A Caltech Library Service

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

Joo, Hyun and Arisawa, Akira and Lin, Zhanglin and Arnold, Frances H. (1999) A high-throughput digital imaging screen for the discovery and directed evolution of oxygenases. Chemistry and Biology, 6 (10). pp. 699-706. ISSN 1074-5521. doi:10.1016/S1074-5521(00)80017-4.

Full text is not posted in this repository. Consult Related URLs below.

Use this Persistent URL to link to this item:


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.

Item Type:Article
Related URLs:
URLURL TypeDescription
Arnold, Frances H.0000-0002-4027-364X
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.
Funding AgencyGrant Number
Biotechnology Research and Development CorporationUNSPECIFIED
Office of Naval Research (ONR)UNSPECIFIED
Korea Research FoundationUNSPECIFIED
Subject Keywords:cytochrome P450; fluorescence digital imaging; horseradish peroxidase; high-throughput screening; toluene dioxygenase
Issue or Number:10
Record Number:CaltechAUTHORS:20180822-141412954
Persistent URL:
Official Citation:Hyun Joo, Akira Arisawa, Zhanglin Lin, Frances H Arnold, A high-throughput digital imaging screen for the discovery and directed evolution of oxygenases, Chemistry & Biology, Volume 6, Issue 10, 1999, Pages 699-706, ISSN 1074-5521, (
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:89060
Deposited By: Tony Diaz
Deposited On:22 Aug 2018 21:24
Last Modified:16 Nov 2021 00:31

Repository Staff Only: item control page