Journal
PROTEIN EXPRESSION AND PURIFICATION
Volume 41, Issue 2, Pages 433-440Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.pep.2005.01.012
Keywords
phosphotriesterase; directed evolution; organophosphate; pathway engineering
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We devised a growth-based strategy for screening phosphotriesterase mutant libraries for variants with enhanced activity towards organophosphates that generate dimethyl phosphate when hydrolysed. Phosphotriesterase mutants were screened for activity by growing transformed Escherichia coli on agar plates containing methyl paraoxon as a sole phosphorus source. E coli is capable of growth under these conditions when coexpressing the phosphotriesterase from Agrobacterium radiobacter P230 (OpdA) and the glycerophosphodiester phosphodiesterase from Enterobacter aerogenes (GpdQ). The latter enzyme can hydrolyse the dimethyl phosphate produced by the phosphotriesterase to methyl phosphate, which can then be used by E coli as a source of phosphate. Phosphotriesterase was expressed from the lac promoter at levels such that its activity was growth-rate limiting. Cultures of the largest colonies (1 % of the transformants) were assayed for activity towards paraoxon spectrophotometrically in microtitre plates. This process produced E coli variants with higher whole cell activity than wild-type, which was found to be a consequence of increased protein expression rather than any increase in enzymatic activity. The mutations present in these mutant enzymes with increased expression were exclusively in the coding region, suggesting the improvement occurs post-transcriptionally. (c) 2005 Elsevier Inc. All rights reserved.
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