Continuous colorimetric screening assay for detection of d-amino acid aminotransferase mutants displaying altered substrate specificity |
| |
Authors: | Janet E.B. Barber Adam M. DamryGuido F. Calderini Curtis J.W. WaltonRoberto A. Chica |
| |
Affiliation: | Department of Chemistry and Centre for Catalysis Research and Innovation, University of Ottawa, Ottawa, Ontario K1N 6N5, Canada |
| |
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. |
| |
Keywords: | Transaminases Coupled enzyme assay High-throughput screening Mutagenesis Substrate specificity Enzyme kinetics |
本文献已被 ScienceDirect 等数据库收录! |
|