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Laboratory Evolution of Toluene Dioxygenase To Accept 4-Picoline as a Substrate
- 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.
- Subjects :
- Stereochemistry
Toluene dioxygenase
Applied Microbiology and Biotechnology
Substrate Specificity
chemistry.chemical_compound
Biotransformation
Escherichia coli
Picoline
Amino Acid Sequence
Saturated mutagenesis
Base Sequence
Ecology
Mutagenesis
Substrate (chemistry)
Sequence Analysis, DNA
Physiology and Biotechnology
Directed evolution
Toluene
Biodegradation, Environmental
chemistry
Picolines
Oxygenases
Directed Molecular Evolution
Oxidation-Reduction
Caltech Library Services
Food Science
Biotechnology
Subjects
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