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Item Open Access Catalytic hydroxylation in biphasic systems using CYP102A1 mutants(2005) Maurer, Steffen Christian; Kühnel, Katja; Kaysser, Leonard A.; Eiben, Sabine; Schmid, Rolf D.; Urlacher, Vlada B.Cytochrome P450 monooxygenases are biocatalysts that hydroxylate or epoxidise a wide range of hydrophobic organic substrates. To date their technical application is limited to a small number of whole-cell biooxidations. The use of the isolated enzymes is believed to be impractical due to the low stability of this enzyme class, to the stochiometric need of the expensive cofactor NADPH, and due to the low solubility of most substrates in aqueous media. To overcome these problems we have investigated the application of a bacterial monooxygenase (mutants of CYP102A1) in a biphasic reaction system supported by cofactor recycling with NADP+-dependent formate dehydrogenase from Pseudomonas sp 101. Using this experimental setup, cyclohexane, octane and myristic acid were hydroxylated. To reduce the process costs a novel NADH-dependent double mutant of CYP102A1 was designed. For recycling of NADH during myristic acid hydroxylation in a biphasic system NAD+-dependent FDH was used. Stability of the monooxygenase under the reaction conditions is quite high as revealed by total turnover numbers of up to 12850 in NADPH-dependent cyclohexane hydroxylation and up to 30000 in NADH-dependent myristic acid oxidation.Item Open Access Cloning, expression and characterisation of CYP102A2, a self-sufficient P450 monooxygenase from Bacillus subtilis(2004) Budde, Michael; Maurer, Steffen Christian; Schmid, Rolf D.; Urlacher, Vlada B.The gene encoding CYP102A2, a novel P450 monooxygenase from Bacillus subtilis, was cloned and expressed in Escherichia coli. The recombinant enzyme formed was purified by immobilised metal chelat affinity chromatography (IMAC) and characterised. CYP102A2 is a 119 kDa self-sufficient monooxygenase, consisting of an FMN/FAD–containing reductase domain and a heme domain. The deduced amino acid sequence of CYP102A2 exhibits a high level of identity with the amino acid sequences of CYP102A1 from Bacillus megaterium (59%) and CYP102A3 from Bacillus subtilis (60%). In reduced, CO-bound form, the enzyme shows a typical Soret band at 450 nm. It catalyses the oxidation of even- and odd-chain saturated and unsaturated fatty acids. In all reactions investigated, the products were the respective ù-3, ù-2 and ù-1 hydroxylated fatty acids. Activity was highest towards oleic and linoleic acid (KM=17.4 ± 1.4 ìM, kcat= 2244 ± 72 min-1), linoleic acid (KM=12.25 ± 1.8 ìM, kcat= 1950 ± 84 min-1). Comparison of CYP102A2 homology model to CYP102A1 crystal structure revealed significant differences in the substrate access channels, which might explain the differences in catalytic properties of these two enzymes.Item Open Access Immobilisation of P450 BM-3 and an NADP+ cofactor recycling system : towards a technical application of heme-containing monooxygenases in fine chemical synthesis(2003) Maurer, Steffen Christian; Schulze, Holger; Schmid, Rolf D.; Urlacher, Vlada B.Cytochrome P450 monooxygenases are potentially a very useful class of hydroxylation catalysts; they are able to introduce oxygen at activated and non-activated carbon-hydrogen bonds and thus lead to regio- and/or stereochemically pure compounds. However, this potential is lowered by their intrinsic low activity and inherent instability. P450-catalysed biotransformations require a constant supply of NAD(P)H, making the process an expensive one. To render these catalysts more suitable for industrial biocatalysis, the immobilisation of P450 BM-3 (CYP 102A1) from Bacillus megaterium in a sol-gel matrix was combined with a cofactor recycling system based on NADPƒy-dependent formate dehydrogenase (EC 1.2.1.2) from Pseudomonas sp. 101 and tested for practical applicability. This approach was used for the conversion of £]-ionone, octane and naphthalene to the respective hydroxy-compounds with DMSO as cosolvent using sol-gel immobilised P450 BM-3 mutants.