The synthesis of enantiopure compounds can be achieved by using dehydrogenases as biocatalysts. For instance, reduction reactions of prochiral compounds (ketones, aldehydes and nitriles) into chiral compounds can be achieved by dehydrogenases. These dehydrogenases are cofactor dependent where cofactor is Nicotinamide Adenin Dinucleotite having some restrictions that confines usage of dehydrogenases in organic synthesis including instability of cofactor in water and high cost. Therefore, suitable recycling methods are required and developed which are enzymatic and electrochemical. We will use an electrochemical approach for the regeneration of reduced co-factors. All active compounds / mediator, cofactor and enzyme, will be immobilized on the electrode surface of the constructed reactor surface. Therefore only educts and products will exist in the reactor medium. A gas diffusion electrode will be employed as a counter electrode / which delivers clear protons to the system. Mediator will carry electrons to the cofactor for cofactor regeneration. Then, enzyme will utilize the cofactor and change the substrates to the products in high stereoselectivity. Our aim in this project is the synthesis of mediators and suitable linkers for enzyme, cofactor and mediator immobilization. In the first part of the study, mediators were synthesized which are pentamethylcyclopentadienyl rhodium bipyridine complexes. In the second part of the study, a conductive monomer (SNS) and linker were synthesized for immobilization of the enzyme. In the last part of the study, the reaction of galactitol dehydrogenase with monomer (SNS) was achieved.

Protein microarrays are becoming powerful tools to screen and identify tumor markers for cancer diagnosis, because of the multiplex detection and minute volume of sample requirement. Due to the diversity and variation in different cancers, no single tumor marker is sensitive and specific enough to meet strict diagnostic criteria. Therefore, a combination of tumor markers is required to increase sensitivity and to establish distinct patterns to increase specificity. To obtain reliable tests, the development of reproducible surface chemistry and immobilization procedure are crucial steps in the elaboration of efficient protein microarrays. In this thesis, 3D micro-structured glass slides were functionalized with various surface chemistries like silane monolayer (amino, epoxy and carboxy), and polymer layers of Jeff amine, chitosan, carboxymethyl dextran (CMD), maleic anhydride-alt-methyl vinyl ether copolymer (MAMVE) for physical adsorption or covalent binding with proteins. Surface characterizations, such as X-ray photoelectron spectroscopy (XPS) and Attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR), confirmed the monolayer/polymer grafting on the glass slides. Colorimetric assay for determining amine density of three aminated surfaces demonstrated that APDMES had more grafting density than Jeffamine and chitosan. Contact angle measurements show that polymer surfaces were more hydrophilic than monolayer surfaces due to the increasing dosages of polar functional groups. Moreover, the parameters such as additives and pH of spotting buffer, probe concentration, blocking procedures etc, were optimized for tumor marker detection. Under the optimized conditions, antibody microarrays were validated with purified tumor antigens. The best analytical performances obtained for each tumor antigen tested were strongly dependent on functionalized surfaces, e.g. MAMVE exhibited best analytical performances for CEA andHsp60 while NHS leads to best results for PDI and CA19-9. Besides, the implemented antibody microarrays were applied to tumor marker detection from colorectal cancer sera. This evaluation shows the interest to combine several tumor markers on the same surface and the combination of tumor markers on their specific surface lead to remarkably increase the positive responses of tested cancer sera (even up to 100 %). A second type of microarrays (tumor-associated antigens - TAA microarrays) was designed to discriminate breast cancer patients from healthy donors through the detection of tumor autoantibodies. This study included a cohort of 29 breast cancer patients' and 28 healthy donors' sera. A panel of fiveTAAs (Hsp60, p53, Her2, NY-ESO-1 and Hsp70) immobilized on their respective optimized surface chemistry allowed to specifically detect over 82% of breast cancer patients.

This thesis aims at presenting the use and development of original catalytic systems based on lipases immobilized on clays which are efficient for the synthesis of biopolyesters and allowing the preparation of organic/inorganic nanohybrids based on clay nanoparticles (sepiolite and montmorillonite) grafted with such polyesters. These nanoclays were used as lipase supports and the clay-immobilized forms of Candida antarctica lipase B (CALB) were tested for ε-caprolactone and lactide isomers polymerization. Polymerization kinetics and characterization of resulting materials have shown that lipases immobilized on montmorillonite show better performances compared to the ones immobilized on sepiolite. Clay surface organo-modification has proved to greatly enhance the catalytic activity of the corresponding systems. CALB immobilized on montmorillonite allowed the elaboration of organic/inorganic nanohybrids as evidenced by the effective grafting of polyester chains from the clay surface. Finally, random PCL/PLA copolyesters were successfully obtained by lipase-catalyzed copolymerization of D-lactide with ε-caprolactone.

The objective of this work was to develop a novel enzyme immobilization scheme for supported lipase sol-gels and to evaluate the potential of the immobilized biocatalyst for the production of biodiesel in a packed bed reactor. Two sources of lipase (EC 3.1.1.3 triacylglycerol hydrolase) were used in this study and the transesterification of methanol and triolein to produce glycerol and methyl oleate was used as a model reaction of biodiesel production. A commercially available form of immobilized lipase, Novozym® 435, was used as a basis for comparison to the literature. Upon establishing a lipase sol-gel formulation technique, the experimental methodology for the transesterification reaction using Novozyme® 435 was developed. Subsequently, a series of inert materials were considered based on their suitability as supports for immobilized lipase sol-gels and the synthesis of methyl oleate. The value of a supported lipase sol-gel is to improve the activity and stability of the enzyme and develop an immobilized biocatalyst that is practical for use under packed bed reactor conditions. Of the six support materials considered (6-12 mesh silica gel, Celite® R633, Celite® R632, Celite® R647, anion exchange resin, and Quartzel® felt), the diatomaceous earth supports (Celite® R633, R632 and R647) exhibited high enzymatic activity, were thermally stable, and possessed high sol-gel adhesion. From the three types of diatomaceous earth considered, Celite® R632 supported lipase sol-gels were identified as the most promising supported lipase sol-gels for methyl oleate production via transesterification. Upon further evaluation, the Celite® R632 lipase sol-gels were found to achieve high methyl oleate percent conversions, glycerol-water absorption was only significant at glycerol levels higher than 75%, and the immobilized lipase had high stability upon storage at 4°C for 1.5 years. To determine the effects of methanol and glycerol inhibition as well as temperature on the reaction kinetics, a ping-pong bi-bi kinetic model was developed and validated over a range of methanol concentrations and temperatures. The optimal methanol concentration for the conditions tested was in the range of 1.3 M to 2.0 M, and increased with increasing temperature. The model developed was consistent with the experimental data and confirmed that glycerol inhibition and the presence of products had significant effects on the reaction kinetics. The methyl oleate production capabilities of the Celite® supported lipase sol-gel were investigated using a packed bed reactor and compared with Novozym® 435 under similar operating conditions. A kinetic and mass transfer based model was developed for the reactor system using a novel efficiency correlation to account for the effect of glycerol on the enzymatic activity. Increasing the flow rate (1.4 mL/min to 20 mL/min) increased the reaction rate, presumably due to the reduction of the glycerol inhibition effect on the immobilized biocatalyst. The Celite® supported lipase sol-gel was found to have superior performance over Novozym® 435 both under batch stirred tank reaction conditions and in a packed bed reactor (83% conversion for Celite® sol-gel vs. 59% conversion for Novozym® 435 at 20 mL/min in the packed bed reactor). Based on the results obtained, Celite® supported lipase sol-gels exhibited good performance for the transesterification of triolein with methanol to produce methyl oleate in both batch and packed bed reactors, and warrant further exploration for the enzymatic production of biodiesel.

ABSTRACT Covalent Immobilization of Glucose Isomerase on Poly (2-hydroxyethyl methacrylate) Particles Yildiz, Hakan &Uuml / mit M.S., Department of Chemistry Supervisor: Prof. Dr. Nesrin Hasirci July 2004, 54 pages In this study, poly (2-hydroxyethyl methacrylate), P(HEMA), particles were prepared by suspension polymerization of the monomer 2-hydroxyethyl methacrylate with addition of ethylene glycol dimethyacrylate, EGDMA, as cross linker. Glucose isomerase, GI, enzyme was covalently immobilized on the prepared P(HEMA) particles after activation of the particles with cyanuric chloride. The activities of the free and immobilized enzymes were measured with Ethanol-Carbazole method. The immobilization of GI on P(HEMA) particles promoted enzyme stability and as a result, the enzyme became more stable to temperature, storage, and reuse. For maximum substrate conversion, optimum temperature was determined as 70 oC for free GI and this value shifted to 60 oC for immobilized enzyme. Optimum pH for maximum substrate conversion was found to be 7.0 for free GI and 8.0 for immobilized GI. The change of enzyme activity with substrate concentration were determined to calculate Km and Vmax values of the free and immobilized enzymes. Km values were found to be 1.7x10-2 mol/L and 3.1x10-1 mol/L while Vmax values were 1.01x10-4 mol/L.min, 1.65x10-3 mol/L.min for free and immobilized GI, respectively. Reuse capability of immobilized GI on P(HEMA) particles was measured and compared with commercial GI. Both systems retained 80 % of their original activities after 40th use, within 6 days. The change of enzyme activities upon storage were detected at certain time intervals for the samples stored in buffer solution at 4 oC. Immobilized enzyme was retained 60% of its original activitiy in 60 days of storage at 4 oC. Immobilized GI and commercial GI both retained 90% of their activities under continuous flow after 180 mL of substrate solution passed through the column.

ABSTRACT IMMOBILIZATION OF INVERTASE, POLYPHENOL OXIDASE AND GLUCOSE OXIDASE IN CONDUCTING COPOLYMERS OF THIOPHENE-CAPPED POLYTETRAHYDROFURAN AND PYRROLE B&ouml / y&uuml / kbayram, AySe Elif Ph.D., Department of Chemistry Supervisor: Prof. Dr. Levent Toppare January 2005, 123 pages Immobilization of invertase, polyphenol oxidase (PPO) and glucose oxidase (GOD) enzymes were performed in electrochemically synthesized two types of conducting copolymers. One end and two end thiophene-capped polytetrahydrofuran (TPTHF-1 and TPTHF-2) were copolymerized with pyrrole under conditions of constant potential electrolysis. The copolymers were characterized by thermal, spectroscopic and scanning electron microscopy analyses. Immobilization was carried out via entrapment of enzymes in two types of matrices during the copolymerization of pyrrole with the insulating polymers in the presence of sodium dodecyl sulphate (SDS). Kinetic parameters: Maximum reaction rate (Vmax) and Michaelis-Menten constant (Km) were determined for the enzyme electrodes. Temperature optimization, pH optimization, operational stability and shelf-life of the enzyme electrodes were investigated. Enzyme electrodes of polyphenol oxidase and glucose oxidase were used to determine the amount of their substrates in samples. Polyphenol oxidase converts mono and diphenols to quinone. Amount of phenolic compounds in two kinds of wines were determined by analyzing the quinone amount. Glucose oxidase converts &amp / #61538 / -D-glucose to D-glucono-1,5-lactone. Glucose amount was determined in two kind of factory-produced orange juices by analyzing D-glucono-1,5-lactone.

Thesis (Ph. D.)--Miami University, Dept. of Geology, 2007. / Title from second page of PDF document. Includes bibliographical references.

La réalisation d'une transformation chimique dans un espace confiné constitue pour les chimistes molécularistes un véritable défi. Des recherches récentes ont montré que des réactions se déroulant dans une poche ou une cavité moléculaire étaient de nature à engendrer des sélectivités nouvelles et faciliter des réactions thermodynamiquement défavorables. L'association métal-cavité permet également le déroulement de processus catalytiques en milieu aqueux, dès lors que la cavité a été rendue hydrosoluble.L’objectif de cette thèse était de préparer des ligands originaux intégrant une ou plusieurs cavités moléculaires de type calix[4]arène et d’en étudier les propriétés complexantes. L’ensemble des calixarènes synthétisés sont porteurs d’un ou plusieurs groupes PPh2 directement liés au bord supérieur du macrocycle. Plusieurs types de molécules ont été préparées : (i) des bis-calixarènes formant, après complexation, des métallo-capsules; (ii) des calixar!ène-diphosphines adaptées à la formation de complexes bimétalliques dans lesquels les centres métalliques sont placés entre deux coquilles se faisant face. L’activité catalytique de certains de ces métallo-capsules est jusqu’à 40 fois supérieurs à celle observé pour un catalyseur classique.La dernière partie de cette thèse a pour objectif de mettre à la disposition des spécialistes de la chimie des surfaces des phosphacalixarènes originaux destinés à la confection de supports solides P(III)-fonctionnalisés et donc de nouveaux catalyseurs supportés. / The realization of a chemical transformation in a confined space is for molecularist chemists a challenge. Recent research has shown that reactions occurring in a pocket or a molecular cavity were such as to generate selectivities and facilitate new thermodynamically unfavorable reactions.The metal-cavity association allows also the course of catalytic processes in aqueous medium, when the cavity has been made water soluble.The objective of this thesis was to prepare original ligands incorporating one or more cavities of molecular type calix[4]arene and to study their complexing properties. All the synthesized calixarenes are carriers of one or more groups PPh2 directly related to the upper rim of the macrocycle.Several types of molecules were prepared: (i) bis-calixarenes forming, after complexation, metallocapsules, (ii) calixarene-diphosphines suitable for the formation of bimetallic complexes in which the metal centers between two shells are placed facing each other. The catalytic activity of some of these metallo-capsules is 40 times higher than that observed for a conventional catalyst.The last part of this thesis aims to make available experts in surface chemistry of the original phosphacalixarenes for making solid supports P (III)-functionalized and thus new supported catalysts

Nesse trabalho foram produzidas lipases da levedura Pseudozyma hubeiensis (HB85A) em reator de 14 L. Após produção da enzima, a lipase foi imobilizada por adsorção em suporte hidrofóbico por processo contínuo em reator de leito fixo. As melhores condições de imobilização foram: tempo de imobilização de 2 h e 29 min., pH de 4,76 e quantidade de enzima livre adicionada por grama de suporte de 1282 U/ g de suporte, sendo que, a máxima atividade da lipase imobilizada obtida foi de 143 U/g de suporte. O sobrenadante contendo lipase e o biocatalisador heterogênio foram caracterizados por planejamento fatorial. A máxima atividade da enzima imobilizada (71 U/g de suporte) foi obtida em pH 6,0 à temperatura de 52 °C. A imobilização da lipase resultou em um aumento na estabilidade dessa enzima em temperaturas altas, pH ácidos e neutros, presença de detergentes não-iônicos e altas concentrações de solventes orgânicos como iso-propanol, metanol e acetona. Foi possível a reutilização da lipase imobilizada por apenas uma vez na reação de hidrólise, havendo uma perda de 72 % da atividade após o primeiro reuso. Analisou-se ainda a estabilidade da lipase livre e imobilizada durante 40 dias de armazenamento a 4 °C. Durante o período de armazenamento, a lipase imobilizada manteve 50 % de sua atividade original e a lipase livre apresentou 80 %. O catalisador heterogêneo foi testado quanto a sua eficácia na produção de biodiesel. A reação de transesterificação foi realizada na ausência de co-solvente utilizando-se como matérias-primas metanol, etanol e iso-propanol e quatro fontes diferentes de triglicerídeo (óleo de soja, óleo de mamona, óleo residual de restaurante e a gordura bovina). A partir dos testes realizados, obteve-se um rendimento máximo quanto à produção de biodiesel de 3,15 % utilizando-se óleo de mamona e iso-propanol como matéria-prima pelo período de 24 h. A produção de biodiesel utilizando diferentes quantidades de lipase imobilizada e também a lipase livre como catalisador foi testada na presença de hexano, iso-propanol e óleo de mamona pelo período de 24 h nas temperaturas de 40, 50 e 60 °C. No entanto, não houve produção de biodiesel nas condições analisadas. / In this work, lipases from yeast Pseudozyma hubeiensis (strain HB85A) were produced in a 14 L reactor. After lipase from yeast P. hubeiensis (strain HB85A) production, the enzyme was immobilized by adsorption in polyestyrene divinylbenzene hydrophobic support in a packet bed column. The best conditions for lipase immobilization were: 2 h and 29 min. immobilizing time, pH 4.76 and rate of free enzyme added per gram of support equal to 1282 U/g. The maximum activity of immobilized lipase was reached of 143 U/g. The lipases of P. hubeiensis (HB85A) supernatant culture and the heterogeneous catalyst were characterized through response surface methodology by factorial design. The maximum activity of immobilized lipase was reached for a support rate of 71 U/g, with pH 6.0 and temperature of 52 °C. It was detected that lipase immobilization increased enzyme stability under high temperatures, neutral and acid pH levels, non-ionic detergent and high concentration of organic solvent like iso-propanol, methanol and acetone. The reuse of immobilized lipase was possible only once for hydrolysis reaction, with activity losses of 72 % after first re-use. Also, it was tested lipase stability in a period of 40 days, under 4 °C storage conditions. During storage period, immobilized lipase kept 50 % of its original activity. Free lipase kept 80 %. After the development of heterogeneous catalyst, its efficiency as catalyst for biodiesel production was analyzed in this study. The transesterification reaction was tested in co-solvent absence using as raw material three differents sources of alcohols (methanol, ethanol and iso-propanol) and four differents triglicerides source (soybean oil, castor oil, waste cooking oil and bovine fat) and as catalystis the immobilized lipase. Based in test results, the maximum biodiesel production yield was 3.15 % using castor oil and methanol as raw material for 24 h. The biodiesel production was also tested with different amount of immobilized lipase and with free lipase as catalystis at the presence of methanol, castor oil and the co-solvent hexane for 24 h at 40, 50 e 60 °C. However there was no biodiesel production at the tested conditions.

Catalytic systems based on zirconoces which are able to polymerize ethene have been studied. Structural modifications on the basic catalytic precursor, diphenylmethylidene(η5- cyclopentadienyl)(η5-9-fluorenyl) zirconium dichloride (5a) have been carried out. The catalytic precursors 5a and bis(para-fluorophenyl)methylidene(η5-cyclopentadienyl)(η5-9- fluorenyl)zirconium dichloride (5b) were synthesized, as well the pre-ligand (1- cyclopentadienyl)(9-fluorenyl)[1,1-bis(para-methoxyphenyl)]methane (3c). Complex Ph2C(Cp)(Flu)ZrCl2 was supported on silica by two different techniques. i) directly on silica, grafting, and ii) on silica modified with MAO. Catalytic tests on homopolymerization of ethene and copolymerization of ethene/1- hexene reactions, using the catalytic precursor 5a, were carried out either on homogeneous and heterogeneous media. The polymers obtained were characterized and their properties were correlated with the reaction polymerization conditions. Three different reactors, under different reaction conditions, were employed to carried out polymerization reactions. Several analytical techniques were employed to characterize the pre-ligands, catalytic precursors, polymers, and supports obtained. / Conselho Nacional de Desenvolvimento Científico e Tecnológico / Neste trabalho foram estudados sistemas catalíticos, a base de metalocenos de zircônio, capazes de promover reações de polimerização de eteno. Foram realizadas de modificação estrutural do precursor catalítico base, difenilmetilideno(η5-ciclopentadienil)(η5- 9-fluorenil)zircônio (5a). Os precursores catalíticos metalocênicos 5a e dicloreto de bis(parafluorofenil) metilideno(η5-ciclopentadienil)(η5-9-fluorenil)zircônio (5b) foram sintetizados, como também o pré-ligante (1-ciclopentadienil)(9-fluorenil)[1,1-bis(para-metoxifenil)] metano (3c). O complexo Ph2C(Cp)(Flu)ZrCl2 (5a) foi suportado em sílica através de duas técnicas: i) diretamente em sílica, grafting e ii) em sílica modificada por metilaluminoxano (MAO). Foram realizados testes catalíticos empregando-se o precursor catalítico 5a, tanto em meio homogêneo, quanto heterogêneo, frente a reações de homopolimerização de eteno e copolimerização de eteno/1-hexeno. Os polímeros obtidos foram caracterizados e suas propriedades foram correlacionadas com as condições da reação de polimerização. As reações de polimerização foram realizadas em três diferentes reatores, sob diferentes condições reação. Várias técnicas analíticas foram empregadas para caracterização dos pré-ligantes, precursores catalíticos, polímeros e suportes obtidos.