JPH06505144A - A novel bacteriocin from Lactococcus lactis subspice cremoris - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Novel bacterium from Lactococcus lactis subspice cremoris Riosin The present invention relates to novel bacteriocins and their isolation, synthesis and uses.

The present inventors have discovered that Lactococcus lactis subspicis cremoris (L. actococcus 1actis 5ubsp, cremoris) A novel bacteriocin was isolated from the strain. This microorganism has not been described in the literature so far. It was.

The microorganisms mentioned above are autolytic when the cell concentration is high; Microorganisms are bacteria found extracellularly in a growth medium, e.g. M17 medium. discovered that it produces ions. This bacterial autolysis is due to the lytic properties of bacteriocin. It can be shown as being due to gender. Bacteriolysis is a process that breaks down bacteriocins. This can be prevented by adding tease.

As used herein, the term "bacteriocin" refers to are released by bacteria that kill not only the producing microorganism itself but also other bacterial strains. It is used to include substances that are Therefore, there is no novel bug related to the claimed invention. Teriocin is a known bacteriocin called diplococin. 1aCtOC containing strains that produce cin and nisin It is known to inhibit the growth of over 120 strains of the genus OCCuS. However, production Living microorganisms themselves confer resistance to bacteriocins to prevent indiscriminate lysis. This bacterium carries a gene encoding an immune factor that appears to lyse its producing microorganisms only at high cell concentrations.

The present inventors determined the amino acid sequence of a novel bacteriocin. Main departure According to Ming's one point of view, the following amino acid sequence 16-Asn Thr Asn Thr His Lys Tyr Val T yr　GIr+　Gln　nv　Gin　Asn　Ala46-ALa　Ala Has or contains GlyGlyPhe Gly Leu His His Polypeptides and derivatives or fragments thereof having bacteriocin activity provided.

The above structure is different from that of nisin, and is based on the 5WISS PROT data database. Known polypeptide sequences of links and significant streaks It has no homologues. The bacteriolytic activity of the bacteriocin of the present invention is well known. The activity is substantially greater than that of the 1aCtiS bacteriocin.

The new bacteriocin is thermostable and remains active even after boiling in water for 30 minutes. keeping. This property has been demonstrated in industrial methods using L.actis microorganisms, e.g. It is valuable, for example, in allowing its use in the production of cheese and yoghurt.

This bacteriocin is thought to kill 1actococcus HA bacteria through bacteriolysis. be exposed. 1 Promoting the lysis of Aetoeoccus bacteria promotes the ripening of cheese. The novel bacteriocins are therefore particularly useful for the production of cheese. I can stay.

The lytic activity of this bacteriocin also applies to the production of cell wall preparations or the release of nucleic acid material. Can be used.

Some bacteria, such as Gram-negative bacteria, are resistant to bacteriocins. , by using the bacteriocins of the invention, e.g. in starter cultures for fermentation. Starter culture of a type of cell and a bacteriocin invented by a fallen tree from a mixed cell population When added to substances, it helps to eliminate foreign microorganisms and also, for example, spore-forming c. Against undesirable strains of lostridium spp., IaCtiS It is valid.

The present bacteriocins are advantageously used in relatively late stages of cheese or yogurt fermentation. In, lactic acid, protease and flavor by L. Iactis microorganism can be added after the production of

Place the producing strain in either a starter culture or in milk or other media. By keeping the ingredients clean and pure, uniform production can be improved.

This bacteriocin selects lactic acid-producing bacterial strains in beer fermentation and distillation fermentation. It can also be used to kill targets. This is because these strains are not pasteurized according to the literature. It is considered the main cause of spoilage in beer that has not been fermented, and also has the highest rate of infection during fermentation. This is because it is caused by

The present invention particularly uses the present bacterium as an inhibitor of contaminating 1actococcus species. Includes a starter culture of a microorganism containing Osin. Such microorganisms are For example, strains resistant to this bacteriocin, 1actis strains, e.g. producing microorganisms. starter culture used in beer fermentation or distillation fermentation. Only undesirable microorganisms are removed from the bacteria or yeast. A starter culture like this The nutrients are usually in lyophilized form.

In view of its high selectivity, this bacteriocin is suitable for 1actococcus species. It can be used as a classification tool in identification.

The new bacteriocin is Lactococcus 1actis 5ubsp , from a culture of P. eremoris by fractionation of the growth medium. In this way, a fraction rich in this bacteriocin is collected. Known separation By applying this technique, it is possible to obtain this bacteriocin with electrophoretic purity. Wear. Thus, for example, by growing the microorganism in a suitable medium, for example M17 broth, After fractional precipitation of the supernatant with, for example, ammonium sulfate, a phosphate buffer solution is added. Chromatography on carboxymethyl agarose and/or Phenyl is gradient eluted with phosphate buffer containing increasing concentrations of ethanol. Work up by chromatography on Superose.

The present inventors have developed a two-gene operon (bloc) from L. Iactis cremoris. encodes this bacteriocin in raw form), as well as another protein Considered to be an immune factor that confers resistance to autolysis by this bacteriocin. It was possible to clone the sea fencing (see below) and determine the sea fence. of this operon The complete sea fence is shown in Figure 1. This figure shows the corresponding proteins, i.e. Also shown is the bacteriocin and the immune factor Seafence. this Operons begin with regions that regulate gene expression. This corresponds to areas -35 and -IO It is thought to contain a promoter located in the molecule and a liposome binding site. Pro Thief The gene encoding the sequence is from base 312 to base 374. Book The gene encoding bacteriocin is from base 375 to base 536. be. The genes encoding this putative immune factor have different readings. From base 554 to base 847 in the frame. The three putative promoters are , designated as Pl, P2 and P3 in region -35 and -1O, The liposome binding site is designated as RBS.

According to another aspect of the invention, the present bacteriocin and the present immunophage, respectively DNA encoding the vector is provided. Ami according to claim 1 The entire amino acid sequence and/or code for this bacteriocin. Knowledge of the DNA sequence that encodes the mature batateriocin It is recognized that no indication of the codon position is given.

Therefore, the present invention is applicable not only to the DNA sequence shown in Figure 1, but also to the degeneracy of the code. Therefore, it also includes Seafence, which can also code for the protein in question. do.

The present invention also relates to the mature bacteriocin and/or the immune factor. Also includes cloning vectors and expression vectors containing DNA encoding do. Expression vectors suitable for L. 1actis are particularly preferred.

Furthermore, the present invention was transformed with such a vector.

Also included are strains of S. lactis.

The immune factor of the present invention eliminates the action of 1.1 actis bacteriocin, e.g. For example, it can be used to suppress the action of the bacteriocin of the present invention.

The gene encoding this immune factor was cloned into a food-grade cloning vector. as a selective marker, e.g. instead of an antibiotic, in the subsequent construction of vectors. Can be used.

The operon shown in Figure 1 is a fragment of L. actis cremoris. The mature bacteriocin of the DNA strand shown in Figure 1 was extracted from the converted plasmid DNA. A protein containing all or part of a non-coding DNA strand that corresponds to a coding portion. Obtained using lobes. Regarding this mature bacteriocin or this immune factor The encoding DNA can be inserted into any conventional cloning vector for amplification and For example, in an expression vector for transformation of a host microorganism such as Iactis, Cloning vector pIL 253 (A, Chopin, Bioch ime 70.1988.59-566). suitable culture When grown under nutrient conditions, this bacteriocin is produced in the growth medium, and this medium The present bacteriocin can be isolated from the teachings of the above-mentioned literature.

A plasmid containing the NA sequence necessary to provide an improved strain or Multiple copies of other vectors can be transformed. Such improved stocks are When used in cheese making, it accelerates the ripening of cheese as it provides faster lysis. . In particular, 1, which produces this bacteriocin and therefore also carries the resistance gene. Strains of S. 1actis can be obtained with such multiple copies of the vector. subordinate This bacteriocin allows this strain to grow without being degraded before maturity. .

Novel bacteriocins are advantageously synthesized by chemical synthesis, e.g. using solid phase synthesis. can be produced using commercially available polypeptide synthesizers. this In such syntheses, the active side chain groups of each amino acid (e.g. amino group or carboxyl group) is protected, the protecting group is removed in the final step, and/or the During the reaction, the polypeptide is removed from the inert support to which it was attached.

When multiplying peptide chains, C-terminal initiation operations are generally used, but as a general rule, can start from either the C-terminus or the N-terminus.

Thus, for example, a suitable derivative of histidine can be combined with a protected suitable derivative of leucine. It is possible to start from the C-terminus by reacting with the body. histidine induction one reactant has a free α-amino group, while the other reactant has a free or activated It has a carboxyl group and a protected amino group. After coupling, compare the intermediates can be purified by chromatography and then subjected to another N-protection selectively N-deprotected to allow the addition of free or activated amino acid residues. can be protected. Continue this operation until the required amino acid sequence is completed. continue.

Carboxylic acid-activated substituents that can be used include, for example, symmetrical or mixed anhydrides, or activated esters such as p-nitrophenyl ester, 2,4,5. - Lichlorophenyl ester, N-human sevenxybenzotriazole ester (OB t), N-human heptoxysuccinimidyl ester (O3u), or pen Tafluorophenyl ester (OFP) is mentioned.

The coupling of a free amino group with a carboxyl group can be achieved, for example, with synchrohexyl This can be carried out using carbodiimide (DCC).

Other coupling agents that may be used include N-ethoxycarbonyl-2-ethoxy Ci-1,2-dihydroquinoline (EEDQ) is mentioned.

Generally, the coupling reaction is carried out at a low temperature, such as from -20°C to ambient temperature, advantageously at a suitable temperature. Solvent-based, e.g. tetrahydrofuran, dioxane, dimethylformamide, chloride Preference is given to working in methylene or a mixture of these solvents.

It may be further advantageous to carry out the synthesis on a solid resin support.

Chloromethylated polystyrene (crosslinked with 1% divinylbenzene) is a useful mold support. In this case, for example, N-protected histidine is coupled to the support. The synthesis is started from the C-terminus.

Many suitable solid phase techniques are described in the following literature: Er1cAthreto nChristopyer J, longan and Robert C , 5heppard, J.

Chem, Soc, Perkin 1.538-46 (1981); J ames P, Tam, Foe S.

Tjoekin, and R.B., Merrifield, J.J., Che m, Soc, 102.6117-27 (1980); James P, T am, Richard D, Dimarchi and R,B.

Merrifi61d 1nj1. J, Peptide Protein R es, 16. 412-25 (1980); Manfred Mutter and Dieter Be1lof, He1vetica ChimicaA Cta 67, 2009”16 (1984).

It is known that protecting groups for amino acids can be selected from a wide range of groups, such as the following: Some of the literature can be cited. 5 chroeder, E., and Luebke.

K,, The Peptides, Vols, 1 and 2. Aca Dernic Press, New York and London, 196 5 and 1966; Pettit, G.R., 5yntheticPep tides, Vats, l-4, Van No5trand, Re1nh old, New York, 1970゜1971, 1975 and 19 76; Houben-Weyl, Methoden der Organi schenChemie, 5ynthese von Peptiden, Band　15. Georg Thieme Verlag Stuttgar t, NY, 1983; The Peptides, Analysis, 5 synthesis.

biology I-7, Ed: Erhard Gross, Johanne s Meienhofer.

Academic Press, NY, San Francisco, L ondon; 5 solid phase peptide 5 synthesis 2nd ed, John M, Stewaet, Janis D, Y owning.

Piece Chemical CompanyAmine protecting groups that can be used accordingly For example, carbobenzoxy (Z-), t-butoxybonyl (Boc- ), 4-methoxy-2,3,6°-trimethylbenzenesulfonyl (Mtr- ) and 9-fluorenylmethoxycarbonyl (Fmoc-). C When building a peptide from the end, the α-amino group of each new residue added amine protecting groups are present and are selectively removed before the next coupling step There is a need. Particularly useful groups for such temporary amine protection are pinpoints in breeding solvents. It is an Fmoc group that can be selectively removed by treatment with peridine. .

The carboxyl protecting groups used include, for example, easily cleavable ester groups, e.g. Babe], Jill (-0BZI), p-21.1 Kobe5 Jill (-0NB) or t-butyl (-tOBu), as well as coupling to a solid support, e.g. An example is a methyl group bonded to polystyrene.

There are a wide range of other groups, e.g. It is within the scope of the present invention to use the groups of in the above-described method.

There is a wide range of procedures for removing amine and carboxyl protecting groups. deer However, these procedures must be coordinated with the synthetic strategy employed. Side chain protection The protecting group is used to remove the temporary α-amino protecting group before the next coupling. It should be stable under 7 conditions.

The amine protecting group with Boc and the carboxyl protecting group with tOBu can be removed by acid treatment. , for example, by treatment with trifluoroacetic acid. Pep When increasing the tide chain, a C-terminal initiation procedure is generally used or, in principle, It can start from either the C-terminus or the N-terminus.

Thus, for example, a suitable derivative of histidine can be combined with a protected suitable derivative of leucine. It is possible to start from the C-terminus by reacting with the body. histidine induction one reactant has a free α-amino group, while the other reactant has a free or activated It has a carboxyl group and a protected amino group. After coupling, compare the intermediates can be purified by chromatography and then subjected to another N-protection. -) Selective N-detachment to allow addition of free or activated amino acid residues. It can be protected. Repeat this operation until the required amino acid sequence is completed. Continue with.

Carboxylic acid-activated substituents that can be used include, for example, symmetrical or mixed anhydrides, or activated esters, such as p-nitrophenyl esters, 2.4.5. -1 -lichlorophenyl ester, N-hyaloquine benzotriazole ester ( OBt), N-hydroxysuccinimidyl ester (O3u), or Examples include pentafluorophenyl ester (OPFP).

The coupling between a free amino group and a carboxyl group can be achieved, for example, with dicyclohexyl This can be carried out using carbodiimide (DCC).

Other coupling agents that can be used include N-ethoxycarbonyl-2-ethoxy Ci-1,2-dihydroquinoline (EEDQ) is mentioned.

Generally, the coupling reaction is carried out at low temperatures, e.g. -20"C to ambient temperature, advantageously suitable solvent systems such as tetrahydrofuran, dioxane, dimethylformamide, salts Preference is given to working in methylene chloride or mixtures of these solvents.

It may be further advantageous to carry out the synthesis on a solid resin support.

Chloromethylated polystyrene (crosslinked with 1% divinylbenzene) is a useful mold support. of the body, in which case, for example, N-protected histidine is coupled to the support. The synthesis is started from the C-terminus.

Many suitable solid phase techniques are described in the following literature: Er1cAthreto nChristopyer J, longan and Robert C , 5heppard, J.

Chem, Soc, Perkin I, 538-46 (1981); James P, Tam, Foe S.

Tjoekin, and R.B., Merrifield, J.A., Ch. em, Soe, 102.6117-27 (1980); James P. Tam, Richard D, Dimarchi and R,B.

Merrifield [nt,, J, Peptide Protein Res, 16.412-25 (1980); Manfred Mutter and Dieter Be1lof, He1vetica Chimica Acta 67, 2009-16 (1984).

It is known that protecting groups for amino acids can be selected from a wide range of groups, such as: Some of the literature can be cited. 5 chroeder, E., and Luebke.

K,, The Peptides, Vols, 1 and 2. Aca demic Press, New York and London, 1965 and 1966; Pettit, G.R., 5yntheticPept ides, Vols, 1-4. Van No5trand, Re1nhol d, New York, 1970゜1971, 1975 and +976 ; Houben-Weyl, Methoden der Organiseh enChemie, 5ynthese van Peptiden, Band 15. Georg Thieme Verlag Stuttgart, NY , 1983; The Peptides, Analysis, 5ynt hesis.

biology l-7, Ed: Erhard Gross, Johanne s Meienhofer.

Academic Press, NY, San Francisco, L ondon; 5 solid phase peptide 5 synthesis 2nd ed, John M, Stewaet, Janis D, Y owning.

Piece Chemical CompanyAmine protecting groups that can be used accordingly For example, carbobenzoxy (Z-), t-butoxycaponyl (Boc- ), 4-methoxy-2,3,6°-trimethylbenzenesulfonyl (Mtr) and 9-fluorenylmethoxycarbonyl (Fmoc-). C- When building a peptide from the end, the α-amino group of each new residue added An amine protecting group is present in the molecule and is selectively removed before the next coupling step. There is a need. Particularly useful groups for such temporary amine protection are It is an Fmoc group that can be selectively removed by treatment with peridine.

Carboxyl protecting groups used include, for example, easily cleavable ester groups, e.g. babenzyl (-0BZI), rim; trobenzyl (-0NB) or t-bu (-tOBu), as well as coupling to solid supports, e.g. Examples include methyl groups bonded to ren.

There are a wide range of other groups, such as those detailed in the above-mentioned literature, and all such It is within the scope of the invention to use the groups in the above-described methods.

There is a wide range of procedures for removing amine and carboxyl protecting groups. deer However, these manipulations must be compatible with the synthetic strategy employed. side chain protection group used to remove the temporary α-amino protecting group before the next coupling. It should be stable to the conditions in which it is applied.

Amine protecting groups such as Boc and carboxyl protecting groups such as tOBu can be removed by acid treatment. , for example by treatment with trifluoroacetic acid.

The examples below are given by way of illustration only.

Example 1 Bacterial strains, media, plasmids and enzymes. Bacterial strains, plasmids and plasmids used The age are listed in Table 1. All 1actOcOcca1 strains are M17 cultured in broth (44) and in M17 broth containing 10% glycerol - Stored as a frozen stock at 80°C. Escherichia coli DH5a was used for propagation of pUc18 and its derivatives. M13 vector - and its clones are treated with restriction endonuclease, T4 DNA ligase, T4 Polynucleotide kinase and DNA molecular weight standards were obtained from Bethesda. Re5erch Laboratories, Inc. RG, Md, ). Bovine intestinal alkaline phosphatase, sequence The trypsin and endoprotease glu-C of Carex violacea were prepared by Boehri hger GmbH (Mannheim.

Germany). Sequenase is U nitedStates Biochemical Corp, (C1eve LAND, 0hio).

Plasmid curing aL, 1acjls 5ubsp, CremOrl S LMG 2130 in the presence of 0.1 Mg novobiocin per ml, 38 The cells were grown in M17 broth containing 1% glucose at 10°C. Dilution of this culture Aliquots were spread onto M17 broth 1% glucose plates and incubated at 30°C. Cultured. Colonies were counted as a sample of bacteriocin production.

Bacteriocin assay. Three methods to determine bacteriocin activity was used. (i) Placing colonies of bacteria capable of producing bacteriocin on agar overnight. Grown on a plate. M17 soft cold containing 100 μm of fresh culture of indicator microorganism A lawn equivalent to 3 ml of turquoise (0.7%) was poured onto the plate.

After culturing overnight at 30°C, colonies were examined to evaluate growth inhibition zones. .

(ii) Form wells with a diameter of 4 mm in M17 soft agar, and Filled with solution. After the liquid has been completely absorbed into the gel, add the M17 soft gel containing the indicator microorganisms. Place the quality agar on this plate and let it show bacteriocin activity as described above. Ta. (iii) The activity of bacteriocin was determined according to Gels et al. (15). Therefore, it was quantified using the disclosed method. However, in this case, 200 μl of M17 broth A microtiter plate was used with wells containing bacteriocin One unit (BU) of activity is 1 actis 5 ubs in this assay. 50% growth inhibition of P. cremoris IMNC18 (containing bacteriocin) It was defined as the amount required to produce a turbidity of 50% of that of the control sample with no turbidity.

Table 1 Bacterial strains, plasmids, and phage bacterial strains used in the examples 4-kb HindI [1 fragment of this example] containing pONlltn^ pUc18 with This example fragment of J-kb Hindll containing pON 21cn^ pUcI8+ with: 1.2 containing plL 253polf T lcn^ -kb R+l1-Hindn [pHc18 with this example fragment: Purification of pIL 253LCN-A. L, 1actis 5ubsp, cr Bacteriocin was purified from 1 liter of culture of Emoris LMG 2130. Manufactured. Various steps were performed at 4°C unless otherwise indicated. Initialize the cells. After growing to constant stage, the bacteria were isolated by centrifugation at 10,000×g for 10 minutes. removed. Add 280 g of ammonium sulfate per liter to culture supernatant. bacteriocin was precipitated. Centrifuge at 101,000 x g for 30 min. After that, dissolve the pellet in water and add 0.5M NatHP○4 to pH 7, Adjusted to 3. This solution was equilibrated with mM sodium phosphate (pH 7.3). Pharmacia Uppsala, 10m1 Sweden). Add this column to 40ml of 20mM sodium phosphate. (pH 7,3) and then 20 ml of the same buffer containing 0.3 M NaCl. The bacteriocin was eluted. Bacteriocins, fast protein solution chroma Reversed phase chromatography by Tokrafi apparatus (Pharmacia) I put it on. The eluate from the cation exchanger was purified with 10 mM sodium phosphate (pH 7). , 3) equilibrated with a 1 ml phenylsperose column (Phenyl-3u perose, Pharmacia). 10mM sodium phosphate After washing with ethanol (pH 7,3), the ethanol concentration was adjusted linearly at a flow rate of 0.3 ml/min. Elution was performed by varying the concentration between 0% and 60%. Purified LCN-A is 60% Stored at -20°C in ethanol-25mM sodium phosphate (pH 7,3). Ta. Protein concentration was measured by spectrophotometer at 2 aonm.

Primary structure of amino acids. Applied Biosys for determining the primary structure of amino acids tems (Foster C1ty Calif,) 477 A sequencer - was used.

Phenyldiohydantoin-derived amino acid residues are available from Applied Biosys Automatically measured using a te + ns l 20 phenylthiohydantoin analyzer did. The C-terminal part of the sea fence is based on Bornstein and Ga1ia The Asn-GIN' bond was synthesized with human glycosylation at pH 9 as disclosed in (3). Obtained after cleavage with amine.

DNA0 isolation, analysis and manipulation. Disclosed by Klaenhammer (23) Sumid DNA was prepared. Disclosed in Maniatis et al. (25) The larger plasmid was isolated from E. coli by the alkaline cleavage method. I let go. From a 1.5 ml culture infected as described in (2a) above, Thief An M13 plus-strand DNA template was prepared for the determination of the genetic background.

DNA manipulation enzymes were used according to the manufacturer's instructions. used for cloning The plasmid DNA from LMo 2130 strain was subjected to CsCl isopycnic centrifugation (33 ).

Isolate restriction fragments suitable for cloning and perform cloning using Gene-Clean. and purified from 0.7% agar.

The DNA cloned in E. coli was transformed into 1act as shown below. It was subcultured into ocoeci. pUc18 plasmid with insert DNA pIL25 was obtained by EcoRI DNA digestion reaction and DNA ligation reaction. It was merged into 3. The resulting product was transformed into L coli. clone is resistant to erythromycin (300μg/ml) and ampicillin (500μg/ml). g/ml) was obtained by selectivity of resistance. Claw: plasmid extracted from / DNA is based on the technology 1. disclosed in Hole and Nes (20).・Ritorobo ration (eleetroporat 1on)...)te 1aetoco Nucleic acid hybridization and nucleic acid hybridization for transforming cci. doshi fencing. Based on the sea fence following amino acid 25 of L CN-A and prepared a 64-fold-denatured synthetic oligodeoxynucleotide probe as follows: (Apl)lied Biosystem 381A DNA synthesizer ). 3'-ATIGT (T/C) GT (T/C) TG (I/ C) TG (T/C) TTICG (I/C) AAICC-5'.

As disclosed in Hanahan and Meselson (18), Ronnie hybridization was performed. DNA digested with endonuclease (separated with 0.7% agar) using GeneScreen Plus Menplan x (N ENResearch Products, Dupont, Boston, Mass, ) to vacuum transfer (Vacugene; Pharmacia) By performing the Southern blot method (42), Church and G11 bert (7), with 261 oligodeoxynucleotides. Hybridization was performed. In M13mp18 and M13mp19 For cloned restriction fragments, the deoxynucleotide method (37) Nucleotide sea fencing was performed using The label includes [α-”S]dAT P (600Ci/mmol; Amersham International 1, Amersham, Llnited Kngdom) was used.

Nucleotide sea fence accession number. NucL nootide seeds presented herein Quence is certified under EMBL accession number M63675.

Cultivation - Hi Clear 1.000 + 4.6 15 102.8 1 100 During growth in 17 medium, it was found to essentially produce bacteriocins. . We developed a method to purify bacteriocins from culture supernatants. purification scheme It is shown in Table 2. This protein contains approximately 95 amino acids, as determined by amino acid sequence analysis. % purity. The amino acid sequence of the purified bacteriocin was as described above. It is. This bacteriocin contains 54 amino acid residues, with a total of 5.778 It was recognized that the compound had a calculated molecular weight. This molecular weight was confirmed by mass spectrometry This means that there is virtually no glycolysis or methylation. It shows. 5w1ss-Prot or in the NBRF database No significant sea fence similarities were observed with either the protein or the putative gene product. I couldn't.

The present inventors named this new bacteriocin LCN-A.

This protein contains alanine residues (8 out of 54) and glycine residues (54 It contains only three charged amino acid residues. This Bakte The calculated isoelectric point of riosin was 9.2. Extinction of LCN-A at 280 nm The coefficient is calculated from its tryptophan and tyrosine content and is 1. ’　2 X10'cm-'M-1 (4). Therefore, purified LCN-A is approximately 4.9 It had a specific activity of XIO5BU/mg. LCN-A activity decreases during purification If we consider that the LM02130 strain does not contain 3 mg per liter (dipp lococein) was observed to have been produced (1O). This purified bacterio Syn was not very soluble in water. When stored in aqueous buffer at 4°C, this buffer Cteriocin formed an inactive precipitate. However, purified LCN-A is 2.5mM sodium phosphate (pH 7.3) without any appreciable decrease in activity. ) in 60% ethanol at -20°C for more than 6 months.

Effect of protease. LCN-A contains the highly specific endoprotease gla-C and Its activity is lost by contacting it with various proteases such as trypsin and . In phosphate buffer (pH 7,8), the endoprotease glu-C is This bacterium at one site between amino acid residues 12 (Asp) and 13 (Leu) trypsin can cleave this bacteriocin into two degradable It was possible to cleave at the carboxyl group of residues (l and 21).

Inhibition spectrum and mode of action. More than 120 strains were detected by agar diffusion assay. 1actis 5ubsp, 1actis andri, 1actis 5u bsp.

The sensitive taste was rapidly sterilized by this bacteriocin. grow exponentially The number of surviving cultures of rMN C28 was 200 BU/ml in M17 medium at 30°C. After 5 minutes of exposure to (bacteriocin), it decreased from 2×10”l/ml.

Table 3: Susceptibility of I&claccocu strains to LCN-A MNC 185 LMG 2141 1,00O NCDO6071,3 NCDO9241,00O NCDO11980,4 BC10150 BC101 (pON2) 5,000 BC101 (pON7) 5,000 Shukoku 1 magazine 5 ubsp, Time 5 NCDO60430 Engineering L 140 pieces 0+4 Engineering L 1403 (pON2) 1,500 Engineering L 140 pieces (pON7) 1,5 0ONCDO176 (biovar diacetylactis) 20L Dan Tactococcus NCD02155 5,000 Table 3 shows various tactococcus The susceptibility of strains of the genus to LCN-A is shown. There is wide variation in sensitivity Admitted. The most sensitive strains tested were found to be more sensitive to milk than in M17 broth. Shows greater susceptibility to bacteriocins when grown in broth (14), the calculated LCN-A concentration was 40 pg/m in milk broth. 1 or 7 pM, 50% growth inhibition of strain NCD0II98 was observed. this amount corresponds to 400 molecules of LCN-A per CPU in the assay.

Of the strains tested, only two, namely the bacteriocin producing strain (1MG2130) Body, L, 1actis 5ubsp, 1actis biovar dia cetylactisN [ZO4-25 was resistant. However, this latter strain was found not to produce bacteriocin. Nisin production tested sensitive to strain LCN-A and inhibitory to 1MG2130; The bacteriocin is 1. Weak inhibitory effect of garviase NCD02155 The results were shown (Table 3).

Identification and cloning of genetic determinants of LCN-A. Amino acid to LCN- Sequence-based oligodeoxynucleotide probes are used to identify genes in diization analysis. LNG21 When plasmid DNA from 30 strains was detected, it corresponded to a 55-kb plasmid. One signal was observed. LM02130 strain is plasmid curing It was attached to. One isolated population that did not produce LNG-A, namely LMG2131. loses the 55-kb plasmid and gives no signal in the Southern blot. There was found. Additionally, Southern analysis of LMGl 30 plasmid DNA digests showed that 4-kb HindIII fragment, 1.2-kb HindIII I-R Signals from the sa17 fragment and the 0.6-kb DraI fragment appeared. 4 of LMG2130 plasmid DNA digested with Hind-III -kb fraction was extracted in E.coli as a vector using pUCl8. was cloned. 10 out of 1,400 clones or oligonucleotides It was found to be positive through probe screening. These 10 cubes The recombinant plasmid (pONl) from one of the loans was transferred to Dral and Rsa Further restriction digestion was performed. The fragment hybridized to the probe, i.e. 4-kb HindITT fragment, 1.2-kb Hindlll-Rs al fragment and the 0.6-kb Dral fragment are M13Mp1 8 and Ml 3mp 19, both orientations inserts were produced.

1cnA nucleotide sea fence o Hind I I l-R5al fl We decided on a Sea Fence for Gument. two of 625 and 292 nucleotides The nucleotide sequence of consecutive Dral fragments is shown in Figure 1. 1c The entire nA gene or 0. Contained with a 6-kb Dral fragment there was. Computer of six possible open reading frames (ORFs) Analysis revealed long ORFs in only one of the DNA strands. 54 a Mature LCN-A, consisting of amino acid residues, begins at nucleotide positions 316-477. encoded by a DNA segment. It means the only possible synthesis ing. The start codon is a possible Shine-Dalgarno sequence 3' It is preceded by AGGAGA5' (40). Three putative promoter elements (All E, coliσ70 consensus and 5treptcoccat pro This liposome binding site (RBS) (which shows considerable similarity to motors) (Fig. 2) (27, 35).

A second ORF, 0RF2, was discovered downstream of 1cnA. Nucleochi Assuming that the translation initiation site is at ATG at position 495, this OFR is Encodes a amino acid polypeptide. Possible RBS sequences, i.e. 5° GA GGATTGA3' appeared 7 nucleotides from the Met codon. two of 0RF2 extending from nucleotide positions 803 and 896 in the two-fold axis of symmetry region of could be formed from a lower stem-loop structure (45). those distant stems The uridine content of these structures suggests that they are Rho-independent terminators of 1cnA transcription. It is suggested to configure the Estimated terminator sequence or pro Motor sea fence is not recognized between IcnA and 0RF2, and this indicates that 1cnA and 0RF2 may constitute an operon. .

Any DNA sequence in the EMBL database can be used as the DNA sequence of the present invention. It did not show a high degree of DNA homology. In this database, the closest was 57.4% identical to the 122'' general sequence.

Cloning in L. lactis. The IcnA gene is present in 1actis. I cloned it. A 4-kb Hindlrl fragment and a 12-kb Hindlrl fragment. PIL growing Hind I I l-R5a 1 fragment, 253: : The pUC18 constructs did not bind pON2 and pON7, respectively. but However, when in BC101, both pON2 and pON7 are LCN- Provided resistance to A. Both plasmids caused 50% growth inhibition. Strain LCN-A concentration increased from 50 to 5,0 OOBU/ml (Table 3). child This result was not seen in transformants containing only the cloning vector.

Similar results were observed for other strains of L. 1actis (data not shown). not present). Among the strains tested, a CN-A producing strain transformed with the 1cnA gene One LMG2130 produced 1,500 BU/ml.

Susceptibility to T., CN-A appears to be common among L. actis strains. There it is. This bacteriocin is so specific that L.

1actis strain. I can do two things. LCN-A is a hydrophobic protein It is. This hydrophobic property has a high affinity for phenyl-superose. indicated by. This matrix is suitable for hydrophobic interaction chromatography. most proteins only bind to this at high salt concentrations. match. I, CN-A binds to this column even in the absence of salt and is less polar than water. It could only be eluted as an active bacteriocin with a clear solvent.

The toxic effects of nisin have been attributed to its ability to form pores in the cytoplasmic membrane (3). 6). The hydrophobic properties of LCN-A make the cytoplasmic membrane a target for this bacteriocin. It suggests that there is something like that. Ra.

and by calculations as disclosed by Argos (34), the amino acid of LCN-A The range of amino acids 30 to 52 forms a helix that connects the membrane (me+++brain-spanni). nghelix) (data not shown). It has not been). The idea that LCN-A acts on membranes is based on this bacteriocin. or leakage of intracellular components even in hypertonic sucrose-containing media. Further support is provided by findings (unpublished data).

Secreted proteins usually contain a short N-terminal portion called a signal peptide. This part is synthesized as a precursor that contains a certain amount of enzymes, and this part promotes secretion and removed by the element (1.43.49°50,52). Mature LCN-A Comparison of genetically derived sea fences and direct amino acid sequence data This shows that LCN-A is synthesized as a 75 amino acid precursor. 21 a The LCN-A leader peptide, consisting of amino acids, has a positively charged N-terminus; Next, the typical hydrophobic range (stret) as a signal peptide for Durham-positive bacteria was ch) exists (1). Mature 1, CN-A, as its N-terminal amino acid Contains lysine. In the LCN-A precursor, A1a-As is added to the second lysine. n-G 1 y-G l y Sea fence or 7 ahead. Von He1j According to the “-3,-1” rule of ne(49,50), there are two signal peptidase Can cleave between glycine (-2, -1), but between glycine and lysine (-1, +1) It is not possible to cut the gap. This theory suggests that first a 20 amino acid peptide, then glycine was removed from the N-terminus, resulting in mature LCN-A consisting of 54 amino acids. This may suggest a gradual process of procera song.

A putative promoter element was found immediately upstream of the 1cnA gene (Fig. 1). Ta However, transcription initiation occurs at points 5 to 9 downstream of the putative Pribnow box. nucleotides and will generate a leader of 17 to 33 nucleotides cormorant. The overlap of 10 regions of the putative promoter element has a stem-loop structure. This is the reverse repeating sea fence obtained (Figure 1). This structure is -9,6kcal/ m.

], (-40,2kJ/mol) (45), and there is a calculated ΔG of 0. It will represent the Rho-independent terminator of RF1.

The LMG3121 strain that lost the IcnA gene was sensitive to LCN-A. . This result suggests that either the production mechanism or the gene encoding immunity to bacteriocins may be affected. It suggests that it is hidden. IL14 with recombinant plasmid pON7 Strain 03 produces LCN-A and is (necessarily) resistant to bacteriocins. It was. Thus, the 1.2-kb Rs a l-Hlnd I I 17 lag Mento not only contains the gene encoding LCN-A, but also the genetic determinants of resistance. That's because I have it. The DNA sequence of this fragment was In addition to the gene, - only one complete ORF, i.e. within the same operon as 1cnA, and a position on the downstream side thereof 2. This is 0RF2. I wanted to make this clear. The co-copied OR, F2 seems to be a candidate encoding the LCN-A immune function. be. A close approximation of the bacteriocytic arch/gene and its corresponding immune gene The mechanism has been shown for several E. coli bacteriocins (2.2 6).

There is a Met at position 495 of Tsuk1 note, and the possible RBS sequence 5'G 0RF2 preceded by GATTAG3' is a hypothetical polypeptide of 98 amino acids. Codes petit de. Alternatively, it has L eu at nucleotide position 540. and is preceded by a possible RBS sequence 5' AAGAAG3' and a hypothetical There may also be ORF2, which encodes an 83 amino acid polypeptide. But why , codon usage for the 15 N-terminal amino acids of a 98-amino acid polypeptide The formula represents the remainder of the 0RF2 polypeptide and the compiled codons of LCN-A. This correlates well with the usage pattern, indicating that ORF2 or the 98 amino acid polypeptide This shows that it codes for petit de.

-One Its six N-terminal residues (Met-Lys-Lys-G1.=y)-=IIe) are , very similar to the signal peptide of Durham-positive bacteria.

7.9 and G11] in 11th place, this estimated signal thief ence retains hydrophobic properties spanning amino acid positions 5-20. VonH According to the -3,-1 rule of eijne, Ala-Thr at amino acid position 20 - There are possible signal peptidase cleavage positions after Ala. Abrahams 14 Durham positive signal sheets compiled by en et at, (1) Seven of the sequences contained Ala-X-Ala at the cleavage site. Furthermore, A la-Thr-AI or Bacillus 5ubtilis β-glucanar 7 -3, -1 amino acid sequence of the signal peptidase cleavage site of was found (29).

This suggests a mature 0RF2 protein consisting of 79 amino acids. . ○The polypeptide encoded by RF2 is secreted into the membrane or anchored. It remains to be shown how the

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261:10176-10181゜ PCT/EP　91101109 Continuation of front page (51) Int, cL,” Identification symbol Internal office reference number C07K 7/10 ZNA 8318-4HC12N 1106 7236-48 C12P 7106 7432-4B //(C12P 21102 C12R1:01) (81) Designated countries EP (AT, BE, CH, DE.

DK, ES, FR, GB, GR, IT, LU, NL, SE), AU, CA, FI, JP, MG, No, USI

Claims (11)

[Claims] 1. Amino acid sequence below [There is an array] or an inducer thereof having bacteriocin activity. conductor or fragment. 2. Amino acid sequence below [There is an array] or a polypeptide having or containing bacteriocin immunoreactivity. derivatives or fragments of. 3. used in a microbial method, comprising a polypeptide according to claim 1; A starter culture in which said microorganism is resistant to said polypeptide. 4. The star according to claim 1, wherein the microorganism is lactic acid bacteria or yeast. tar culture. 5. In order to lyse lactic acid bacteria, the polypeptide according to claim 1 is added. A method for producing cheese or yogurt, comprising: 6. The polypeptide according to claim 1 is used to selectively kill contaminant strains of lactic acid bacteria. A fermentation method for producing ethanol using Tido. 7. A culture of a microorganism expressing the polypeptide according to claim 1 is separated. , thereby collecting a fraction enriched in said polypeptide. A method for isolating the polypeptide according to item 1. 8. The microorganism mentioned above is Lactococcus lactis subspice cremoris ( Lactococcus lactis subsp. cremoris) 8. The method according to claim 7. 9. The polypeptide according to claim 1 and/or claim 2 The DNA sequence that codes for it. 10. Polypeptide according to claim 1 and/or claim 2 Lactococcus transformed with a vector containing DNA encoding for - A strain of L. lactis. 11. Deprotect or remove the corresponding protected or immobilized polypeptide. or separated from an inert support, as claimed in claim 1 or claim 2. A method for producing the polypeptide described above.