Applied Microbiology and Biotechnology

, Volume 73, Issue 5, pp 1065–1072

Cloning, expression, and characterization of a Baeyer–Villiger monooxygenase from Pseudomonas fluorescens DSM 50106 in E. coli

  • Anett Kirschner
  • Josef Altenbuchner
  • Uwe T. Bornscheuer
Biotechnologically Relevant Enzymes and Proteins


A gene encoding a Baeyer–Villiger monooxygenase (BVMO) identified in Pseudomonas fluorescens DSM 50106 was cloned and functionally expressed in Escherichia coli JM109. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis showed an estimated 56 kDa-size protein band corresponding to the recombinant enzyme. Expression in BL21 (DE3) resulted mainly in the formation of inclusion bodies. This could be overcome by coexpression of molecular chaperones, especially the DnaK/DnaJ/GrpE complex, leading to increased production of soluble BVMO enzyme in recombinant E. coli. Examination of the substrate spectra using whole-cell biocatalysis revealed a high specificity of the BVMO for aliphatic open-chain ketones. Thus, octyl acetate, heptyl propionate, and hexyl butyrate were quantitatively formed from the corresponding ketone substrates. Several other esters were obtained in conversion >68%. Selected esters were also produced on preparative scale.


Baeyer–Villiger monooxygenase Aliphatic ketones Pseudomonas fluorescens Cloning 


  1. Britton LN (1984) Microbial degradation of aliphatic hydrocarbons. In: Gibson DT (ed) Microbial degradation of organic compounds, vol 13. Marcel Dekker, New York, pp 89–129Google Scholar
  2. Britton LN, Markovetz AJ (1977) A novel ketone monooxygenase from Pseudomonas cepacia. J Biol Chem 252:8561–8566PubMedGoogle Scholar
  3. Brzostowicz PC, Blasko MS, Rouviere PE (2002) Identification of two gene clusters involved in cyclohexanone oxidation in Brevibacterium epidermidis strain HCU. Appl Microbiol Biotechnol 58:781–789PubMedCrossRefGoogle Scholar
  4. Brzostowicz PC, Walters DM, Thomas SM, Nagarajan V, Rouviere PE (2003) mRNA differential display in a microbial enrichment culture: simultaneous identification of three cyclohexanone monooxygenases from three species. Appl Environ Microbiol 69:334–342PubMedCrossRefGoogle Scholar
  5. Carrea G, Redigolo B, Riva S, Colonna S, Gaggero N, Battistel E, Bianchi D (1992) Effects of substrate structure on the enantioselectivity and stereochemical course of sulfoxidation catalyzed by cyclohexanone monooxygenase. Tetrahedron: Asymmetry 3:1063–1068CrossRefGoogle Scholar
  6. de Gonzalo G, Torres Pazmino DE, Ottolina G, Fraaije MW, Carrea G (2006) 4-Hydroxyacetophenone monooxygenase from Pseudomonas fluorescens ACB as an oxidative biocatalyst in the synthesis of optically active sulfoxides. Tetrahedron: Asymmetry 17:130–135CrossRefGoogle Scholar
  7. Donoghue NA, Norris DB, Trudgill PW (1976) The purification and properties of cyclohexanone oxygenase from Nocardia globerula CL1 and Acinetobacter NCIB 9871. Eur J Biochem 63:175–192PubMedCrossRefGoogle Scholar
  8. Fraaije MW, Kamerbeek NM, van Berkel WJH, Janssen DB (2002) Identification of a Baeyer–Villiger monooxygenase sequence motif. FEBS Lett 518:43–47PubMedCrossRefGoogle Scholar
  9. Fraaije MW, Kamerbeek NM, Heidekamp AJ, Fortin R, Janssen DB (2004) The prodrug activator EtaA from Mycobacterium tuberculosis is a Baeyer–Villiger monooxygenase. J Biol Chem 279:3354–3360PubMedCrossRefGoogle Scholar
  10. Fraaije MW, Wu J, Heuts DPHM, van Hellemond EW, Spelberg JHL, Janssen DB (2005) Discovery of a thermostable Baeyer–Villiger monooxygenase by genome mining. Appl Microbiol Biotechnol 66:393–400PubMedCrossRefGoogle Scholar
  11. Gagnon R, Grogan G, Levitt MS, Robets SM, Wan PWH, Willetts AJ (1994) Biological Baeyer–Villiger oxidation of some monocyclic and bicyclic ketones using monooxygenases from Acinetobacter calcoaceticus NCIMB 9871 and Pseudomonas putida NCIMB 10007. J Chem Soc Perkin Trans 1:2537–2543CrossRefGoogle Scholar
  12. Griffin M, Trudgill PW (1976) Purification and properties of cyclopentanone oxygenase of Pseudomonas NCIB 9872. Eur J Biochem 63:199–209PubMedCrossRefGoogle Scholar
  13. Grogan G, Roberts S, Willetts A (1992) Biotransformations by microbial Baeyer–Villiger monooxygenases stereoselective lactone formation in vitro by coupled enzyme systems. Biotechnol Lett 14:1125–1130CrossRefGoogle Scholar
  14. Grogan G, Roberts S, Wan P, Willetts AJ (1993) Camphor-grown Pseudomonas putida, a multifunctional biocatalyst for undertaking Baeyer–Villiger monooxygenase-dependent biotransformations. Biotechnol Lett 15:913–918CrossRefGoogle Scholar
  15. Hedges AR (1998) Industrial applications of cyclodextrins. Chem Rev 98:2035–2044PubMedCrossRefGoogle Scholar
  16. Hildebrandt P, Musidlowska A, Bornscheuer UT (2002) Cloning, functional expression and biochemical characterization of a stereoselctive alcohol dehydrogeenase from Pseudomonas fluorescens DSM50106. Appl Microbiol Biotechnol 59:483–487PubMedCrossRefGoogle Scholar
  17. Ikura K, Kokubu T, Natsuka S, Ichikawa A, Adachi M, Nishihara K, Yanagi H, Utsumi S (2002) Co-overexpression of folding modulators improves the solubility of the recombinant guinea pig liver transglutaminase expressed in Escherichia coli. Prep Biochem Biotechnol 32:189–205PubMedCrossRefGoogle Scholar
  18. Jones KH, Smith RT, Trudgill PW (1993) Diketocampane enantiomer-specific ‘Baeyer–Villiger’ monooxygenases from camphor-grown Pseudomonas putida ATCC 17453. J Gen Microbiol 139:797–805PubMedGoogle Scholar
  19. Kamerbeek NM, Mooen MJH, van der Ven JGM, van Berkel WJH, Fraaije MW, Janssen DB (2001) 4-Hydroxyacetophenone monooxygenase from Pseudomonas fluorescens ACB. Eur J Biochem 268:2547–2557PubMedCrossRefGoogle Scholar
  20. Kamerbeek NM, Olsthoorn JJ, Fraaije MW, Janssen DB (2003) Substrate specificity and enantioselectivity of 4-hydroxyacetophenone monooxygenase. Appl Environ Microbiol 69:419–426PubMedCrossRefGoogle Scholar
  21. Khalameyzer V, Fischer I, Bornscheuer UT, Altenbuchner J (1999) Screening, nucleotide sequence and biochemical characterization of an esterase from Pseudomonas fluorescens with high activity toward lactones. Appl Environ Microbiol 65:477–482PubMedGoogle Scholar
  22. Kostichka K, Thomas SM, Gibson KJ, Nagarajan V, Cheng Q (2001) Cloning and characterization of a gene cluster for cyclododecanone oxidation in Rhodococcus ruber SC1. J Bacteriol 183:6478–6486PubMedCrossRefGoogle Scholar
  23. Kyte BG, Rouviere PE, Cheng Q, Stewart JD (2004) Assessing the substrate selectivities and enantioselectivities of eight novel Baeyer–Villiger monooxygenases toward alkyl-substituted cyclohexanones. J Org Chem 69:12–17PubMedCrossRefGoogle Scholar
  24. Laemmli UK (1970) Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature 27:680–685CrossRefADSGoogle Scholar
  25. Lee DH, Kim MD, Lee WH, Kweon DH, Seo JH (2004) Consortium of fold-catalyzing proteins increases soluble expression of cyclohexanone monooxygenase in recombinant Escherichia coli. Appl Microbiol Biotechnol 63:549–552PubMedCrossRefGoogle Scholar
  26. Malito E, Alfieri A, Fraaije MW, Mattevi A (2004) Crystal structure of a Baeyer–Villiger monooxygenase. Proc Natl Acad Sci USA 101:13157–13162PubMedCrossRefADSGoogle Scholar
  27. Mihovilovic MD, Müller B, Stanetty P (2002) Monooxygenase-mediated Baeyer–Villiger oxidations. Eur J Org Chem:3711–3730Google Scholar
  28. Nishihara K, Kanemori M, Kitagawa M, Yanagi H, Yura T (1998) Chaperone coexpression plasmids: differential and synergistic roles of DnaK-DnaJ-GrpE and GroEL-GroES in assisting folding of an allergen of Japanese cedar polen, Cryj2, in Escherichia coli. Appl Environ Microbiol 64:1694–1699PubMedGoogle Scholar
  29. Nishihara K, Kanemori M, Yanagi H, Yura T (2000) Overexpression of trigger factor prevents aggregation of recombinant proteins in Escherichia coli. Appl Environ Microbiol 66:884–889PubMedCrossRefGoogle Scholar
  30. Pasta P, Carrea G, Gaggero N, Grogan G, Willets A (1996) Enantioselective oxidations catalyzed by diketocamphene monooxygenase from Pseudomonas putida with macromolecular NAD in a membrane reactor. Biotechnol Lett 18:1123–1128CrossRefGoogle Scholar
  31. Saenger W (1980) Cyclodextrin-Einschlussverbindungen in Forschung und Industrie. Angew Chem 92:343–361Google Scholar
  32. Stewart JD (1998) Cyclohexanone monooxygenase: a useful reagent for asymmetric Baeyer–Villiger reactions. Curr Org Chem 2:211–232Google Scholar
  33. Stumpp T, Wilms B, Altenbuchner J (2000) Ein neues, L-Rhamnose-induzierbares Expressionssystem für Escherichia coli. BIOspektrum 6:33–36Google Scholar
  34. Tanner A, Hopper DJ (2000) Conversion of 4-hydroxyacetophenone into 4-phenyl acetate by a flavin adenine dinucleotide-containing Baeyer–Villiger-type monooxygenase. J Bacteriol 182:6565–6569PubMedCrossRefGoogle Scholar
  35. Taylor DG, Trudgill PW (1986) Camphor revisited: studies of 2,5-diketocamphane 1,2-monooxygenase from Pseudomonas putida ATCC 17453. J Bacteriol 165:489–497PubMedGoogle Scholar
  36. Trudgill PW (1984) Microbial degradation of the alicyclic ring. In: Gibson DT (ed) Microbial degradation of organic compounds, vol 13. Marcel Dekker, New York, pp 131–180Google Scholar
  37. van Beilen JB, Li Z, Duetz WA, Smits THM, Witholt B (2003a) Diversity of alkane hydroxylase systems in the environment. Oil Gas Sci Technol Rev IFP 58:427–440CrossRefGoogle Scholar
  38. van Beilen JB, Mourlane, Seeger MA, Kovac J, Li Z, Smits THM, Fritsche U, Witholt B (2003b) Cloning of Baeyer–Villiger monooxygenases from Comamonas, Xanthobacter and Rhodococcus using polymerase chain reaction with highly degenerate primers. Environ Microbiol 5:174–182PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2006

Authors and Affiliations

  • Anett Kirschner
    • 1
  • Josef Altenbuchner
    • 2
  • Uwe T. Bornscheuer
    • 1
  1. 1.Department of Biotechnology and Enzyme Catalysis, Institute of BiochemistryGreifswald UniversityGreifswaldGermany
  2. 2.Institute of Industrial GeneticsStuttgart UniversityStuttgartGermany

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