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Tuyển tập báo cáo các nghiên cứu khoa học quốc tế ngành hóa học dành cho các bạn yêu hóa học tham khảo đề tài: Recombinant expression and purification of the 2,5-diketocamphane 1,2-monooxygenase from the camphor metabolizing Pseudomonas putida strain NCIMB 10007 | Kadow et al. AMB Express 2011 1 13 http www.amb-express.eom content 1 1 13 o AMB Express a SpringerOpen Journal ORIGINAL Open Access Recombinant expression and purification of the 2 5-diketocamphane 1 2-monooxygenase from the camphor metabolizing Pseudomonas putida strain NCIMB 10007 Maria Kadow Stefan SaB Marlen Schmidt and Uwe T Bornscheuer Abstract 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. Keywords Baeyer-Villiger monooxygenases camphor Pseudomonas putida NCIMB 10007 2 5-diketocamphane 1 2-monooxygenase bicyclic ketones Introduction The discovery of the enzymatic Baeyer-Villiger reaction is closely connected to the exploration of the biodegradation of camphor 1 in Pseudomonads Figure 1 . Initial studies on the microbial decomposition of -1 by Pseudomonas putida NCIMB 10007 isolated from sewage sludge were already carried out in 1959 Bradshaw et al. 1959 and the involved enzymes were separated and characterized during the following decade. In studies of the enzymatic lactonization of the intermediate 2 5-diketocamphane 3 from the .
