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Applied and Environmental Microbiology, April 2007, p. 2682-2689, Vol. 73, No. 8
0099-2240/07/$08.00+0 doi:10.1128/AEM.02523-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.
Generation by a Widely Applicable Approach of a Hybrid Dioxygenase Showing Improved Oxidation of Polychlorobiphenyls
,
Beatriz Cámara,1,
Michael Seeger,1
Myriam González,1
Christine Standfuß-Gabisch,2
Silke Kahl,2 and
Bernd Hofer2,
*
Laboratorio de Microbiología Molecular y Biotecnología Ambiental, Departamento de Química and Millennium Nucleus of Microbial Ecology and Environmental Microbiology and Biotechnology, Universidad Técnica Federico Santa María, Valparaíso, Chile,1 Division of Microbiology, Helmholtz-Zentrum für Infektionsforschung, Braunschweig, Germany2
Received 30 October 2006/ Accepted 16 February 2007
Recently, a sequence-based approach has been developed for the fast isolation and characterization of class II aryl-hydroxylating dioxygenase activities (S. Kahl and B. Hofer, Microbiology 149:1475-1481, 2003). It comprises the PCR amplification of segments of alpha subunit genes of unknown sequence that encode the catalytic center and their fusion with sequences of the bphA gene cluster of Burkholderia xenovorans LB400. One of the resulting chimeric enzymes, harboring the core segment of a dioxygenase from Pseudomonas sp. strain B4-Magdeburg, has now been characterized with respect to the oxidation of chlorobiphenyls (CBs). Its substrate and product specificities differed favorably from those of the parental dioxygenase of strain LB400. The hybrid possessed a higher regiospecificity and yielded less unproductive dioxygenations at meta and para carbons. It attacked ortho-, meta-, and para-chlorinated rings with comparable efficiencies. It gave significantly higher yields in ortho,meta-dioxygenation of recalcitrant congeners containing a doubly ortho-chlorinated ring. While the parental enzyme yielded mainly unproductive meta, para dioxygenation of 2,5,4'-CB, the hybrid predominantly converted this congener into an ortho,meta-dioxygenated product. The subsequent enzymes of the LB400 catabolic pathway were able to transform most of the metabolites formed by the novel dioxygenase, indicating that the substrate ranges of these biocatalysts are not adapted to that of their initial pathway enzyme. Some of the catabolites, however, were identified as problematic for further degradation. Our results demonstrate that the outlined approach can successfully be applied to obtain novel dioxygenase specificities that favorably complement or supplement known ones.
* Corresponding author. Mailing address: Helmholtz-Zentrum für Infektionsforschung, Abteilung Chemische Biologie, Inhoffenstraße 7, D-38124 Braunschweig, Germany. Phone: (49-531) 61814200. Fax: (49-531) 61813499. E-mail: bernd.hofer{at}helmholtz-hzi.de
Published ahead of print on 23 February 2007.
Supplemental material for this article may be found at http://aem.asm.org/.
Present address: Division of Microbiology, Helmholtz-Zentrum für Infektionsforschung, Braunschweig, Germany.
Present address: Department of Chemical Biology, Helmholtz-Zentrum für Infektionsforschung, Braunschweig, Germany.
Applied and Environmental Microbiology, April 2007, p. 2682-2689, Vol. 73, No. 8
0099-2240/07/$08.00+0 doi:10.1128/AEM.02523-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.
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