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.

In this study various microbial and enzymatic methods developed for enantioselective acyloin synthesis for preparation of some pharmaceutically important intermediates. By performing Aspergillus flavus (MAM 200120) mediated biotransformation, enantioselective bio-oxidation of meso-hydrobenzoin was achieved with a high ee value (76%). Racemic form of hydrobenzoin was also employed for the same bio-oxidation process and this bioconversion was resulted in accumulation of meso form (&gt / 90% yield) confirming the suggested mechanism of oxidation-reduction sequence of hydrobenzoin. Wieland-Miescher ketone (3,4,8,8a-tetrahydro-8a-methylnaphthalene-1,6(2H,7H)-dione) is an important starting material for bioactive compounds like steroids and terpenoids. Many synthetic approaches include enantioselective reduction of this compound. In this study Aspergillus niger (MAM 200909) mediated reduction of Wieland-Miescher ketone was achieved with a high yield (80%), de (79%) and ee (94%) value and these results were found much more superior than previously reported studies. Carboligating enzymes benzaldehyde lyase (BAL) (EC 4.1.2.38) and benzoiyl formate decarboxilase (BFD) (E.C. 4.1.1.7) are used for biocatalytic acyloin synthesis. These enzymes are immobilized to surface modified superparamagnetic silica coated nanoparticles by using metal ion affinity technique. With this system recombinant histidine tagged BAL and BFD purified and immobilized to magnetic particles by one-pot purification-immobilization procedure. SDS page analysis showed that our surface modified magnetic particles were eligible for specific binding of histidine tagged proteins. Conventional BAL and BFD catalyzed benzoin condenzation reactions and some representative acyloin reactions were performed with this system with a high enantioselectivity (99-92%) and yield. Results obtained with magnetic particle-enzyme system were also found comparable with that of free enzyme catalyzed reactions.

The production of enantiopure compounds can be achieved by using dehydrogenases as biocatalysts catalyzing reduction reactions of prochiral compounds such as ketones, aldehydes and nitriles. These dehydrogenases are cofactor dependent enzyme where cofactor is Nicotinamide dinucleotite having some restrictions that limits usage of dehydrogenases in organic synthesis including instability of cofactor in water and high cost. Therefore suitable regeneration method is needed and developed which are enzymatic and electrochemical. We will use an electrochemical approach for the regeneration of reduced co-factors which has been shown in principal with mediators like pentamethylcyclopentadienyl rhodium bipyridine complexes or ferrocenes. This project is European Union project, whose name is Development of Electrochemical Reactors Using Dehydrogenases for Enantiopure Synthon Preparations. All active compounds / mediator, cofactor and enzyme, will be immobilized on the electrode surface of the constructed reactor surface. Therefore only educts and v 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 use the cofactor and convert substrates to the product in high stereoselectivity. Our part in this project is the synthesis of mediator and suitable linkers for enzyme, cofactor and mediator immobilization. In the first part of the study, Linkers which contain thiol group and disulfide linkage were synthesized because working electrode made of by gold nano particles and immobilization carried out by the help of these groups on gold nano surface. In the second part of the study, mediators were synthesized which are pentamethylcyclopentadienyl rhodium bipyridine complexes and ferrocene derivatives. Synthesized mediators were reacted with linkers by using Click Chemistry and by imine formation in order to convert mediator to the thiol functionalized form.

By mixing different stoichiometric ratios of chitosan with alginic acid (AA) and chitosan with p(toluenesulfonicacid) (PTSA), two new polymer networks were prepared. FT-IR spectroscopy results show the protonation of chitosan by AA and PTSA. Elemental analysis (EA) results show the composition of the networks. Thermal gravimetry analysis (TGA) and differential scanning calorimetry (DSC) results were used to characterize the thermal stability of the networks. Then, cholesterol oxidase (ChOx) enzyme were immobilized in these networks and checked for potential use of these enzyme entrapped polymer networks (EEPN) for enzyme immobilization. Additionally, the maximum reaction rate (Vmax) and Michaelis-Menten constant (Km) were evaluated for immobilized ChOx in these two polymer networks. Also, temperature and pH optimization, operational stability, shelf-life and the proton conductivity of these networks were investigated.

Automated and continuous processes are the future trends in downstream protein purification. A functionalized nanometer-scale membrane electrode system, mimicking the function of cell wall transporters, can selectively capture genetically modified proteins and subsequently pump them through the system under programmed voltage pulses. Numerical study of the two-step pulse pumping cycles coupled with experimental His-GFP releasing study reveals the optimal 14s/1s pumping/repel pulse pumping condition at 10 mM bulk imidazole concentration in the permeate side. A separation factor for GFP: BSA of 9.7 was achieved with observed GFP electrophoretic mobility of 3.1×10-6 cm2 s-1 V-1 at 10 mM bulk imidazole concentration and 14 s/1 s pumping/repel duration. The purification of His6-OleD Loki variant directly from crude E. coli extracts expression broth was demonstrated using the pulse pumping process, simplifying the separation process as well as reducing biopharmaceutical production costs. The enzymatic reactions showed that His6-OleD Loki was still active after purification. A nanoporous membrane/electrode system with directed flow carrying reagents to sequentially attached enzymes to mimic nature’s enzymes-complex system was demonstrated. The substrates residence time on the immobilized enzyme can be precisely controlled by changing the pumping rate and thereby prevent a secondary hydrolysis reaction. Immobilized enzyme showed long term storage longevity with activity half-life of 50 days at 4℃ and the ability to be regenerated. One-step immobilization and purification of His-tagged OleD Loki variant directly from expression broth, yielded 98% Uridine Diphosphate glycosylation and 80% 4-methylumbelliferone glycosylation conversion efficiency for the sequential reaction. A flow-through electroporation system, based on a novel membrane/electrode design, for the delivery of membrane-impermeant molecules into Model Leukocyte cells was demonstrated. The ability to apply low voltage between two short distance electrodes contributes to high cell viability. The flow-through system can be easily scaled-up by varying the micro-fluidic channel geometry and/or the applied voltage pulse frequency. More importantly, the system allows the electrophoretical pumping of molecules from the reservoir across the membrane/electrode system to the micro-fluidic channel for transfection, which reduces large amount of reagents used.

While considerable progress has been made towards understanding the effect that membrane-based layer-by-layer (LbL) immobilizations have on the activity and stability of enzymatic catalysis, detailed work is required in order to fundamentally quantify and optimize the functionalization and operating conditions that define these properties. This work aims to probe deeper into the nature of transport mechanisms by use of pressure-induced, flow-driven enzymatic catalysis of LbL-functionalized hydrophilized poly(vinyldiene) (PVDF)-poly(acrylic acid) (PAA)-poly(allylamine hydrochloride) (PAH)-glucose oxidase (GOx) membranes. These membranes were coupled in a sealed series following cellulose acetate (CA) membranes for the elimination of product accumulation within the feed-side solution during operation. At pH = 6 and T = 21oC, the enzymatic catalysis of LbL-immobilized GOx from Aspergillus niger performed remarkably well in comparison to the homogeneous-phase catalysis within an analogous aqueous solution. On average, the enzymatic turnover was 0.0123 and 0.0076 mmol/(mg-GOx)(min) for the homogeneous-phase catalysis and the LbL-immobilized catalysis, respectively. Multiple consecutive permeations resulted in replicable observed kinetic results with R2 > 0.95. Permeations taking place over the course of a three week trial period resulted in a retention of >90% normalized activity when membranes were removed when not in use and stored at -20oC, whereas the homogenous-phase kinetics dropped below 90% normalized activity in under one day.

Synthesis and characterization of conducting copolymers were achieved by using thiophene-3-yl acetic acid cholesteryl ester (CM) and poly (3-methylthienyl methacrylate) (PMTM). A new polythiophene containing a cholesteryl side chain in the &amp / #946 / -position was chemically polymerized in nitromethane/carbon tetrachloride using FeCl3 as the oxidizing agent. Polymerization was also achieved by constant current electrolysis in dichloromethane. Subsequently, conducting copolymers of thiophene-3-yl acetic acid cholesteryl ester (CM), PCM1 (obtained from chemical polymerization method), PCM4 (obtained from constant current electrolysis) with pyrrole were synthesized. Thiophene functionalized methacrylate monomer (MTM) was synthesized via esterification of the 3-thiophene methanol with methacryloyl chloride. The methacrylate monomer was polymerized by free radical polymerization in the presence of azobis (isobutyronitrile) (AIBN) as the initiator. Graft copolymers of poly (3-methylthienyl methacrylate)/polypyrrole, (PMTM2/PPy) and poly (3-methylthienyl methacrylate)/polythiophene, (PMTM2/PTh) were synthesized by constant potential electrolyses. PMTM2 coated Pt electrodes were utilized as the anode in the polymerization of pyrrole and thiophene. Moreover, oxidative polymerization of PMTM1 was studied by galvanostatic and chemical techniques. Characterizations of the samples were performed by CV, FTIR, NMR, DSC, TGA and SEM analyses. Electrical conductivities were measured by the four-probe technique. Immobilization of invertase in conducting copolymer matrices, poly (3-methylthienyl methacrylate) with pyrrole and thiophene was achieved by constant potential electrolysis using the sodium dodecyl sulfate as the supporting electrolyte. Polythiophene was also used for immobilization matrices. Cholesterol oxidase has been immobilized in conducting copolymer of thiophene-3-yl acetic acid cholesteryl ester with polypyrrole (CM/PPy) and polypyrrole (PPy) by the electropolymerization method. p-Toluene sulfonic acid was used as a supporting electrolyte. Kinetic parameters (Kinetic parameters / Vmax and Michaelis-Menten constant / Km) and operational stability of enzyme electrodes were investigated. Surface morphology of the films was also examined.

abstract: Enzymes which regulate the metabolic reactions for sustaining all living things, are the engines of life. The discovery of molecules that are able to control enzyme activity is of great interest for therapeutics and the biocatalysis industry. Peptides are promising enzyme modulators due to their large chemical diversity and the existence of well-established methods for library synthesis. Microarrays represent a powerful tool for screening thousands of molecules, on a small chip, for candidates that interact with enzymes and modulate their functions. In this work, a method is presented for screening high-density arrays to discover peptides that bind and modulate enzyme activity. A viscous polyvinyl alcohol (PVA) solution was applied to array surfaces to limit the diffusion of product molecules released from enzymatic reactions, allowing the simultaneous measurement of enzyme activity and binding at each peptide feature. For proof of concept, it was possible to identify peptides that bound to horseradish peroxidase (HRP), alkaline phosphatase (APase) and â-galactosidase (â-Gal) and substantially alter their activities by comparing the peptide-enzyme binding levels and bound enzyme activity on microarrays. Several peptides, selected from microarrays, were able to inhibit â-Gal in solution, which demonstrates that behaviors selected from surfaces often transfer to solution. A mechanistic study of inhibition revealed that some of the selected peptides inhibited enzyme activity by binding to enzymes and inducing aggregation. PVA-coated peptide slides can be rapidly analyzed, given an appropriate enzyme assay, and they may also be assayed under various conditions (such as temperature, pH and solvent). I have developed a general method to discover molecules that modulate enzyme activity at desired conditions. As demonstrations, some peptides were able to promote the thermal stability of bound enzyme, which were selected by performing the microarray-based enzyme assay at high temperature. For broad applications, selected peptide ligands were used to immobilize enzymes on solid surfaces. Compared to conventional methods, enzymes immobilized on peptide-modified surfaces exhibited higher specific activities and stabilities. Peptide-modified surfaces may prove useful for immobilizing enzymes on surfaces with optimized orientation, location and performance, which are of great interest to the biocatalysis industry. / Dissertation/Thesis / Ph.D. Chemistry 2010

As β-glicosidades são enzimas que catalisam a hidrólise de ligações glicosídicas. São amplamente encontradas na natureza em plantas, frutas e animais. Possuem diversas aplicações biotecnologicas podendo ser amplamente empregadas na indústria de alimentos e bebidas afim de melhorar a qualidade de aroma, sabor, coloração e viscosidade do produto. Este estudo apresenta o processo de imobilização de uma β-glicosidase comercial em suporte de quitosana e a obtenção de um derivado ativo e estável, para ser aplicado no processamento de vinhos aumentando a complexidade aromática de vinhos joven. A imobilizaçãpo foi realizada em suporte de quitosana, reticulado com glutaraldeído, atingindo 100% de eficiência na imobilização com 50mg de proteína por grama de suporte e 65% de atividade recuperada no derivado imobilizado. A imobilização além de contribuir para um maior controle do processo, alterou algumas características da β-glicosidase, a qual demonstrou manter uma atividade mais alta em faixas mais amplas de pH, quando comparada a enzima livre. A β-glicosidase imobilizada apresentou grande estabilidade podendo ser reutilizada por mais de 30 ciclos, mantendo sua atividade inicial. A aplicação da β-glicosidase no vinho foi realizada em batelada, por um tempo de 90 min, sob agitação. A análise por SPME/GC-MS revelou um aumento na concentração terpenos, quando comparada a amostras não tratadas. Houve um aumento na concentração de geraniol, citronelol, linalol e nerol. A aplicação da β-glicosidase foi bem sucedida, liberando os compostos aromáticos em um curto períuodo de tempo de contato. O processo de reutilização mostra que o biocatalisador imobilizado é uam ferramenta vantajosa para a indústria de bebidas. / β-glucosidases are enzymes that catalyze the hydrolysis of glycosidic bonds. They are widely found in nature at plants, fruits and animals. They have various biotechnological applications being largely used in food and beverage industry for the enhance the product viscosity, coloration, flavour and aroma qualities. This study presents a commercial β-glucosidase immobilization in chitosan support in order to obtain an active and stable derivative, enabling its application in winemaking, enhancing the aromatic complexity in young wines. The immobilization process was conducted in chitosana support, cross-linked with glutaraldehyde, reaching 100% efficiency in immobilization with 50 mg of protein per gram of support and 65% recovered activity in imobilized derived. The immobilization of the enzyme contributes to greater control of the process, changed some features of β-glucosidase, which proved to be more stable at pH changes when compared to free enzyme. Also the immobilized β-glucosidase showed great operational stability been reused for more than 30 cycles maintaining its initial activity. The application of β-glucosidase in the wine was held in batch for 90 minutes under stirring. The analyzis by SPME / GC-MS revelead a increasement in terpens concentration when compared to the sample without treatment. Was noticed a increase in geraniol, citronellol, linalool and nerol concentration. Apliccation of β-glucosidase was sucesfull, releasing aromatic compounds in contact for a short period of time. The reuses process showed that the immobilized biocatalyst is a advantageous tool for the beverage industry.

Lipases (EC 3.1.1.3) são um grupo de enzimas que catalisam a hidrólise e síntese de triacilgliceróis. Estas enzimas apresentam estabilidade em diversos solventes orgânicos, podendo ser aplicadas como biocatalisadores em vários processos anteriormente realizados apenas por catalisadores químicos. Este trabalho teve como objetivo produzir, purificar e imobilizar lipases de Staphylococcus warneri EX17, cepa capaz de utilizar glicerol como fonte de carbono. Inicialmente, as condições de cultivo para produção de lipases foram otimizadas através de duas ferramentas de planejamento experimental: delineamento Placket Burman (P-B) e delineamento composto central rotacional (DCCR). Determinou-se que as melhores condições para produção desta enzima são: temperatura de 36 °C; pH 8,1; 30 g/L de glicerol; 3,0 g/L de óleo de oliva e 2,5 g/L de óleo de soja. Também se verificou ser possível a utilização de glicerol residual, oriundo da síntese enzimática de biodiesel como fonte de carbono. O extrato enzimático mostrou-se estável em três solventes orgânicos testados (metanol, etanol e η-hexano). Ainda visando a otimização das condições de cultivo, foram realizados cultivos submersos em biorreatores a fim de estudar a influência da taxa volumétrica de transferência de oxigênio (kLa) e do controle do pH, na produção da enzima. A maior produção de lipases ocorreu quando aplicado um kLa de 38 h-1 e com o pH controlado em 7,0 ao longo do cultivo, o que permitiu aumentar a produção da enzima em 5 vezes, em relação ao obtido nas condições anteriormente empregadas. A purificação da lipase foi realizada baseando-se no mecanismo de ativação interfacial destas enzimas sobre superfícies hidrofóbicas. Duas resinas foram testadas, octil-Sepharose e butil- Toyopearl. A lipase produzida foi purificada em apenas um passo utilizando esta última resina. Foi estudada a hiperativação da lipase purificada na presença de detergentes, a atividade lipolítica foi aumentada em 2,5 vezes na presença de 0,1% de Triton X-100. A lipase purificada foi imobilizada através de três estratégias: adsorção em suporte hidrofóbico; união covalente unipontual e união covalente multipontual. A influência da imobilização na modulação das propriedades da enzima foi estudada. A lipase apresentou maior estabilidade quando imobilizada multipontualmente. A hidrólise de distintos ésteres quirais pelos diferentes biocatalisadores obtidos também foi estudada. Os ésteres utilizados foram: (±) mandelato de metila, (±)-2-O-butiril-2-fenilacético e (±)-2-hidroxi-4-fenilbutirato de etilo. A especificidade da enzima foi muito dependente do método de imobilização, sendo que a lipase imobilizada unipontualmente foi mais específica para o substrato (±)-2-hidroxi-4-fenilbutirato de etilo, enquanto que para os outros dois substratos foi a lipase adsorvida hidrofobicamente. Este estudo demonstrou que a lipase de S. warneri EX17 pode ser produzida utilizando glicerol residual como fonte de carbono, levando a diminuição do custo na produção da enzima, que apresenta propriedades bastante interessantes para sua aplicação em biocatálise. / Lipases (EC 3.1.1.3) constitute a group of enzymes that catalyze the hydrolysis and synthesis of triacylglycerols. These enzymes show stability in many organic solvents, being able to be used as biocatalysts in some processes that were once carried out only by chemical catalysys. The aim of this research was the production, purification, and immobilization of lipase by Staphylococcus warneri strain EX17 using glycerol as carbon source. Initially, the cultivation conditions for the production of lipases have been optimized through two statistical procedures, Plackett-Burman statistical design (PB) and central composite design (CCD). It was determined that the best conditions for this enzyme production are: temperature, 36 °C; pH, 8.1; glycerol, 30 g/L; olive oil, 3.0 g/L; and soybean oil, 2.5 g/L. It was also studied the use of raw glycerol from enzymatic synthesis of biodiesel as carbon source, and stability studies showed that this lipase from S. warneri EX17 was stable in methanol, ethanol and nhexane. Moreover, experiments were conducted in submerged bioreactors in order to study the influence of oxygen volumetric mass transfer rate (kLa) and the control of pH in the production of the enzyme. The higher lipase production occurred when the microorganism was submitted to a kLa of 38 h-1 and the pH controlled at 7.0 during the cultivation, which improved 5-fold the enzyme production, compared to the results obtained in shaker flasks. The lipase purification was carried out based on mechanisms of interfacial activation of these enzymes on hydrophobic surface. Two supports were tested, octyl-Sepharose and butyl-Toyopearl. The lipase produced was purified 20-fold in only one step of purification. The purified lipase was immobilized on cyanogens bromide activated agorese and its hyperactivation in the presence of detergents was studied. The lipolytic activity increased 2.5-fold in presence of 0.1% of Triton X-100. After this, lipase was immobilized by three strategies: adsorption on hydrophobic support, mild covalent attachment, and multipoint covalent attachment. The stability over thermal, organic solvent and detergent inactivation was verified, as well as the influence of the immobilization protocol in the modulation of the properties of the enzyme. The lipase showed higher stability when multipointly immobilized on glyoxyl agarose. The hydrolysis of different chiral esters by the three biocatalysts obtained was also studied. The esters used were: (±) methyl mandelate, ((±) methyl mandelate, (±)-2-O-butyryl-2-phenylacetic acid, (±)-2-hydroxy-4-phenyl-butyric acid ethyl ester. The specificity of the enzyme was highly dependent of the protocol of immobilization, and the lipase mildly immobilized on cyanogen bromide agarose was more specific to the hydrolysis of (±)-2-hydroxy-4-phenyl-butyric acid ethyl ester, while for the other two substrates was the lipase adsorbed on octyl agarose. This study demonstrated that the lipase from S. warneri EX17 can be produced using raw glycerol as carbon source, contributing to the reduction in production costs of the enzyme, and the enzyme, when immobilized on different supports, presented quite interesting properties that may be usefull as biocatalysts.