Affiliation:
1. Institut National de la Recherche Scientifique INRS-Institut Armand-Frappier, Université du Québec, Pointe-Claire, H9R 1G6 Quebec, Canada
Abstract
ABSTRACT
In this work we used a new strategy designed to reduce the size of the library that needs to be explored in family shuffling to evolve new biphenyl dioxygenases (BPDOs). Instead of shuffling the whole gene, we have targeted a fragment of
bphA
that is critical for enzyme specificity. We also describe a new protocol to screen for more potent BPDOs that is based on the detection of catechol metabolites from chlorobiphenyls. Several BphA variants with extended potency to degrade polychlorinated biphenyls (PCBs) were obtained by shuffling critical segments of
bphA
genes from
Burkholderia
sp. strain LB400,
Comamonas testosteroni
B-356, and
Rhodococcus globerulus
P6. Unlike all parents, these variants exhibited high activity toward 2,2′-, 3,3′-, and 4,4′-dichlorobiphenyls and were able to oxygenate the very persistent 2,6-dichlorobiphenyl. The data showed that the replacement of a short segment (
335
TFNNIRI
341
) of LB400 BphA by the corresponding segment (
333
GINTIRT
339
) of B-356 BphA or P6 BphA contributes to relax the enzyme toward PCB substrates.
Publisher
American Society for Microbiology
Subject
Molecular Biology,Microbiology
Reference39 articles.
1. Influence of Chlorine Substituents on Rates of Oxidation of Chlorinated Biphenyls by the Biphenyl Dioxygenase of
Burkholderia
sp. Strain LB400
2. Asturias, J. A., E. Diaz, and K. N. Timmis. 1995. The evolutionary relationship of biphenyl dioxygenase from gram-positive Rhodococcus globerulus P6 to multicomponent dioxygenases from gram-negative bacteria. Gene156:11-18.
3. Barriault, D., and M. Sylvestre. 1993. Factors affecting PCB degradation by an implanted bacterial strain in soil microcosms. Can J. Microbiol.39:594-602.
4. Barriault, D., C. Simard, H. Chatel, and, M. Sylvestre. 2001. Characterization of hybrid biphenyl dioxygenases obtained by recombining Burkholderia sp. strain LB400 bphA with the homologous gene of Comamonas testosteroni strain B-356. Can. J. Microbiol.47:1025-1032.
5. Barriault, D., M. Vedadi, J. Powlowski, and M. Sylvestre. 1999. cis-2,3-Dihydro-2,3-dihydroxybiphenyl dehydrogenase and cis-1,2-dihydro-1,2-dihydroxynaphthalene dehydrogenase catalyze dehydrogenation of the same range of substrates. Biochem. Biophys. Res. Commun.260:181-187.
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