Universität Stuttgart

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Start date: 2005End date: 2005Author: search.filters.author.Lange, Stefan

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    Screening, cloning and biochemical characterisation of novel esterases from bacillus sp. associated with the marine sponge aplysina aerophoba
    (2005) Karpushova, Anna Alexandrovna; Brümmer, Franz; Lange, Stefan; Schmid, Rolf D.
    Two novel esterases (EstB1 and EstB2) were isolated from a genomic library of Bacillus sp. associated with the marine sponge Aplysina aerophoba. EstB1 shows low identity (26-44 %)with the published hydrolases of the genus Bacillus, whereas EstB2 shows high identity (73-74 %) with the carboxylesterases from B. cereus and B. anthracis. Both esterases were efficiently expressed in Escherichia coli under the control of T7 promoter using the vector pET-22b(+). Recombinant EstB1 was purified in a single step to electrophoretic homogeneity by IMAC. A method for the refolding of inclusion bodies formed by the recombinant EstB2 was established to obtain active enzyme. Substrate specificity of the two enzymes towards pnitrophenyl and methyl esters and the respective kinetic parameters Km and Vmax were determined. The temperature optima of EstB1 and EstB2 were determined to be in the range of 30-50°C and 20-35°C, respectively. The pH optima were found to be in the range of 6.5-7.5 and 6.5-8.0, respectively. Both enzymes showed the highest stability in up to 50 % (v/v) DMSO followed by methanol, ethanol and 2-propanol. The influence of high NaCl and KCl concentrations was tested. The inhibition effect of 10-50 mM Zn2+ and 50 mM Mg2+ and Ca2+ ions was observed for both esterases. 1-5 mM PMSF deactivated the enzymes, whereas β-mercaptoethanol, DTT and EDTA had no effect on the enzymes activity.
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    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.