WO2004055202A2

WO2004055202A2 - Screening method for identifying substances active against halitosis, gingivitis or parodontitis

Info

Publication number: WO2004055202A2
Application number: PCT/EP2003/013814
Authority: WO
Inventors: Dirk Bockmühl; Julia Boy; Roland Breves
Original assignee: Henkel Kommanditgesellschaft Auf Aktien
Priority date: 2002-12-17
Filing date: 2003-12-06
Publication date: 2004-07-01
Also published as: AU2003296620A1; WO2004055202A3; DE10260891A1; AU2003296620A8

Abstract

The invention relates to a screening method for identifying substances acting against halitosis, gingivitis or parodontitis, test-kits for locating substances acting against halitosis, gingivitis or parodontitis, the use of methionine-gamma-lyase (METase) for locating substances acting against halitosis, gingivitis or parodontitis. The invention also relates to a test method for detecting the effectiveness of substances acting against halitosis, gingivitis or parodontitis and to a method for producing a pharmaceutical preparation against halitosis, gingivitis or parodontitis.

Description

Screening procedure for the identification of effective substances against halitosis, gingivitis or periodontitis

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.

Bad breath, also called halitosis or "foetor ex ore", can have different causes. In healthy people, however, bad breath is in most cases caused by bacteria in the mouth or throat that metabolize body cells or food residues, whereby as a breakdown products of proteins u. a. Volatile sulfur compounds (VSC) are created, which are held responsible for the bad smell.

These sulfur compounds are in particular: H ₂ S = hydrogen sulfide (about 30%), OH3-SH = methyl mercaptan (about 60%) and CH3-S-CH3 = dimethyl sulfide (about 10%).

The volatile sulfur compounds are sometimes very aggressive and can damage the gums and 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 Jul; 63 (7): 994-7; It is known that hydrogen sulfide and methyl mercaptan increase the permeability of the oral mucosa and can thus promote gum disease.

Gum disease is a high risk factor for health. According to studies by the World Health Organization (WHO), over 50% suffer of adult Germans with periodontal diseases that require urgent treatment.

Gum disease is popularly called 'periodontitis'. This expression is incorrect because the ending "-ose" describes a normal, age-related decline in an organ. The terms "periodontitis" or "inflammatory periodontopathy" are correct.

In addition to caries, it is the most widespread oral disease and mainly occurs in adults.

Periodontitis is an inflammatory change in the tissue surrounding the tooth, especially the jawbone, caused by bacteria. Periodontitis (= with involvement of the gum pockets and bone) always develops from gingivitis and first affects the interdental spaces that are most difficult to clean. The bacteria in the gum pocket have a destructive effect on the gums, tooth structure and bones through their metabolic products, in particular through the VSC you produce, thus constantly increasing the existing periodontitis.

This health problem is very widespread: Studies show that more teeth are lost through periodontal disease (gum disease) than from tooth decay from around the age of 35.

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 immune. 2000 Dec; 68 (12): 6912-6; an L-methionine-alpha-deamino-gamma-mercaptomethane lyase (METase) from P. gingivalis, which produces large amounts of methyl mercaptan.

However, METase from bacteria in the oral cavity has not been 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 bac- terial growth by trifluoromethionine "; Biochem Biophys Res Commun. 2002 Apr 12; 292 (4): 964-8; Page 964, right column, first paragraph;

The diagnosis of bad breath is usually carried out by bacterial cultures, sulfide detection methods and organoleptic methods (smelling), or by 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 usually been made to combat halitosis, gingivitis or periodontitis using bactericidal active ingredients, although some of these have strong side effects. Another approach involves removing the VSCs using zinc-containing compounds. However, the latter procedure results in a short-term decrease in odor formation and does not prevent gingivitis or periodontitis.

There is therefore a considerable need to provide new active substances against halitosis, gingivitis or periodontitis.

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

The present invention therefore relates to a screening method for identifying active 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 in synthesized in vitro, b) the nucleic acid coding for a methionine gamma lyase and cloned and expressed in suitable host cells or the methionine gamma lyase synthesized in vitro, c) the methionine gamma lyase expressing host cells in spatially separated approaches in the presence of a Sulfur source and a suitable neten detection agent for reaction products cultivated 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 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

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 microorganisms present in the oral cavity, without thieves. For this purpose, all substances are discarded which inhibit the formation of VSC, but at the same time kill the bacterial host cell.

In this way, side effects that are free of side effects or at least have few side effects can be found that do not damage the natural and useful bacterial colonization of the oral cavity.

Methionine-γ-lyases (METases) belong to an enzyme family that occur in pro and eukaryotes (but not in mammals) and catalyze the conversion of methionine to ammonia, 2-oxobutyrate and methyl mercaptan. Pyridoxal phosphate is required as a cofactor, which on a conserved region with the consensus sequence [DQ] - [LIMVF] -X (3) - [STAGC] - [STAGCI] -TK- [FYWQ] - [LIVMF] -XG- [HQ] - [SGNH] binds, where K acts as a pyridoxal phosphate binding site. X denotes any amino acids, while the amino acids in brackets describe the possible alternatives for this position, ie [DQ] means that either D or Q can be at this position in the consensus sequence. The METases from the protozoon Trichomonas vaginalis and the bacteria Pseudomonas putida and Porphyromonas gingivalis have already been identified described and characterized. For Fusobacterium nucleatum, the annotation of the genome identified the METase gene.

The applicant has succeeded in elucidating the sequence of the oral bacterium Treponema denticola coding for the METase protein and thus showing for the first time that METases also occur in spirochetes.

The base sequence of the mgll 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, an expression of the form "at least X%" means "X% to 100% (including the basic values X and 100 and all integer and non-integer percentage values in between)".

For the purposes of the present application, a protein is to be understood as a polymer which is composed of the natural amino acids and has a largely linear structure and usually assumes a three-dimensional structure to perform its function. In the present application, the 19 proteinogenic, naturally occurring L-amino acids are designated with the internationally used 1- and 3-letter codes.

For the purposes of the present application, an enzyme is to be understood as a protein which has a specific biochemical function. Methionine gamma lyases are proteins that catalyze the conversion of methionine to ammonia, 2-oxobutyrate and methyl mercaptan. Numerous proteins are formed as so-called pre-proteins, i.e. together with a signal peptide. This is to be understood as the N-terminal part of the protein, the function of which mostly consists in ensuring that the protein formed is discharged from the producing cell into the periplasm or the surrounding medium and / or that it is correctly folded. Subsequently, the signal peptide is split off from the rest of the protein under natural conditions by a signal peptidase, so that this exerts its actual catalytic activity without the N-terminal amino acids initially present.

Due to their enzymatic activity, the mature peptides, that is to say the enzymes processed after their production, are preferred over the pre-proteins for technical applications.

Pro-proteins are inactive precursors to proteins. Their precursors with signal sequences are called pre-pro proteins.

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 code for the linear amino acid sequence 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 of both complementary strands must be taken into account in all three possible reading frames. It should also be taken into account that different codon triplets can code for the same amino acids, so that a certain amino acid sequence can be derived from several different and possibly only slightly identical nucleotide sequences (degeneracy of the genetic code). In addition, different organisms show differences in the use of these codons. For these reasons, both amino acid sequences and nucleotide sequences have to be included in the consideration of the protected area, and specified nucleotide sequences are only to be regarded as exemplary coding for a specific amino acid sequence.

The information unit 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 of recombinant proteins. According to the invention, this includes all genetic engineering or microbiological processes which are based on the genes for the proteins of interest being introduced into a host organism suitable for production and being transcribed and translated by the latter. The genes in question are suitably introduced via vectors, in particular expression vectors; but also via those that cause the gene of interest in the host organism to be inserted into an existing genetic element such as the chromosome or other vectors. According to the invention, the functional unit consisting of gene and promoter and any further counterent elements is referred to as an expression cassette. However, it does not necessarily have to be a physical unit.

It is possible for a person skilled in the art to use methods which are generally known today, such as chemical synthesis or the polymerase chain reaction (PCR) in conjunction with standard molecular biological and / or protein chemical methods, using known DNA and / or amino acid sequences to determine the corresponding nucleic acids up to complete genes manufacture. Such methods are known, for example, from the "Lexicon of Biochemistry", Spektrum Akademischer Verlag, Berlin, 1999, Volume 1, pp. 267-271 and Volume 2, pp. 227-229.

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

Fragments are understood to mean all proteins or peptides that are smaller than natural proteins or those that correspond to fully translated genes and that can also be obtained synthetically, for example. Based on their amino acid sequences, they can be assigned to the relevant complete proteins. For example, they can take on the same structures or have proteolytic or sub-activities, such as the complexation of a substrate. Fragments and deletion variants of parent proteins are basically the same; while fragments are rather smaller fragments, the deletion mutants tend to lack only short areas, and thus only partial functions.

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

For the purposes of the present application, chimeras or hybrid proteins are understood to mean those proteins which are composed of elements which naturally come from different polypeptide chains from the same organism or from different organisms. This procedure is also called shuffling or fusion mutagenesis. The purpose of such a fusion can, for example, be to bring about or modify a certain enzymatic function with the aid of the fused-in protein part. In the context of the present invention, it is immaterial whether such a chimeric protein consists of a single polypeptide chain or several subunits, over which different functions can be distributed. For realizing tion of the latter alternative, it is possible, for example, to separate a single chimeric polypeptide chain into several post-translationally or only after a purification step by means of a targeted proteolytic cleavage.

Proteins obtained by insertion mutation are to be understood as those variants which have been obtained by methods known per se by inserting a nucleic acid or protein fragment into the starting sequences. 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 is lower in such insert-mutated proteins than in chimeric proteins.

Inversion mutagenesis, i.e. a partial reversal of the sequence, can be viewed as a special form of both deletion and insertion. The same applies to a regrouping of different parts of the molecule that deviates from the original amino acid sequence. It can be viewed both as a deletion variant, as an insertion variant, and as a shuffling variant of the original protein.

For the purposes of the present application, derivatives are understood to mean those proteins whose pure amino acid chain has been chemically modified. Such derivatizations can take place, for example, biologically in connection with protein biosynthesis by the host organism. Molecular biological methods can be used for this. However, they can also be carried out chemically, for example by chemically converting a side chain of an amino acid or by covalently binding another compound to the protein. Such a compound can also be, for example, other proteins which are bound to proteins according to the invention, for example, via bifunctional chemical compounds. Such modifications can, for example, influence the substrate specificity or the binding strength to the substrate or can temporarily block the enzymatic activity if the coupled substance is a Inhibitor acts. This can be useful, for example, for the period of storage. Derivatization should also be understood to mean the covalent bond to a macromolecular carrier.

Proteins can also be grouped into groups of immunologically related proteins by reaction with an antiserum or a specific antibody. The members of a group are distinguished by the fact that they have the same antigenic determinant recognized by an antibody.

For the purposes of the present invention, all enzymes, proteins, fragments and derivatives, unless they need to be explicitly addressed as such, are summarized under the generic term proteins.

For the purposes of the present invention, vectors are understood to mean elements consisting of nucleic acids which contain a gene of interest as the characteristic nucleic acid region. They are able to establish this in a species or a cell line over several generations or cell divisions as a stable genetic element that replicates independently of the rest of the genome. Vectors are special plasmids, in particular circular genetic elements, when used in bacteria. In genetic engineering, a distinction is made between those vectors that are used for storage and thus also to a certain extent also for genetic engineering work, the so-called cloning vectors, and those that fulfill the function of realizing the gene of interest in the host cell, that is, the To enable expression of the protein in question. These vectors are called expression vectors.

The enzymatic activity of an enzyme under consideration can be deduced from the amino acid or nucleotide sequence by comparison with known enzymes, which are stored, for example, in generally accessible databases. This can be qualitatively or quantitatively modified by other areas of the protein that are not involved in the actual reaction. This could affect, for example, enzyme stability, activity, reaction conditions or substrate specificity. Such a comparison takes place in that similar sequences in the nucleotide or amino acid sequences of the proteins under consideration are assigned to one another. This is called homologation. A tabular assignment of the relevant positions is called alignment. When analyzing nucleotide sequences, both complementary strands and all three possible reading frames must be taken into account; likewise the degeneracy of the genetic code and the organism-specific codon usage. Alignments are now being created using computer programs, such as the FASTA or BLAST algorithms; this procedure is described, for example, by DJ Lipman and WR Pearson (1985) in Science, volume 227, pp. 1435-1441.

A compilation of all positions that match in the compared sequences is referred to as a consensus sequence.

Such a comparison also allows a statement to be made about the similarity or homology of the compared sequences to one another. This is expressed in percent identity, that is, the proportion of identical nucleotides or amino acid residues in the same positions. A broader concept of homology includes the conserved amino acid exchanges in this value. The percent similarity is then mentioned. Such statements can be made about entire proteins or genes or only about individual areas.

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, two amino acid sequences, which, when largely identical, have two different amino acids each at only two different locations, thus establish a sequence space of four different amino acid sequences. A very large sequence space is obtained if too individual sequences of a room, further homologous sequences can be found. Such high homologies, which exist in pairs, can also be used to recognize very low homologous sequences as belonging to a sequence space.

Homologous regions of different proteins are those with the same functions, which can be recognized by matches in the primary amino acid sequence. It goes up to complete identities in the smallest areas, so-called boxes, which contain only a few amino acids and mostly perform functions essential for overall activity. The functions of the homologous areas are to be understood as the smallest sub-functions of the function performed by the entire protein, such as, for example, the formation of individual hydrogen bonds for complexing a substrate or transition complex.

In step b) of the method according to the invention, the nucleic acid is suitably cloned into a vector. The molecular biological dimension of the invention thus consists of vectors with the genes for the corresponding proteins. These can include, for example, those derived from bacterial plasmids, from viruses or from bacteriophages, or predominantly synthetic vectors or plasmids with elements of various origins. With the other genetic elements present in each case, vectors are able to establish themselves as stable units in the host cells concerned over several generations. It is irrelevant in the sense of the invention whether they establish themselves extrachomosomally as separate units or integrate into a chromosome. Which of the numerous systems known from the prior art is chosen depends on the individual case. Decisive factors are, for example, the number of copies that can be achieved, the selection systems available, including above all antibiotic resistance, or the cultivability of the host cells capable of taking up the vectors.

The vectors form suitable starting points for molecular biological and biochemical investigations of the gene or associated protein in question and for further developments according to the invention and ultimately for amplification and production of proteins according to the invention. They represent embodiments of the present invention in that the sequences of the nucleic acid regions according to the invention contained in each case lie within the homology ranges specified in more detail above.

Preferred embodiments of the present invention are cloning vectors. In addition to the storage, the biological amplification or the selection of the gene of interest, these are suitable for the characterization of the gene in question, for example by creating a restriction map or sequencing. Cloning vectors are also preferred embodiments of the present invention because they represent a transportable and storable form of the claimed DNA. They are also preferred starting points for molecular biological techniques that are not bound to cells, such as the polymerase chain reaction.

Expression vectors are chemically similar to the cloning vectors, but differ in the partial sequences that enable them to replicate in the host organisms optimized for the production of proteins and to express the gene contained there. Preferred embodiments are expression vectors which themselves carry the genetic elements necessary for expression. Expression is influenced, for example, by promoters which regulate the transcription of the gene. For example, expression can be carried out by the natural promoter originally located in front of this gene, but also after genetic engineering fusion both by a promoter of the host cell provided on the expression vector and by a modified or a completely different promoter from another organism.

Preferred embodiments are those expression vectors which can be regulated via changes in the culture conditions or the addition of certain compounds, such as, for example, the cell density or special factors. Expression vectors enable the associated protein to be produced heterologously, that is to say in an organism other than that from which it can be obtained naturally. Also a homologous protein extraction from a gene naturally expressing host organism via an appropriate vector is within the scope of the present invention. This can have the advantage that natural modification reactions associated with the translation are carried out on the resulting protein in exactly the same way as they would occur naturally.

Embodiments of the present invention can also be cell-free expression systems in which the protein biosynthesis is reproduced in vitro. Such expression systems are also established in the prior art.

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 manageability. 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 those host cells whose activity can be regulated on the basis of genetic regulatory elements which are provided, for example, on the expression vector but which may also be present in these cells from the outset. For example, by the controlled addition of chemical compounds that serve as activators, by changing the cultivation conditions or when a certain cell density is reached can be stimulated for expression. This enables the proteins of interest to be produced very economically.

Preferred host cells are prokaryotic or bacterial cells. Bacteria are usually distinguished from eukaryotes by shorter generation times and lower demands on the cultivation conditions. In this way, inexpensive methods for obtaining proteins according to the invention can be established. In Gram-negative bacteria, such as E. coli, a large number of proteins are secreted into the periplasmic space, that is, into the compartment between the two membranes enclosing the cells. This can be advantageous for special applications. Gram-positive bacteria, such as Bacilli or Actinomycetes or other representatives of the Actinomycetes, on the other hand, have no outer membrane, so that secreted proteins are immediately released into the nutrient medium surrounding the cells, from which, according to another preferred embodiment, the expressed proteins according to the invention are purified directly to let.

Expression systems represent a variant of this experimental principle in which additional genes, for example those which are made available on other vectors, influence the production of proteins according to the invention. These can be modifying gene products or those that are to be purified together with the protein according to the invention, for example in order to influence its enzymatic function. These can be, for example, other proteins or enzymes, inhibitors or elements that influence the interaction with different substrates.

Based on the extensive experience that one has, for example with regard to molecular biological methods and the cultivability with coliform bacteria, these represent preferred embodiments of the present invention. Particularly preferred are those of the genera Escherichia coli, in particular non-pathogenic strains suitable for biotechnological production. Representative representatives of these genera are the K12 derivatives and the B strains of Escherichia coli. Strains that can be derived from them according to known genetic and / or microbiological methods, and thus can be regarded as their derivatives, are of greatest importance for genetic and microbiological work and are preferably used to develop methods according to the invention. Such derivatives can be modified, for example, via deletion or insertion mutagenesis with regard to their requirements for the culture conditions, have different or additional selection markers or express other or additional proteins. In particular, these can be derivatives which, in addition to the protein produced according to the invention, express further economically interesting proteins.

Also preferred are those microorganisms which are characterized in that they have been obtained after transformation with one of the vectors described above. These can be cloning vectors, for example, which have been introduced into any bacterial strain for storage and / or modification. Such steps are common in the storage and development of relevant genetic elements. Since the relevant genetic elements can be directly transferred from these microorganisms into gram-negative bacteria suitable for expression, the transformation products mentioned above are also implementations of the subject matter of the invention.

Eukaryotic cells can also be suitable for the production of proteins according to the invention. Examples include fungi such as Actinomycetes or yeasts such as Saccharomyces or Kluyveromyces. This can be particularly advantageous, for example, if the proteins are to undergo specific modifications in connection with their synthesis which enable such systems. These include, for example, the binding of small molecules 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, a suitable nutrient medium is inoculated with the recombinant bacterial strains and the product is harvested from the medium after an experimentally determined period. Continuous fermentations are characterized by achieving a flow equilibrium in which cells die off telephonically over a comparatively long period of time, but also regrow and product can be removed from the medium at the same time.

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

All fermentation processes which are based on one of the processes for producing the recombinant proteins set out above represent correspondingly preferred embodiments of this subject of the invention.

Here, the optimal conditions for the production processes used, for the host cells and / or the proteins to be produced must be determined experimentally on the basis of the previously optimized culture conditions of the strains concerned to the knowledge of the person skilled in the art, for example with regard to fermentation volume, media composition, oxygen supply or stirrer speed.

Fermentation processes, which are characterized in that the fermentation is carried out via a feed strategy, are also suitable. Here, the media components that are consumed by the ongoing cultivation are fed; one also speaks of a feeding strategy. As a result, considerable increases can be achieved both in the cell density and in the dry biomass and / or above all the activity of the protein of interest.

Analogously, the fermentation can also be designed in such a way that undesired metabolic products are filtered out or neutralized by adding buffer or suitable counterions. The protein produced can subsequently be harvested from the fermentation medium. This fermentation process is preferred to product preparation from dry matter, but requires the provision of suitable secretion markers and transport systems.

Without secretion, the purification of the protein from the cell mass is necessary; various methods are also known for this, such as precipitation, for example by ammonium sulfate or ethanol, or chromatographic purification, if necessary, until homogeneous. However, the majority of the technical processes described should manage with an enriched, stabilized preparation.

All of the elements already explained above can be combined to processes in order to produce proteins according to the invention. A large number of possible combinations of process steps is conceivable for each protein according to the invention. The optimal procedure must be determined experimentally for each specific individual case.

In step a), a nucleic acid coding for a methionine gamma lyase is preferably isolated or synthesized, which is selected from METase genes from the protozoan 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 by 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.

In step a), a nucleic acid coding for a methionine gamma lyase is also preferably isolated or synthesized, the nucleotide sequence of which corresponds to that in Seq. 6 (METase gene from Pseudomonas putida) specified nucleotide sequence to 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%.

In step a), a nucleic acid coding for a methionine gamma lyase is also preferably isolated or synthesized, the nucleotide sequence of which corresponds to that in Seq. 5 (METase gene from Fusobacterium nucleatum) specified nucleotide sequence corresponds to 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%.

In step a), a nucleic acid coding for a methionine gamma lyase is particularly preferably isolated or synthesized, the nucleotide sequence of which corresponds to that in Seq. 4 (METase gene from Treponema denticola) specified nucleotide sequence corresponds to 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%.

In step a) it is also 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 the sequences shown 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 is 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 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 the sequences shown 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 amino acid sequences given are identical to at least 70%, preferably at least 80%, in particular at least 90%, particularly preferably at least 95% and very particularly preferably 100%.

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 contains a part which corresponds to the consensus sequence [DQ] - [LIMVF] -X (3) - [STAGC ] - [STAGCI] -TK- [FYWQ] - [LIVMF] -XG- [HQ] - [SGNH] (Seq. ID 7) to at least 96%, in particular at least 97%, preferably at least 98%, preferably to is at least 99%, particularly preferably 100% identical.

In step c) of the method according to the invention, a methionine gamma lyase is preferably expressed or used, the amino acid sequence of which corresponds to that in Seq. 3 (METase from Pseudomonas putida) specified amino acid sequence corresponds to at least 50%, preferably at least 70%, in particular at least 80%, particularly preferably at least 90% and very particularly preferably 100%.

In step c) of the method according to the invention, preference is likewise given to expressing or using a methionine gamma lyase whose amino acid sequence matches that in Seq. 2 (METase from Fusobacterium nucleatum) specified amino acid sequence corresponds to at least 50%, preferably at least 70%, in particular at least 80%, particularly preferably at least 90% and very particularly preferably 100%.

In step c) of the method according to the invention, a methionine gamma lyase is particularly preferably expressed or used, the amino acid sequence of which corresponds to that in Seq. 1 (METase from Treponema denticola) specified amino acid sequence corresponds to at least 50%, preferably at least 70%, in particular at least 80%, particularly preferably at least 90% and very particularly preferably 100%. Host cells suitable according to the invention for the expression in step b) are microorganisms, in particular bacteria, particularly preferably E. coli.

Detection agents suitable according to the invention for reaction products catalyzed by methionine gamma-lyase reactions are, for example, salts of metals which form sparingly soluble salts with sulfide ions with a solubility product of <10-20, preferably a heavy metal salt which precipitates as a sparingly soluble compound with sulfide ions. For example, lead acetate is suitable (preferred concentration: about 4 mg / l, broadest concentration: about 0.1 to about 10 mg / l). Salts of Hg, Ni, Sb, Sn, Zn, Ag, Cu, Co, Cd are also possible, Ag and Cu being preferred in addition to Pb. 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-containing amino acids (in particular cysteine) and organic sulfur compounds (glutathione) can also serve as the sulfur source (preferred concentration: about 500 mg / l, furthest concentration: about 50 to about 1000 mg / l). Sulphides formed by the cells due to the METase activity from methionine as a sulfur source are precipitated as lead sulfide and lead to the colony turning black.

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 METase).

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. Further subjects of the present invention are cloning vectors and expression vectors which contain nucleic acids isolated or synthesized according to the invention.

Another object of the present invention is a test kit for finding effective substances against halitosis, gingivitis or periodontitis, which comprises means for performing the method according to the invention.

Another object of the present invention is the use of methionine gamma lyases (METase) to find 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 in synthesized in vitro, b) the nucleic acid coding for a methionine-gamma-lyase and cloned and expressed in suitable host cells or the methionine-gamma-lyase synthesized in vitro, c) the methionine-gamma-lyase-expressing host cells in spatially separated approaches in the presence of a Cultivated 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 batches are mixed with substances which possibly inhibit the function of the methionine gamma lyase, e) for each of them spatially separated approaches determined the formation of reaction products by reactions catalyzed by methionine-gamma-lyase and thus leads to the proof of the effectiveness of substances against halitosis gingivitis or periodontitis. 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 ingredients (inhibitors of METase) are determined with the aid of the screening method according to the invention or the use according to the invention and b) active substances found to be effective (inhibitors of METase) are mixed with pharmacologically suitable and compatible carriers.

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

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

The β-elimination reaction on which the release of sulfur-containing compounds is based generates a double bond which leads to deamination in free amino acids by subsequent rearrangement, but alternatively leads to the generation of a hydroxyamino acid in bound peptide amino acids by addition of water. Ultimately, serum and methio- nin threonine. Thus, a METase protein can be used to desulfurize a sulfur-containing protein, e.g. B. a cosmetic raw material can be used.

Another object of the present invention is thus the use of one of the above-mentioned METases, in particular a METase, the amino acid sequence of which with the in Seq. 1 (METASE from Treponema denticola) specified amino acid sequence corresponds to at least 50%, preferably at least 70%, in particular at least 80%, particularly preferably at least 90% and very particularly preferably 100%, for the desulfurization of a sulfur-containing protein, for. B. a cosmetic raw material.

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

As an additive for animal feed, methionine is of considerable technical importance and is produced in complex processes.

Another object of the present invention is consequently the use of one of the abovementioned METases, in particular a METase, the amino acid sequence of which corresponds to that in Seq. 1 (METASE from Treponema denticola) specified amino acid sequence corresponds to at least 50%, preferably at least 70%, in particular at least 80%, particularly preferably at least 90% and very particularly preferably 100%, for the synthesis of the amino acids cysteine and methionine.

In order to provide the METase protein for use in technical, cosmetic or pharmaceutical preparations, all processes for the production of recombinant proteins can be considered. The following examples illustrate the invention without, however, restricting it thereto:

Example 1: heterologous expression of the mgll gene from T. denticola

The coding sequence of the mgll 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. co // 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, but requires incubation periods of 5-10 days.

The inhibitory effect of the substance to be tested is evident from the lack of blackening of the colonies. The advantage here is that a growth-inhibiting property of the substance can be checked at the same time, since the expression of the mgll gene alone has no influence on the growth.

Example 2: Enzymatic tests with purified Mgl proteins

The mgf / gene from T. denticola is provided at the 5 'end with a sequence which codes for six histidine residues and is constitutive in an expression vector expressed promoter cloned (pGEM). The cells transformed with this plasmid are grown overnight in LB medium, then a further 50 ml culture with an OD ₆ oo = 0.05 from the previous culture is inoculated and at 37 ° C. up to an OD ₆ oo = 0, 2 dressed. The culture is centrifuged off, resuspended in 1 ml of lysis buffer (50 mM NaH ₂ PO ₄ ; 300 mM NaCl; 10 mM imidazole; pH 8) and frozen at -70 ° C. at least 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 (Röche, 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 can be measured indirectly by converting 2-oxobutyrate and NADH2 to 2-hydroxybutyrate and NAD ⁺ in a photometer at 340 nm (absorption maximum of NADH ₂ ) by decreasing the absorbance. Alternatively, the conversion in cuvettes can also be measured under direct observation in the photometer. Incubation time 10-30 min at 25 ° C.

Another method for determining the enzyme activity is the reaction of the methyl mercaptan formed with 5,5'-dithio-bis-2-nitrobenzoic acid (DTNB; Sigma Aldrich). For this purpose, the batch of the enzyme assay described above is mixed with DTNB (final concentration 0.1 mM) in the cuvette and the conversion to an aryl mercaptan is monitored photometrically at 412 nm. 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 Sθ ₃ .

sequences:

An accession number is also given for some of the genes or gene products listed below. The respective genes or gene products are disclosed in the database of the National Center for Biotechnology Information (NCBI) under their UniGene Accession Number. This database is accessible on the Internet at the following address: http://www.ncbi.nlm.nih.gov/. The genes or gene products are also available at http://www.ncbi.nlm.nih.gov/UniGene/Hs.Home.html or http://www.ncbi.nlm.nih.gov/genome/ guide directly accessible.

Seq. 1 (METase, Treponema denticola):

NRKELEKLGFASKQIHAGSIKNKYGALATPIYQTSTFAFDSAEQGGRRFALEEEGYIYTR LGNPTTTVVEEKLACLENGEAC SASSGIGAVTSCIWSIVNAGDHIVAGKTLYGCTFAFLN HGLSRFGVDVTFVDTRDPENVKKALKPNTKIVYLETPANPNMYLCDIAAVSKIAHAHNPEC KVIVDNTYMTPYLQRPLDLGADVVLHSATKYLNGHGDVIAGFVVGKKEFIDQVRFVGVKDM TGSTLGPFEAYLIGRGMKTLDIRMEKHCANAQKVAEFLEKHPAVESIAFPGLKSFPQYELA KKQMKLCGAMIAFTVKGGLEAGKTLINSVKFATIAVSLGDAETLIQHPASMTHSPYTPEER AASDIAEGLVRLSVGLEDAEDIIADLKQALDKLVK

Seq. 2 (METase, Fusobacterium nucleatum):

EMKKSGLGTTAIHAGTLKNLYGTLAMPIYQTSTFIFDSAEQGGRRFALEEAGYIYTRLGN PTTTVLENKIAALEEGEAGIÄ SSGMGAISSTL TVLKAGDHWTDKTLYGCTFALMNHGL TRFGVEVTFVDTSNLEEVKNAMKKNTRVVYLETPANPNLKIVDLEALSKIAHTNPNTLVIV DNTFATPYMQKPLKLGVDIVVHSATKYLNGHGDVIAGLWTRQELADQIRFVGLKDMTGAV LGPQEAYYIIRGLKTFEIRMERHCKNARTIVDFLNKHPKVEKVYYPGLETHPGYEIAKKQM KDFGAMISFELKGGFEAGKTLLNNLKLCSLAVSLGDTETLIQHPAS THSPYTKEEREVAG ITDGLVRLSVGLENVEDIIADLEQGLEKI

Seq.3 (METase, Pseudomonas putida): MHGSNKLPGFATRAIHHGYDPQDHGGALVPPVYQTATFTFPTVEYGAACFAGEQAGHFYSR ISNPTLNLLEARMASLEGGEAGLALASGMGAITSTLWTLLRPGDEVLLGNTLYGCTFAFLH HGIGEFGVKLRHVDMADLQALEAAMTPATRVIYFESPANPNMHMADIAGVAKIARKHGATV VVDNTYCTPYLQRPLELGADLWHSATKYLSGHGDITAGIVVGSQALVDRIRLQGLKDMTG AVLSPHDAALLMRGIKTLNLRMDRHCANAQVLAEFLARQPQVELIHYPGLASFPQYTLARQ QMSQPGGMIAFELKGGIGAGRRFMNALQLFSRAVSLGDAESLAQHPASMTHSSYTPEERAH YGISEGLVRLSVGLEDIDDLLADVQQALKASA

Seq. 4 (nucleic acid sequence, METase, Treponema denticola):

ATGAATAGAA AAGAATTGGA AAAGTTGGGA TTTGCTTCAA AACAGATTCA CGCAGGAAGC ATTAAGAACA AGTACGGTGC TTTAGCTACA CCTATTTATC AAACTTCAAC ATTTGCATTT GATTCGGCAG AACAAGGCGG CAGAAGATTT GCCTTAGAGG AAGAAGGTTA TATCTACACC CGTTTAGGTA ACCCTACGAC TACGGTTGTT GAAGAAAAAC TTGCCTGCCT TGAAAACGGG GAAGCCTGTA TGTCCGCGAG CTCCGGTATA GGTGCCGTTA CCTCATGCAT TTGGTCAATT GTAAATGCAG GAGACCATAT CGTTGCCGGA AAGACTCTTT ACGGCTGTAC ATTTGCGTTT TTAAATCACG GTCTGTCACG ATTCGGTGTT GATGTAACCT TCGTTGATAC CCGTGATCCT GAAAACGTTA AAAAAGCTTT AAAACCGAAT ACAAAAATCG TTTATTTGGA AACGCCAGCC AACCCGAACA TGTATCTTTG CGACATTGCA GCAGTAAGTA AAATTGCCCA TGCTCATAAT CCCGAATGTA AGGTAATAGT AGATAATACT TATATGACTC CCTATCTTCA AAGGCCTCTT GATTTAGGTG CAGACGTCGT TCTTCATTCT GCAACAAAAT ACCTAAACGG CCATGGAGAC GTTATTGCAG GTTTTGTTGT CGGGAAAAAA GAATTCATCG ATCAGGTACG CTTTGTAGGT GTTAAGGATA TGACAGGTTC TACATTAGGT CCATTTGAAG CTTACTTAAT AGGCCGAGGT ATGAAGACCC TCGATATCCG AATGGAAAAG CACTGCGCCA ATGCTCAAAA AGTTGCAGAG TTCTTGGAAA AACATCCGGC AGTTGAGTCC ATCGCCTTCC CCGGTCTTAA ATCCTTCCCG CAATATGAGC TTGCAAAAAA ACAGATGAAA CTTTGCGGAG CTATGATTGC CTTTACCGTT AAGGGCGGAT TGGAAGCCGG TAAAACCCTT ATAAACTCGG TTAAATTTGC AACCATTGCA GTAAGCTTAG GCGÄTGCCGA AACCCTTATT CAGCATCCTG CAAGTATGAC CCACTCTCCG TATACCCCTG AAGAAAGAGC CGCTTCGGAT ATTGCCGAAG GTTTGGTACG TCTTTCTGTC GGTCTTGAAG ATGCGGAAGA TATTATTGCC GATTTAAAAC AAGCCTTGGA TAAACTTGTT AAA

Seq. 5 (nucleic acid sequence, METase, Fusobacterium nucleatum): (Accession: AE010647): atggaaatga aaaaatctgg tttaggaaca actgctatac atgcaggaac tttaaaaaat 61 ttatatggaa ctcttgcaat gcctatatat caaacttcta cttttatatt tgattcagca 121 gaacaaggag gaagaagatt tgcccttgaa gaagctggat atatttacac aagactaggc 181 aatcctacaa caacagtgtt agaaaataaa attgctgctc ttgaagaagg tgaagctgga 241 atagctatgt catctggtat gggagctatc tcttcaacat tgtggactgt attaaaagct 301 ggagatcatg ttgttacaga taaaacttta tatggttgta cttttgcttt gatgaatcat 361 ggacttacaa gatttggagt tgaagttact tttgttgata cttctaattt agaagaagtt 421 aaaaatgcta tgaaaaaaaa tacaagagtt gtttatcttg aaactcctgc caatccaaat 481 ttaaaaatag ttgatttaga agctttatct aaaattgctc acacaaatcc aaatactttg 541 gttatagtag ataatacttt tgcaactcca tatatgcaaa aacctttaaa attaggtgta 601 gatattgttg tacactctgc aactaaatat ttgaatggac atggagatgt aatagcaggt 661 cttgttgtaa caagacaaga acttgcagat caaatccgtt ttgttggatt aaaagatatg 721 acaggagctg ttttaggacc tcaagaagca tattacatta taagaggatt gaaaacattt 781 gaaattcgta tggaaagaca ctgtaaaaat gcaagaacta ttgtagattt cttaaataaa 841 catccaaaag ttgaaaaagt ttattatcct ggacttgaga ctcatcctgg ttatgaaata 901 gctaaaaaac aaatgaaaga ttttggagca atgatttcat ttgaattäaa aggtggcttt 961 gaagcaggta aaactttatt aaataattta aaactttgtt cattagcagt ttcattagga 1021 gatactgaaa ctcttattca acacccagca tctatgacac actctcctta tacaaaggaa 1081 gaaagagaag ttgctggaat cactgatggt ttagttagat tatcagttgg acttgaaaat 1141 gttgaagata ttatagctga tttagaacaa ggactagaaa aaatttaa

Seq. 6 (nucleic acid sequence, METase, Pseudomonas putida): (Accession: L43133):

ataggatggc ctggtagcca gtgatatagc cgttgtcttc cagcagcttg acccggcgcc 61 agcaggggcg aggtggtcaa tgccacctgg tcggcaagtt cggcgacggt taggcgggcg 121 ttgtcctgca aggcggcgag cagggcgcgg tcggtgcggt cgaggcttga aggcatgttt 181 tgccctcctg gtccgttaat tattgttttt gttccagcaa gcacgcagat gcgtgggcaa 241 ttttggaaaa aatcgggcag ctcggtggca taagcttata acaaaccaca agaggctgtt 301 gccatgcgcg actcccataa caacaccggt ttttccacac gggccattca ccacggctac 361 gacccgcttt cccacggtgg tgccttggtg ccaccggtgt accagaccgc gacctatgcc 421 ttcccgactg tcgaatacgg cgctgcgtgc ttcgccgggg aggaggcggg gcacttctac 481 agccgcatct ccaaccccac cctggccttg ctcgagcaac gcatggcctc gttggagggt 541 ggtgaggcgg gattggcgct ggcgtcgggg atgggagcca ttacttcgac cctctggacc 601 ctgctgcggc ctggtgatga gctgatcgtg gggcgcacct tgtatggctg cacctttgcg 661 ttcctgcacc atggcattgg cgagttcggg gtcaagatcc accatgtcga ccttaacgat 721 gccaaggccc tgaaagcggc gatcaacagc aaaacgcgga tgatctactt cgaaacaccg 781 gccaacccca acatgcaact ggtggatata gcggcggtcg tcgaggcagt gcgggggagt 841 gatgtgcttg tggtggtcga caacacctac tgcacgccct acctgcagcg gccactggaa 901 ctgggggcag acctggtggt gcattcggca accaagtacc tcagtggcca tggcgacatc 961 actgcgggcc tggtggtggg gcgcaaggct ttggtcgacc gcattcggct ggaagggctg 1021 aaagacatga ccggggcagc cttgtcaccg catgacgctg cgttgttgat gcgcggcatc 1081 aagaccctgg cgctgcgcat ggaccggcat tgcgccaacg ccctggaggt cgcgcagttc 1141 ctggccgggc agccccaggt ggagctgatc cactacccgg gcttgccgtc gtttgcccag 1201 tacgaactgg cacagcggca gatgcgtttg ccgggcggga tgattgcctt tgagctcaag 1261 ggcggtatcg aggccgggcg cggcttcatg aatgccctgc agctttttgc ccgtgcggtg 1321 agcctggggg atgccgagtc gctggcacag cacccggcga gcatgacgca ctccagttac 1381 acgccacaag agcgggcgca tcacgggata tcagaggggc tggtgaggtt gtcagtgggg 1441 ctggaggatg tggaggacct gctggcagat atcgagttgg cgttggaggc gtgtgcatga 1501 acttgccttg caggatcggg aacacttgcc caatgcctca cgggatcagg cgatggcact 1561 ttggatgagc tggtgaattg gccggcttat ccaagaggag tttaaaatga ccgta

Seq. 7 (METase consensus sequence)

[DQ] - [LIMNF] -X (3) - [STAGC] - [STAGCI] -T-K- [FYWQ] - [LIVMF] -X-G- [HQ] - [SGNH]

CLUSTALW sequence alignment: t.denticola MSTRKE: - EKLGFASKQI- ^ GS-IKNKYG p. gingivalis MRSGFATRAIHGGA-IENAFGCLATPIYQTSTFVFDTAEQGGRRFAGEEDGYIY f.nucleatum MEMKKSGLGTTAIHAGT-LKNLYGTLAMPIYQTSTFIFDSAEQGGRRFALEEAGYIY p. utida -MHGSNKLPGFATRAIHHGYDPQDHGGALVPPVYQTATFTFPTVEYGAACFAGEQAGHFY t.denticola TI .GNPTTTVVEEK - ACI-ENGEACMSASSGIGAVTSCI SIVNAGDHIVAGKTLYGCTFA p. gingivalis TRLGNPNCTQVEEKLAMLEGGEAAASASSGIGAISSAI VCVKAGDHIVAGKTLYGCTFA f.nucleatum TRLGNPTTTVLENKIAALEEGEAGIAMSSGMGAISSTL TVLKAGDHWTDKTLYGCTFA p.putida SRISNPTLGLCTLLASASGGLGGLLLASG denticola F --- SmGLSR Gv-- tTFVDTRDPE ^ VKK-- ^ p. gingivalis FLTHGLSRYGVEVTLVDTRHPEEVEAAIRPNTKLVYLETPANPNMYLTDIKAVCDIAHKH f.nucleatum LMNHGLTRFGVEVTFVDTSNLEEVKNAMKKNTRVVYLETPANPNLKIVDLEALSKIAHTN P.putida FLHHGIGEFGVKLRHVDMADLQALEAAMTPATRVIYFESPANPNMHMADIAGVAKIARKH .denticola NPECKVIVDNTYMTPY QRP - D-GADVV HSATKYI ISIGHGDVIAGFVVGIKEFIDQVI-FV p. gingivalis -EGVRVMVDNTYCTPYICRPLELGADIWHSATKYLNGHGDVIAGFWGKEDYIKEVKLV f.nucleatum -PNTLVIVDNTFATPY QKPLKLGVDIWHSATKYLNGHGDVIAGLWTRQELADQIRFV p .putida - ^• GATVWDNTYCTPYLQRPLELGADLWHSATKYLSGHGDITAGIWGSQALVDRIRLQ .denticola GvT GST GPFEAYLIGRG- ^ TI-DIRMEK ^^ p. gingivalis GVKDLTGANMSPFDAYLISRGMKTLQIRMEQHCRNAQTVAEFLEKHPAVEAVYFPGLPSF f.nucleatum GLKDMTGAVLGPQEAYYIIRGLKTFEIRMERHCKNARTIVDFLNKHPKVEKVYYPGLDRHLLPHVKHG denticola PQYE - AKKQM-OiCG- ^ I - ^ - 7VKGGI-EAGKT INSVKFA-? IAVS GDAET IQHPASMTHS p. gingivalis PQYELAKKQMALPGAMIAFEVKGGCEAGKKLMNNLHLCSLAVSLGDTETLIQHPASMTHS f.nucleatum PGYEIAKKQMKDFGAMISFELKGGFEAGKTLLNNLKLCSLAVSLGDTETLIQHPASMTHS P. putida PQYTLARQQMSQPGGMIAFELKGGIGAGRRF NALQLFSRAVSLGDAESLAQHPASMTHS , denticola PYTPEERAASDIAEGLVR - 1SVGLEDAEDIIADLKQAI-DKLVK p. gingivalis PYTPEERAASDISEGLVRLSVGLENVEDIIADLKHGLDSLI- f .nucleatum PYTKEEREVAGITDGLVRLSVGLENVEDIIADLEQGLEKI— p.putida SYTPEERAHYGISEGLVRLSVGLEDIDDLLADVQQALKAS

Claims

claims

1. 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) the for a nucleic acid encoding 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 in spatially separated 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 spatially separated batches in the presence of methionine, cysteine or glutathione in an in vitro test system, d) the batches with possibly the function of the methionine-gamma-lyase inhibiting substances, 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

2. Screening method according to claim 1, characterized in that in step a) a nucleic acid coding for a methionine gamma lyase is isolated or synthesized, which is selected from METase genes from the protozoan Trichomonas vaginalis and the bacteria Treponema denticola, Pseudomonas putida, Fusobacterium nucleatum, particularly preferably Treponema denticola.

3. Screening method according to claim 1 or 2, characterized in that in step a) a nucleic acid coding for a methionine gamma lyase is isolated or synthesized, the nucleotide sequence of which in Seq. 6 (METase gene from Pseudomonas putida) given 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%.

4. Screening method according to one of the preceding claims, characterized in that in step a) a nucleic acid coding for a methionine gamma-lyase is isolated or synthesized, the nucleotide sequence of which in Seq. 5 (METase gene from Fusobacterium nucleatum) specified nucleotide sequence corresponds to 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%.

5. Screening method according to one of the preceding claims, characterized in that in step a) a nucleic acid coding for a methionine gamma-lyase is isolated or synthesized, the nucleotide sequence of which in Seq. 4 (METase gene from Treponema denticola) specified nucleotide sequence corresponds to 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%.

6. Screening method according to one of the preceding claims, characterized in that in step a) a nucleic acid is isolated or synthesized, which codes for a methionine gamma lyase, the amino acid sequence with one of those 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 amino acid sequences given are at least 70%, preferably at least 80%, in particular at least 90%, particularly preferably at least 95% and very particularly preferably 100% identical.

, Screening method according to one of the preceding claims, characterized in that in step a) a nucleic acid is isolated or synthesized which codes for a methionine gamma lyase, the amino acid sequence of which is 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 is at least 70%, preferably at least 80%, in particular at least 90%, particularly preferably at least 95% and very particularly preferably 100% identical.

8. Screening method according to one of the preceding claims, characterized in that in step a) a nucleic acid is isolated or synthesized which codes for a methionine gamma lyase, the amino acid sequence of which contains a part which corresponds to the consensus sequence [DQ ] - [LIMVF] -X (3) - [STAGC] - [STAGCI] -TK- [FYWQ] - [LIVMF] -XG- [HQ] - [SGNH] (Seq. ID 7) at least 96%, in particular is at least 97%, preferably at least 98%, preferably at least 99%, particularly preferably 100% identical.

9. 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 in Seq. 3 (METase from Pseudomonas putida) specified amino acid sequence corresponds to at least 50%, preferably at least 70%, in particular at least 80%, particularly preferably at least 90% and very particularly preferably 100%.

10. 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 in Seq. 2 (METase from Fusobacterium nucleatum) specified amino acid sequence corresponds to at least 50%, preferably at least 70%, in particular at least 80%, particularly preferably at least 90% and very particularly preferably 100%.

11. 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 in Seq. 1 (METase from Treponema denticola) specified amino acid sequence corresponds to at least 50%, preferably at least 70%, in particular at least 80%, particularly preferably at least 90% and very particularly preferably 100%.

12. Screening method according to one of the preceding claims, characterized in that in step b) of the method, host cells are used for the expression, which are selected from microorganisms, in particular bacteria, particularly preferably E. coli.

13. Screening method according to one of the preceding claims, characterized in that in step c) of the method as detection agents for reaction products catalyzed by methionine-gamma-lyase reactions, lead acetate and salts of Hg, Ni, Sb, Sn, Zn, Ag, Cu, Co and Cd, preferably uses salts of Pb, Ag and Cu.

14. Screening method according to one of the preceding claims, characterized in that methionine, cysteine or glutathione is used as the sulfur source in step c) of the method.

15. Screening method according to one of claims 1 to 12, characterized in that in step c) the determination of reaction products by methionine-gamma-lyase-catalyzed reactions is carried out by detecting the product formed α-ketobutyrate.

16. Cloning vectors or expression vectors which, according to step a), contain nucleic acids isolated or synthesized from one of the preceding claims.

17. Host cells containing cloning vectors or expression vectors according to claim 16.

18. Test kit for finding effective substances against halitosis, gingivitis or periodontitis, which comprises means for performing the method according to any one of claims 1 to 15.

19. Use of methionine gamma lyases (METase) to find effective substances against halitosis, gingivitis or periodontitis.

20. Test method for detecting the efficacy of 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 Nucleic acid encoding 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 in spatially separated approaches in the presence of a sulfur source and a suitable detection agent for reaction products Cultured methionine-gamma-lyase catalyzed reactions or; the methionine-gamma-lyase is introduced into an in vitro test system in spatially separate batches in the presence of methionine, cysteine or glutathione, d) the batches are mixed with substances which possibly inhibit the function of methionine-gamma-lyase, e) for each of the two spatially separated approaches determined the formation of reaction products by reactions catalyzed by methionine-gamma-lyase and so proves the effectiveness of substances against halitosis gingivitis or periodontitis.

21. A method for producing a pharmaceutical preparation against halitosis, gingivitis or periodontitis, which is characterized in that a) active ingredients (inhibitors of METase) using the screening method according to any one of claims 1 to 15, or the use according to the invention Claim 19 determines and b) active ingredients found to be effective (inhibitors of METase) mixed with pharmacologically suitable and compatible carriers.

22. Use of a METase as defined in one of claims 1 to 15, in particular a METase, the amino acid sequence of which in Seq. 1 (METASE from Treponema denticola) specified amino acid sequence corresponds to at least 50%, preferably at least 70%, in particular at least 80%, particularly preferably at least 90% and very particularly preferably 100%, for the structuring treatment of sulfur-containing fibers, such as e.g. Hair, feathers and wool.

23. Use of a METase as defined in one of claims 1 to 15, in particular a METase, the amino acid sequence of which in Seq. 1 (METASE from Treponema denticola) specified amino acid sequence corresponds to at least 50%, preferably at least 70%, in particular at least 80%, particularly preferably at least 90% and very particularly preferably 100%, for the desulfurization of a sulfur-containing protein, preferably a cosmetic raw material.

24. Use of a METase as defined in one of claims 1 to 15, in particular a METase, the amino acid sequence of which in Seq. 1 (METASE from Treponema denticola) specified amino acid sequence to at least 50%, preferably at least 70%, in particular at least 80%, particularly preferably at least 90% and very particularly preferably 100% matches, for the synthesis of the amino acids cysteine and methionine.