Premium
Towards Preparative Scale Steroid Hydroxylation with Cytochrome P450 Monooxygenase CYP106A2
Author(s) -
Zehentgruber Daniela,
Hannemann Frank,
Bleif Sabrina,
Bernhardt Rita,
Lütz Stephan
Publication year - 2010
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/cbic.200900706
Subject(s) - monooxygenase , adrenodoxin , hydroxylation , chemistry , bacillus megaterium , steroid , biocatalysis , cofactor , substrate (aquarium) , cytochrome p450 , pregnenolone , stereochemistry , biochemistry , ferredoxin , enzyme , catalysis , biology , bacteria , ecology , ionic liquid , hormone , genetics
Cytochrome P450 monooxygenases are of outstanding interest for the synthesis of pharmaceuticals and fine chemicals, due to their ability to hydroxylate CH bonds mainly in a stereo‐ and regioselective manner. CYP106A2 from Bacillus megaterium ATCC 13368, one of only a few known bacterial steroid hydroxylases, enables the oxidation of 3‐keto‐4‐ene steroids mainly at position 15. We expressed this enzyme together with the electron‐transfer partners bovine adrenodoxin and adrenodoxin reductase in Escherichia coli. Additionally an enzyme‐coupled cofactor regeneration system was implemented by expressing alcohol dehydrogenase from Lactobacillus brevis . By studying the conversion of progesterone and testosterone, the bottlenecks of these P450‐catalyzed hydroxylations were identified. Substrate transport into the cell and substrate solubility turned out to be crucial for the overall performance. Based on these investigations we developed a new concept for CYP106A2‐catalyzed steroid hydroxylations by which the productivity of progesterone and testosterone conversion could be increased up to 18‐fold to yield an absolute productivity up to 5.5 g L −1 d −1 . Product extraction with absorber resins allowed the recovery of quantitative amounts of 15β‐OH‐progesterone and 15β‐OH‐testosterone and also the reuse of the biocatalyst.