DE10260891A1 - Screening procedure for the identification of effective substances against halitosis, gingivitis or periodontitis - Google Patents

Abstract

The present invention relates to a screening method for identifying active substances against halitosis, gingivitis or periodontitis, test kits for finding active substances against halitosis, gingivitis or periodontitis, the use of methionine gamma-lyases (METase) for finding active substances against halitosis , Gingivitis or periodontitis; furthermore a test method for proving the effectiveness of substances against halitosis, gingivitis or periodontitis and a method for producing a pharmaceutical preparation for halitosis, gingivitis or periodontitis.

Description

The present invention relates to a screening process to identify active substances against halitosis, gingivitis or periodontitis, test kits to find effective substances against halitosis, gingivitis or periodontitis, the use of methionine gamma lyases (METase) for detection effective substances against halitosis, gingivitis or periodontitis; also a test procedure to prove the effectiveness of substances against halitosis, gingivitis or periodontitis as well as a procedure for the production of a pharmaceutical preparation against halitosis, Gingivitis or periodontitis.

Bad breath, also halitosis or "Foetor called ex ore " can have different causes. In healthy people bad breath however in most cases caused by bacteria in the mouth or throat, the body cells or metabolize food residues, being used as breakdown products of proteins u. a. volatile Sulfur compounds ("volatile sulfur compounds ", VSC) arise for the bad Smell to be held responsible.

These sulfur compounds are in particular: H ₂ S = hydrogen sulfide (about 30%), CH ₃ -SH = methyl mercaptan (about 60%) and CH ₃ -S-CH ₃ = dimethyl sulfide (about 10%).

The volatile sulfur compounds are sometimes very aggressive and can damage the gums and the Damage to the oral mucosa. For example, Ng W, Tonzetich J .; "Effect of hydrogen sulfide and methyl mercaptan on the permeability of oral mucosa "; J Dent Res. 1984 July; 63 (7): 994-7; known to be hydrogen sulfide and methyl mercaptan the permeability the mouth increases mucous membrane and so can promote gum disease.

Gum disease is one high risk factor for represents health. According to studies by the World Health Organization (WHO) suffer over 50% of adult citizens to those in urgent need of treatment Periodontal disease.

Gum disease is in the Popularly called 'periodontitis'. This Expression is not correct because the ending "-ose" one normal age-related decline describes an organ. The terms "periodontitis" or "inflammatory" are correct Periodontal disease ".

In addition to caries, it is wide most common oral cavity disease and occurs mainly in adults on.

Periodontitis is one Inflammatory change in the area around the tooth caused by bacteria Tissue, especially the jawbone. Periodontitis (= with Involvement of the gum pockets and bone) always comes from an inflammation of the gums (gingivitis) and affects first the interdental spaces that are most difficult to clean. The Bacteria in the gum pocket work through their metabolic products, especially those you produce VSC, destructive to gums, tooth structure and bones strengthen so constantly the existing periodontitis.

This is a health problem very widespread: Studies show that around the age of 35 more teeth from periodontal diseases (gum disease) than from caries get lost.

Yoshimura M, Nakano Y, Yamashita Y, Oho T, Saito T and Koga T. describe in “Formation of methyl mercaptan from L-methionine by Porphyromonas gingivalis "; Infect Immun. 2000 Dec; 68 (12): 6912-6; an L-methionine-alpha deamino-gamma-mercaptomethane lyase (METase) from P. gingivalis, the great one Amounts of methyl mercaptan produced.

METase from oral cavity bacteria was but not yet described in connection with halitosis, such as the publication by Yoshimura M, Nakano Y and Koga T; "L-Methionine-gamma-lyase, as a target to inhibit malodorous bacterial growth by trifluoromethionine "; Biochem Biophys Res Commun. 2002 Apr 12; 292 (4): 964-8; Page 964, right column, first paragraph; can be seen.

The diagnosis of bad breath is usually carried out using bacterial cultures, sulfide detection methods and organoleptic methods (smelling), or using measuring devices such as the "Halimeter ^® " described on the Internet at http://www.halithose.de/halimtr.htm (as of December 16, 2002). ,

So far, attempts have mostly been made Halitosis, gingivitis or periodontitis due to bactericidal agents fight, which, however, sometimes have strong side effects. Another approach intends to remove the VSCs using zinc-containing compounds. The latter procedure, however, only has a short-term acceptance the development of odors and prevents gingivitis or periodontitis not before.

There is therefore a significant one Need to provide new agents against halitosis, gingivitis or periodontitis.

Surprisingly it was found that methionine gamma lyases (METases), in particular a methionine gamma lyase from Treponema denticola, can be used to advantage against new active ingredients Find halitosis, gingivitis or periodontitis.

• a) eine für eine Methionin-Gamma-Lyase codierende Nukleinsäure aus einem pro- oder eukaryotischen Organismus isoliert oder in vitro synthetisiert,
• b) die für eine Methionin-Gamma-Lyase codierende Nukleinsäure kloniert und in geeigneten Wirtszellen exprimiert oder die Methionin-Gamma-Lyase in vitro synthetisiert,
• c) die Methionin-Gamma-Lyase exprimierenden Wirtszellen in voneinander räumlich getrennten Ansätzen in Gegenwart einer Schwefelquelle und eines geeigneten Nachweisagens für Reaktionsprodukte durch Methionin-Gamma-Lyase katalysierter Reaktionen kultiviert; oder die Methionin-Gamma-Lyase in voneinander räumlich getrennten Ansätzen in Gegenwart von Methionin, Cystein oder Glutathion in ein in vitro Testsystem einbringt,
• d) die Ansätze mit möglicherweise die Funktion der Methionin-Gamma-Lyase hemmenden Substanzen versetzt,
• e) für jeden der voneinander räumlich getrennten Ansätze die Bildung von Reaktionsprodukten durch Methionin-Gamma-Lyase katalysierter Reaktionen bestimmt und so

wirksame Substanzen gegen Halitosis, Gingivitis oder Parodontitis identifiziert
Vorteilhafterweise ermöglicht das erfindungsgemäße Screening-Verfahren, wenn es mit Methionin-Gamma-Lyase produzierenden bakteriellen Wirtszellen durchgeführt wird, Substanzen zu identifizieren, die die VSC-Bildung verhindern, ohne alle im Mundraum vorhandenen Mikroorganismen abzutöten. Man verwirft hierzu alle Substanzen, die zwar die Bildung von VSC inhibieren, gleichzeitig aber die bakterielle Wirtszelle abtöten.The present invention therefore relates to a screening method for identifying active substances against halitosis; Gingivitis or periodontitis, which is characterized in that one

• a) a nucleic acid coding for a methionine gamma lyase from a pro- or eukaryotic Or ganism isolated or synthesized in vitro,
• b) the nucleic acid coding for a methionine gamma lyase is cloned and expressed in suitable host cells or the methionine gamma lyase is synthesized in vitro,
• c) the host cells expressing methionine-gamma-lyase are cultivated in spatially separate batches in the presence of a sulfur source and a suitable detection agent for reaction products by reactions catalyzed by methionine-gamma-lyase; or the methionine gamma lyase is introduced into an in vitro test system in spatially separated batches in the presence of methionine, cysteine or glutathione,
• d) the approaches are mixed with substances that possibly inhibit the function of methionine gamma lyase,
• e) for each of the spatially separated approaches, the formation of reaction products determined by reactions catalyzed by methionine-gamma-lyase, and so on

effective substances against halitosis, gingivitis or periodontitis identified
Advantageously, the screening method according to the invention, when carried out with bacterial host cells producing methionine gamma-lyase, makes it possible to identify substances which prevent VSC formation without killing all microorganisms present in the oral cavity. For this purpose, all substances are discarded which inhibit the formation of VSC, but at the same time kill the bacterial host cell.

So side effects or at least active substances with little side effects are found, which the natural and useful Germination of the oral cavity do not damage.

Methionine-γ-lyases (METases) belong to one Enzyme family found in pro- and eukaryotes (but not in mammals) and the conversion of methionine to ammonia, 2-oxobutyrate and methyl mercaptan catalyze. Pyridoxal phosphate is necessary as a cofactor on a conserved region with the consensus sequence [DQ] - [LIMVF] -X (3) - [STAGC] - [STAGCI] -T-K- [FYWQ] - [LIVMF] -X-G- [HQ] - [SGNH] binds, where K acts as a pyridoxal phosphate binding site. The METases from the protozoon Trichomonas vaginalis and the bacteria Pseudomonas putida and Porphyromonas gingivalis have already been described and characterized. For Fusobacterium nucleatum was created by annotating the genome METase gene identified.

The applicant has succeeded in for the Sequence of the oral bacterium Treponema encoding METase protein to clear up denticola and thus to show for the first time that METases also in spirochetes occurrence.

The base sequence of the mgl1 gene from T. denticola is 64.5% identical to the sequence from P. gingivalis and contains the pyridoxal phosphate binding region, which, in contrast to the other Mgl proteins at position 209, is a Leu instead of one Val has (CLUSTALW alignment):
Of particular interest in this context is the exchange of Glu to Thr at position 320, which could indicate an additional phosphorylation site in the Mgl protein from T.denticola, which may possibly serve as a target for a specific inhibitor.

In the following means an expression the form “at least X% "" X% to 100% (including the basic parameters X and 100 and all integer and non-integer Percentages in between) ".

Under a protein is in mind of the present application a composed of the natural amino acids, largely linear, mostly to perform its function to understand three-dimensional structure-adopting polymer. In the In the present application, the 19 proteinogenic, naturally occurring L-amino acids with the internationally used 1 and 3 letter codes.

An enzyme is in the sense of present application to understand a protein that a certain has a biochemical function. Under methionine gamma lyases proteins are to be understood, which convert methionine to ammonia, Catalyze 2-oxobutyrate and methyl mercaptan.

Numerous proteins are called pre-proteins, thus formed together with a signal peptide. Then underneath to understand the N-terminal part of the protein, its function mostly consists in the removal of the protein formed from the producing cell into the periplasm or the surrounding To ensure medium and / or its correct folding. Then will the signal peptide under natural Cleaved conditions from a signal peptidase from the rest of the protein, so this its actual catalytic activity without the existing ones N-terminal amino acids exercises.

For technical applications are due to their enzymatic activity Peptides, that is the enzymes processed after their production compared to the pre-proteins prefers.

Pro proteins are inactive precursors of proteins. Their predecessor with signal sequence are called pre-pro proteins designated.

For the purposes of the present application, nucleic acids are understood to mean the molecules which are naturally built up from nucleotides and serve as information carriers and which are responsible for the linear amino acid sequence encode in proteins or enzymes. They can be present as a single strand, as a single strand complementary to this single strand or as a double strand. As the naturally more permanent information carrier, the nucleic acid DNA is preferred for molecular biological work. In contrast, an RNA is formed for the implementation of the invention in a natural environment, such as, for example, in an expressing cell, which is why RNA molecules essential to the invention also represent embodiments of the present invention.

In DNA, the sequences are both complementary strands in all three possible To take into account reading frames. It must also be taken into account that different Codon triplets for the same amino acids can code, so that one certain amino acid sequence of several different and possibly only minor identities Nucleotide sequences can be derived (degeneracy of the genetic Codes). Moreover different organisms show differences in the use of these codons on. For these reasons have to both amino acid sequences as well as nucleotide sequences when considering the protection area are included and the specified nucleotide sequences are in each case only as an exemplary coding for a particular amino acid sequence to watch.

The unit of information corresponding to a protein is also referred to as a gene in the sense of the present application.

The method according to the invention comprises the production recombinant proteins. According to the invention, these include all genetic engineering or to understand microbiological processes based on that the Genes for the proteins of interest into a suitable one for production Host organism introduced and transcribed and translated by this become. The relevant ones are suitably introduced Genes over Vectors, especially expression vectors; but also about those that cause the gene of interest in the host organism into an existing one genetic element such as the chromosome or other vectors can be inserted can. The functional unit consisting of gene and promoter and any Further counter-elements are used according to the invention as an expression cassette designated. However, you must not necessarily as a physical unit.

To a professional it is about nowadays generally known methods, such as chemical synthesis or the polymerase chain reaction (PCR) in connection with molecular biological and / or protein-chemical standard methods possible, based on known DNA and / or amino acid sequences the corresponding nucleic acids to complete To produce genes. Such methods are, for example, from the "Lexicon biochemistry ", Spektrum Akademischer Verlag, Berlin, 1999, Volume 1, pp. 267-271 and Volume 2, pp. 227-229.

amendments the nucleotide sequence, as known, for example, from molecular biological methods can be brought about referred to as mutations. Depending on the type of change, one knows, for example Deletion, insertion or substitution mutations or such, in which different genes or parts of genes fused together (shuffling); these are gene mutations. The associated organisms are called mutants. Those derived from mutant nucleic acids Proteins are called variants. For example, deletion, Insertion, substitution mutations or fusions to deletion, insertion-substituted mutation or fusion genes and at the protein level corresponding deletion, insertion or substitution variants, or fusion proteins.

Fragments are all proteins or understood peptides that are smaller than natural proteins or those that are complete correspond to translated genes, and for example also synthetically can be obtained. Because of their amino acid sequences can the relevant complete Proteins are assigned. For example, they can be the same Accept structures or perform proteolytic activities or partial activities, such as for example the complexation of a substrate. Fragments and deletion variants of starting proteins are basically the same; during fragments rather smaller fragments represent, the deletion mutants tend to lack only short areas, and thus only individual sub-functions.

The fragments correspond at the nucleic acid level the partial sequences.

Among chimeric or hybrid proteins in the sense of the present application, such proteins are to be understood which are composed of elements that are naturally different Polypeptide chains from the same organism or from different Organisms originate. This procedure is also called shuffling or fusion mutagenesis called. The purpose of such a merger can be, for example, with the help of the fused protein part a certain enzymatic Function or to modify. It is within the meaning of the present invention irrelevant whether such a chimeric protein from a single polypeptide chain or multiple subunits exists on which different functions can be distributed. to Realization of the latter alternative is possible, for example, post-translational or only after a purification step by a targeted proteolytic Cleavage a single chimeric Breaking down the polypeptide chain into several.

Proteins obtained by insertion mutation are to be understood as those variants which are known per se by inserting a nucleic acid or protein fragment into the Aus have been obtained. Because of their principle similarity, they can be assigned to the chimeric proteins. They differ from those only in the size ratio of the unchanged protein part to the size of the entire protein. The proportion of foreign protein in such insertion-mutated proteins is lower than in chimeric proteins.

Inversion mutagenesis, i.e. a partial one Reverse sequence, can be a special form of deletion, as well of the insertion. The same goes for one of the original ones amino acid sequence different regrouping of different molecular parts. It can be used both as Deletion variant, as an insertion variant, as well as a shuffling variant of the original Proteins are viewed.

Derivatives are defined in the sense of present application understood such proteins, their pure amino acid chain has been chemically modified. Such derivatizations can, for example biologically in connection with protein biosynthesis by the Host organism. Therefor can molecular biological methods are used. But you can also carried out chemically through the chemical conversion of a side chain of a amino acid or by covalently linking another compound to the protein. Such a connection can also be others, for example Proteins act, for example, via bifunctional chemical Compounds bound to proteins of the invention become. Such modifications can, for example, the substrate specificity or binding strength affect the substrate or temporarily block the enzymatic activity bring, if the coupled substance is an inhibitor. For example, for the period of storage may be useful. Likewise is under derivatization understand the covalent bond to a macromolecular support.

Proteins can also react via a Antiserum or a specific antibody to groups immunologically related proteins can be summarized. The relatives of a Groups are characterized by the fact that they are recognized by an antibody have antigenic determinants.

In the sense of the present invention all enzymes, proteins, fragments and derivatives, provided they do not need to be explicitly addressed as such under summarized the generic term proteins.

Under vectors in the sense of present invention understood elements consisting of nucleic acids, which as the characteristic nucleic acid region contain a gene of interest. You can do this in one species or a cell line across more generations or cell divisions than from the rest of the genome independently to establish a replicating, stable genetic element. vectors are special plasmids, especially when used in bacteria, circular genetic elements. A distinction is made in genetic engineering on the one hand between such vectors, the storage and thus so to speak also serve genetic engineering work, the so-called cloning vectors, and on the other hand, those who fulfilled the function, the thing of interest Realize gene in the host cell, that is, the expression of the subject To enable proteins. These vectors are called expression vectors.

By comparison with known enzymes, which are stored, for example, in generally accessible databases are, can from the amino acid or nucleotide sequence the enzymatic activity of a particular enzyme conclude. This can be caused by other areas of the protein that are not are involved in the actual reaction, qualitatively or quantitatively be modified. this could for example enzyme stability, the activity, the reaction conditions or the substrate specificity.

Such a comparison happens because that similar Sequences in the nucleotide or amino acid sequences the proteins under consideration are assigned to one another. This calls one homologation. A tabular assignment of the concerned Positions are called alignment. When analyzing nucleotide sequences are both complementary strands and all three possible To consider reading frames; likewise the degeneracy of the genetic code and the organism-specific one Codon usage. Alignments are now created using computer programs, such as through the algorithms FASTA or BLAST; this The procedure is described, for example, by D. J. Lipman and W. R. Pearson (1985) in Science, volume 227, pp. 1435-1441.

A compilation of all in the matching sequences Positions is called the consensus sequence.

Such a comparison also allows a statement about the similarity or homology of the compared sequences to one another. This will in percent identity, this means the proportion of identical nucleotides or amino acid residues reproduced in the same positions. A broader concept of homology relates the conserved amino acid exchanges in this value. The percent similarity is then mentioned. Such Statements can be made about whole Proteins or genes or just about individual areas are hit.

The creation of an alignment is the first step in defining a sequence space. This hypothetical space encompasses all sequences to be derived by permutation in individual positions, which result taking into account all variations occurring in the relevant individual positions of the alignment. Every hypothetically possible protein molecule forms a point in this sequence space. For example, there are two amino acid sequences which, if largely identical, are based on only two different ones Sites each have two different amino acids, thus a sequence space of four different amino acid sequences. A very large sequence space is obtained if additional homologous sequences are found for individual sequences in a space. Such high homologies, which exist in pairs, can also be used to recognize very low homologous sequences as belonging to a sequence space.

Homologous areas of different Proteins are those with the same functions, which are characterized by matches in the primary amino acid sequence reveal. It goes down to complete identities in the smallest Areas, so-called boxes, which contain only a few amino acids and mostly for the total activity perform essential functions. The smallest sub-functions are among the functions of the homologous areas that of all protein Understand function, such as the formation of individual hydrogen bonds for complexing a substrate or transition complex.

In step b) of the method according to the invention becomes the nucleic acid suitably cloned into a vector. The molecular biological Dimension of the invention thus consists in vectors with the genes for the corresponding Proteins. You can do this such as include which differ from bacterial plasmids, from viruses or from bacteriophages deduce, or predominantly synthetic vectors or plasmids with elements of the most varied Origin. With the other existing genetic elements fortune vectors, in the host cells concerned over several generations to establish as stable units. It is in the spirit of the invention irrelevant whether they establish themselves as separate units extrachomosomally or integrate into a chromosome. Which of the numerous from the State-of-the-art systems selected depend on the individual case. decisive can for example, the number of copies that can be achieved Selection systems, including above all antibiotic resistance, or the cultivability of the host cells capable of receiving the vectors his.

The vectors form suitable starting points for molecular biological and biochemical studies of the gene or protein in question and for Further developments according to the invention and ultimately for the amplification and production of proteins according to the invention. In this respect, they pose embodiments of the present invention as the sequences of the contained nucleic acid regions according to the invention each within the above closer designated homology ranges.

Preferred embodiments of the present Invention are cloning vectors. These are suitable in addition to the Storage, biological amplification or selection of the interesting gene for the characterization of the gene in question, for example via the Create a restriction map or sequencing. cloning vectors are therefore also preferred embodiments of the present Invention because it is a transportable and storable form represent the claimed DNA. They are also preferred starting points for molecular biology Techniques that are not bound to cells, such as the polymerase chain reaction.

Expression vectors are chemical similar to the cloning vectors, but differ in the partial sequences that enable them to the for replicate the production of protein-optimized host organisms and to express the contained gene there. preferred Embodiments are Expression vectors, which themselves are the genetic necessary for expression Wear items. Expression is used, for example, by promoters affected which regulate the transcription of the gene. So expression through the natural, originally promoter localized in front of this gene, but also after genetic engineering Fusion both by one provided on the expression vector Promoter of the host cell as well as by a modified or one completely another promoter of another organism.

Preferred embodiments are such expression vectors the about changes the culture conditions or addition of certain compounds, such as for example, the cell density or special factors, adjustable are. Allow expression vectors that this associated Protein heterologous, i.e. in an organism other than that which it is natural can be won, is produced. Also a homologous protein extraction from one the gene naturally expressing host organism via a matching vector is within the scope of the present Invention. This can have the advantage of being natural, with translation related modification reactions to the resulting Protein done the same way as they are naturally would expire.

Embodiments of the Present Invention can can also be cell-free expression systems in which protein biosynthesis is reproduced in vitro. Such expression systems are in State of the art also established.

The in vivo synthesis of an enzyme according to the invention, that is to say by living cells, requires the transfer of the associated gene into a host cell, the so-called transformation. In principle, all organisms are suitable as host cells, that is to say prokaryotes, eukaryotes or cyanophyta. Preferred are host cells that are genetically easy to handle, for example as regards transformation with the expression vector and its stable establishment, for example unicellular fungi or bacteria. In addition, preferred host cells are characterized by good microbiological and biotechnological handling out. This applies, for example, to easy cultivation, high growth rates, low demands on fermentation media and good production and secretion rates for foreign proteins. Often the optimal expression systems for the individual case must be determined experimentally from the abundance of different systems available according to the prior art. In this way, each protein according to the invention can be obtained from a large number of host organisms.

Preferred embodiments are such Host cells represent that due to genetic regulatory elements that provided for example on the expression vector will be present in these cells from the start can, in their activity are adjustable. For example, through the controlled addition of chemical compounds that serve as activators by change the cultivation conditions or when a certain one is reached Cell density can these are stimulated for expression. This enables a very economical one Production of the proteins of interest.

Preferred host cells are prokaryotic or bacterial cells. Bacteria are distinguished from eukaryotes usually by shorter ones Generation times and lower demands on the cultivation conditions out. This allows inexpensive Methods for obtaining proteins according to the invention are established. Gram-negative bacteria such as E. coli a large number of proteins are secreted into the periplasmic space, that is, in the compartment between the two membranes enclosing the cells. This can be for special applications may be advantageous. Gram positive bacteria, such as Bacilli or Actinomyceten or other representatives the Actinomycetales, in contrast, have no outer membrane, so that secreted Proteins are immediately released into the nutrient medium surrounding the cells, from which emerges according to another preferred embodiment the expressed proteins of the invention let it clean up directly.

A variant of this experimental principle represent expression systems in which additional genes, for example those that are provided on other vectors, production proteins of the invention influence. This can be modifying gene products act or those with the protein of the invention are to be cleaned up together, for example for its enzymatic Influence function. For example, it may be others Proteins or enzymes, inhibitors or such elements, that affect the interaction with different substrates.

Because of the extensive experience, for example with regard to molecular biological methods and which can be cultivated with coliform bacteria, these represent preferred embodiments of the present invention. Those are particularly preferred of the genera Escherichia coli, especially non-pathogenic, for the biotechnological Production of suitable strains.

Representative representatives of this Genera are the K12 derivatives and the B strains from Escherichia coli. strains which are based on known genetic and / or microbiological Let methods be derived from them, and thus as their derivatives can be viewed own for genetic and microbiological work of the greatest importance and are preferred for the development of methods according to the invention used. Such derivatives can for example about Deletion or insertion mutagenesis with regard to their requirements changed to the cultural conditions be other or additional Have selection markers or express other or additional proteins. In particular, these can be derivatives that are additional to the manufactured according to the invention Protein express other economically interesting proteins.

Such microorganisms are also preferred, which are characterized in that after transformation with one of the vectors described above have been obtained. there can be, for example, cloning vectors that for storage and / or modification in any bacterial strain have been introduced. Such steps are in storage and in the further development of relevant genetic elements in general common. Because from these microorganisms the relevant genetic elements immediately transferred into gram-negative bacteria suitable for expression can be are also the predicted transformation products for realizations of the subject matter of the invention.

Eukaryotic cells can also develop for the production of proteins according to the invention suitable. Examples of this are mushrooms like Actinomycetes or yeasts like Saccharomyces or Kluyveromyces. This can be particularly advantageous, for example, if the Proteins specific modifications related to their synthesis experience that enable such systems. These include, for example the binding of low molecular weight compounds such as membrane anchors or Oligosaccharides.

The host cells of the method according to the invention are cultivated and fermented in a manner known per se, for example in discontinuous or continuous systems. In the first Case becomes a suitable nutrient medium inoculated with the recombinant bacterial strains and the product after an experimentally determined period from the medium harvested. Continuous fermentations are characterized by reaching a steady state from where about for a comparatively long period of time, cells die telly but also regrow and at the same time product is removed from the medium can be.

Fermentation processes are known per se from the prior art and represent the actual large-scale production step; followed by a suitable purification method.

All fermentation processes that on one of the above Processes for the production of the recombinant proteins are based correspondingly preferred embodiments this subject of the invention.

The manufacturing processes used, for the Host cells and / or the proteins to be produced are each optimal Conditions based on the previously optimized culture conditions of the concerned strains to the knowledge of the person skilled in the art, for example with regard to fermentation volume, Media composition, oxygen supply or stirrer speed can be determined experimentally.

Fermentation process thereby are marked that the Fermentation over carried out an inflow strategy will also be considered. Here, the media components, that are consumed by ongoing cultivation, fed; you also speaks of a feeding strategy. hereby can considerable Increases in cell density as well as in dry biomass and / or especially the activity of the protein of interest.

Similarly, the fermentation also be designed so that unwanted metabolites filtered out or by adding buffer or suitable in each case Counterions are neutralized.

The protein produced can subsequently be made the fermentation medium are harvested. This fermentation process is opposite the product preparation from the dry matter preferred, required however the provision suitable secretion markers and transport systems.

Without secretion, the purification of the protein from the cell mass is required, and there are also several Traffickers known as precipitation e.g. by ammonium sulfate or ethanol, or the chromatographic Cleaning if necessary until homogeneous. The majority of those described technical process is likely however with an enriched, stabilized preparation get along.

All the elements already mentioned above can can be combined into processes to produce proteins according to the invention. It is there for any protein of the invention a variety of possible combinations possible process steps. The best practice must be for everyone concrete individual case can be determined experimentally.

Preferably isolated or synthesized one in step a) one for a nucleic acid encoding methionine gamma lyase which is selected among METase genes from the protozoon Trichomonas vaginalis and the bacteria Treponema denticola, Pseudomonas putida, Clostridium sporogenes and Fusobacterium nucleatum, particularly preferably Treponema denticola.

A Clostridium sporogenes METase is described for example in the publication of Kreis W and Hession C; "Isolation and purification of L-methionine-alpha-deamino-gamma-mercaptomethane-lyase (L-methioninase) from Clostridium sporogenes "; Cancer Res. 1973 Aug; 33 (8): 1862-5.

It is also preferred in step a) one for isolated or synthesized a nucleic acid encoding methionine gamma lyase, whose nucleotide sequence with that in Seq. 6 (Pseudomonas METase gene putida) specified nucleotide sequence at least 50%, at least 75%, at least 80%, preferably at least 85%, in particular at least 90%, particularly preferably at least 95% and very particularly preferably 100% matches.

It is also preferred in step a) one for isolated or synthesized a nucleic acid encoding methionine gamma lyase, whose nucleotide sequence with that in Seq. 5 (METase gene from Fusobacterium nucleatum) specified nucleotide sequence at least 50%, at least 75%, at least 80%, preferably at least 85%, in particular at least 90%, particularly preferably at least 95% and very particularly preferably 100% matches.

In step is particularly preferred a) one for isolated or synthesized a nucleic acid encoding methionine gamma lyase, whose nucleotide sequence with that in Seq. 4 (Treponema METase gene denticola) specified nucleotide sequence at least 50%, at least 75%, at least 80%, preferably at least 85%, in particular at least 90%, particularly preferably at least 95% and entirely: particularly preferably 100% matches.

It is also preferred in step a) a nucleic acid isolated or synthesized for a methionine gamma lyase encoded whose amino acid sequence with one of the in Seq. 1 to 3 specified amino acid sequences to at least 70%, preferably at least 80%, in particular at least 90%, particularly preferably at least 95% and very particularly preferably is 100% identical, or whose amino acid sequence contains a part which with one of the in Seq. 1 to 3 specified amino acid sequences to at least 70%, preferably at least 80%, in particular at least 90%, particularly preferably at least 95% and very particularly preferably is 100% identical.

In step a) it is particularly preferred to isolate or synthesize a nucleic acid which codes for a methionine gamma lyase, the amino acid sequence of which corresponds to one of those described in Seq. 1 to 3 specified amino acid sequences are at least 70%, preferably at least 80%, in particular at least 90%, particularly preferably at least 95% and very particularly preferably 100% identical, or the amino acid sequence of which contains a part which corresponds to one of those described in Seq. 1 to 3 specified amino acid sequences to at least 70%, preferably at least 80%, in particular at least 90%, particularly preferably at least 95% and very particularly preferably 100% identical.

In step is particularly preferred a) a nucleic acid isolated or synthesized for a methionine gamma lyase encoded whose amino acid sequence contains a part with the consensus sequence [DQ] - [LIMVF] -X (3) - [STAGC] - [STAGCI] -T-K- [FYWQ] - [LIVMF] -X-G- [HQ] - [SGNH] (Seq. ID 7) at least 96%, in particular at least 97%, preferably at least 98%, preferably at least 99%, particularly is preferably 100% identical.

Preferably in step c) inventive method expresses or uses a methionine gamma lyase whose amino acid sequence with the one in Seq. 3 (METase from Pseudomonas putida) given amino acid sequence at least 50%, preferably at least 70%, in particular at least 80%, particularly preferably at least 90% and very particularly preferred 100% matches.

It is also preferred in step c) the method according to the invention expresses or uses a methionine gamma lyase whose amino acid sequence with the one in Seq. 2 (METase from Fusobacterium nucleatum) amino acid sequence at least 50%, preferably at least 70%, in particular at least 80%, particularly preferably at least 90% and very particularly preferred 100% matches.

In step is particularly preferred c) the method according to the invention expresses or uses a methionine gamma lyase whose amino acid sequence with the one in Seq. 1 (METASE from Treponema denticola) given amino acid sequence at least 50%, preferably at least 70%, in particular at least 80%, particularly preferably at least 90% and very particularly preferred 100% matches.

Host cells suitable for expression according to the invention in step b), microorganisms, in particular bacteria, are special preferably E. coli.

Detection agents suitable according to the invention for reaction products Examples are reactions catalyzed by methionine gamma lyase Salts of metals, the salts which are sparingly soluble with sulfide ions with a solubility product form from <10 - 20, preferably a heavy metal salt that is heavy with sulfide ions soluble Connection fails. For example, lead acetate is suitable (preferred concentration: about 4 mg / l widest concentration: about 0.1 to about 10 mg / l). Possible are also salts of Hg, Ni, Sb, Sn, Zn, Ag, Cu, Co, Cd, where in addition to Pb, Ag and Cu are preferred. Since the solubility of sulfide ions in Acid is particularly low, a slightly acidic medium is preferred (pH less than or equal to 7). In addition to methionine, other sulfur sources can also be used sulfur-containing amino acids (esp. cysteine) and organic sulfur compounds (glutathione) serve (preferred concentration: about 500 mg / l widest concentration: about 50 to about 1000 mg / l). From the cells due to the METase activity from methionine Sulfides formed as a sulfur source are precipitated as lead sulfide and to lead to a black color the colony.

The determination of reaction products by methionine-gamma-lyase catalyzed reactions can be carried out by one of the above-mentioned detection agents or z. B. by detection of the products formed in the following reaction: Methionine → methyl mercaptan + α-ketobutyrate + ammonia (catalyzes ME-Tase).

The enzyme activity of the METase is measured indirectly, for example, by converting α-ketobutyrate and NADH ₂ to 2-hydroxybutyrate and NAD ⁺ in a photometer at 340 nm (absorption maximum of NADH ₂ ) by reducing the absorbance.

Other objects of the present invention are cloning vectors and expression vectors isolated according to the invention or synthesized nucleic acids contain.

Another subject of the present Invention is a test kit for finding effective substances against Halitosis, gingivitis or periodontitis, the means of carrying out the inventive method includes.

• a) eine für eine Methionin-Gamma-Lyase codierende Nukleinsäure aus einem pro- oder eukaryotischen Organismus isoliert oder in vitro synthetisiert,
• b) die für eine Methionin-Gamma-Lyase codierende Nukleinsäure kloniert und in geeigneten Wirtszellen exprimiert oder die Methionin-Gamma-Lyase in vitro synthetisiert,
• c) die Methionin-Gamma-Lyase exprimierenden Wirtszellen in voneinander räumlich getrennten Ansätzen in Gegenwart einer Schwefelquelle und eines geeigneten Nachweisagens für Reaktionsprodukte durch Methionin- Gamma-Lyase katalysierter Reaktionen kultiviert oder; die Methionin-Gamma-Lyase in voneinander räumlich getrennten Ansätzen in Gegenwart von Methionin, Cystein oder Glutathion in ein in vitro Testsystem einbringt,
• d) die Ansätze mit möglicherweise die Funktion der Methionin-Gamma-Lyase hemmenden Substanzen versetzt,
• e) für jeden der voneinander räumlich getrennten Ansätze die Bildung von Reaktionsprodukten durch Methionin-Gamma-Lyase katalysierter Reaktionen bestimmt und so

den Nachweis der Wirksamkeit von Substanzen gegen Halitosis Gingivitis oder Parodontitis führt.Another object of the present invention is the use of methionine gamma lyases (METase) for finding effective substances against halitosis, gingivitis or periodontitis;
Another object of the present invention is a test method for detecting the effectiveness of substances against halitosis gingivitis or periodontitis, which is characterized in that

• a) a nucleic acid coding for a methionine gamma lyase is isolated from a pro- or eukaryotic organism or synthesized in vitro,
• b) the nucleic acid coding for a methionine gamma lyase is cloned and expressed in suitable host cells or the methionine gamma lyase is synthesized in vitro,
• c) the host cells expressing methionine-gamma-lyase are cultivated in spatially separate batches in the presence of a sulfur source and a suitable detection agent for reaction products or reactions catalyzed by methionine-gamma-lyase; the methionine gamma lyase in spatially separated batches in the presence of methionine, cysteine or glutathione in an in vitro test system brings in,
• d) the approaches are mixed with substances that possibly inhibit the function of methionine gamma lyase,
• e) for each of the spatially separated approaches, the formation of reaction products determined by reactions catalyzed by methionine-gamma-lyase, and so on

proves the effectiveness of substances against halitosis gingivitis or periodontitis.

• a) wirksame Wirkstoffe (Hemmstoffe der METase) mit Hilfe des erfindungsgemäßen Screening-Verfahrens, oder der erfindungsgemäßen Verwendung bestimmt und
• b) als wirksam befundene Wirkstoffe (Hemmstoffe der METase) mit pharmakologisch geeigneten und verträglichen Trägern vermischt.

Another object of the present invention is a method for producing a pharmaceutical preparation for halitosis, gingivitis or periodontitis, which is characterized in that

• a) active substances (inhibitors of METase) are determined using the screening method according to the invention or the use according to the invention and
• b) active ingredients found to be effective (inhibitors of METase) mixed with pharmacologically suitable and compatible carriers.

An enzyme preparation of the overexpressed METase protein can be used to in a technical application from methionine methyl mercaptan or from cysteine hydrogen sulfide release. The advantages of this process are the controlled, gentle release of aggressive, volatile products. An enzymatic Process using methionine as a substrate can be used for structuring treatment of sulfur-containing fibers, e.g. Serve hair, feathers and wool. The resulting free SH group opens the tertiary structure the protein fiber stabilizing disulfide bridges and can e.g. as replacement for the use of thioglycolic acid serve.

Another subject of the present The invention is therefore the use of one of the above-mentioned METases, in particular a METase, the amino acid sequence of which in Seq. 1 (METASE from Treponema denticola) given amino acid sequence at least 50%, preferably at least 70%, in particular at least 80%, particularly preferably at least 90% and very particularly preferred 100% matches for the structuring treatment of sulfur-containing fibers, such as e.g. Hair, feathers and wool.

The release of sulfur Compounds underlying β-elimination reaction creates a double bond in free amino acids by subsequent rearrangement leads to deamination, alternatively in bound peptide amino acids but through water addition leads to the generation of a hydroxyamino acid. Ultimately, it can Cysteine serine and methionine threonine. So can a METase protein for the desulfurization of a sulfur-containing protein, z. B. a cosmetic raw material can be used.

Another subject of the present The invention is therefore the use of one of the above-mentioned METases, in particular a METase, the amino acid sequence of which in Seq. 1 (METASE from Treponema denticola) given amino acid sequence at least 50%, preferably at least 70%, in particular at least 80%, particularly preferably at least 90% and very particularly preferred 100% matches for desulfurization of a sulfur-containing protein, e.g. B. one cosmetic raw material.

Finally, in a synthetic process by reversing the enzymatic reaction (expediently under high pressure) using gaseous hydrogen sulfide or methyl mercaptan and ammonia from pyruvate or a-ketobutyrate the valuable amino acids Cysteine and methionine are generated.

Methionine has an additive for animal Feed is of considerable technical importance and is used in complex Process manufactured.

Another subject of the present The invention is therefore the use of one of the above-mentioned METases, in particular a METase, the amino acid sequence of which in Seq. 1 (METASE from Treponema denticola) given amino acid sequence at least 50%, preferably at least 70%, in particular at least 80%, particularly preferably at least 90% and very particularly preferred 100% matches for the synthesis of amino acids Cysteine and methionine.

To use the METase protein in technical, cosmetic or pharmaceutical preparations To provide, all methods for the production of recombinant come Proteins into consideration.

The following examples illustrate the Invention without, however, restricting it to:

Example 1: heterologous Expression of the mgl1 gene from T. denticola

The coding sequence of the mgl1 gene (METase gene) from T. denticola was amplified using PCR using database comparisons and cloned into a commercially available vector (pGEM-TEasy, Promega). Expression in E. coli can be used for screening for active substances according to the invention as follows:
The E. coli cells transformed with the vector containing the mgl1 gene are grown on solid nutrient media (for example LB medium, 1% tryptone, 0.5% yeast extract, 0.5% NaCl, 1-2% agar). To detect the formation of sulfur-containing compounds, a sulfur source (methionine; preferred concentration: 350 mg / l, furthest concentration: 50-1000 mg / l) and a heavy metal salt, which precipitates as a poorly soluble compound with sulfide ions, are added to the medium. In the example, lead acetate was added to the medium (preferred concentration: 4 mg / l broadest concentration: 0.1-10 mg / l).

The bacteria are grown at 37 ° C for 2-7 Days (preferably 3 days) under anaerobic conditions in petri dishes or multiwell plates. Aerobic incubation is also possible, required but incubation periods of 5-10 days.

The inhibitory effect of those to be tested Substance becomes visible through the lack of blackening of the colonies. It is advantageous that at the same time a growth-inhibiting Property of the substance to be checked can, since the expression of the mgl1 gene alone has no influence that has growth.

Example 2: Enzymatic Tests with purified Mgl proteins

The mgl1 gene from T. denticola is provided at the 5 'end with a sequence which codes for six histidine residues and clones into an expression vector with a constitutively expressed promoter (pGEM). The cells transformed with this plasmid are grown overnight in LB medium, then a further 50 ml culture with an OD ₆₀₀ = 0.05 from the previous culture is inoculated and grown at 37 ° C. to an OD ₆₀₀ = 0.2 , The culture is centrifuged off, resuspended in 1 ml of lysis buffer (50 mM NaHP ₂ O ₄ ; 300 mM NaCl; 10 mM imidazole; pH 8) and frozen at least at -70 ° C. overnight. The digestion takes place after addition of lysozyme (final concentration 1 mg / ml) and an incubation step of 30 min. on ice under native conditions using ultrasound (in total 2 min. with 5 sec. pulses and 5 sec. pause). The lysate is kept for 20-30 min. Centrifuged at 13,000 rpm and 600 μl of the supernatant purified on a Ni-NTA column (Qiagen) according to the manufacturer's instructions.

The actual enzyme assay for screening for active substances according to the invention is carried out as follows:
10-100 μg (1-1000μg) of the purified enzyme are dissolved in 50 mM Tris-HCl buffer (pH 7 (6-8)) together with 100 mM methionine (10-1000 mM), 2 mM (1-10 mM) NADH, 100 μM pyridoxal phosphate and 10U lactate dehydrogenase Roche, preferably from cardiac muscle cells, isoenzyme LDH-1) or hydroxybutyrate dehydrogenase (TEso Diagnostics) incubated at 25 ° C (in Eppendorf tubes or microtiter plates). The enzyme activity of the METase is measured indirectly via the conversion of 2-oxobutyrate and NADH ₂ to 2-hydroxybutyrate and NAD ⁺ in a photometer at 340 nm (absorption maximum of NADH ₂ ) via the decrease in absorbance. Alternatively, the conversion in cuvettes can be measured under direct observation in the photometer. Incubation time 10-30 min at 25 ° C.

With the help of this assay, substances can be tested for their inhibitory effect on the METase. A control in which the substance is tested for its inhibitory effect on LDH is important. To do this, the above experiment is carried out without methionine and METase, but in the presence of 2 mM pyruvate. Any inhibitory effects can be eliminated by adding Na ₂ SO ₃ .

page 6

Claims (24)

Screening method for identifying active substances against halitosis, gingivitis or periodontitis, characterized in that a) a nucleic acid coding for a methionine gamma-lyase is isolated from a pro- or eukaryotic organism or synthesized in vitro, b) that for a methionine Nucleic acid encoding gamma-lyase is cloned and expressed in suitable host cells or the methionine-gamma-lyase is synthesized in vitro, c) the host cells expressing methionine-gamma-lyase in spatially separated approaches in the presence of a sulfur source and a suitable detection agent for reaction products by methionine -Gamma-lyase catalyzed reactions cultured; or the methionine-gamma-lyase is introduced into an in vitro test system in spatially separated batches in the presence of methionine, cysteine or glutathione, d) the batches are mixed with substances which possibly inhibit the function of the methionine-gamma-lyase, e) for each of the spatially separated approaches determine the formation of reaction products by reactions catalyzed by methionine-gamma-lyase and thus identify active substances against halitosis, gingivitis or periodontitis Screening method according to claim 1, characterized in that he in step a) one for isolated or synthesized a nucleic acid encoding methionine gamma lyase, the selected is among METase genes from the protozoon Trichomonas vaginalis and the bacteria Treponema denticola, Pseudomonas putida, Fusobacterium nucleatum, particularly preferably Treponema denticola. Screening method according to claim 1 or 2, characterized characterized that one in step a) one for one Nucleic acid encoding methionine gamma lyase isolated or synthesized, whose nucleotide sequence with that in Seq. 6 (Pseudomonas METase gene putida) specified nucleotide sequence at least 50%, at least 75%, at least 80%, preferably at least 85%, in particular at least 90%, particularly preferably at least 95% and very particularly preferably 100% matches. Screening method according to one of the preceding Expectations, characterized in that one in Step a) one for a methionine gamma lyase coding nucleic acid isolated or synthesized, the nucleotide sequence of which in Seq. 5 (METase gene from Fusobacterium nucleatum) indicated nucleotide sequence at least 50%, at least 75%, at least 80%, preferably at least 85%, in particular at least 90%, particularly preferred at least 95% and very particularly preferably 100%. Screening method according to one of the preceding Expectations, characterized in that one in Step a) one for a methionine gamma lyase coding nucleic acid isolated or synthesized, the nucleotide sequence of which in Seq. 4 (METASE gene from Treponema denticola) specified nucleotide sequence at least 50%, at least 75%, at least 80%, preferably at least 85%, in particular at least 90%, particularly preferred at least 95% and very particularly preferably 100%. Screening method according to one of the preceding Expectations, characterized in that one in Step a) a nucleic acid isolated or synthesized for a methionine gamma lyase encoded whose amino acid sequence with one of the in Seq. 1 to 3 specified amino acid sequences to at least 70%, preferably at least 80%, in particular at least 90%, particularly preferably at least 95% and very particularly preferably is 100% identical, or whose amino acid sequence contains a part which with one of the in Seq. 1 to 3 specified amino acid sequences to at least 70%, preferably at least 80%, in particular at least 90%, particularly preferably at least 95% and very particularly preferably to Is 100% identical. Screening method according to one of the preceding Expectations, characterized in that one in Step a) a nucleic acid isolated or synthesized for a methionine gamma lyase encoded whose amino acid sequence with one of the in Seq. 1 to 3 specified amino acid sequences to at least 70%, preferably at least 80%, in particular at least 90%, particularly preferably at least 95% and very particularly preferably is 100% identical, or whose amino acid sequence contains a part which with one of the in Seq. 1 to 3 specified amino acid sequences to at least 70%, preferably at least 80%, in particular at least 90%, particularly preferably at least 95% and very particularly preferably to Is 100% identical. Screening method according to one of the preceding Expectations, characterized in that one in Step a) a nucleic acid isolated or synthesized for a methionine gamma lyase encoded whose amino acid sequence contains a part with the consensus sequence [DQ] - [LIMVF] -X (3) - [STAGC] - [STAGCI] -T-K- [FYWQ] - [LIVMF] -X-G- [HQ] - [SGNH] (Seq. ID 7) at least 96%, in particular at least 97%, preferably at least 98%, preferably at least 99%, particularly is preferably 100% identical. Screening method according to one of the preceding Expectations, characterized in that one in Step c) of the method allows a methionine gamma lyase to be expressed or uses their amino acid sequence with the one in Seq. 3 (METase from Pseudomonas putida) given amino acid sequence at least 50%, preferably at least 70%, in particular at least 80%, particularly preferably at least 90% and very particularly preferred 100% matches. Screening method according to one of the preceding Expectations, characterized in that one in Step c) of the method allows a methionine gamma lyase to be expressed or uses their amino acid sequence with the one in Seq. 2 (METase from Fusobacterium nucleatum) amino acid sequence at least 50%, preferably at least 70%, in particular at least 80%, particularly preferably at least 90% and very particularly preferred 100% matches. Screening method according to one of the preceding claims, characterized in that in step c) of the method a methionine gamma lyase is expressed or used, the amino acid sequence of which corresponds to that in Seq. 1 (METASE from Treponema denticola) specified amino acid sequence to at least at least 50%, preferably at least 70%, in particular at least 80%, particularly preferably at least 90% and very particularly preferably 100%. Screening method according to one of the preceding Expectations, characterized in that one in Step b) of the method uses host cells for the expression, the selected are particularly preferred among microorganisms, especially bacteria E. coli. Screening method according to one of the preceding Expectations, characterized in that one in Step c) of the method as detection agents for reaction products by methionine gamma lyase catalyzed reactions lead acetate and salts of Hg, Ni, Sb, Sn, Zn, Ag, Cu, Co and Cd, preferably salts of Pb, Ag and Cu. Screening method according to one of the preceding Expectations, characterized in that one in Step c) of the process as a sulfur source methionine, cysteine or uses glutathione. Screening method according to one of claims 1 to 12, characterized in that one in step c) the determination of reaction products by methionine gamma lyase catalyzed reactions by detection of the product α-ketobutyrate formed performs. Cloning vectors or expression vectors that according to step a) isolated or synthesized from one of the preceding claims nucleic acids contain. Host cells, the cloning vectors or expression vectors according to claim 16 included. Test kit to find effective substances against Halitosis, gingivitis or periodontitis, the means of carrying out the Method according to one of the claims 1 to 15 comprises. Use of methionine gamma lyases (METase) for Finding effective substances against halitosis, gingivitis or Periodontitis. Test procedure to prove the effectiveness of substances against halitosis gingivitis or periodontitis, characterized in that that he a) one for a nucleic acid encoding a methionine gamma lyase from a pro- or eukaryotic Organism isolated or synthesized in vitro, b) that for a methionine gamma lyase coding nucleic acid cloned and expressed in suitable host cells or the methionine gamma lyase synthesized in vitro, c) the host cells expressing methionine gamma lyase in each other spatial separate approaches in the presence of a sulfur source and a suitable detection agent for reaction products reactions catalyzed by methionine gamma lyase or; the methionine gamma lyase in each other spatially separate approaches in the presence of methionine, cysteine or glutathione in an in vitro Test system, d) the approaches with possibly the function of Methionine gamma lyase inhibiting substances added, e) for each of the spatially separated approaches the formation of reaction products catalyzed by methionine gamma lyase Reactions determined and such proof of the effectiveness of Substances against halitosis gingivitis or periodontitis. Process for the manufacture of a pharmaceutical Preparation against halitosis, gingivitis or periodontitis caused by this is characterized in that one a) active substances (inhibitors of METase) with the help of the screening process after a of claims 1 to 15, or the use according to the invention determined according to claim 19 and b) active ingredients found to be effective (Inhibitors of METase) with pharmacologically suitable and tolerable carriers mixed. Use of a METase as defined in one of the Expectations 1 to 15, in particular a METase, its amino acid sequence with the one in Seq. 1 (METASE from Treponema denticola) given amino acid sequence at least 50%, preferably at least 70%, in particular at least 80%, particularly preferably at least 90% and very particularly preferred 100% matches for the structuring treatment of sulfur-containing fibers, such as e.g. Hair, feathers and wool. Use of a METase as defined in one of the Expectations 1 to 15, in particular a METase, its amino acid sequence with the one in Seq. 1 (METASE from Treponema denticola) given amino acid sequence at least 50%, preferably at least 70%, in particular at least 80%, particularly preferably at least 90% and very particularly preferred 100% matches for desulfurization of a sulfur-containing protein, preferably of a cosmetic raw material. Use of a METase as defined in one of the Expectations 1 to 15, in particular a METase, its amino acid sequence with the one in Seq. 1 (METASE from Treponema denticola) given amino acid sequence at least 50%, preferably at least 70%, in particular at least 80%, particularly preferably at least 90% and very particularly preferred 100% matches for the synthesis of amino acids Cysteine and methionine.