Publicação
Fungal bioconversion of Agro-Industrial by-products and modeling of Laccase Kinetics
| Resumo: | The study of enzymatic processes involved in fungal bioconversion of lignocellulosic byproducts is the main objective of this thesis. Since cellulose and lignin are the main components of most of these byproducts, the mathematical modeling of bioconversion processes can be used to predict/evaluate the behavior of the interveners in these processes allowing their optimization. In a first phase (Chapter 2), the bioconversion of two byproducts, wheat straw and chestnut shell, was evaluated in its enzymatic component and effect on the hydrolysis of the pretreated substrate, using a commercial mix of holocellulases. Lignocellulolytic enzymes, as fundamental catalysts in bioconversion processes, should be among the study priorities, not only to clarify their mechanisms, but also to search for multiple inhibition relationships. Following this perspective, the lignocellulolytic activity of the fungi and the enzymatic saccharification were evaluated. To clarify the relationships within the general mechanisms of bioconversion/saccharification, the effect of some variables (hydrolysis time, enzymatic activity and holocellulase concentration) was estimated whiles a general predictive model of saccharification. The findings showed that Trametes versicolor was able to significantly increase saccharification in both pretreated substrates. The covariance analysis showed a significant effect between lignin peroxidase and increased straw saccharification. A high consistency was also found, relating the effects of xylanase and laccase activities on the final release of reducing sugars from the chestnut shell. These results can be used for optimization planning in saccharification of this substrate. In a second phase (Chapter 3), the development of the work focused on one of the major enzymes of bioconversion, laccase. Several Michaelis-Menten models (linear and nonlinear) were applied to clarify the inhibition mechanisms that may influence the optimization of the processes. For the mathematical modeling of the inhibition mechanism, the oxidation of a lignin-derived natural substrate, (ferulic acid) in the presence of chlorine, was used. The models were submitted to a ranking methodology, using as estimator, of the relative quality of the different kinetic models, the Akaike Information Criterion (AIC) with respective Akaike Weights. At the end of this chapter it was concluded that, analysis of AIC with Akaike weights allowed the discrimination and quantification of probabilities associated with each model, with only one inhibitor concentration, confirming a competitive inhibition of laccase in the presence of chlorine. Continuing the study of laccase and possible inhibitions (Chapter 4), Bi-Bi substrate models were tested in two features of substrate inhibition: no inhibition and inhibition by reducing and/or oxidizing substrate. During delignification, many intermediates (e.g. 2,6-dimethoxyphenol, DMP) may serve as substrates and inhibitors at the same time. Generally, in the laccase oxidation, the dioxygen is assumed under saturating conditions, simplifying the mechanism and its study. It is also commonly assumed, that the reducing substrate and dioxygen are related via ping-pong mechanism. In order to clarify the laccase mechanism under non-saturating oxygen conditions, two substrates: ABTS (2,2'-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) and DMP, were used. The discrimination of the models was done based on the ranking methodology previously described (AIC). It was concluded that the most likely models for ABTS and DMP oxidation are Ping-Pong and Theorell-Chance, respectively. The catalytic efficiency of oxygen conversion to water appears to be compatible with a process relatively independent of reducing substrates and the bisubstrate mechanism type. In addition, in the DMP oxidation mechanism laccase is not oxygen-saturated which, in turn, may lead to potential increased reaction rates resulting from the use of oxygen-pressurized bioreactors. The Chapter 5 describes the development of a new methodology and a new integrated equation, which allows the determination of kinetic parameters for two mutually non-exclusive inhibitors, when one of them is produced during the reaction. The determination of kinetic parameters in these circumstances, without the integrated Michaelis-Menten equations, was referred as containing increased errors in parameter estimation. Phosphate and urea, as alkaline phosphatase inhibitors, were used to illustrate the study of this methodology. The results obtained were subjected to a comparative analysis of previously published data to assess: (i) whether the inhibitors were mutually exclusive or non-exclusive inhibitors; ii) if non-exclusive inhibitors, determine the nature of the interaction (facilitation, impediment or independence); iii) determine whether they were non-exclusive unique only to E (enzyme) or ES (enzymesubstrate complex), or both enzyme forms; iv) the type of inhibition and v) the kinetic constants of each inhibitor. The methodology developed, with the new integrated equation, allowed not only to obtain the inhibition constants of each inhibitor, but also to determine the interaction between the two inhibitors. This interaction study is not possible using the Michaelis-Menten initial velocity equations whenever one of the inhibitors is a reaction product. |
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| Autores principais: | Pinto, Paula Alexandra da Costa Sousa Botelho |
| Assunto: | Laccase pretreatment |
| Ano: | 2020 |
| País: | Portugal |
| Tipo de documento: | tese de doutoramento |
| Tipo de acesso: | acesso restrito |
| Instituição associada: | Universidade de Trás-os-Montes e Alto Douro |
| Idioma: | inglês |
| Origem: | Repositório da UTAD |
| Resumo: | The study of enzymatic processes involved in fungal bioconversion of lignocellulosic byproducts is the main objective of this thesis. Since cellulose and lignin are the main components of most of these byproducts, the mathematical modeling of bioconversion processes can be used to predict/evaluate the behavior of the interveners in these processes allowing their optimization. In a first phase (Chapter 2), the bioconversion of two byproducts, wheat straw and chestnut shell, was evaluated in its enzymatic component and effect on the hydrolysis of the pretreated substrate, using a commercial mix of holocellulases. Lignocellulolytic enzymes, as fundamental catalysts in bioconversion processes, should be among the study priorities, not only to clarify their mechanisms, but also to search for multiple inhibition relationships. Following this perspective, the lignocellulolytic activity of the fungi and the enzymatic saccharification were evaluated. To clarify the relationships within the general mechanisms of bioconversion/saccharification, the effect of some variables (hydrolysis time, enzymatic activity and holocellulase concentration) was estimated whiles a general predictive model of saccharification. The findings showed that Trametes versicolor was able to significantly increase saccharification in both pretreated substrates. The covariance analysis showed a significant effect between lignin peroxidase and increased straw saccharification. A high consistency was also found, relating the effects of xylanase and laccase activities on the final release of reducing sugars from the chestnut shell. These results can be used for optimization planning in saccharification of this substrate. In a second phase (Chapter 3), the development of the work focused on one of the major enzymes of bioconversion, laccase. Several Michaelis-Menten models (linear and nonlinear) were applied to clarify the inhibition mechanisms that may influence the optimization of the processes. For the mathematical modeling of the inhibition mechanism, the oxidation of a lignin-derived natural substrate, (ferulic acid) in the presence of chlorine, was used. The models were submitted to a ranking methodology, using as estimator, of the relative quality of the different kinetic models, the Akaike Information Criterion (AIC) with respective Akaike Weights. At the end of this chapter it was concluded that, analysis of AIC with Akaike weights allowed the discrimination and quantification of probabilities associated with each model, with only one inhibitor concentration, confirming a competitive inhibition of laccase in the presence of chlorine. Continuing the study of laccase and possible inhibitions (Chapter 4), Bi-Bi substrate models were tested in two features of substrate inhibition: no inhibition and inhibition by reducing and/or oxidizing substrate. During delignification, many intermediates (e.g. 2,6-dimethoxyphenol, DMP) may serve as substrates and inhibitors at the same time. Generally, in the laccase oxidation, the dioxygen is assumed under saturating conditions, simplifying the mechanism and its study. It is also commonly assumed, that the reducing substrate and dioxygen are related via ping-pong mechanism. In order to clarify the laccase mechanism under non-saturating oxygen conditions, two substrates: ABTS (2,2'-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) and DMP, were used. The discrimination of the models was done based on the ranking methodology previously described (AIC). It was concluded that the most likely models for ABTS and DMP oxidation are Ping-Pong and Theorell-Chance, respectively. The catalytic efficiency of oxygen conversion to water appears to be compatible with a process relatively independent of reducing substrates and the bisubstrate mechanism type. In addition, in the DMP oxidation mechanism laccase is not oxygen-saturated which, in turn, may lead to potential increased reaction rates resulting from the use of oxygen-pressurized bioreactors. The Chapter 5 describes the development of a new methodology and a new integrated equation, which allows the determination of kinetic parameters for two mutually non-exclusive inhibitors, when one of them is produced during the reaction. The determination of kinetic parameters in these circumstances, without the integrated Michaelis-Menten equations, was referred as containing increased errors in parameter estimation. Phosphate and urea, as alkaline phosphatase inhibitors, were used to illustrate the study of this methodology. The results obtained were subjected to a comparative analysis of previously published data to assess: (i) whether the inhibitors were mutually exclusive or non-exclusive inhibitors; ii) if non-exclusive inhibitors, determine the nature of the interaction (facilitation, impediment or independence); iii) determine whether they were non-exclusive unique only to E (enzyme) or ES (enzymesubstrate complex), or both enzyme forms; iv) the type of inhibition and v) the kinetic constants of each inhibitor. The methodology developed, with the new integrated equation, allowed not only to obtain the inhibition constants of each inhibitor, but also to determine the interaction between the two inhibitors. This interaction study is not possible using the Michaelis-Menten initial velocity equations whenever one of the inhibitors is a reaction product. |
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