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Laboratory Evolution of Toluene Dioxygenase To Accept 4-Picoline as a Substrate

Authors :
Takeshi Sakamoto
Frances H. Arnold
John M. Joern
Akira Arisawa
Source :
Applied and Environmental Microbiology. 67:3882-3887
Publication Year :
2001
Publisher :
American Society for Microbiology, 2001.

Abstract

We are using directed evolution to extend the range of dioxygenase-catalyzed biotransformations to include substrates that are either poorly accepted or not accepted at all by the naturally occurring enzymes. Here we report on the oxidation of a heterocyclic substrate, 4-picoline, by toluene dioxygenase (TDO) and improvement of the enzyme's activity by laboratory evolution. The biotransformation of 4-picoline proceeds at only ∼4.5% of the rate of the natural reaction on toluene. Random mutagenesis, saturation mutagenesis, and screening directly for product formation using a modified Gibbs assay generated mutant TDO 3-B38, in which the wild-type stop codon was replaced with a codon encoding threonine. Escherichia coli -expressed TDO 3-B38 exhibited 5.6 times higher activity toward 4-picoline and ∼20% more activity towards toluene than wild-type TDO. The product of the biotransformation of 4-picoline is 3-hydroxy-4-picoline; no cis -diols of 4-picoline were observed.

Details

ISSN :
10985336 and 00992240
Volume :
67
Database :
OpenAIRE
Journal :
Applied and Environmental Microbiology
Accession number :
edsair.doi.dedup.....b282572dfb4bee5899e95e474ec1cc90
Full Text :
https://doi.org/10.1128/aem.67.9.3882-3887.2001