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APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 0099-2240/01/$04.00 0 DOI: 10.1128/AEM.67.9.38823887.2001
 

Summary: APPLIED AND ENVIRONMENTAL MICROBIOLOGY,
0099-2240/01/$04.00 0 DOI: 10.1128/AEM.67.9.38823887.2001
Sept. 2001, p. 38823887 Vol. 67, No. 9
Copyright 2001, American Society for Microbiology. All Rights Reserved.
Laboratory Evolution of Toluene Dioxygenase To Accept
4-Picoline as a Substrate
TAKESHI SAKAMOTO, JOHN M. JOERN, AKIRA ARISAWA, AND FRANCES H. ARNOLD*
Division of Chemistry and Chemical Engineering, California Institute of Technology,
Pasadena, California 91125
Received 31 January 2001/Accepted 31 May 2001
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 di-
rectly 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 prod-
uct of the biotransformation of 4-picoline is 3-hydroxy-4-picoline; no cis-diols of 4-picoline were observed.
Dioxygenase enzymes involved in the catabolism of aromatic

  

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

 

Collections: Chemistry; Biology and Medicine