Engineering oxidative enzymes Enzymes act as catalysts in chemical reactions to produce useful chemicals. Selectively oxidative enzymes are particularly useful in industrial applications but issues with availability, reliability, yield and robustness need to be addressed. Climate Change and Environment © Thinkstock The EU-funded project 'Effective redesign of oxidative enzymes for green chemistry' (OXYGREEN) developed tools to design oxidative enzymes for industrial and biotechnological applications. They investigated three families of biocatalysts — non-heme iron alpha-ketoglutarate dependent dioxygenases (KGDOs), Baeyer-Villiger monooxygenases (BVMOs) and mammalian P450 monooxygenases. Significant milestones were achieved through a deeper insight into enzyme function and structure. Ten new protein structures were elucidated and entered into the publicly available Protein Data Bank . The knowledge garnered was also used to generate a movie depicting at an atomic level how an enzyme performs selective oxidation. OXYGREEN also built a freely available web-based tool called MuteinDB that provides comprehensive information on muteins (mutant forms of enzymes). Methodologies for constructing and analysing high-quality enzyme mutant libraries were successfully developed. The developed methods include the electrochemical microtitre plate for screening P450 monooxygenases, enzyme-coupled screening for BVMOs and a product-based screening for KGDOs. Researchers also optimised the patented four-step sequence saturation mutagenesis method and the 3D computational tool Mutagenesis Assistant Program. Interesting enzyme mutants were identified after screening the selected enzyme families. The generated P450 monooxygenases showed good performance in an industrial setting and have already been patented. Hydroxyproline is an amino acid that could not be efficiently produced on an industrial scale until the KGDO-based whole-cell biocatalytic process was developed. This promising bioprocess is currently being pilot tested and technology transfer has already been executed. BVMOs have wide-ranging industrial applications such as synthesis of 3-hydroxypropionic acid, fragrant lactones and monomers of polyamide-9. Several of the newly synthesised BVMOs are under consideration for patent application and some are currently commercially available. Enzyme engineering knowledge and technology are offered on the Oxygreen Services website . Such academia–industry collaborations will facilitate further research in enzyme engineering and ensure rapid commercialisation of products. EU competitiveness will be enhanced through such green, sustainable and cost-effective bioprocesses obtained from renewable resources. Potential applications include production of biofuels, textiles, fragrances, fertilisers, food additives, packaging materials and drugs. Keywords Oxidative enzyme, enzyme, catalyst, chemical reaction, green chemistry, biotechnological, alpha-ketoglutarate dependent dioxygenases, Baeyer-Villiger monooxygenases, P450 monooxygenases, mutein, hydroxyproline, polyamide-9
