DE102007048432A1 - A transglutaminase substrate which inhibits cysteine- and serine-protease and shows antibiotic properties, used e.g. for plant protection, treatment of infections and production of antibiotic film or surgical sponge - Google Patents

Abstract

A transglutaminase substrate (I) which inhibits cysteine and serine proteases and shows antibiotic properties.

Description

The The present invention relates to a novel transglutaminase substrate, which inhibits cysteine and serine proteases and antibiotic properties and methods of making the protein. Farther be protein sequences of the invention Transglutaminasesubstrats provided.

Transglutaminases (protein glutamine: amine γ-glutamyltransferases, EC 2.3.2.13) are ubiquitous enzymes that catalyze the covalent cross-linking of proteins via the side chains of the amino acids glutamine and lysine (see 1 ). Furthermore, in the presence of a transglutaminase, primary amines and omega-hydroxy fatty acids can be linked to glutamine dinor proteins, whereby the alkylation of a protein on the glutamine side chain in question occurs by transamidation or esterification. When the amine is attached via a spacer of typically 2-5 methylene groups to another functional moiety, for example a fluorescent dye, the transglutaminase substrate acquires novel properties, in particular a self-fluorescence. Biological functions of transglutaminases are essentially only known in mammals, plants and parasites ( Mehta and Eckert (ed.), 2005, in: Prog. Exp. Tumor Res. (Bertino, ed.) Vol. 38, Karger, Basel ). For example, in the presence of calcium ions and after activation by thrombin, the blood coagulation factor XIII stabilizes the blood clot formed by crosslinking the fibrin threads. The role of bacterial transglutaminase in the life cycle of streptomycetes has not yet been clarified. However, because of their higher stability, calcium-independent activity and simple production processes, bacterial enzymes have great economic importance, especially in the food sector (Motoki et al., 1992, US 5,156,956 (October 20, 2002)). The enzymatic cross-linking reaction allows in simple applications the improvement of product properties such as texture, gel strength or viscosity ( Nielsen, 1995, Food Biotechnol., Pp. 119-156 ). However, bacterial transglutaminase can also be used in the production of emulsifiers and diagnostic kits, in immobilization processes and in the production of a wide variety of biomaterials.

Actinomycetes are known to produce most natural antibiotics, including a variety of protease inhibitors. Protease inhibitors that are exported by Streptomycetes are usually small molecules (molar mass less than 1,000 daltons), which have a peptidic structure and very specific inhibit proteases. Proteases are categorized by their catalytic mechanism or after the construction of the catalytic center in four major classes, these are serine proteases, cysteine proteases, aspartyl proteases and Metallopoteasen. Each type of protease combines different enzyme clades and enzyme families under its roof. The serine proteases include, for example, trypsin, chymotrypsin and elastase-like proteases as well as aminopeptidases, to the cysteine proteases papain-like proteases and dipeptidyl peptidases, to the aspartyl proteases pepsin-like proteases and to the metalloproteases thermolysin-like proteases, collagenases or carboxypeptidases. Accordingly, leupeptin inhibits trypsin-like proteases ( Kim et al., 1998, Biochem. J. 331, pp. 539-545 ), Chymostatin chymotrypsin-like proteases ( Umezawa et al., 1970, J. Antibiot. (Tokyo) 23, pp. 425-427 ), Elastatinal elastase-like proteases ( Umezawa et al., 1973, J. Antibiot. (Tokyo) 26, pp. 787-789 ), Bestatin ( Umezawa et al., 1976, J. Antibiot. (Tokyo) 29, pp. 97-99 ), Lapstatin ( Repic Lampret et al., 1999, Arch. Microbiol. 171, p. 397-404 ), Leucinal ( Sträter et al., 1995, Biochemistry 34, pp. 14792-14800 ), Phebestin ( Nagai et al., 1997, J. Antibiot. 50, pp. 82-84 ), Probestin ( Yoshida et al., 1990, J. Antibiot. 43, pp. 149-153 ) Aminopeptidases, antipain trypsin and papain-like proteases ( Hanlon and Liener, 1986, Camp. Biochem. Physiol. B, 84, pp. 53-57 ; Kadowaki et al., 2003, Biol. Chem. 384, pp. 911-920 ), Pepstatin pepsin-like proteases ( Kunimoto et al., 1972, J. Anibiot. (Tokyo) 25, pp. 251-255 ), Piperastatin A ( Murakami et al., 1996, J. Enzyme Inhib. 10, pp. 93-103 ) and piperastatin B ( Murakami et al., 1996, J. Enzyme Inhib. 11, pp. 51-66 ), Histargin ( Umezawa et al., 1984, J. Antibiot. (Tokyo) 37, pp. 1088-1090 ) as well as (S) -alpha-benzyl-malic acid ( Tanaka et al., 1984, J. Antibiot. (Tokyo) 37, pp. 682-684 ) Carboxypeptidases. Small antibiotic protease inhibitors are not substrates of transglutaminases and therefore can not be enzymatically cross-linked, modified or covalently incorporated into protein scaffolds.

Furthermore, it is known that streptomycetes can be used to prepare two types of proteinogenic protease inhibitors: the thermolysin inhibitor SMPI (US Pat. Tate et al., 1998, J. Mol. Biol. 282, pp. 435-446 ) and various subtilisin inhibitors (e.g. Taguchi et al., 1998, J. Biochem. (Tokyo) 124, pp. 804-810 ), which form their own large inhibitor family, the Streptomyces subtilisin inhibitor (SSI) family, and also neutral metalloproteases such as SGMPII ( Kumazaki et al., 1993, J. Biochem. (Tokyo) 114, pp. 570-575 ) or TAMEP ( Zotzel et. al., 2003, Eur. J. Biochem. 270, pp. 3214-3222 ) can inhibit. SMPI and SSI family proteins have a molecular weight of about 14,000 daltons, with SSI molecules being described as 28,000 Da molecular weight dimers. Two intramolecular cystine bridges ensure that SSI proteins can be exposed to high temperatures (> 80 ° C) without loss of activity ( Kojima et al., 1993, J. Mol. Biol. 230, pp. 395-399 ). They inhibit the growth of mold fungi ( Vernekar et al., 1999, Biochem. Biophys. Res. Commun. 262, pp. 702-707 ) and viruses ( Angelova et al., 2006, Biochim. Biophys. Acta 1760, pp. 1210-1216 ). According to the current state of knowledge, the known proteinogenic inhibitors of actinomycetes can not be cross-linked by transglutaminase.

The The object of the present invention was protease inhibitors to produce antibiotic properties with transglutaminases can be enzymatically crosslinked or functionalized. to Solution of the problem were inventively strains of Actinomycetes cultured for several days. Cell supernatants of liquid cultures or aqueous extracts of Agar cultures were grown using various proteases Inhibitor activity examined. Surprisingly became a novel antibiotic transglutaminase substrate with inhibitory activity against cysteine and serine proteases, preferably against Papain from Papaya carica. The invention Transglutaminase substrate is hereinafter referred to as Streptomyces papaininhibitor (SPI).

From The inventors have observed that SPI is about 30 hours after The seeding is detectable in liquid cultures and his highest activity reached after 48 hours.

It has also been observed by the inventors that Streptomyces mobaraensis, a strain formerly belonging to the genus Streptoverticillium, produces the highest amount of SPI in liquid media (see 2 ).

The present invention further provides a process for producing SPI. The production is possible from cell-free supernatants of liquid cultures or filtered extracts of agar cultures. In a preferred embodiment, SPI is prepared with liquid cultures of Streptomyces mobaraensis in 40-48 hours. Purification to homogeneity is then carried out from cell-free supernatants by ethanol precipitation and cation exchange chromatography on Fractogel EMD SO ₃ ^- (see 3 ).

The purified product is untreated according to the invention or after desalting as a lyophilisate, ammonium sulfate precipitate or in buffered solution after addition of 30-50 Percent glycerin, 10-30% maltodextrin, 5-30% polyethylene glycol or 2-20 mM glutathione at -80 ° C to + 4 ° C stored.

• A) N-Terminale Sequenz von SPI. Die mit dem mutmaßlichen Protein von Streptomyces lavendulae identischen Aminosäuren sind grau unterlegt:
  
• B) Nukleinsäuresequenz eines mutmaßlichen Proteins von Streptomyces lavendulae (EMBL-EBI: AAD32751)
  
• C) Aus der Nukleinsäuresequenz von Streptomyces lavendulae übersetzte Proteinsequenz eines mutmaßlichen Proteins mit unbekannter Funktion (SwissProt Entry Name: Q9X5U4_STRLA). Aminosäuren, die mit SPI identisch sind, sind grau unterlegt:
  
  

The present invention further provides information about the structure of SPI. By SDS gel electrophoresis, an apparent molecular mass of 12,000 daltons was determined for SPI. SPI thus has a molecular mass similar to the two known proteinogenic Streptomyces protease inhibitors SMPI and SSI. The SPI protein band is significantly below the TAMEP inhibitor band. The determination of the isoelectric point of SPI gave a value of 7.3. Like representatives of the SSI family, SPI is heat stable (see 4 ). The sequence information below was obtained by Edman degradation. The primary structure of SPI is homologous (identity of 70%) to a putative protein product of unknown function of Streptomyces lavendulae translated from the coding DNA:

• A) N-terminal sequence of SPI. The amino acids identical to the putative protein of Streptomyces lavendulae are highlighted in gray:
  
• B) Nucleic acid sequence of a putative protein of Streptomyces lavendulae (EMBL-EBI: AAD32751)
  
• C) Protein sequence of a putative protein of unknown function translated from the nucleic acid sequence of Streptomyces lavendulae (SwissProt Entry Name: Q9X5U4_STRLA). Amino acids that are identical to SPI are highlighted in gray:
  
  

The putative protein obviously has next to the mature one Domain a signal peptide (Met → Ala29 / Ala30, underlined Amino acids), which are extracellular Streptomyces proteins typical length of 29 to 30 amino acids having. The calculated molecular weight without signal peptide is 11,944 or 12,015 daltons, which is fine with the experimentally found apparent molecular mass of SPI matches. The calculated pI of the unknown protein is higher at 8.45 by one pH unit as the experimentally determined pI of SPI.

labeling experiments of purified SPI with the specific labeling reagent monobiotinylcadaverine (MBC) and transglutaminase surprisingly revealed that SPI has reactive glutamine residues which are the inventive Enable cross-linking and modification of SPI with transglutaminases.

Furthermore, it was observed by the inventors that the reactivity of the glutamine residues of SPI by structural modulators of the general formula R ¹ -CO-XR ³ (with X = NR ² , O and R ¹ , R ² , R ³ as in the German Patent 17.09 .2007) can be increased without loss of inhibitory activity (see 5 ). The concentration of acylamido compounds is 0-0.2% (m / v), preferably 0-0.07% (m / v).

labeling experiments of purified SPI with the specific labeling reagent N-biotinyl- (6-N'-carbobenzoxy-L-glutaminylglycyl) hexanediamine (ZQG-HDA-biotin) and transglutaminase surprisingly revealed that SPI has reactive lysine residues which are the inventive Enable cross-linking of SPI with transglutaminases.

Furthermore, it was observed by the inventors that the reactivity of the lysine residues of SPI by structural modulators of the general formula R ¹ -CO-XR ³ (with X = NR ² , O and R ¹ , R ² , R ³ as in the German Patent 17.09 .2007) can be increased (acylpolyamines) or reduced (acylamino acids) without loss of the inhibiting activity. The concentration of acylamido compounds is included 0-0.2% (m / v), preferably 0-0.07% (m / v).

The Crosslinking of SPI according to the invention with transglutaminase carried out. It was the origin of the inventors oligomeric and polymeric aggregates observed by SPI. The attempt further shows that SPI for enzymatic incorporation in novel biomaterials with antibiotic properties.

The present invention further provides information on the biological function of SPI. According to the invention, SPI inhibits cysteine and serine proteases, preferably members of the papain family, but in particular papaya of papaya carica (see 6 and Tab. 2).

The present invention further provides information on the antibiotic property of SPI. The inventors have observed that spraying Cuscuta reflexa with an SPI solution results in the death of the tobacco plant parasite (see 7 ).

According to the invention was furthermore observed that the emergence of mold on sausage slices through SPI is inhibited.

According to the invention was continues to observe that SPI growth of multicellular Actinomycetes inhibits.

The The following figures and examples illustrate the invention.

Description of the pictures

1 : Reaction Scheme of Transglutaminases. The gamma-carboxamide group of protein-bound glutamine is transferred to a primary amine releasing ammonia. When the glutamyl acceptor is the epsilon amino function of a protein-bound lysine residue, an inter- or intramolecular isopeptide bond is formed, depending on whether a second or the same protein is involved.

2 : Cultivation course of Streptomyces mobaraensis. Determination of the residual activity of papain after incubation for 30 minutes with culture supernatant at RT.

3 : Purification of SPI from the supernatant of cell-free culture broth of Streptomyces mobaraensis
Silver-stained SDS-polyacrylamide gel
Lane M: Molecular weight marker proteins: 66 kDa, 45 kDa, 29 kDa, 0 kDa, 14.2 kDa; Lane 1: Cell-free culture broth supernatant; Lane 2: supernatant of a 1: 1 mixture of cell-free culture broth supernatant and ethanol; Lane 3: United fractions of ion exchange chromatography (SPI pool); Lane 4: SPI pool after dialysis through a 10 kDa membrane.

4 : Thermostability of SPI: filled points, SPI activity; open points, papain activity

• A) Silbergefärbtes SDS-Polyacrylamidgel
• B) Auf Nitrocellulose transferierte Proteine einer SDS-Polyacrylamidgelelektrophorese angefärbt mit einem Streptavidin-Alkalische Phosphatase-Konjugat Spuren 1, 2, Inkubation für 1 und 3 Stunden

5 : Labeling of purified SPI with monobiotinylcadaverine (MBC) and transglutaminase to identify reactive glutamine residues

• A) Silver-stained SDS-polyacrylamide gel
• B) Nitrocellulose-transferred proteins of SDS-polyacrylamide gel electrophoresis stained with streptavidin-alkaline phosphatase conjugate lanes 1, 2, incubation for 1 and 3 hours

6 : Inhibition of Papain with SPI

• A) Unbehandelte Tabakpflanze mit dem Parasiten
• B) Mit SPI behandelte Tabakpflanze mit abgestorbenem Parasiten

7 : Biocidal effect of SPI on the tobacco parasite Cuscuta reflexa.

• A) Untreated tobacco plant with the parasite
• B) SPI-treated tobacco plant with dead parasite

The The following examples serve to explain the present Invention.

Example I

Cultivation of streptomycetes

a) plate cultures for inoculating Liquid media and for root preservation

The cultivation of the microorganisms for the inoculation of liquid media as well as the establishment of permanent cultures was carried out on GYM agar plates ( Shirling and Gottlieb, 1966, Int. J. Syst. Bacteriol. 16, pp. 313-340 ). Starting from pure cultures of the DSMZ (German Collection of Microorganisms and Cell Cultures GmbH), Streptomycetes strains were taken up in sterile water. The suspension was transferred to the GYM plates with a pipette and spread with a Drigalski spatula.

In front Inoculations of liquid cultures were precultures created on a new agar plate. To this were bacterial colonies struck with a loop on the plate and at Incubated at 28 ° C for at least 5 days until next to the substrate mycelium also sporulating aerial hyphae were to be seen.

to Stem conservation was the Streptomycetenstämme after about 30 days on a new GYM agar plate inoculated. The Petri dishes were subsequently covered with a plastic film closed and incubated at 28 ° C.

b) liquid cultures for preparative Production of SPI

to preparative production of SPI were 1 L Erlenmeyer culture flasks filled with 110 ml of liquid medium. After autoclaving The culture media was filtered through a sterile filter 1 ml of trace element solution fed and pre-cultured on GYM agar plates was inoculated, inoculated.

The Cultivation was carried out at 28 ° C until reaching the desired SPI product. The oxygen input needed for growth The aerobic bacteria is needed, carried out by intensive Movement of the liquid culture on a cross shaker with a frequency of 110 per minute. After three to four days For example, the SPI-containing culture medium was permeated by the microorganisms Centrifugation and filtration separated, directly further processed or stored at -20 ° C.

c) Cultivation of Streptomyces mobaraensis

The cultivation and root preservation of Streptomyces mobaraensis was carried out on agar plates, starting from a pure culture of the DSMZ. The submerged cultivation for SPI production was carried out as described in Example I b) in a complex medium which was additionally enriched with trace elements ( Voelskow, 19, Biotechnology Yearbook, Carl Hanser Verlag, Munich, pp. 341-361 ). After a cultivation period of 40-48 hours, the cell mass was separated by centrifugation and filtration from the liquid medium into which the bacteria secrete the product. The amount of SPI produced averaged 15-20 milligrams per liter of culture broth.

Example II

Determination of inhibitory activity from SPI

a) pre-incubation with papain

Of the based inhibitor activity tests for SPI is based on a preincubation of the inhibiting protein with papain for the duration of 30 minutes at room temperature and determination of proteolytic Residual activity with the activity test described under 2b).

b) Modified Ansontest

• ( Yang and Wang, 1999, Bot. Bull. Acad. Sin. 40, pp. 259-265 )

200 μl of alkali-soluble casein (10 mg / ml) in 0.1 M citrate pH 6.5 and 200 μl of a suitable protease-SPI mixture with 100 μg papain were incubated at 37 ° C for 10 min. After addition of 600 μl of trichloroacetic acid, centrifugation was carried out and the absorbance of the supernatant was measured at 280 nm against a non-papain blank in 1 cm cuvettes. The concentration of absorbing amino acids and peptides that Residual activity of the protease in the supernatant is summarily recorded as "free tyrosine." The extinction coefficient for tyrosine is 0.9611 mM ^-1 cm ^-1 under the conditions chosen, with a modified Anson unit (AE) corresponding to the release of One micromole of tyrosine per minute The reduction of an Anson unit by SPI is referred to as an inhibition unit (IU).

c) Paranitroanilide test

100 μl of 2 mM Cbz-Phe-Arg-pNA in ethanol and 400 μl of 0.1 M citrate pH 6.5 with 25 μg papain were incubated for 10 min at 37 ° C. After adding 500 μl of 5 mM PMSF in dimethyl sulfoxide, centrifugation was carried out and the absorbance in the supernatant was measured at 405 nm against a blank without Papain. The extinction coefficient for released para-nitroaniline is 14.253 mM ^-1 cm ^-1 under the chosen conditions.

Example III

Purification of SPI from the culture broth of Streptomyces mobaraensis

a) Preparation of a cell-free supernatant

Streptomyces mobaraensis was, as described in Example I c), in one Liquid medium cultured for 40-48 hours. The educated Cell mass was purified by centrifugation (10,000 g, 4 ° C, 10 min) and filtration separated, and the supernatant on processed.

b) Accumulation of SPI by ethanol precipitation

Cell-free supernatant was cooled to 4 ° C, with the same volume mixed at -18 ° C cold ethanol and 30 minutes incubated. After separation of the precipitated proteins by Centrifugation (10,000 g, 4 ° C, 10 min) were the remaining Proteins of the supernatant separated by chromatography.

c) Column chromatographic method

Full purification of SPI was accomplished by low pressure liquid chromatography on a strong cation exchange material. Before starting, the ethanol supernatant was freed of very fine particles by filtration through a 0.45 μ filter. The buffers used for the chromatography were prepared with Milli-Q water, sterile filtered before use and degassed in an ultrasonic bath. Table 1: Elution conditions for SPI function Buffer A Buffer B Volume (ml) Order of the protein solution 200 ml Elution of non-binding proteins 100 about 200 ml plateau 90 10 about 50 ml Linear gradient 90-0 10-100 100 ml

Chromatography was carried out at room temperature. All protein-containing fractions were characterized by inhibitor activity assay, SDS-PAGE, and protein determinations. Fractions containing SPI were optionally pooled, left untreated, or desalted at -20 ° C with desalting columns or by dialysis, lyophilized, precipitated with ammonium sulfate, or conditioned by the addition of glycerine, maltodextrin, polyethylene glycol, or glutathione. equipment Amersham-Pharmacia Stationary phase Fractogel EMD SO ₃ ^- (S) diameter 1.6 cm bed height 10.3 cm bed volume 20 ml Mobile phases 50 mM Tris-acetate pH 6.0 (buffer A) 1 M NaCl in 50 mM Tris-acetate pH 6.0 (buffer B) flow rate 1 ml / min Injected volume about 200 ml detection Continuous measurement of the absorption at λ = 280 nm

Example IV

Characterization of SPI

a) Determination of the molecular size

The SDS polyacrylamide gel demonstrates complete purification of SPI from Streptomyces mobaraensis (see 3 ). The apparent molecular weight determined by the marker proteins is 12,000 daltons.

b) Determination of the isoelectric point

Of the isoelectric point of SPI was using a prefabricated acrylamide gel Serva (Servalyt Prenet) according to the manufacturer. For calibration, cytochrome (horse, pI of 10.7), ribonuclease A (bovine, 9.5), lectin (lens culinaris, 8.3, 8.0, 7.8), myoglobin (Horse, 7.4, 6.9), carbonic anhydrase (bovine, 6.0), β-lactoglobulin (Bovine, 5.3, 5.2), trypsin inhibitor (soybean, 4.5), glucose oxidase (Aspergillus niger, 4,2) and amyloglucosidase (Aspergillus niger, 3,5) used. The found isoelectric point of SPI lies at 7.3.

c) Determination of protein sequences

Purified SPI was separated by gel electrophoresis ( Poduslo, 1981, Anal. Biochem. 114, pp. 131-139 ), transferred to polyvinylidene difluoride (PVDF) -membanes ( Khyse-Andersen, 1984, J. Biochem. Biophys. Methods 10, pp. 203-209 ) and stained with Coomassie blue ( Matsudaira, 1987, J. Biol. Chem. 262, pp. 10035-10038 ). The protein band was excised and analyzed by Edman degradation.

Example V

Biotinylation of SPI with transglutaminase

a) Labeling with monobiotinylcadaverine

SPI labeling for reactive glutamine side chains was performed in 0.1M N- (2-hydroxyethyl) piperazine-N '- (2-ethanesulfonic acid) (HEPES) pH 7.5. 100 μl buffered solution contained 25 μl SPI (300 μg / ml), 2 mM monobiotinyl cadaverine (MBC), 0.06% lauroyl sarcosine and 5.4 μl transglutaminase (0.5 mg / ml). Incubation was at 37 ° C for up to 24 hours. The reaction mixture was separated by SDS-polyarylamide gel electrophoresis, transferred to a nitrocellulose membrane and stained with an avidin-alkaline phosphatase conjugate. For this, non-specific binding sites of the nitrocellulose membrane were blocked with fat-free milk after protein transfer. After multiple washes with 10 mM Tris-HCl / 150 mM NaCl / 0.05% Tween pH 8.0, incubation with the avidin-conjugate (50 ng / ml) for 1.5 hours at room temperature, rinsing again and adjusting the membrane to pH 9.5 with 0.1 M Tris-HCl / 0.1 M NaCl / 5 mM MgCl ₂ was stained with 5-bromo-4-chloroindolyl phosphate and 2,2'-di-p-nitrophenyl-5,5'- diphenyl (3,3'-dimethoxy-4,4'-diphenylene) -ditetrazolium chloride (nitrotetrazolium blue).

b) Labeling with N-biotinyl- (5-N'-carbobenzoxy-L-glutaminylglycyl) cadaverine

The enzymatic labeling methods of SPI for the determination of reactive Lysine residues were carried out as described under 5a). Only contained the Reaction mixture instead of MBC 0.13 mM N-biotinyl- (6-N'-carbobenzoxy-L-glutaminylglycyl) hexanediamine (ZQG HDA biotin).

Example VI

Crosslinking of SPI with transglutaminase

The Crosslinking of SPI by transglutaminase was carried out as in 5a) described in HEPES buffer pH 7.5 with 0.05% N-lauroyl-3-N ', N'-dimethylpropanediamine and without labeling reagent. The reaction mixture was up to Incubated for 24 hours, separated by SDS-polyacrylamide gel electrophoresis and silver colored.

Example VII

Determination of specificity of SPI

The inhibitory activity of SPI against various proteases was determined by the analysis method described under 2a). For serine and metalloproteases, the procedure was modified such that 0.1 M Tris / HCl pH 7.5 with 2 mM CaCl _{2 was} used as the buffer. It showed the high activity of SPI against papain, bromelain, trypsin, proteinase K and subtilisin. Furthermore, dispase and thermolysin are inhibited at high concentration (Table 2). Table 2: Inhibitory activity of SPI on endoproteases protease origin SPI: Protease (M / M) Residual activity (%) cysteine proteases papain Carica papaya 0.25 0.8 2.0 5 85 50 16 7 bromelain Pineapple comosus 0.8 2.0 72 18 serine proteases trypsin Bos taurus 0.8 2.0 36 7 α-Chymotrypsin Bos taurus 0.8 2.0 100 100 subtilisin Bacillus globigii 0.8 2.0 92 10 Proteinase K Tritirachium album 0.8 2.0 3 metalloproteases Dispase I Bacillus polymyxa 0.8 2.0 100 21 collagenase Clostridium histolyticum 0.8 2.0 100 100 thermolysin Bacillus thermoproteolyticus rocco 0.8 2.0 100 45

It follows Sequence listing according to WIPO St. 25. This can of the official publication platform of the DPMA become.

QUOTES INCLUDE IN THE DESCRIPTION

This list The documents listed by the applicant have been automated generated and is solely for better information recorded by the reader. The list is not part of the German Patent or utility model application. The DPMA takes over no liability for any errors or omissions.

Cited patent literature

• US 5156956 [0002]

Cited non-patent literature

• - Mehta and Eckert (ed.), 2005, in: Prog. Exp. Tumor Res. (Bertino, ed.) Vol. 38, Karger, Basel [0002]
• - Nielsen, 1995, Food Biotechnol., Pp. 119-156 [0002]
• Kim et al., 1998, Biochem. J. 331, pp. 539-545 [0003]
• Umezawa et al., 1970, J. Antibiot. (Tokyo) 23, pp. 425-427 [0003]
• Umezawa et al., 1973, J. Antibiot. (Tokyo) 26, pp. 787-789 [0003]
• Umezawa et al., 1976, J. Antibiot. (Tokyo) 29, pp. 97-99 [0003]
• Repic Lampret et al., 1999, Arch. Microbiol. 171, p. 397-404 [0003]
• Sträter et al., 1995, Biochemistry 34, pp. 14792-14800 [0003]
• Nagai et al., 1997, J. Antibiot. 50, pp. 82-84 [0003]
• Yoshida et al., 1990, J. Antibiot. 43, pp. 149-153 [0003]
• - Hanlon and Liener, 1986, Camp. Biochem. Physiol. B, 84, pp. 53-57 [0003]
• Kadowaki et al., 2003, Biol. Chem. 384, pp. 911-920 [0003]
• Kunimoto et al., 1972, J. Anibiot. (Tokyo) 25, pp. 251-255 [0003]
• Murakami et al., 1996, J. Enzyme Inhib. 10, pp. 93-103 [0003]
• Murakami et al., 1996, J. Enzyme Inhib. 11, pp. 51-66 [0003]
• Umezawa et al., 1984, J. Antibiot. (Tokyo) 37, pp. 1088-1090 [0003]
• Tanaka et al., 1984, J. Antibiot. (Tokyo) 37, pp. 682-684 [0003]
• Tate et al., 1998, J. Mol. Biol. 282, pp. 435-446 [0004]
• Taguchi et al., 1998, J. Biochem. (Tokyo) 124, pp. 804-810 [0004]
• Kumazaki et al., 1993, J. Biochem. (Tokyo) 114, pp. 570-575 [0004]
• - Zotzel et. al., 2003, Eur. J. Biochem. 270, pp. 3214-3222 [0004]
• Kojima et al., 1993, J. Mol. Biol. 230, pp. 395-399 [0004]
• Vernekar et al., 1999, Biochem. Biophys. Res. Commun. 262, pp. 702-707 [0004]
• - Angelova et al., 2006, Biochim. Biophys. Acta 1760, pp. 1210-1216 [0004]
• - Shirling and Gottlieb, 1966, Int. J. Syst. Bacteriol. 16, pp. 313-340 [0030]
• - Voelskow, 19, Jahrbuch Biotechnologie, Carl Hanser Verlag, Munich, pp. 341-361 [0035]
• - Yang and Wang, 1999, Bot. Bull. Acad. Sin. 40, pp. 259-265 [0036]
• - Poduslo, 1981, Anal. Biochem. 114, pp. 131-139 [0045]
• Khyse-Andersen, 1984, J. Biochem. Biophys. Methods 10, pp. 203-209 [0045]
• - Matsudaira, 1987, J. Biol. Chem. 262, pp. 10035-10038 [0045]

Claims (39)

Transglutaminase substrate, the cysteine and serine proteases inhibited and has antibiotic properties. Transglutaminase substrate according to claim 1, characterized in that the N-terminal protein sequence may have the following amino acids:

Transglutaminase substrate according to claim 1 characterized characterized in that the primary structure homologous with the of a putative protein (full sequence under EMBL-EBI: AAD32751 and SwissProt: Q9X5U4_STRLA) unknown Function of Streptomyces lavendulae. Transglutaminase substrate according to claim 1 characterized characterized in that the protein has an apparent molecular weight of 12,000 Dalton and a pI of 7.3. Transglutaminase substrate according to claim 1 characterized characterized in that the protein possesses glutamine residues that its Enable crosslinking, alkylation and esterification with transglutaminases. Transglutaminase substrate according to claim 1 characterized characterized in that the protein possesses lysine residues which limit its cross-linking with transglutaminases. Transglutaminasesubstrat according to claim 1, characterized in that a compound of the general structure R ¹ -CO-XR ³ (with X = NR ² , O and R ¹ , R ² , R ³ as in the German patent application "structural modulator of proteins" from 17. September 2007) favored crosslinking, alkylation or esterification by transglutaminases without loss of inhibitory activity. Transglutaminase substrate according to claim 1 characterized characterized in that N-lauroyl-3-N ', N'-dimethylpropanediamine the Crosslinking, alkylation or esterification by transglutaminases favored. The concentration of acylpolyamine is included 0-0.2% (m / v), preferably 0-0.07% (m / v). Transglutaminase substrate according to claim 1 characterized characterized in that N-lauroyl sarcosine alkylation or esterification favored by transglutaminases. The concentration of Acylamino acid is 0-0.2% (m / v), preferably at 0-0.07% (m / v). Transglutaminasesubstrat according to claims 1-8 characterized in that crosslinking and modification reactions take place with a Ca ²⁺ -independent transglutaminase. Transglutaminase substrate according to claim 1 characterized characterized in that cysteine and serine proteases are inhibited. Transglutaminase substrate according to claim 1 characterized characterized in that papain is inhibited by papaya carica. Transglutaminase substrate according to claim 1 characterized characterized in that bromelain is inhibited by pineapple comosus. Transglutaminase substrate according to claim 1 characterized characterized in that trypsin is inhibited. Transglutaminase substrate according to claim 1 characterized characterized in that proteinase K of Tritirachium album inhibits becomes. Transglutaminase substrate according to claim 1 characterized characterized in that subtilisin inhibited by Bacillus globigii becomes. Transglutaminase substrate according to claim 1 characterized characterized in that plant parasites are inhibited in growth or be killed. Transglutaminase substrate according to claim 1 characterized characterized in that the tobacco plant parasite Cuscuta reflexa in Growth is inhibited or killed. Transglutaminase substrate according to claim 1 characterized characterized in that bacteria, fungi and viruses are inhibited in growth or be killed. Transglutaminase substrate according to claim 1 characterized characterized in that the inhibitory protein is the growth of Mold inhibits. Transglutaminase substrate according to claim 1 characterized characterized in that the inhibitory protein is the growth of Streptomycetes inhibits. Transglutaminase substrate according to the claims 1-16, characterized in that the preparation by Cultivation of bacteria, preferably done by actinomycetes. Transglutaminase substrate according to the claims 1-16, characterized in that the preparation by Cultivation of Streptomyces, preferably Streptomyces mobaraensis he follows. Transglutaminase substrate according to the claims 1-16, characterized in that the production of a active product by cultivating Streptomyces mobaraensis and separating the product-containing culture broth from the cell mass by centrifugation, filtration or other common Separation process takes place. Transglutaminase substrate according to the claims 1-16, characterized in that the production of a active product by cultivating Streptomyces mobaraensis, Separation of the product-containing culture broth from the Cell mass and precipitation of contaminating protein with Ammonium sulfate, polyethylene glycol, ethanol, acetone or isopropanol he follows. Transglutaminase substrate according to the claims 1-16 and 25 characterized in that the precipitation of contaminating protein with 50% by volume ethanol. Transglutaminase substrate according to the claims 1-16, characterized in that the production of a highly purified product by cultivating Streptomyces mobaraensis, Separation of the product-containing culture broth from the Cell mass, ethanol precipitation and chromatographic methods he follows. Transglutaminasesubstrat according to claims 1-16 and 27, characterized in that the preparation of a highly purified product by cation exchange chromatography on Fractogel EMD SO ₃ ^- takes place. Transglutaminase substrate according to the claims 1-16 characterized in that the according to claims 22-28 product prepared as lyophilisate, ammonium sulfate precipitate or in buffered solution after addition of 30-50 Percent glycerin, 10-30% maltodextrin, 5-30% polyethylene glycol or 2-20 mM glutathione at -80 ° C to + 4 ° C is stored. Transglutaminase substrate according to the claims 1-21 characterized in that the protein as biological Plant protection product is used. Transglutaminase substrate according to the claims 1-21 characterized in that the protein as a preservative is used. Transglutaminase substrate according to the claims 1-21 characterized in that the protein as a medical device is used. Transglutaminase substrate according to the claims 1-21 characterized in that the protein for treatment used by infections. Transglutaminase substrate according to the claims 1-21 characterized in that the protein for treatment used by skin and wound infections. Transglutaminase substrate according to the claims 1-21 characterized in that the protein for treatment used by periodontitis. Transglutaminasesubstrat according to claims 1-10, characterized in that it with the aid of Transglutaminases can be incorporated into suitable biomaterials without loss of inhibitory activity. Transglutaminase substrate according to the claims 1-21 characterized in that the inhibitory protein used for the production of antibiotic films and patches. Transglutaminase substrate according to the claims 1-21 characterized in that the inhibitory protein for making surgical sponges with antibiotic Properties is used. Transglutaminase substrate according to the claims 1-21 characterized in that the inhibitory protein for the production of scaffold material with antibiotic properties is used for replacement cartilage and bone.