The classical chemical ozonolysis represents one of the most important alkene cleavage reactions in organic chemistry. It is widely used in academia, but scarcely applied in industry due to major safety concerns. Ozone is toxic and the reaction intermediates are highly explosive. The second most important chemical method relies on the diol formation with subsequent oxidative cleavage. This method requires stoichiometric amounts of toxic metal oxidants. Our aim is to investigate novel enzymes for the cleavage of C=C double bonds by a mild and environmentally benign method.

Research question: How much chemical space can be covered by alkene cleaving enzymes and can it be a safe and more sustainable substitute for the chemical ozonolysis?

Our approach to solve this synthetically relevant problem combines in silico based enzyme discovery (BLAST search, sequence similarity network analysis & sequence selection by reported function), an in-house developed fluorescence-based high throughput assay (HTA) for the discovery, identification and quantification of enzymatic alkene cleavage activity, state of the art multivariate enzyme characterization by ‘design of experiments’ (DoE), structure-function relationship studies and the biocatalytic synthesis of aldehydes.

We aim to establish novel alkene cleaving enzymes to substitute the chemical and environmentally problematic ozonolysis or diol-cleavage. We will perform an in-depth characterization (kinetically and structurally) of putative candidates to gain deeper insights into the structure and their molecular mechanism. The focus will be on the scope and limitations of the tryptophan dioxygenases, carotenoid cleavage- and the cupin-family enzymes. Parameter optimization will be realized in a multi-variate approach by exploiting a HTA assay for aldehydes recently developed within our collaboration. Selected enzyme candidates will be applied in the synthesis of specific aldehydes.