Towards robust radiobiocatalytic transformations combining radiolytic H2O2 generation and selective peroxygenasecatalysis

• Autor(a) principal: Gil, Inês de Oliveira
• Data de Publicação: 2023
• Tipo de documento: Dissertação
• Idioma: eng
• Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
• Texto Completo: http://hdl.handle.net/10362/161535
• topic: Unspecific Peroxygenase; Radioactivity; Physical separation; Flow Biotransformation; Radiolytic H2O2; Enzyme stability; Domínio/Área Científica::Engenharia e Tecnologia::Outras Engenharias e Tecnologias

Unspecific peroxygenases (UPO) have recently gained attention for their capability to catalyse oxidative reactions, a challenging task in organic chemistry. These enzymes offer higher activity, enhanced stability, and a broad substrate scope. Nonetheless, a significant limitation is their inactivation under high H2O2 concentrations. To address this, strategies like in situ H2O2 generation systems, have been explored, such as the novel approach employing water radiolysis for H2O2 generation. In a prior study, a major hurdle was encountered, with the enzyme becoming inactivated after only 30 minutes of direct radiation exposure. Thus, the goal of this project was to overcome this limitation through the application of flow chemistry. To achieve this, a suitable setup was assembled, using a non-reactive plastic column to ensure H2O2 stability inside. To retain the enzyme in the column and separate it from the radiation source, enzyme immobilisation was employed. Surprisingly, this study revealed that a neutral pH is better for immobilisation, contrary to the literature. At pH 7, the immobilisation yield was 58.8%, compared to 55.1% at pH 8. The activity of the immobilised enzyme was also slightly higher at neutral pH (0.041 U µL-1), compared to pH 8 (0.036 U µL-1). Enzyme stability studies, in flow chemistry, were performed using supplemented H2O2 and the radiation source (radiolytic H2O2). The supplemented H2O2 strategy demonstrated higher enzyme stability, maintaining constant substrate conversion for several days and, even, extending to weeks. However, with the introduction of the radiolytic H2O2, stability was reduced to only 7 days, possibly due to H2O2 generation within the radiation source. Notably, the enzyme's stability was reduced to 24 hours when directly exposed to the radiation source. Consequently, it was deduced that the physical separation of the enzyme from the radiation source provided the solution to overcome the stability challenges encountered in previous study.