Orientador: Paulo Roberto Bueno / Banca: Ronaldo Censi Faria / Banca: Paulo Olivi / Resumo: Este trabalho está centrado no desenvolvimento, caracterização e utilização de uma superfície composta por monocamada auto-organizada (self assembled monolayer - SAM) de L-cisteína, funcionalizada com a enzima metalo-β-lactamase (MβL). A função designada a este sensor foi de detecção da velocidade da cinética enzimática, em tempo real, da reação da penicilinase com seu substrato, um antibiótico β-lactâmico, por técnica piezelétrica, microbalança a cristal de quartzo (QCM), e por técnicas eletroquímicas. As curvas de progresso foram obtidas pela variação da frequência no cristal de quartzo, demonstrando a cinética em tempo real. Não foram obtidas curvas com perfil hiperbólico, como esperado pelo modelo de Michaelis-Menten, por ambas as técnicas devido às baixas concentrações de substrato utilizadas nas análises. Foram obtidos gráficos com padrões lineares e a partir deles foi possível calcular a constante de Michaelis-Menten ( ). A obtida a partir da técnica eletroquímica mostrou-se mais próxima ao valor fornecido pelo fabricante da enzima. No entanto, pela técnica piezelétrica a foi maior, refletindo em perda de sensibilidade da enzima pelo substrato. / Abstract: The aim of this study was to develop, characterize, and apply the functionalized tranducer imobilized with metalo-β-lactamase (MβL) enzyme using appropriate procedures based on L-cysteine self-assembling monolayer-SAM. The enzymatic activity of MBL under β-lactam antibiotic was measured, on real time, by piezoeletric quartz crystal microbalance (QCM) and eletrochemical techniques. The progress curves were obtained by varying the frequency of quartz crystal. The hyperbolic pattern corresponding to the pattern set on the model of the Michaelis-Menten was not obtained due to low concentration of substrate. On the other side, linear graphic was obtained and the Km was calculated. The Km obtained from electrochemical analyses showed a value similar to manufacturer's instructions of fabricant, and the Km obtained by QCM showed higher, reflecting the lost of sensibility. / Mestre

Na maioria dos estudos de digestibilidade de aminoácidos em suínos usam-se animais de peso igual ou superior a 20 kg, isso se deve a menor dificuldade na implantação de cânulas íleo-cecais e a melhor recuperação pós-cirúrgica na fase do crescimento. No entanto, avaliar ingredientes com leitões mais jovens torna-se importante, visto que há limitações fisiológicas do trato gastrointestinal nesse período que podem afetar o seu desenvolvimento. Assim, 175 leitões desmamados, divididos em 7 ensaios experimentais com 25 animais cada, foram usados para determinar o balanço de nutrientes e da energia, a digestibilidade ileal aparente (AID) e estandardizada (SID) dos aminoácidos (AA) de sete ingredientes usualmente utilizados em dietas de leitões, com ou sem suplementação de multi-carboidrase (MC) e fitase (F). Os leitões foram desmamados aos 23 dias de idade e alojados em gaiolas para estudo de digestibilidade e metabolismo, permanecendo no experimento até os 45 dias de idade. Adaptação, coleta total de fezes e urina ocorreram do 10° ao 20° dia do período experimental e as amostragens do conteúdo ileal se deu ao abate, no 22° dia (45 dias de idade). Foi utilizado o delineamento experimental inteiramente casualisado com 4 tratamentos e cinco repetições. O leitão foi considerado como unidade experimental. As dietas experimentais consistiram de ingrediente teste sem enzimas e combinado a MC, F ou MC + F. Dois tipos de dieta referência foram usados como base para os cálculos do balanço nutricional e os coeficientes de digestibilidades aparente e estandardizada dos aminoácidos. Óxido de cromo (0,3%) foi usado como marcador indigestível nas avaliações dos aminoácidos. A enzima MC apresentava 10% de galactomananase, 10% de xilanase, 10% de β-glucanase, 60% de cevada maltada e 10% de α-galactosidase. A F era proveniente da fermentação de Saccharomyces cerevisiae com atividade de 10.000 FTU/g. Os ingredientes testados foram: farelo de arroz integral, soja integral micronizada, farelo de trigo, quirera de arroz, sorgo, milho e farinha de soja texturizada. Os resultados indicaram efeitos da combinação ingrediente e enzima exógena e as evidencias se deram de acordo com as características bromatológicas de cada um / Most of the studies, involving AA digestibility in pigs are used animals with 20 kg minimal weight or higher than. This is due difficulty to implant a simple T-cannula on distal ileum of younger pigs, besides a better post-surgical recovery in growing phase. However, ingredient evaluations with young pigs become important because there are physiological limitations in gastrointestinal tract that may affect its performance. Thus, 175 weaned pigs, divided into seven experimental trials with 25 animals each, were used to determine the nutrients and energy balance, the apparent (AID) and standardized (SID) ileal digestibility of amino acids (AA) from seven ingredients usually used in pig diets, with or without multi-carbohydrase (MC) and phytase (Phy) supplementation. Weaned piglets at 23 d old were housed in cages to studies digestibility and metabolism, remaining in the experiment until 45 d of age. Pig adaptation to feces and urine collection was 10 to 20 d experimental period and ileal content sampling at slaughter at 22 d (45 d old). A completely randomized experimental design was used with 4 treatments and 5 replicates. The pig was considered as an experimental unit. The experimental diets consisted of test ingredient as the sole source of protein with or without MC, Phy or MC+Phy. Two kind of reference diet were used to calculate the nutrient balance or AID and SID of AA. Titanium dioxide (0.3%) was used as indigestible marker. The MC enzyme had: galactomannanase, 10%; xylanase, 10%; beta-glucanase, 10%; malted barley, 60% and α-galactosidase, 10%. The Phy was obtained from the Saccharomyces cerevisiae fermentation with 10,000 FTU/g activity. The ingredients used were: rice bran, micronized full fat soybean meal, wheat bran, broken rice, sorghum, corn and texturized soybean meal. The results indicated effects from ingredients and enzymes combinations and the evidences occurred according to bromatological characteristics of each one

A transferência de oxigênio para o meio, durante o cultivo de Candida kefyr DSM 70106, mostrou ter grandE influência na produção de inulinase extracelular. O KLa (coeficiente volumétrico de transferência de oxigênio) de 43 h-1 foi o que proporcionou a obtenção de atividade enzimática mais elevada (37,5 U.mL-1). Este parâmetro foi utilizado como critério para ampliação de escala do cultivo em biorreator de 15 litros para 300 litros. O volume de meio obtido foi congelado para utilização nos processos seguintes de separação de células e purificação da inulinase. Para isto foram utilizados três processos diferentes, a saber, filtração (microfiltração, diafiltração e ultrafiltração), extração líquido-líquido por micela reversa e cromatografia de troca iônica em leito fluidizado. Durante a separação de células, a membrana de microfiltração tipo HVLP com 0,45 µm de diâmetro de poro foi a que possibilitou a passagem de maior fluxo de filtrado (324 L.h-1.m-2) quando comparada com outras membranas de poros e materiais diferentes. Associado a este fluxo obteve-se ainda a passagem pela membrana de 99% da enzima presente. Foram também estudados diferentes tipos de módulos de filtração. Os filtros rotativos foram os que apresentaram melhores desempenhos, com destaque para o tipo \"Biodruckfilter\". Fluxo de filtrado de 300 L.h-1.m-2 e transmissão de 99% da enzima foram obtidos neste filtro. Os filtros tipo cassete e os tubulares apresentaram desempenhos, em geral, inferior a 50% quando comparados em termos de fluxo de filtrado. O processo de filtração em escala ampliada, realizada no filtro cassete, apresentou fluxo de filtrado 45% inferior ao obtido na pequena escala. Os estudos de extração líquido-líquido por micela reversa foram feitos utilizando um agente tensoativo catiônico (BDBAC- [N-Benzyl-N-Dodecyl-N bis(2-hydroxyethyl) ammonium chloride] e um aniônico (AOT- sodium-di-2ethyl-hexyl-sulphosuccinate). O BDBAC proporcionou recuperação de 91 % da enzima presente inicialmente. Na ampliação de escala do processo foi verificado um rendimento de 77%, e o aumento da atividade enzimática específica, nos dois casos, foi de aproximadamente 2,8 vezes. Em comparação com os dois processos anteriores, o processo de purificação da inulinase com separação simultânea de células por cromatografia de trocaiônica em leito fluidizado foi o que proporcionou maior aumento da atividade enzimática específica (da ordem de 4,3 vezes). Após adsorção e eluição do leito cromatográfico, 93,1% da enzima foi recuperada. / It has been observed that the production of extracellular inulinase by Candida kefyr DSM 70106 was markedly affected by the rate of oxygen transfer to the medium. The highest inulinase activity (37,5 U.Ml-1) was attained at a KLa value (volumetric coefficient of oxygen transfer) equal to 43 h-1. As such, KLa was chosen as a criterion for scaling-up the bioreactor capacity from 15 L to 300 L. Once the fermentation ended, the whole fermented broth was frozen for further cell separation and inulinase purification. For that, three different processes were used, namely filtration (microfiltration, diafiltration and ultrafiltration), liquid-liquid extraction by reversed micelles and ion-exchange chromatography in fluidized bed. Microfiltration membranes differing in the chemical nature and on the pores diameter were used in cell separations. The highest filtrate flow (FF = 324 L.h-1.m-2) occurred when HVLP type membrane (0,45 µm pore diameter) was employed. Moreover, 99% of inulinase passed through the membrane. Several types of filtration modules were also studied. The rotatory filter, such as the \"Biodruckfilter\" (FF=300 L.h-1.m-2 and 99% of inulinase transmission), presented remarkable performance when compared with cassette and tubulartype filters, in which FF was lower than 50%. Furthermore, filtration carried out with a scaled-up cassette module presented a FF 45% lower than that observed on a smaller scale. Studies on liquid-liquid extraction by reverse micelles were made utilizing a cationic surfactant (BDBAC- [N-Benzyl-N-Dodecyl-N-bis(2-hydroxyethyl) ammonium chloride] and an anionic surfactant (AOT-sodium-di-2-ethyl-hexylsulphosuccinate). The use of BDBAC resulted in a recuperation of 91 % of the enzyme initially present, which decreased about 77% when the process was scaled-up. Neverthless, in both cases the increase in specific enzymatic activity was about 2.8 times. In comparison with the aforementioned processes, the ion-exchange chromatography in fluidized bed, in which inulinase purification and cell separation take place simultaneously, provided. an increase in specific enzymatic activity of about 4.3 times. After adsorption and elution of the chromatographic bed about 93% of the inulinase was recovered.

O processamento de celulose em moinho de bolas de porcelana produz um pó no qual se detecta a presença de radicais livres, por Ressonância Paramagnética Eletrônica. O espectro obtido e complexo e pode ser decomposto em pelo menos dois sinais: uma linha instável, eliminada apôs alguns dias de exposição ao ar, e outra estável, que decai lentamente por aquecimento a 400-500ºC, e desaparece a 900ºC. A moagem simultânea de celulose com amido, acrilamida, azul de dextrana e sacarose produz celulose quimicamente modificada. Por exemplo, no caso da acrilamida, dosagem pelo método kjeldahl revela a presença de ate 0,65% de nitrogênio no material obtido apôs lavagens exaustivas. A celulose moída na presença de vários reagentes foi submetida a ensaios de hidrólise e em alguns casos, mostrou-se significativamente mais suscetível ao ataque enzimático do que a celulose simplesmente moída. Os resultados deste trabalho mostram que a conversão enzimática de celulose em açucares redutores e sensível a uma pequena modificação no pré-tratamento mecanoquímico. / Cellulose processing on porcelain ball mill produce a powder in which one can detect free radicals by means of Electronic Paramagnetic Ressonance. The spectrum attained is complex and it may be decomposed in at least two signals: an unstable line that is eliminated after some days of exposition tc air and another stable line, which slowly decays due to heating at 400-500ºC and disapears at 900ºC. Cellulose simultaneously milled with starch, acrylamide, dextran blue and saccharose produce chemically modified cellulose. For instance, when using acrylamide one can detect, by means of the Kjeldhal method, the existence of nitrogen up to 0.65% in the material obtained after exhaustive washings. In some cases cellulose milled with several reactants seemed to be significantly more susceptible to the enzymatic attack during hydrolysis than cellulose milled without any reactant. The results of this work show that the enzymatic conversion of cellulose into reducible sugars is sensible to a little change in the mechano-chemical pre-treatment.

by Wong Yuk Hang. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1994. / Includes bibliographical references (leaves 110-114). / Abstract --- p.i / Acknowledgement --- p.iv / Dedication --- p.v / Table of Contents --- p.vi / List of Tables --- p.xi / List of Figures --- p.xii / Chapter Chapter 1 --- Introduction and literature review / Chapter 1.1 --- Introduction --- p.2 / Chapter 1.2 --- Literature review --- p.5 / Chapter 1.2.1 --- General considerations --- p.5 / Chapter 1.2.2 --- Nature of glucosinolate --- p.6 / Chapter 1.2.3 --- Degradation of glucosinolates by myrosinase --- p.7 / Chapter 1.2.4 --- Toxicology of glucosinolate and hydrolysis products --- p.8 / Chapter 1.2.5 --- Plant myrosinase --- p.9 / Chapter 1.2.6 --- Fungal myrosinase --- p.11 / Chapter 1.2.7 --- Purification and properties of fungal myrosinase --- p.11 / Chapter Chapter 2 --- Screening of fungi with myrosinase activity and physiological studies of myrosinase production in Aspergillus oryzae / Chapter 2.1 --- Introduction --- p.15 / Chapter 2.2 --- Materials and methods --- p.16 / Chapter 2.2.1 --- Fungal strains --- p.16 / Chapter 2.2.2 --- Media --- p.16 / Chapter 2.2.3 --- Screening --- p.17 / Chapter 2.2.4 --- Enzyme assay and protein determination --- p.18 / Chapter 2.2.4.1 --- Myrosinase assay --- p.18 / Chapter 2.2.4.2 --- Definition of myrosinase unit and specific activity --- p.19 / Chapter 2.2.4.3 --- Protein determination --- p.19 / Chapter 2.2.5 --- Physiological studies of myrosinase production in Aspergillus oryzae --- p.19 / Chapter 2.2.5.1 --- Incubation time --- p.20 / Chapter 2.2.5.2 --- Inducer concentration --- p.20 / Chapter 2.3 --- Results --- p.21 / Chapter 2.3.1 --- Screening --- p.21 / Chapter 2.3.1.1 --- Degradation of sinigrin in culture medium --- p.21 / Chapter 2.3.1.2 --- Confirmation of myrosinase activity --- p.21 / Chapter 2.3.2 --- Physiological studies of myrosinase production in Aspergillus oryzae --- p.21 / Chapter 2.3.2.1 --- Incubation time --- p.21 / Chapter 2.3.2.2 --- Inducer concentration --- p.22 / Chapter 2.4 --- Discussion --- p.23 / Chapter 2.4.1 --- Fungi selection in screening programme --- p.23 / Chapter 2.4.2 --- Medium composition --- p.23 / Chapter 2.4.3 --- Screening --- p.24 / Chapter 2.4.4 --- Physiological studies of myrosinase production in Aspergillus oryzae --- p.25 / Chapter 2.4.4.1 --- Incubation time --- p.25 / Chapter 2.4.4.2 --- Inducer concentration --- p.25 / Chapter Chapter 3 --- Purification and characterization of myrosinase in Aspergillus oryzae / Chapter 3.1 --- Introduction --- p.33 / Chapter 3.2 --- Materials and methods --- p.35 / Chapter 3.2.1 --- Reagents --- p.35 / Chapter 3.2.2 --- Fungal propagation --- p.35 / Chapter 3.2.3 --- Purification of the fungal myrosinase --- p.36 / Chapter 3.2.3.1 --- Preparation of crude extract --- p.36 / Chapter 3.2.3.2 --- Dialysis --- p.37 / Chapter 3.2.3.3 --- DEAE-Sepharose CL-6B ion-exchange chromatography --- p.37 / Chapter 3.2.3.4 --- Sephacryl S-200 molecular sieving chromatography --- p.37 / Chapter 3.2.3.5 --- FPLC Phenyl Superose hydrophobic interaction chromatography --- p.38 / Chapter 3.2.3.6 --- FPLC Mono P chromatofocusing --- p.38 / Chapter 3.2.4 --- Myrosinase assay and protein concentration determination --- p.39 / Chapter 3.2.4.1 --- Spot test for myrosinase activity --- p.39 / Chapter 3.2.4.2 --- Standard end-point assay --- p.40 / Chapter 3.2.4.3 --- Determination of protein concentration --- p.42 / Chapter 3.2.5 --- Physicochemical characterization of the myrosinase isozymes --- p.42 / Chapter 3.2.5.1 --- Sodium dodecyl sulfate polyacrylamide gel electrophoresis --- p.42 / Chapter 3.2.5.2 --- Protein staining and glycoprotein detection --- p.43 / Chapter 3.2.5.3 --- Chromatofocusing --- p.43 / Chapter 3.2.5.4 --- Gel filtration with FPLC Superose 6 --- p.44 / Chapter 3.2.6 --- Enzymatic properties --- p.44 / Chapter 3.2.6.1 --- Effect of pH on crude enzyme stability --- p.44 / Chapter 3.2.6.2 --- Effect of substrate concentration on enzyme activity --- p.45 / Chapter 3.2.6.3 --- Effect of pH on enzyme activity --- p.45 / Chapter 3.2.6.4 --- Effect of temperature on enzyme activity --- p.46 / Chapter 3.2.6.5 --- Effects of metallic ions on enzyme activity --- p.46 / Chapter 3.2.6.6 --- Effects of various compounds on enzyme activity --- p.46 / Chapter 3.2.6.7 --- Effects of various buffers on enzyme activity --- p.47 / Chapter 3.3 --- Results --- p.48 / Chapter 3.3.1 --- Fungal propagation --- p.48 / Chapter 3.3.2 --- Purification of fungal myrosinase in Aspergillus oryzae --- p.48 / Chapter 3.3.2.1 --- Extraction of the enzyme --- p.48 / Chapter 3.3.2.2 --- Dialysis --- p.49 / Chapter 3.3.2.3 --- DEAE-Sepharose ion-exchange chromatography --- p.49 / Chapter 3.3.2.4 --- Sephacryl S-200 molecular sieving chromatography --- p.50 / Chapter 3.3.2.5 --- FPLC Phenyl Superose hydrophobic interaction chromatography --- p.50 / Chapter 3.3.2.6 --- FPLC Mono P chromatofocusing --- p.51 / Chapter 3.3.3 --- Physicochemical characterization --- p.52 / Chapter 3.3.3.1 --- Sodium dodecyl sulfate polyacrylamide gel electrophoresis --- p.52 / Chapter 3.3.3.2 --- Chromatofocusing --- p.53 / Chapter 3.3.3.3 --- Gel filtration --- p.53 / Chapter 3.3.4 --- Enzymatic properties --- p.53 / Chapter 3.3.4.1 --- Effect of pH on the crude enzyme stability --- p.53 / Chapter 3.3.4.2 --- Effect of substrate concentration on enzyme activity --- p.54 / Chapter 3.3.4.3 --- Effect of pH on enzyme activity --- p.54 / Chapter 3.3.4.4 --- Effect of temperature on enzyme activity --- p.55 / Chapter 3.3.4.5 --- Effects of metallic ions on enzyme activity --- p.55 / Chapter 3.3.4.6 --- Effects of various compounds on enzyme activity --- p.56 / Chapter 3.3.4.7 --- Effects of various buffers on enzyme activity --- p.57 / Chapter 3.4 --- Discussion --- p.58 / Chapter 3.4.1 --- Purification of Aspergillus oryzae myrosinase --- p.58 / Chapter 3.4.1.1 --- Dialysis --- p.58 / Chapter 3.4.1.2 --- Enzyme purification --- p.58 / Chapter 3.4.2 --- Physicochemical properties --- p.60 / Chapter 3.4.2.1 --- Glycoprotein --- p.60 / Chapter 3.4.2.2 --- Molecular weights --- p.60 / Chapter 3.4.2.3 --- Isoelectric points --- p.61 / Chapter 3.4.3 --- Enzymatic properties --- p.61 / Chapter 3.4.3.1 --- pH and temperature optima --- p.61 / Chapter 3.4.3.2 --- Substrate affinity --- p.62 / Chapter 3.4.3.3 --- Inhibitions by various compounds and metallic ions --- p.63 / Chapter 3.4.3.4 --- Inhibitions by various buffer systems --- p.64 / Chapter Chapter 4 --- Summary --- p.106 / References --- p.110

by Tam Yan Kit. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1992. / Includes bibliographical references (leaves 102-114). / Abstract --- p.1 / Acknowledgement --- p.3 / Table of Contents --- p.5 / List of Tables --- p.7 / List of Figures --- p.8 / List of Plates --- p.8 / Chapter Chapter1 --- Introduction --- p.9 / Chapter Chapter2 --- Literature Review / Chapter 2.1 --- The study of enzyme polymorphism --- p.11 / Chapter 2.2 --- The zymogram technique --- p.20 / Chapter 2.3 --- Molecular approaches to systematic studies --- p.24 / Chapter 2.4 --- Genetic variation and systematic studies of decapod Crustacea --- p.29 / Chapter 2.5 --- Taxonomy of Penaeus and Metapenaeus --- p.33 / Chapter Chapter3 --- Materials and Methods / Chapter 3.1 --- Sample collection and storage --- p.43 / Chapter 3.2 --- Extract preparation --- p.44 / Chapter 3.3 --- Starch gel preparation --- p.45 / Chapter 3.4 --- Starch gel electrophoresis --- p.46 / Chapter 3.5 --- Gel slicing --- p.47 / Chapter 3.6 --- Enzyme staining --- p.47 / Chapter 3.7 --- Data collection --- p.48 / Chapter 3.8 --- Data analysis --- p.49 / Chapter Chapter4 --- Results / Chapter 4.1 --- Genetic interpretation of zymograms --- p.65 / Chapter 4.2 --- Conformity to Hardy-Weinberg equilibrium distribution --- p.67 / Chapter 4.3 --- Level of genetic variability --- p.67 / Chapter 4.4 --- Interspecific and intergeneric genetic differentiation --- p.68 / Chapter Chapter5 --- Discussion / Chapter 5.1 --- Enzyme polymorphism in Penaeus and Metapenaeus --- p.84 / Chapter 5.2 --- Level of genetic variability --- p.85 / Chapter 5.3 --- Phylogenetic relationships --- p.91 / Chapter 5.4 --- Biochemical and molecular systematic studies of Penaeus and Metapenaeus --- p.96 / Chapter Chapter6 --- General Conclusions --- p.99 / References --- p.102

In heterogeneous catalysis, the phase of a catalytic agent, which is responsible for reducing the activation energy of a reaction, is different from the phase of its reactants or substrates. Often, soluble catalysts are tightly associated with an inert carrier in order to artificially alter their phase. Applying this concept to biocatalysis yields a system in which enzyme molecules are immobilized on a solid support. This often serves to stabilize the enzyme, as well as enhance the recyclability of the enzyme since it is no longer soluble. In this dissertation, two methods of enzyme immobilization are evaluated: adsorption to a solid surface and whole-cell biocatalysis. The latter is then engineered for improved kinetics and functional activity using principles of synthetic biology. Adsorption of a protein to a solid surface is driven by the same thermodynamic factors that are responsible for the folding of a protein. Hydrophobic interactions, ionic interactions, covalent bonding, and weak forces all contribute to minimizing the free energy of a protein, which defines its secondary, tertiary, and quaternary structures. Upon introduction to a surface, these different forces rearrange across the surface of the substrate to minimize the free energy of the system. Many factors influence this behavior, including particle curvature, material properties of the surface, and the stability of the protein. In the preexisting body of work, much of the research performed regarding the effects of thermal stability on adsorption were performed using mutant proteins whose structures were intentionally altered for a range of stabilities. In Chapter 2, we evaluate the effects of thermal stability on adsorption behavior using naturally evolved enzymes from the AKR superfamily, namely AdhD and hAR. These enzymes were selected for their structural homology, but vastly different thermal stabilities. Using these proteins, we demonstrate that the previously held theories of thermostable protein adsorption behavior are not entirely applicable to naturally evolved proteins that are not artificially stabilized. We also propose a modification to the classic 4-state adsorption/desorption model by introducing new pathways and protein states based on our experiments. In addition to physisorption, whole-cell biocatalysis was explored as an enzyme immobilization platform. In general, this can be accomplished by cytosolic expression, periplasmic expression, or surface display. After weighing these options, we chose periplasmic expression in E. coli for our biocatalysts. As for the catalytic component, we selected carbonic anhydrase (CA), a class of Zn+2-binding metalloenzymes that are capable of catalyzing the reversible hydration of CO2. This enzyme was selected for the breadth of applications it can be used for, as well as its ubiquity in nature and extremely fast kinetics. Two isoforms were selected (Cab and Cam) for their respective benefits and were periplasmically expressed using 2 different leader peptides, which we discuss in Chapter 3. The enzyme loading in the periplasm, kinetics, thermal stability, and functional activity are all reported for the resulting whole-cell biocatalysts. We also describe a new method for the measurement of the operational stability of CA-based biocatalysts. Modifications to the whole-cell biocatalysts are described in Chapter 4 and Chapter 5. In Chapter 4, we demonstrate that expression of a viral envelope protein enhances the permeability of the outer membranes of E. coli cells. We characterize this improvement by measuring small-molecule permeance, whole-cell kinetics, and functional activity of the modified biocatalysts. We also quantify this enhancement by applying concepts of porous chemical catalysts to our whole-cells. In doing so, we show improvements in parameters such as the effectiveness factor, Thiele modulus, diffusivity, and permeability. Finally, in Chapter 5 we show enhancement of the functional activity of the whole-cell biocatalysts by displaying small peptides on the outer surfaces of the cells. The modified cells are shown to enhance precipitation of calcium carbonate, a common end product of carbon mineralization. Improved solid formation rates are also reported and possible explanations for these effects are discussed. Overall, this dissertation explores immobilization of enzymes to create heterogeneous biocatalysts. First, the effects of immobilization on enzyme structure, stability, and activity are shown for two different immobilization techniques: adsorption to a solid surface and periplasmic expression in E. coli cells. After characterization, engineering of the whole-cell biocatalysts for improved properties is presented. Finally, future directions for this work are discussed which would advance our understanding of heterogeneous biocatalysts, as well as enhance their utility.

Dois experimentos foram realizados no Departamento de Zootecnia da Universidade de São Paulo (USP), na faculdade de Zootecnia e Engenharia de Alimentos (FZEA), em Pirassununga/SP, com o objetivo de estudar o desempenho de frangos de corte alimentados com rações suplementadas com enzimas exógenas. A ração experimental foi constituída de milho, farelo de soja, vitaminas, minerais e enzimas exógenas (50 g / 100 kg). No experimento I foram utilizados 504 pintainhos Cobb distribuídos em um delineamento inteiramente casualizado em esquema fatorial 2x3: 2 sexos - fêmeas e machos, 3 níveis nutricionais - Reduzido, reduzido + complexo enzimático e convencional, totalizando 6 tratamentos com 7 repetições de 12 animais cada. Os resultados revelaram que machos e fêmeas, recebendo dietas com níveis nutricionais reduzidos com adição de enzima, apresentaram maiores peso médio e ganho de peso aos 21 e 42 dias de idade quando comparados aos demais tratamentos (P<0,05). Não foram encontradas diferenças significativas para as características de carcaças. No experimento II foram utilizados 1.080 frangos de corte, machos, de sete dias de idade, Cobb 500, distribuídos em um delineamento inteiramente casualizado em esquema fatorial 3X3: sendo três níveis nutricionais - convencional, reduzido e reduzido + complexo enzimático e três níveis de restrição - 0%, 2% e 4% - totalizando 9 tratamentos com 10 repetições de 12 animais. Os resultados encontrados revelaram um efeito significativo sobre o desempenho das aves com a redução do ganho de peso dos animais e conversão alimentar. Não foram observados efeitos sobre a mortalidade e incidência de transtornos metabólicos. Não foram encontradas diferenças significativas para as características de carcaças e percentagem de gordura abdominal, porém, ocorreu um aumento no rendimento de peito para aves alimentadas com o complexo enzimático na dieta. / Two experiments were conducted at the Department of Animal Science, University of São Paulo (USP), College of Animal Science and Food Engineering (FZEA) in Pirassununga / SP, with the aim of studying the performance of broilers fed diets supplemented with exogenous enzymes. The experimental diets consisted of corn, soybean meal, vitamins, minerals and exogenous enzymes (50 g / 100 kg). In the first experiment were used Cobb 504 chicks distributed in a completely randomized 2x3 factorial: two sexes - males and females, three nutritional levels - Low, low + enzyme complex and conventional, totaling 6 treatments with seven replicates of 12 animals each. The results revealed that males and females, fed diets with reduced nutrient levelswith the addition of enzyme, showed higher average weight and weight gain at 21 and 42 days of age when compared to other treatments (P<0.05). There were no significant differences for carcass traits. In experiment II were used 1080 broilers, males, seven-day-old Cobb 500, distributed in a completely randomized design in factorial 3X3: with three levels of nutrition - conventional, reduced and reduced + enzyme complex and threelevels of restriction - 0%, 2% and 4% - a total of nine treatments with 10 replicates of 12animals. The results showed a significant effect on broiler performance with reducedweight gain and feed the animals. No effects on mortality and incidence of metabolic disorders. There were no significant differences for carcass traits and abdominal fat percentage, however, there was an increase in breast yield for birds fed the enzyme complex in the diet.

Catalytic, selective C-H bond activation for the oxidative hydroxylation RH → ROH of simple or complex compounds is of significant interest in synthetic organic chemistry. One of the major classes of enzymes used for C-H bond activation are cytochrome P450 monooxygenases (EC 1.14.X.X), which can promote chemo-, regio- and stereoselective oxidations under mild reaction conditions. For the current study, catalytically self-sufficient forms of biocatalyst P450cam-RhFRed were investigated. These self-sufficient P450 systems were previously created by fusing the reductase domain of P450 RhF (CYP116B2, RhFRed from Rhodococcus sp.) with the catalytic domain of P450cam (CYP101A1, Pseudomonas putida), thus mimicking the natural fusion of P450 RhF. The generation of 93 P450cam-RhFRed variants has expanded the synthetic toolbox to serve as a basis for exploring the substrate scope towards ethylbenzenes, substituted alkylbenzenes, 4-ethylphenol and (+)-pleuromutilin. To select for active mutants from this library of 93, high throughput screening methods were developed. A pooling approach was applied in order to express P450s and analyse them against a panel of non-natural substrates, such as ethylbenzene, 4-ethylphenol and (+)-pleuromutilin in whole cell biotransformation reactions. The concentration of P450 enzymes was determined using CO difference spectroscopy in whole cells. The assay was significantly improved both in terms of speed and safety by using carbon monoxide releasing molecules as a source of CO rather than the gas CO itself. These screening studies served as starting point to identify P450cam-RhFRed mutants for specific reactions. In particular, a systematic investigation of this library showed mutants that generated chiral benzyl alcohols with good enantioselectivities. To interpret these results on a structural basis, molecular dynamics simulations were used to estimate enantioselectivity of selected mutants for the regio-isomers of methylated ethylbenzene derivatives. The results from the molecular dynamics simulations were broadly consistent with experimentally determined data and identified the importance of conformational changes and flexibility of mutant-substrate complexes to enforce enantioselectivity.

Made available in DSpace on 2014-06-11T19:22:15Z (GMT). No. of bitstreams: 0 Previous issue date: 2012-01-13Bitstream added on 2014-06-13T19:27:20Z : No. of bitstreams: 1 jadoski_cj_me_botfca.pdf: 475805 bytes, checksum: 4e8e389e5955869014b117a71c01ee93 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / A deficiência hídrica destaca-se como um dos principais fatores limitantes na produção vegetal. A fotossíntese possui relação direta na produtividade das plantas e é um dos processos fisiológicos afetados pelo estresse hídrico. Há uma interação entre a cultura, elementos ambientais e manejo. Neste último, a aplicação de fungicidas vem apresentando resultados significativos nos processos metabólicos das plantas, além do controle de doenças. O fungicida piraclostrobina possui efeito verdejante, influências na regulação hormonal, assimilação de carbono e nitrogênio e retardo na senescência. Baseado nos efeitos fisiológicos que as estrobilurinas desenvolvem nas plantas, em função de seu modo de ação na fotossíntese; este trabalho teve por objetivo avaliar seus efeitos em plantas sob diferentes tensões de água no solo. O delineamento experimental utilizado parcelas subdivididas no esquema fatorial 4x4, quatro doses de piraclostrobina (zero, 0.050, 0.075 e 0.1 L.ha-1) e quatro tensões de água no solo (-10, -20, -30 e -40 kPa) em dois momentos, inicio da fase reprodutiva R5 e no inicio da fase de enchimento de grãos R7 na cultura do feijoeiro cultivar Carioca precoce (Phaseolus vulgaris L.) de crescimento determinado e ciclo médio de 70 a 90 dias. A semeadura ocorreu em canteiros de 2X5 m, na qual a população de plantas utilizada foi de 250.000 plantas.ha-1 com espaçamento de 40 cm entre linhas e 12,5 sementes por metro. O sistema de irrigação foi constituído de uma linha principal de onde saíam às fitas gotejadoras diretamente na linha de semeadura, na qual a irrigação era controlada por tensiômetros. Na primeira aplicação da piraclostrobina (R5) aplicou-se as tensões de irrigação e fizeram-se coletas... / The water deficit stands out as one of the major limiting factors in crop production. Photosynthesis has a direct relationship in the productivity of plants and is one of the physiological processes affected by water stress. There is an interaction between culture, environmental factors and management. In the latter application of fungicides has shown significant results in the metabolic processes of plants beyond the control of diseases. The fungicide pyraclostrobin has green effect, influences the hormonal regulation, carbon and nitrogen assimilation and delayed senescence. Based on the physiological effects that strobilurins develop in plants, due to its mode of action in photosynthesis; before that this work was to evaluate its effects on plants under water stress. The experimental design was split-plot factorial design in four doses of pyraclostrobin (zero, 0,050, 0,075 and 0.1 L.ha-1) and four soil water tensions (-10, -20, -30 and -40 kPa) in two stages, beginning of reproductive stage (R5) and during early grain filling stage R7 in the bean crop to cultivate early Carioca (Phaseolus vulgaris) Growth determined and average cycle of 70 to 90 days. Sowing occurred in beds of 2x5 m, where the plant population of 250,000 was used plants.ha-1 with 40 cm spacing between rows and 12.5 seeds per meter. The irrigation system consisted of a main line to the left of where drip tape directly in the row in which irrigation was controlled by tensiometers. In the first application of pyraclostrobin (R5) was applied tensions irrigation where they were collecting material for the enzymatic determination. Three days after the application of... (complete abstract click electronic access below)