RESUMO
There is an increasing interest in the application of peroxygenases in biocatalysis, because of their ability to catalyse the oxyfunctionalisation reaction in a stereoselective fashion and with high catalytic efficiencies, while using hydrogen peroxide or organic peroxides as oxidant. However, enzymes belonging to this class exhibit a very low stability in the presence of peroxides. With the aim of bypassing this fast and irreversible inactivation, we study the use of a gradual supply of hydrogen peroxide to maintain its concentration at stoichiometric levels. In this contribution, we report a multienzymatic cascade for in situ generation of hydrogen peroxide. In the first step, in the presence of NAD+ cofactor, formate dehydrogenase from Candida boidinii (FDH) catalysed the oxidation of formate yielding CO2. Reduced NADH was reoxidised by the reduction of the flavin mononucleotide cofactor bound to an old yellow enzyme homologue from Bacillus subtilis (YqjM), which subsequently reacts with molecular oxygen yielding hydrogen peroxide. Finally, this system was coupled to the hydroxylation of ethylbenzene reaction catalysed by an evolved peroxygenase from Agrocybe aegerita (rAaeUPO). Additionally, we studied the influence of different reaction parameters on the performance of the cascade with the aim of improving the turnover of the hydroxylation reaction.
Assuntos
Proteínas de Bactérias/química , FMN Redutase/química , Formiato Desidrogenases/química , Proteínas Fúngicas/química , Peróxido de Hidrogênio/síntese química , Oxigenases de Função Mista/química , Agrocybe/química , Agrocybe/enzimologia , Bacillus subtilis/química , Bacillus subtilis/enzimologia , Proteínas de Bactérias/metabolismo , Derivados de Benzeno/química , Derivados de Benzeno/metabolismo , Biocatálise , Candida/química , Candida/enzimologia , Dióxido de Carbono/química , Dióxido de Carbono/metabolismo , Coenzimas/química , Coenzimas/metabolismo , FMN Redutase/metabolismo , Mononucleotídeo de Flavina/química , Mononucleotídeo de Flavina/metabolismo , Formiato Desidrogenases/metabolismo , Formiatos/química , Formiatos/metabolismo , Proteínas Fúngicas/metabolismo , Peróxido de Hidrogênio/metabolismo , Hidroxilação , Cinética , Oxigenases de Função Mista/metabolismo , NAD/química , NAD/metabolismo , Oxirredução , Oxigênio/química , Oxigênio/metabolismo , EstereoisomerismoRESUMO
An increasing number of biocatalytic oxidation reactions rely on H2 O2 as a clean oxidant. The poor robustness of most enzymes towards H2 O2 , however, necessitates more efficient systems for inâ situ H2 O2 generation. In analogy to the well-known formate dehydrogenase to promote NADH-dependent reactions, we here propose employing formate oxidase (FOx) to promote H2 O2 -dependent enzymatic oxidation reactions. Even under non-optimised conditions, high turnover numbers for coupled FOx/peroxygenase catalysis were achieved.