Applied Microbiology and Biotechnology

, Volume 75, Issue 5, pp 1055–1061

Construction of a thermostable cytochrome P450 chimera derived from self-sufficient mesophilic parents

  • Sabine Eiben
  • Heike Bartelmäs
  • Vlada B. Urlacher
Biotechnologically Relevant Enzymes and Proteins

DOI: 10.1007/s00253-007-0922-z

Cite this article as:
Eiben, S., Bartelmäs, H. & Urlacher, V.B. Appl Microbiol Biotechnol (2007) 75: 1055. doi:10.1007/s00253-007-0922-z

Abstract

The P450 monooxygenases CYP102A1 from Bacillus megaterium and CYP102A3 from Bacillus subtilis are fusion flavocytochromes comprising of a P450 heme domain and a FAD/FMN reductase domain. This protein organization is responsible for the extraordinary catalytic activities making both monooxygenases promising enzymes for biocatalysis. CYP102A1 and CYP102A3 are fatty acid hydroxylases that share 65% identity, and their mutants are able to oxidize a wide range of substrates. In an attempt to increase the process stability of CYP102A1, we exchanged the more unstable reductase domain of CYP102A1 with the more stable reductase domain of CYP102A3. Stability of the chimeric fusion protein was determined spectrophotometrically as well as by measuring the hydroxylation activity towards 12-para-nitrophenoxydodecanoic acid (12-pNCA) after incubation at elevated temperatures. In the reaction with 12-pNCA, the new chimeric protein exhibited 88 and 38% of the activity of CYP102A3 and CYP102A1, respectively, but was able to hydroxylate substrates within a wider temperature range compared with the parental enzymes. Maximum activity was obtained at 51°C, and the half-life at 50°C was with 100 min more than ten times longer than that of CYP102A1 (8 min).

Keywords

Cytochrome P450 monooxygenase Thermostability Chimera Self-sufficient 

Copyright information

© Springer-Verlag 2007

Authors and Affiliations

  • Sabine Eiben
    • 1
  • Heike Bartelmäs
    • 1
  • Vlada B. Urlacher
    • 1
  1. 1.Institute of Technical BiochemistryUniversity of StuttgartStuttgartGermany

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