Development of a novel high-throughput screening assay and its application to racemases

• Main Author: Morgan, Jemma
• Published: University of Edinburgh 2008
• Subjects: 572.7
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.659582

A high-throughput hydrogen peroxide-based colorimetric screen used to detect oxidase activity was extended to detect racemase activity through the production of the specific substrate for an enantioselective oxidase enzyme. A two-plasmid system (encoding the racemase and oxidase) was shown to be successful and could have possible applications for many different classes of enzymes that produce an oxidase substrate. In order to validate the screening technology, three amino acid racemase genes were cloned from genomic DNA and placed into expression vectors. The screen was used to confirm the substrate specificities of each racemase. The amino acid racemases were then subjected to random mutagenesis using the mutator strain method and error-prone polymerase chain reaction. Libraries of the variant racemase enzymes were screened for novel activities towards substrates not accepted by the wild type enzymes: L-arginine; L-lysine and L-leucine. Novel activity was discovered in selected <i>Streptomyces coelicolor </i>alanine racemase variants. Sequencing revealed that the best variant had three point mutations I195T, N223D and I374N. Computer modelling suggested that the I374N mutation was a key mutation and so the variant containing the double mutation (I195T and N223D) was prepared. Partial purification of the wild type enzyme, the variants containing the double and the triple mutations was carried out to compare the substrate specificities. The I195T/N223D variant was shown to be ten times more active towards L-arginine that the wild type enzyme, and the variant containing all three mutations was shown to be 100 times more active towards L-arginine than the wild type enzyme. Both variants displayed novel activity towards L-arginine only.