Recombinant expression and purification of the 2,5-diketocamphane 1,2-monooxygenase from the camphor metabolizing Pseudomonas putida strain NCIMB 10007 英文参考文献.docVIP

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Recombinant expression and purification of the 2,5-diketocamphane 1,2-monooxygenase from the camphor metabolizing Pseudomonas putida strain NCIMB 10007 英文参考文献.doc

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Recombinant expression and purification of the 2,5-diketocamphane 1,2-monooxygenase from the camphor metabolizing Pseudomonas putida strain NCIMB 10007 英文参考文献

Kadowetal.AMBExpress2011,1:13 /content/1/1/13 ORIGINAL OpenAccess Recombinantexpressionandpurificationofthe 2,5-diketocamphane1,2-monooxygenasefrom thecamphormetabolizingPseudomonasputida strainNCIMB10007 MariaKadow,StefanSa?,MarlenSchmidtandUweTBornscheuer* Abstract ThreedifferentBaeyer-Villigermonooxygenases(BVMOs)werereportedtobeinvolvedinthecamphormetabolism byPseudomonasputidaNCIMB10007.During(+)-camphordegradation,2,5-diketocamphaneisformedservingas substrateforthe2,5-diketocamphane1,2-monooxygenase.ThisenzymeisencodedontheCAMplasmidand dependsonthecofactorsFMNandNADHandhencebelongstothegroupoftypeIIBVMOs.Wehaveclonedand recombinantlyexpressedtheoxygenatingsubunitofthe2,5-diketocamphane1,2-monooxygenase(2,5-DKCMO)in E.colifollowedbyHis-tag-basedaffinitypurification.ArangeofcompoundsrepresentingdifferentBVMOsubstrate classesweretheninvestigated,butonlybicyclicketoneswereconvertedby2,5-DKCMOusedascrudecellextract orafterpurification.Interestingly,also(-)-camphorwasoxidized,butconversionwasabout3-foldlowercompared to(+)-camphor.Moreover,activityofpurified2,5-DKCMOwasobservedintheabsenceofanNADH- dehydrogenasesubunit. Keywords:Baeyer-Villigermonooxygenases,camphor,PseudomonasputidaNCIMB10007,2,5-diketocamphane1,2- monooxygenase,bicyclicketones Introduction subunit was claimed to be a ketolactonase. Since ThediscoveryoftheenzymaticBaeyer-Villigerreaction mechanistic similarities to thechemical Baeyer-Villiger iscloselyconnectedtotheexploration ofthebiodegra- oxidationofbicyclicketones(MeinwaldandFrauenglass dation of camphor (1) in Pseudomonads (Figure 1). 1960)weredetected,thenomenclatureoftheketolacto- Initial studies on the microbial decomposition of(+)-1 nasewaschanged toaBVMO. In1965asecondlacto- by Pseudomonas putida NCIMB 10007 isolated from nizing system for the degradation of (-)-1 was found sewage sludge were already carried out in 1959 (Conrad et al. 1965a). Thus it was claimed that (+)-1 (Bradshaw et al. 1959) and the involved enzymes