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 chelate 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 B. megaterium (59%) and CYP102A3 from B. subtilis (60%). In reduced, CO-bound form, the enzyme shows a typical Soret band at 449 nm. It catalyses the oxidation of even- and odd-chain saturated and unsaturated fatty acids. In all reactions investigated, the products were the respective ω-3, ω-2 and ω-1 hydroxylated fatty acids. Activity was highest towards oleic acid (KM=17.36±1.4 μM, kcat=2,244±72 min−1) and linoleic acid (KM=12.25±1.8 μM, kcat=1,950±84 min−1). Comparison of a CYP102A2 homology model with the CYP102A1 crystal structure revealed significant differences in the substrate access channels, which might explain the differences in the catalytic properties of these two enzymes.

Cloning, expression and characterisation of CYP102A7, a self-sufficient P450 monooxygenase from Bacillus licheniformis

Whereas most products of the chemical industry are based on petrochemical feedstocks, considerable efforts have been made during the past few decades to use renewable resources as industrial raw materials Besides polysaccharides and sugars, plant oils and animal fats play an important role in such programmes because of their ready availability (present production is >100 million t/a, which could be increased on demand) of which the lion’s share is used for nutrition (~85 million t/a), whereas ~ 15 – 20 million t/a are used for the synthesis of polymers, surfactants, emollients, lubricants, bio-diesel, emulsifiers, etc) 
From a chemical point of view, most natural triglycerides offer just two reactive sites, the ester group and the double bonds of unsaturated fatty acids In fact, the chemistry of fats and oils is largely focused on the ester group which can be hydrolyzed or catalytically reduced, leading to glycerol and fatty acids or fatty alcohols, respectively Reactions involving the alkyl chain or double bonds of triglycerides, fatty acids, fatty alcohols or their derivatives represent far less than 10% of today’s oleochemistry, with the production of sulfonated fatty alcohols and their derivatives being a major process of this kind Oxidation reactions at the alkyl or alkenyl chains would be highly desirable as they would lead to oleochemicals with new properties, but the methods available today lack selectivity and require harsh conditions Notable exceptions are the epoxidation of unsaturated plant oils and the synthesis and use of a few hydroxy fatty acids

Biocatalysts for the epoxidation and hydroxylation of fatty acids and fatty alcohols

Cytochrom P450-Monooxygenasen sind in der Natur weit verbreitet und besitzen Schlusselfunktionen im Metabolismus exogener und endogener Verbindungen. Funk-tionell ist allen P450-Enzymen gemeinsam, Sauerstoffatome auf nicht aktivierte 
aliphatische oder aromatische C-H, N-H oder S-H-Bindungen zu ubertragen. Die funktionelle Vielfalt der durch P450-Systeme katalysierten Monooxygenierungen von auf chemischem Wege haufig nur schwer zuganglichen Verbindungen bietet ein enormes biotechnologisches und pharmakologisches Potential. Eine wichtige Voraussetzung zur Nutzung dieser Potentiale ist die Entwicklung geeigneter Expressions-, Reinigungs- und Aktivitatsnachweissysteme, die erlauben P450-Systeme zu charakterisieren und Enzymvarianten mit verbesserten Eigenschaften aufzufinden.

Es existieren bereits zahlreiche Untersuchungen zur bakteriellen Fettsaurehydroxylase CYP102A1 aus Bacillus megaterium, die viel versprechende Eigenschaften in Bezug auf Aktivitat und Stabilitat im Vergleich zu eukaryontischen Systemen aufweist. Im Rahmen dieser Arbeit wurde eine weitere bakterielle Monooxygenase der P450-Familie CYP102 kloniert und in E. coli funktionell exprimiert. Bei der Charakterisierung der P450-Monooxygenase CYP102A3 aus Bacillus subtilis wurde entdeckt, dass sie hohere Stabilitat bei Raumtemperatur und in Losungsvermittlern aufweist als CYP102A1 und damit fur technische Anwendungen geeigneter erscheint. Durch Mutagenese wurde die Substratspezifitat stark erweitert und auf Alkane und Arene ausgedehnt. Um die Regioselektivitat der n-Octan-Hydroxylierung durch Mutagenese von rein subterminaler zu terminaler Hydroxylierung zu andern, wurde ein Hochdurchsatzassay entwickelt, der selektiv fur terminale Hydroxylierungen an aliphatischen Verbindungen ist. Er beruht auf der Oxidation des entstandenen primaren Alkohols zum Aldehyd durch eine Hefe-Alkoholdehydrogenase.

Mit Hilfe des Assays und der Methode der gerichteten Evolution konnte aus 6500 Mutanten eine terminal hydroxylierende Variante identifiziert werden, deren Sequenzinformationen die Erstellung einer stabilen Doppelmutante auf der Basis von CYP102A3 ermoglichte, welche bei der Hydroxylierung von n-Octan einen Produktanteil von 48% 1-Octanol liefert.
 
P450 monooxygenases play a key role in primary and secondary metabolic pathways and in drug detoxification. Cytochromes P450 are heme-thiolate proteins which are widely distributed in animals, plants and microorganisms. Their unique hydroxylation capacity provides the opportunity to use these biocatalysts for the synthesis of fine chemicals, as they even act on non-activated carbon-hydrogen bonds and hydroxylate such compounds as alkanes, fatty acids, terpenes and steroids, often exhibiting high regio- and stereoselectivity. A very important premise for using this potential is the development of expression, purification and activity test systems that provide the possibility to characterize enzyme variants with enhanced properties.

There exist many publications about the bacterial fatty-acid hydroxylating P450 CYP102A1 from Bacillus megaterium. This enzyme shows very promising properties regarding activity and stability compared to eukaryotic systems. During the work presented here another bacterial monooxygenase of the CYP family 102 was cloned and actively expressed in E. coli. The characterization of this P450 enzyme (CYP102A3) from Bacillus subtilis showed higher stability at room temperature and in co-solvents than CYP102A1. This makes the enzyme more suitable for technical applications. Using mutagenesis, the substrate specificity was enlarged and incorporates now also alkanes and arenes. To change the regioselectivity of the octane hydroxylation from subterminal to terminal position, a HTS assay was developed that is selective for terminal hydroxylation of aliphatic compounds. It is based on the oxidation of the primary alcohol developed during the hydroxylation reaction by yeast alcohol dehydrogenase.

Using this assay combined with directed evolution, a gene bank of 6,500 enzyme variants was screened. A terminal hydroxylating mutant was discovered and sequenced. The sequence information was used to create a new, more stable variant of CYP102A3 which yields up to 48% 1-octanol after octane hydroxylation.

Klonierung, Expression und Charakterisierung der Cytochrom P450-Monooxygenase CYP102A3 aus Bacillus subtilis sowie Veränderung ihrer Regioselektivität durch gerichtete Evolution

Bioconversion of progesterone with cell preparations with Aspergillus niger 171.

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Bioconversion of progesterone with cell preparations with Aspergillus niger 171.