The production of hydrogen from low-cost abundant renewable biomass would be vital to sustainable development. Cell-free (in vitro) biosystems comprised of synthetic enzymatic pathways would be a promising biomanufacturing platform due to several advantages, such as high product yield, fast reaction rate, easy control and access, and so on. However, it is essential to produce (purified) enzymes at low costs and stabilize them for long periods to decrease biocatalyst costs. Thermophilic recombinant enzymes as building blocks were discovered and developed: fructose 1,6-bisphosphatase (FBP) from Thermotoga maritime, phosphoglucose isomerase (PGI) from Clostridium thermocellum, triose phosphate isomerase (TIM) from Thermus thermophiles and fructose bisphosphate aldolase (ALD) from T. maritima and T. thermophilus. The recombinant proteins were over-expressed in E. coli, purified and characterized. For purification and stabilization of enzymes, one-step, simple, low-cost purification and immobilization methods were developed based on simple adsorption of cellulose-binding module (CBM)-tagged protein on the external surface of high-capacity regenerated amorphous cellulose. Also, a simple, low-cost purification method of thermophilic enzymes was developed utilizing a combination of heat and ammonium sulfate precipitation. For development of cascade enzymes as building modules (biocatalyst modules), it was discovered that the presence of other enzymes/proteins had a strong synergetic effect on the stabilization of the thermolabile enzyme (e.g., PGI) due to the in vitro macromolecular crowding effect. And substrate channeling among CBM-tagged self-assembled three-enzyme complex (synthetic matabolon) immobilized on the easily-recycled cellulose-containing magnetic nanoparticles can not only increase cascade reaction rates greatly, but also decrease enzyme cost in cell-free biosystems. The high product yield and fast reaction rate of dihydrogen from sucrose was validated in a batch reaction containing fifteen enzymes comprising a non-natural synthetic pathway. The yield of dihydrogen production from 2 mM of sucrose was 96.7 % compared to theoretical yield at 37 °C. The maximum rate was increased 3.1 fold when the substrate concentration was increased from 2 to 50 mM in a fed-batch reaction. The research and development of cell-free biosystems for biomanufacturing require more efforts, especially in low-cost recombinant thermostable enzymes as building blocks, efficient cofactor recycling, enzyme and cofactor stabilization, and fast reaction rates. / Ph. D.

Este trabalho teve como objetivo sintetizar e caracterizar uma matriz híbrida estável de poli(estireno-co-divinilbenzeno) magnetizado pela adição de magnetita (Fe3O4) e avaliar seu potencial como suporte para a imobilização de lipases. A matriz híbrida foi sintetizado pela técnica de polimerização em suspensão utilizando dos monômeros de estireno e divinilbenzeno e ao qual foram adicionadas partículas de magnetita preparadas por coprecipitação dos íons Fe+2 e Fe+3. A caracterização foi realizada pelas técnicas de microscopia eletrônica de varredura (MEV), espectroscopia na região do infravermelho por transformada de Fourier (FTIR), difratometria de raios-X (DRX) e magnetização de amostra vibrante (VSM), comparando os materiais magnetizados e não magnetizados. Os biocatalisadores foram preparados pela imobilização da lipase de Candida rugosa (LCR) e lipase PS Burkholderia cepacia (LPS) via adsorção física e foram caracterizados em função da influência de pH e temperatura na atividade hidrolítica, parâmetros cinéticos, estabilidade térmica, estabilidade operacional e estabilidade de estocagem. O derivado de LCR foi aplicado em reações de esterificação e o derivado de LPS em reações de transesterificação. Os resultados obtidos pelas análises de FTIR, DRX e VSM confirmaram que a magnetita foi incorporada ao polímero, gerando atração das partículas por um campo magnético externo. A caracterização bioquímica indicou forte influência do pH na atividade hidrolítica, apresentando ponto ótimo próximo a 8,0 tanto para as lipases livres quanto imobilizadas. Os biocatalisadores magnetizados preparados apresentaram bom desempenho em todos os aspectos, o derivado da lipase de Candida rugosa alcançou conversões entre 89-94% nas reações de esterificação, revelando tempo de meia vida de t1/2=52 dias na estabilidade operacional. O derivado de Burkholderia cepacia atingiu rendimentos próximos a 80% nas reações de transesterificação com t1/2=40 dias. A imobilização aumentou a estabilidade térmica das lipases em 50 vezes no caso da LCR e em 2,3 vezes para a LPS. Estes resultados indicam que o material híbrido magnetizado sintetizado possui grande potencial para ser utilizado como suporte na imobilização de enzimas com aplicação em reações de interesse industrial. / This study aimed to synthesize and characterize a stable hybrid matrix of poly (styrene-codivinylbenzene) magnetized by the addition of magnetite (Fe3O4) and evaluate its potential for application in the immobilization of lipases, by characterization of the prepared biocatalysts. The support was synthesized by the suspension polymerization technique by applying styrene and divinylbenzene monomers and adding magnetite particles synthesized by co-precipitation of Fe + 2 and Fe + 3. The characterization of the material was performed by scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), vibrating sample magnetization (VSM), by comparison of the magnetized and the not magnetized particles. The biocatalysts were prepared by immobilization of lipase from Candida rugosa (CRL) and lipase from Burkholderia cepacia (Lipase PS) via physical adsorption and they were characterized according to the influence of pH and temperature on the hydrolytic activity, kinetic parameters, thermal stability, operational stability and storage stability. The CRL derivative was applied in esterification reactions and the lipase PS derivative was applied in transesterification reactions. The results obtained by the analysis FTIR, XRD and VSM confirmed the magnetite was successfully incorporated into the polymer and generated the atraction for an external magnetic field. Biochemical characterization indicated a strong influence of pH on the hydrolytic activity, showing better results on pH around 8,0 for free and both immobilized lipases. The magnetized biocatalysts prepared had good performance in all respects, derivative from Candida rugosa lipase reached 89-94% conversion in esterification reactions showing half-life of operational stability t1 / 2 = 52 days. The immobilized lipase from Burkholderia cepacia reached yields close to 80% in transesterification reactions presenting t1/2 = 40 days. Immobilization increased the thermal stability of lipase by 50 times in the case of CRL and 2,3 times for Lipase PS. These results indicate that the magnetized hybrid material synthesized has great potential to be used as a support for the immobilization of enzymes for use in reactions of industrial interest.

Bien que les matériaux poreux soient nombreux dans la nature, la synthèse en laboratoirede matériaux présentant une porosité multi-échelle ou hiérarchisée est toujours délicate. Enutilisant la matière molle (émulsions concentrées, auto-assemblages, mésophases lyotropes, etc)et le procédé sol-gel, il est possible d’obtenir une grande variété de matériaux monolithiques, àporosité hiérarchisée, composés d’un squelette silicique. La porosité de ces matériaux peut êtreoptimisée en jouant avec la nature de l’émulsion, le tensioactif utilisé, ou avec l’ajout d’agentd’extérieur comme le sel. En combinant ces méthodes, des matériaux possédant une mésoporositéhexagonale ont été obtenus. Grâce à leur surface riche en silanols, ces matériaux poreux ont étéfonctionnalisés par greffage post-synthèse de molécules organiques. Dès lors, l’immobilisationd’entités biologiques comme les enzymes au sein de la structure poreuse a permis d’utiliser cesmatériaux pour des réactions d’hydrolyse, de synthèse ou de décoloration en milieu aqueux dansune approche de « chimie verte ». Enfin, des micro-organismes ont été piégés dans ces matériauxporeux qui ont été recouverts d’une coque en silice. Les micro-organismes peuvent s’y développersans restriction et leur croissance est très différente de celle observée dans les cultures classiques.La coque en silice, formée en surface, est donc imperméable au passage des bactéries (taillemicrométrique) mais perméable à la diffusion des substrats et des réactifs. Cette diffusion a étémise à profit pour réaliser des réactions enzymatiques en cascade. Ces matériaux se positionnentcomme des biocatalyseurs très prometteurs pour de nombreuses applications. / Although porous materials are numerous in nature, the laboratory synthesis of materials withmulti-scale or hierarchical porosity is always difficult. By using soft matter (concentrated emulsions,self-assemblies, lyotropic mesophases, etc.) and the sol-gel process, it is possible to obtaina wide variety of monolithic materials with hierarchical porosity composed of a silicic skeleton.The porosity of these materials can be optimized by playing with the nature of the emulsion,the surfactant used, or with the addition of external agents such as salt. By combining these methods,materials with hexagonal mesoporosity have been obtained. Thanks to their silanol-richsurface, these porous materials have been functionalized by post-synthesis grafting of organicmolecules. Therefore, the immobilization of biological entities such as enzymes within the porousstructure has made it possible to use these materials for hydrolysis, synthesis or discolorationreactions in aqueous media in a "green chemistry" approach. Finally, microorganisms were trappedin these porous materials which were covered with a silica shell. Microorganisms can growthere without restriction and their growth is very different from that observed in conventionalcultures. The silica shell formed on the surface is therefore impermeable to the passage of bacteria(micrometric size) but permeable to diffusion of substrates and reagents. This diffusion wasused to carry out cascade enzymatic reactions. These materials are positioned as very promisingbiocatalysts for many applications.

Melanina é um polímero constituído por uma grande heterogeneidade de monômeros tendo como característica comum a presença de grupos indóis. Por outro lado, a eumelanina produzida pela oxidação enzimática da tirosina é um polímero mais simples constituído principalmente de monômeros 5,6-dihidroxindol (DHI) e de indol-5,6-quinona (IQ). Tirosinase é a enzima chave na produção de melanina, sendo que a sua atividade cinética é medida em função da formação do intermediário dopacroma. Nanopartículas (NPs) de sílica são partículas nanométricas compostas de oxido de silício e são obtidas pelo processo sol-gel desenvolvido por Stöber de hidrólise e condensação de tetraetilortosilicato (TEOS), usando etanol como solvente em meio alcalino. As NPs foram funcionalizadas com 3-Aminopropiltrietoxissilano (ATPES) e depois com glutaraldeído. Este último permitiu a imobilização da tirosinase na superfície da sílica. Caracterizamos as NPs antes e após a reação da enzima, a atividade catalítica da enzima ligada à NP e o mecanismos de formação de melanina na superfície da sílica. As NPs foram caracterizadas por espectrofotometria de absorção e de reflectância, termogravimetria e microscopia eletrônica. A síntese da NP de sílica retornou partículas esféricas com 55nm de diâmetro e a funcionalização da partícula mostrou modificar eficientemente a sua superfície. A imobilização da tirosinase por ligação covalente foi de 99,5% contra 0,5% da adsorção física. A atividade da tirosinase foi caracterizada pela formação de dopacroma. O Km da enzima imobilizada não sofreu alteração em comparação com a tirosinase livre, mas a eficiência catalítica - que considera a eficiência recuperada - foi de apenas 1/3 para a enzima ligada covalentemente, significando que 2/3 das enzimas ligadas não estão ativas. Obtivemos NPs revestidas com melanina a partir de oxidação de tirosina solubilizada em duas preparações: NP com tirosinase ligada covalentemente na superfície e NP funcionalizada com glutaraldeido dispersa em solução de DHI e IQ. O revestimento de melanina foi na forma de um filme fino com espessura ~1,9nm, conferindo perfil de absorção luminosa equivalente ao da própria melanina. Mostramos que o mecanismo de polimerização passa pela oxidação da tirosina pela tirosinase, que gera intermediários oxidados (principalmente DHI e IQ) que vão para solução (mesmo quando a tirosinase está ligada covalentemente na sílica). Estes intermediários ligam-se ao glutaraldeido e a superfície da sílica passa a funcionar como ambiente de polimerização da melanina. / Melanin is a polymer consisting of a large heterogeneity of monomers having as a common feature the presence of indole groups. Contrarily, eumelanin produced by enzymatic oxidation of tyrosine is a simpler polymer consisting mainly of 5,6-dihidroxindol (DHI) and indole-5,6-quinone (IQ) monomers. Tyrosinase is the key enzyme in melanin production, and its kinetic activity is measured by the formation of the intermediate dopacroma. Nanoparticles (NPs) are made of silica nanoparticles of silicon oxide and are obtained by sol-gel method developed by Stöber of hydrolysis and condensation of tetraethylorthosilicate (TEOS), using ethanol as solvent in an alkaline medium. NPs were functionalized with 3-Aminopropyltriethoxysilane (ATPES) and then with glutaraldehyde. The latter allows the immobilization of tyrosinase on the silica surface. We characterized NPs before and after the reaction of the enzyme, the catalytic activity of the enzyme bound to the NP and melanin-forming mechanisms on the silica surface. NPs were characterized by absorption spectrophotometry and reflectance, electron microscopy and thermogravimetric analysis. The synthesis of silica NP returned spherical particles of 55nm diameter and particle functionalization showed efficiently modify its surface. The immobilization of tyrosinase by covalent bond was 99.5% versus 0.5% by physical adsorption. The activity of tyrosinase was characterized by the formation of dopacroma. The Km of the immobilized enzyme did not change compared to the free tyrosinase, but the catalytic efficiency - considering the recovered efficiently - was only 1/3 for the enzyme covalently bound, meaning that 2/3 of the enzymes are not connected active. We obtained melanin coated NPs from tyrosine oxidation in two preparations: NP with covalently bound tyrosinase in the NP surface and NP functionalized with glutaraldehyde dispersed in DHI and IQ solution. The melanin coating was in the form of a thin film with the thickness of ~ 1,9 nm, giving light absorption profile equivalent to that of melanin itself. We showed that the polymerization mechanism involves the oxidation of tyrosine by tyrosinase, which generates oxidized intermediates (especially DHI and lQ) that go into solution (even when tyrosinase is covalently bound to the silica). These intermediates bind the glutaraldehyde and the surface of the silica begins to function as an environment for melanin polymerization.

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / O objetivo deste trabalho foi estudar a preparaÃÃo de biocatalisadores utilizando lipase de Candida antarctica tipo B (CALB) imobilizada covalentemente em quitosana, uma matÃria-prima abundante e de baixo custo no CearÃ, em quitosana-alginato e em agarose, com o intuito de utilizÃ-los na sÃntese de Ãsteres de vitamina A. Diversas estratÃgias de imobilizaÃÃo foram realizadas com o intuito de obter um derivado com elevada atividade enzimÃtica e com alta estabilidade tÃrmica e operacional. TrÃs tipos de suportes (agarose, quitosana e quitosana-alginato) foram preparados a partir de tais estratÃgias, sendo que um estudo aprofundado foi realizado com dois desses suportes (quitosana e quitosana-alginato). Apenas uma estratÃgia de imobilizaÃÃo foi realizada com agarose para testÃ-lo na sÃntese de palmitato de retinila, juntamente com dois derivados comerciais (lipase imobilizada de Thermomyces lanuginosus (Lipozyme TL IM) e lipase imobilizada de Mucor miehei (Lipozyme RM IM)), com o objetive de definir algumas condiÃÃes operacionais. Uma condiÃÃo avaliada que apresentou bons resultados na sÃntese foi o uso de peneira molecular para a retirada de Ãgua no meio reacional, sendo, portanto, utilizada nos estudos posteriores. ApÃs os estudos de imobilizaÃÃo e estabilidade tÃrmica a 60 ÂC, dois derivados (J8: quitosana ativada com glicidol seguido de etilenodiamina (EDA) e glutaraldeÃdo, e G10: quitosana-alginato ativada com glutaraldeÃdo) foram escolhidos, por apresentarem maiores atividades especÃficas (422,44 Â 50,4 U/g e 378,30 Â 34,7 U/g, respectivamente) e melhores estabilidades tÃrmicas (fatores de estabilizaÃÃo de 10,25 e 29,00, respectivamente), para estudos de estabilidade operacional de hidrÃlise e para sÃntese de palmitato de retinila. O derivado que apresentou melhor estabilidade tÃrmica a 60ÂC foi o G10, CALB imobilizada em quitosana-alginato, sendo aproximadamente 29 vezes mais estÃvel que a enzima solÃvel, e mais de 2 vezes mais estÃvel do que a enzima comercial Novozyme 435. PorÃm, o derivado J8 apresentou melhor estabilidade operacional de hidrÃlise, semelhante ao derivado comercial Novozyme 435. Um planejamento experimental 22 foi realizado para se avaliar a sÃntese de palmitato de retinila. Avaliou-se a influÃncia da temperatura (37 ÂC e 45 ÂC) e da razÃo entre os substratos, retinol:Ãcido palmÃtico (1:3 e 1:5), no rendimento de sÃntese, catalisada pelo derivado J8. Uma reaÃÃo utilizando o derivado G10 utilizando a melhor condiÃÃo do planejamento experimental foi realizada para ver o comportamento desse derivado. Com uma anÃlise estatÃstica dos resultados, pÃde-se observar que a razÃo entre os substratos teve efeito significativo no rendimento de sÃntese. Maiores foram obtidos quando a razÃo entre substratos foi igual a 1:5. Como os resultados nas temperaturas de 37 ÂC e 45 ÂC foram semelhantes, selecionou-se a temperatura de 37 ÂC para reaÃÃes posteriores, por necessitar de um menor gasto de energia para atingi-la / The objective of this work was to study the preparation of biocatalysts using lipase of Candida antarctica type B (CALB) covalently immobilized in agarose, chitosan, an abundant and low cost raw material, to be used in the synthesis of ester of Vitamin A. Several strategies of immobilization were studied in order to obtain a biocatalyst with good enzymatic activity and high thermal and operational stabilities. Three types of supports (agarose, chitosan and chitosanalginate) were activated by different strategies, but most of attention was given to the supports chitosan and chitosan-alginate. Only one derivative was prepared by immobilizing CALB in agarose and results of synthesis were compared to commercial derivatives (immobilized lipase of Thermomyces lanuginosus - Lipozyme TL IM - and immobilized lipase of Mucor miehei - Lipozyme RM IM), for the definition of some operational conditions. The operational condition that presented good results in the synthesis was used in further studies, such as removal of water from the reacional media by molecular sieves. After immobilization and thermal stabilities at 60 ÂC tests, two derivatives (J8: chitosan actived with glicidol follow by EDA and glutaraldehyde; G10: chitosan-alginate actived with glutaraldehyde) were selected: the ones that presented higher specific activities (422.44 Â 50.4 U/g and 378.30 Â 34.7 U/g, respectively) and best thermal stabilities (factors of stabilization of 10.25 and 29.0, respectively). Operational hydrolytic stabilities and the performance of these biocatalysts on the synthesis of retinyl palmitate were evaluated. One factorial design 22 was carried out to evaluate the synthesis of retinyl palmitate. The influence of the temperature (37 ÂC and 45 ÂC) and ratio between substrates concentration, retinol: palmitic acid (1:3 and 1:5), in the yield of synthesis, catalyzed for the J8 derivative, were evaluated. A statistical analysis of the results showed that the the most significant effect was the rate of substrates concentration. Higher yields of synthesis were obtained when the ratio of substrates concentration was equal to 1:5. Results of reaction yields at 37ÂC and 45 ÂC were very similar. Therefore, 37 ÂC was selected for further studies. Best results for thermal stability at 60ÂC were obtained for G10, CALB immobilized in chitosan-alginate, being approximately 29-fold more stable than soluble enzyme, and 2-fold more stable than the commercial enzyme (Novozyme 435). On the other hand, J8, CALB immobilized in chitosan, presented higher operational hydrolysis stability, with a similar deactivation profile to Novozyme 435

Este trabalho teve como objetivo sintetizar e caracterizar uma matriz híbrida estável de poli(estireno-co-divinilbenzeno) magnetizado pela adição de magnetita (Fe3O4) e avaliar seu potencial como suporte para a imobilização de lipases. A matriz híbrida foi sintetizado pela técnica de polimerização em suspensão utilizando dos monômeros de estireno e divinilbenzeno e ao qual foram adicionadas partículas de magnetita preparadas por coprecipitação dos íons Fe+2 e Fe+3. A caracterização foi realizada pelas técnicas de microscopia eletrônica de varredura (MEV), espectroscopia na região do infravermelho por transformada de Fourier (FTIR), difratometria de raios-X (DRX) e magnetização de amostra vibrante (VSM), comparando os materiais magnetizados e não magnetizados. Os biocatalisadores foram preparados pela imobilização da lipase de Candida rugosa (LCR) e lipase PS Burkholderia cepacia (LPS) via adsorção física e foram caracterizados em função da influência de pH e temperatura na atividade hidrolítica, parâmetros cinéticos, estabilidade térmica, estabilidade operacional e estabilidade de estocagem. O derivado de LCR foi aplicado em reações de esterificação e o derivado de LPS em reações de transesterificação. Os resultados obtidos pelas análises de FTIR, DRX e VSM confirmaram que a magnetita foi incorporada ao polímero, gerando atração das partículas por um campo magnético externo. A caracterização bioquímica indicou forte influência do pH na atividade hidrolítica, apresentando ponto ótimo próximo a 8,0 tanto para as lipases livres quanto imobilizadas. Os biocatalisadores magnetizados preparados apresentaram bom desempenho em todos os aspectos, o derivado da lipase de Candida rugosa alcançou conversões entre 89-94% nas reações de esterificação, revelando tempo de meia vida de t1/2=52 dias na estabilidade operacional. O derivado de Burkholderia cepacia atingiu rendimentos próximos a 80% nas reações de transesterificação com t1/2=40 dias. A imobilização aumentou a estabilidade térmica das lipases em 50 vezes no caso da LCR e em 2,3 vezes para a LPS. Estes resultados indicam que o material híbrido magnetizado sintetizado possui grande potencial para ser utilizado como suporte na imobilização de enzimas com aplicação em reações de interesse industrial. / This study aimed to synthesize and characterize a stable hybrid matrix of poly (styrene-codivinylbenzene) magnetized by the addition of magnetite (Fe3O4) and evaluate its potential for application in the immobilization of lipases, by characterization of the prepared biocatalysts. The support was synthesized by the suspension polymerization technique by applying styrene and divinylbenzene monomers and adding magnetite particles synthesized by co-precipitation of Fe + 2 and Fe + 3. The characterization of the material was performed by scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), vibrating sample magnetization (VSM), by comparison of the magnetized and the not magnetized particles. The biocatalysts were prepared by immobilization of lipase from Candida rugosa (CRL) and lipase from Burkholderia cepacia (Lipase PS) via physical adsorption and they were characterized according to the influence of pH and temperature on the hydrolytic activity, kinetic parameters, thermal stability, operational stability and storage stability. The CRL derivative was applied in esterification reactions and the lipase PS derivative was applied in transesterification reactions. The results obtained by the analysis FTIR, XRD and VSM confirmed the magnetite was successfully incorporated into the polymer and generated the atraction for an external magnetic field. Biochemical characterization indicated a strong influence of pH on the hydrolytic activity, showing better results on pH around 8,0 for free and both immobilized lipases. The magnetized biocatalysts prepared had good performance in all respects, derivative from Candida rugosa lipase reached 89-94% conversion in esterification reactions showing half-life of operational stability t1 / 2 = 52 days. The immobilized lipase from Burkholderia cepacia reached yields close to 80% in transesterification reactions presenting t1/2 = 40 days. Immobilization increased the thermal stability of lipase by 50 times in the case of CRL and 2,3 times for Lipase PS. These results indicate that the magnetized hybrid material synthesized has great potential to be used as a support for the immobilization of enzymes for use in reactions of industrial interest.

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.

In this study, glucose oxidase and polyphenol oxidase were immobilized in conducting copolymer poly(N-(4-(3-thienyl methylene)-oxycarbonylphenyl)maleimide)-co-pyrrole(P(MBThi-co-Py)). A copolymer was electrochemically synthesized by using sodium dodecyl sulfate (SDS) as supporting electrolyte and characterized by FTIR, scanning electron microscopy (SEM) and conductivity measurements. Immobilization of glucose oxidase (GOD) and polyphenol oxidase (PPO) enzymes were performed in conducting PPy and P(MBThi-co-Py) matrices by electropolymerization. Kinetic parameters, maximum reaction rate (Vmax) and Michaelis-Menten constant (Km) were determined for the enzyme electrodes by help of Lineweaver-Burk plot. Effect of temperature and pH on GOD and PPO activity was examined. Operational stability and long term stability of the enzyme electrodes were investigated. The immobilized GOD and PPO electrodes were used for determination of glucose amount in Turkish orange juices and analyzing the concentration of phenolic compounds in Turkish red wines respectively.

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.

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.