Orientador: Rubens Monti / Banca: Edwil Aparecida de Lucca Gattás / Banca: Adriane Maria Ferreira Milagres / Banca: Raquel de Lima Camargo Giordano / Banca: Benevides Costa Chitunda Pessela / Resumo: Neste trabalho estudou-se a possibilidade de imobilização de uma amilase produzida por cepa de Neurospora crassa (mutante exo-1), e produção de derivados ativos e estabilizados. Foram testados diferentes suportes sólidos, incluindo-se diferentes suportes de agarose e suportes epóxidos preparados com Eupergit e Sepabeads. Além da agarose 10BCL foi utilizada a agarose 4BCL para que se verificasse possíveis dificuldades difusionais do substrato desta enzima, o amido. O acompanhamento das cinéticas de imobilização com a maltose como alternativa ao amido, também colaborou em evidenciar a dificuldade de difusão do amido através de ambos os suportes glioxil-agarose. O derivado obtido com agarose 10BCL, assim como aquele produzido com uso de suporte Eupergit foram os derivados mais estáveis, capazes de manter 100% de suas atividades após 12 horas de incubação à temperatura de 60°C, quando na forma solúvel a enzima conservou apenas 12% de sua atividade inicial. Quando incubados a 70°C destacou-se o derivado de glioxil-agarose (10BCL) como mais estável, mantendo cerca de 30% de sua atividade inicial após 4 horas de incubação. Quando testada a utilização de uma agarose comercial alternativa, sem percentual de crosslink conhecido, de menor custo, sua aplicação mostrou-se promissora e os derivados produzidos além de ativos se apresentam estáveis frente à temperatura. Em conjunto, as informações contidas no presente estudo indicam que a amilase de Neurospora crassa apresenta-se promissora em comparação às amilases de mercado aqui estudadas, tanto em sua utilização na forma solúvel quanto no que se relaciona a produção de derivados estáveis. / Abstract: In this work were studied the immobilization of amylase from Neurospora crassa (Mutant Exo-1). This amylase showed easily production, purification and high capacity of immobilization on agarose and epoxy supports. It was used crosslinked agarose with two polymer concentration: 4 and 10%. The 4BCL agarose presents bigger porous diameter than 10 BCL agarose, so, in this case, possible diffusion problems of the starch across the supports could be reduced. Also, in this study, we have tested two epoxy supports for this amylase immobilization, using Eupergit and Sephabeads supports. The activies of the obtained derivatives were measured using two substrates - maltose and starch. Both glyoxyl agarose support prepared with 4BCl and 10BCL agarose present diffusion problems when the starch was used as substrate to measure the immobilization course. The 10BCL glyoxyl derivative presented the highest thermal stability when comparing the others derivatives. Among the epoxy derivatives the Eupergit one were better than the derivatives obtained using Sepabeads as support. In a confrontation between the two best derivatives, that is, the glyoxyl 10 BCL and Eupergit derivatives, both of them were stable at 60º incubation, maintaining 100% of activities for 12 hours, while the soluble amylase preserved about 12% of initial activity. These two amylase derivatives only showed differences at 70ºC incubation, when the glyoxyl 10BCL amylase derivative was more thermally stable, preserving about 30% of the initial activity after 4 hours. / Doutor

The limited availability of fossil resourses mandates the development of new energy vectors, which is one of the Grand Challenges of the 21st Century [1]. Biocatalytic energy conversion is a promising solution to meet the increased energy demand of industrialized societies. Applications of biocatalysis in the gas-phase are so far limited to production of fine chemicals and pharmaceuticals. However, this technology has the potential for large scale biocatalytic applications [2], e.g. for the formation of novel energy carriers. The so-called solid/gas biocatalysis is defined as the application of a biocatalyst immobilized on solid-phase support acting on gaseous substrates [3]. This process combines the advantages of bio-catalysis (green chemistry, mild reaction conditions, high specicity & selectivity) and heterogeneous dynamic gas-phase processes (low diffusion limitation, high conversion, simple scale-up). This work presents the modifications of a PID Microactivity Reference reactor in order to make it suitable for solid/gas biocatalysis. The reactor design requirements are based on previously published laboratory scale solid/gas systems with a feed of saturated vapors [4]. These vapors are produced in saturation flasks, which were designed and optimized during this project. Other modifications included relocation of the gas mixing chamber, redesigning the location and heating mechanism for the reactor tube, and heating of the outlet gas line. The modified reactor system was verified based on the Candida antarctica lipase B catalyzed transesterication of ethyl acetate with 1-hexanol to hexyl acetate and ethanol and results were compared to liquid-phase model reactions. Products were analyzed on line by a gas chromatograph with a flame ionization detector. C. antarc- tica physisorbed on silica particles produced a 50% conversion of hexanol at 40 C in the gas-phase. A commercial immobilized lipase from Iris Biotech produced 99% and 97% conversions of hexanol in similar experiments. This project achieved its goal to design, establish and successfully verify a solid/- gas biocatalysis reactor. Future work will target optimization of the reactor's operating conditions and the development of whole cell catalysts for energy production reactions. Potential experiments include the study of hydrogenolytic carbon dioxide reduction to methanol by free enzymes or methanogenic organisms [5], and the investigation of hydrogen production by water splitting of algae or cyanobacteria.

Immobilization of Electrocatalytically Active Gold Nanoparticles on Nitrogen-Doped Carbon Fiber Electrodes ABSTRACT Recently, immobilization of single metal nanoparticles on nanometer-sized electrodes has been demonstrated as a means to electrochemically probe the relationship between nanoparticle structure and function. Such studies of individual, isolated nanoparticles enable investigation of electrochemical behavior and electrocatalytic properties in the absence of complicating factors like interparticle distance and nanoparticle loading that are typically associated with collections of particles distributed on electrode supports. However, interpretation of electrochemical data obtained from single nanoparticle immobilization experiments can also be difficult since the underlying nanoelectrode platform can sometimes contribute to the measured current or the immobilization strategy may have adverse effects on electron transfer. Here we report immobilization of gold nanoparticles on relatively catalytically inert carbon fiber ultramicro- and nanoelectrodes through a modification method based on recently reported soft nitriding process found to be effective in attaching ligand-free ultrasmall noble metal catalysts to activated carbons. X-ray photoelectron spectroscopy results reveal that the nitriding of carbon fibers provides 3.5 times increase in surface nitrogen content, introducing mostly pyridinic and amine nitrogen groups. The nitrogen-containing surface sites proved to be beneficial to the deposition of gold nanoparticles (AuNPs), as sodium borohydride reduction of tetrachloroaurate resulted in attachment of AuNPs on nitrided carbon fiber ultramicroelectrodes (N-CF-UMEs) in as little as 10 seconds while immobilization of AuNPs on unmodified CF-UMEs required at least 12 hours. A recently reported electrochemical method was employed to characterize immobilized AuNPs, and AuNP size was found to be directly related to deposition time. AuNPs immobilized on N-CF-UMEs also exhibited electrocatalytic activity towards methanol oxidation. Reduction of electrode size will enable this strategy to be employed to investigate electrochemical behavior of individual gold nanoparticles, while the ligand-free nature of the immobilized particles also provides the opportunity to investigate effects of surface capping agents on electrocatalytic properties.

No description available.

No description available.

The purpose of this study was to determine the effects of short-term (14d) unilateral leg immobilization using a simple knee brace (60° flexion)/crutchmediated model on muscle function and morphology in men (M, N=13) and women (W, N=14). Isometric and isokinetic (concentric SLOW, 0.52 rad•s-1 and FAST, 5.24 rad•s-1) knee extensor peak torque was determined at three time points (PRE, DAY -2, and DAY -14). At the same time points, magnetic resonance imaging was used to measure the cross-sectional area of the quadriceps femoris and DEXA scanning was used to calculate leg lean mass. Muscle biopsies were taken from vastus lateralis at PRE and DAY-14 for myosin ATPase and myosin heavy chain analysh Women showed greater decreases (PRE vs. DAY-14) compared to men in specific strength (N•cm-2) for isometric (M=3.1±13.3, W=17.1±15.9%; p= 0.055, [mean±SD]) and concentric SLOW (M=4.7±11.3, W=16.6±18.4%; p<0.05) contractions. There were no immobilization-induced sex-specific differences in the decrease in quadriceps femoris cross-sectional area (M=5.7±5.0, W=5.9±:5.2%) or leg lean mass (M=3.7±4.2, W=2.7±2.8%). There were no fiber type transformations, and the decrease in Type I (M=4.8±5.0, W=5.9±3.4%), IIa (M=7.9±9.9, W=8.8±8.0%) and Ilx (M=10.7±10.8, W=10.8±12.1 %) fiber areas was similar between sexes. These findings indicate that immobilization-indt: ced loss of knee extensor muscle strength is greater in women compared tc men in spite of a similar extent of atrophy at the myofiber and whole muscle levels after only 14d of unilateral leg immobilization. Furthermore, we have described an effective and safe method of knee immobilization that results in significant reductions in quadriceps muscle strength and fiber size. / Thesis / Master of Science (MS)

photosynthetic bacteria is attractive field as production is fueled by solar energy. Hydrogen production potential of two photosynthetic bacteria R.capsulatus (DSM1710 wild type and R.capsulatus YO3 Hup- uptake hydrogenase deleted mutant strain) were examined in agar immobilized systems. In the present work agar and glutamate concentrations were optimized for immobilization of bacteria while feeding bacteria with 40/2-4 mM acetate/ glutamate. Immobilized bacteria produced hydrogen for 420-1428 hours covering 5-7 rounds. Optimizing of acetate concentration indicated that 60 mM produced the highest observed yield around 90-95%. Results shown that 2.5 mg dry cell weight/mL is the optimum cell concentration for wild type strain while 5 mg dry cell weight/mL was optimum for YO3 strain. Using either glycerol or sodium dithionite caused decrease in hydrogen production capacity of immobilized bacteria. It was observed that agar provided protection against inhibition effect of ammonium. Co- v immobilization of bacteria with packed cells of H. salinarium increased total hydrogen production capacity by about 1.14-1.41 folds. Hydrogen production by immobilized bacteria in panel photobioreactor was achieved by a novel system which allowed long term hydrogen production. Immobilized R. capsulatus DSM 1710 in panel reactor worked for about 67-82 days covering 4-5 rounds while immobilized R. capsulatus YO3 worked for 69-72 days covering seven rounds.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / Immobilization of enzymes provides advantages such as increased in: stability, level of process control, yield and purity of the final product, in addition to allowing the use of different reactor configurations. In this study, two types of support, an organic polymer (polimetilmetacrilato- PMMA) and other inorganic (silica) were used for the immobilization of lipase Candida antarctica type B (CALB). Different alcohols (ethanol, iso-propanol, n-butanol) were evaluated as agents for optimization of the immobilization process, the concentrations of 5, 10 and 15% (volume of alcohol à 100 / solution volume immobilization). Immobilization of PMMA was conducted using two different protocols here denominated first and second protocol generation. The best results of hydrolytic activity and esterification to the derivative of the first generation was 3,91  0,09 UpNPB/g. of catalyst e 409,5  19,7 Uoleic acid/g. of catalyst, respectively. When using the protocol of the second generation, the best results were obtained when aa immobilization was carried out in the presence of 5% Triton X-100 2 mM, rising rate of the esterification reaction between the derivatives studied was highly significant with a higher ratio of 3,81 times to the derivative prepared with Triton X-100. The CALB was also immobilized for the macroporous silica support (IB S60S - Chiralvision), the activate agent used was Triethoxy(octyl)silane (C8-TEOS). The results observed for immobilization in the presence of 5% n-butanol were the best for both hydrolytic activity, being four times higher than the activity for the immobilized derivative without alcohol, as for the esterification activity was 985,1  5,6 Uoleic acid/g. of catalyst . All observed derivatives supported on silica and PMMA reached a level conversion for equilibrium esterification reaction for a reaction time of 24 h the best results varying between 80 to 90%. / A imobilizaÃÃo das enzimas traz vantagens como: aumento da estabilidade, do nÃvel de controle do processo, do rendimento e da pureza do produto final, alÃm de possibilitar o uso de reatores de diferentes configuraÃÃes. Neste trabalho, dois tipos de suporte, um polÃmero orgÃnico (polimetilmetacrilato- PMMA) e outro inorgÃnico (sÃlica), foram utilizados para a imobilizaÃÃo da lipase do tipo B de Candida antarctica (CALB). Diferentes Ãlcoois (etanol, iso-propanol, n-butanol) foram avaliados como agentes de otimizaÃÃo do processo de imobilizaÃÃo, nas concentraÃÃes de 5, 10 e 15 % (volume de Ãlcool Ã100 / volume da soluÃÃo de imobilizaÃÃo). A imobilizaÃÃo em PMMA foi conduzida atravÃs de dois protocolos diferentes, aqui denominados de protocolo de primeira e segunda geraÃÃo. Os melhores resultados de atividade hidrolÃtica e de esterificaÃÃo para o derivado de primeira geraÃÃo foi de 3,91  0,09 UpNPB/g. de catalisador e 409,5  19,7 UÃc. olÃico/g. de catalisador, respectivamente. Quando se utilizou o protocolo de segunda geraÃÃo, melhores resultados foram obtidos quando a a imobilizaÃÃo foi conduzida na presenÃa de n-butanol 5 % e Triton X-100 2mM; o aumento da velocidade da reaÃÃo de esterificaÃÃo entre os derivados estudados foi bastante significativo, sendo 3,8 vezes maior para o derivado preparado com Triton X-100. A CALB foi imobilizada tambÃm para o suporte sÃlica macroporosa (IB S60S - Chiralvision), o agente ativante utilizado foi o octiltrietoxisilano (C8-TEOS). Os resultados observados para imobilizaÃÃo na presenÃa de n-butanol 5 % foram os melhores tanto para a atividade hidrolÃtica, sendo quatro vezes maior que a atividade para o derivado imobilizado sem Ãlcool, quanto para a atividade de esterificaÃÃo que foi de 985,1  5,6 UÃc. olÃico/g. de catalisador. Todos os derivados observados suportados em PMMA e sÃlica atingiram um patamar de conversÃo do equilÃbrio para reaÃÃo de esterificaÃÃo por um perÃodo de reaÃÃo de 24 h com os melhores resultados variando entre 80 a 90%.

The demand for economical, efficient protein production, reuse, and recovery has never been greater due to their versatility in a large variety of applications ranging from industrial chemical manufacturing to pharmaceutical drug production. The applications for naturally and artificially produced proteins include protein drugs and other pharmaceutical products, as biocatalysts in environmentally friendly chemical manufacturing, as enzymes for food processing purposes, and as an essential component in many biomedical devices. However, protein production suffers from many challenges, which include the cost of production, protein stability especially under harsh conditions, and recoverability and reusability of the proteins. The combination of two developing technologies, cell-free protein synthesis systems (CFPS) and unnatural amino acid incorporation, provides solutions to these protein production challenges.This dissertation reports on the use of cell-free protein synthesis systems and unnatural amino acid incorporation to develop new proteins and enzyme immobilization techniques that significantly increase activity and stability while simplifying recoverability and reuse.

Amine transaminases (ATA) catalyse the transfer of an amino group from one molecule and replaces a ketone or aldehyde with the amino group, the amino group on the amino-donor is replaced with a ketone or aldehyde. This enzyme, ATA from Chromobacterium violaceum, has previously been used to catalyse the reaction involving amphetamine, therefore, it might be possible to use this enzyme to convert amphetamine and the product absorbs in the UV spectrum and can therefore be measured spectrophotometrically. The aim of the project was to explore the possibility of using ATA in a portable biosensor for the detection of amphetamine. A literature study of commercially available portable biosensors was performed, activity of the free enzyme was tested against two substrates, methylbenzylamine (MBA) and amphetamine. Research on immobilization techniques, materials, and surface functionalization was done to chose suitable methods for immobilizing ATA. Two immobilization methods were suggested and one of the methods, ionic immobilization through His-tag towards Ni2+ on the surface, was tested for enzyme activity toward MBA. The enzyme activity of the free enzyme in solution towards MBA was comparable to previously reported enzyme activity, however, no enzyme activity towards amphetamine was observed. No activity was observed for the immobilized enzyme, but it might be due to the experimental design, more experiments need to be performed to draw conclusions. / Amintransaminaser (ATA) katalyserar överförandet av en amingrupp från en molekyl och ersätter en keton eller aldehyd med den amingruppen, amingruppen på amin-donatorn ersätts med en keton eller aldehyd. Det här enzymet, ATA från Chromobacterium violaceum (CvATA), har tidigare använts för att katalysera en reaktion som involverar amfetamin, därför skulle detta enzym kunna användas på amfetamin. Produkten av reaktionen absorberar i UV spektrumet och kan mätas med en spektrofotometer. Målet med projektet var att utforska möjligheten av att använda CvATA i en biosensor för att detektera amfetamin. En litteraturstudie på kommersiellt tillgängliga bärbara biosensorer genomfördes, aktiviteten av det fria enzymet testades mot två substrat, metylbenzylamin (MBA) och amfetamin. Information samlades om immobiliseringstekniker, material, och ytfunktionalisering gjordes för att välja ut lämpliga metoder för immobilisering av CvATA. Två immobiliseringsmetoder föreslogs och en av metoderna, immobilisering via enzymets His6-tagg och Ni2+ joner på ytan, testades för enzymaktivitet mot MBA. Enzymaktiviteten av det fria enzymet i lösning mot MBA var i samma storleksordning som tidigare rapporterad enzymaktivitet, men ingen enzymaktivitet mot amfetamin kunde observeras. Ingen aktivitet kunde observeras för det immobiliserade enzymet, men det kan vara på grund av designen på experimentet, fler experiment behöver göras för att kunna dra några fler slutsatser.