Universität Stuttgart

Permanent URI for this communityhttps://elib.uni-stuttgart.de/handle/11682/1

Browse

Institute: search.filters.UBSInstitut.Sonstige EinrichtungAuthor: search.filters.author.Lange, Stefan

Search Results

Now showing 1 - 2 of 2
  • Thumbnail Image
    ItemOpen Access
    Durch Überexpression in der Hefe Pichia pastoris zu erhöhter Enantioselektivität : ein neues Kapitel in der Anwendung von Schweineleberesterase
    (2001) Musidlowska, Anna; Lange, Stefan; Bornscheuer, Uwe Theo
    Lipases and Esterases can be used as efficient biocatalysts for the preparation of a wide variety of optically pure compounds. Whereas a range of lipases - especially of microbial origin - are commercially available, only a few esterases can be obtained for the kinetic resolution of racemates or desymmetrization. In the majority of publications, pig liver esterase (PLE) is used, which is isolated from pig liver by extraction. Although it could be demonstrated, that this preparation can convert a broad range of compounds at partially very high stereoselectivity, its application is encountered with a number of disadvantages.
  • Thumbnail Image
    ItemOpen Access
    Recombinant production of human microsomal cytochrome P450 2D6 in the methylotrophic yeast Pichia pastoris
    (2005) Dietrich, Matthias; Grundmann, Lisa; Kurr, Katja; Valinotto, Laura; Richter, Tanja; Schmid, Rolf D.; Lange, Stefan
    Microsomal cytochrome P450 monooxygenases of the groups 1 – 3 are mainly expressed in liver and play a crucial role in phase 1 reactions of the xenobiotics metabolism. The cDNAs encoding human CYP2D6 and human NADPH-P450 oxidoreductase (CPR) were transformed into the methylotrophic yeast Pichia pastoris and expressed under control of the methanol inducible AOX1 promotor. The determined molecular weights of the recombinant CYP2D6 and CPR closely matched the calculated values of 55.8 and 76.6 kDa. CPR activity was detected by conversion of cytochrome c using isolated microsomes. Nearly all the recombinant CYP is composed of the active holo-enzyme as confirmed by reduced CO-difference spectra which showed a single peak at 450 nm. Only by co-expression of human CPR and CYP, CYP2D6 activity was obtained. Microsomes containing human CPR and CYP2D6 converted different substrates, such as 3-cyano-7-ethoxycoumarin, parathion, and dextrometorphan. The kinetic parameters of the dextrometorphan conversion closely matched those of CYP2D6 from other recombinant expression systems as well as from human microsomes. The endogenous NADPH-P450 oxidoreductase of Pichia pastoris seems to be incompatible with human CYP2D6, as expression of CYP2D6 without human CPR did not result in any CYP-activity. These recombinant strains provide a novel, easy-to-handle and cheap source for the biochemical characterization of single microsomal cytochromes as well as their allelic variants.