Two different systems involving the dye rhodamine 6G (R6G) were studied using absorption spectrophotometric techniques. The first is a compound that forms when R6G, HgCl$\sb2,$ and KI are combined in aqueous solution at concentrations near 10$\sp{-5}$ m. The second involves the formation of an addition complex between 10$\sp{-5}$m R6G and 10$\sp{-3}$ to 10$\sp{-2}$m $\beta$-cyclodextrin ($\beta$-CD) in aqueous solution. When R6G, HgCl$\sb2,$ and KI are combined, the absorption spectrum shows a shoulder around 571 nm that is attributed to the formation of a complex. The formula for this compound was determined by two methods: (1) the mole ratio method, which results in a 1:1 ratio between Hg(II) and R6G, and (2) the method of continuous variations, which this work has extended for a three reactant system, resulting in a ratio of 3:1 between iodide and R6G. Therefore, the formula for the complex is proposed to be R6GHgI$\sb3$ in the range of concentrations under study. Thermodynamic constants for the formation of the R6GHgI$\sb3$ compound are determined. Mole ratio plots at temperatures ranging from 15$\sp\circ$C to 30$\sp\circ$C are used to determine the mole fraction equilibrium constants. The values for the constants are 6.74 $\times$ 10$\sp{26}$, 5.53 $\times$ 10$\sp{26},$ 1.67 $\times$ 10$\sp{26},$ and 1.31 $\times$ 10$\sp{26}$ at 15.0, 18.0, 25.0, and 30.0$\sp\circ$C, respectively. The changes in enthalpy, $\Delta$H$\sb{\rm x}$, and free energy, $\Delta$G$\sb{\rm x}$, are then determined from the mole fraction equilibrium constant. $\Delta$H$\sb{\rm x}$ is $-$80.8 kJ/mol, $\Delta$G$\sb{\rm x}$ is 150 $\pm$ 2 kJ/mol, and $\Delta$S$\sb{\rm x}$ is 233 $\pm$ 2 J/mol. The addition of R6G to $\beta$-CD results in a red shift of the absorption peak of R6G from 526 nm to 529 nm. When the concentration of $\beta$-CD is varied, an isosbestic point is observed at 528 nm. Two different methods were used to calculate the equilibrium constant of the R6G - $\beta$-CD complex. Both methods use the same absorbance data, which is obtained by varying the concentration of $\beta$-CD and keeping the R6G concentration constant. One involves a double reciprocal plot using the Benesi-Hildebrand equation. The second uses a non-linear least squares fit of the absorbance data to the hypothetical equilibrium equation to calculate the equilibrium constant. The equilibrium constant determined by the Benesi-Hildebrand equation is 36 $\pm$ 44. The non-linear least squares equilibrium constant is (1.2 $\pm$ 0.4) $\times$ 10$\sp2.$

Oral drug administration remains the most popular route of drug delivery. Absorption of the dissolved drug through the intestinal epithelial membrane is a prerequisite to systemic bioavailability and drug efficacy. In efforts to reduce the long lead times, attrition rates, and costs of drug discovery and development, computational models have been developed to predict the membrane permeability and absorption efficiency of a dosed drug. Many models utilize various molecular descriptors to correlate with in vitro permeability or human intestinal absorption data. It is widely accepted that the two most significant physicochemical properties that control a compound's passive transport process are its aqueous solubility and lipophilicity characteristics.This work will discuss the theoretical background of passive transport, a number of computational models developed to predict in vitro permeability, other models that predict human fraction of dose absorbed, and predicting absorption efficiency relative to a maximum dose. A newly developed prediction method is also presented, that reveals an interesting relationship between fraction absorbed and the melting point of the drug.

Activated Carbon (AC) is an amorphous carbon-based material characterized with a large surface area (~ 1,000 m2/g) and consists primarily of graphitic (sp2 hybrid) layers. Its amphoteric chemical property results because of the chemical treatment of the surface of AC with oxidizing agents, reducing agents, and grafting agents. β-cyclodextrin (β-CD) is a very interesting carbohydrate oligomer that provides very strong binding ability for small organic guest molecules in its inner cavity (6.0 ~ 6.5 Å) by van der Waals interactions and hydrogen bond formation between the guest molecules and the host. <p>Surface modification of AC with β-CD was synthesized by chemical methods: oxidation with HNO3, reduction with LiAlH4, and grafting β-CD onto the surface of AC via organic linkers such as glutaraldehyde and 1,4-phenylene diisocyanate. This surface grafted AC with β-CD, then, was evaluated for its surface area and sorption performance by using a solution dye sorption method using dye adsorbates. <p>Surface functional groups produced from oxidation (carboxylic acid, lactone, quinine, phenol, and nitro groups), reduction (alcohol and amine groups), and grafting (imine, hemiacetal, and urethane bonds) methods including microscopy of untreated, surface modified, and grafted ACs were characterized by various surface characterization methods: Diffuse Reflectance Infra-red Fourier Transform Spectroscopy (DRIFTS), Scanning Electron Microscopy (SEM), Raman spectroscopy, X-ray photoelectron spectroscopy (XPS), Thermogravimetric analysis (TGA), Differential thermogravimetry (DTG), Matrix Assisted Laser Desorption Ionization Time of Flight mass spectrometry (MALDI TOF MS), and Electron spin resonance (ESR) spectroscopy. A chemical method, the Boehm method, was used for identifying surface bound acidic and basic functional groups. Nitrogen porosimetry was used to analyze the surface area and pore structure characteristics of AC, surface modified ACs, and grafted ACs. <p>p-nitrophenol (PNP) and methylene blue (MB) were used as adsorbates for the dye sorption method. PNP and MB were used to measure the sorption performance of grafted ACs at equilibrium using UV-vis spectrophotometry in aqueous solution. Sorption capacity (Qe), surface area (m2/g), and binding affinity characteristics [KF (L/g), KL (g/mol), and KBET (L/g)] were determined at equilibrium conditions using fundamental sorption models such as Langmuir, Freundlich, and BET isotherms. The sorption performance of grafted ACs and granular AC were different according to the difference in surface area and pore structure characteristics of each material.

Activated Carbon (AC) is an amorphous carbon-based material characterized with a large surface area (~ 1,000 m2/g) and consists primarily of graphitic (sp2 hybrid) layers. Its amphoteric chemical property results because of the chemical treatment of the surface of AC with oxidizing agents, reducing agents, and grafting agents. β-cyclodextrin (β-CD) is a very interesting carbohydrate oligomer that provides very strong binding ability for small organic guest molecules in its inner cavity (6.0 ~ 6.5 Å) by van der Waals interactions and hydrogen bond formation between the guest molecules and the host. <p>Surface modification of AC with β-CD was synthesized by chemical methods: oxidation with HNO3, reduction with LiAlH4, and grafting β-CD onto the surface of AC via organic linkers such as glutaraldehyde and 1,4-phenylene diisocyanate. This surface grafted AC with β-CD, then, was evaluated for its surface area and sorption performance by using a solution dye sorption method using dye adsorbates. <p>Surface functional groups produced from oxidation (carboxylic acid, lactone, quinine, phenol, and nitro groups), reduction (alcohol and amine groups), and grafting (imine, hemiacetal, and urethane bonds) methods including microscopy of untreated, surface modified, and grafted ACs were characterized by various surface characterization methods: Diffuse Reflectance Infra-red Fourier Transform Spectroscopy (DRIFTS), Scanning Electron Microscopy (SEM), Raman spectroscopy, X-ray photoelectron spectroscopy (XPS), Thermogravimetric analysis (TGA), Differential thermogravimetry (DTG), Matrix Assisted Laser Desorption Ionization Time of Flight mass spectrometry (MALDI TOF MS), and Electron spin resonance (ESR) spectroscopy. A chemical method, the Boehm method, was used for identifying surface bound acidic and basic functional groups. Nitrogen porosimetry was used to analyze the surface area and pore structure characteristics of AC, surface modified ACs, and grafted ACs. <p>p-nitrophenol (PNP) and methylene blue (MB) were used as adsorbates for the dye sorption method. PNP and MB were used to measure the sorption performance of grafted ACs at equilibrium using UV-vis spectrophotometry in aqueous solution. Sorption capacity (Qe), surface area (m2/g), and binding affinity characteristics [KF (L/g), KL (g/mol), and KBET (L/g)] were determined at equilibrium conditions using fundamental sorption models such as Langmuir, Freundlich, and BET isotherms. The sorption performance of grafted ACs and granular AC were different according to the difference in surface area and pore structure characteristics of each material.

Cyclodextrin glucanotransferase (EC 2.4.1.19) from the alkaliphilic Bacillus sp. G-825-6 converts starch mainly to c-cyclodextrin (CD8). A combination of error-prone PCR and DNA shuffling was used to obtain variants of this enzyme with higher product specificity for CD8 and a broad pH activity range. The variant S54 with seven amino acid substitutions showed a 1.2-fold increase in CD8-synthesizing activity and the product ratio of CD7:CD8 was shifted to 1:7 compared to 1:3 of the wild-type enzyme. Nine amino acid substitutions of the cyclodextrin glucanotransferase were performed to generate the variant S35 active in a pH range 4.0–10.0. Compared to the wild-type enzyme which is inactive below pH 6.0, S35 retained 70% of its CD8-synthesizing activity at pH 4.0.

The influences of loading cholesterol into stallion spermatozoa membranes prior to cold storage or cryopreservation were determined using cholesterol loaded cyclodextrin (CLC) before preservation, followed by the unloading of cholesterol after preservation using methyl beta cyclodextrin (MBCD). Experiment I: dose response trials determining optimal amounts of CLC and MBCD based on percentages of progressively motile spermatozoa (PMS) following preservation. Experiment II: influences of CLC and MBCD on PMS, the percentages of live intact (LI) and live non-intact (LNI) spermatozoa following cold storage. Experiment III: influences of CLC before cryopreservation and MBCD on PMS, LI, and LNI post-thaw. Addition of CLC improved (P&lt;0.05) PMS and LI following preservation when compared to the control. Unloading cholesterol using MBCD does not alter PMS, LI nor LNI. Addition of CLC is beneficial to survival of spermatozoa following preservation and addition of MBCD in small amounts does not negatively influence PMS, LI or LNI.

Multicomponent nonaqueous phase liquids (NAPL) contaminating the subsurface can significantly inhibit remediation. One method of enhancing the rate of remediation of NAPL constituents, compared to pump-and-treat, involves source zone treatment with enhanced solubilization agents (ESAs) including cyclodextrins. Equilibrium cyclodextrin enhanced solubilization of simple 1, 2, and 3 component NAPL mixtures was examined to evaluate the applicability of Raoult's Law. The results suggest that Raoult's Law may be used to estimate equilibrium and early-time dynamic concentrations in contact with ideal NAPL mixtures, and Raoult's Law may be used to estimate cyclodextrin enhanced groundwater concentrations for ideal NAPL mixtures. Solubility enhancement of NAPL compounds was dependent on the cyclodextrin concentration and independent of NAPL composition. Column experiments and numerical modeling were used to evaluate the dissolution behavior of the NAPL mixtures in water and a cyclodextrin solution to estimate mass transfer rates. The aqueous multicomponent dissolution followed Raoult's Law, and the model-estimated lumped rate coefficients were independent of the NAPL composition. Addition of the cyclodextrin enhanced the dissolution and removal of compounds from residual NAPL due to an increase in the driving force (i.e. concentration gradient) and the mass transfer coefficient. The model results suggest that Raoult's Law is applicable for ideal NAPL mixture dissolution in water, but potential nonideality was observed and caused the model simulation to deviate from the dissolution behavior for NAPL mixture cyclodextrin experiments. The cyclodextrin dissolution experiments were less rate-limited than aqueous dissolution, and the mass transfer coefficients were quantified with the model. The results of the model suggest that NAPL mixture nonideality and intra-NAPL diffusion may also impact enhanced dissolution behavior. Additionally, the importance of NAPL mixture characterization was illustrated by evaluation of a mixture of PCE (tetrachloroethene) and diesel fuel collected from a site in Tucson, Arizona. A sample from the site was used to create mixtures with increasing PCE in the NAPL. Chemical evaluation of the complex NAPL was conducted, and physical property and phase partitioning testing was performed, which demonstrated the effect of NAPL composition on its distribution, interphase mass transfer, and potential mobilization.

Made available in DSpace on 2014-06-11T19:32:54Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-12-23Bitstream added on 2014-06-13T20:04:37Z : No. of bitstreams: 1 cucolo_gr_dr_rcla.pdf: 912012 bytes, checksum: bb2f52ae986d8910d4d4ba1e27a6db3e (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Ciclodextrina-glicosil-transferase (CGTase, EC 2.4.1.19) é uma enzima capaz de formar ciclodextrinas (CDs), tendo o amido como substrato. A enzima é de grande interesse industrial, principalmente nas indústrias alimentícias, cosméticas, químicas e farmacêuticas. CDs são moléculas cíclicas formadas por monômeros de glicoses, unidos por ligações α-1,4. Os tipos mais comuns de ciclodextrinas, α-, β- e γ- CD, consistem de 6, 7 e 8 monômeros de glicose, respectivamente. A molecula de CD possui uma estrutura única com a cavidade interna hidrofóbica e a região externa hidrofílica, sendo possível a formação de complexos de inclusão com uma variedade de compostos, que pode melhorar ou proteger as propriedades físicoquímicas da molécula encapsulada. Este estudo foi dividio em três capitulos, o primeiro foi uma revisão bibliografica, e os outros dois são trabalhos de pesquisa. No primeiro trabalho estudou-se a produção de CDs pela CGTase do Bacillus clausii subgrupo E16, tanto em relação ao efeito da concentração do amido na produção das CDs, como em relação a utilização de fontes alternativas de amido (amido solúvel de batata PA, de milho, de trigo e de mandioca) como substrato. Observouse que a enzima produziu preferencialmente β-CD nos diferentes tipos de substrato utilizados. O mais alto grau de conversão de CDs foi obtido com o uso de amido de mandioca, tendo convertido 95,30% (p/p) em β-CD, seguido de 86,60% (p/p) do amido solúvel. A concentração de 1% de amido de mandioca apresentou o melhor rendimento na produção de CDs. No segundo trabalho de desquisa, o objetivo foi a produção de CDs pela CGTase do Bacillus clausii E16 em fontes alternativas de substrato (farelo de trigo, farelo de mandioca; farinhas de milho, trigo e mandioca). Observou-se que a enzima produziu β-CD nos diferentes tipos de substratos utilizados. O grau de conversão de CDs... / Cyclodextrin glycosyltransferase (CGTase, EC 2.4.1.19) is an enzyme capable of converting starch into cyclodextrins (CDs) molecule. CDs are becoming increasingly popular in pharmaceutical, chemical, cosmetics and food industries. CDs are cyclic oligosaccharides compounded of glucose units jointed by α-1,4 linkages. The most common types of cyclodextrins, α-, β- and γ- CD, consist of 6, 7 and 8 glucose units, respectively. A CD molecule has a unique torus-shaped structure with a hydrophobic internal cavity and a hydrophilic external surface. As a result, CDs can form inclusion complexes with many hydrophobic guest molecules and thereby change their physical and chemical properties. This study was divided in three chapters, the first is about one bibliographic review, and the other are about two research approaches. The aim of the first work was to verify the effect of different starch botanical sources and the concentration of starch in the production of cyclodextrins by CGTase enzyme from Bacillus clausiii E16. It was noticed that the enzyme mainly produced β-CD with the different types of starch (soluble potato starch, corn starch, wheat starch and cassava starch) used substrate. The higher degree of conversion of CDs was obtained with the use cassava starch, of which 95,30% (w/w) was converted in β-CD, followed by 86,60% (w/w) of soluble starch. The concentration 1% of cassava starch showed the best efficiency in the production of CDs. In the second research work, the aim was studied the application of a CGTase from Bacillus clausiii E16 in alternative substrate sources (cassava bran, wheat bran; corn meal, wheat flour e cassava flour) for production of cyclodextrins. It was noticed that the enzyme produced β-CD with the different types of used substrate. The degree of conversion of CDs obtained with the use cassava bran was 40,48% (w/w) in β-CD and preliminary studies... (Complete abstract click electronic access below)

Magister Scientiae - MSc / Glucose oxidase (GOx) and horseradish peroxidase (HRP) are important enzymes for the development of amperometric enzyme linked immunosensors. The selectivity of each enzyme towards its analyte deepens its importance in determining the sensitivity of the resultant immunosensor. In designing immunosensors that have customized transducer surfaces, the incorporation with FAD and iron based enzymes ensures that electron kinetics remains optimal for electrochemical measurement. Various different immobilization strategies are used to produce response signals directly proportional to the concentration of analyte with minimal interferences. The combination of self-assembled monolayers and supramolecular chemistry affords stability and simplicity in immunosensor design. In this work, two electrochemical strategies for the detection of human chorionic gonadotropin (hCG) is presented. This involves the modification of a gold surface with a thiolated β- cyclodextrin epichlorohydrin polymer (βCDPSH) to form a supramolecular inclusion complex with ferrocene (Fc)-functionalised carboxymethyl cellulose polymer (CMC). Cyclic voltammetry indicated that ferrocene is in close proximity to the electrode surface due to the supramolecular complex formed with βCDPSH. Furthermore, strategy (a) for the detection of hCG used α-antihCG labelled (HRP) as reporter conjugate. Strategy (b) maintained the CMC bifunctionalised with Fc and recognition antibody for hCG hormone. However, the system was functionalised with a HRP enzyme and detection is done by using GOx reporter conjugates for in situ production of hydrogen peroxide. The reduction of H2O2 was used for the amperometric detection of hCG by applying a potential of 200 mV. The sensitivity and limit of detection of both strategies were calculated from calibration plots. For strategy (a) the LOD was found to be 3.7283 ng/mL corresponding to 33.56 mIU/mL and a sensitivity of 0.0914 nA ng-1 mL-1. The corresponding values for strategy (b) are 700 pg/mL (6.3 mIU/mL) and 0.94 nA ng-1 mL-1.

Made available in DSpace on 2014-06-11T19:27:23Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-06-10Bitstream added on 2014-06-13T20:35:51Z : No. of bitstreams: 1 blanco_kc_me_rcla.pdf: 835926 bytes, checksum: 46db69ad433dc1b354710697a08da977 (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Este trabalho tem como objetivo estudar a produção da enzima ciclodextrina glicosiltransferase (CGTase) empregando culturas de Bacillus sp subgrupo alcalophilus utilizando como fonte de carbono fécula de mandioca (polvilho) proveniente de uma fecularia de mandioca. Nos ensaios foram empregados Bacillus sp subgrupo alcalophilus, isolado de água residuária de uma fecularia de mandioca. Para avaliar a produção de CGTase mediante a atividade enzimática pelo Bacillus sp subgrupo alcalophilus foi utilizado um planejamento fatorial a dois (2) níveis, estudando como variáveis as concentrações da fonte de carbono (polvilho), de nitrogênio e de carbonato de sódio. Os experimentos foram realizados em Erlenmeyers de 300 mL de capacidade contendo 100 mL do meio de produção com pH inicial de 9,2, a 150 rpm e temperatura de 35 ± 1ºC durante 72 horas de fermentação. A produção de CGTase foi monitorada pela determinação da atividade enzimática (U/mL). Após os ensaios realizados em frascos foram realizados experimentos em biorreator de 5 L de volume útil, contendo 2 L do meio de produção, pH de 9,20, agitação de 150 rpm e temperatura de 35 ± 1ºC durante 72 h de fermentação, utilizando um planejamento fatorial a dois (2) níveis, estudando como variáveis as taxas de aeração e a velocidade de agitação. A otimização das concentrações da fonte de carbono, nitrogênio e carbonato de sódio foram obtidas a partir de um planejamento experimental composto central (PCC) e seus resultados analisados pelas superfícies de resposta. Os melhores resultados do planejamento encontrados no ponto central, corresponderam a 6,96 g/L da fonte de carbono, 8,07 g/L de nitrogênio e 9,45 g/L de carbonato de sódio. A maior produtividade obtida de CGTase após 72 horas de fermentação, foi 98,86 U/mL com valor teórico de 98,87 U/mL. A partir do melhor resultado obtido no PCC, determinou-se... / The aim of this study was to investigate the Cyclodextrin glycosyltransferase (CGTase) enzyme production by Bacillus sp subgroup alcalophilus using cassava starch (manioc flour) as a carbon source. Bacillus sp subgroup alcalophilus was isolated from wastewater of cassava flour industry. To evaluate the assays results, two (2) levels of complete factorial experimental design was used, studying the variables: carbon source, nitrogen and sodium carbonate concentrations. The experiments were performed in 300 mL erlenmeyer flasks containing 100 mL of medium production with initial pH of 9.2, at 150 rpm and 35±1ºC, during 72 hour. CGTase production was monitored by measurements of enzymatic activity (U/mL). After the flasks experiments, assays was running in 5 L containing 2 L of medium production, pH 9.2, 35 ± 1ºC during 72-hours using a factorial design at two (2) levels for aeration and agitation rate. The optimization of carbon source, nitrogen and sodium carbonate concentrations was obtained by a central composite design and their results analyzed by surface response. The best results was located on the central point, 6.96 g/L of carbon source, 8.07 g/L of nitrogen and 9.45 g/L of sodium carbonate. The CGTase activity predicted by model was 98.86 U/mL and the experimental activity obtained was 98,87 U/mL. After the best results obtained by PCC, the conditions of aeration (vvm) and agitation (rpm) rates was determined in bioreactor using a complete factorial experimental design. The best conditions, of 2.18 vvm and agitation of 157.07 rpm gave a experimental predicted CGTase activity of 130.36 U/mL, which was very close to the theoric CGTase activity of 130.33 U/mL.