Protein having peptidoglycan-degrading activity, DNA encoding the protein, microbial decomposition preparation, and microbial decomposition method

Abstract

Provided is a protein derived from Tumebacillus sp. NITE BP-02779 that has peptidoglycan-degrading activity. Provided is a protein comprising the amino acid sequence corresponding to positions 1-164 of SEQ ID NO: 2 or the amino acid sequence corresponding to positions 1-493 of SEQ ID NO: 4, or a protein comprising an amino acid sequence having a substitution, deletion, insertion or addition of 1-10 amino acid residues in these amino acid sequences, or an amino acid sequence having a sequence identity of at least 90% to these amino acid sequences, wherein the protein has peptidoglycan-degrading activity.

Description

Technical Field

[0001] The present invention relates to a protein having peptidoglycan-degrading activity, DNA encoding the protein, a microbial decomposition preparation, and a microbial decomposition method.

Background Art

[0002] When sewage is purified by the activated sludge method, the removed organic matter becomes flocs containing microorganisms (bacteria), and sludge called excess sludge is generated. Excess sludge discharged from wastewater treatment facilities accounts for more than 20% of industrial waste. Generally, excess sludge is dehydrated, dried, and then incinerated (Non-Patent Document 1: Report on the Survey of the Discharge and Treatment Status of Industrial Waste in the 30th Year of Heisei, Results in the 28th Year of Heisei (Summary Edition), Non-Patent Document 2: Masayuki Yamamoto, "On the Combustion Technology of Sewage", Journal of the Japan Fuel Society, The Combustion Society of Japan, 2011, Vol. 53, No. 164, p91-96).

Prior Art Documents

Non-Patent Documents

[0003]

Non-Patent Document 1

Non-Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0004] However, since incineration of excess sludge generates greenhouse gases, consideration for the environment requires reduction of the volume of excess sludge. As a method for reducing the volume of excess sludge, a method of decomposing the microorganisms constituting the excess sludge has been proposed.

[0005] An object of the present invention is to provide a novel protein capable of decomposing peptidoglycan, DNA encoding the protein, a microbial decomposition preparation, and a microbial decomposition method.

Means for Solving the Problems

[0006] The present invention relates to [1] to

[11] exemplified below. [1] A protein derived from Tumebacillus sp. NITE BP-02779 and having peptidoglycan-degrading activity. [2] The protein according to [1], which is a secreted protein. [3] Any of the following proteins (A1) to (A3): (A1) A protein comprising the amino acid sequence of positions 1 to 164 of SEQ ID NO: 2 and having peptidoglycan-degrading activity, (A2) A protein comprising an amino acid sequence in which 1 to 10 amino acid residues are substituted, deleted, inserted, or added in the amino acid sequence of positions 1 to 164 of SEQ ID NO: 2 and having peptidoglycan-degrading activity, (A3) A protein comprising an amino acid sequence having 90% or more identity to the amino acid sequence of positions 1 to 164 of SEQ ID NO: 2 and having peptidoglycan-degrading activity. [4] The protein according to any one of [1] to [3], wherein the peptidoglycan-degrading activity is an activity of decomposing a glycosidic bond between N-acetylmuramic acid and N-acetylglucosamine. [5] Any of the following proteins (a1) to (a4): (a1) A protein consisting of the amino acid sequence of positions 1 to 164 of SEQ ID NO: 2, (a2) A protein consisting of an amino acid sequence in which Ala at position 1 is substituted with Met in the amino acid sequence of positions 1 to 164 of SEQ ID NO: 2, (a3) A protein consisting of an amino acid sequence in which Ala at position 1 in the amino acid sequence at positions 1 to 164 of SEQ ID NO: 2 is replaced with Met, and the amino acid sequence set forth in SEQ ID NO: 21 is added to the N-terminus of the Met, (a4) A protein consisting of an amino acid sequence in which Met is added to the N-terminus of Ala at position 1 in the amino acid sequence at positions 1 to 164 of SEQ ID NO: 2. [6] Any one of the following proteins (B1) to (B3): (B1) A protein comprising the amino acid sequence at positions 1 to 493 of SEQ ID NO: 4 and having peptidoglycan-degrading activity, (B2) A protein comprising an amino acid sequence in which 1 to 10 amino acid residues are substituted, deleted, inserted, or added in the amino acid sequence at positions 1 to 493 of SEQ ID NO: 4 and having peptidoglycan-degrading activity, (B3) A protein comprising an amino acid sequence having 90% or more identity to the amino acid sequence at positions 1 to 493 of SEQ ID NO: 4 and having peptidoglycan-degrading activity. [7] The protein according to [1], [2], or [6], wherein the peptidoglycan-degrading activity is N-acetylmuramoyl-L-alanine amidase activity. [8] Any one of the following proteins (b1) to (b4): (b1) A protein consisting of the amino acid sequence at positions 1 to 493 of SEQ ID NO: 4, (b2) A protein consisting of an amino acid sequence in which Glu at position 1 in the amino acid sequence at positions 1 to 493 of SEQ ID NO: 4 is replaced with Met, (b3) A protein consisting of an amino acid sequence in which Glu at position 1 in the amino acid sequence at positions 1 to 493 of SEQ ID NO: 4 is replaced with Met, and the amino acid sequence set forth in SEQ ID NO: 21 is added to the N-terminus of the Met, (b4) A protein consisting of an amino acid sequence in which Met is added to the N-terminus of Glu at position 1 in the amino acid sequence at positions 1 to 493 of SEQ ID NO: 4. [9] DNA encoding the protein according to any one of [1] to [8].

[10] A vector containing the DNA according to [9]. A transformant containing the DNA described in

[11] or the vector described in

[10] .

[12] A microbial decomposition preparation comprising at least one selected from the group consisting of the protein described in any one of [1] to [8], the transformant described in

[11] , and its culture.

[13] A microbial decomposition method comprising a step of allowing at least one selected from the group consisting of the protein described in any one of [1] to [8], the transformant described in

[11] , and its culture to act on a target microorganism. [Effect of the Invention]

[0007] According to the present invention, it is possible to provide a novel protein capable of decomposing peptidoglycan, DNA encoding the protein, a microbial decomposition preparation, and a microbial decomposition method. [Brief Description of the Drawings]

[0008]

Figure 1

Figure 2

Figure 3

Figure 4

[0009] Hereinafter, the modes for carrying out the present invention will be described in detail. Note that the present invention is not limited to the following embodiments.

[0010] [Protein derived from Tumebacillus sp. NITE BP-02779] The protein according to one embodiment of the present invention is a protein derived from Tumebacillus sp. NITE BP-02779 (hereinafter sometimes referred to as "NITE BP-02779") and having peptidoglycan-degrading activity. The protein may be a synthesized protein or a purified or isolated protein. Whether a certain protein is the protein according to the present invention can be determined based on the results of the protein identification method and the peptidoglycan-degrading activity measurement method described below. A protein derived from NITE BP-02779 and having peptidoglycan-degrading activity can decompose target microorganisms, and thus can reduce the volume of excess sludge.

[0011] NITE BP-02779 was internationally deposited with the Patent Microorganisms Depositary, National Institute of Technology and Evaluation (Room 122, 5-8 Kazusa Kamashita 2-chome, Kisarazu-shi, Chiba-ken 292-0818, Japan) on September 11, 2018, and a deposit receipt and a certificate of viability for the original deposit were issued on September 26, 2018. NITE BP-02779 is considered to be a new species of the genus Tumebacillus. Since NITE BP-02779 can decompose various target microorganisms such as Bacillus, Micrococcus, and Staphylococcus, as well as excess sludge, it is useful for excess sludge treatment. The mycological properties of NITE BP-02779 are shown in Tables 1 to 4 of the examples described below. NITE BP-02779 is a bacterium having a 16S rRNA gene containing the nucleotide sequence set forth in SEQ ID NO: 10.

[0012] "Derived from NITE BP-02779" may mean that a substance is synthesized or produced by NITE BP-02779, or may be based on the genomic sequence of NITE BP-02779. For example, based on the genomic sequence of NITE BP-02779, a substance may be artificially synthesized or produced within the cells of other organisms. Examples of substances derived from NITE BP-02779 include biomolecules such as proteins, nucleic acids, sugars, and metabolite products. These may be localized within the cell after being produced, or may be secreted outside the cell.

[0013] As a method for examining whether a certain protein is a protein derived from NITE BP-02779, for example, the following method can be mentioned. First, decode the genomic sequence of NITE BP-02779 using a next-generation sequencer. Trim the adapter sequence from the obtained base sequence and perform de novo assembly. Analyze the scaffold sequence obtained by de novo assembly, predict gene regions and perform annotation to create a protein database for mass spectrometry. Next, subject the target protein to mass spectrometry and compare it with the above protein database to determine whether the target protein is a protein derived from NITE BP-02779.

[0014] As another method for examining whether a certain protein is a protein derived from NITE BP-02779, the amino acid sequence of the target protein may be decoded using an amino acid sequencer and compared with the above protein database.

[0015] A protein derived from NITE BP-02779 and having peptidoglycan-degrading activity is preferably a secreted protein. A secreted protein can function extracellularly. In order to reduce the volume of excess sludge, it is useful for a protein having peptidoglycan-degrading activity to have peptidoglycan-degrading activity in the extracellular environment. A secreted protein having peptidoglycan-degrading activity may be recovered from the culture supernatant of the cells producing the protein, or may be recovered by destroying the cells producing the protein.

[0016] Peptidoglycan is a molecule in which polysaccharides in which N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc) are alternately β1,4-linked are cross-linked with oligopeptides. Figure 1 shows the structure of a typical peptidoglycan. "Peptidoglycan-degrading activity" refers to a state in which the reaction in which peptidoglycan is changed into two or more substances is promoted, and a "protein having peptidoglycan-degrading activity" (hereinafter sometimes referred to as "peptidoglycan-degrading enzyme") refers to a protein that promotes the above reaction.

[0017] Peptidoglycan is efficiently decomposed by the decomposition of glycosidic bonds between sugars constituting the polysaccharide, peptide bonds in oligopeptides, etc. Sites that can be decomposed by peptidoglycan-degrading enzymes are indicated by arrows in Figure 1. Examples of proteins having the activity of decomposing peptidoglycan include (1) a protein having the activity of decomposing the glycosidic bond between N-acetylglucosamine and N-acetylmuramic acid, (2) a protein having the activity of decomposing the glycosidic bond between N-acetylmuramic acid and N-acetylglucosamine, (3) a protein having the activity of decomposing the amide bond between N-acetylmuramic acid and L-alanine, and (4) a protein having the activity of decomposing each peptide bond.

[0018] Specifically, examples of such enzymes include: (1) N-acetylglucosaminidase; (2) N-acetylmuramidase, lytic transglycosylase; (3) N-acetylmuramoyl-L-alanine amidase; and (4) L,D-endopeptidase, D,L-endopeptidase, carboxypeptidase, D,D-endopeptidase.

[0019] As a method for examining whether a protein has peptidoglycan-degrading activity, for example, a method of reacting a target protein with peptidoglycan in a buffer for a certain period of time and then measuring the degradation of peptidoglycan can be mentioned. The method for examining the degradation is not particularly limited, and examples include a method of measuring the turbidity of peptidoglycan, a method of detecting peptidoglycan with an SLP reagent, high performance liquid chromatography (HPLC); mass spectrometry (MS); thin layer chromatography (TLC); nuclear magnetic resonance (NMR); gas chromatography (GC), etc., to detect degradation products derived from peptidoglycan.

[0020] As a method for examining whether a protein has the activity of degrading the glycosidic bond between (2) N-acetylmuramic acid and N-acetylglucosamine among the peptidoglycan-degrading activities, a method using Micrococcus lysodeikticus as a substrate can be mentioned. Specifically, cultured cells of Micrococcus lysodeikticus may be used as a substrate, or Lysozyme activity kit (manufactured by Sigma-Aldrich), EnzChek Lysozyme assay kit (manufactured by ThermoFisher Scientific), etc. may be used.

[0021] As a method for examining amidase activity that hydrolyzes the amide bond between (3) N-acetylmuramic acid and L-alanine among peptidoglycan-degrading activities, a method using L-aniline-p-nitroanilide hydrochloride (manufactured by Sigma-Aldrich), which is a substrate of N-acetylmuramoyl-L-alanine amidase, as described in Appl Microbiol Biotechnol. 2015, Oct, 99(20):8563-73, can be mentioned.

[0022] [The protein described in SEQ ID NO: 2 or 4] The protein according to one embodiment of the present invention is any of the following proteins. (A1) A protein comprising the amino acid sequence of positions 1 to 164 of SEQ ID NO: 2 and having peptidoglycan-degrading activity (B1) A protein comprising the amino acid sequence of positions 1 to 493 of SEQ ID NO: 4 and having peptidoglycan-degrading activity

[0023] The amino acid sequence of positions 1 to 164 of SEQ ID NO: 2 and the amino acid sequence of positions 1 to 493 of SEQ ID NO: 4 are the amino acid sequences of the mature forms of the proteins predicted from the genomic sequence of NITE BP-02779. Whether a protein has peptidoglycan-degrading activity can be determined, for example, by the method described above. A protein having peptidoglycan-degrading activity can decompose target microorganisms, so the volume of excess sludge can be reduced.

[0024] The protein according to one embodiment of the present invention may be a variant of a protein having the amino acid sequence of positions 1 to 164 of SEQ ID NO: 2 or the amino acid sequence of positions 1 to 493 of SEQ ID NO: 4 as long as the peptidoglycan-degrading activity is maintained. A variant in which the peptidoglycan-degrading activity is maintained may be referred to as a "conservative variant". In the present invention, the proteins specified as proteins having peptidoglycan-degrading activity include, in addition to the above-described proteins, their conservative variants, and fusion proteins of those proteins with other peptides. Hereinafter, conservative variants of peptidoglycan-degrading enzymes will be exemplified.

[0025] The protein according to one embodiment of the present invention may be a protein having an amino acid sequence in which one or several amino acids are substituted, deleted, inserted, or added in the amino acid sequence of positions 1 to 164 of SEQ ID NO: 2 or the amino acid sequence of positions 1 to 493 of SEQ ID NO: 4, as long as the peptidoglycan-degrading activity is maintained. residue "One or several" may be, for example, 1 to 10, 1 to 8, 1 to 5, or 1 to 3, although it varies depending on the position and type of amino acid residues in the three-dimensional structure of the protein.

[0026] The protein according to one embodiment of the present invention may be a protein having an amino acid sequence with high identity, for example, 90% or more, 93% or more, 95% or more, 97% or more, or 99% or more identity, to the amino acid sequence of positions 1 to 164 of SEQ ID NO: 2 or the amino acid sequence of positions 1 to 493 of SEQ ID NO: 4, as long as the peptidoglycan-degrading activity is maintained.

[0027] The mutations of the above amino acid residues are conservative mutations that maintain the normal function of the protein. Representative conservative mutations are conservative substitutions. A conservative substitution means that when the substitution site is an aromatic amino acid, it is between Phe, Trp, and Tyr; when the substitution site is a hydrophobic amino acid, it is between Leu, Ile, and Val; when the substitution site is a polar amino acid, it is between Gln and Asn; when the substitution site is a basic amino acid, it is between Lys, Arg, and His; when the substitution site is an acidic amino acid, it is between Asp and Glu; and when the substitution site is an amino acid with a hydroxyl group, it is between Ser and Thr. Specific substitutions considered to be conservative substitutions include substitution of Ala with Ser or Thr, substitution of Arg with Gln, His, or Lys, substitution of Asn with Glu, Gln, Lys, His, or Asp, substitution of Asp with Asn, Glu, or Gln, substitution of Cys with Ser or Ala, substitution of Gln with Asn, Glu, Lys, His, Asp, or Arg, substitution of Glu with Gly, Asn, Gln, Lys, or Asp, substitution of Gly with Pro, substitution of His with Asn, Lys, Gln, Arg, or Tyr, substitution of Ile with Leu, Met, Val, or Phe, substitution of Leu with Ile, Met, Val, or Phe, substitution of Lys with Asn, Glu, Gln, His, or Arg, substitution of Met with Ile, Leu, Val, or Phe, substitution of Phe with Trp, Tyr, Met, Ile, or Leu, substitution of Ser with Thr or Ala, substitution of Thr with Ser or Ala, substitution of Trp with Phe or Tyr, substitution of Tyr with His, Phe, or Trp, and substitution of Val with Met, Ile, or Leu. The mutations of amino acid residues also include mutations caused by naturally occurring mutations (mutants or variants) based on individual differences or species differences of the organisms from which the protein is derived.

[0028] The protein according to an embodiment of the present invention may be a fusion protein with another peptide. Examples of the other peptide include a marker peptide (marker protein), a peptide tag, and a pro sequence or prepro sequence, such as a secretion signal peptide. The peptide linked to the peptidoglycan-degrading enzyme may be only one kind, or two or more peptides. When the peptidoglycan-degrading enzyme is a fusion protein with a signal peptide, after being expressed in a secretable host cell, the signal peptide can be cleaved and only the peptidoglycan-degrading enzyme portion (mature protein) can be secreted extracellularly. When the peptidoglycan-degrading enzyme is a fusion protein, the above identity is the identity in the mature protein portion excluding the other peptide portion.

[0029] The marker peptide is not particularly limited as long as it can function as a marker, and examples thereof include alkaline phosphatase, the Fc region of an antibody, HRP, GFP, etc. The peptide tag is not particularly limited, and examples thereof include conventionally known peptide tags such as Myc tag, His tag, FLAG tag, and GST tag. As the secretion signal peptide, in addition to the secretion signal peptide of the peptidoglycan-degrading enzyme itself possessed by NITE BP-02779, a secretion signal peptide that can function in the host expressing the peptidoglycan-degrading enzyme can be used. The fusion protein can be produced by a conventional method.

[0030] The protein according to an embodiment of the present invention may be any of the following proteins (a1) to (a4). (a1) A protein consisting of the amino acid sequence of positions 1 to 164 of SEQ ID NO: 2 (a2) A protein consisting of an amino acid sequence in which Ala at position 1 is substituted with Met in the amino acid sequence of positions 1 to 164 of SEQ ID NO: 2 A protein consisting of an amino acid sequence in which Ala at position 1 in the amino acid sequence at positions 1 to 164 of SEQ ID NO: 2 is replaced with Met, and the amino acid sequence set forth in MetGlySerSerHisHisHisHisHisHisSerSerGlyLeuValProArgGlySerHis (SEQ ID NO: 21) is added to the N-terminus of the Met. A protein consisting of an amino acid sequence in which Met is added to the N-terminus of Ala at position 1 in the amino acid sequence at positions 1 to 164 of SEQ ID NO: 2.

[0031] The protein according to one embodiment of the present invention may be any of the following proteins (b1) to (b4). A protein consisting of the amino acid sequence at positions 1 to 493 of SEQ ID NO: 4. A protein consisting of an amino acid sequence in which Glu at position 1 in the amino acid sequence at positions 1 to 493 of SEQ ID NO: 4 is replaced with Met. A protein consisting of an amino acid sequence in which Glu at position 1 in the amino acid sequence at positions 1 to 493 of SEQ ID NO: 4 is replaced with Met, and the amino acid sequence set forth in SEQ ID NO: 21 is added to the N-terminus of the Met. A protein consisting of an amino acid sequence in which Met is added to the N-terminus of Glu at position 1 in the amino acid sequence at positions 1 to 493 of SEQ ID NO: 4.

[0032] The protein consisting of the amino acid sequence at positions 1 to 164 of SEQ ID NO: 2 is considered to have the activity of decomposing the glycosidic bond between (2) N-acetylmuramic acid and N-acetylglucosamine among the above-mentioned peptidoglycan-degrading activities. (A1) A protein containing the amino acid sequence at positions 1 to 164 of SEQ ID NO: 2 and having peptidoglycan-degrading activity, (A2) A protein containing an amino acid sequence in which 1 to 10 amino acid residues are substituted, deleted, inserted, or added in the amino acid sequence at positions 1 to 164 of SEQ ID NO: 2 and having peptidoglycan-degrading activity, (A3) A protein containing an amino acid sequence having 90% or more identity to the amino acid sequence at positions 1 to 164 of SEQ ID NO: 2 and having peptidoglycan-degrading activity preferably has the activity of decomposing the glycosidic bond between (2) N-acetylmuramic acid and N-acetylglucosamine among the above-mentioned peptidoglycan-degrading activities.

[0033] The protein consisting of the amino acid sequence at positions 1 to 493 of SEQ ID NO: 4 is considered to have the activity of (3) N-acetylmuramoyl-L-alanine amidase among the above-mentioned peptidoglycan-degrading activities. (B1) A protein containing the amino acid sequence at positions 1 to 493 of SEQ ID NO: 4 and having peptidoglycan-degrading activity, (B2) A protein containing an amino acid sequence in which 1 to 10 amino acid residues are substituted, deleted, inserted, or added in the amino acid sequence at positions 1 to 493 of SEQ ID NO: 4 and having peptidoglycan-degrading activity, (B3) A protein containing an amino acid sequence having 90% or more identity to the amino acid sequence at positions 1 to 493 of SEQ ID NO: 4 and having peptidoglycan-degrading activity preferably has the activity of (3) N-acetylmuramoyl-L-alanine amidase among the above-mentioned peptidoglycan-degrading activities.

[0034] (A1)-(A3) and (B1)-(B3) related proteins may be synthesized proteins, and may be purified or isolated proteins. (A1)-(A3) and (B1)-(B3) related proteins may be proteins derived from NITE BP-02779, and may also be artificial or proteins synthesized or produced within the cells of NITE BP-02779 or other organisms.

[0035] [DNA encoding a peptidoglycan-degrading enzyme] The DNA according to the present invention is DNA encoding the above-mentioned proteins. Specifically, DNA encoding the amino acid sequence of positions 1 to 164 of SEQ ID NO: 2, DNA encoding the amino acid sequence of positions 1 to 493 of SEQ ID NO: 4, and DNA encoding conservative variants of proteins having these amino acid sequences can be mentioned. The DNA according to one embodiment of the present invention may be DNA having the base sequence of positions 205 to 696 of SEQ ID NO: 1 or the base sequence of positions 85 to 1563 of SEQ ID NO: 3. SEQ ID NOs: 1 and 3 are base sequences based on the genomic sequence of NITE BP-02779 and encode the mature form of a protein having peptidoglycan-degrading activity. The DNA according to the present invention may contain a start codon and / or a base sequence encoding another peptide on the 5'-end side of the above base sequence, and may contain a stop codon at the 3'-end of the above base sequence. The DNA may be recombinant DNA. The DNA may be synthesized DNA and may be complementary DNA (cDNA). The DNA may be purified or isolated DNA.

[0036] The DNA according to the present invention is not limited to DNA consisting of the sequence possessed by NITE BP-02779, and may be DNA containing a base sequence in which codons encoding each amino acid in the coding region are replaced with other equivalent codons encoding the same amino acid. The DNA according to one embodiment of the present invention may be DNA containing a base sequence in which the codon usage is changed so as to improve the expression of a protein having peptidoglycan-degrading activity.

[0037] The DNA according to an embodiment of the present invention includes a probe having a base sequence complementary to the above base sequence, or a DNA that hybridizes with a probe that can be prepared from the above complementary base sequence under stringent conditions and encodes a protein having peptidoglycan-degrading activity. Stringent conditions refer to conditions under which so-called specific hybrids are formed and non-specific hybrids are not formed. For example, DNA molecules with high identity, such as DNA molecules with an identity of 50% or more, 65% or more, 80% or more, preferably 90% or more, more preferably 95% or more, still more preferably 97% or more, and particularly preferably 99% or more, hybridize with each other, and DNA molecules with lower identity do not hybridize with each other, or conditions corresponding to the washing conditions of normal Southern hybridization, such as 60 °C, 1×SSC, 0.1% SDS, preferably 60 °C, 0.1×SSC, 0.1% SDS, and more preferably 68 °C, 0.1×SSC, 0.1% SDS, and washing once, preferably 2 to 3 times can be mentioned. Further, for example, when a DNA fragment having a length of about 300 bp is used as a probe, the washing conditions for hybridization include 50 °C, 2×SSC, 0.1% SDS.

[0038] The percentage of identity between two arrays can be determined, for example, using a mathematical algorithm. Examples of mathematical algorithms include the algorithm described in Myers and Miller (1988) CABIOS, 4:11-17, the local homology algorithm described in Smith et al (1981) Adv. Appl. Math. 2:482, the homology alignment algorithm described in Needleman and Wunsch (1970) J. Mol. Biol. 48:443-453, the method for searching for similarity described in Pearson and Lipman (1988) Proc. Natl. Acad. Sci. 85:2444-2448, and the improved algorithm of Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA, 90:587-5877, which is an improvement over Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA, 87:2264.

[0039] Array comparison (alignment) for determining array identity can be performed using a program based on these mathematical algorithms. The program can be executed by a computer as appropriate. Such programs are not particularly limited, but include, for example, CLUSTAL of the PC / Gene program (available from Intelligenetics, Mountain View, Calif.), as well as the ALIGN program (Version 2.0), GAP, BESTFIT, BLAST, FASTA, and TFASTA of the Wisconsin Genetics Software Package, Version 8 (available from Genetics Computer Group (GCG), 575 Science Drive, Madison, Wis., USA). Alignment using these programs can be performed, for example, using initial parameters. The CLUSTAL program is well described in HigGlns et al. (1988) Gene 73:237-244, HigGlns et al. (1989) CABIOS 5:151-153, Corpet et al. (1988) Nucleic Acids Res. 16:1088190, Huang et al. (1992) CABIOS 8:155-65, and Pearson et al. (1994) Meth. Mol. Biol. 24:307-331.

[0040] To obtain a nucleotide sequence having identity with the nucleotide sequence encoding the target protein, for example, a BLAST nucleotide search can be performed using the BLASTN program, score = 100, word length = 12. To obtain an amino acid sequence having identity with the target protein, for example, a BLAST protein search can be performed using the BLASTX program, score = 50, word length = 3. For BLAST nucleotide search and BLAST protein search, refer to http: / / www.ncbi.nlm.nih.gov. Gapped BLAST (BLAST 2.0) can be used to obtain an alignment with gaps added for comparison purposes. PSI-BLAST (BLAST 2.0) can be used to perform iterative searches for detecting distant relationships between sequences. For Gapped BLAST and PSI-BLAST, refer to Altschul et al. (1997) Nucleic Acids Res. 25:3389. When using BLAST, Gapped BLAST, or PSI-BLAST, the initial parameters of each program (for example, BLASTN for nucleotide sequences and BLASTX for amino acid sequences) can be used. The alignment may be performed manually.

[0041] The identity between two sequences is calculated as the ratio of residues that match between the two sequences when the two sequences are aligned to maximize the match.

[0042] The DNA according to the present invention can be obtained by chemical synthesis or by PCR or the like from NITE BP-02779.

[0043] [Vector] The vector according to the present invention contains the above DNA. A vector is a nucleic acid molecule capable of amplifying and maintaining DNA, and can be an expression vector or a cloning vector. The vector according to the present invention may be constructed by incorporating the above DNA into a basic vector according to ordinary genetic engineering techniques. The basic vector is, for example, capable of replicating autonomously in a host cell, can be isolated and purified from the host cell, and has a detectable marker. The DNA according to the present invention is inserted into, for example, an expression vector available in a host cell into which a gene is introduced, and the vector can express a protein having peptidoglycan-degrading activity in the host cell by being introduced into the host cell.

[0044] The basic vector may be, for example, a vector derived from a bacterial plasmid, a vector derived from a yeast plasmid, a viral vector, a cosmid vector, a phagemid vector, an artificial chromosome vector, etc. Examples of the basic vector include pBR322, pUC plasmid vectors, pET series plasmid vectors, etc. Specifically, when Escherichia coli is used as the host cell, for example, vectors such as pUC19, pUC18, pUC119 (manufactured by Takara Shuzo Co., Ltd.), phagemid pBluescriptII (manufactured by Stratagene), pET28a(+) vector and pET22b(+) vector (Merck Millipore) can be mentioned. When budding yeast is used as the host cell, for example, vectors such as pGBT9, pGAD424, pACT2 (manufactured by Clontech) can be mentioned. When mammalian cells are used as the host cell, for example, vectors such as pRc / RSV, pRc / CMV (manufactured by Invitrogen), bovine papillomavirus vector pBPV (manufactured by Amersham Pharmacia Biotech), Epstein-Barr virus vector pCEP4 (manufactured by Invitrogen), vaccinia virus vector, retrovirus vector, lentivirus vector, adenovirus vector, adeno-associated virus vector, etc. can be mentioned. When insect cells are used as the host cell, for example, a baculovirus vector can be mentioned.

[0045] When constructing the vector according to the present invention using a basic vector having an autonomous replication origin (ori), the vector is retained intracellularly as an episome when introduced into a host cell. When a vector incorporated with the ori of SV40 is introduced into, for example, COS cells transformed with an SV40 genome lacking ori, the copy number of the vector can be greatly increased intracellularly.

[0046] The expression vector may have a promoter sequence and a terminator sequence for expressing the incorporated gene. The promoter is not particularly limited as long as it is a promoter that functions in the host cell. The "promoter that functions in the host" refers to a promoter that has promoter activity in the host and can control the expression of the incorporated gene. Generally, the DNA sequence incorporated into the basic vector is inserted downstream of the promoter in a state where the promoter can function. For example, in the basic vector, a cloning site is provided downstream of the promoter sequence. The basic vector may have a selectable marker sequence.

[0047] The promoter may be a promoter derived from the host or a promoter derived from a heterologous source. The promoter may be a promoter specific to the peptidoglycan-degrading enzyme gene or a promoter of another gene. When the host cell is Escherichia coli, the promoter may be, for example, the promoter of the lactose operon of Escherichia coli (lacP), the promoter of the tryptophan operon (trpP), the promoter of the arginine operon (argP), the promoter of the galactose operon (galP), the tac promoter, the T7 promoter, the T3 promoter, the promoter of lambda phage (lambda-pL, lambda-pR), etc. When the host cell is an animal cell or fission yeast, the promoter may be, for example, the Rous sarcoma virus (RSV) promoter, the cytomegalovirus (CMV) promoter, the early or late promoter of simian virus (SV40), the mouse mammary tumor virus (MMTV) promoter, etc. When the host cell is budding yeast, the promoter may be, for example, the ADH1 promoter, etc. The ADH1 promoter can be prepared by ordinary genetic engineering methods from, for example, the yeast expression vector pAAH5 [available from the Washingtn Research Fundation, Ammerer et al., Method in Enzymology, 101 part (p.192-201)] which holds the ADH1 promoter and its terminator. The vector pACT2 having the ADH1 promoter can highly express the target gene in budding yeast such as CG1945 (manufactured by Clontech) if the target gene is inserted downstream of the ADH1 promoter.

[0048] [Transformant] The transformant according to the present invention contains the DNA according to the present invention or the vector according to the present invention. The transformant containing the DNA according to the present invention or the vector according to the present invention can express a protein having peptidoglycan-degrading activity.

[0049] As the host cell into which the DNA according to the present invention or the vector according to the present invention is introduced, for example, cells of eukaryotes or prokaryotes can be used, and examples include bacteria, fungi, plant cells, animal cells, and insect cells. The host cell is preferably Escherichia coli. By introducing the DNA or vector into the host cell, the host cell can be transformed to produce a transformant.

[0050] The DNA according to the present invention or the vector according to the present invention may be retained extrachromosomally in the host cell or may be integrated into the chromosome. In the transformant, the DNA according to the present invention or the vector according to the present invention is preferably retained in a state where the gene can be expressed under the control of a promoter that functions in the host cell.

[0051] As a method for introducing the DNA according to the present invention or the vector according to the present invention into a host cell, a normal introduction method according to the host cell may be applied. When Escherichia coli is used as the host cell, for example, gene introduction methods such as the calcium chloride method and electroporation method described in J. Sambrook, E. F. Frisch, T. Maniatis "Molecular Cloning 2nd edition", published by Cold Spring Harbr Laboratory (1989) can be mentioned. When mammalian cells or insect cells are used as the host cell, for example, gene introduction methods such as the calcium phosphate method, DEAE dextran method, electroporation method, and lipofection method can be mentioned. When yeast is used as the host cell, for example, gene introduction methods such as the lithium method used in Yeast transformation kit (manufactured by Clontech) can be mentioned. When a virus is used as the vector, in addition to introducing the genome of the virus into which the DNA according to the present invention is inserted into the host cell by the above gene introduction method, the DNA according to the present invention can be introduced into the host cell by infecting the host cell with virus particles containing the genome of the virus.

[0052] In order to select a transformant into which the DNA according to the present invention or the vector according to the present invention has been introduced, a selection marker may be used. For example, the DNA or vector according to the present invention and a selection marker gene are introduced into a host cell at the same time, and the host cell is cultured by a method according to the property of the selection marker. For example, when the selection marker gene is a gene that confers drug resistance to a selection drug that exhibits lethal activity in the host cell, the host cell into which the DNA or vector according to the present invention has been introduced may be cultured using a medium supplemented with the selection drug. Examples of the combination of the gene that confers drug resistance and the selection drug include the combination of the neomycin resistance-conferring gene and neomycin, the combination of the hygromycin resistance-conferring gene and hygromycin, and the combination of the blasticidin S resistance-conferring gene and blasticidin S. When the marker gene is a gene that complements the auxotrophy of the host cell, the host cell into which the DNA or vector according to the present invention has been introduced may be cultured using a minimal medium that does not contain the nutrient corresponding to the auxotrophy. Transformants can also be selected based on the activity of the enzyme expressed from the DNA or vector according to the present invention.

[0053] To obtain a transformant in which the DNA according to the present invention is located in the chromosome of a host cell, for example, first, the vector according to the present invention and a vector having a marker gene are linearized by digestion with a restriction enzyme or the like, and then these are introduced into the host cell by the above-described gene introduction method. After culturing the introduced cells for several weeks, the target transformant may be obtained based on the expression level of the marker gene. When a gene that confers resistance to a selection drug is used as the marker gene, after introducing the vector according to the present invention and the vector having the marker gene into the host cell by the above-described gene introduction method, the cells are subcultured in a medium supplemented with the selection drug for several weeks or more, and the selection drug-resistant clones that survived in a colony form are purified and cultured, whereby a transformant in which the DNA according to the present invention is introduced into the chromosome of the host cell can be obtained. To confirm that the DNA according to the present invention has been integrated into the chromosome of the host cell, the genomic DNA of the cell is prepared according to a usual genetic engineering method, and PCR, Southern hybridization, etc. are performed using DNA having a partial base sequence of the introduced DNA according to the present invention as a primer or a probe, and the presence of the DNA according to the present invention may be detected. The transformant in which the DNA according to the present invention is integrated into the chromosome of the host cell can be cryopreserved and can be revived and used as needed, so that the labor of preparing transformants for each experiment can be saved, and it is also possible to conduct tests using transformants whose properties and handling conditions have been confirmed in advance.

[0054] [Production of the protein according to the present invention] The protein according to the present invention can be obtained, for example, from NITE BP-02779. To obtain it from NITE BP-02779 means to obtain it from within the cells or the culture supernatant of NITE-BP02779. For example, when obtaining the protein according to the present invention from within the cells, the cells can be appropriately disrupted, lysed, or extracted, etc., and the protein according to the present invention can be recovered. The cells can be recovered from the culture by centrifugation or the like. Disruption, lysis, or extraction of the cells, etc. can be carried out by known methods. Such methods include, for example, ultrasonic disruption method, Dynomill method, bead disruption, French press disruption, lysozyme treatment. These methods can be used alone or in appropriate combination of two or more. When the protein according to the present invention accumulates in the culture supernatant, the culture supernatant can be obtained by centrifugation or the like, and the protein according to the present invention can be recovered from the culture supernatant.

[0055] Purification of the protein according to the present invention can be carried out by known methods used for enzyme purification. Such methods include, for example, ammonium sulfate fractionation, ion exchange chromatography, hydrophobic chromatography, affinity chromatography, gel filtration chromatography, isoelectric precipitation. These methods can be used alone or in appropriate combination of two or more. Purification of the protein according to the present invention can be carried out to the desired degree.

[0056] The protein according to the present invention can also be produced using a transformant into which the DNA encoding them has been introduced into an appropriate host. The DNA is preferably incorporated into a vector and used for transformation. The protein according to the present invention may be artificially synthesized in a cell-free system.

[0057] When producing the protein according to the present invention using a transformant, the culture of the transformant may be carried out by the method of culturing the host cell. When the transformant is a microorganism, for example, it can be cultured using various media appropriately containing carbon sources, nitrogen sources, organic or inorganic salts, etc. usually used for culturing in microorganisms.

[0058] Examples of carbon sources include saccharides such as glucose, dextrin, and sucrose; sugar alcohols such as glycerol; organic acids such as fumaric acid, citric acid, and pyruvic acid; animal oils, vegetable oils, and molasses. The addition amount of these carbon sources to the medium is usually about 0.1 to 30% (w / v) with respect to the culture solution.

[0059] Examples of nitrogen sources include natural organic nitrogen sources such as meat extract, peptone, yeast extract, malt extract, soybean powder, corn steep liquor, cottonseed meal, dried yeast, and casamino acid; amino acids; inorganic acids such as sodium nitrate salt ammonium salts of inorganic acids such as ammonium chloride, ammonium sulfate, and ammonium phosphate; ammonium salts of organic acids such as ammonium fumarate and ammonium citrate; and urea. Among these, ammonium salts of organic acids, natural organic nitrogen sources, amino acids, etc. can often be used also as carbon sources. The addition amount of these nitrogen sources to the medium is usually about 0.1 to 30% (w / v) with respect to the culture solution.

[0060] Examples of organic salts or inorganic salts include chlorides, sulfates, acetates, carbonates, and phosphates of potassium, sodium, magnesium, iron, manganese, cobalt, zinc, etc. Specifically, sodium chloride, potassium chloride, magnesium sulfate, ferrous sulfate, manganese sulfate, cobalt chloride, zinc sulfate, copper sulfate, sodium acetate, calcium carbonate, monopotassium hydrogen phosphate, and dipotassium hydrogen phosphate can be mentioned. The addition amount of these organic salts and / or inorganic salts to the medium is usually about 0.0001 to 5% (w / v) with respect to the culture solution.

[0061] In the case of a transformant in which a promoter induced by allolactose such as the tac promoter, trc promoter, or lac promoter is connected to the DNA according to the present invention and a gene is expressed, as an inducer for producing the protein according to the present invention, for example, a small amount of isopropylthio-β-D-galactoside (IPTG) can be added to the medium.

[0062] The culture of the transformant according to the present invention may be carried out according to the method usually used for culturing host cells. Examples include liquid culture and solid culture such as test tube shaking culture, reciprocating shaking culture, Jar Fermenter culture, and tank culture. The culture temperature can be appropriately changed within the range in which the transformant can grow, but is usually about 15°C to about 40°C. The pH of the medium is preferably in the range of about 6.0 to about 8.0. The culture time may be about 1 day to about 5 days, although it varies depending on the culture conditions.

[0063] By culturing the transformant according to the present invention, a culture containing the protein according to the present invention can be obtained. The protein according to the present invention can accumulate, for example, in the cells and / or the culture supernatant of the transformant. The production of the protein according to the present invention can be confirmed, for example, by measuring the peptidoglycan-degrading activity according to the above method for an appropriate fraction such as the culture supernatant or the intracellular extract.

[0064] The protein according to the present invention may be obtained by appropriately disrupting, lysing, extracting, and purifying the transformant by the same method as the above-described NITE BP-02779. The protein according to the present invention is not limited to the purified protein according to the present invention, and any fraction containing the protein according to the present invention may be used as the "protein according to the present invention" for uses such as degradation of peptidoglycan. The fraction containing the protein according to the present invention is not particularly limited as long as the protein according to the present invention is contained so as to be able to act on peptidoglycan. Such fractions include, for example, transformants, culture supernatants of transformants, disrupted products, lysates, extracts (cell-free extracts), etc., partial purifications (crude purifications) thereof, and combinations thereof. Any of these fractions may be used alone for uses such as degradation of peptidoglycan, or may be used together with the purified protein according to the present invention. Other enzymes different from the protein according to the present invention may also be produced and accumulated in the culture. The protein according to the present invention may be recovered as a mixture with such other enzymes, or may be recovered separately from such other enzymes.

[0065] [Microbial decomposition preparation] The microbial decomposition preparation includes at least one selected from the group consisting of the protein according to the present invention, the transformant according to the present invention, and a culture thereof. The culture of the transformant includes the cultured transformant, the culture supernatant of the transformant, disrupted products, lysates, cell extracts (cell-free extracts), etc., partial purifications (crude purifications) thereof, and combinations thereof.

[0066] The microbial decomposition preparation preferably contains a protein consisting of the amino acid sequence of positions 1 to 164 of SEQ ID NO: 2 and / or a protein consisting of the amino acid sequence of positions 1 to 493 of SEQ ID NO: 4. The microbial decomposition preparation may contain one enzyme having peptidoglycan-degrading activity, or may contain two or more in combination. As long as it does not inhibit the peptidoglycan-degrading activity, it may contain other components. The transformant that may be contained in the microbial decomposition preparation may be one type, or two or more types.

[0067] The content of the protein according to the present invention, the transformant according to the present invention, and the culture thereof in the microbial decomposition preparation is not particularly limited, and may be appropriately set according to the type and concentration of the target microorganism, and the use environment of the microbial decomposition preparation (volume of the reaction system, temperature, etc.).

[0068] Since the microbial decomposition preparation contains a protein having peptidoglycan-degrading activity or a transformant capable of expressing a protein having peptidoglycan-degrading activity, it can decompose peptidoglycan. The microbial decomposition preparation can decompose a target microorganism having peptidoglycan. The target microorganism is preferably a bacterium constituting excess sludge. The target microorganism may be a dead bacterium or a live bacterium. The microbial decomposition preparation can contribute to the volume reduction of excess sludge.

[0069] Whether the microorganism is decomposed can be confirmed by a method commonly used by those skilled in the art. Examples of such a confirmation method include a method of detecting the decomposition of the target microorganism in a medium or buffer solution after reacting the microbial decomposition preparation and the target microorganism in an appropriate medium or buffer solution for a certain period of time. The method of detecting the decomposition of the target microorganism is not particularly limited, but examples include a method of measuring the turbidity of the target microorganism, a method of detecting the target microorganism with an SLP reagent, a method of detecting the DNA of the target microorganism by PCR, a method of measuring the dry cell weight of the target microorganism, a method of detecting decomposition products derived from the target microorganism using high performance liquid chromatography (HPLC); mass spectrometry (MS); thin layer chromatography (TLC); nuclear magnetic resonance (NMR); gas chromatography (GC), etc. It is also possible to examine whether the target microorganism is decomposed by the method for examining the above-mentioned peptidoglycan-degrading activity.

[0070] When examining the decomposition of target microorganisms by turbidity, decomposing the target microorganisms means that the turbidity after the reaction is significantly lower than the turbidity before the reaction. For example, the turbidity after the reaction is 80% or less of the turbidity before the reaction, preferably 50% or less, and more preferably 30% or less. When examining the decomposition of target microorganisms by dry cell weight, decomposing the target microorganisms means that, for example, the dry cell weight after the reaction is significantly lower than before the reaction. For example, the dry cell weight after the reaction is 95% or less of that before the reaction, preferably 90% or less.

[0071] The dosage form of the microbial decomposition preparation is not particularly limited as long as the functions of the protein according to the present invention, the transformant according to the present invention, and its culture are not lost. Examples of the dosage form include liquids, suspensions, powders, solids, encapsulated bodies, or frozen or lyophilized products thereof. In formulating the preparation, additives such as excipients, binders, disintegrants, lubricants, stabilizers, diluents, and surfactants can be used.

[0072] The microbial decomposition preparation can be used by being introduced into a sludge treatment device, a wastewater treatment device, etc. that generate excess sludge. The microbial decomposition preparation can also be used by being introduced into a tank or the like in which excess sludge or target microorganisms are accumulated.

[0073] One aspect of the present invention is at least one use selected from the group consisting of the protein according to the present invention, the transformant according to the present invention, and its culture in the production of a microbial decomposition preparation.

[0074] [Microbial decomposition method] The microbial decomposition method includes a step of allowing at least one selected from the group consisting of the protein according to the present invention, the transformant according to the present invention, and a culture thereof to act on a target microorganism. The culture of the transformant includes the cultured transformant, the culture supernatant of the transformant, disrupted products, lysates, cell extracts (cell-free extracts), etc., partially purified products (crude purified products) thereof, and combinations thereof. The step of allowing at least one selected from the protein according to the present invention, the transformant according to the present invention, and a culture thereof to act on a target microorganism may be performed using the above-described microbial decomposition preparation.

[0075] Allowing the protein according to the present invention to act on a target microorganism means, for example, bringing the protein according to the present invention into contact with the target microorganism. Allowing the transformant according to the present invention to act on a target microorganism means, for example, culturing the transformant according to the present invention in the presence of the target microorganism. Allowing the culture of the transformant according to the present invention to act on a target microorganism means, for example, bringing the culture of the transformant, preferably a protein having peptidoglycan-degrading activity contained in the culture, into contact with the target microorganism.

[0076] According to the microbial decomposition method of the present invention, a target microorganism having peptidoglycan can be decomposed. The target microorganism is preferably a bacterium constituting excess sludge. The target microorganism may be a dead bacterium or a live bacterium. According to the microbial decomposition method, it is possible to contribute to the volume reduction of excess sludge.

[0077] The microbial decomposition method preferably includes a step of allowing a protein consisting of the amino acid sequence of positions 1 to 164 of SEQ ID NO: 2 and / or a protein consisting of the amino acid sequence of positions 1 to 493 of SEQ ID NO: 4 to act on a target microorganism. In this step, one kind of protein having peptidoglycan-degrading activity may be used, or two or more kinds may be used in combination. The transformant allowed to act on the target microorganism in the microbial decomposition method may be one kind or two or more kinds.

[0078] The step of allowing the protein according to the present invention, the transformant according to the present invention, and the culture thereof to act on the target microorganism is not particularly limited as long as the protein according to the present invention is not lost or the peptidoglycan-degrading activity of the protein according to the present invention is not lost, or the transformant is not killed and the protein can be synthesized. This step may be carried out, for example, under conditions where the temperature is 20 to 35°C, or may be carried out under conditions where the temperature is 25 to 30°C. The pH may be under conditions of 5.5 to 8.0, or may be under conditions of 6.0 to 7.5.

[0079] The amounts of the protein according to the present invention, the transformant according to the present invention, and the culture thereof used in the microbial decomposition method can be appropriately set in consideration of the type and concentration of the target microorganism, the volume of the reaction system, the reaction temperature, and the like.

[0080] Whether the target microorganism is decomposed can be confirmed by the method described in the column of the microbial decomposition preparation.

[0081] One aspect of the present invention is at least one use selected from the group consisting of the protein according to the present invention, the transformant according to the present invention, and the culture thereof for the decomposition of a target microorganism. One aspect of the present invention is the use of a microbial decomposition preparation containing at least one selected from the group consisting of the protein according to the present invention, the transformant according to the present invention, and the culture thereof for the decomposition of a target microorganism.

Examples

[0082] Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples.

[0083] [Experiment 1. Isolation of Microbial Decomposing Bacteria] (Method) Using a medium with bacteria of the genus Micrococcus as a carbon source, the microbial community present in the environment (water) was cultured to enrich and culture the microorganisms that decompose bacteria of the genus Micrococcus. Next, several strains with good growth were isolated from the enriched microbial community.

[0084] (Results) When the ability to decompose bacteria of the genus Micrococcus was examined for each isolated strain, one strain showed the ability to decompose bacteria of the genus Micrococcus. Hereinafter, the strain showing the ability to decompose bacteria of the genus Micrococcus may be referred to as "strain A".

[0085] [Experiment 2. Identification of Strain A] (Materials) · Forward primer for cloning (27F: SEQ ID NO: 5) · Reverse primer for cloning (1492R: SEQ ID NO: 6) · Primers for sequence analysis (339F: SEQ ID NO: 7, 536R: SEQ ID NO: 8, 907F: SEQ ID NO: 9)

[0086] (Methods) The identification of strain A was carried out by 16S rRNA gene analysis, morphological observation, and physiological and biochemical property tests.

[0087] The 16S rRNA gene analysis was performed according to the following procedure. Genomic DNA was extracted from strain A, and using the obtained genomic DNA as a template, PCR amplification of the 16S rRNA gene was performed using a forward primer for cloning (27F) and a reverse primer for cloning (1492R). PCR amplification was carried out using KOD FX (manufactured by Toyobo Co., Ltd.), and the amplification product after PCR was purified.

[0088] The cycle sequencing reaction was performed using the purified amplification product after PCR. The cycle sequencing reaction was carried out using the BigDye Terminator v3.1 Cycle Sequencing Kit. The obtained reaction solution was purified, and the purified solution was subjected to DNA sequence analysis (3730xl DNA Analyzer) to determine the nucleotide sequence of the 16S rRNA gene of the template DNA extracted from strain A.

[0089] Morphological observation and physiological and biochemical property tests were performed by morphological observation using an optical microscope, the method of BARROW et al. (Cowan and Steel’s Manual for the Identification of Medical Bacteria 3rd Edition 1993, Cambridge University Press.), and API50CHB (manufactured by bioMerieux, Lyon, France).

[0090] (Results) The nucleotide sequence of the obtained 16S rRNA gene (SEQ ID NO: 10) was subjected to homology analysis against the international nucleotide sequence databases (DDBJ / ENA (EMBL) / GenBank). Among the reference strains, it showed 98.1% identity to the nucleotide sequence of the 16S rRNA gene of Tumebacillus permanentifrigoris Eurl_9.5. However, there was no microorganism having a 16S rRNA gene that completely matched the obtained nucleotide sequence. Also, strain A did not grow at 10°C, but such a characteristic was not observed in Tumebacillus permanentifrigoris Eurl_9.5, which had the highest identity of the 16S rRNA gene. Therefore, it was suggested that strain A was a new species different from the conventional Tumebacillus. Strain A was internationally deposited as Tumebacillus sp. NITE BP-02779.

[0091] The results of morphological observation and physiological and biochemical property tests of NITE BP-02779 are shown in Tables 1 to 4.

[0092]

Table 1

[0093]

Table 2

[0094]

Table 3

[0095]

Table 4

[0096] [Experiment 3. Evaluation of the degradation ability of NITE BP-02779 against excess sludge (dead bacteria)-1] (Materials) ·R2A medium: R2A Broth, DAIGO (manufactured by Nippon Pharmaceutical Co., Ltd.) was dissolved at a ratio of 3.2 g in 1000 mL of ultrapure water, and the medium was autoclaved ·Inorganic medium containing excess sludge (dead bacteria): A medium prepared by mixing 986 mL of substrate solution, 3.0 mL of Solution A, 3.0 mL of Solution B, 3.0 mL of Solution C, 3.0 mL of Solution D, and 1.8 mL of 1% phosphoric acid The following were used as the substrate solution and Solutions A to D Substrate solution: After washing the excess sludge, it was mixed with 986 mL of ultrapure water to a turbidity (OD660) of 0.2, and the solution was autoclaved Solution A: A solution prepared by dissolving 4.35 g of dipotassium hydrogen phosphate, 1.70 g of potassium dihydrogen phosphate, 8.92 g of disodium hydrogen phosphate dodecahydrate, and 0.34 g of ammonium chloride in ultrapure water, adjusting the volume to 200 mL, and autoclaving Solution B: A solution prepared by dissolving 4.50 g of magnesium sulfate heptahydrate in ultrapure water, adjusting the volume to 200 mL, and autoclaving Solution C: A solution prepared by dissolving 5.50 g of anhydrous calcium chloride in ultrapure water, adjusting the volume to 200 mL, and autoclaving Solution D: A solution prepared by dissolving 0.05 g of iron(III) chloride hexahydrate in ultrapure water, adjusting the volume to 200 mL, and filtering and sterilizing with a 0.2-μm syringe filter

[0097] (Method) NITE BP-02779 was inoculated into R2A medium and cultured at 25 °C for 24 to 48 hours. After the culture, 50 μL of the NITE BP-02779 culture solution and 5.0 mL of the inorganic medium containing excess sludge (dead bacteria) were added to a test tube and reacted at 25 °C and 200 rpm. The turbidity (OD660) of the test tube was measured over time with a simple turbidimeter (Simple OD Monitor miniphoto 518R, manufactured by TAITEC). The number of days it took for the turbidity (OD660) of the inorganic medium containing excess sludge (dead bacteria) to reach 50% of the turbidity (OD660) of the negative control was calculated. The negative control used the turbidity (OD660) of the inorganic medium containing the target bacteria (dead bacteria) without the addition of NITE BP-02779.

[0098] (Results) By adding NITE BP-02779, a decrease in the turbidity of the excess sludge (dead bacteria) was observed, and it reached 50% of the turbidity of the negative control in 3.2 days. Therefore, NITE BP-02779 was able to decompose the excess sludge (dead bacteria).

[0099] [Experiment 4. Evaluation of the degradation ability of NITE BP-02779 against excess sludge (dead bacteria) - 2] (Materials) The same materials as in Experiment 3 were used.

[0100] (Method) The reaction was carried out in the same manner as in Experiment 3. The reaction was performed in quintuplicate, and on the 4th day after the start of the reaction, the entire amount of the excess sludge remaining in the test tube was collected. After drying the collected excess sludge, its weight was measured, and a significance test (t-test, one-sided) was performed between the negative control group and the NITE BP-02779-added group.

[0101] (Results) The results are shown in Fig. 2. A significant decrease in the dry weight of excess sludge (p < 0.01) was observed in the NITE BP-02779-added group compared with the negative control group. Therefore, it was possible to reduce excess sludge by adding NITE BP-02779.

[0102] [Experiment 5. Determination of the genomic sequence of NITE BP-02779] (Method) The genomic sequence of NITE BP-02779 was analyzed by a next-generation sequencer according to the following procedure. NITE BP-02779 was inoculated into 50 mL of R2A medium and cultured with shaking at 25 °C and 130 rpm for 2 days. The obtained cells were collected by centrifugation, and genomic DNA was extracted using a genomic DNA extraction kit (QIAamp DNA Mini Kit (250), manufactured by QIAGEN). Using the extracted DNA as a template, sequencing was performed by the Paired-End method using a next-generation sequencer, HiSeq 2500 (manufactured by Illumina). Next, the adapter sequence was trimmed from the obtained nucleotide sequence, and de novo assembly was performed using Velvet. The scaffold sequences obtained by de novo assembly were analyzed for gene region prediction and annotation. Rapid Annotations using Subsystems Technology (RAST) was used for gene region prediction and annotation.

[0103] (Results) As a result of de novo assembly, 149 scaffolds were obtained, the total length of the scaffold sequences was 4.44 Mbp, and the presence of 4446 genes was predicted.

[0104] [Experiment 6. Identification of a protein (ID2839) with peptidoglycan-degrading activity] (1) Preparation of the NITE BP-02779 culture supernatant (Method) NITE BP-02779 was inoculated into 5 mL of R2A medium and pre-cultured at 25°C. 5 mL of the obtained pre-culture solution was inoculated into 1.5 L of R2A medium and cultured with shaking at 25°C and 130 rpm. After 48 hours, the culture solution was centrifuged (4°C, 8000×g, 10 minutes), and the obtained culture supernatant was filter-filtered (0.20 μm). This filter-filtered culture supernatant was concentrated using a centrifugal filter unit (manufactured by Merck Millipore) and used as a starting material for enzyme purification.

[0105] (2) Fractionation by column chromatography Fractionation and purification of the enzyme were performed by column chromatography shown in the following (a) to (c). The peptidoglycan-degrading activity of each fractionated fraction was measured using a Lysozyme activity kit (manufactured by Sigma-Aldrich). The proteins contained in the obtained fractions were electrophoresed using a 4-20% Mini-PROTEAN TGX Precast Gel (manufactured by Bio-Rad) and confirmed by CBB staining.

[0106] (a) Cation exchange chromatography (Materials) · Sample: Concentrated NITE BP-02779 culture supernatant

[0107] (Method) The buffer in the sample was exchanged with 50 mM phosphate buffer (pH 5.8) using a centrifugal filter unit (manufactured by Merck Millipore). This sample was applied to a HiTrap Q HP, 5 mL column (manufactured by GE Healthcare) equilibrated with the same buffer, and after washing the column with the same buffer, the adsorbed proteins were eluted with a linear concentration gradient of 0 to 1.0 M sodium chloride.

[0108] (Results) When the peptidoglycan-degrading activity was examined, activity was observed in the unadsorbed fraction, so the unadsorbed fraction was subjected to hydrophobic interaction chromatography.

[0109] (b) Hydrophobic interaction chromatography · Sample: Unadsorbed fraction of cation exchange chromatography

[0110] (Method) Using a centrifugal filter unit (manufactured by Merck Millipore), (a) the buffer in the unadsorbed fraction of cation exchange chromatography was exchanged with 50 mM phosphate buffer (pH 5.8) containing 1 M ammonium sulfate. This sample was applied to a Hitrap HIC, 5 mL column (manufactured by GE Healthcare) equilibrated with the same buffer. After washing the column with the same buffer, the adsorbed protein was eluted with a linear concentration gradient of ammonium sulfate from 1.0 to 0 M.

[0111] (Results) When the peptidoglycan-degrading activity was examined, activity was observed in the adsorbed fraction. Therefore, the fraction with activity was subjected to gel filtration chromatography.

[0112] (c) Gel filtration chromatography · Sample: Adsorbed fraction of hydrophobic interaction chromatography

[0113] (Method) Using a centrifugal filter unit (manufactured by Merck Millipore), (b) the buffer in the adsorbed fraction of hydrophobic interaction chromatography was exchanged with 66 mM potassium phosphate (pH 6.24). This sample was applied to a SuperdexTM 75 10 / 300 GL (manufactured by GE Healthcare) equilibrated with the same buffer and eluted with the same buffer.

[0114] (Results) When the peptidoglycan-degrading activity was examined, as shown in Figure 3, activity was observed in the eluted fraction.

[0115] (3) Identification of the protein having peptidoglycan-degrading activity by mass spectrometry (Materials) · Sample: Eluted fraction of gel filtration chromatography

[0116] (Method) The elution fraction showing activity was concentrated using a centrifugal filter unit (manufactured by Merck Millipore), and electrophoresis was performed using a 4-20% Mini-PROTEAN TGX Precast Gel (manufactured by Bio-Rad). As a result, as shown in Figure 4, it was confirmed that the protein-derived band contained in the elution fraction was a single band. (c) The purified fraction of gel filtration chromatography and the band separated by electrophoresis were each subjected to in-solution digestion or in-gel digestion using trypsin, and the digestion solution was desalted. The desalted sample was subjected to Data Dependent MS / MS Acquisition (DDA) measurement using nano LC-MS (manufactured by Thermo Scientific, Q Exactive HF, nano column: Acclaim PepMap RSLC C18 (manufactured by Thermo Scientific)). After the measurement, a Mascot search (MS / MS ion search) was performed via Proteome Discoverer 2.1 against the protein sequence database created from the genomic information of NITE BP-02779.

[0117] (Result) When searching for trypsin-cleaved peptides detected by mass spectrometry, the amino acid sequences of SEQ ID NOs: 11 to 17, which are partial sequences of ID2839, were detected. These partial sequences correspond to a part of the amino acid sequence at positions 15 to 164 of SEQ ID NO: 2. From the above, it was suggested that the protein ID2839 expressed by NITE BP-02779 has peptidoglycan-degrading activity.

[0118] (4) Identification of the N-terminal cleavage site of ID2839 (Method) Furthermore, the DDA measurement data file in which ID2839 was identified was searched to determine whether it contained a peptide whose N-terminal side was cleaved at any site from positions -11 to 14 of SEQ ID NO: 2.

[0119] (Result) The subsequence of SEQ ID NO: 18 was obtained as the peptide having the highest intensity signal value. The subsequence of SEQ ID NO: 18 corresponds to positions 1 to 10 of SEQ ID NO: 2. Therefore, the entire amino acid sequence of SEQ ID NO: 2 was considered to be the amino acid sequence of the precursor (pro sequence) of ID2839. The amino acid sequence of the mature form of ID2839 was positions 1 to 164 of SEQ ID NO: 2 and was considered to be encoded by the nucleotide sequence of positions 205 to 696 of SEQ ID NO: 1. Positions -68 to -1 of SEQ ID NO: 2 were considered to be the signal peptide. Hereinafter, the protein consisting of the amino acid sequence of positions 1 to 164 of SEQ ID NO: 2 may be referred to as "ID2839 mature form", and the protein consisting of the entire amino acid sequence of SEQ ID NO: 2 (positions -68 to 164) may be referred to as "ID2839 precursor". When the signal peptide is cleaved from the ID2839 precursor, it becomes the ID2839 mature form, and the ID2839 mature form is considered to be secreted outside the cell and capable of degrading peptidoglycan. The molecular weight of the ID2839 precursor was about 25 kDa, and the molecular weight of the ID2839 mature form was about 18 kDa.

[0120] [Experiment 7. Measurement of specific activity of ID2839 mature form] (Materials) · ID2839 mature form (elution fraction of gel filtration chromatography)

[0121] (Methods) The activity of the ID2839 mature form (Units / mL) and protein quantification (mg / mL) were performed, and the specific activity (Units / mg) was calculated. Two commercially available lysozymes (manufactured by Sigma-Aldrich) were used as controls. The peptidoglycan-degrading activity was evaluated by the activity of degrading the glycosidic bond between N-acetylmuramic acid and N-acetylglucosamine using a Lysozyme activity kit (manufactured by Sigma-Aldrich).

[0122] (Results) The results are shown in Table 5. The ID2839 mature form was shown to have the activity of degrading the glycosidic bond between N-acetylmuramic acid and N-acetylglucosamine, and its specific activity was higher than that of commercially available mutanolysin and lysostaphin.

[0123] [Table 5]

[0124] [Preparation of ID2839 mature form in E. coli in Experiment 8] (1) Construction of ID2839 mature form expression strain (Materials) · Primers F and R for ID2839 cloning (SEQ ID NOs: 19 and 20) · LB agar medium: Agar medium prepared by dissolving LB Ager (manufactured by Sigma-Aldrich) at a ratio of 10 per 500 mL of ultrapure water, autoclaving, and dispensing into petri dishes

[0125] (Method) Using the genomic DNA of NITE BP-02779 as a template, PCR amplification of the region containing the nucleotide sequence encoding ID2839 mature form (with the Ala at position 1 in SEQ ID NO: 2 substituted with Met) was performed using primers F and R for ID2839 cloning. The PCR amplification was carried out using KOD FX (manufactured by Toyobo Co., Ltd.). The reaction solution was prepared according to the composition attached to the kit, and was performed at (1) 94°C for 2 minutes, (2) 98°C for 10 seconds, (3) 50°C for 30 seconds, and (4) 68°C for 1.5 minutes. The steps of (2) to (4) were repeated 25 cycles. The obtained PCR fragment and the pET-28a(+) vector (manufactured by Merck Millipore) were digested with NdeI and XhoI respectively, and ligated using ligation high Ver.2 (manufactured by Toyobo Co., Ltd.). E. coli DH5α was transformed with this ligation reaction solution, and the target plasmid was extracted from the kanamycin-resistant strains. Using this plasmid, E. coli BL21(DE3) was transformed to obtain an expression strain of ID2839 mature form (sometimes referred to as "BL21(DE3) / pET28a-ID2839 mature form"). In BL21(DE3) / pET28a-ID2839 mature form, a protein with an amino acid sequence (MetGlySerSerHisHisHisHisHisHisSerSerGlyLeuValProArgGlySerHis: SEQ ID NO: 21) containing a His tag and a thrombin cleavage site added to the N-terminus of the mature protein (with the Ala at position 1 in SEQ ID NO: 2 substituted with Met) was expressed. A strain that does not express ID2839 mature form (sometimes referred to as "BL21(DE3) / pET28a") was also obtained as a control strain.

[0126] (2) Induction of expression of ID2839 mature form and preparation of soluble fraction (Materials) · LB liquid medium: LB Broth, 1.1G PER TABLET (manufactured by Sigma-Aldrich) was dissolved at a ratio of 10 tablets per 500 mL of ultrapure water, and the medium was autoclaved. · Transformant: BL21(DE3) / pET28a-ID2839 mature form · Transformant (control): BL21(DE3) / pET28a

[0127] (Method) Each transformant was inoculated into 5 mL of LB medium containing 50 mg / L of kanamycin and pre-cultured at 37°C. 1.0 mL of the obtained pre-culture solution was inoculated into 100 mL of LB medium containing 50 mg / L of kanamycin, and shake culture was performed using a baffled flask. IPTG was added (final concentration 0.1 mM) when OD660 reached 0.6, and the culture was further continued at 18°C for 16 hours. After completion of the culture, the cells were collected from the obtained culture solution by centrifugation, and a certain amount of BugBuster (manufactured by Novagen) was added per wet cell mass to disrupt the cells. The cell residue was removed from the disrupted solution by centrifugation, and the obtained supernatant was used as the soluble fraction. From BL21(DE3) / pET28a-ID2839 mature form, a soluble fraction containing ID2839 mature form was obtained. From BL21(DE3) / pET28a, a soluble fraction not containing ID2839 was obtained.

[0128] (3) Activity evaluation of soluble fraction (Materials) · Soluble fraction derived from BL21(DE3) / pET28a-ID2839 mature form · Soluble fraction derived from BL21(DE3) / pET28a

[0129] (Method) Using a Lysozyme activity kit (manufactured by Sigma-Aldrich), the activity (Units / mL) of decomposing the glycosidic bond between N-acetylmuramic acid and N-acetylglucosamine was calculated. The activity (UNITS / mL) was calculated using the following formula. Activity of decomposing the glycosidic bond between N-acetylmuramic acid and N-acetylglucosamine (UNITS / mL) = {ΔABS450nm / min (target bacteria-containing phosphate buffer added with soluble fraction) - ΔABS450nm / min (target bacteria-containing phosphate buffer without soluble fraction)} / (0.001×0.1)

[0130] (Results) The results are shown in Table 6. Activity for decomposing the glycosidic bond between N-acetylmuramic acid and N-acetylglucosamine was observed in the soluble fraction derived from BL21(DE3) / pET28a-ID2839 mature form, while almost no activity for decomposing the glycosidic bond between N-acetylmuramic acid and N-acetylglucosamine was observed in the soluble fraction derived from the control BL21(DE3) / pET28a. The ID2389 mature form was found to have peptidoglycan-degrading activity and activity for decomposing the glycosidic bond between N-acetylmuramic acid and N-acetylglucosamine.

[0131] [Table 6]

[0132] [Experiment 9. Identification of a protein (ID1644) with peptidoglycan-degrading activity] (1) Identification from genomic information From the results of decoding the genomic sequence of NITE BP-02779, a gene for an enzyme with high identity to N-acetylmuramoyl-L-alanine amidase was found. This enzyme was named ID1644. ID1644 was secreted into the culture supernatant of NITE BP-02779. The entire amino acid sequence of SEQ ID NO: 4 was considered to be the amino acid sequence of the precursor (pro sequence). The amino acid sequence of the mature protein was positions 1 to 493 of SEQ ID NO: 4, and was considered to be encoded by the nucleotide sequence of positions 85 to 1563 of SEQ ID NO: 3. Positions -28 to -1 of SEQ ID NO: 4 were considered to be the signal peptide. Hereinafter, the protein consisting of the amino acid sequence of positions 1 to 493 of SEQ ID NO: 4 may be referred to as "ID1644 mature form", and the protein consisting of the entire amino acid sequence (-28 to 493 positions) of SEQ ID NO: 4 may be referred to as "ID1644 precursor". When the signal peptide is cleaved from the ID1644 precursor, it becomes the ID1644 mature form, and the ID1644 mature form is secreted outside the cell and is considered to be able to degrade peptidoglycan. The molecular weight of the ID1644 precursor was approximately 56 kDa, and the molecular weight of the ID1644 mature form was approximately 53 kDa.

[0133] [Experiment 10. Preparation with Mature E. coli, ID1644] (1) Construction of ID1644 Mature Expression Strain (Materials) · Primers F and R for ID1644 cloning (SEQ ID NOs: 22 and 23) · LB agar medium

[0134] (Method) Using the genomic DNA of NITE BP-02779 as a template, PCR amplification of the region containing the nucleotide sequence encoding ID1644 mature was performed using primers F and R for ID1644 cloning. PCR amplification was performed using KOD FX (manufactured by Toyobo Co., Ltd.). The reaction solution was prepared according to the composition attached to the kit, and carried out at (1) 94°C for 2 minutes, (2) 98°C for 10 seconds, (3) 50°C for 30 seconds, and (4) 68°C for 1.5 minutes. The steps of (2) to (4) were repeated 25 cycles. The obtained PCR fragment and the pET-22b(+) vector (manufactured by Merck Millipore) were digested with NdeI and XhoI respectively, and ligated using ligation high Ver.2 (manufactured by Toyobo Co., Ltd.). E. coli DH5α was transformed with this ligation reaction solution, and the target plasmid was extracted from the ampicillin-resistant strains. Using this plasmid, E. coli BL21(DE3) was transformed to obtain an expression strain of ID1644 mature (sometimes referred to as "BL21(DE3) / pET22b-ID1644 mature"). In BL21(DE3) / pET22b-ID1644 mature, a protein with methionine added to the N-terminus of the mature protein (positions 1 to 493 of SEQ ID NO: 4) is expressed. As a control strain, a strain that does not express ID1644 mature (sometimes referred to as "BL21(DE3) / pET22b") was also obtained.

[0135] (2) Induction of ID1644 Mature Expression and Preparation of Soluble Fraction (Materials) · LB liquid medium · Transformant: BL21(DE3) / pET22b-ID1644 mature · Transformant (control): BL21(DE3) / pET22b

[0136] (Method) Each transformant was inoculated into 5 mL of LB medium containing 100 mg / L of ampicillin and pre-cultured at 37°C. 1.0 mL of the obtained pre-culture solution was inoculated into 100 mL of LB medium containing 100 mg / L of ampicillin, and shake culture was performed using a baffle flask. When OD660 reached 0.6, IPTG was added (final concentration 0.1 mM), and the culture was further continued at 18°C for 16 hours. After completion of the culture, the cells were collected from the obtained culture solution by centrifugation, and a certain amount of BugBuster (manufactured by Novagen) was added per wet cell mass to disrupt the cells. The cell residue was removed from the disrupted solution by centrifugation, and the obtained supernatant was used as the soluble fraction. From BL21(DE3) / pET22b-ID1644 mature form, a soluble fraction containing ID1644 mature form was obtained. From BL21(DE3) / pET22b, a soluble fraction not containing ID1644 mature form was obtained.

[0137] (3) Activity evaluation of soluble fraction - 1 (Materials) · Soluble fraction derived from BL21(DE3) / pET22b-ID1644 mature form · Soluble fraction derived from BL21(DE3) / pET22b · Peptidoglycan solution: A solution prepared by dissolving peptidoglycan (manufactured by Sigma-Aldrich) at a ratio of 10 mg in 50 mL of phosphate buffer (pH 7.0)

[0138] (Method) 5 mL of the peptidoglycan solution and 50 μL of the soluble fraction were mixed, and OD450 was measured over time to calculate the peptidoglycan-degrading activity (Units / mL). The peptidoglycan-degrading activity (UNITS / mL) was calculated using the following formula. Peptidoglycan-degrading activity (UNITS / mL) = {ΔABS450nm / min (peptidoglycan-containing phosphate buffer with soluble fraction added) - ΔABS450nm / min (peptidoglycan-containing phosphate buffer without soluble fraction added)} / (0.001 × 0.05)

[0139] (Results) The results are shown in Table 7. Peptidoglycan-degrading activity was observed in the available fraction derived from BL21(DE3) / pET22b-ID1644 mature form, but not in the soluble fraction derived from the control BL21(DE3) / pET22b. The ID1644 mature form was found to have peptidoglycan-degrading activity.

[0140] [Table 7]

[0141] (4) Activity evaluation of soluble fraction - 2 (Materials) · Soluble fraction derived from BL21(DE3) / pET22b-ID1644 mature form · Soluble fraction derived from BL21(DE3) / pET22b · Substrate for N-acetylmuramoyl-L-alanine amidase: L-aniline-p-nitroanilide hydrochloride (manufactured by Sigma-Aldrich)

[0142] (Methods) 10 μL of the soluble fraction, 10 μL of the 1 mg / mL substrate solution, and 80 μL of 100 mM Tris-HCl (pH 7.6) were allowed to stand at 37°C for 10 minutes, and the absorbance (405 nm) was measured using a microplate reader (manufactured by Molecular Device).

[0143] (Results) The results are shown in Table 8. The soluble fraction derived from BL21(DE3) / pET22b-ID1644 mature form showed a higher value of ABS405nm than the soluble fraction derived from BL21(DE3) / pET22b. The ID1644 mature form was found to have the activity of N-acetylmuramoyl-L-alanine amidase.

[0144] [Table 8]

[0145] [Experiment 11.ID2839 Preparation in E. coli of the mature form - 2] (1) Construction of the ID2839 mature form - 2 expression strain (Materials) · Primers F and R for ID2839 cloning (SEQ ID NOs: 24 and 25) · LB agar medium

[0146] (Method) Using the genomic DNA of NITE BP - 02779 as a template, PCR amplification of the region containing the nucleotide sequence encoding ID2839 mature form - 2 (a protein consisting of the amino acid sequence in which Met is added to the N - terminus of Ala at position 1 in the amino acid sequence of positions 1 to 164 of SEQ ID NO: 2) was performed using primers for ID2839 cloning (SEQ ID NOs: 24 and 25). The PCR amplification was carried out using KOD plus NEO (manufactured by Toyobo Co., Ltd.). The reaction solution was prepared according to the composition attached to the kit and carried out at (1) 94°C for 2 minutes, (2) 98°C for 10 seconds, and (3) 68°C for 30 seconds, and the steps of (2) - (3) were repeated 30 cycles. The obtained PCR fragment and the fragment obtained by digesting the pET - 28a(+) vector (manufactured by Merck Millipore) with NcoI and XhoI were ligated using the In - Fusion HD Cloning Kit (manufactured by Takara Bio Inc.). E. coli DH5α was transformed with this ligation reaction solution, and the target plasmid was extracted from the kanamycin - resistant strains. Using this plasmid, E. coli BL21(DE3) was transformed to obtain an expression strain of ID2839 mature form - 2 (sometimes referred to as "BL21(DE3) / pET28a - ID2839 mature form - 2"). In BL21(DE3) / pET28a - ID2839 mature form - 2, the mature protein (a protein consisting of the amino acid sequence in which Met is added to the N - terminus of Ala at position 1 in the amino acid sequence of positions 1 to 164 of SEQ ID NO: 2) is expressed.

[0147] (2) Induction of ID2839 mature form - 2 expression and preparation of the soluble fraction (Materials) · LB liquid medium · Transformant: BL21(DE3) / pET28a - ID2839 mature form - 2 · Transformant (control): BL21(DE3) / pET28a

[0148] (Method) Each transformant strain was inoculated into 5 mL of LB medium containing 50 mg / L of kanamycin and pre-cultured at 37°C. 1.0 mL of the obtained pre-culture solution was inoculated into 100 mL of LB medium containing 50 mg / L of kanamycin, and shaking culture was performed using a baffle flask. IPTG was added (final concentration 1 mM) when OD660 reached 0.6, and the culture was further continued at 24°C for 20 hours. After the culture was completed, the soluble fraction of the cells was obtained by the same method as in Experiment 8.

[0149] (3) Activity evaluation of the soluble fraction (Materials) · BL21(DE3) / pET28a-ID2839 mature form-2 soluble fraction derived from · Soluble fraction derived from BL21(DE3) / pET28a

[0150] (Method) It was carried out in the same manner as in Experiment 8.

[0151] (Results) The results are shown in Table 9. Peptidoglycan-degrading activity was observed in the soluble fraction of BL21(DE3) / pET28a-ID2839 mature form-2.

[0152]

Table 9

[0153] [Preparation in E. coli of Experiment 12.ID2839 mature form-3] (1) Construction of the ID2839 mature form-3 expression strain (Materials) · Primers F and R for ID2839 cloning (SEQ ID NO: 26 and 25) · LB agar medium

[0154] (Method) An expression strain of ID2839 mature form-3 (sometimes referred to as "BL21(DE3) / pET28a-ID2839 mature form-3") was obtained in the same manner as in Experiment 11, except that the cloning primers described in SEQ ID NOs: 26 and 25 were used. In BL21(DE3) / pET28a-ID2839 mature form-3, a mature protein (a protein consisting of an amino acid sequence in which Ala at position 1 is replaced by Met in the amino acid sequence of positions 1 to 164 of SEQ ID NO: 2) is expressed.

[0155] (2) Induction of expression of ID2839 mature form -3 and preparation of soluble fraction (Materials) · LB liquid medium · Transformant: BL21(DE3) / pET28a-ID2839 mature form-3 · Transformant (control): BL21(DE3) / pET28a

[0156] (Methods) The soluble fraction of the bacterial cells was obtained in the same manner as in Experiment 11.

[0157] (3) Activity evaluation of soluble fraction (Materials) · Soluble fraction derived from BL21(DE3) / pET28a-ID2839 mature form-3 · Soluble fraction derived from BL21(DE3) / pET28a

[0158] (Methods) It was carried out in the same manner as in Experiment 8.

[0159] (Results) The results are shown in Table 10. Peptidoglycan-degrading activity was observed in the soluble fraction derived from BL21(DE3) / pET28a-ID2839 mature form-3.

[0160]

Table 10

Claims

1. A protein derived from Tumebacillus sp. NITE BP-02779 and having peptidoglycan-degrading activity.

2. The protein according to claim 1, which is a secreted protein.

3. A protein according to any one of the following (A1) to (A3): (A1) A protein comprising the amino acid sequence of positions 1 to 164 of SEQ ID NO: 2 and having peptidoglycan-degrading activity; (A2) A protein comprising an amino acid sequence in which 1 to 10 amino acid residues are substituted, deleted, inserted, or added in the amino acid sequence of positions 1 to 164 of SEQ ID NO: 2 and having peptidoglycan-degrading activity; (A3) A protein comprising an amino acid sequence having 93% or more identity to the amino acid sequence of positions 1 to 164 of SEQ ID NO: 2 and having peptidoglycan-degrading activity.

4. The protein according to any one of claims 1 to 3, wherein the peptidoglycan-degrading activity is an activity of degrading a glycosidic bond between N-acetylmuramic acid and N-acetylglucosamine.

5. A protein according to any one of the following (a1) to (a4): (a1) A protein consisting of the amino acid sequence of positions 1 to 164 of SEQ ID NO: 2; (a2) A protein consisting of an amino acid sequence in which Ala at position 1 is substituted with Met in the amino acid sequence of positions 1 to 164 of SEQ ID NO: 2; (a3) A protein consisting of an amino acid sequence in which Ala at position 1 is substituted with Met and the amino acid sequence set forth in SEQ ID NO: 21 is added to the N-terminus of the Met in the amino acid sequence of positions 1 to 164 of SEQ ID NO: 2; (a4) A protein consisting of an amino acid sequence in which Met is added to the N-terminus of Ala at position 1 in the amino acid sequence of positions 1 to 164 of SEQ ID NO:

2.

6. A protein according to any one of the following (B1) to (B3): (B1) A protein comprising the amino acid sequence of positions 1 to 493 of SEQ ID NO: 4 and having peptidoglycan-degrading activity; (B2) A protein comprising an amino acid sequence in which 1 to 10 amino acid residues are substituted, deleted, inserted, or added in the amino acid sequence of positions 1 to 493 of SEQ ID NO: 4 and having peptidoglycan-degrading activity; (B3) A protein comprising an amino acid sequence having 90% or more identity to the amino acid sequence of positions 1 to 493 of SEQ ID NO: 4 and having peptidoglycan-degrading activity.

7. The protein according to claim 1, 2 or 6, wherein the peptidoglycan-degrading activity is N-acetylmuramoyl-L-alanine amidase activity.

8. A protein according to any one of the following (b1) to (b4): (b1) A protein consisting of the amino acid sequence of positions 1 to 493 of SEQ ID NO: 4; (b2) A protein consisting of an amino acid sequence in which Glu at position 1 is substituted with Met in the amino acid sequence of positions 1 to 493 of SEQ ID NO: 4; (b3) A protein consisting of an amino acid sequence in which Glu at position 1 is substituted with Met and the amino acid sequence described in SEQ ID NO: 21 is added to the N-terminus of the Met in the amino acid sequence of positions 1 to 493 of SEQ ID NO: 4; (b4) A protein consisting of an amino acid sequence in which Met is added to the N-terminus of Glu at position 1 in the amino acid sequence of positions 1 to 493 of SEQ ID NO:

4.

9. DNA encoding the protein according to any one of claims 1 to 8.

10. A vector containing the DNA according to claim 9.

11. A transformant containing the DNA according to claim 9 or the vector according to claim 10.

12. A microbial decomposition preparation comprising at least one selected from the group consisting of the protein according to any one of claims 1 to 8, the transformant according to claim 11, and a culture thereof.

13. A microbial decomposition method comprising the step of allowing at least one selected from the group consisting of the protein according to any one of claims 1 to 8, the transformant according to claim 11, and a culture thereof to act on a target microorganism.

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