title: Efficient screening for new amino acid dehydrogenase activity: directed evolution of Bacillus sphaericus phenylalanine dehydrogenase towards activity with an unsaturated non-natural amino acid.
abstract: 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.
authors.affiliation: School of Biomolecular and Biomedical Science, Conway Institute, University College Dublin, Belfield, Dublin 4, Ireland.
authors.forename: Sihong
authors.initials: S
authors.lastname: Chen
authors.forename: Paul C
authors.initials: PC
authors.lastname: Engel
mesh_terms: D000480:Alkynes / Q000378:metabolism*; D000594:Amino Acid Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology* / Q000235:genetics; D055162:Biocatalysis; D020134:Catalytic Domain; D019020:Directed Molecular Evolution; D005998:Glycine / Q000031:analogs & derivatives* / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D016415:Sequence Alignment; D013379:Substrate Specificity
chemical_list: D000480:Alkynes; C009055:propargylglycine; D000594:Amino Acid Oxidoreductases; C022342:phenylalanine oxidase; D005998:Glycine