Abstract
The wide use of pesticides is a threat to the public health. An attractive tool for bioremediation of pesticides pollution is the bacterial pesticides degrading enzyme, phosphotriesterase (OPH), which is able to hydrolyze a wide range of organophosphate compounds. Yet, engineering of a more potent hydrolytic machinery into this enzyme could yield an advantageous bioremediation tool.
We undertook the in-vitro evolution strategy in order to improve the hydrolyzing power of phosphotriesterase towards certain organophosphates. The opd gene, coding for the phosphotriesterase enzyme, was amplified from Flavobacterium by PCR, and introduced into pmalE gene fusion system, expressed in E. coli, to allow simple procedure for purification of the recombinant mature native (and future mutant) enzyme. Random mutagenesis of the opd gene was carried out by error-prone PCR, and controlled low level mutageriesis was adopted. Mutagenesis products served for generation of an expression library, using a modified pUC 18 as an expression vector. A screening system was developed to allow monitoring of enzymatic kinetics with whole expressing cells, and to isolate phosphotriesterase mutants with altered enzymatic properties.
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© 1999 Springer Science+Business Media New York
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Flashner, Y. et al. (1999). Directed Evolution of a Bacterial Pesticides Degrading Enzyme. In: Fass, R., Flashner, Y., Reuveny, S. (eds) Novel Approaches for Bioremediation of Organic Pollution. Springer, Boston, MA. https://doi.org/10.1007/978-1-4615-4749-5_13
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DOI: https://doi.org/10.1007/978-1-4615-4749-5_13
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