Mr C.R. Otey, California Institute of Technology, USA

Mr Otey, from the group of Frances Arnold, discussed work on making unnatural P450 by combinatorial techniques for use in the oxidation of unactivated C-H bonds. There are various approaches to generating P450 chimeras, with a huge potential protein space to explore, but by breaking existing enzymes into smaller sections and recombining these the chances of finding active enzymes is increased and this is coupled with selective mutations that are less likely to disrupt the protein structure. The number of contacts broken by recombination is called the E value and the prediction is that chimeric sequences with low E values will be more likely to fold and so produce active enzymes. A rapid screening technique for activity on a variety of substrates was designed based on colorimetric differentiation. 8-(4-Nitrophenoxy)octanoic acid is one such substrate which is oxidised at C-8 to generate a hemi-acetal which breaks down under the test conditions to generate 4-nitrophenol, which reacts with 4-aminoantipyrine to produce a red colour. Chimeric P450s with specific activity for oxidation of 2-phenoxyethanol were found.