RU2285044C2 - Novel cytochrome p-450 monooxygenases and their using for oxidation of organic compounds - Google Patents

Abstract

FIELD: biotechnology, biochemistry, enzymes, microbiology.

SUBSTANCE: invention proposes a method for microbiological oxidation of N-, O- or S-heterocyclic mono- or multinuclear aromatic compounds. The method involves culturing the recombinant microorganism that expresses cytochrome P-450-dependent monooxygenase BM-3 from Bacillus megaterium with amino acid sequence represented in SEQ ID NO:2 that comprises at least one functional mutation in region 86-88 and, if necessary, at least one functional mutation in one of regions 73-82, 172-224, 39-43, 48-52, 67-170, 300-335 and 352-356. The prepared oxidation product is isolated from medium, Invention provides carrying out oxidation of organic compounds with enhanced degree of effectiveness.

EFFECT: enhanced effectiveness of enzymes.

17 cl, 1 tbl, 7 ex

Description

The present invention relates to new cytochrome P450 monooxygenases with altered substrate specificity, suitable for the oxidation of organic substrates, for example, N-heterocyclic aromatic compounds encoded for this nucleotide sequence containing this sequence of expression constructs and vectors, and thus transformed microorganisms, a method of microbiological oxidation of various organic substrates, such as N-heterocyclic aromatic compounds and, in particular, ba manufacture of indigo and indirubin.

Enzymes with new functions and properties can be obtained either by screening natural samples or by changing the protein of known enzymes. Under certain circumstances, the latter method may be preferable in obtaining properties that are unbelievable in the way of natural selection. Despite various attempts to create enzymes, there are still few successful studies on stimulating the catalytic activity of enzyme mutants relative to a specific substrate (1-10). In these known cases, the substrates are structurally closely related to the natural substrates of the corresponding enzyme. There are still no reports of the successful creation of enzymes that, after modification, would catalyze the conversion of a compound that is completely different from the natural substrate of the enzyme.

The cytochrome P450 monooxygenase isolated from the Bacillius megaterium bacterium usually catalyzes the subterminal hydroxylation of long chain saturated acids and their corresponding amides and alcohols or the epoxidation of medium chain unsaturated long chain fatty acids (11-13). The optimal chain length of saturated fatty acids is from 14 to 16 carbon atoms. Fatty acids with a chain length of less than 12 are not hydroxylated (11).

The structure of the P450 BM-3 heme domain was determined using x-ray diffraction analysis (14-16). The binding sites of the substrate are in the form of a long tunnel-shaped opening, which goes from the surface of the molecule to the heme molecule and is limited almost exclusively to hydrophobic amino acid residues. Individual charged residues on the surface of the heme domain are Arg47 and Tur51 residues. It is assumed that they participate in the binding of the carboxylate group of the substrate by the formation of a hydrogen bond (14). The mutation of Arg47 to Glu causes the deactivation of the arachidonic acid enzyme (13), but increases its activity with respect to the C ₁₂ -C ₁₄ compounds of alkyltrimethylammonia. The use of a substrate for aromatic compounds, in particular for one-, two- or polycyclic, in particular heterocyclic aromatic hydrocarbons, alkanes, alkenes, cycloalkanes and cycloalkenes, has not been described with respect to this enzyme. Until now, it has been accepted among specialists that organic substrates other than those described here, such as indole, cannot be substrates due to obvious structural differences with the natural substrates P450 VM-3, in particular due to the lack of functional groups, which could bind the above residues in the pocket of the substrate.

Therefore, the objective of the present invention is the preparation of new cytochrome P450 monooxygenases with altered substrate specificity or altered substrate profile. In particular, mutants of mono-oxygenase must be prepared, which, in comparison with the non-mutated natural enzyme, must be enzymatically active with structurally significantly different substrates.

"Modified substrate profile" should be distinguished from the claimed mutants in relation to natural enzymes. For the respective mutants, in particular, an improvement in reactivity is observed, for example, an increase in the specific activity (expressed in nmol of the converted substrate / per minute / k nmol of the P450 enzyme), and / or at least the kinetic parameter selected among K _cat , K _m or K _cat / K _m (for example, at least 1%, from 10% to 1000%, from 10% to 500%, or from 10% to 100%) when converting at least one defined in groups a) to d ) oxidizable compounds. The inventive oxidation reaction encompasses enzymatic-catalytic oxidation of at least one exogenous (i.e., added to the reaction medium) or endogenous (already existing in the reaction medium) compound. In particular, the claimed oxidation reaction encompasses mono- or polyhydroxylation, such as, for example, mono- and / or dihydroxylation with respect to an aliphatic or aromatic C-H group, or epoxidation with respect to a predominantly non-aromatic C = C group. Combinations of the above reactions are also possible. The direct reaction product can be converted, in addition, as part of a non-enzymatic sequential or adverse reaction. Combinations of this kind of enzymatic and non-enzymatic processes are also the subject of the invention.

The abovementioned problems can be solved in an unobvious way by using new cytochrome P450 monooxygenases, which, for example, are capable of oxidizing N-heterocyclic two- or multi-link aromatic compounds.

In particular, the subject of the invention is such monooxygenases in which the substrate-binding region is capable of functionally perceiving new, for example, N-heterocyclic substrates, by local specific mutagenesis.

In a preferred embodiment of the invention, the novel monooxygenases are soluble, i.e. do not exist in membrane bound form and are enzymatically active in this form.

The inventive monooxygenases are mainly derived from cytochrome P450 monooxygenase of bacterial origin, such as, in particular, a derivative of cytochrome P450 monooxygenase BM-3 from Bacillicus megaterium with an amino acid sequence according to SEQ ID NO: 2, which has at least one functional, t .e. contributing to the oxidation of new organic substrates (see, in particular, groups of compounds defined from a) to d), such as, for example, N-heterocyclic one-, two- or multi-link aromatic compounds, a mutation in the region of the amino acid sequence 172-224 (closed F / G region), 39-43 (β-band 1), 48-52 (β-band 2), 67-70 (β-band 3), 330-335 (β-band 5), 352-356 ( β-band 8), 73-82 (spiral 5), 86-88 (spiral 6).

The cytochrome P450 monooxygenase mutants made according to the invention are preferably at least capable of one of the following reactions:

a) oxidizing, if necessary, substituted N-, O-, or S-heterocyclic single-, double- or multi-link aromatic compounds;

b) oxidizing, if necessary, substituted single or multi-link aromatic hydrocarbons;

c) the oxidation of unbranched or branched alkanes and alkenes, and

d) oxidizing optionally substituted cycloalkanes and cycloalkenes.

Preferred monooxygenase mutants have at least one functional mutation, in particular amino acid substitution in at least one of the regions of the sequence 73-82, 86-88 and 172-224. So, for example, Phe87 may be substituted by an amino acid with an aliphatic side chain, such as Ala, Val, Leu, in particular Val, Leu188 may be substituted by an amino acid with an amide side chain, such as Asn or, in particular, Gln; and Ala74 may be replaced by another amino acid with an aliphatic side chain, such as, for example, Val and, in particular, Gly.

Particularly preferred monooxygenase mutants of this type are characterized in that they have at least one or multiple amino acid substitutions:

1) Phe87Val;

2) Phe87Val, Leu188Gln; or

3) Phe87Val, Leu188Gln; Ala74Gly;

as well as their functional equivalents. The numerical value here denotes the position of the mutation; the numerical value is preceded by the initial, the numerical value is the newly introduced amino acid.

"Functional equivalents" or analogues of specifically manifested mutants in this regard are various mutants that subsequently possess the desired specificity of the substrate in the framework of at least one of the aforementioned oxidation reactions a) -d), i.e., for example, with respect to heterocyclic aromatic hydrocarbons and, for example, hydroxyl indole, or subsequently, compared with natural enzymes, they find a "modified substrate profile".

According to the invention, “functional equivalents” also mean mutants that find, at least in one of the above positions of the sequence, other than the specifically named amino acid substitution, but despite this, leading to a mutant, which also, like the specifically named mutants , in comparison with natural enzymes, demonstrate a “modified substrate profile” and catalyze at least one of the above oxidation reactions. The functional equivalent is set, in particular, also when changes in the substrate profile qualitatively coincide, i.e., for example, when identical substrates are transformed at different rates.

"Functional equivalents" encompass, of course, also mutants of P450 monooxygenase, which are accessible by mutating P450 enzymes from other organisms in the same manner as specifically named P450 BM-3. For example, by sequentially comparing regions, homologous regions of sequences can be established. Using modern methods of molecular modeling, then, based on the specific objectives of the invention, it is possible to tackle the equivalent mutations that affect the sample reaction.

"Functional equivalents" also encompass mutants obtained by one or more additions, substitutions, deletions and / or transformations of amino acids, wherein said additional changes can occur at each position of the sequence as long as they lead to a mutant with a modified substrate profile in the above sense.

The substrates of group a) oxidizable according to the invention are optionally substituted heterocyclic single, double or multi-aromatic compounds; in particular, oxidizable or hydroxylated N-, O-, or S-heterocyclic one-, two- or multi-link aromatic compounds. They cover mainly two or three, in particular two, from four to seven, in particular six or five link condensed rings, with at least one, mainly all rings have an aromatic appearance and at least one of the aromatic rings carries from one to three, preferably one N-, O- or S-heteroatom. In the general ring structure, if necessary, there can be one or two other identical or different heteroatoms. Aromatic compounds may also carry from 1 to 5 ring carbon substituents or heteroatoms. Examples of suitable substituents are C ₁ - to C ₄ -alkyl, as methyl, ethyl, n- or i-propyl, or n-, i- or t-butyl, or C ₂ - to C ₄ -alkenyl, as ethenyl , 1-propenyl, 2-propenyl, 1-butenyl, 2-butenyl or 3-butenyl, hydroxyls and halogens such as F, Cl or Br. The aforementioned alkyl or alkenyl substituents, if necessary, may also have a keto or aldehyde group, such as, for example, propan-2-one-3-yl, butan-2-ol-4-yl, 3-buten-2-ol-4 -il. Non-limiting examples of suitable heterocyclic substrates are, in particular, two-unit heterocycles such as indole, N-methylindole and their analogs substituted with one to three above-mentioned substituents of carbon atoms, such as, for example, 5-chloro- or 5-bromo-indole; as well as quinoline and quinoline derivatives, such as, for example, 8-methylquinoline, 6-methylquinoline and quinaldine; benzothiophene and its analogues substituted with one to three of the above-mentioned substituents of carbon atoms.

Substrates of group b) oxidized according to the invention are optionally substituted with single or multi-link aromatic hydrocarbons, such as benzene and naphthalene. Aromatic compounds, if necessary, can be once or repeatedly substituted and, for example, have from 1 to 5 substituents of carbon atoms of the ring. Examples of suitable substituents are C ₁ - to C ₄ -alkyl, like methyl, ethyl, n- or isopropyl, or n-, iso- or tert-butyl, or from C ₂ - to C ₄ -alkenyl, like ethenyl, 1 propenyl, 2-propenyl, 1-butenyl, 2-butenyl or 3-butenyl, hydroxyls and halogens such as F, Cl or Br. The aforementioned alkyl or alkenyl substituents, if necessary, may also have a keto or aldehyde group, such as, for example, propan-2-one-3-yl, butan-2-ol-4-yl, 3-buten-2-ol-4 -il. The aromatic hydrocarbon, if necessary, can be condensed with a four-seven-membered non-aromatic ring. The non-aromatic ring, if necessary, can detect one or two double bonds C = C, one or many times be replaced by the above substituents and, if necessary, carry one or two ring heteroatoms. Examples of particularly useful aromatic hydrocarbons are single-unit, such as cumene, as well as two-unit substrates, such as indene and naphthalene, as well as substituted by one to three of the above-mentioned substituents of carbon atoms.

The substrates of group c) oxidized according to the invention are unbranched or branched alkanes and alkenes with 4-15, mainly 6-12 carbon atoms. Examples include n-butane, n-pentane, n-hexane, n-heptane, n-octane, n-nonane, n-decane, n-undecane and n-dodecane, as well as single or multiple branched analogues these compounds, such as, for example, analogs of compounds with 1-3 lateral methyl groups; or once or repeatedly, for example, once unsaturated analogues of the above alkanes.

Substrates of group d) oxidizable according to the invention are optionally substituted cycloalkanes and cycloalkenes with 4-8 ring carbon atoms. Examples of this kind are cyclopentane, cyclopentene, cyclohexane, cyclohexene, cycloheptane and cycloheptene. The ring structure may contain one or more, for example, from 1 to 5 substituents according to the above definition for compounds of group a) and b). Non-limiting examples for this are ionones, such as α-, β- and γ-ionones, as well as the corresponding methylionones and isomethylionones. Particularly preferred are α- and β-ionones.

The subject of the invention are also nucleic acid sequences encoded for the claimed monooxygenase. Preferred sequences are derived from SEQ ID NO: 1, which has at least one nucleotide substitution leading to one of the functional amino acid mutations described above. The subject of the invention is, in addition, functional analogs of nucleic acids obtained by the addition, substitution, insertion and / or removal of single or several nucleotides, which subsequently encode a monooxygenase with the desired substrate specificity, such as, for example, with indole oxidizing activity.

In accordance with the invention, nucleotide sequences are also included that include the so-called mute mutations, or are altered accordingly to use for encoding a special organism of origin or a host organism compared to the specifically named sequence, as well as previous natural variants. The invention also encompasses varieties of nucleic acid sequences obtained by degeneration of the genetic code (i.e., without changing the corresponding amino acid sequence) or conservative substitution of nucleotides (i.e., the corresponding amino acids are replaced by other amino acids of the same charge, magnitude, polarity and / or solubility), as well as those altered by addition, insertion, inversion or removal of nucleotides, that encode the claimed monooxygenase "with changes profile of the substrate, as well as corresponding complementary sequences.

The subject of the invention is, in addition, expression constructs containing, during genetic control of regulatory nucleic acid sequences, at least one nucleic acid sequence encoding the claimed mutants, as well as vectors spanning at least one of these expression constructs.

The constructs of the invention encompass the 5'-up promoter along the length of the corresponding coding sequence and the 3'-down terminator sequence along the length, as well as, if necessary, other conventional regulatory elements, namely, operatively linked to the coding sequence. By operative binding is meant the sequential arrangement of a promoter, a coding sequence, a terminator, and, if necessary, other regulatory elements of this kind, so that each of the regulatory elements can consistently perform its function in the expression of the coding sequence. Examples of operatively linked sequences are target sequences, as well as a translation amplifier, multiplier, polyadenylation signals, and others. Other regulatory elements include selective marker, gain signals, replicas, and the like.

In addition to artificial regulatory sequences, there may still be a natural regulatory sequence in front of the original structural gene. Through genetic change, this natural regulation can turn off and increase or decrease gene expression. The gene construct can be constructed more simply, which means that no additional regulatory signals can be inserted in front of the structural gene, and the natural promoter with its regulation is not removed. Instead, the natural regulatory sequence is mutated so that no longer needs to be adjusted, and gene expression is increased or decreased. Nucleic acid sequences may be contained in a gene construct in one or many copies.

Examples of useful promoters are: cos-, tac-, trp-, tet-, trp-tet-, lpp-, lac-, lpp-lac-, lacIg-, T7-, T5-, T3-, gal-, trc- , ara-, SP6-, 1-PR or 1-PL-promoters, which are mainly used in gram-negative bacteria, as well as gram-positive promoters amy and SPO2, yeast promoters ADCl, MFa, AC, P-60, CYC1, GAPDH or plant promoters CaMV / 35S, SSU, OCS, lib4, usp, STLS1, B33, nos or ubiquitin or phaseolin promoters. Especially preferred is the use of inducible promoters, such as, for example, light and, especially, thermally induced promoters, such as P _r P _l . Fundamentally, all natural promoters can be used with their regulatory sequences. Based on this, synthetic promoters can also be used to advantage.

Said regulatory sequences should provide targeted expression of nucleic acid sequences and expression of proteins. This, for example, may, depending on the host organism, mean that the gene is expressed or re-expressed only after induction, or it is immediately expressed or re-expressed.

Regulatory sequences, respectively, factors can predominantly positively influence expression and thereby increase or decrease it. Thus, amplification of regulatory elements can be carried out mainly in the transcription plane, and strong transcriptional signals, such as promoters and / or “amplifiers”, are used. Along with this, translation is also possible, and, for example, mRNA stability is improved.

The expression cassette is made by fusing a suitable promoter with a suitable monooxygenase nucleotide sequence, as well as a terminator or polyadenylation signal. To do this, use the available recombination and cloning technique described in T. Maniatis, EFFrinsch, J. Sambrook, Manual Cloning: A Labaratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York (1989), as well as in TJ SIlhavy , MLBerman, LWEnquist, Experiments with Gene fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York (1984), and Ausubel, FM et al .. Current Protocols in Molecular Biology, Greene Publishing Assoc. and Wiley Interscience (1987).

A recombinant nucleic acid construct, respectively, a gene construct is introduced to expand into a suitable host organism in an advantageous manner in the host specific vector, which enables optimal expression of the gene in the host. Vectors are known to those skilled in the art and can be taken from "Cloning Vectors" (Pouwels P.H. et al., Hrsg, Elsevier, Amsterdam-New York-Oxford, 1985). These vectors, in addition to plasmids, should also be understood as all other vectors known to the person skilled in the art, such as phages, viruses such as SV40, CMV (cytomegalovirus), baculovirus and adenovirus, transposons, IS elements (transposable elements), plasmids, cosmids and linear or circular DNA. These vectors can replicate either autonomously in the host organism, or chromosomally.

Using the vectors described in the invention, recombinant microorganisms are produced that are transformed, for example, by at least one vector described in the invention and which can be used to produce mutants. Advantageously, the above-described described inventive recombinant constructs are introduced into a suitable host system and then expressed. In this case, the specialists mainly use known methods of cloning and transfection in order to insert the named nucleic acids into the corresponding expression system for expression. Suitable systems are described, for example, in Carrent Protocols in Molecular Biology, F. Ausubel et al., Hrsg., Wiley Interscience, New York 1997.

As organisms of the host, all organisms that make possible the expression of the claimed nucleic acids, variants of their alleles, their functional equivalents or derivatives are fundamentally suitable. Under the host organisms should be understood, for example, bacteria, fungi, yeast, plant or animal cells. Preferred host organisms are bacteria, such as bacteria from the genus Escherichia (e.g., Escherichia coli), streptomycetes, bacilli or pseudomonads, eukaryotic microorganisms, such as Saccharimyces cerevisiae, Aspergillus, higher-order eukaryotic cells of animal or plant origin, e.g., Sf9 or CHO.

If desired, the gene product can be introduced for expression also in transgenic organisms, such as transgenic animals, in particular in mice, sheep or transgenic plants. In the case of transgenic organisms, we can also talk about the so-called Knock-Out animals or plants in which the corresponding endogenous gene was turned off, for example, by mutation or partial or complete removal.

Selection of successfully transformed organisms is carried out by a marker gene, which is also contained in a vector or expression cassette. An example of such marker genes are genes for resistance to antibiotics and enzymes that catalyze a color reaction that causes staining of transformed cells. The latter can then be selected automatically. Microorganisms successfully transformed with a vector that carry the antibiotic resistance gene (e.g. G418 or hydromycin) can be selected using antibiotic-containing media or nutrient soils. Marker proteins that are present on the surface of cells can be used for selection by affinity chromatography.

The combination of host organisms with vectors suitable for organisms, such as plasmids, viruses or phages, for example plasmids with a polymerase / RNA promoter system, λ, μ phages, or other mild phages or transposons and / or other advantageous regulatory sequences forms an expression system. For example, the term "expression system" is understood to mean a combination of mammalian cells, like CHO cells, and vectors, like the pcDNA3neo vector.

As already mentioned, the gene product can advantageously be introduced for expression in transgenic animals, for example in mice, in sheep or in transgenic plants. It is also possible to program cell-free translation systems with nucleic acid derived RNA.

A further subject of the invention is a method for the manufacture of the claimed monooxygenase, when the microorganism producing the monooxygenase is cultured, if necessary, the expression of the monooxygenase is induced and the monooxygenase is isolated from the culture. The inventive monooxygenase, if necessary, can be produced on an industrial scale.

Microorganisms can be cultured and fermented by known methods. Bacteria can multiply, for example, in a TV or LB medium and at a temperature of from 20 ° C to 40 ° C and a pH value of from 6 to 9. Details of suitable cultivation conditions are described in T. Maniatis, E.F. Frinsch, J. Sambrook, Manual Cloning: A Labaratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York (1989).

In the case when monooxygenase is not released into the culture medium, the cells decompose, and monooxygenase is extracted from the lysate by the method of protein isolation. Cells can be decomposed by choice: high-frequency ultrasound, high pressure, as, for example, in a French pressure cell, by osmolysis, by exposure to detergents, lytic enzymes or organic solvents, using homogenizers, or by a combination of several of the above methods. Monooxygenase purification can be achieved by known chromatographic methods such as molecular sieve chromatography (gel filtration), such as Q-Sepharose chromatography, ion exchange chromatography and hydrophobic chromatography, as well as other conventional methods such as ultrafiltration, crystallization, salting out, dialysis and native gel electrophoresis . Suitable methods are described, for example, in Cooper F.G., Biochemische arbeitsmenhoden, Verlag Walter de Gruyter, Berlin, New York or Scopes R., Protein Purification, Springer Verlag, New York, Heidelberg, Berlin.

It is especially advantageous to use a vector system or oligonucleotides for the isolation of a recombinant protein, which extend the cDNA by a specific nucleotide sequence and thereby encode extended polypeptides or mixed proteins, which serve for easier purification. Appropriate modifications of this kind are, for example, acting as an immobilizer, the so-called "tags", such as, for example, the modification known as the hexahistidine immobilizer or epitope, which can be recognized as an antibody antigen (described, for example, in Harlow E. and Lane D., 1988 Antibodies: A Laboratory Manual. Cold Spring Harbor (NY) Press). These immobilizers can serve to bind the protein on solid supports, for example polymer matrices, which, for example, can be embedded in a chromatographic column, or can be used on a microtiter plate, or other carriers.

At the same time, these immobilizers can be used to recognize proteins. For the recognition of proteins, in addition, conventional markers such as fluorescent dyes, enzyme markers, which, after reaction with the substrate, form a detectable reaction product, or radioactive markers alone or in combination with immobilizers for protein derivation can be used.

The invention also relates to a method for microbiological oxidation of organic compounds, such as, for example, N-heterocyclic one-, two- or multi-link aromatic compounds, which are characterized in that

A1) cultivate the above-defined recombinant microorganism in culture in the presence of an exogenous (externally introduced) or intermediate oxidizing substrate of the inventive monooxygenase, preferably in the presence of oxygen (i.e., aerobically); or

A2) incubate the substrate-containing reaction medium with the claimed enzyme, mainly in the presence of oxygen and an electron donor; and

b) isolate the resulting oxidation product or a sequential product thereof from the medium.

The oxygen required for the conversion is supplied to the reaction medium from ambient air or may be introduced, if required, in a known manner.

Preferably, the oxidizable substrate is selected among

a) optionally substituted N-heterocyclic one-, two- or multi-link aromatic compounds as defined above;

b) optionally substituted single or multi-link aromatic hydrocarbons;

c) unbranched or branched alkanes and alkenes;

d) optionally substituted cycloalkanes and cycloalkenes.

A preferred embodiment of the method is directed to the formation of indigo / indirubin and is characterized in that the substrate is an indole intermediate formed in the culture, and the indigo and / or indirubin formed, which was obtained by oxidation of the intermediate hydroxyindoles, is isolated from the culture medium.

If the oxidation of the present invention is carried out using a recombinant microorganism, the cultivation of the microorganisms is carried out mainly in the presence of oxygen in a complex medium, such as, for example, a TV or LB medium at a cultivation temperature of about 20-40 ° C and a pH value of from 6 to 9, until a sufficient cell density is reached. The addition of exogenous indole is usually not required, since it is formed by the microorganism as an intermediate product. When converting other substrates, however, the addition of an exogenous substrate may be required. In order to better control the oxidation reaction, it is preferable to use an inducible, in particular, temperature-induced promoter. The temperature is raised to the point of induction, for example, 42 ° C with the promoter P _r P _l , it is maintained for a sufficient time, for example, from 1 to 10 or from 5 to 6 hours, for expression of active monooxygenase, and then the temperature is reduced to 30 -40 ° C. In this case, cultivation continues in the presence of oxygen from 12 hours to 3 days. In particular, during the oxidation of indole, the pH value due to the addition of NaOH can increase, for example, to 9-10, which additionally promotes the formation of indigo, respectively, indirubin by oxidation of the enzymatically formed oxidation products of 2- and 3-hydroxyindole in air.

The formation of indigo / indirubin according to the present invention is illustrated by the following reaction scheme:

If the oxidation according to the invention, on the contrary, is carried out using purified or enriched enzyme mutants, then the claimed enzyme is dissolved in an exogenous substrate, for example indole, in a medium (about 0.01-10 mm, or 0.05-5 mm) in the presence of oxygen, at a temperature of from about 10 ° C to 50 ° C, for example from 30 ° C to 40 ° C, and a pH value of from about 6 to 9 (which, for example, is set at 100-200 mm phosphate or TRIS buffer ), as well as in the presence of a reducing agent, wherein the substrate-containing medium contains, rum addition, with respect to oxidizable substrate about 1-100 or 10-100-fold molar excess reducing equivalents. A preferred reducing agent is NADP. Additive reducing agent can, if necessary, be carried out in portions.

In a similar manner, n-hexane, n-octane, n-decane, n-dodecane, cumene, 1-methylindole, 5-Cl or Br-indole, indene, benzotriophene, α, β and γ-ionone, acridine are preferred as substrates , naphthalene, 6-methyl or 8-methylquinoline, quinoline and quinaldine.

For example, the claimed enzymatic oxidation reaction may be carried out under the following conditions:

Substrate Concentration: from 0.01 to 20 mm Enzyme Concentration: 0.1 to 10 mg / ml Reaction temperature: from 10 ° С to 50 ° С pH: from 6 to 8 Buffer: from 0.05 to 0.2 M potassium phosphate or TRIS / HCl Electron donor: preferably added in portions (initial concentration of about 0.1 to 2 mg / ml)

Before starting the reaction, for example, by adding an electron donor (for example, NADPH), a short-term (1-5 minutes) preliminary incubation (approximately 20-40 ° C) can be carried out. The conversion is carried out aerobically, if necessary, with the additional introduction of oxygen.

With the inventive oxidation method, the resulting oxygen or added oxygen is split enzymatically and reductively. The required reducing equivalent is supplied by an added reducing agent (electron donor).

The resulting oxidation product is then separated from the medium and purified in the usual way, such as by extraction or chromatography.

Further subjects of the invention are bioreactors comprising the claimed enzyme or the claimed recombinant organism in immobilized form.

The last subject of the invention relates to the use of the claimed cytochrome P450 monooxygenase or the claimed vector or microorganism for the biological oxidation of a substrate from groups a) -d), in particular, N-heterocyclic one, two or multi-link aromatic compounds and, preferably, for the formation of indigo and / or indirubin.

The invention is described in more detail in light of the following examples.

Example 1

Randomization of special codons P450 VM-3

The experiments were largely carried out as described in (19). Three positions (Phe87, Leu188, and Ala74) were randomized using locally-specific mutagenesis using the Stratagene QuikChange kit (La Jolla, California, USA). The following PCR examples were used in individual positions:

Phe87: 5'-gcaggagacgggttgnnnacaagctggacg-3 '(SEQ ID NO: 3)

5'-cgtccagcttgtnnncaacccgtctcctgc-3 '(SEQ ID NO: 4)

Leu188: 5'-gaagcaatgaacaagnnncagcgagcaaatccag-3 '(SEQ ID NO: 5)

5'-ctggatttgctcgctgnnncttgttcattgcttc-3 '(SEQ ID NO: 6)

Ala74: 5'-gctttgataaaaacttaaagtcaannncttaaatttgtacg-3 '(SEQ ID NO: 7)

5'-cgtacaaatttaagnnnttgacttaagtttttatcaaagc-3 '(SEQ ID NO: 8)

The conditions for PCR were identical for all three positions. In particular, 50 μl of the reaction volume, 17.5 pmol of each example, 20 pmol of the DNA plasmid template, 3 units were used. Pfu polymerase and 3.25 nmol for each dNTP (deoxynucleoside-5'-triphosphate). The PCR reaction was started at 94 ° C / 1 min, and then the following cycle was repeated 20 times: 94 ° C, 1 min; 46 ° C, 2.5 min; 72 ° C, 17 min. After 20 cycles, the reaction was continued for 15 minutes at 72 ° C. After PCR, the DNA template reacted with 20 units. Dpni at 37 ° C for 3 hours. Next, E. coli DH5α cells were transformed. Transformed E. coli DH5α cells were placed on LB agar plates that contained 150 mg / ml ampicillin. After that, 18 hours at 37 ° C, incubation was performed.

Example 2

Expression and purification of P450 VM-3 and its mutants and production of blue pigment

The P450 VM-3 gene and its mutants were experimentally controlled by the strong temperature-induced promoter P _R P _L plasmid pCYTEXP1 in E. coli DH5α, as already described (20). The colonies were taken with a sterile toothed sampler and transferred to microtiter plates with 96 cells containing 200 μl of TB medium and 100 μg / ml ampicillin per cell. After that, incubation was performed all night at 37 ° C. 40 μl of the cell culture of each cell were then transferred to a culture tube that contained 2 ml of TB medium with 100 μg / ml ampicillin. After this, cultivation was carried out for 2 hours at 37 ° C. Then, for induction, the temperature increased to 42 ° C for 6 hours. After this, cultivation continued all night at 37 ° C, whereby a blue pigment was obtained.

Preparative production of the enzyme or blue pigment was carried out on the basis of 300 ml of cell culture (OD _{578 nm} = 0.8-1.0). To isolate the enzyme, the cells were centrifuged for 10 min at 4000 rpm, then resuspended in 0.1 M K _x PO ₄ buffer, pH 7.4. Cells cooled to ice were carefully decomposed using a Branson Sonifiers W25 device (Daitsebach, Germany) at an output power of 80 W by irradiating three times for 2 min each. Suspensions were centrifuged for 20 min at 32750 g. The crude extract was used to determine activity, respectively, to purify the enzyme. The enzyme was purified as described in (21), to which reference is made here. The concentration of the purified enzyme was determined by considering the difference in absorbance at 450 and 490 nm, as already described in (11) using the extinction coefficient of ε = 91 mM ^-1 cm ^-1.

Example 3

Isolation of mutants that produce large quantities of blue pigment

From each position, 100 mutant colonies were allocated, respectively, which were obtained by randomizing the codon of the corresponding position. These colonies were cultured in culture tubes for the production of blue pigment. After washing the cells with water and many slow centrifugation steps (500 rpm), the blue pigment was extracted with dimethyl sulfoxide (DMSO). The solubility of blue pigment in DMSO was greatest. The absorption of the extract was determined at 677 nm. Those mutants that produced the greatest amount of pigment with respect to all mutants of a certain position were used for DNA sequencing (ABI DNA Sequenzierungs-Kit; ABI Prism ^TM 377 DNA Sequencer) and, in addition, were used as a template for locally-specific randomized mutagenesis.

Example 4

Activity Test for Indole Hydroxylation

Indole hydroxylation activity was tested in a solution that contained 8 μl of a 10-500 mM indole solution in DMSO, 850 μl TRIS / HCl buffer (0.1 M, pH 8.2) and 0.6 nmol P450 VM-3 natural or mutants in a final volume of 1 ml. The mixture was preincubated for 9 minutes before the reaction was started by adding 50 μl of an aqueous 1 mM NADP solution. The reaction was stopped after 20 s by adding 60 μl of 1.2 M KOH. Within 5-30 s (under aerobic conditions), the enzyme products were completely converted to indigo ([Δ2,2'-biindolin] -3,3'-dione) and indirubin ([Δ2,2'-biindolin] -3,3 '-dion). Indigo products were determined by their absorption at 670 nm. The calibration curve for pure indigo at this wavelength gives an extinction coefficient of 3.9 mM ^-1 cm ^-1. The linear course of the calibration curve was obtained for the production of indigo through a reaction time of 40 s using 0.6 nmol of the natural type, respectively, of the mutant P450 VM-3 and from 0.05 to 5.0 mM indole. Indirubin has a very weak absorption at 670 nm, and the resulting amounts of indirubin are much less than the amount of idigo. In determining the kinetic parameters, the formation of indirubin was not taken into account. NADP consumption was determined by measuring in the region of 340 nm and calculating using an extinction coefficient of 6.2 mM ^-1 cm ^-1 , as described in (17).

Example 5

Purification of indigo and indirubin

After washing the cells with water and repeated centrifugation at 500 g, the resulting blue residue was extracted with tetrahydrofuran (THF). The extract was evaporated almost to a dry state, and the red pigment was repeatedly extracted with absolute ethanol. The remaining blue solid was dissolved in THF and analyzed by thin layer chromatography (TLC). The ethanol solution was evaporated and purified by silica gel chromatography (DC 60, Merck, Darmstadt, Germany; 2 cm × 30 cm) before it was washed with THF and petroleum ether in a 1: 2 ratio. The resulting red solution was evaporated and analyzed by TLC. The absorption spectra of blue and red pigment were obtained using an Ultraspec 3000 spectrophotometer (Pharmasia, Uppsala, Sweden) in the region from 400 to 800 nm. In addition, the blue and red dye was analyzed using mass spectroscopy and NMR spectroscopy.

Experiment Results

1. Increased production of blue pigment by mutagenesis P450 VM-3

Natural P450 BM-3 does not have the ability to produce blue indigo pigment, respectively, intermediate products - 2-, respectively, 3-hydroxyquinol. To obtain a sufficient amount of blue pigment, P450 BM-3 was subjected to targeted conversion. All mutants that produce the blue pigment have been sequenced. It was found that at least one of the following three positions mutated: Phe87, Leu188 and Ala74. Therefore, it was assumed that these three positions play a decisive role for the activity of P450 VM-3 in the production of blue pigment. From the structure of the heme domain of cytochrome P450 BM-3, complexed with palmitooleic acid, it can be seen that Phe87 protects the substrate from the near shift of the heme group (14). The Phe87Val mutant exhibits high regio- and stereoselectivity during epoxidation of (14S, 15R) -arachidonic acid (13), and the Phe87Ala mutant moves the hydroxylation positions ω-1, ω-2, and ω-3 (22). Position 87 was therefore selected first for locally-specific randomized mutagenesis using PCR. In test cultures, 7 colonies were obtained which, after induction, produced a small amount of blue pigment. The colony that produced the most blue pigment was selected for DNA sequencing. These sequences gave the substitution of Phe87 on Val. The Phe87Val mutant was eventually used as a template on the second round of locally-specific randomized mutagenesis at position Leu188. The structure of the heme domain complexed with palmitooleic acid shows that repositioning of the F and G helices leads the Leu188 residue to direct contact with the substrate (14). Therefore, this position can play an important role in binding or orienting the substrate. After a second screening, 31 colonies were observed that produced blue pigment. The mutant that produced the most pigment contained the substitutions Phe87Val and Leu188Gln. This mutant was eventually mutated at position Ala74 in the third pass of locally-specific randomized mutagenesis. Three-fold F87L188A74 mutants (Phe87Val, Leu188Gln and Ala74Gly) were obtained, which produced many mg of blue pigment in a two-liter flask containing 300 ml of TV medium. This amount was enough to isolate and determine the blue pigment.

2. Isolation and determination of blue pigment

After washing the cells, the blue residue was extracted with THF and analyzed by thin layer chromatography. The blue pigment was divided into fast moving blue components and slowly moving red components. Both components have the same mobility parameters as the commercial indigo sample components.

After purification, the absorption spectra of both components in DMSO were recorded. The blue component has the same spectrum as a commercial indigo sample. The purified blue and red components, respectively, were analyzed using mass spectrometry. The mass spectra of both pigments have a strong ion-molecular peak at m / z = 262 and 2 fragment peaks at m / z = 234 and 205 (relative intensity, respectively, 10%). This sample is typical of indigo compounds. The elemental composition of these ions was determined using high resolution mass spectroscopy as C ₁₆ H ₁₀ N ₂ O ₂ , C ₁₅ H ₁₀ N ₂ O, respectively, C ₁₄ H ₉ N ₂ . This is also characteristic of structures of indigo types. Thus, blue pigment was identified as indigo, and red as indirubin. To confirm the structure, the NMR of both pigments in a DMSO-D ₆ solution was checked. The results are consistent with published data.

3. Indigo production with isolated enzymes

It is known that indigo can be obtained from indole by microbiological transformation (24-26). However, none of these microbiological systems contains P450 monooxygenase. According to the invention, first of all, the catalytic activity of a pure enzyme with respect to indole must be determined. The mutant F87L188A74 was mixed with indole. No color reaction was observed. Only after adding NADP to the reaction mixture after about 20 minutes did a blue pigment form. By adjusting the pH of the reaction mixture to 11, 30 seconds after the addition of NADP, a blue color became visible within a few seconds. Control experiments using natural P450 VM-3 were always negative, even when using elevated concentrations of the enzyme, indole, and NADP. The blue pigment was extracted with ethyl acetate and analyzed by thin layer chromatography. The blue pigment is again divided into a rapidly advancing blue and slowly advancing red component. X-ray fluorescence values and absorption spectra were identical to the same values as in the fermentation broth extract. Mutants F87L188A74 P450 BM-3 are indole hydroxylase.

So far, only 2 pathways of enzymatic transformation of indole into indigo have been described. One pathway catalyzes with the help of dioxygenase, the second one - styrene monooxygenase (24, 25). The stoichiometry of NADP in both cases is 2. Therefore, it was accepted that, in contrast to dioxigenase, the claimed indole mutants F87L188A74 are hydroxylated in only one position to form oxindole (2 hydroxyindole) or indoxyl (3-hydroxyindole).

4. Kinetic parameters of indole hydroxylation

To determine the kinetic parameters of indole hydroxylation, pure samples of the natural enzyme P450 BM-3 and mutants Leu188Gln, Phe87Val, F87L188, and F87L188A74 were used. The results are presented in the following Table 1.

Table 1.
Kinetic parameters of hydroxylation of indole mutants P450 VM-3 Mutants K _cat (c ^-1 ) K _m (mm) K _cat / K _m (M ^-1 s ^-1 ) Wt - ^a) - - Leu188Gln but. ^b) but. but. Phe87val 2.03 (0.14) 17.0 (1.0) 119 F87L188 2.28 (0.16) 4.2 (0.4) 543 F87L188A74 2.73 (0.16) 2.0 (0.2) 1365 ^{a) no} activity was observed
^b) not determined (activity was too small to be measurable)

Even with an excess of purified enzyme and a high concentration of indole, the natural enzyme is not able to oxidize indole. The Leu188Gln mutant exhibits negligible activity. The Phe87Val mutant has a catalytic activity of 119 M ^-1 s ^-1 for hydroxylation of indole. The catalytic efficiency of the F87L188 double mutants (Phe87Val, Leu188Gln) was increased to 543 M ^-1 s ^-1 and was increased due to the introduction of other Ala74Gly substitutions to 1365 M ^-1 s ^-1 . K _cat values increase from Phe87Val to triplicate mutants by about 35%, while K _m values decrease by about 7 times. This indicates that Ala74Gly and Leu188Gln are involved in substrate binding.

The reversibility fraction of indole (K _cat = 2.73 s ^-1 ) for the three-fold F87L188A74 mutants is more than 10 times higher than for most P450 enzymes (18).

Example 6

Hydroxylation of n-octane with modified cytochrome P450 monooxygenase

The transformation was carried out using the mutant monooxygenase P450 BM-3, which contains the following mutations: Phe87Val, Leu188Gln, Ala74Gly.

N-octane was chosen as a substrate. The following reaction set was used for the hydroxylation of n-octane:

mutants P450 VM-3: 17.5 mg (lyophilisate) buffer: 961 ml (potassium phosphate buffer, 50 mM, pH 7.5) substrate: 50 μl of a solution of 60 mm (in acetone) temperature: 25 ° C

The enzyme lyophilisate was dissolved in 500 μl of buffer and immediately with the substrate and buffer was incubated for 5 min at room temperature. Then followed by the addition of 300 μl of a NADP solution (5 mg / ml). The addition of NADP was repeated twice. The progress of the reaction was monitored by measuring absorbance at 340 nm, so that a decrease in NADP could be observed. In this case, 300 μl was added to NADP, since a high concentration of NADP leads to deactivation of the enzyme. Then, to isolate the product, the reaction solution was extracted 3 times with 5 ml of diethyl ether. The combined organic phases were dried with MgSO ₄ and collected. The products were then analyzed by thin layer chromatography (DC), gas chromatography with mass spectroscopic completion (GC / MS) and NMR.

GC / MS - analysis of the reaction mixture gave the following results:

Compound Rt (min) ¹⁾ Conversion (%) 4-octanol 13.51 37 3-octanol 14.08 47 2-octanol 14.26 16 ¹⁾ Temperature program: 40 ° С 1 min isotherms / 3 ° С / min 95 ° С / 10 ° С / min 275 ° С; Finnigan MAT 95 hardware; GC: HP 5890 Series II Slit Injector; column: HP-5MS (methylsiloxane) 30 m × 0.25 mm; carrier gas: 0.065 ml / min He.

An extraction product was not found.

Example 7

Hydroxylation of aromatic hydrocarbons, heteroaromatic hydrocarbons, trimethylcyclohexenyl compounds

a) Example 6 was repeated, but instead of n-octane, naphthalene was taken as a substrate. The products were 1-naphthanol and cis-1,2-dihydroxy-1,2-dihydronaphthalene. Introduced naphthalene was converted by 88%.

Analysis of naphthalene reactions

Gc :

Instrumentation: Carlo Erba Strumentazion type HRGC 4160 on a column injector; column: DB5 30 m × 0.2 mm; material: 5% diphenyl - 95% dimethylpolysiloxane; carrier gas: 0.5 bar H ₂ ; temperature program: 40 ° С 1 min isotherms / 10 ° С / min up to 300 ° С; Rt (1-naphthanol) = 16.68.

NMR:

1-naphthanol and cis-1,2-dihydroxy-1,2-dihydronaphthalene were identified.

b) Example 6 was repeated, however, instead of n-octane, 8-methylquinoline was taken as a substrate. As the main product, 5-hydroxy-8-methylquinoline was identified, among other derivatives (product ratio 5: 1). The introduced product was converted by 35%.

c) Example 6 was repeated, however, instead of n-octane, α-ionone was taken as a substrate. As the main product, 3-hydroxy-α-ionone was identified, among other derivatives (product ratio 76:24). The introduced product was converted by 60%.

d) Example 6 was repeated, however, instead of n-octane, cumene (iso-propylbenzene) was taken as a substrate. Five monohydroxide products and one dihydroxide product were identified. The introduced product was converted by 70%.

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Claims (20)

1. The method of microbiological oxidation of N-, O- or S-heterocyclic mono- or multinuclear aromatic compounds, characterized in that A1) cultivate a recombinant microorganism that expresses cytochrome P450-monooxygenase of bacterial origin in a culture medium in the presence of an exogenous or intermediate forming substrate; or A2) incubate the substrate-containing reaction medium with cytochrome P450-monooxygenase of bacterial origin; b) isolate the resulting oxidation product or its derivative from the medium, moreover, monooxygenase is a derivative of cytochrome P450-monooxygenase BM-3 from Bacillus megaterium with the amino acid sequence according to SEQ ID NO: 2, which has at least one functional mutation in the region of amino acid sequence 86-88 and, if necessary, additionally, at least one functional mutation in one of the regions of the amino acid sequence 73-82, 172-224, 39-43, 48-52, 67-70, 330-335 and 352-356. 2. The method according to claim 1, characterized in that the exogenous or intermediate-forming substrate is selected among unsubstituted or substituted N-, O- or S-heterocyclic mono- or multinuclear aromatic compounds. 3. The method according to claim 1 or 2, characterized in that they use a mutant having at least the following single or multiple amino acid substitutions: 1) Phe87Val; 2) Phe87Val, Leu188Gln; or 3) Phe87Val, Leu188Gln; Ala74Gly. 4. The method according to one of claims 1 to 3, characterized in that as an exogenous substrate, at least one compound from the group of unsubstituted or substituted N-, O- or S-heterocyclic mono-, dual- or multicore aromatic compounds is added into the medium, and the oxidation is carried out by enzymatic conversion of the substrate-containing medium in the presence of oxygen at a temperature of from about 20 ° C to 40 ° C, and a pH value of from about 6 to 9, and the medium containing the substrate contains, in addition, about 10-100 molar zbytok reducing equivalents. 5. The method according to claim 4, characterized in that as an exogenous substrate use a compound selected among the following: indole, 1-methylindole, 5-chloro or bromo-indole, indene, benzothiophene, acridine, 6-methyl- or 8 -methylquinoline, quinoline and quinaldine. 6. The method of microbiological oxidation of unsubstituted or substituted mono- or multinuclear aromatic hydrocarbons, linear or branched alkanes and alkenes, or unsubstituted or substituted cycloalkanes and cycloalkenes, characterized in that A1) cultivate a recombinant cytochrome P450 monooxygenase producing microorganism in a culture medium in the presence of an exogenous or intermediate forming substrate; or A2) incubate the substrate-containing reaction medium with cytochrome P450 monooxygenase capable of oxidizing the aforementioned compounds; and b) isolate the resulting oxidation product or its derivative from the medium, moreover, monooxygenase is capable of oxidizing the aforementioned compounds and is a derivative of cytochrome P450-monooxygenase BM-3 from Bacillus megaterium with the amino acid sequence according to SEQ ID NO: 2, which has at least one functional mutation in the region of amino acid sequence 86-88 and if necessary, additionally has at least one functional mutation in one of the regions of the amino acid sequence 73-82, 172-224, 39-43, 48-52, 67-70, 330-335 and 352-356. 7. The method according to claim 6, characterized in that the exogenous or intermediate-forming substrate is selected among b) unsubstituted or substituted mono- or multicore aromatic hydrocarbons; c) linear or branched alkanes and alkenes; d) unsubstituted or substituted cycloalkanes and cycloalkenes. 8. The method according to claim 6 or 7, characterized in that they use a mutant having at least the following single or multiple amino acid substitutions: 1) Phe87Val; 2) Phe87Val, Leu188Gln; or 3) Phe87Val, Leu188Gln; Ala74Gly. 9. The method according to one of claims 6 to 8, characterized in that at least one compound is selected as an exogenous substrate from the groups of unsubstituted or substituted mono- or multinuclear aromatic hydrocarbons defined above, linear or branched alkanes or alkenes and unsubstituted or substituted cycloalkanes and cycloalkenes are added to the medium, and oxidation is carried out by enzymatic conversion of the substrate containing medium in the presence of oxygen at a temperature of from about 20 ° C to 40 ° C and a pH value of from about 6 to 9, pr than the substrate containing medium contains, in addition, with respect to the substrate about 10-100-fold molar excess of reduction equivalents. 10. The method according to claim 9, characterized in that as an exogenous substrate using a compound selected among the following: n-hexane, n-octane, n-decane, n-dodecane, cumene, α-, β- and γ-ionone and naphthalene. 11. A method of microbiological production of indigo and / or indirubin, characterized in that a1) cultivates a recombinant microorganism producing an indole oxidizing cytochrome P450 monooxygenase in a culture medium in the presence of an exogenous or intermediate indole; or a2) the indole-containing reaction medium is incubated with cytochrome P450 monooxygenase oxidizing indole, and b) the resulting oxidation product or its sequential product is isolated from the medium, the monooxygenase being a derivative of cytochrome P450 monooxygenase BM-3 from Bacillus megaterium according to the amino acid sequence SEQ ID NO: 2, which has at least one functional mutation in the region of amino acid sequence 86-88 and, if necessary, additionally has at least one functional mutation in one regions of amino acid sequence 73-82, 172-224, 39-43, 48-52, 67-70, 330-335 and 352-356. 12. The method according to claim 11, characterized in that the resulting indigo and / or indirubin obtained by oxidation of the intermediate indole is isolated from the medium. 13. The method according to p. 12, characterized in that the oxidation of indole is carried out by culturing the microorganism in the presence of oxygen at a temperature of about 20-40 ° C and a pH value of from 6 to 9. 14. The method according to one of claims 11 to 13, characterized in that a mutant is used having at least the following single or multiple amino acid substitutions: 1) Phe87Val; 2) Phe87Val, Leu188Gln; or 3) Phe87Val, Leu188Gln; Ala74Gly. 15. Cytochrome P450-monooxygenase, which is capable of at least one of the following reactions: a) oxidation of unsubstituted or substituted N-, O-, or S-heterocyclic mono-, dual- or multicore aromatic compounds; b) the oxidation of unsubstituted or substituted mono- or multicore aromatic hydrocarbons; c) the oxidation of linear or branched alkanes and alkenes; and d) the oxidation of unsubstituted or substituted cycloalkanes and cycloalkenes, moreover, monooxygenase is a derivative of cytochrome P450-monooxygenase BM-3 from Bacillus megaterium with an amino acid sequence according to SEQ ID NO: 2, which has at least one functional mutation in the region of amino acid sequence 86-88 and at least one functional a mutation in one of the regions of the amino acid sequence 73-82 and 172-224 and, if necessary, additionally has one functional mutation in one of the regions of the amino acid sequence 39-43, 48-52, 67-70, 330-335 and 352-356. 16. Monooxygenase according to claim 15, characterized in that it has at least one functional mutation in the region of amino acid sequence 86-88 and has at least one functional mutation in one of the regions of amino acid sequence 73-82 and 172 -224. 17. Monooxygenase according to claim 15, characterized in that it has at least multiple amino acid substitutions: a) Phe87Val, Leu188Gln; or b) Phe87Val, Leu188Gln; Ala74Gly, as well as their functional equivalents, which are capable of at least one of the above oxidation reactions. Priority on points: 07.27.1999 - p.15 of the claims in the part of Cytochrome P450-monoxygenase, which is capable of at least one of the reactions under subparagraphs a) - and d), except for the oxidation of O-heterocyclic aromatic compounds, and which has a functional mutation at the position amino acid sequence 87 and at least one functional mutation at one of the positions of amino acid sequence 74-188 and, if necessary, additionally has a functional mutation at the position of amino acid sequence 354; claim 16 in the Cytochrome P450 monooxygenase part, which is capable of at least one of the reactions according to subparagraphs a) -d) claim 15, except for the oxidation of O-heterocyclic aromatic compounds, and which has a functional mutation at the amino acid position sequences 87 and at least one functional mutation at one of the positions of amino acid sequence 74 and 188; 17 of the claims in the Cytochrome P450 monooxygenase part according to subparagraph a) or b), which is capable of at least one of the reactions of subparagraphs a) -d) of paragraph 15 of the formula, except for the oxidation of O-heterocyclic aromatic compounds. 01/18/1999 - claims 1-5 of the claims in terms of microbiological oxidation of N-heterocyclic multinuclear aromatic compounds; PP.11-14 claims; p. 15 of the claims in the part of Cytochrome P450 monooxygenase, which is capable of oxidizing N-heterocyclic two- or multicore aromatic compounds according to subparagraph a), with the exception of Cytochrome P450 monooxygenase, which has a functional mutation at the amino acid sequence 87 and at least at least one functional mutation at one of the positions of amino acid sequence 74 and 188 and, if necessary, additionally has a functional mutation at the position of amino acid sequence 354; p. 16 of the claims in the part of Cytochrome P450 monooxygenase, which is capable of oxidizing N-heterocyclic two- or multicore aromatic compounds according to subparagraph a) of clause 15 of the formula, with the exception of Cytochrome P450 monooxygenase, which has a functional mutation at the amino acid sequence position 87 and at least one functional mutation in one of the positions of the amino acid sequence 74 and 188. 03/20/2000 according to the filing date of DE 10014085.8, filed with the German Patent Office - claims 1-5 of the claims in terms of microbiological oxidation of O- or S-heterocyclic mono- or multicore aromatic compounds and oxidation of N-heterocyclic mononuclear aromatic compounds; claims 6-10 of the claims; p. 15 of the claims in the part of Cytochrome P450-monooxygenase, which is capable of at least one of the reactions under subparagraphs a) -d), with the exception of Cytochrome P450-monooxygenase, which is capable of at least one of the reactions of subparagraphs a ) -d), in addition to the oxidation of O-heterocyclic aromatic compounds, and which has a functional mutation at the position of amino acid sequence 87 and at least one functional mutation at one of the positions of amino acid sequence 74 and 188 and, if necessary, additionally has a function an ionic mutation at the position of amino acid sequence 354 and with the exception of Cytochrome P450 monooxygenase, which is capable of oxidizing N-heterocyclic bicarbonyl or multinuclear aromatic compounds according to subparagraph a); claim 16 in the Cytochrome P450 monooxygenase part, which is capable of at least one of the reactions according to subparagraphs a) -d) claim 15, except for Cytochrome P450-monooxygenase, which is capable of at least one of reactions according to subparagraphs a) to d) of clause 15 of the formula, in addition to the oxidation of O-heterocyclic aromatic compounds, and which has a functional mutation at the position of amino acid sequence 87 and at least one functional mutation at one of the positions of amino acid sequence 74 and 188 and except for cytochro m P450-monooxygenase, which is capable of oxidizing N-heterocyclic binders or multicore aromatic compounds according to subparagraph a); 17 a claim in the Cytochrome P450 monooxygenase portion according to subparagraph a) or b), which is capable of oxidizing O-heterocyclic aromatic compounds.

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