Caracterização estrutural de enzimas lignocelulolíticas com potencial de aplicação em biorrefinaria
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2020-09-04
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Universidade Estadual Paulista (Unesp)
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O cenário atual do uso de biocatalisadores em processos industriais movimenta a busca por novas enzimas, principalmente as que são capazes de degradar material lignocelulósico potencialmente utilizado na produção de biocombustíveis e outros produtos de alto valor agregado. O etanol de segunda geração é considerado um substituto sustentável aos combustíveis fósseis, devido ao processo ser desenvolvido a partir de resíduos agroindustriais. No entanto, a produção deste biocombustível detém diversos interferentes próprios do processo de produção, que contribuem para impulsionar a busca e o aperfeiçoamento de enzimas a fim de torná-las mais estáveis e eficientes. As enzimas β-glicosidase, β-xilosidase e lacase apresentam um amplo potencial biotecnológico, principalmente no que diz respeito à sacarificação da biomassa lignocelulósica. A resolução da estrutura tridimensional destas enzimas é de grande relevância, pois permite compreender alguns dos inúmeros mecanismos biológicos destas macromoléculas, como a especificidade da enzima pelo substrato, relação estrutura-função, arquitetura proteica e centros catalíticos. Deste modo, o presente estudo teve como objetivo expressar, purificar e caracterizar estruturalmente as enzimas β-glicosidase, β-xilosidase e lacase. A enzima β-glicosidase tolerante e estimulada pela glicose (Bg10) foi expressa e purificada em alta concentração e submetida a ensaios de cristalografia e difração de raios-X que forneceram dados de difração a uma resolução de 2,3 Å e permitiram determinar sua estrutura tridimensional. A estrutura proteica apresentou características estruturais conservadas entre as β-glicosidases GH1 tolerantes e estimuladas pela glicose. Além disso, seus dados estruturais forneceram percepções importantes sobre alguns dos mecanismos biológicos envolvidos na tolerância e estímulo pela glicose desta molécula, tais como a configuração do canal do sítio ativo e a presença de resíduos “gatekeepers”, os quais regulam a passagem e a orientação da glicose. A enzima β-xilosidase (BXil), por sua vez, teve sua caracterização estrutural realizada através de abordagens in silico e estudos de ancoragem molecular, os quais forneceram informações importantes sobre esta molécula, tais como a arquitetura de domínios composta pela repetição de dois domínios típicos de GH43 conservados e até hoje não caracterizados; a disposição do seu sítio catalítico e os principais resíduos envolvidos em interações com diferentes substratos. A expressão da enzima BXil ocorreu em corpos de inclusão e, assim, sua extração foi realizada através de métodos desnaturantes com otimização de protocolo e submetida a ensaios de cristalografia, no entanto, não ocorreu a formação de cristais. Sendo assim, novas abordagens para obtenção da proteína solúvel estão sendo realizadas, a fim de sustentar a caracterização in silico e desvendar a função e a interação entre seus domínios a partir de caracterização cinética e estrutural. Por último, a enzima lacase (LacMeta), também foi submetida à ensaios de cristalografia e difração de raios-X com dados coletados a uma resolução de 1,8 Å. Apesar disso, não foi possível determinar a estrutura da LacMeta através do método de substituição molecular devido à falta de um modelo de fase adequado no Banco de Dados de Proteínas. Diante disso, será necessária a realização de novos ensaios para obtenção de cristais derivados com selenometionina para obtenção das fases e determinação da estrutura. Este trabalho de caracterização permitiu elucidar algumas características estruturais destas macromoléculas, conhecimento este que poderá ser útil para potencializar suas propriedades catalíticas de acordo com propósitos específicos para aplicação biotecnológica em biorrefinaria.
The current scenario of the use of biocatalysts in industrial processes moves the research in the search for new enzymes, especially those that are capable of degrading lignocellulosic material potentially used in the production of biofuels and other products with high added value. Second generation ethanol is considered a sustainable substitute for fossil fuels, due to the process being developed from agro-industrial waste. However, the production of this biofuel has several interferents hat part of the production process, which contribute to boost the search and the improvement of enzymes in order to make them more stable and efficient. The enzymes β-glycosidase, β-xylosidase and laccase have a wide biotechnological potential, mainly with regard to the saccharification of lignocellulosic biomass. The resolution of the three-dimensional structure of these enzymes is of great relevance, as it will allow us to understand some of the innumerable biological mechanisms of these macromolecules, such as the substrate specificity, structure-function relationship, protein architecture and catalytic centers. Thus, the present study aimed to express, purify and structurally characterize the enzymes β-glycosidase, β-xylosidase and laccase. The glucose-stimulated and tolerant β-glucosidase enzyme (Bg10) was expressed and purified in high concentration and subjected to crystallography and X-ray diffraction tests that provided diffraction data for a resolution of 2.3 Å and allowed to determine its three-dimensional structure. The protein structure showed structural characteristics conserved among tolerant and glucose-stimulated GH1 β-glycosidases. In addition, its structural data provided important insights into some of the biological mechanisms involved in glucose tolerance and stimulation of this molecule, such as the configuration of the active site channel and the presence of gatekeeper’s residues, which regulate the passage and orientation glucose. The β-xylosidase enzyme (BXil), in turn, had its structural characterization carried out through in silico approaches and molecular docking studies, which provided important information about this molecule, such as the domain architecture composed by the repetition of two typical domains of conserved and not yet characterized GH43; the disposition of its catalytic site and the main residues involved in interactions with different substrates. The expression of the BXil enzyme occurred in inclusion bodies and, therefore, its extraction was performed through denaturing methods with protocol optimization and subjected to crystallography tests, however, crystal formation did not occur. The factors that may explain the absence of crystals may be related to the characteristics of the sample obtained from denaturing extractions, being homogeneity, concentration and even its intrinsic characteristics. Therefore, new approaches to obtain the soluble protein are being carried out, in order to support the characterization in silico and unveil the function and the interaction between its domains based on kinetic and structural characterization. Finally, the laccase enzyme (LacMeta) was also subjected to crystallography and X-ray diffraction tests with data collected to a resolution of 1.8 Å. Despite this, it was not possible to determine the structure of LacMeta using the molecular substitution method, due to the lack of a suitable phasing model in the Protein Data Bank. Therefore, it will be necessary to carry out new tests to obtain crystals derivatized with selenomethionine to obtain the phases and determine the structure. This characterization work allowed to elucidate some structural characteristics of these macromolecules, a knowledge that could be useful to enhance their catalytic properties according to specific purposes for biotechnological application in biorefinery.
The current scenario of the use of biocatalysts in industrial processes moves the research in the search for new enzymes, especially those that are capable of degrading lignocellulosic material potentially used in the production of biofuels and other products with high added value. Second generation ethanol is considered a sustainable substitute for fossil fuels, due to the process being developed from agro-industrial waste. However, the production of this biofuel has several interferents hat part of the production process, which contribute to boost the search and the improvement of enzymes in order to make them more stable and efficient. The enzymes β-glycosidase, β-xylosidase and laccase have a wide biotechnological potential, mainly with regard to the saccharification of lignocellulosic biomass. The resolution of the three-dimensional structure of these enzymes is of great relevance, as it will allow us to understand some of the innumerable biological mechanisms of these macromolecules, such as the substrate specificity, structure-function relationship, protein architecture and catalytic centers. Thus, the present study aimed to express, purify and structurally characterize the enzymes β-glycosidase, β-xylosidase and laccase. The glucose-stimulated and tolerant β-glucosidase enzyme (Bg10) was expressed and purified in high concentration and subjected to crystallography and X-ray diffraction tests that provided diffraction data for a resolution of 2.3 Å and allowed to determine its three-dimensional structure. The protein structure showed structural characteristics conserved among tolerant and glucose-stimulated GH1 β-glycosidases. In addition, its structural data provided important insights into some of the biological mechanisms involved in glucose tolerance and stimulation of this molecule, such as the configuration of the active site channel and the presence of gatekeeper’s residues, which regulate the passage and orientation glucose. The β-xylosidase enzyme (BXil), in turn, had its structural characterization carried out through in silico approaches and molecular docking studies, which provided important information about this molecule, such as the domain architecture composed by the repetition of two typical domains of conserved and not yet characterized GH43; the disposition of its catalytic site and the main residues involved in interactions with different substrates. The expression of the BXil enzyme occurred in inclusion bodies and, therefore, its extraction was performed through denaturing methods with protocol optimization and subjected to crystallography tests, however, crystal formation did not occur. The factors that may explain the absence of crystals may be related to the characteristics of the sample obtained from denaturing extractions, being homogeneity, concentration and even its intrinsic characteristics. Therefore, new approaches to obtain the soluble protein are being carried out, in order to support the characterization in silico and unveil the function and the interaction between its domains based on kinetic and structural characterization. Finally, the laccase enzyme (LacMeta) was also subjected to crystallography and X-ray diffraction tests with data collected to a resolution of 1.8 Å. Despite this, it was not possible to determine the structure of LacMeta using the molecular substitution method, due to the lack of a suitable phasing model in the Protein Data Bank. Therefore, it will be necessary to carry out new tests to obtain crystals derivatized with selenomethionine to obtain the phases and determine the structure. This characterization work allowed to elucidate some structural characteristics of these macromolecules, a knowledge that could be useful to enhance their catalytic properties according to specific purposes for biotechnological application in biorefinery.
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Biotecnologia, Enzimas, Cristalografia de raios-X