WP2 – Heme-dependent monooxygenases

WP2 – Heme-dependent monooxygenases (RWTH – ESR4, TUG – ESR5 and SSB – ESR6)

The research will focus on prevention of undesired side reactions (uncoupling) by enzyme engineering and developing an efficient technical approach to apply such monooxygenases. Three existing heme-dependent monooxygenases will be optimised and investigated for the conversion of heterocyclic compounds (RWTH – ESR4). The SeSaM libraries generated at SSB (ESR6) will be used to identify the amino acid positions that govern selectivity. The identified positions will then be subjected to site saturation mutagenesis and subsequent beneficial variants will be characterised. The YASARA software package will assist in designing and analysing the libraries and can also be used for the modelling of the heme-dependent monooxygenases studied by TUG and SSB. The work conducted by the ESR at TUG (ESR5) will generate novel heme-monooxygenases that will be produced and evaluated (UNIPV), engineered and screened (RWTH), coexpressed in controlled bioreactor experiments and scaled up and evaluated for industrial biotransformations. Finally, ESR6 will work on the generation of random mutagenesis libraries to be used by RWTH and TUG, as well as the other partners. Further, she/he will produce the enzymes and the variants obtained at TUG and RWTH and will use the identified improved enzymes for substrate testing and application in hydroxylation kits.