10181342Enhancing Enzyme Quality and Yield to Unlock Sustainable Biocatalytic NitrationActiveGrant for R&DInnovate UKOur project, Novel Biocatalysts for Sustainable Manufacture of Pharmaceuticals, is developing a greener, safer way to produce important pharmaceutical chemicals. Traditional chemical processes for adding nitro groups to molecules require harsh conditions and generate hazardous waste. We are replacing these methods with enzymes called nitrating cytochrome P450s (NCP450s), which can perform the same transformations under mild, water-based conditions. A key challenge is producing these enzymes consistently at high quality. The enzymes need a molecule called heme to function properly, and standard production methods can result in a mixture of active and inactive proteins. This variability makes it difficult to scale up the process for industrial use. To solve this, we are working with the Centre for Process Innovation (CPI) to identify the best microbial hosts for producing fully functional NCP450 enzymes. By testing different bacteria, including specially engineered E. coli strains and naturally heme-producing species, we aim to find a system that reliably produces large amounts of active enzyme. This will ensure our biocatalysts perform consistently and can be scaled up safely and efficiently. The project will generate new knowledge about which microbes are best suited for producing these enzymes, develop methods to measure enzyme quality accurately, and provide practical guidance for industrial use. These outputs will strengthen our existing programme, support regulatory compliance, and accelerate the adoption of environmentally friendly manufacturing processes in the pharmaceutical industry. By overcoming this bottleneck, our work will help make pharmaceutical manufacturing safer, more sustainable, and less wasteful, supporting the UK's leadership in green biomanufacturing and innovation.