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Published October 1999 | public
Journal Article

A high-throughput digital imaging screen for the discovery and directed evolution of oxygenases

Abstract

Background: Oxygenases catalyze the hydroxylation of a wide variety of organic substrates. An ability to alter oxygenase substrate specificities and improve their activities and stabilities using recombinant DNA techniques would expand their use in processes such as chemical synthesis and bioremediation. Discovery and directed evolution of oxygenases require efficient screens that are sensitive to the activities of interest and can be applied to large numbers of crude enzyme samples. Results: Horseradish peroxidase (HRP) couples the phenolic products of hydroxylation of aromatic substrates to generate colored and/or fluorescent compounds that are easily detected spectroscopically in high-throughput screening. Coexpression of the coupling enzyme with a functional mono- or dioxygenase creates a pathway for the conversion of aromatic substrates into fluorescent compounds in vivo. We used this approach for detecting the products of the toluene-dioxygenase-catalyzed hydroxylation of chlorobenzene and to screen large mutant libraries of Pseudomonas putida cytochrome P450_(cam) by fluorescence digital imaging. Colors generated by the HRP coupling reaction are sensitive to the site of oxygenase-catalyzed hydroxylation, allowing the screen to be used to identify catalysts with new or altered regiospecificities. Conclusions: The coupled oxygenase-peroxidase reaction system is well suited for screening oxygenase libraries to identify mutants with desired features, including higher activity or stability and altered reaction specificity. This approach should also be useful for screening expressed DNA libraries and combinatorial chemical libraries for hydroxylation catalysts and for optimizing oxygenase reaction conditions.

Additional Information

© 1999 Elsevier Science Ltd. Under an Elsevier user license. Received 11 June 1999, Accepted 20 July 1999, Available online 20 March 2000. The authors thank John H. Richards (Caltech) for encouragement and support and Paul Ortiz de Montellano (University of California, San Francisco) for helpful discussions and the P450_(cam) gene and pCWori(+) vector. This work was supported by the Biotechnology Research and Development Corporation and by the Office of Naval Research. H.J. received partial support from the Korea Research Foundation.

Additional details

Created:
August 22, 2023
Modified:
October 18, 2023