Aplicação do método de Monte Carlo para simular a hidrólise de oligômeros de ácido D-galacturônio por uma endopoligalacturonase

Orientador : Prof. Dr. David Alexander MitchellCoorientador : Prof. Pós-Dr. Ivo NeitzelDissertação (mestrado) - Universidade Federal do Paraná, Setor de Tecnologia, Programa de Pós-Graduação em Engenharia Química. Defesa: Curitiba, 31/03/2015Inclui referências : f.51-55Resumo: A hidrólise da pectina por misturas de enzimas pectinoliticas é uma etapa importante em biorrefinarias de resíduos cítricos. No entanto, apesar da hidrólise enzimática da pectina ter sido estudada por muitos anos, há relativamente poucos modelos matemáticos que descrevam a cinética deste processo. O desenvolvimento de modelos cinéticos com base nas equações diferenciais é bastante difícil devido à estrutura complexa da pectina e a mistura complexa de enzimas pectinoliticas envolvidas na sua degradação. Um modelo capaz de descrever a degradação enzimática de substratos pécticos será uma importante ferramenta para o projeto de um processo de hidrólise de pectina em uma biorrefinaria de polpa cítrica. Com isso o objetivo do presente trabalho foi de iniciar o desenvolvimento de um modelo de Monte Carlo com um sistema mais simples, ou seja, um modelo com uma enzima trabalhando com oligômeros de ácido D-galacturônico de cadeia linear. Os dados detalhados da hidrólise dos oligômeros pela enzima endopoligalacturonase foram obtidos da literatura (Bonnin et al., 2001, Study of the mode of action of endopolygalacturonase from Fusarium moniliforme, Biochimica et Biophysica Acta 1526, 301-309). A partir destes dados foi implementado um modelo que descreve as preferências de uma enzima endopoligalacturonase do Fusarium moniliforme durante a hidrólise dos oligômeros de ácido D-galacturônico com grau de polimerização (GP) entre 2 a 7, estes substratos foram referidos de acordo com o seu GP, como monômeros (S = GalA), dímeros (S = GalA2), trímeros (S = GalA3), tetrâmeros (S = GalA 4), pentâmeros (S = GalA5), hexâmeros (S = GalA6) e heptâmeros (S = GalA7) de ácido D-galacturônico, onde o "S" representa substrato. Os valores dos parâmetros relativos as constantes de especificidade, foram determinados usando a função "fminsearch" do MATLAB, que utiliza o método simplex para minimização de uma função objetivo. Neste caso particular, a sub-rotina que foi usada para calcular a função objetivo continha um programa que realizou cinco simulações Monte Carlo. A grande vantagem da análise realizada, é que ela permite atribuir as preferências para as várias reações que são possíveis para cada oligômero, isto é, ao contrario de Bonnin et al. (2001) que atribuiu uma constante de especificidade para cada oligômero. Ao se determinar uma constante de especificidade para cada reação de encontro da enzima com o substrato, é possível identificar qual é o tipo de substrato que a enzima prefere, bem como o tipo de ligação que a enzima prefere para fazer a reação de hidrólise, ou seja, com a definição de mais de uma constante de especificidade foi possível perceber que a enzima prefere o centro da molécula de substrato. Com este estudo foi possível identificar que a aplicação do modelo desenvolvido é viável, pois o ajuste foi realizado como todos os dados simultaneamente, e seria interessante continuar o estudo para estender o modelo para descrever situações mais complexas envolvendo substratos ramificados e misturas de enzimas com diferentes ações. Uma vez desenvolvido o modelo final será uma ferramenta útil na concepção e otimização de biorreatores enzimáticos para hidrólise de pectina em ácido D-galacturônico. Palavras-chave: Método de Monte Carlo. Pectina. Ácido D-galacturônico. Endopoligalacturonase. Constante de especificidade.Abstract: The hydrolysis of the pectin mixtures of pectinolytic enzymes is an important step in biorefinery citrus waste. However, despite pectin enzymatic hydrolysis has been studied for many years, there are relatively few mathematical models describing the process kinetics. The development of kinetic models based on differential equations is very difficult due to the complex structure of pectin and the mixture of pectinolytic complex enzymes involved in its degradation. Modelling the enzymatic degradation of pectic substrates will be important for the design of a pectin hydrolysis process in citrus pulp biorefineries. Therefore, the objective of this study is to start the development of a Monte Carlo model with a simpler system, in other words, a model with an enzyme working with acid oligomers D-galacturonic straight chain. The detailed data of the oligomers by the hydrolysis of endopolygalacturonase enzyme were obtained from the literature (Bonnin et al., 2001, Study of the mode of action of endopolygalacturonase from Fusarium moniliforme, Biochimica et Biophysica Acta 1526, 301-309). From these data, a model is implemented which describes the preferences of a Fusarium moniliforme endopolygalacturonase enzyme for the hydrolysis of oligomers of D-galacturonic acid with a degree of polymerisation (DP) between 2 and 7, these substrates were listed according to their GP, as monomers (S = GalA), dimers (S = GalA2), trimers (S = GalA3), tetramers (S = GalA4), pentamers (S = GalA5), hexamers (S = GalA6) and heptamers (S = GalA7) of D-galacturonic acid, where "S" represents a substrate. The parameter values for the constants of specificity were determined using the tool "fminsearch" MATLAB, which uses the simplex method to minimize an objective function. In this particular case, the subroutine that was used to calculate the objective function contained a program that held five Monte Carlo simulations. The great advantage of the analysis that was made is that it allows you to assign preferences for the various reactions that are possible for each oligomer, that is, as opposed to Bonnin et al. (2001) attributed a specificity constant for each oligomer. When determining a specificity constant for each enzyme against reaction with the substrate, it is possible to identify what kind of substrate the enzyme prefers, as well as the type of connection that the enzyme prefers to make the hydrolysis reaction, or that is, the definition of more than one specificity constant was revealed that the enzyme prefers the center of the substrate molecule. In this study it was identified that the application of the developed model is viable, because the adjustment is performed with all data simultaneously and continue the study would be interesting to extend the model to describe situations involving more complex branched substrates and enzyme mixtures with different actions. Once developed the final model will be a useful tool in the design and optimization for bioreactors enzymatic hydrolysis of pectin as D-galacturonic acid. Keywords: Monte Carlo method. Pectin. D-galacturonic acid. Endopolygalacturonase. Specificity constant

The invisible fish: hydrodynamic constraints for predator-prey interaction in fossil fish Saurichthys compared to recent actinopterygians

Recent pike-like predatory fishes attack prey animals by a quick strike out of rest or slow movement. This fast-start behaviour includes a preparatory, a propulsive and a final phase, and the latter is crucial for the success of the attack. To prevent prey from escape, predators tend to minimise the duration of the interaction and the disturbance caused to surrounding water in order to not be detected by the prey's lateral line sensory system. We compared the hydrodynamic properties of the earliest fossil representative of the pike-like morphotype, the Triassic actinopterygian Saurichthys, with several recent pike-like predators by means of computational fluid dynamics (CFD). Rainbow trout has been used as a control example of a fish with a generalist body shape. Our results show that flow disturbance produced by Saurichthys was low and similar to that of the recent forms Belone and Lepisosteus, thus indicative of an effective ambush predator. Drag coefficients are low for all these fishes, but also for trout, which is a good swimmer over longer distances but generates considerable disturbance of flow. Second-highest flow disturbance values are calculated for Esox, which compensates the large disturbance with its extremely high acceleration performance (i.e. attacks at high speeds) and the derived teleostean protrusible mouth that allows prey catching from longer distances compared to the other fishes. We show CFD modelling to be a useful tool for palaeobiological reconstruction of fossil fishes, as it allows quantification of impacts of body morphology on a hypothesised lifestyle

Size does matter: The use of fish motion for improving human swimming simulations

In this study we evaluated the length and volume inconsistencies that occur when applying simple motion functions onto surface models of swimming fish and human, respectively. The larger the size (diameter and length) of the body, the larger are these inconsistencies. To eliminate these errors, different surface correction algorithms are introduced. Additional terms are implemented to overcome structural insufficiencies through over-stretching and overlapping in sensible body parts. For human undulatory swimming these models combine the motion of rigid extremities with the smooth surface bending at joints. The advantages of this hybrid model can be crucial for Computational Fluid Dynamics, where deviation from natural behaviour results in unrealistic surface pressures and artificial vortex structures. The structural improvements are also promising for computational graphic programming, where skin-like stretching surfaces mediate a natural impression

Unsteady flow phenomena in human undulatory swimming: a numerical approach

The undulatory underwater sequence is one of the most important phases in competitive swimming. An understanding of the recurrent vortex dynamics around the human body and their generation could therefore be used to improve swimming techniques. In order to produce a dynamic model, we applied human joint kinematics to three-dimensional (3D) body scans of a female swimmer. The flow around this dynamic model was then calculated using computational fluid dynamics with the aid of moving 3D meshes. Evaluation of the numerical results delivered by the various motion cycles identified characteristic vortex structures for each of the cycles, which exhibited increasing intensity and drag influence. At maximum thrust, drag forces appear to be 12 times higher than those of a passive gliding swimmer. As far as we know, this is the first disclosure of vortex rings merging into vortex tubes in the wake after vortex recapturing. All unsteady structures were visualized using a modified Q-criterion also incorporated into our methods. At the very least, our approach is likely to be suited to further studies examining swimmers engaging in undulatory swimming during training or competition

Forschend lernen - studentische Umsetzung eines kooperativen Forschungsprojekts in Eigenverantwortung

Das Forschende Lernen stellt für das Fach Forschungsmethoden einen höchst interessanten Ansatz dar, der Wissensermittlung, Wissenserfahrung und fächerübergreifende Kompetenzen miteinander vereint. Der Werkstattbericht beschäftigt sich mit der Erarbeitung und Umsetzung des Konzeptes des Forschenden Lernens in den Bewegungswissenschaften sowie dem Maschinenbau und dem Umweltengineering

Sandy Everlasting (Helichrysum arenarium (L.) Moench): Botanical, Chemical and Biological Properties

Sandy everlasting [Helichrysum arenarium (L.) Moench] is herbaceous perennial plant belonging to Asteraceae family and it is native to Europe, Central Asia, and China. It belongs to the section HELICHRYSUM (Asteraceae family, genus Helichrysum) along with H. plicatum DC. Prodr., which very similar phenolic profile and H. italicum (Roth), which is widely used for essential oil extraction. Its flowers have a long tradition in European ethnomedicine as a cholagogue, choleretic, hepatoprotective, and detoxifying herbal drug. The flowers are rich in phenolic compounds including flavonoids, chalcones, phenolic acids, coumarins, and pyrones. Apart frompolyphenols, other compounds such as sterols, lignans, and glycosides of aromatic compounds have been also isolated from H. arenarium. The majority of authors confirm that the most important group of compounds responsible for biological activities is flavonoids. Moreover, significant activities of naringenin, one of the main flavonoids of H. arenarium, were reported. On the other hand, there are no clinical data about testing the extracts or preparations based on H. arenarium. Although H. arenarium is well known in phytotherapy for its potential in the treatment of gallbladder disease and are classified as endangered species in a number of European countries, very few data about its cultivation are available in the literature

Bacterial Biodegradation of 4-Monohalogenated Diphenyl Ethers in One-Substrate and Co-Metabolic Systems

The use of diphenyl ether (DE) and its 4-monohalogenated derivatives (4-HDE) as flame retardants, solvents, and substrates in biocide production significantly increases the risk of ecosystem contamination. Their removal is important from the point of view of environmental protection. The aim of this study was to evaluate the degradation processes of DE and 4-HDE by enzymes of the environmental bacterial strains under one-substrate and co-metabolic conditions. The study is focused on the biodegradation of DE and 4-HDE, the enzymatic activity of microbial strains, and the cell surface properties after contact with compounds. The results show that the highest biodegradation (96%) was observed for 4-chlorodiphenyl ether in co-metabolic culture with P. fluorescens B01. Moreover, the activity of 1,2-dioxygenase during degradation of 4-monohalogenated diphenyl ethers was higher than that of 2,3-dioxygenase for each strain tested. The presence of a co-substrate provoked changes in dioxygenase activity, resulting in the increased activity of 1,2-dioxygenase. Moreover, the addition of phenol as a co-substrate allowed for increased biodegradation of the diphenyl ethers and noticeable modification of the cell surface hydrophobicity during the process. All observations within the study performed have led to a deeper understanding of the contaminants’ biodegradation processes catalyzed by environmental bacteria

Profesor Andrzej Litwornia 1943–2006. Materiały do bibliografii

Professor Andrzej Litwornia 1943–2006. Materials for a biobibliography This publication was created during the inventory making and preparation of “Professor Andrzej Litwornia 1943–2006 archives in Udine” in June 2008. “Materials for a biobibliography” include a few-page “Biographical note” presenting facts from the professor’s life in a chronological order as well as information about his academic and didactic work, “Bibliography of publications” extended description of 115 titles and “Filmography” 8 original titles which complements the literature. Finally, there is a “List of printed sources” 86 items: interviews, publications, websites of scientific associations, reports, ending in 2009, still incomplete, especially when it comes to the Italian sources. The format of the present work has been infl uenced by “Bibliografi a di Andrzej Litwornia” 2005, reviewed and accepted by Professor Litwornia, which was prepared during the promotion of his last book and later added to a book that became a posthumous tribute to him 2007.Professor Andrzej Litwornia 1943–2006. Materials for a biobibliography This publication was created during the inventory making and preparation of “Professor Andrzej Litwornia 1943–2006 archives in Udine” in June 2008. “Materials for a biobibliography” include a few-page “Biographical note” presenting facts from the professor’s life in a chronological order as well as information about his academic and didactic work, “Bibliography of publications” extended description of 115 titles and “Filmography” 8 original titles which complements the literature. Finally, there is a “List of printed sources” 86 items: interviews, publications, websites of scientific associations, reports, ending in 2009, still incomplete, especially when it comes to the Italian sources. The format of the present work has been infl uenced by “Bibliografi a di Andrzej Litwornia” 2005, reviewed and accepted by Professor Litwornia, which was prepared during the promotion of his last book and later added to a book that became a posthumous tribute to him 2007

Listy Jana Czeczota. Faktografia w manuskryptach

The goal of this study is a presentation of a recently discovered, unknown letter of Jan Czeczot from 1845 against a biographical and historical background. According to the fact that an evaluation of the sources, mainly manuscripts and range of their use in the available biographical works is still far from a monographic level, a more complex view on the Philomath’s figure must be postponed until the publication collecting the entity of the materials (Association — related, literary and epistolographical) of Filareci’s trial, his exile and patriotic activity after his return . The presented, even partial study, seems to reveal that Jan Czeczot stayed true to the Philomaths Association’s devotion to “national duties” and was not enshadowed by Adam Mickiewicz or Filareci’s legend of his times. We ought to see him more widely: he was one of the first important folklorist poets, the author of valuable ethnographic works, confirming the exceptional persistence of Filarets’ organisational bonds with his attitude.The goal of this study is a presentation of a recently discovered, unknown letter of Jan Czeczot from 1845 against a biographical and historical background. According to the fact that an evaluation of the sources, mainly manuscripts and range of their use in the available biographical works is still far from a monographic level, a more complex view on the Philomath’s figure must be postponed until the publication collecting the entity of the materials (Association — related, literary and epistolographical) of Filareci’s trial, his exile and patriotic activity after his return . The presented, even partial study, seems to reveal that Jan Czeczot stayed true to the Philomaths Association’s devotion to “national duties” and was not enshadowed by Adam Mickiewicz or Filareci’s legend of his times. We ought to see him more widely: he was one of the first important folklorist poets, the author of valuable ethnographic works, confirming the exceptional persistence of Filarets’ organisational bonds with his attitude