Caracterização bioquímica e biofísica de enzima ácido ferúlico descarboxilase e aplicação na produção de 4-vinilguaiacol a partir de ácido ferúlico
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Data
2023-03-07
Autores
Navarrete, Vitória Gonçalves
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Universidade Estadual Paulista (Unesp)
Resumo
A crescente valorização dos processos industriais sustentáveis e limitações na obtenção de alguns produtos de importância industrial têm tornado a tecnologia enzimática uma alternativa cada vez mais oportuna. A biossíntese de compostos permite produção contínua, sem impacto ou interferência sazonal, maior controle e otimização de parâmetros e maior especificidade dos produtos. A enzima ácido ferúlico descarboxilase (FADase) catalisa a descarboxilação não oxidativa do ácido ferúlico (AF) em 4-vinilguaiacol (4VG), um composto fenólico volátil utilizado como aromatizante em alimentos e bebidas. O 4VG, composto com aroma picante de cravo, é um produto de interesse industrial cuja demanda não é suprida por fontes naturais e com alto custo de produção por via química. Com a finalidade de entender a dinâmica estrutural e parâmetros funcionais da FADase heteróloga, derivada do gene da bactéria Klebsiella pneumoniae, para aplicação na produção de 4VG, foram realizados estudos bioquímicos e biofísicos da enzima. Para futura aplicação em processos industriais de produção, também foram realizadas tentativas de imobilização da enzima. Os testes de capacidade de conversão de AF em 4VG pela enzima purificada e em extrato bruto tiveram consumo de substrato e formação de produto foram confirmados por HPLC. A temperatura e pH ótimos determinados foram 40 oC e pH 5,5, e a faixa de estabilidade variou entre 35 oC e 50 oC e pH 5,0 a 5,5. O melting point de 60 oC foi determinado por Differential Scanning Calorimetry (DSC) e a investigação de um possível estágio intermediário de desenovelamento foi feito por Circular Dichroism (CD) nas faixas de 218 e 222nm, que apontou apenas dois estágios. A modelagem computacional da enzima possibilitou a confirmação da estrutura, seu sítio catalítico e sugeriu compatibilidade com glutaraldeído como agente reticulante a ser utilizado para imobilização. Nos ensaios de imobilização com CLEAs (cross-linked aggregates) e m-CLEAs (magnetic cross-linked aggregates), observou-se perda da atividade da enzima. Visando o entendimento deste resultado, a interação da FADase com o ligante foi analisada por espectroscopia de fluorescência, a partir da qual foram determinados dois sítios de ligação e uma constante de associação muito baixa para que a ligação fosse estável. Entretanto, estes resultados ainda são inconclusivos e novos ensaios são necessários para se definir as condições de imobilização adequadas para a enzima.
The growing appreciation of sustainable industrial processes and limitations in obtaining some industrially important products have made enzymatic technology an increasingly opportune alternative. The biosynthesis of compounds allows continuous production, without seasonal interference or impact, greater control and optimization of parameters and greater product specificity. The enzyme ferulic acid decarboxylase (FADase) catalyzes the non-oxidative decarboxylation of ferulic acid (FA) to 4-vinylguaiacol (4VG), a volatile phenolic compound used as a flavoring in foods and beverages. 4VG, a compound with a spicy clove aroma, is a product of industrial interest whose demand is not met by natural sources and with a high cost of chemical production. In order to understand the structural dynamics and functional parameters of the heterologous FADase, derived from the gene of the bacterium Klebsiella pneumoniae, for application in the production of 4VG, biochemical and biophysical studies of the enzyme were carried out. For future application in industrial production processes, attempts were also made to immobilize the enzyme. Tests on the ability to convert FA into 4VG by the purified enzyme and crude extract had substrate consumption and product formation confirmed by HPLC. The optimal temperature and pH determined were 40oC and pH 5.5, and the stability range varied between 35 and 50oC and pH 5.0 to 5.5. The melting point of 60oC was determined by Differential Scanning Calorimetry (DSC) and the investigation of a possible intermediate stage of unfolding was carried out by Circular Dichroism (CD) in the ranges of 218 and 222 nm, which identified only two stages. The computational modeling of the enzyme enabled confirmation of the structure, its catalytic site and suggested compatibility with glutaraldehyde as a cross-linking agent to be used for immobilization. In immobilization assays with CLEAs (cross-linked aggregates) and m-CLEAs (magnetic cross-linked aggregates), loss of enzyme activity was observed. Aiming at understanding this result, the interaction of the FADase with the ligand was analyzed by Fluorescence Spectroscopy, from which two binding sites and a very low association constant were determined for the binding to be stable. However, these results are still inconclusive and new tests are needed to define the appropriate immobilization conditions for the enzyme.
The growing appreciation of sustainable industrial processes and limitations in obtaining some industrially important products have made enzymatic technology an increasingly opportune alternative. The biosynthesis of compounds allows continuous production, without seasonal interference or impact, greater control and optimization of parameters and greater product specificity. The enzyme ferulic acid decarboxylase (FADase) catalyzes the non-oxidative decarboxylation of ferulic acid (FA) to 4-vinylguaiacol (4VG), a volatile phenolic compound used as a flavoring in foods and beverages. 4VG, a compound with a spicy clove aroma, is a product of industrial interest whose demand is not met by natural sources and with a high cost of chemical production. In order to understand the structural dynamics and functional parameters of the heterologous FADase, derived from the gene of the bacterium Klebsiella pneumoniae, for application in the production of 4VG, biochemical and biophysical studies of the enzyme were carried out. For future application in industrial production processes, attempts were also made to immobilize the enzyme. Tests on the ability to convert FA into 4VG by the purified enzyme and crude extract had substrate consumption and product formation confirmed by HPLC. The optimal temperature and pH determined were 40oC and pH 5.5, and the stability range varied between 35 and 50oC and pH 5.0 to 5.5. The melting point of 60oC was determined by Differential Scanning Calorimetry (DSC) and the investigation of a possible intermediate stage of unfolding was carried out by Circular Dichroism (CD) in the ranges of 218 and 222 nm, which identified only two stages. The computational modeling of the enzyme enabled confirmation of the structure, its catalytic site and suggested compatibility with glutaraldehyde as a cross-linking agent to be used for immobilization. In immobilization assays with CLEAs (cross-linked aggregates) and m-CLEAs (magnetic cross-linked aggregates), loss of enzyme activity was observed. Aiming at understanding this result, the interaction of the FADase with the ligand was analyzed by Fluorescence Spectroscopy, from which two binding sites and a very low association constant were determined for the binding to be stable. However, these results are still inconclusive and new tests are needed to define the appropriate immobilization conditions for the enzyme.
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Palavras-chave
Ácido ferúlico descarboxilase, Ácido ferúlico, 4-vinilguaiacol, Ferulic acid decarboxylase, Ferulic acid, 4-vinylguaiacol