Efficient screening for new amino acid dehydrogenase activity: directed evolution of Bacillus sphaericus phenylalanine dehydrogenase towards activity with an unsaturated non-natural amino acid.

Mutants of phenylalanine dehydrogenase (PheDH) from Bacillus sphaericus with improved activity and specificity towards an unsaturated non-natural amino acid of commercial interest, propargylglycine, have been obtained by directed evolution after screening 10,000 colonies. An effective high-throughput screening assay for the detection of amino acid dehydrogenase activity using spectrophotometric measurement of formazan colour has been developed and explored. Parallel over-expression of the target protein library in 96-well plates was optimized by using auto-induction medium. Using these methodologies and medium/high mutation frequency error-prone polymerase chain reaction, combinations of mutations and multi-site saturation mutagenesis targeting two "hot-spots" close to the active site of the enzyme, one mutant was obtained with 7.4-fold improved catalytic efficiency with the target substrate together with 612-fold improvement of selectivity between the target substrate and the natural one. In the high mutation frequency epPCR and saturation mutagenesis, a robotic colony picker and automation workstation were employed for the high-throughput screening.