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

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

 

The present invention relates to a new cytochrome P450-monooxygenases with an altered substrate specificity, suitable for oxidation of organic substrates, such as N-heterocyclic aromatic compounds encoded by this nucleotide sequences containing this sequence expression constructs and vectors, and thereby transformed microorganisms, methods of microbiological oxidation of various organic substrates, such as N-heterocyclic aromatic compounds and, in particular, the method of manufacturing Indigo and indirubin.

Enzymes with new functions and properties can be obtained, or by screening of natural samples or by modifying a protein of known enzymes. Under certain circumstances, the latter method may be preferable in obtaining properties that are incredible in the way of natural selection. Despite various attempts to create enzymes, still few good studies on the stimulation of catalytic activity of the mutant enzymes relative to a specific substrate (1-10). In these known cases, the substrates are structurally closely related to the natural substrates of the respective enzyme. So far there are no reports about the successful creation of enzymes that after modification catalyzed the transformation is unity, which is completely different from the natural substrate of the enzyme.

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

The structure of the heme-domain of P450 BM-3 was determined by x-ray analysis (14-16). Binding sites of the substrate are in the form of long channelvideo opening from the surface of the molecule goes to the heme-molecule and is limited almost exclusively hydrophobic amino acid residues. Individual charged residues on the surface of the heme domain are remnants of Arg47 and Tug. It is assumed that they are involved in binding the carboxylate group of the substrate through the formation of hydrogen bonds (14). Mutation Arg47 to Glu causes deactivation of the enzyme of arachidonic acid (13), but increases its activity against With12-C14connections of alkyltrimethylammonium. The use of the substrate for aromatic compounds, in particular for one-, two - or polycyclic, in particular, heterocyclic aroma is practical hydrocarbons, alkanes, alkenes, cycloalkanes and cycloalkenes regarding this enzyme has not been described. Still among experts it was decided that other than described here, the organic substrates, such as indole, may not be substrates for the obvious reason of structural differences with the natural substrates of P450 BM-3, in particular, due to the lack of functional groups that could link above remains in the pocket of the substrate.

Therefore, the present invention is to prepare new cytochrome P450 monooxygenases with an altered substrate specificity or a modified profile of the substrate. In particular, should be prepared mutants mono-oxygenase that in comparison with neutrophase natural enzyme should be enzymatically active with structurally significantly different substrates.

"The changed profile of the substrate" should be distinguished from the claimed mutants relative to natural enzymes. For the corresponding mutants is observed, in particular, the improvement of the reactivity, for example the increase of the specific activity (expressed as nmol of converted substrate/min/nmol P450 enzyme), and/or, at least, a kinetic parameter, selected among the Kcat, Kmor Kcat/Km(for example, at least 1%, from 10% to 1000%, from 10% to 500% or from 10% to 100%) when the mo is a cation, at least one identified in groups of a) to d) oxidizable compounds. Declare the oxidation reaction comprises enzyme-catalytic oxidation of at least one exogenous (i.e. added to reaction medium) or endogenous (already present in the reaction medium) compound. In particular, we state the oxidation reaction comprises mono - or polyhydroxyalkane, as, for example, mono - and/or dihydroxypropane towards aliphatic or aromatic C-H group, or epoxidation in relation to the predominantly non-aromatic C=C-group. Possible combinations of the above reactions. The immediate reaction product may be converted, in addition, in the framework of sequential or non-enzymatic reactions. The combination of such enzymatic and non-enzymatic processes are also the subject of invention.

The above tasks in a non-obvious way can be solved by using a new cytochrome P450-monooxygenases, which, for example, is able to oxidize N-heterocyclic two - or multi-tier aromatic compounds.

In particular, the subject invention are monooxygenase, in which the substrate binding region is capable of through local-specific mutagenesis functionally to perceive new, for example, N-heterocyclics is their substrates.

In a preferred form of execution of the invention the new monooxygenase soluble, i.e. do not exist in a membrane-bound form and in this form enzymatically active.

The claimed monooxygenase mainly derived from cytochrome P450-monooxygenase of bacterial origin, such as, in particular, the derivative of cytochrome P450-monooxygenase cm-3 of Bacillicus B. megaterium with the amino acid sequence according to SEQ ID NO:2, which has at least one functional, i.e. contributing to the oxidation of organic substrates (see, in particular, defined from a) to d) group of compounds), such as N-heterocyclic one-, two - or multi-tier aromatic compounds, mutation in the region of amino acid sequence 172-224 (closed area F/G), 39-43 (βlane 1), 48-52 (βlane 2), 67-70 (βlane 3), 330-335 (βlane 5), 352-356 (βlane 8), 73-82 (helix 5), 86-88 (helix 6).

Manufactured according to the invention mutants of cytochrome P450-monooxygenase preferably at least able to one of the following reactions:

a) oxidation if necessary substituted by N-, O-or S-heterocyclic, one -, two - or multi-tier aromatic compounds;

b) oxidation, if necessary, substituted single or multi-tier aromatic hydrocarbons;

c) oxidation unbranched Il is branched alkanes and alkenes, and

d) oxidation, if necessary, substituted cycloalkanes and cycloalkenes.

Preferred mutants of monooxygenase have at least one functional mutation, in particular, the substitution of amino acids at least one of the areas of sequence 73-82, 86-88 and 172-224. For example, Phe87 may be substituted by amino acids with aliphatic side chain, such as Ala, Val, Leu, in particular Val, Leu188 may be substituted amino acid with amide side chain, such as Asn or, in particular, Gln; and l74 may be replaced by another amino acid with an aliphatic side chain, such as Val and, in particular, Gly.

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

1) Phe87Val;

2) Phe87Val, Leu188Gln; or

3) Phe87Val, Leu188Gln; Ala74Gly;

as well as their functional equivalents. The numeric value refers to the position of the mutation; before the numerical value is initial, for the numerical value of new amino acid.

"Functional equivalents" or analogs of the specifically manifested mutants in this regard, various mutants, which possess the desired specificity of the substrate within at least one of the above oxidation reactions (a)-(d), i.e, for example, in relation to the heterocycle is ical aromatic hydrocarbons and for example, gidroksilnuyu indole, or in the future in comparison with natural enzymes find "the changed profile of the substrate".

Under the "functional equivalents" are understood in accordance with the invention also mutants that detect at least one of the above positions sequence other than specifically mentioned the substitution of amino acids, but in spite of this, resulting in the mutant, which is also exactly how these mutants, compared with natural enzymes demonstrate "changed profile of the substrate and catalyze at least one of the above oxidation reactions. A functional equivalent is defined, in particular, when changes in the profile of the substrate qualitatively the same, ie, for example, when the same substrate is converted with a different speed.

"Functional equivalents" includes, of course, also mutants of P450-monooxygenase that are available by mutation of P450 enzymes from other organisms in the same way, as specifically called P450 BM-3. For example, by successive comparison of the fields you can set the homologous regions of the sequences. Using modern methods of molecular modeling can then, based on the specific objectives of the present invention, to be equivalent affecting the sample reaction Muta the AI.

"Functional equivalents" also cover the mutants obtained by one or more additions, substitutions, deletions and/or transformations of amino acids, and called for more changes can occur in each position in the sequence, until they lead to a mutant with a modified profile of the substrate in the above sense.

Oxidized according to the invention, the substrates of the group and, if necessary, are substituted heterocyclic, one -, two - or multi-tier aromatic compounds; in particular, oxidized or hydroxytyramine N-, O-or S-heterocyclic, one -, two - or multi-tier aromatic compounds. They cover mainly two or three, in particular two, from four to seven, in particular six, or five-condensed ring, and at least one, substantially all of the rings are aromatic and at least one of the aromatic rings carries from one to three, mainly one N-, O - or S-heteroatom. In General the circular structure can be, if necessary, one or more identical or different heteroatom. Aromatic compounds can also carry 1 to 5 substituents of the ring carbon or heteroatoms. Examples of suitable substituents are from C1up With4-alkyl, the AK methyl, ethyl, n - or i-propyl, or n-, i - or t-butyl, or from C2up With4alkenyl as ethynyl, 1-propenyl, 2-propenyl, 1-butenyl, 2-butenyl or 3-butenyl, hydroxyl and halogen, as F, Cl or Br. Named alkalemia or alkenilovyh alternates, if necessary, may also have keto - or aldehyde group, such as, for example, propane-2-on-3-yl, butane-2-ol-4-yl, 3-butene-2-ol-4-yl. Non-limiting examples of suitable heterocyclic substrates are, in particular, two heterocycles, as indole, N-methylindole and their analogs, substituted from one to three videopreteen substituents of the carbon atoms, as, for example, 5-chloro - or 5-bromo-indole, and quinoline and quinoline derivatives, such as 8-methylinosine, 6-methylinosine and heraldin; benzothiophen and its analogs, substituted from one to three videopreteen substituents of the carbon atoms.

Oxidized according to the invention, the substrates of group b), if necessary, are replaced by one - or multi-tier aromatic hydrocarbons as benzene and naphthalene. Aromatic compounds, if necessary, can be substituted one or more times and, for example, be from 1 to 5 substituents of the carbon atoms of the ring. Examples of suitable substituents are from C1up With4-alkyl, like methyl, ethyl, n - or isopropyl or n-, ISO - or tert-Buti is, or from C2up With4alkenyl as ethynyl, 1-propenyl, 2-propenyl, 1-butenyl, 2-butenyl or 3-butenyl, hydroxyl and halogen, as F, Cl or Br. Named alkalemia or alkenilovyh alternates, if necessary, may also have keto - or aldehyde group, such as, for example, propane-2-on-3-yl, butane-2-ol-4-yl, 3-butene-2-ol-4-yl. Aromatic hydrocarbons, if necessary, can be condensed four-semiclean non-aromatic ring. Non-aromatic ring, if necessary, can detect one or two double bonds C=C, one or more times to be substituted by the above substituents and, if necessary, to carry one or two ring heteroatoms. Examples of particularly common aromatic hydrocarbons are single, as cumene, and two substrates, as inden and naphthalene, and substituted from one to three videopreteen substituents of the carbon atoms.

Oxidized according to the invention the substrates) is unbranched or branched alkanes and alkenes with 4-15, mostly 6-12 carbon atoms. As examples can be called n-butane, n-pentane, n-hexane, n-heptane, n-octane, n-nonan, n-decane, n-undecane and n-dodecane, as well as single or multiply branched analogs of these compounds, as, for example, analogs of compounds with 1-3 lateral methylsilicone; or one or more times, for example, one unsaturated analogs of the aforementioned alkanes.

Oxidized according to the invention are substrates of group d) are, if necessary, substituted cycloalkanes and cycloalkenes from 4 to 8 ring carbon atoms. Such examples are cyclopentane, cyclopentene, cyclohexane, cyclohexene, Cycloheptane and cyclohepten. The ring structure may contain one or more, for example, from 1 to 5 substituents according to above definition of the compounds of groups a) and b). Non-limiting examples for this are ionone as α-, β- and γ-ionone, as well as relevant methylionone and isometries. Especially preferred αand β-ionone.

The subject of the invention are nucleic acid sequences that coded for the claimed monooxygenase. The preferred sequence derived from SEQ ID NO:1, which at least one nucleotide substitution, leading to one of the above functional amino acid mutations. The subject of the invention are, in addition, the functional analogues of nucleic acids obtained by the addition, substitution, insertion and/or deletion of single or multiple nucleotides, which encode the monooxygenases with the desired specificity of the substrate, as, for example, oxidizing indole activity.

In accordance with the invention makes it possible to detect such sequences of nucleotides, which include the so-called silent mutations, or changed, respectively, to use for encoding special organism of origin or the host organism in comparison with specifically named sequence, as well as previous natural versions. The invention encompasses also variants of the sequences of nucleic acids obtained by degeneration of the genetic code (i.e. without changing the corresponding amino acid sequence) or a conservative substitution of nucleotides (i.e. the corresponding amino acids are replaced with other amino acids of the same charge, size, polarity and/or solubility), and modified by addition, insertion, inversion, or deletion of nucleotide sequences that encode the claimed monooxygenase "with a modified profile of the substrate", and the corresponding complementary sequences.

The subject of the invention are, in addition, expression constructs containing in genetic control of regulatory sequences of nucleic acids of at least one encoding the claimed mutant sequence of nucleic acids, and is also vectors, covering at least one of these expression constructs.

Corresponding to the invention, the constructs comprise a promoter 5'-up the length of the corresponding coding sequence and a termination sequence to the 3'down the length, and, if necessary, other common regulatory elements operatively linked to the coding sequence. Under the operational linking understand the sequential arrangement of promoter, coding sequence, terminator and, if appropriate, other regulatory elements such that each of the regulatory elements could consistently perform its function in the expression of the coding sequence. Examples operatively linked sequences are sequences of targets, as well as public address amplifier, multiplier, polyadenylation signals, and others. Other regulatory elements cover a selective marker, signal amplification, replicas, and so forth.

In addition to the artificial regulatory sequences before the initial structural gene may be more natural regulatory sequence. By genetic changes in this natural setting can be turned off to increase or decrease gene expression. The gene construct may be on train easier that means that before the structural gene may not be inserted more regulatory signals, and the natural promoter with its regulation is not removed. Instead, the natural adjustment sequence motirola so that there was no more adjustment, and gene expression is increased or decreased. Nucleic acid sequences can be included in the gene construct in one or many copies.

Examples of commonly used 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 used mainly in gram-negative bacteria and gram-positive promoters amy and SPO2, in the yeast promoters ADCl, MFa, AC, P-60, CYC1, GAPDH or vegetable promoters CaMV/35S, SSU, OCS, lib4, usp, STLS1, B33, nos or ubiquitinate or phaseolinae promoters. Especially preferred is the use of inducible promoters, such as light and, especially, thermoinsulating promoters, as PrPl. In principle, all natural promoters can be used with their regulatory sequences. On this basis, can also with advantage be applied synthetic promoters.

Called regulatory sequences should provide a meaningful expression of the sequences of nucleic acids and the expression of proteins. Uh what about the can, for example, depending on the host organism to mean that the gene exprimarea or preexperiments only after induction, or he immediately exprimarea or preexperiments.

Regulatory sequences are, respectively, the factors can mainly positive influence on the expression and thereby to raise or lower it. Thus, strengthening of the regulatory elements can be carried out predominantly in the plane of transcription, and apply strong transcription signals, as promoters and/or enhancers". Along with this it is also possible strengthening of the broadcast, for example, improving the stability of the mRNA.

Production of expression cassettes is carried out by fusion of a suitable promoter with a suitable nucleotide sequence of monooxigenase and signal terminator or polyadenylation. For this purpose apply available techniques recombination and cloning described in T.Maniatis, E.F.Frinsch, J.Sambrook, Manual Cloning: A Labaratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1989), and T.J SIlhavy, M.L.Berman, L.W.Enquist, Experiments with Gene fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1984)and in Ausubel, F.M. et al., Current Protocols in Molecular Biology, Greene Publishing Assoc. and Wiley Interscience (1987).

Recombinant nucleic acid construct, respectively, the gene construct is introduced to extend in proximity to the body of the sm master predominantly in specific host vector, allowing for optimal gene expression in the host. Vectors known in the art and can be found in "Cloning Vectors" (Pouwels P.H. et al., Hrsg, Elsevier, Amsterdam - New York - Oxford, 1985). Under these vectors in addition to the plasmids should be understood also all other known specialist vectors, such as phages, viruses like SV40, CMV (cytomegalovirus), baculovirus and adenovirus, transposons, IS-elements (transposable elements), plasmids, Comedy and linear or circular DNA. These vectors can replicate or offline in the host organism, or the chromosome.

Using the steps described in the invention vectors are recombinant microorganisms which are transformed, for example, at least one described in the invention is a vector, and can be used for the production of mutants. Mostly the above described inventive recombinant constructs are introduced into suitable system owner, then expriments. The specialists are primarily known methods cloning and transfection to insert named nucleic acid into an appropriate 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 master fundamentally fit all bodies the isms, which make possible the expression of the claimed nucleic acids, variants of their alleles, their functional equivalents or derivatives. Under the host organisms should be understood, for example, bacteria, fungi, yeast, plant or animal cells. Preferred host organisms are bacteria, as bacteria of the genus Escherichia (e.g., Escherichia coli), a streptomycete, bacilli or found, eukaryotic microorganisms, as Saccharimyces cerevisiae, Aspergillus, eukaryotic cells of a higher order of animal or vegetable origin, for example, Sf9 or SNO.

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

Selection of successfully transformed organisms to be a marker gene, which is also contained in the vector or expression cassette. Examples of such marker genes are genes of resistance to antibiotics and enzymes, which catalyze a color reaction, causing the color of transformed cells. The latter can then otber the sterile automatically. Microorganisms that are successfully transformed with vectors that carry the gene of resistance to antibiotics (e.g., G418 or hygromycin), you can discriminates by media containing antibiotics, or nutrient soils. Marker proteins, which are presented on the cell surface can be used for selection by affinity chromatography.

The combination of their host organisms with vectors suitable for organisms, such as plasmids, viruses or phages, such as plasmids with polymerase/promoter RNA phages λ, μor other temperate phages or transposons and/or others having the advantage of a regulatory sequence forms an expression system. For example, the term "expression system" should be understood a combination of mammalian cells like Cho cells, and vectors as vector pcDNA3neo.

As already mentioned, the gene product can mainly be entered for expresii in transgenic animals, such as mice, sheep, or in transgenic plants. It is also possible to program free cell translation system derived from the nucleic acid RNA.

A further object of the invention is a method of manufacturing the inventive monooxygenase, when culturing a microorganism that produces the monooxygenases, if necessary, induce the expression of monoxid is called and allocate the regioselectivity of the culture. The claimed monooxygenase if necessary, can be produced on an industrial scale.

The microorganisms may be cultured and ermentrout by known methods. Bacteria can reproduce, for example, in the TV or LB medium and at a temperature of from 20°C to 40°and the pH value from 6 to 9. Detail suitable cultivation conditions 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 not secreted into the culture medium, the cells are decomposed, and monooxygenase is extracted from the lysate by the method of isolation of the protein. Cells can decompose to choose from: high-frequency ultrasound, by high pressure, as, for example, in a cell pressure of the French, by osmolysis, through action of detergents, lytic enzymes or organic solvents, by using a homogenizer or by a combination of several of these methods. Cleaning monooxygenase can be achieved by such well-known chromatographic methods as chromatography with molecular sieve (gel filtration), as chomatography Q-Sepharose, ion-exchange chromatography and hydrophobic chromatography, and other conventional methods, such as ultrafiltration, crystallization, salting out, dialysis and native gel electrophoresis. Suitable methods are described, for example,in F.G. Cooper, Biochemische arbeitsmenhoden, Verlag Walter de Gruyter, Berlin, New York or in Scopes, R., Protein Purification, Springer Verlag, new York, Heidelberg, Berlin.

Especially advantageous to use for isolation of recombinant protein vector system or oligonucleotides which extend the cDNA by a specific sequence of nucleotides and thereby encode extra long polypeptides or mixed proteins, which serve for easier cleaning. Appropriate modifications of this kind are, for example, acting as an immobilizer so-called "tags", as, for example, known as exegetically immobilizer modification or epitope that can be recognized as an antigen antibodies (described, for example, E. Harlow and D. Lane, 1988, Antibodies: A Laboratory Manual. Cold Spring Harbor (N.Y.) Press). These absorbers can be used for binding of the protein on solid media, such as polymer matrices, which, for example, can be covered in a chromatographic column, or may be applied on a microtiter plate, or other media.

Simultaneously, these absorbers can be used for detection of proteins. For detection of proteins can be used, in addition, such conventional markers, as fluorescent dyes, enzyme markers, which after reaction with the substrate to form the detected reaction product, or radioactive markers alone or in to the munali with immobilization for derivatization of proteins.

The invention relates, furthermore, how the microbiological oxidation of organic compounds, such as N-heterocyclic one-, two - or multi-tier videopreteen aromatic compounds, which are characterized in that

A1) were cultured videopreteen recombinant microorganism in culture in the presence of exogenous (introduced) or formed by oxidizing intermediate substrate claimed monooxygenase, mainly in the presence of oxygen (i.e. aerobic); or

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

b) allocate the resulting oxidation product or a sequential product of this environment.

Required to convert oxygen enters the reaction medium from the ambient air or may be entered, if required, in a known manner.

Preferably, the oxidizable substrate is selected among the

a) if necessary substituted N-heterocyclic one-, two - or multi-tier videopreteen aromatic compounds;

b) if necessary substituted single or multi-tier aromatic hydrocarbons;

c) an unbranched or branched alkanes and alkenes;

d) if necessary substituted cycloalkanes, cyclea is kenow.

A preferred variant of the method is aimed at the formation of Indigo/indirubin and differs in that the substrate is an intermediate formed in the culture of the indole, and from culture medium emit produced Indigo and/or indirubin, which was obtained by oxidation of an intermediate formed hydroxyindoles.

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 environment, such as a TV or LB-medium at a temperature of cultivation is approximately 20-40°and the pH value from 6 to 9, until it reaches a sufficient density of cells. The addition of exogenous indole usually not required, because it is formed by the microorganism as an intermediate product. When converting other substrates may, however, require the addition of exogenous substrate. To better control the oxidation reaction, preferably using induced, in particular, induced by the temperature of the promoter. When this temperature increases to the point of induction, for example, 42°when the promoter PrPlsupport for a sufficient time, for example from 1 to 10, or from 5 to 6 hours, for the expression of active monooxygenase, and then reduce the temperature to 0-40° C. When the cultivation is continued in the presence of oxygen from 12 hours to 3 days. In particular, the oxidation of indole pH value due to the additive NaOH may be increased, for example, up to 9-10, thanks to advanced sposobstvuete education Indigo, respectively, of indirubin by oxidation in air enzymatically formed oxidation products 2 - and 3-hydroxyindole.

Education Indigo/indirubin according to the present invention is illustrated by the following reaction scheme:

If the oxidation according to the invention, in contrast, is carried out using purified or enriched mutant enzyme, the claimed enzyme dissolved in containing exogenous substrate, such as indole, medium (about 0.01 to 10 mm, or 0.05-5 mm) and carry out the conversion mainly in the presence of oxygen, at a temperature of from about 10°C to 50°With, for example, from 30°C to 40°and the pH value from about 6 to 9 (what, for example, is set at 100-200 mm phosphate or TRIS buffer), and in the presence of a reducing agent, and containing substrate medium contains, in addition, with respect to the oxidizable substrate approximately 1-100-fold or 10-100-fold molar excess regenerative equivalents. The preferred reducing agent is NADP. The addition of the reducing agent may, if the mu is a necessity, implemented portions.

In a similar way as preferred substrates: n-hexane, n-octane, n-decane, n-dodecane, cumene, 1-methylindole, 5-Cl, or Br-indole, inden, benzothiophen, α, β and γ-ionon, acridine, naphthalene, 6-methyl or 8-methylinosine, quinoline and hinanden.

For example, the inventive enzymatic oxidation reaction can be conducted under the following conditions:

The substrate concentration:from 0.01 to 20 mm
The concentration of enzyme:from 0.1 to 10 mg/ml
The reaction temperature:10°C to 50°
pH:from 6 to 8
Buffer:from 0.05 to 0.2 M potassium phosphate or TRIS/HCl
The electron donor:preferably added in portions (initial concentration of from about 0.1 to 2 mg/ml)

Before the reaction, for example, by adding an electron donor (e.g., NADP) may be short-term (1-5 minutes) pre-incubation (approximately 20-40°). The transformation is carried out aerobically, if necessary with additional introduction of oxygen.

When the claimed method of oxidation resulting in the reaction medium or added oxygen is cleaved enzymatically and reduction is. Required restoration equivalent comes the added reducing agent (electron donor).

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

Further objects of the invention are bioreactors, including the claimed enzyme or the claimed recombinant organism in immobilizovannoi form.

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

The invention is described in more detail with the following examples.

Example 1

Randomization special codons P450 BM-3

Experiments to a significant extent were carried out as described in (19). Three positions (Phe87, Leu188 and l74) were randomized using metropacific mutagenesis using set Stratagene QuikChange kit (La JOLLA, CA, USA). In some positions, we used the following PCR-examples:

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)

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

Example 2

Expression and purification of P450 BM-3 and its mutants, and the production of blue pigment

Gene P450 BM-3 and its mutants was exprimarea under the control of a strong temperature-induced promoter PRPLplasmid pCYTEXP1 in E.coli DH5αas already described (20). Colonies were taken with sterile gear odbornikom and transferred to microtiter plates with 96 cells containing 200 μl of medium TV and 100 μg/ml of ampicillin per cell. Then overnight at 37°incubation was carried out. 40 μl of cell culture b the th cells were then transferred into a culture tube, which contained 2 ml TV environment with 100 μg/ml ampicillin. Then within 2 hours of the cultivation was carried out at 37°S. Then for the induction temperature was increased to 42°With 6 hours. Thereafter, cultivation was continued overnight at 37°and there was obtained a blue pigment.

Preparative production of the enzyme or blue pigment was carried out on the basis of 300 ml of cell culture (OD578 nm=0,8-1,0). For isolation of the enzyme, the cells were subjected to centrifugation for 10 min at 4000 rpm, then resuspendable in 0.1 MxPO4-buffer, pH 7.4. Chilled to ice the cells was carefully decomposed with the help of the device Branson Sonifiers W25 (Dietzenbach, Germany) at an output power of 80 watts through three irradiation for 2 minutes Suspension were centrifuged 20 min at 32750 g. The crude extract was used for determination of activity, respectively, for the purification of the enzyme. Purification of the enzyme was carried out as described in (21), on which there is a compelling link. The concentration of purified enzyme was determined taking into account the difference in extinction at 450 and 490 nm, as already described in (11), using the extinction coefficient ε=91 mm-1cm-1.

Example 3

The selection of mutants that produce large amounts of blue pigment

From each position were selected respectively 100 colonies of mutants to the e were obtained by randomization of the codon corresponding position. These colonies were cultivated in culture tubes for the production of blue pigment. After washing the cells with water and many slow steps of centrifugation (500 rpm) blue pigment was extracted with dimethylsulfoxide (DMSO). The solubility of the blue pigment in DMSO was the greatest. The absorbance of the extract was determined in the area of 677 nm. Those mutants that produced the greatest amount of pigment in relation to all mutants certain position, were used to sekvestirovanija DNA (set ABI DNA Sequenzierungs-Kit; ABI PrismTM377 DNA Sequencer) and, in addition, was used as a template for metropacific randomized mutagenesis.

Example 4

Test activity for hydroxylation of indole

Activity hydroxylation of indole was tested in solution, which contained 8 μl of a solution of indole 10-500 mm in DMSO, 850 μl of buffer TRIS/HCl (0.1 M, pH 8,2) and 0.6 nmol P450 BM-3 natural type or mutants in a final volume of 1 ml of the Mixture was pre-incubated for 9 min before start the reaction by adding 50 ál of water 1 mm solution of NADP. The reaction was stopped after 20 seconds by adding 60 µl of 1.2 M KOH. Within 5-30 (under aerobic conditions) enzyme products have been fully converted into Indigo ([Δ2,2'-bayingolin]-3,3'-dione), indirubin ([Δ2,2'-bayingolin]-3,3'-dione). Products Indigo would be settled and determined by their absorption in the region of 670 nm. Calibration curve for pure Indigo at this wavelength gives the extinction coefficient of 3.9 mm-1cm-1. Linear travel of the calibration curve obtained for the production of Indigo through a reaction time of 40 s at the application of 0.6 nmol natural type, respectively, mutant P450 BM-3 and from 0.05 to 5.0 mm indole. Indirubin has a very weak absorption at 670 nm, and the resulting number of indirubin significantly less than the number of Idaho. When determining the kinetic parameters of the education of indirubin was not taken into account. Consumption of NADP was determined by measuring in the region of 340 nm and calculation using the extinction coefficient of 6.2 mm-1cm-1as described in (17).

Example 5

Clean Indigo and indirubin

After washing the cells with water and repeated centrifugation at 500 g formed the blue residue was extracted with tetrahydrofuran (THF). The extract was evaporated until almost dry, 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 was evaporated and purified using silikagelevye chromatography (DC 60, Merck, Darmstadt, Germany; 2cm × 30 cm)before it was washed with THF and petroleum ether in the ratio 1:2. The obtained cu is sny the solution was evaporated and analyzed by TLC method. Absorption spectra of the blue and red pigment were obtained using a spectrophotometer Ultraspec 3000 (Pharmacia, Uppsala, Sweden) in the region from 400 to 800 nm. In addition, the blue and red dye were analyzed using mass spectroscopy and NMR spectroscopy.

The results of experiments

1. Increase the production of blue pigment by mutagenesis P450 BM-3

Natural P450 BM-3 does not have the ability to manufacture containing blue Indigo pigment, respectively, intermediate products - 2-respectively 3-hydroxyindole. In order to obtain a sufficient amount of the blue pigment, P450 BM-3 was subjected to purposeful transformation. All the mutants that produce the blue pigment were sekvenirovan. It was found that the mutated at least one of the following three positions: Phe87, Leu188 and l74. So I accepted that these three positions play a crucial role for the activity of P450 BM-3 in the production of blue pigment. From the structure of the heme domain of cytochrome P450 BM-3, complexional palmitoleic acid, it is seen that Phe87 protects the substrate from the middle of the shift of the heme group (14). Mutant Phe87Val detects high Regio - and stereoselectivity when epoxidase (14S, 15R)-arachidonic acid (13), and the mutant Phe87Ala moves the position of hydroxylation ω-1, ω-2 ω-3 (22). Position 87 was therefore the selected first for locally-specific random mutagenesis using PCR. In vitro cultures were obtained 7 colonies that after the induction produced a small amount of blue pigment. The colony, which produced the greatest amount of the blue pigment was selected for sekvenirovaniya DNA. Data sequences gave the substitution Phe87 on Val. Mutant Phe87Val was eventually used as template for the second round of locally-specific randomized mutagenesis at position Leu188. The structure of the heme domain, complexional palmitoleic acid shows that the repositioning of F - and G-helix leads the rest Leu188 in direct contact with the substrate (14). Therefore, this position may play an important role in linking or orientation of the substrate. After the second screening was observed 31 colony, which produced a blue pigment. The mutant, which produced the greatest amount of pigment contained substitution Phe87Val and Leu188Gln. This mutant was eventually mutated at position l74 in the third pass locally-specific random mutagenesis. When this was received triple mutants F87L188A74 (Phe87Val, Leu188Gln and Ala74Gly), which produced many mg blue pigment in a two-liter flask containing 300 ml of a TV environment. This amount was sufficient for the selection and definition of a blue pigment.

2. The selection and definition of a blue pigment

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

After treatment were recorded absorption spectra of both components in DMSO. The blue component has the same range as the commercial sample Indigo. Treated blue and red components, respectively, were analyzed using mass spectrometry. Mass spectra of both pigments have a strong ion-molecular peak at m/z=262 and 2 fragmentary peak at m/z=234 and 205 (relative intensity, respectively, 10%). This sample is typical for indigoid compounds. The basic composition of these ions was determined using mass spectroscopy high-resolution as C16H10N2O2C15H10N2O, respectively, With14H9N2. This is also true for structures indigoids types. Thus, the blue pigment was identified as Indigo, and red as indirubin. To confirm the structure was verified by NMR of both pigments in a solution of DMSO-D6. The results are consistent with literature data.

3. The production of Indigo with isolated enzymes

It is known that Indigo can be obtained from indole by microbiological Tran the formation (24-26). However, none of these microbial systems does not contain monooxygenase P450. According to the invention, first and foremost, must be defined catalytic activity of pure enzyme with respect to the indole. Mutant F87L188A74 was mixed with indole. There wasn't any color reaction. Only after the addition NADP in the reaction mixture after about 20 min formed a blue pigment. By bringing the pH of the reaction mixture to a value of 11, after 30 s after addition of NADP in a few seconds became visible blue color. Control experiments using natural P450 BM-3 were always negative, even when using higher concentrations of the enzyme, indole and NADP. The blue pigment was extracted with ethyl acetate and analyzed by thin layer chromatography. The blue pigment was again divided into fast-moving blue and slow moving red component. X-ray fluorescence values and absorption spectra were identical to the same values as in the extract of fermentation broth. Mutants F87L188A74 P450 BM-3 represent hydroxylase indole.

So far been described only 2 ways enzymatic transformation of indole to Indigo. One way catalyzes using dioxygenase, and the second styrene-monooxygenase (24, 25). The stoichiometry of NADP in both cases is 2. The poet is mu was taken, in contrast to the dioxygenase claimed mutants F87L188A74 hydroxylated indole only in one position to form oxindole (2 hydroxyindole) or indoxyl (3-hydroxyindole).

4. Kinetic parameters hydroxylation of indole

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

Table 1.

Kinetic parameters hydroxylation of indole mutants of P450 BM-3
MutantsKcat(c-1)Tom(mm)Kcat/Km(M-1c-1)
WT-a)--
Leu188Glnthe concentration isb)the concentration isthe concentration is
Phe87Val2,03 (0,14)17,0 (1,0)119
F87L1882,28 (0,16)4,2 (0,4)543
F87L188A742,73 (0,16)2,0 (0,2)1365
a)activity was not observed

b)not defined (the activity was too small to be able to change the shape)

Even with an excess of purified enzyme and high concentrations of indole natural enzyme is not able to oxidize indole. Mutant Leu188Gln detects an insignificant activity. Mutant Phe87Val has catalytic activity 119 M-1with-1for hydroxylation of indole. The catalytic efficiency of double mutants F87L188 (Phe87Val, Leu188Gln) increased to 543 M-1with-1and was enhanced by the introduction of other substitutions Ala74Gly to 1365 M-1with-1. Values of Kcatrising from Phe87Val to triple mutants, approximately 35%, while values Formdecrease approximately 7 times. This indicates that Ala74Gly and Leu188Gln involved in substrate binding.

The share of reversibility indole (Kcat=2,73-1for triple mutants F87L188A74 more than 10 times higher than for most P450 enzymes (18).

Example 6

Hydroxylation of n-octane modified cytochrome P450-monooxygenase

Transformation was performed using the mutant monooxygenase P450 BM-3, which contains the following mutations: Phe87Val, Leu188Gln, Ala74Gly.

As the substrate was selected n-octane. For hydroxylation of n-octane was used next reaction set:

mutants of P450 BM-3:of 17.5 mg (lyophilized)
buffer:961 ml (buffer of FOS is ATA potassium, 50 mm, pH 7.5)
substrate:50 μl of a solution of 60 mm (in acetone)
temperature:25°

The lyophilized enzyme was dissolved in 500 µl of buffer and once with the substrate and the buffer was incubated 5 min at room temperature. Followed by adding 300 ál of NADP solution (5 mg/ml). Adding NADP was repeated twice. The reaction course was monitored by measuring the absorbance at 340 nm, due to which it was possible to observe the reduction of NADP. NADP was added to 300 µl, since a high concentration of NADP leads to deactivation of the enzyme. Then to separate the product of the reaction solution was extracted 3 times with 5 ml of diethylether. The combined organic phases were dried with MgSO4and collected. Then the products were analyzed by thin layer chromatography (DC), gas chromatography with mass spectroscopy finish (GC/MS) and NMR.

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

ConnectionRt (min)1)Conversion (%)
4-octanol13,5137
3-octanol14,0847
2-octanol14,2616
1)Temperature program: 40°1 min isotherm/ 3 °C/min 95°/10 °C/min to 275°Apparatus; Finnigan MAT 95; GC: HP 5890 series II slot injector; column: HP-5MS (methylsiloxane) 30 m × 0.25 mm; carrier gas: 0,065 ml/min Not.

Product extraction was not found.

Example 7

Hydroxylation of aromatic hydrocarbons, heteroaromatic hydrocarbons, compounds trimethylcyclohexane

(a) Example 6 was repeated, but instead of n-octane as the substrate was taken naphthalene. The products were 1-nattonal and CIS-1,2-dihydroxy-1,2-dihydronaphthalene. Introduced naphthalene was converted to 88%.

The analysis of reactions of naphthalene

GC:

Equipment: Carlo Erba Strumentazion type HRGC 4160 on column injector; column: DB5 30 m × 0.2 mm; material: 5% diphenyl - 95% dimethylpolysiloxane; carrier gas: 0.5 bar H2; temperature program: 40°1 min isotherm/10°C/min to 300°; Rt (1-nattonal)=16,68.

NMR:

Were identified 1-nattonal and CIS-1,2-dihydroxy-1,2-dihydronaphthalene.

b) Example 6 was repeated, but instead of n-octane as the substrate was taken 8-methylinosine. As the main product was identified 5-hydroxy-8-methylinosine, in addition to other derivatives (the ratio of product 5:1). Entered the product was converted to 35%.

c) Example 6 was repeated, however, this is m instead of n-octane as the substrate was taken α -ionan. As the main product was identified by 3-hydroxy-α-ionon, in addition to other derivatives (the ratio of the product 76:24). Entered the product was converted to 60%.

d) Example 6 was repeated, but instead of n-octane as the substrate was taken cumene (ISO-propylbenzyl). Identified 5 products monohydrates and one product digiraatii. Entered the product was converted to 70%.

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2. Zhang, J.-H., Dawes, G. and Stenner, W.P.C. (1997) Proc. Natl. Acad. Sci. USA 94, 4504-4509.

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7. Tucker, C.L., Hurley, J.H., Miller, T.R., and Hurley, I.B. (1998) Proc. Natl. Acad. Sci. USA 95, 5993-5997.

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9. Marsden, A-F.A., Wilkinson, C., Cortes, J., Dunster, N.J., Staunton, I Leadlay, P.F. (1998) Science 279, 199-201.

10. Chen, R., Greer, A., and Dean, A.M. (1998) Proc. Natl. Acad. Sci. USA 95, 11666-11670.

11. Boddupalli, S.S., Estabrook, R.W. and Peterson, J.A. (1990) J. Biol. Chem. 265, 4233-4239.

12. Capdevila, J.H., Wie, S., Helvig, C., Faick, J.R., Belosludtsev, Y., Truan, G., Graham-Lorence, S.E. and Peterson, J.A. (1996) J. Biol. Chem. 271, 22663-22671.

13. Graham-Lorence, S., Truan, G., Peterson, J.A., Flack, J. R., Wel, S., Helvig, C., Capdevilla, J.H. (1997) J. Biol. Chem. 272, 1127-1135.

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1. The way microbiological oxidation of N-, O - or S-heterocyclic one-or polynuclear aromatic compounds, characterized in that

A1) culturing a recombinant microorganism, which will exprimere cytochrome P450-monooxygenases of bacterial origin in the culture medium in the presence of exogenous or intermediate formed substrate; or

A2) incubated containing substrate reaction medium with the cytochrome P450-monooxygenases of bacterial origin;

b) allocate the resulting oxidation product or derived product from the environment

when the eating monooxygenase is derived from cytochrome P450-monooxygenase BM-3 from Bacillus B. 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 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, wherein the exogenous or intermediate formed substrate choose among unsubstituted or substituted by N-, O - or S-heterocyclic one-or polynuclear aromatic compounds.

3. The method according to claim 1 or 2, characterized in that the use of mutant having at least the following single or multiple substitutions of amino acids:

1) Phe87Val;

2) Phe87Val, Leu188Gln; or

3) Phe87Val, Leu188Gln; Ala74Gly.

4. The method according to one of claims 1 to 3, characterized in that the exogenous substrate, at least one compound from the group of unsubstituted or substituted by N-, O - or S-heterocyclic, one -, two - or polynuclear aromatic compounds, is added to the environment, and the oxidation is carried out by enzymatic conversion containing substrate medium in the presence of oxygen at a temperature of from about 20°C to 40°and the pH value from about 6 to 9, and containing substrate medium contains, in addition, with respect to the substrate approximately 10-100-fold molar excess restorative equivalent the clients.

5. The method according to claim 4, characterized in that the exogenous substrate is used as a compound selected from among the following: indole, 1-methylindole, 5-chloro - or bromo-indole, indene, benzothiophene, acridine, 6-methyl - or 8-methylinosine, quinoline and rinaldina.

6. The way microbiological oxidation of unsubstituted or substituted one or polynuclear aromatic hydrocarbons, linear or branched alkanes and alkenes or unsubstituted or substituted cycloalkanes and cycloalkenes, characterized in that

A1) were cultured producing recombinant cytochrome P450-monooxygenases microorganism in a culture medium in the presence of exogenous or intermediate formed substrate; or

A2) incubated containing substrate reaction medium with the cytochrome P450-monooxygenases capable of oxidation of the aforementioned compounds; and

b) allocate the resulting oxidation product or derived product from the environment

and monooxygenase is capable of oxidation of the above-mentioned compounds and is derived from cytochrome P450-monooxygenase BM-3 from Bacillus B. 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, also eat, at least one functional mutation in 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, wherein the exogenous or intermediate formed substrate choose among b) unsubstituted or substituted one or polynuclear aromatic hydrocarbons; (c) a 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 the use of mutant having at least the following single or multiple substitutions of amino acids:

1) Phe87Val;

2) Phe87Val, Leu188Gln; or

3) Phe87Val, Leu188Gln; Ala74Gly.

9. The method according to one of p-8, characterized in that the exogenous substrate choose at least one connection among the above defined groups unsubstituted or substituted with one or polynuclear aromatic hydrocarbons, linear or branched alkanes or alkenes and unsubstituted or substituted cycloalkanes and cycloalkenes add to the environment, and the oxidation is carried out by enzymatic conversion containing substrate medium in the presence of oxygen at a temperature of from about 20°C to 40°and the pH value from about 6 to 9, and containing substrate medium contains, in addition to also, with respect to the substrate approximately 10-100-fold mole of the hydrated excess regenerative equivalents.

10. The method according to claim 9, characterized in that the exogenous substrate is used as a compound selected from among the following: n-hexane, n-octane, n-decane, n-dodecane, cumene, α-, β- and γ-ionone and naphthalene.

11. The way the microbiological production of Indigo and/or indirubin, wherein A1) culturing a recombinant microorganism that produces in culture medium oxidizing indole cytochrome P450-monooxygenase in the presence of exogenous or intermediate formed indole; or A2) incubated containing indole reaction medium with the cytochrome P450-monooxygenases, oxidizing indole, and (b) allocate the resulting oxidation product or a serial product of the environment, and monooxygenase is derived from cytochrome P450-monooxygenase BM-3 from Bacillus B. 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 the 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 environment allocate formed Indigo and/or indirubin obtained by oxidation of an intermediate formed indole.

13. The method according to the .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°and the pH value from 6 to 9.

14. The method according to one of § § 11 to 13, characterized in that the use of mutant having at least the following single or multiple substitutions of amino acids:

1) Phe87Val;

2) Phe87Val, Leu188Gln; or

3) Phe87Val, Leu188Gln; Ala74Gly.

15. Cytochrome P450-monooxygenase which is capable, at least one of the following reactions: a) oxidation of unsubstituted or substituted by N-, O-or S-heterocyclic, one -, two - or polynuclear aromatic compounds; (b) oxidation of the unsubstituted or substituted one or polynuclear aromatic hydrocarbons; (c) oxidation of linear or branched alkanes and alkenes, and (d) oxidation of unsubstituted or substituted cycloalkanes and cycloalkenes,

and monooxygenase is derived from cytochrome P450-monooxygenase BM-3 from Bacillus B. 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 at least one functional mutation in the amino acid sequence 73-82 and 172-224 and, if necessary, additionally has one functional mutation in one of the area of the 39-43 amino acid sequence, 48-52, 67-70, 330-335 and 352-356.

16. Monooxygenase indicated in paragraph 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 the amino acid sequence 73-82 and 172-224.

17. Monooxygenase indicated in paragraph 15, characterized in that it has at least a few times, substitution of amino acids:

a) Phe87Val, Leu188Gln; or

b) Phe87Val, Leu188Gln; Ala74Gly,

as well as their functional equivalents, which are able, at least one of the above oxidation reactions.

Priority items:

27.07.1999 - 15 claims in part Cytochrome P450-monooxygenase which is capable, at least one of the reactions (a)and (d), in addition to the oxidation of O-heterocyclic aromatic compounds, and which has a functional mutation in the position of the amino acid sequence 87 and at least one functional mutation in one of the positions of the amino acid sequence 74-188 and, if necessary, additionally has a functional mutation in the position of the amino acid sequence 354;

article 16 of the claims in part Cytochrome P450-monooxygenase which is capable, at least one of the reactions (a)-(d) of clause 15 of the formulas, in addition to the oxidation of O-heterocycle is ical aromatic compounds, and which has a functional mutation in the position of the amino acid sequence 87 and at least one functional mutation in one of the positions of the amino acid sequences 74 and 188;

17 claims in part Cytochrome P450-monooxygenase pursuant to subparagraph (a) or (b)which is capable, at least one of the reactions (a)-(d) of paragraph 15 of the formula, in addition to the oxidation of O-heterocyclic aromatic compounds.

18.01.1999 - claims 1 to 5 of the claims in part microbiological oxidation of N-heterocyclic polynuclear aromatic compounds;

§ § 11-14 claims;

15 claims in part Cytochrome P450-monooxygenase, which is capable of oxidation of N-heterocyclic two - or polynuclear aromatic compounds (a), except for Cytochrome P450-monooxygenase, which has a functional mutation in the position of the amino acid sequence 87 and at least one functional mutation in one of the positions of the amino acid sequences 74 and 188 and, if necessary, additionally has a functional mutation in the position of the amino acid sequence 354;

article 16 of the claims in part Cytochrome P450-monooxygenase, which is able to oxidize N-heterocyclic two - or polynuclear aromatic is soedinenii in subparagraph (a) of clause 15 of the formula, with the exception of Cytochrome P450-monooxygenase, which has a functional mutation in the position of the amino acid sequence 87 and at least one functional mutation in one of the positions of the amino acid sequences 74 and 188.

20.03.2000 according to the filing date DE 10014085.8 filed in the Patent office Germany - claims 1 to 5 of the claims in part microbiological oxidation of O-, or S-heterocyclic one - or polynuclear aromatic compounds and oxidation of N-heterocyclic mononuclear aromatic compounds;

PP-10 claims;

15 claims in part Cytochrome P450-monooxygenase which is capable, at least one of the reactions (a)-(d), except for Cytochrome P450-monooxygenase which is capable, at least one of the reactions (a)to (d), in addition to the oxidation of O-heterocyclic aromatic compounds, and which has a functional mutation in the position of the amino acid sequence 87 and at least one functional mutation in one of the positions of the amino acid sequences 74 and 188 and, if necessary, additionally has a functional mutation at position amino acid sequence 354 and with the exception of Cytochrome P450-monooxygenase, which is able to oxidize N-heterocyclic two or mnogoe hernych aromatic compounds in subparagraph (a);

article 16 of the claims in part Cytochrome P450-monooxygenase which is capable, at least one of the reactions (a)-(d) of clause 15 of the formula, except for the Cytochrome P450-monooxygenase which is capable, at least one of the reactions (a)to (d) of clause 15 of the formulas, in addition to the oxidation of O-heterocyclic aromatic compounds, and which has a functional mutation in the position of the amino acid sequence 87 and at least one functional mutation in one of the positions of the amino acid sequences 74 and 188 and with the exception of Cytochrome P450-monooxygenase which is able to oxidize N-heterocyclic two - or polynuclear aromatic compounds in subparagraph (a);

17 claims in part Cytochrome P450-monooxygenase pursuant to subparagraph (a) or (b), which is able to oxidize O-heterocyclic aromatic compounds.



 

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