using amplex ultrared (AU) as a fluorogenic substrate. In the presence of H2O2, the reaction pH between Fe3O4 NPs and AU was similar to the reaction of oxidase and the substrate. The catalytic activity of Fe3O4 NPs with AU was nearly unchanged following modification with poly(diallyldimethylammonium chloride) (PDDA). Based on these features, we prepared a composite of PDDA-modified Fe3O4 NPs and oxidase for the quantification of specific substrates through the H2O2-mediated oxidation of AU. By monitoring fluorescence intensity at 587 nm of oxidized AU, the minimum detectable concentrations of glucose, galactose, and choline were found to be 3, 2, and 20 £gM using glucose oxidase-Fe3O4, galactose oxidase-Fe3O4, and choline oxidase-Fe3O4 composites, respectively. The identification of glucose in blood was selected as the model to validate the applicability of this proposed method. The second study follows the first one. Using the catalytic activity of Fe3O4 NPs with AU to detect four kinds of neurotransmitter, such as dopamine, L-DOPA, adrenaline (epinephrine) and noradrenaline (norepinephrine). Because of there is specific interaction between Fe3O4 NPs and catecholamines (CAs), the Fe3O4 NPs will form CAs-Fe3O4 NPs composites in presence of CAs. The CAs on the Fe3O4 NPs surface must shelter the reaction between AU and H2O2, cause the fluorescence to be turned-off. The CAs just like a inhibitor, to inhibit the catalytic activity of Fe3O4 NPs. Therefore, we could use this inhibited system to detect the CAs compound concentration in the real sample.

ABSTRACT PREPARATION OF CROSS-LINKED TYROSINASE AGGREGATES Aytar, Burcu Selin M.S., Department of Chemical Engineering Supervisor: Prof. Dr. Ufuk Bakir June 2006, 82 pages The aim of this study was to prepare cross-linked enzyme aggregate (CLEA) from crude mushroom (Agaricus bisporus) extract. However, the optimization of CLEA production was performed by using pure tyrosinase. Important parameters were determined as protein, ammonium sulfate and glutaraldehyde concentrations, CLEA particle size, and cross-linking temperature and period. On the other hand, the order of ammonium sulfate and glutaraldehyde addition did not affect the yield of CLEA. Optimum CLEA preparation conditions were 60 % ammonium sulfate saturation, 2 % (v/v) glutaraldehyde, and 3 hour cross-linking reaction at room temperature. Particle size of the CLEAs should be reduced by mechanical stirring to eliminate mass transfer limitations. Under these circumstances, 100 % recovery was obtained from both pure and crude tyrosinases. Optimum temperature and the activation energy for catechol oxidation were determined as 34 oC and 16.9 kcal/mol for CLEAs, whereas, 32 oC and 12.5 kcal/mol for the free enzyme. Furthermore, the thermostability of CLEAs was significantly higher than the free enzyme. CLEAs, prepared from crude mushroom extract, retained 72 % of its maximum activity in eight months storage at 4 oC. Moreover, changing the storage temperature from 4 oC to room temperature did not decrease CLEAs stabilities.

ABSTRACT BIOSENSOR BASED ON INTERPENETRATED POLYMER NETWORK OF ALGINIC ACID AND POLY (1-VINYLIMIDAZOLE) Kartal, M&uuml / jgan M.S., Department of Chemistry Supervisor : Prof. Dr. Levent Toppare January 2008, 63 pages A new proton conductor polymer was prepared using alginic acid (AA) and poly (1-vinylimidazole) (PVI). The polymer network was obtained by mixing AA and PVI at various stoichiometric ratios, x (molar ratio of the monomer repeat units). The AA/PVI network was characterized by elemental analysis (EA) and FT-IR spectroscopy. Potential use of this network in enzyme immobilization was studied. Enzyme entrapped polymer networks (EEPN) were produced by immobilizing invertase and tyrosinase (PPO) in the AA/PVI network. Additionally, the maximum reaction rate (Vmax) and Michaelis-Menten constant (Km) were investigated for the immobilized invertase and enzymes. Also, temperature and pH optimization, operational stability and shelf life of the polymer network were examined.

Phenolic compounds are the chemicals which are used by many different industries and as a result of this spread to the environment. These compounds can be absorbed easily through the human and animal skin and through the mucosal membrane, mix in to the blood circulation and thus create a toxic effect on several tissue and organs including, liver, lung and kidneys. For this reason, determination of phenolic compounds emitted to environment is a very important issue. In fact, there are standard methods for the determination of these compounds like HPLC, Spectrophotometric and calorimetric methods however, these are time consuming methods and requires to be expertise. On the other hand, there are also different types of biosensors developed for the phenolic compound detection. In this study, a new, disposable, cheap and convenient tyrosinase biosensor was developed for the phenolic compound detection. By means of absorption method, the enzyme tyrosinase and the chromophore MBTH were immobilized on the support material and as a model substrate L- dopa was used. As a result of optimization studies 1mg/ml tyrosinase concentration and 1.5mM MBTH concentration were determined for using in biosensor construction. Detection limit of l-dopa, model substrate, found as 0,064 mM and for other phenolic compounds, 4-chlorophenol, catechol, m-cresol and p-cresol, detection limit was obtained 0.032 mM, 0.032 mM, 0.128 mM, 0.128 mM, respectively. In addition, we found that the biosensor response was not affected by pH changes ranging from 3 to 11. The stability of biosensor which is one of the important parameter for commercialization was not change through 70 days at room temperature and 4&deg / C when compared to at the beginning response.

The main object of the presented study was investigation of the use of cross-linked enzyme (tyrosinase) aggregates (CLTA) obtained from crude mushroom extract for a rapid phenolic content analysis in wines. In addition, a comparison of phenolic characteristics of Turkish red wines was performed. Reproducible and reliable results in total phenolic measurement were obtained with CLTAs similar to pure tyrosinase and tyrosinase obtained from crude mushroom extract. Measurement of total phenolic content is possible both in standard solutions and in complex matrices, such as wine. In a very short time period, 10 seconds, phenolics content in red and white wines produced from grapes of Turkey were investigated by using CLTAs. Results were consistent when compared to a well known phenolic measurement method, Folin-Ciocalteau. CLTAs exhibited very high operational stability and retained more than 90% of its activity after 30th use. Moreover, it showed good shelf-life stability for about 2 months storage by maintaining 90% of its maximum activity. So, use of CLTAs prepared from crude mushroom extract is an effective, fast and cheap alternative in total phenolics measurements in wines. Moreover, a novel catalase phenoloxidase (CATPO) produced by a fungal microorganism, Scytalidium thermophilum, was studied to check its capabilities in phenolics measurements. This novel catalase phenol oxidase showed similarly good results, exhibiting widesubstrate selectivity.

The goal of this study is to utilize polymers as macromolecular ligands for the construction of catalysts by formation of coordination complexes with transition metals with the main focus on complexes of Cu(II) and Fe(III) and further determine (a) their catalytic efficiency (b) mechanism of action (c) similarities to enzymatic systems and synthetic metal complexes. The reactions of interest are (1) hydrolytic cleavage of a series of phosphoesters(2) oxidation of catechol type of substrates (3) hydroxylation of phenolic substrates and chlorinated phenols (4) activation of molecular oxygen and/ or hydrogen peroxide (5)oxidative cleavage of DNA plasmid. The major premise of the study is that by mimicking the macromolecular nature and some structural features of enzymes, polymers can in principle, catalyze chemical transformations with similar efficiencies and specificities and can offer alternatives to peptide based catalysts or simple metal complexes with the advantage of a wider range of building blocks, increased stability and the potential of reusability. The crosslinked resins used contained the functional groups iminodiacetate (chelex resin), diethylenetriamine and tris(2-aminomethylamine) and were based on styrene-divinylbenzene backbone. The catalytic proficiencies of the Fe(III) and the Cu(II) complexes of chelex resin and diethylenetriamine approached 100 and 1000 respectively towards the model phosphodiester BNPP at pH 8.0 and 25°C. Moreover, the Fe(III) complexes of linear copolymers with repeating unit of three vinylpyridines to one acrylamide (P1) showed selectivity towards phosphodiester hydrolysis over monoesters and phosphonate esters and exhibited catalytic proficiencies approaching 50,000 towards BNPP hydrolysis. Further exploration of the catalytic capabilities of copolymer P1 revealed that Cu(II) complexes of this macromolecular ligand are potentially capable of assembling to active dicopper intermediates found in the catalytic pathways of copper oxygenases like tyrosinase and catechol oxidase and thus were able to accelerate catechol oxidation to ortho-quinones with rate accelerations approaching 10,000 and hydroxylate phenols with rate accelerations close to one million. The results suggest that these Cu(II)-polymer systems can potentially be used as model systems to further understand metal centered reactive oxygen species (ROS) generated in vivo and can be very promising remediation agents for the dechlorination of persistant chlorine containing pollutants.

Cancer immunotherapy seeks to boost the host’s immune system to respond to tumor antigens. The adaptive immune system comprises of two arms, one that elicits a cellular immune response and one that elicits a humoral immune response. Cytotoxic T lymphocytes (CTL) recognize short antigenic peptides presented to them in the context of class I major histocompatibility complex (MHC) molecules and are capable of killing tumor cells. CTL are educated to discriminate between foreign and self-antigen. Tumors frequently express self-antigen which usually makes them poorly immunogenic. Because tumors are genetically unstable, they may present excess self peptides and/or peptides in a reading frame different from wild type self proteins. These frameshift (FS) peptides, are caused by an insertion or deletion of nucleotides that disrupt translation of the normal reading frame and alters the protein produced such that it is non-self. Binding affinity, dissociation rate and the overall stability of the peptide/MHC/β₂-microglobulin complex are important considerations in determining the immunogenicity of a given peptide. Interaction between the anchor residues in a peptide and binding pockets in MHC are essential, but this interaction is not always strong enough to stimulate T cell responses. This indicates that not all amino acids of the peptide ligand bound to MHC are equally important for the functional outcome of the receptor engagement and that other amino acid residues in the sequence are important for binding. Optimized peptide ligands (OPL) are analogues derived from natural wild type antigenic peptides that contain amino acid substitutions at anchor and auxiliary residues. OPL can be rationally designed to generate a more robust immune response compared to that of the wild-type peptide. Active immunotherapy using OPL of tumor antigen epitopes are designed to elicit tumor-specific CTL that can overcome tolerance and either re-awaken or elicit new T-cell responses to an antigen. The work and principles presented here using brain tumor-derived peptides demonstrates that HLA-A*0201-restricted CTL generated against wild type, frameshift and OPL peptides elicit CTL that were able to recognize and respond to wild type, tumorderived peptides. The response was donor dependent in that not all individuals responded more strongly to OPL; a minority responded better to wild type peptide. This data further suggests that the rational design and testing of multiple peptides for the same epitope should elicit a broader response among different individuals than single peptide immunization.