WO2009037216A1 - Procédé d'oxydation de groupes méthyle dans des hydrocarbures aliphatiques par utilisation d'un système enzymatique ayant l'activité d'une mono-oxygénase - Google Patents

Procédé d'oxydation de groupes méthyle dans des hydrocarbures aliphatiques par utilisation d'un système enzymatique ayant l'activité d'une mono-oxygénase Download PDF

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WO2009037216A1
WO2009037216A1 PCT/EP2008/062216 EP2008062216W WO2009037216A1 WO 2009037216 A1 WO2009037216 A1 WO 2009037216A1 EP 2008062216 W EP2008062216 W EP 2008062216W WO 2009037216 A1 WO2009037216 A1 WO 2009037216A1
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seq
protein
enzyme system
activity
compounds
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PCT/EP2008/062216
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Roland H. MÜLLER
Uta Breuer
Martin Von Bergen
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Helmholtz-Zentrum Für Umweltforschung Gmbh - Ufz
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Priority to EP08804177A priority Critical patent/EP2205726A1/fr
Publication of WO2009037216A1 publication Critical patent/WO2009037216A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
    • C12N9/0077—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14) with a reduced iron-sulfur protein as one donor (1.14.15)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/62—Carboxylic acid esters
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales

Definitions

  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • MTBE methyl tert-butyl ether
  • ETBE ethyl-tert-butyl ether
  • TAME te / t-amyl methyl ether
  • AIkB the typical and widely used n-alkane monooxygenase
  • 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.
  • 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.
  • 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.
  • 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.
  • HFC hydrogen fluoride
  • CHC CHC, BKW, IKW
  • hydroxy group alcohols
  • alkoxy group Ethers
  • thiol group carbonyl group (aldehydes, ketones)
  • thiocarbonyl group thioaldehydes, thi
  • 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.
  • 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.
  • 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.
  • the enzyme system consists of SEQ ID NO: 1 and SEQ ID NO: 2 as oligomeric protein ,
  • 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.
  • 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.
  • 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).
  • 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.
  • 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).
  • the variants may be synthetically produced sequences. These variants have certain common characteristics, e.g.
  • Enzyme activity 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.
  • 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
  • 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
  • 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.
  • the methyl groups on a secondary carbon atom 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.
  • 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.
  • 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.
  • these are heterotrophic substrates that can be used by unchanged microorganism as a carbon and energy source for growth and propagation.
  • 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
  • PHB Polyhydroxybutyrate
  • 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.
  • the auxiliary substrate used is formic acid or its salts.
  • 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.
  • 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.
  • 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.
  • the said proteins can also be used in enriched, isolated or synthetically produced form.
  • 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.
  • 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.
  • the method can also be carried out as a semicontinuous or batch process.
  • 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.
  • 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 ⁇ g / 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.
  • TBA t-butyl alcohol
  • 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.
  • 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-
  • 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.
  • 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.
  • 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.

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Abstract

La présente invention concerne un procédé enzymatique d'oxydation de groupes méthyle situés dans des hydrocarbures aliphatiques ainsi que de nouvelles protéines et de nouveaux mélanges enzymatiques ayant l'activité enzymatique d'une mono-oxygénase. Selon l'invention, de préférence des composés comprenant des groupes méthyle situés dans des hydrocarbures aliphatiques à chaîne ramifiée, sont oxydés, lesdits hydrocarbures étant en particulier des composés acycliques et/ou des composés aliphatiques comprenant des groupes fonctionnels ou des composés comprenant des groupes méthyle au niveau d'hydrocarbures aliphatiques mono- ou substitués au niveau de l'atome C2, les substituants pouvant être identiques ou différents. L'invention concerne de préférence un processus biotechnologique d'oxydation de groupes méthyle contenus dans des structures à chaîne ramifiée, des micro-organismes qui possèdent ou produisent l'activité de mono-oxygénase souhaitée, étant cultivés dans un système aqueux et convertis en produits cibles correspondants.
PCT/EP2008/062216 2007-09-17 2008-09-15 Procédé d'oxydation de groupes méthyle dans des hydrocarbures aliphatiques par utilisation d'un système enzymatique ayant l'activité d'une mono-oxygénase WO2009037216A1 (fr)

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DE102007045092A DE102007045092A1 (de) 2007-09-17 2007-09-17 Enzymsystem mit der Aktivität einer Monooxygenase und Verfahren zur Oxidation von Methylgruppen in aliphatischen Kohlenwasserstoffen

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