High throughput screening of biocatalysts is of great relevance for the fields of chemical processing, biotransformation, and drug discovery. Continuous progress in molecular biology, especially DNA cloning and mutagenesis, has facilitated access to a number of biocatalysts unpredicted a few years ago. Conventional screening methods practiced by organic chemists are unable to provide answers within the process development timeline for new pharmaceuticals. ThermoGen proposes to develop a comprehensive screening system for hydrolases. This system will use actual substrates instead of fluorogenic/chromogenic substrate analogs and will also perform enantioselectivity measurements. In Phase I we will develop an assay for isolated enzymes based on pH indicators using a solid phase format. We will use fluorescence indicators as an alternative for screening heterogeneous reaction mixtures, like in vivo assay of clones and screening of poorly soluble substrates. In Phase II we will design assays for the synthetic use of hydrolytic enzymes (in anhydrous media) and will develop data analysis software compatible with the mainstream hardware available. Phase III will see commercialization of the solid phase and the data analysis software and the offer of the screening technology to synthetic organic chemists and pharmaceutical manufacturers. PROPOSED COMMERCIAL APPLICATION: ThermoGen proposes to develop a high throughput assay to asses activity and enantioselectivity of hydrolytic enzymes. This assay will shorten and simplify the currently tedious and timeconsuming screening step to discover and implement industrial scale biotransformation processes.
