Recombinant expression and purification of the 2,5-diketocamphane 1,2-monooxygenase from the camphor metabolizing Pseudomonas putida strain NCIMB 10007
Three different Baeyer-Villiger monooxygenases (BVMOs) were reported to be involved in the camphor metabolism by Pseudomonas putida NCIMB 10007. During (+)-camphor degradation, 2,5-diketocamphane is formed serving as substrate for the 2,5-diketocamphane 1,2-monooxygenase. This enzyme is encoded on t...
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Published in | AMB Express Vol. 1; no. 1; p. 13 |
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Main Authors | , , , |
Format | Journal Article |
Language | English |
Published |
Berlin/Heidelberg
Springer Berlin Heidelberg
23.06.2011
BioMed Central Ltd Springer |
Subjects | |
Online Access | Get full text |
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Summary: | Three different Baeyer-Villiger monooxygenases (BVMOs) were reported to be involved in the camphor metabolism by
Pseudomonas putida
NCIMB 10007. During (+)-camphor degradation, 2,5-diketocamphane is formed serving as substrate for the 2,5-diketocamphane 1,2-monooxygenase. This enzyme is encoded on the CAM plasmid and depends on the cofactors FMN and NADH and hence belongs to the group of type II BVMOs. We have cloned and recombinantly expressed the oxygenating subunit of the 2,5-diketocamphane 1,2-monooxygenase (2,5-DKCMO) in
E. coli
followed by His-tag-based affinity purification. A range of compounds representing different BVMO substrate classes were then investigated, but only bicyclic ketones were converted by 2,5-DKCMO used as crude cell extract or after purification. Interestingly, also (-)-camphor was oxidized, but conversion was about 3-fold lower compared to (+)-camphor. Moreover, activity of purified 2,5-DKCMO was observed in the absence of an NADH-dehydrogenase subunit. |
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Bibliography: | ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 |
ISSN: | 2191-0855 2191-0855 |
DOI: | 10.1186/2191-0855-1-13 |