EP2205726A1

EP2205726A1 - Method for the oxidation of methyl groups in aliphatic hydrocarbons using an enzyme system with the activity of a monooxygenase

Info

Publication number: EP2205726A1
Application number: EP08804177A
Authority: EP
Inventors: Roland H. MÜLLER; Uta Breuer; Martin Von Bergen
Original assignee: Helmholtz Zentrum fuer Umweltforschung GmbH UFZ
Current assignee: Helmholtz Zentrum fuer Umweltforschung GmbH UFZ
Priority date: 2007-09-17
Filing date: 2008-09-15
Publication date: 2010-07-14
Also published as: WO2009037216A1; DE102007045092A1

Abstract

The invention relates to an enzymatic method for the oxidation of methyl groups in aliphatic hydrocarbons and to novel proteins and enzyme mixtures with the enzymatic activity of a monooxygenase. According to the invention, preferably compounds with methyl groups are oxidised in branched-chain, aliphatic hydrocarbons, which are essentially acyclic compounds and/or aliphatic compounds having functional groups or compounds with methyl groups at the aliphatic hydrocarbons which are mono- or disubstituted at the C2 atom, wherein the substituents can be the same or different. The invention preferably relates to a biotechnological process for the oxidation of methyl groups in the branched-chain structures, wherein microorganisms which have or produce the desired monooxygenase activity are cultivated in an aqueous system and are converted into the corresponding target products.

Description

Process for the oxidation of methyl groups in aliphatic hydrocarbons using an enzyme system with the activity of a monooxygenase

The invention relates to an enzymatic process for the oxidation of methyl groups in aliphatic hydrocarbons and to novel proteins and enzyme mixtures having the enzymatic activity of a monooxygenase. According to the invention, compounds with methyl groups are preferably oxidized in branched-chain, aliphatic hydrocarbons, which are in particular acyclic compounds and / or aliphatic compounds with functional groups, or compounds with methyl groups on aliphatic or disubstituted aliphatic hydrocarbons on the C2 atom, the substituents being the same or different could be. Preferably, the invention relates to a biotechnological process for the oxidation of methyl groups in the branched-chain structures, wherein microorganisms containing the desired

Possess or produce monooxygenase activity, are cultured in an aqueous system and converted to corresponding target products.

2-Methyl-1,2-dihydroxypropane and secondary products or in C2-substituted propanols and secondary products are of great interest as synthesis intermediates of pharmaceuticals, fine chemicals and the like. For example, By esterification and / or etherification of one or both hydroxy groups of propane-1,2-diol, it is possible to obtain numerous products which can be used as solvents, plasticizers, thickeners or emulsifiers. The preparation of optically active compounds is also of great importance (EP 1 550 730 A1). The optically active (R) - (-) and (S) - (+) - forms are used as chiral building block in organic syntheses and are test substrates for the development of separation methods for enantiomers. (R) - (-) - and (S) - (+) - forms of C2-substituted propanols and secondary products in the form of the corresponding acid, e.g. are active in active ingredients and are used in the pharmaceutical sector or in crop protection and agriculture.

A source of enzyme activities for the oxidation of branched-chain alkane derivatives is seen in bacterial strains, the methyl tert-butyl ether (MTBE) and related compounds such as ethyl-tert-butyl ether (ETBE) and te / t-amyl methyl ether (TAME). able to degrade. These compounds have been used as so-called oxygenates to increase the octane number in fuels since the 1980's. The intensive dealing with these connections has to one Pollution of the environment. Ways of relieving contaminants are generally seen in the metabolic potential of microorganisms because of its universality and versatility. MTBE and related substances, however, prove to be recalcitrant. However, the metabolism of MTBE is only partially elucidated. The degradation begins in principle with a cleavage of the ether bond, this is a monooxygenase mechanism in question. For Rhodococcus ruber IFP2001 or A. tertiaricarbonis L108, this step is catalyzed by a P450-dependent enzyme encoded by the ethABCD gene. The resulting from the cleavage te / t-butoxymethanol leads via chemical dismutation or an appropriate dehydrogenase to te / t-butyl alcohol (TBA). This intermediate is often found in MTBE-degrading cultures, that is, the step in the further metabolism of this compound is rate-limiting in these circumstances.

In Journal of Bacteriology Vol. 189 (5), 1931 -1945 is by Kane, S. R. u. a. the biodegradation of MTBE by Beta-Proteobacterium Methylibium petroleiphilum PM1 described. It is shown that MTBE-degrading key enzymes are located on the PM1 megaplasmid. Statements about the function of these enzymes, the specificity and mediation of reactions remain open.

Recently, it has been shown in Mycobacterium austroafricanum IFP2012 that AIkB, the typical and widely used n-alkane monooxygenase, is indicated in the presence of MTBE (Lopez Ferreira et al., Appl. Microbiol Biotechnol., 2007, 75, 909-919). AIkB as a general n-alkane monooxygenase is known to have a broad substrate spectrum. However, the substrate specificity of AlkB-typical enzymes is variable, but particularly high for n-alkane structures, while branched-chain compounds are reacted rather slowly or not at all by these monooxygenases. This is also evident in the case of strain IFP2012, which has a maximum specific growth rate μ _ma χ on TBA of only 0.039 h ^"1. The fact that the conversion rates are so low suggests that this is also a nonspecific reaction of the For another gram-positive strain, a μ _max of 0.032 h ^{-1 was} determined, but there are no data on the enzymes involved. For Variovorax paradoxus CL-8, similarly small rates of μ _max = 0.042 h ^-1 are obtained (Zaitsev et al., Appl.Microbiol.Biotechnol., 74: 1092). Again, the enzyme catalyzing the initial step is unknown There are no strains of the enzyme involved and few usable data on the realized substrate turnover. It is an object of the present invention to seek other proteins and enzyme systems capable of utilizing branched chain aliphatic compounds and transforming them into target products which are of intermediate or end products of industrial interest.

The invention is based on the surprising finding that in the strain L108 (DSM 18260), owing to its high growth rates on TBA of 0.1 h ^-1 and the resulting high specific TBA conversion rates amounting to up to 210 mmol / h * g biomass One protein could be identified as a nominal phthalate dioxygenase by mass spectrometry and sequenced analysis, and another protein was diagnosed as Fe / S oxidoreductase, which together constitute an enzyme residue the phthalate dioxygenase has a large subunit and Fe / S

Oxidoreductase represent a small subunit of this enzyme complex. The confirmation of the function of the enzyme or its two components in the metabolism of TBA was shown by switching off the relevant genes. The enzyme system surprisingly acts as monooxygenase in the present case.

The invention therefore provides the use of an enzyme system having the enzymatic activity of a monooxygenase which has a protein having hydroxylase activity and a protein having oxidoreductase activity for the oxidation of a methyl group on branched-chain, aliphatic structures, wherein the oxidation at a tertiary or secondary C-atom can take place, as well as methyl groups in compounds which are substituted on the C2 atom, so that after oxidation, compounds with an optically active carbon atom are obtained.

In the context of the invention, branched-chain, aliphatic structures are understood as meaning in particular acyclic compounds and / or aliphatic compounds with functional groups which increase the reactivity and induce a characteristic reaction mode. These include hydrocarbon compounds (HC), such as alkanes, alkenes and alkynes. Aliphatic compounds with functional groups are, for example, HC with C, C double bond (alkenes), C, C triple bond (alkynes), with halogen group (HFC, CHC, BKW, IKW), hydroxy group (alcohols), alkoxy group ( Ethers), thiol group, carbonyl group (aldehydes, ketones), thiocarbonyl group (thioaldehydes, thioketones), carboxy group (carboxylic acids), amino, imino, carboxamide group (Amines, imines, amides), alkoxycarbonyl group (esters), nitrile group, nitro group and sulfonic acid group.

The preferably used enzyme system having the enzymatic activity of a monooxygenase comprises at least one protein having SEQ ID NO: 1, as well as mutants, variants and parts thereof having a sequence homology of at least 50% (assignment by BLAST) and having hydroxylase activity and / or a protein having SEQ ID NO: 2, as well as mutants, variants and parts thereof which have a sequence homology of at least 50% (assignment by BLAST) and have oxidoreductase activity.

It is particularly preferable to use an enzyme system comprising as a large subunit protein sequence SEQ ID NO: 1 and as a small subunit SEQ ID NO: 2 and at least 50% of its homologs having the same properties.

Very particular preference is given to using an oligomeric protein which consists of the sequence SEQ ID NO: 1 and SEQ ID NO: 2 according to the invention.

The invention likewise provides an enzyme system comprising a protein having SEQ ID NO: 1 and an enzyme system which comprises a protein having SEQ ID NO: 2. A preferred enzyme system comprises, as a large subunit, a protein with SEQ ID NO: 1 and, as a small subunit, a protein with SEQ ID NO: 2. Particularly preferably, the enzyme system consists of SEQ ID NO: 1 and SEQ ID NO: 2 as oligomeric protein ,

Also new is the protein having SEQ ID NO: 1, which has hydroxylase activity. The protein with SEQ ID NO: 1 preferably has the conserved motifs SEQ ID NO: 3 and SEQ ID NO: 4 or SEQ ID NO: 10, the conserved motifs SEQ ID NO: 3 for the binding of the Rieske type Cys ₂ His ₂ [2Fe-2S] clusters and SEQ ID NO: 4 and SEQ ID NO: 10, respectively, for the binding of a singular Fe ²⁺ .

The invention also relates to the protein having SEQ ID NO: 2, which has oxidoreductase activity. Preferably, the protein SEQ ID NO: 2 has the conserved motifs SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7, wherein the conserved motifs SEQ ID NO: 5 for the binding of FMN or FAD, SEQ ID NO: 6 for the binding of a plant-specific Cys ₄ [2Fe-2S] cluster and SEQ ID NO: 7 for the binding of ferredoxin. The invention also relates to a nucleic acid molecule encoding an enzyme having the activity of a monooxygenase, selected from the group consisting of a) nucleic acid molecules which encode a protein having the amino acid sequences given under SEQ ID NO: 1 and / or SEQ ID NO: 2; b) Nucleic acid molecules which have the nucleotide sequences shown under SEQ ID NO: 8 and / or SEQ ID NO: 9.

It has been found that the enzyme system used according to the invention preferably represents a heterodimeric protein which comprises the subunits described under SEQ ID NO: 1 and SEQ ID NO: 2 and thus has excellent enzyme activity.

A preferred nucleic acid molecule encodes a protein which, as an oligomeric enzyme composed of two different subunits, has the amino acid sequences given under SEQ ID NO: 1 and SEQ ID NO: 2.

A nucleic acid molecule may be a DNA molecule, preferably cDNA or genomic DNA and / or an RNA molecule. Both nucleic acids and proteins can be isolated from natural sources, preferably from bacterial strain HCM-10 (DSM 18028), Ideonella sp. L108 (DSM 18260) or methylibium sp. R8 (DSM 19669).

However, the proteins can also be chemically chemically synthesized according to methods known per se. be synthesized by means of a solid phase synthesis, genetically engineered by a prokaryotic host or in the broadest sense by directed mutagenesis or enzyme design.

Mutations can be generated in the nucleic acid molecules used according to the invention by molecular biological techniques known per se, which makes it possible to synthesize further enzymes with analogous or similar properties which can likewise be used according to the invention. Mutations can be deletion mutations leading to truncated enzymes. By other molecular mechanisms such as insertions, duplications, transpositions, gene fusion, nucleotide exchange or gene transfer and gene-shuffling between different genes Microorganism strains can also be produced modified enzymes with similar or analogous properties.

The identification and isolation of such nucleic acid molecules can be accomplished using the nucleic acid molecules or portions thereof. The molecules hybridizing with the nucleic acid molecules also include fragments, derivatives and allelic variants of the nucleic acid molecules described above which encode an enzyme useful in the invention. By fragments are meant parts of the nucleic acid molecules that are long enough to encode the described enzyme. Derivative is understood as meaning sequences of these molecules which differ from the sequences of the above-described nucleic acid molecules at one or more positions, but have a high degree of homology to these sequences. Homology here means a sequence identity of at least 40%, in particular an identity of at least 60%, preferably over 80% and particularly preferably over 90%, 95%, 97% or 99% at the nucleic acid level. The degree of homology is determined for both proteins and nucleic acids by augment using BLAST (BlastX or BlastN). The encoded enzymes have a sequence identity to the indicated amino acid sequences of at least 50% or at least 60%, preferably of at least 80%, more preferably of at least 95%, most preferably at least 97% and at least 99% at the amino acid level. The deviations can be caused by deletion, substitution, insertion or recombination. These may be naturally occurring variations, for example sequences from other organisms, or mutations, which mutations may occur naturally or by directed mutagenesis (UV rays, X-rays, chemical agents or others). Furthermore, the variants may be synthetically produced sequences. These variants have certain common characteristics, e.g. Enzyme activity, active enzyme concentration, subunits, functional groups, immunological reactivity, conformation and / or physical properties, such as gel electrophoresis, chromatographic behavior, solubility, sedimentation coefficients, pH optimum, temperature optimum, spectroscopic properties, stability and / or other.

The enzyme system used according to the invention with the enzymatic activity of a monooxygenase is preferably prepared by the proteins having the SEQ ID NO: 1 and / or SEQ ID NO: 2 or their homologs in one prokaryotic host may be cloned and expressed singly or in common, and recovered appropriately or individually after expression.

The enzyme system is preferred in a process for the oxidation of

Methyl groups, which are located on a branched-chain, aliphatic structure, or to compounds which have an optically active carbon atom after oxidation, wherein the oxidation takes place at sec sec or tert-C atom located methyl groups or terminal methyl groups in on the C2 -substituted alkane derivatives. Thus, an aqueous reaction solution which has an enzyme system or protein according to the invention or a microorganism producing it, is incubated with the compounds bearing methyl groups and then the correspondingly converted target compound is obtained, which is used as an intermediate or end product or as a so-called "building block". Possible intermediate or target products or "building blocks" are

Oxidation products, e.g. in the broadest sense, propane derivatives, which means both actual propane derivatives which are substituted on the C2 atom and which after the oxidation give optically active compounds, e.g. as R- and S-enantiomers, respectively, as drug forms in the pharmaceutical industry (preferably the S-enantiomer), e.g. Profene, or in crop protection and agriculture

(preferably the R-enantiomer), e.g. Phenoxyalkanoates are used. However, this also means 2-methyl-alkane structures, which in the sense of the enzymatic mechanism are understood to be synonymous with propane derivatives substituted on C2. Structures with tertiary carbon atoms are thus to be interpreted as C2 di-substituted propane derivatives.

In a preferred embodiment, a hydroxylation of methyl groups on aliphatic compounds, preferably alkanes or hydrocarbons having functional groups, preferably having 3 to 10 C-atoms with secondary and tertiary carbon atoms, performed. This concerns e.g. 2,2-dimethylhexane as a medium-chain branched-chain reaction with tertiary carbon atom or 2,4- or 2,5-dimethylhexane with one or two secondary C atoms.

In a further embodiment, the methyl groups on a secondary carbon atom (C2) are oxidized, preferably compounds are used, which lead after oxidation of the methyl group located on the secondary carbon atom to compounds with an optically active center on the secondary carbon atom. This relates, for example, to 2,5-dimethylhexane or hexene as a medium-chain branched-chain member in saturated or unsaturated form or, for example, 2-chloropropane or 2-hydroxypropane (isopropanol) as the representative with the shortest chain of interest here.

The process according to the invention is in particular characterized in that a microorganism or a crude extract thereof is used. The natural sources are preferably bacterial strain HCM-10 (DSM 18028), Ideonella sp. L108 (DSM 18260) or methylibium sp. R8 (DSM 19669) used. With these strains, preferred suitable biological systems have been found. Strain HCM-10 was deposited under the Budapest Treaty on the deposit of microorganisms for the purposes of patent procedure at the German Collection of Microorganisms and Cell Cultures GmbH, Braunschweig, DE under the number DSM 18028 on 03.03.2006; Strain Ideonella sp. L108 was deposited with the German strain collection for microorganisms and cell cultures in Braunschweig, DE under the number DSM 18260 on 28.04.2006, and strain Methylibium sp. R8 was deposited with the German strain collection for microorganisms and cell cultures in Braunschweig, DE under the number DSM 19669 on 04.09.2007. Strain Methylibium sp. R8 (DSM 19669) is new.

The process is preferably carried out in such a way that, if appropriate, substrates are supplied which ensure the growth of the microorganisms and thus guarantee stability or stabilization of the enzyme system or provide reduction equivalents for the monooxygenase. Preferably, these are heterotrophic substrates that can be used by unchanged microorganism as a carbon and energy source for growth and propagation. In a further preferred variant, the pre-cultivation of the cells is carried out so that products or storage materials, e.g. Polyhydroxybutyrate (PHB), e.g. the nitrogen source in the medium limits growth, and the polymeric storage materials during product formation

Provision and regeneration of reduction equivalents. In a further preferred variant, the cells of the microorganisms are optionally incubated in the presence of an auxiliary substrate, which is suitable for the provision or for the regeneration of reduction equivalents. Preferably, the auxiliary substrate used is formic acid or its salts. Optionally, the gene for a formate dehydrogenase is cloned and expressed. It can also cell-free crude extracts of microorganisms are used, preferably cell-free crude extracts of the microorganism HCM-10.

The process is controlled so that the protein with the oxidoreductase activity leads to the transfer of reducing equivalents from reduced coenzymes to the protein with hydroxylase activity. Reduced coenzymes are usually NADH + H ⁺ and NADPH + H ^+, respectively.

In another embodiment, cell extracts and / or enzyme system with monooxygenase activity after partial or complete isolation from the microorganisms, optionally in purified form and optionally with the addition of further enzymes, e.g. used for the regeneration of reduction equivalents.

A microorganism can be used after cloning and expression of the proteins having the SEQ ID NO: 1 and / or SEQ ID NO: 2 as a whole cell, unchanged, permeabilized or carrier-fixed. In combination with enzyme system and proteins, it is possible to use further enzymes or enzyme systems for the regeneration of reduction equivalents, which can be employed in solution or in a carrier-fixed manner.

The strains used according to the invention preferably produce the proteins having the sequences SEQ ID NO: 1 and / or SEQ ID NO: 2 or comprise the nucleic acid sequences SEQ ID NO: 8 and / or SEQ ID NO: 9 or at least 50% homologs thereof. For the purposes of the invention, the said proteins can also be used in enriched, isolated or synthetically produced form.

As already stated, in a further preferred embodiment variant of the invention, in particular microorganisms, crude extracts, parts thereof and / or the enriched or isolated enzymes are immobilized. By the

Immobilization places enzymes, cell organelles and cells in an insoluble and reaction space limited state. For example, they can be immobilized in a polymer matrix (eg alginate, polyvinyl alcohol or polyacrylamide gel). Immobilization may also be carried out on dissolved or undissolved carrier materials (eg celite) to facilitate catalyst recovery and use. Methods for cell immobilization in a polymer matrix or on a dissolved or undissolved carrier are known to the person skilled in the art and have already been described in detail. The enzyme activities can also be characterized isolated from the microbial cells. These can then be used directly immobilized as a catalyst or in a polymer matrix or on a dissolved or undissolved carrier. The methods necessary for this are known to the person skilled in the art and described, for example, in Methods in Biotechnology, Vol. 1: Immobilization of enzymes and cells, publisher: GF Bickerstaff, Humana Press, Totowa, New Jersey, 1997.

The conversion to the target product preferably takes place in the context of a continuous process which can be carried out in a reactor through which microbial growth and thus product formation takes place. However, a continuous process may also be understood to mean any system of growing cells and catalyzing enzymes, to which nutrient solution is added on the one hand and from which, on the other hand, culture solution, including enzymatically formed target product, is withdrawn. According to the invention, the method can also be carried out as a semicontinuous or batch process.

In connection with the invention, in addition to the monooxygenase optionally enzymes are used which are present in the microorganism or added. The conversion to the target product is optionally carried out in a single process step, i. the transformation of

Starting substrates run simultaneously or slightly delayed in one and the same reaction solution.

The process may be carried out aerobically, preferably using whole cells, or else micro-aerobic, e.g. under nitrogen, preferably when extracts or purified enzymes are used, in which case the oxygen supply is controlled in terms of a substrate supply.

The target products produced according to the invention can be isolated by treatment of the culture medium (after removal of undissolved constituents such as microbial cells) by methods already known. Such methods are in addition to other z. As concentration, ion exchange, distillation, electrodialysis, extraction and crystallization.

SEQ ID NO: 1 shows the phthalate dioxygenase with hydroxylase activity with 470 amino acids (large subunit of the enzyme system with the enzymatic activity of a monooxygenase) from DSM 18028. SEQ ID NO: 2 shows the sequence comprising 337 amino acids with Fe / S-oxidoreductase activity (small subunit of the enzyme system with the enzymatic activity of a monooxygenase) from DSM 18028.

SEQ ID NO: 3 shows the conserved motifs for the binding of the Rieske-type Cys ₂ His ₂ [2Fe-2S] cluster.

SEQ ID NO: 4 and SEQ ID NO: 10 show the conserved motifs for the binding of a singular Fe ²⁺

SEQ ID NO: 5 shows the conserved motifs for the binding of FMN and FAD, respectively.

SEQ ID NO: 6 shows the conserved motifs for the binding of a plant-typical Cys ₄ [2Fe-2S] cluster.

SEQ ID NO: 7 shows the conserved motifs for the binding of ferredoxin.

SEQ ID NO: 8 shows 1413 bp of the coding nucleotide sequence for the phthalate dioxygenase from DSM 18028.

SEQ ID NO: 9 shows 1014 bp of the coding nucleotide sequence for the Fe / S-oxidoreductase from DSM 18028.

Subsequently, the invention will be described in more detail in exemplary embodiments, to which, however, it should not be restricted.

embodiment

Example 1 The strain DSM 18028 was cultured on mineral salt medium (MSM) in the presence of a vitamin mixture. The medium contained (in mg / l): NH ₄ Cl, 350; KH ₂ PO ₄ , 340; K ₂ HPO ₄ , 485; CaCl ₂ ^* 6 H ₂ O, 27; MgSO ₄ ^* 7 H ₂ O, 71.2, and 1 ml / l Spurensalz- stock solution. The trace salt stock solution contained (in g / l): FeSO ₄ .7H ₂ O, 4.98; CuSO ₄ ^* 5H ₂ O, 0.785; CoCl ₂ , 5; MnSO ₄ ^* 4H ₂ O, 0.81; ZnSO ₄ ^* 7 H ₂ O, 12:44; Na ₂ MoO ₄ * 2 H ₂ O, 0.25. The concentration of vitamin in the medium was (in μg / l): biotin, 20; Folic acid, 20; Pyridoxine HCl, 100; Thiamine HCl, 50; Riboflavin, 50; Nicotinic acid, 50; DL-Ca-pantothenate, 50; p-aminobenzoic acid, 50; Lipoic acid, 50, and cobalamin, 50. The growth substrate used was succinate at a concentration of 3 g / l. The Cultivation was continuous or discontinuous. In continuous cultivation, additional t-butyl alcohol (TBA) was added at a concentration of 0.2 g / l. In the case of discontinuous culture, culture TBA was added following growth on succinate to induce monooxygenase activity and the culture was incubated for a further 5 h.

After continuous cultivation, the cells were separated, washed and adjusted to a biomass concentration of 0.5 g / l with phosphate buffer (50 mM, pH 7.0). 2,2-Dimethylhexane was added to this suspension in a concentration of 10 mM and the batch was incubated in a gastight vessel at 30 ^° C. The ratio of liquid to gas volume was 4 to 1. The starting gas was room air. After 24 h, 6.8 mM 2-hydroxymethyl-2-methylhexane were formed under these conditions and using cells which had been cultivated discontinuously; 7.1 mg of 2-hydroxymethyl-2-methylhexane were obtained after 24 hours of incubation using continuously cultivated cells.

Example 2

The strain DSM 18028 was pre-cultured on a medium as indicated in Example 1, but in the presence of 760 mg / l NH ₄ Cl. After culturing, the suspension was prepared for subsequent product formation phase as indicated above in phosphate buffer.

This suspension was added to 2,2-dimethylhexane in a concentration of 10 mM and 1 g / l succinate and the mixture was incubated in a gas-tight vessel at 30 ⁰ C under the conditions given in Example 1. After 24 h, 5.4 mM 2-hydroxymethyl-2-methylhexane were formed under these conditions.

Example 3

The strain DSM 18028 was pre-cultured on a medium as described in Example 1 and after cultivation for a subsequent product formation phase as described above prepared in phosphate buffer accordingly. 2,5-Dimethylhexane was added to this suspension in a concentration of 10 mM and the batch was incubated in a gas-tight vessel at 30 ° C. under the conditions given in Example 1. After 24 h under these conditions, 4.2 mM 2-hydroxymethyl-5-methylhexane and 3.8 mM 2.5-

Dihydroxymethylhexane formed. After 48 h, the concentrations were 2.7 mM 2-hydroxymethyl-5-methylhexane and 5.9 mM 2,5-dihydroxymethylhexane. Example 4

The strain DSM 18028 was pre-cultured on a medium as described in Example 1 and after cultivation for a subsequent product formation phase as described above prepared in phosphate buffer accordingly. 2-Chloropropane was added to this suspension in a concentration of 10 mM and the batch was incubated in a gastight vessel at 30 ^° C. under the conditions given in Example 1. After 24 h, 5.8 mM 2-chloropropanol were formed under these conditions.

Example 5

The strain DSM 18028 was pre-cultured on a medium as described in Example 1 and after cultivation for a subsequent product formation phase as described above prepared in phosphate buffer accordingly. To this suspension was added 2,5-dimethyl-2,4-hexadiene in a concentration of 10 mM was added and the mixture was incubated in a gastight vessel at 30 ⁰ C under the specified conditions of Example 1. After 24 hours, 3.3 mM 2-hydroxymethyl-5-methylhexadiene and 3.1 mM 2,5-dihydroxymethylhexadiene were formed under these conditions. After 48 h, the concentrations were 1.9 mM 2-hydroxymethyl-5-methylhexadiene and 6.4 mM 2,5-dihydroxymethylhexadiene.

Example 6

The strain DSM 19669 was pre-cultured on mineral salt medium as described in Example 1 and after cultivation for a subsequent product formation phase as described above prepared in phosphate buffer accordingly. 2,2-Dimethylhexane was added to this suspension in a concentration of 10 mM and the batch was incubated in a gas-tight vessel at 30 ° C. under the conditions given in Example 1. After 24 hours, 5.3 mM 2-hydroxymethyl-2-methylhexane were formed under these conditions.

SEQ ID NO: 1 (gr UE)

1 MGNREPLAAA GQGTAYSGYR LRDLQNVAPT NLEILRTGPG TPMGEYMRRY WQPVCLSQEL 60

61 TDVPKAIRIL HEDLVAFRDR RGNVGVLHRK CAHRGASLEF GIVQERGIRC CYHGWHFDVD 120

121 GSLLEAPAEP PDTKLKETVC QGAYPAFERN GLVFAYMGPA DRRPEFPVFD GYVLPKGTRL 180

181 IPFSNVFDCN WLQVYENQID HYHTALLHNN MTWGVDAKL ADGATLQGGF GEMPIIDWHP 240 241 TDDNNGMIFT AGRRLSDDEV WIRISQMGLP NWMQNAAIVA AAPQRHSGPA MSRWQVPVDD 300

301 EHSIAFGWRH FNDEVDPEHR GREEECGVDK IDFLIGQTRH RPYEETQRVP GDYEAIVSQG 360

361 PIALHGLEHP GRSDVGVYMC RSLLRDAVAG KAPPDPVRVK AGSTDGQTLP RYASDSRLRI 420

421 RRRPSREADS DVIRKAAHQV FAIMKECDEL PWQRRPHVL RRLDEIEASL 470

SEQ ID NO: 2 (CLU UE)

1 MYQLSHTGKY PKTALNLRVR QITYQGIGIN AYEFVREDGG ELEEFTAGAH VDLYFRDGRV 60

61 RQYSLCNDPA ERRRYLIAVL RDDNGRGGSI AIHERVHTQR LVAVGHPRNN FPLIEGAPHQ 120

121 VLLAGGIGIT PLKAMVHRLE RMGADYTLHY CAKSSAHAAF QEELAPMAAK GRVIMHFDGG 180 181 NPAKGLDIAA LLRRYEPGWQ LYYCGPPGFM EACTRACTNW PAEAVHFEYF VGAPVLPDDG 240

241 VPHDIGSDAL ALGFQIKIAS TGTVLTVPND KSIAQVLGEH GIEVPTSCQS GLCGTCKVRY 300 301 LAGDVEHRDY LLSAEARTQF LTTCVSRSKG ATLVLDL 337

SEQ ID NO: 3 (Rieske)

-RX 1 -CXHRXXXLXXG-X _S -CXYHR-X _Ö -G-

(X values can be +/- 2)

SEQ ID NO: 4 (D) or SEQ ID NO: 10 (E) (singular Fe)

- (D / E) xxxDxxHxxxxH-

SEQ ID NO: 5 (FMN) -RxYSL-x _{_20-22} -RGGS- SEQ ID NO: 6 (NAD) -GGIGxTPxxxM-

SEQ ID NO: 7 (ferredoxin)

-CxxGxCGxCxeGxxxH RDxxLx _5-7 CxS-

SEQ ID NO: 8 (gr UE)

1 tcagaggctc gcttcgatct cgtcgaggcg ccgcaggaca tgcggcctgc gctgcacgac

61 cggcagttcg tcgcactcct tcatgatcgc gaaaacctgg tgcgcggcct tgcggatgac

121 gtcactgtcc gcttcccggc tcggccggcg gcggatccgc agtcgactgt ccgacgcgta

181 tcgcggcagc gtttgcccat cggtcgaccc agccttcacg cgcaccgggt cgggcggcgc 241 cttgccggcc acagcgtcgc gaagcagcga gcgacacatg tacacaccca cgtccgaccg

301 gccgggatgc tcaagaccgt ggagggctat cggcccctgg ctgacgatgg cttcgtagtc

361 gcccggaacc cgctgcgtct cttcataagg ccgatgccgg gtctgaccga tcaggaagtc

421 gatcttgtcg accccgcact cctcttccct tccacggtgc tccgggtcga cctcgtcgtt

481 gaagtggcgc cagccgaagg cgatcgagtg ctcgtcgtcg accggcacct gccagcgtga 541 catcgccggg ccggagtgtc gctgcggagc cgccgccacg atggcggcgt tctgcatcca

601 gttcggcagg cccatctgcg aaattcggat ccagacttcg tcgtccgaca ggcgccggcc

661 gggggtgaag atcatggtgt tgttgtcatc ggtcgggtgc cagtcgatga tcggcatttc

721 gccgaagccc ccctgcagcg tcgcgccgtc ggcgagcttc gcgtccacgc cgacgaccgt

781 catgttgttg tgcagcagcg cggtgtggta gtggtcgatc tgattttcgt agacctgcag 841 ccagttgcag tcgaagacat tggagaacgg gatcaaccgc gttcccttcg gcaacacgta

901 gccgtcgaac accgggaact ctggcctgcg atccgccggc cccatgtagg cgaacaccaa

961 gccgttgcgc tcgaaggccg gataggcgcc ctggcagacg gtttccttca gcttggtgtc

1021 ggggggttcc gccggcgcct ccagcaggct gccgtcgacg tcgaagtgcc aaccgtggta

1081 gcagcaacgg atcccgcgct cctggacgat gccgaactcg agcgaagccc cgcggtgggc 1141 gcacttgcgg tgcagcacgc cgacgttgcc cctgcgatcc ctgaaagcca ccagatcctc

1201 gtgcaggatc cgaatcgcct tgggcacgtc ggtcagctcc tgcgacaggc ataccggctg

1261 ccagtagcgt cgcatgtact cgcccatcgg cgtgccgggg cccgtacgga gaatttccag

1321 gttcgtgggc gcgacattct gcagatctcg cagccggtac ccgctgtagg ctgtgccctg

1381 cccggccgcg gccaaaggct ctctgttacc cat SEQ ID NO: 9 (small UE)

1 tcaaagatca aggacgagcg tcgcgccctt cgagcgcgac acgcaggtcg tcaggaactg

61 cgtgcgtgcc tcggcggaca gcaagtagtc ccgatgctcg acgtcgcccg ccaggtagcg 121 caccttgcac gttccgcaca ggccgctctg gcacgatgtc ggcacttcga tgccgtgctc

181 gccaagcacc tgcgcgatcg acttgtcgtt cggtaccgtc aggaccgttc ccgtgctggc

241 gatcttgatc tggaacccga gcgccagcgc atcgctgccg atgtcgtgag gtactccatc

301 gtcgggaagc accggcgcgc cgacgaagta ctcgaagtgc accgcctcgg cgggccaatt

361 ggtgcaggca cgtgtgcagg cctccatgaa cccggggggg ccgcagtagt agagctgcca 421 acccggctcg taccgccgca gcagcgcagc gatgtccagg cccttggccg gattgccgcc 481 gtcgaagtgc atgatcacgc gccccttggc ggccatcggc gcgagttcct cctggaacgc 541 cgcgtgggcg ctcgacttcg cgcagtagtg cagggtgtag tccgcgccca tcctttccaa 601 ccgatgcacc atggccttca gcggcgtgat gccgatgccg ccggccagca ggacctggtg 661 gggcgcccct tcaatcagcg ggaagttgtt gcgcgggtgt ccgaccgcga cgagccgttg 721 cgtgtgcacg cgttcgtgga tcgcgatgga acccccgcgc ccattgtcgt cgcgcagcac 781 cgcgatcagg tatcgccgac gctcggcggg gtcgttgcac aacgaatact gtcgcacgcg 841 tccgtcgcgg aagtacagat ccacgtgggc cccggcggtg aactcctcca gttcgccgcc 901 gtcctcgcgc acgaattcgt aggcgttgat gccgatcccc tggtaggtga tctgccggac 961 ccgcaggttc agcgccgtct tcgggtactt gccggtgtga ctcaactgat acat

Claims

claims

1 . A process for the oxidation of methyl groups on branched-chain, aliphatic hydrocarbons at the tertiary or secondary carbon atom or in structures derivatized at the C2 atom, characterized in that an enzyme system having the enzymatic activity of a monooxygenase which is a protein having hydroxylase activity and a protein having oxidoreductase activity is used.

2. The method according to claim 1, characterized in that in an aqueous

Reaction solution, the enzyme system having the enzymatic activity of a monooxygenase is incubated with the methyl group-carrying compounds, and then the corresponding converted target compound is recovered, wherein the enzyme system comprises and / or produces a protein having hydroxylase activity and a protein having oxidoreductase activity, containing at least one SEQ ID NO: 1 and SEQ ID NO: 2 and / or at least 50% of which have homologues, and wherein the monooxygenase activity-containing enzyme system is an isolated enzyme, an isolated oligomeric enzyme system and / or a microorganism or a crude extract thereof that has or produces the enzyme system.

3. The method according to claim 1 or 2, characterized in that branched-chain, aliphatic hydrocarbons having methyl groups to be oxidized acyclic compounds and / or aliphatic compounds having functional groups, preferably alkanes, alkenes, alkynes,

Compounds containing a halogen group, alcohols, ethers, thiols, aldehydes, ketones, thioaldehydes, thioketones, carboxylic acids, amines, imines, amides, esters, nitriles, nitro compounds and sulfonic acids.

4. The method according to any one of claims 1 to 3, characterized in that for the oxidation, an enzyme system is used which comprises a protein having the SEQ ID NO: 1 and / or mutants, variants and parts thereof, which have a sequence homology of at least 50% have and have hydroxylase activity and a protein with the SEQ ID NO: 2 and / or mutants, variants and parts thereof, which have a sequence homology of at least 50% and have oxidoreductase activity.

5. The method according to any one of claims 1 to 4, characterized in that an enzyme system is used for the oxidation, which is a large subunit Protein sequence SEQ ID NO: 1 and as a small subunit SEQ ID NO: 2 comprises and their at least 50% homologs having the same properties.

6. The method according to any one of claims 1 to 5, characterized in that an enzyme system is used for the oxidation, which is an oligomeric protein consisting of the sequence SEQ ID NO: 1 and SEQ ID NO: 2.

7. The method according to any one of claims 1 to 6, characterized in that a hydroxylation of methyl groups on alkane compounds or

Alkane compounds having at least one functional group, preferably having 3 to 10 carbon atoms with secondary and tertiary carbon atoms, or to methyl groups in structures that are derivatized at the C2 atom, is performed.

8. The method according to any one of claims 1 to 6, characterized in that the methyl groups on a secondary carbon atom (C2) are oxidized, wherein compounds are preferably used, which after oxidation of the secondary C atom located on the methyl group to compounds with a lead optically active center at the secondary carbon atom.

9. The method according to any one of claims 1 to 6, characterized in that the methyl groups of a compound are oxidized at a tertiary C-atoms.

10. The method according to any one of claims 1 to 6, characterized in that the methyl groups of a compound are oxidized, in which one or 2 H atoms are substituted on the C2 carbon.

1 1 .Method according to one of claims 1 to 10, characterized in that a microorganism selected from bacterial strain HCM-10 (DSM 18028), Ideonella sp. L108 (DSM 18260) or methylibium sp. R8 (DSM 19669) or a crude extract thereof.

12. The method according to any one of claims 1 to 1 1, characterized in that whole cells of the microorganisms are used unchanged, permeabilized or carrier-fixed.

13. The method according to any one of claims 1 to 12, characterized in that the cells of the microorganisms are incubated in the presence of an auxiliary substrate, which is suitable for the provision or for the regeneration of reduction equivalents.

14. The method according to any one of claims 1 to 13, characterized in that are used as auxiliary substrates formic acid or its salts.

15. The method according to any one of claims 1 to 13, characterized in that the auxiliary substrates are heterotrophic substrates which are used by unchanged microorganisms as a carbon and energy source for growth and propagation.

16. The method according to any one of claims 1 to 15, characterized in that the auxiliary substrates as a source for providing or regeneration of

Reduction equivalents are used.

17. The method according to any one of claims 1 to 16, characterized in that the cells are cultured on a heterotrophic substrate for the accumulation of products which are preferably intracellular storage materials and optionally used for the regeneration of reduction equivalents for the monooxygenase reaction.

18. The method according to any one of claims 1 to 17, characterized in that is formed as an intracellular storage material polyhydroxybutyric acid.

19. The method according to any one of claims 1 to 18, characterized in that cell-free crude extracts of the microorganisms are used, preferably cell-free crude extracts of the microorganism DSM18028.

20. The method according to any one of claims 1 to 19, characterized in that the protein with the oxidoreductase activity for the transfer of reduction equivalents from reduced coenzymes to the protein with hydroxylase activity is used.

21. The method according to any one of claims 1 to 20, characterized in that the reduced coenzymes NADH + H ⁺ and NADPH + H ⁺ are.

22. The method according to any one of claims 1 to 21, characterized in that cell extracts and / or the enzyme systems with monooxygenase activity after partial or complete isolation from the microorganisms, optionally in purified form, and optionally with the addition of other enzymes for regeneration of reduction equivalents are used.

23. The method according to any one of claims 1 to 22, characterized in that the microorganism after cloning and expression of the proteins having the SEQ ID NO: 1 and / or SEQ ID NO: 2 is used as an entire cell, unchanged, permeabilized or fixed in support.

24. Bacterial strain Methylibium sp. R8 (DSM 19669).

25. An enzyme system comprising a protein having SEQ ID NO: 1.

26. Enzyme system comprising a protein having SEQ ID NO: 2.

27. Enzyme system which as a large subunit a protein with the SEQ ID NO:

1 and as a small subunit comprises a protein having the SEQ ID NO: 2.

28. An enzyme system as an oligomeric protein, which consists of SEQ ID NO: 1 and SEQ ID NO:

2 exists.

29. Protein with SEQ ID NO: 1.

30. Protein according to claim 29, characterized in that it has the conserved motifs SEQ ID NO: 3 and SEQ ID NO: 4, wherein the conserved motifs SEQ ID NO: 3 for the binding of iron and sulfur derivatives in a Rieske type Cys ₂ His ₂ [2Fe-2S] cluster and SEQ ID NO: 4 and SEQ ID NO: 10, respectively, for the binding of a singular Fe ²⁺ .

31. Protein with SEQ ID NO: 2.

32. Protein according to claim 31, characterized in that the protein has the conserved motifs SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7, wherein the conserved motifs SEQ ID NO: 5 for the binding of FMN or FAD, SEQ ID NO: 6 for the binding of iron and Sulfur derivatives in a plant-specific Cys ₄ [2Fe-2S] cluster and SEQ ID NO: 7 for the binding of ferredoxin.

33. A nucleic acid molecule encoding an enzyme having the activity of a monooxygenase, selected from the group consisting of a) nucleic acid molecules which encode a protein having the amino acid sequences given under SEQ ID NO: 1 and / or SEQ ID NO: 2; b) Nucleic acid molecules which have the nucleotide sequences shown under SEQ ID NO: 8 and / or SEQ ID NO: 9.

34. Nucleic acid molecule according to claim 33, characterized in that it encodes a protein comprising, as an oligomeric enzyme composed of two different subunits, the amino acid sequences given under SEQ ID NO: 1 and SEQ ID NO: 2.

35. Nucleic acid molecule according to claim 33 or 34, which is a DNA molecule.

36. The nucleic acid molecule of claim 35, which is a cDNA or genomic DNA.

37. The nucleic acid molecule of claim 34, which is an RNA molecule.

38. A method for producing an enzyme system having the enzymatic activity of a monooxygenase according to any one of claims 25 to 28 or a protein according to any one of claims 29 to 32, characterized in that the proteins having the SEQ ID NO: 1 and / or SEQ ID NO 2 are cloned and expressed individually or together in a prokaryotic host, and the proteins of SEQ ID NO: 1 and / or SEQ ID NO: 2 are recovered individually or in combination after expression in an appropriate manner.