title: Continuous colorimetric screening assay for detection of d-amino acid aminotransferase mutants displaying altered substrate specificity.
abstract: D-Amino acid aminotransferase (DAAT) catalyzes the synthesis of numerous d-amino acids, making it an attractive biocatalyst for the production of enantiopure d-amino acids. To bolster its biocatalytic applicability, improved variants displaying increased activity toward non-native substrates are desired. Here, we report the development of a high-throughput, colorimetric, continuous coupled enzyme assay for the screening of DAAT mutant libraries that is based on the use of d-amino acid oxidase (DAAO). In this assay, the d-amino acid product of DAAT is oxidized by DAAO with concomitant release of hydrogen peroxide, which is detected colorimetrically by the addition of horseradish peroxidase and o-dianisidine. Using this assay, we measured apparent KM and kcat values for DAAT and identified mutants displaying altered substrate specificity via the screening of cell lysates in 96-well plates. The DAAO coupled assay is sensitive in that it allowed the detection of a DAAT mutant displaying an approximately 2000-fold decrease in kcat/KM relative to wild type. In addition, the DAAO assay enabled the identification of two DAAT mutants (V33Y and V33G) that are more efficient than wild type at transaminating the non-native acceptor phenylpyruvate. We expect that this assay will be useful for the engineering of additional mutants displaying increased activity toward non-native substrates.
authors.affiliation: Department of Chemistry and Centre for Catalysis Research and Innovation, University of Ottawa, Ottawa, Ontario K1N 6N5, Canada.
authors.forename: Janet E B
authors.initials: JE
authors.lastname: Barber
authors.affiliation: Department of Chemistry and Centre for Catalysis Research and Innovation, University of Ottawa, Ottawa, Ontario K1N 6N5, Canada.
authors.forename: Adam M
authors.initials: AM
authors.lastname: Damry
authors.affiliation: Department of Chemistry and Centre for Catalysis Research and Innovation, University of Ottawa, Ottawa, Ontario K1N 6N5, Canada.
authors.forename: Guido F
authors.initials: GF
authors.lastname: Calderini
authors.affiliation: Department of Chemistry and Centre for Catalysis Research and Innovation, University of Ottawa, Ottawa, Ontario K1N 6N5, Canada.
authors.forename: Curtis J W
authors.initials: CJ
authors.lastname: Walton
authors.affiliation: Department of Chemistry and Centre for Catalysis Research and Innovation, University of Ottawa, Ottawa, Ontario K1N 6N5, Canada. Electronic address: rchica@uottawa.ca.
authors.forename: Roberto A
authors.initials: RA
authors.lastname: Chica
mesh_terms: D019943:Amino Acid Substitution; D000596:Amino Acids / Q000378:metabolism; D003124:Colorimetry; D003605:D-Amino-Acid Oxidase / Q000378:metabolism; D003962:Dianisidine / Q000378:metabolism; D006735:Horseradish Peroxidase / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000032:analysis; D007700:Kinetics; D013379:Substrate Specificity; D000637:Transaminases / Q000378:metabolism*
chemical_list: D000596:Amino Acids; D006861:Hydrogen Peroxide; D006735:Horseradish Peroxidase; D003605:D-Amino-Acid Oxidase; D000637:Transaminases; D003962:Dianisidine
keywords: Coupled enzyme assay; Enzyme kinetics; High-throughput screening; Mutagenesis; Substrate specificity; Transaminases