Dottorato in BIOCHIMICA

Titolo della tesi: Biocatalytic Applications of Lathyrus Cicera Amine Oxidase

Biocatalysis refers to the use of enzymes from both natural sources or produced in recombinant form for the eco-sustainable organic synthesis of complex molecules. For quite a long time, only a subset of enzymes, such as lipases or ketoreductases were available for chemical synthesis applications, but this scenario is quickly changing with the development of a diverse toolkit of enzymes for a wider range of biotransformations. In this regard, increasing attention is paid to amine oxidases, enzymes belonging to the class of oxidoreductases that catalyze the oxidative deamination of primary amines into the corresponding aldehydes. These compounds are extremely interesting for their numerous industrial applications. Considering the high demand for “naturally” synthesized aldehydes (especially in food and cosmetic industries), the development of green enzymatic protocols for their production is of great interest. In this frame, we set up a fully enzymatic strategy to synthesize value-added aldehydes by means of a plant extracted amine oxidase (Lathyrus cicera amine oxidase, LCAO). As a first step, we have developed a chromatography-free purification protocol based on cross-flow ultrafiltration, which makes the production of this enzyme easily scalable. Furthermore, we determined the kinetic parameters of LCAO towards 20 differently substituted aliphatic and aromatic primary amines, and we developed a biocatalytic process for their conversion into the corresponding aldehydes. The reaction takes place in aqueous media at neutral pH in the presence of catalase, which removes the hydrogen peroxide produced during the reaction itself, contributing to the recycling of oxygen. A very high conversion rate (> 95%) was achieved within 3 hours for all the tested compounds. Although very promising, the use of this enzyme is challenging due to the possible inactivation that might occur at high product concentrations. One of the ways to overcome this issue could be the immobilization on solid supports, which typically improves the overall catalytic performance, allowing enzyme reuse/recycling. Among the different support materials, magnetic microparticles are considered the future of enzyme immobilization, due to their exceptional ease of handling, recovery, and reuse. We show that LCAO immobilized on this support can be easily recycled, retains its activity, and greatly improves its thermostability. Furthermore, we demonstrate that the immobilized enzyme can be successfully used for scaling up the synthesis of a set of variously substituted phenylacetaldehydes, with yields up to 45 times higher than the previously published method with the free enzyme. Testing free and immobilized LCAO stability in non-aqueous media, including biphasic systems, could be be advantageous for future biocatalytic applications as it would allow testing water insoluble substrates. We demonstrate LCAO is particularly stable in non-aqueous media as immobilized form.