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

Summary: A high-throughput digital imaging screen for the discovery and
directed evolution of oxygenases
Hyun Joo, Akira Arisawa, Zhanglin Lin and Frances H Arnold
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
P450cam 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.

  

Source: Arnold, Frances H. - Division of Chemistry and Chemical Engineering, California Institute of Technology

 

Collections: Chemistry; Biology and Medicine