Metodologias enzimáticas envolvendo nitrilas hidratases e transaminases para aplicações em síntese orgânica
Carregando...
Data
2022-10-10
Autores
Teixeira, Iris dos Santos
Título da Revista
ISSN da Revista
Título de Volume
Editor
Universidade Estadual Paulista (Unesp)
Resumo
A biocatálise consiste no uso de enzimas e outros catalisadores biológicos na transformação de substâncias orgânicas não naturais. Ela é uma ferramenta importante na síntese orgânica, já que as enzimas são seletivas, atuam em condições reacionais brandas e em solventes ambientalmente amigáveis. Entre os biocatalisadores disponíveis, as transaminases (ATAs) são enzimas dependentes de piridoxal-5-fosfato (PLP) que catalisam a transferência de grupos amino para aldeídos e/ou cetonas. Nesse trabalho foram estudadas as reações de aminação redutiva de cetonas proquirais, com foco em avaliar a presença de heterociclos e de um sistema insaturado nos compostos. Foram empregadas um conjunto de cinco enzimas selvagens recombinantes e uma enzima comercial. Quando se avaliou a presença do heterociclo na furilmetilcetona, foi observado que a conversão diminuiu drasticamente quando comparado a cetona acetofenona, indicando que a presença do heteroátomo no anel de cinco membros influenciou negativamente essa reação. Em relação a cetona α-β-insaturada 4-fenilbut-3-en-2-ona, também foi demonstrado que a conversão caiu drasticamente em relação a seu análogo saturado 1-fenil-3-butanona (78 - >99%), e esse sistema foi posteriormente estudado com maior profundidade com o auxílio de técnicas de ancoragem molecular. As nitrilas hidratases (NHases) são enzimas amplamente empregadas industrialmente, que realizam a reação de hidratação enzimática de nitrilas a suas correspondentes amidas. Nesse trabalho primeiramente foi investigada a presença de uma NHase na bactéria Lysinibacillus boronitolerans CBMAI 2094, previamente identificada como um dos microrganismos responsáveis pela biodegradação do herbicida benzonitrilado Totril® através de triagens enzimáticas. Para identificação do gene responsável pela NHase foram usadas duas abordagens: uma abordagem in silico através de técnicas de bioinformática, e também através de reações em cadeia da polimerase (PCR) investigativas. Por meio das duas estratégias não foi possível identificar o gene correspondente a enzima de interesse. Posteriormente a reação de hidratação enzimática com a NHase de Rhodococcus erythropolis foi otimizada com o uso de maior porcentagem de DMSO como cossolvente no meio reacional, melhorando os resultados de conversão para as nitrilas testadas. Por fim a NHase de R. erythropolis foi usada como molde para a obtenção de variantes enzimáticas através de mutagênese sítio dirigida. Com auxílio de ancoragem molecular para determinação da localização das mutações pontuais, foram obtidas três variantes enzimáticas: W118A, W118D e W118H e elas foram empregadas em reações com um conjunto de nitrilas.
Biocatalysis consists of the use of enzymes and other biological catalysts in the transformation of unnatural organic substances. It is an important tool in organic synthesis, since enzymes are selective, act under mild reaction conditions and in environmentally friendly solvents. Among the available biocatalysts, amine transaminases (ATAs) are pyridoxal-5- phosphate (PLP)-dependent enzymes that catalyze the transfer of amino groups to aldehydes and/or ketones. In this work, the reductive amination reactions of prochiral ketones were studied, focusing on evaluating the presence of heterocycles and an unsaturated system in the compounds. A set of five recombinant wild-type enzymes and a commercial enzyme were used. When the presence of the heterocycle in the furylmethylketone was evaluated, it was observed that the conversion decreased dramatically when compared to the ketone acetophenone, indicating that the presence of the heteroatom in the five-membered ring negatively influenced this reaction. Regarding the α-β-unsaturated ketone 4-phenylbut-3-en-2-one, it was also shown that the conversion dropped dramatically compared to its saturated analogue 1-phenyl-3- butanone (78 - >99%), and this system was later studied in greater depth with the aid of molecular anchoring techniques. Nitriles hydratases (NHases) are enzymes widely used industrially, which carry out the enzymatic hydration reaction of nitriles to their corresponding amides. In this work, we first investigated the presence of a NHase in the bacterium Lysinibacillus boronitolerans CBMAI 2094, previously identified as one of the microorganisms responsible for the biodegradation of the benzonitrified herbicide Totril® through enzymatic screening. Two approaches were used to identify the gene responsible for NHase: an in-silico approach through bioinformatics, and also through investigative polymerase chain reactions (PCR). Using the two strategies it was not possible to identify the gene corresponding to the enzyme of interest. Subsequently, the enzymatic hydration reaction with NHase from Rhodococcus erythropolis was optimized using a higher percentage of DMSO as a co-solvent in the reaction medium, improving the conversion results for the tested nitriles. Finally, R. erythropolis NHase was used as a template to obtain enzymatic variants through site-directed mutagenesis. Molecular docking was used to determine the location of point mutations, and three enzymatic variants were obtained: W118A, W118D and W118H which were used in reactions with a set of nitriles.
Biocatalysis consists of the use of enzymes and other biological catalysts in the transformation of unnatural organic substances. It is an important tool in organic synthesis, since enzymes are selective, act under mild reaction conditions and in environmentally friendly solvents. Among the available biocatalysts, amine transaminases (ATAs) are pyridoxal-5- phosphate (PLP)-dependent enzymes that catalyze the transfer of amino groups to aldehydes and/or ketones. In this work, the reductive amination reactions of prochiral ketones were studied, focusing on evaluating the presence of heterocycles and an unsaturated system in the compounds. A set of five recombinant wild-type enzymes and a commercial enzyme were used. When the presence of the heterocycle in the furylmethylketone was evaluated, it was observed that the conversion decreased dramatically when compared to the ketone acetophenone, indicating that the presence of the heteroatom in the five-membered ring negatively influenced this reaction. Regarding the α-β-unsaturated ketone 4-phenylbut-3-en-2-one, it was also shown that the conversion dropped dramatically compared to its saturated analogue 1-phenyl-3- butanone (78 - >99%), and this system was later studied in greater depth with the aid of molecular anchoring techniques. Nitriles hydratases (NHases) are enzymes widely used industrially, which carry out the enzymatic hydration reaction of nitriles to their corresponding amides. In this work, we first investigated the presence of a NHase in the bacterium Lysinibacillus boronitolerans CBMAI 2094, previously identified as one of the microorganisms responsible for the biodegradation of the benzonitrified herbicide Totril® through enzymatic screening. Two approaches were used to identify the gene responsible for NHase: an in-silico approach through bioinformatics, and also through investigative polymerase chain reactions (PCR). Using the two strategies it was not possible to identify the gene corresponding to the enzyme of interest. Subsequently, the enzymatic hydration reaction with NHase from Rhodococcus erythropolis was optimized using a higher percentage of DMSO as a co-solvent in the reaction medium, improving the conversion results for the tested nitriles. Finally, R. erythropolis NHase was used as a template to obtain enzymatic variants through site-directed mutagenesis. Molecular docking was used to determine the location of point mutations, and three enzymatic variants were obtained: W118A, W118D and W118H which were used in reactions with a set of nitriles.
Descrição
Palavras-chave
Química verde, Biocatálise, Enzimas microbianas, Biologia molecular, Expressão gênica