Protein having peptidoglycan-degrading activity and DNA encoding protein, microorganism degrading preparation and method for degrading microorganism

Abstract

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

Description

Protein with peptidoglycan decomposition activity, DNA encoding said protein, microbial decomposition preparation, and microbial decomposition method

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

[0002] When sewage is purified using the activated sludge process, the removed organic matter turns into flocs containing microorganisms (bacteria), generating what is known as excess sludge. The 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: FY2018 Project, Industrial Waste Discharge and Treatment Status Survey Report, FY2016 Results (Summary); Non-Patent Document 2: Yamamoto Masayuki, "On Sewage Combustion Technology," Journal of the Japan Fuel Society, Combustion Society of Japan, 2011, Vol. 53, No. 164, pp. 91-96).

[0003] "Fiscal Year 2018 Project, Industrial Waste Discharge and Treatment Status Survey Report, Fiscal Year 2016 Results (Summary)" [online], March 2019, Waste Control Division, Environmental Regeneration and Resource Recycling Bureau, Ministry of the Environment [Retrieved March 17, 2020], Internet <URL: https: / / www.e-stat.go.jp / stat-search / file-download?statInfId=000031887949&fileKind=2> Masayuki Yamamoto, "On Sewage Combustion Technology," Journal of the Japan Fuel Society, Combustion Society of Japan, 2011, Vol. 53, No. 164, pp. 91-96

[0004] However, since incineration of excess sludge generates greenhouse gases, there is a need to reduce the volume of excess sludge from environmental considerations. As a method for reducing the volume of excess sludge, a method of decomposing the microorganisms that make up the excess sludge has been proposed.

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

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

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

[10] A vector comprising the DNA according to [9].

[11] A transformant comprising the DNA according to [9] or the vector according to

[10] .

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

[11] , and a culture thereof.

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

[11] , and a culture thereof to act on a target microorganism.

[0007] According to the present invention, there are provided a novel protein capable of degrading peptidoglycan, DNA encoding said protein, a microbial degradation preparation, and a microbial degradation method.

[0008]

[0023] Figure 1 shows a representative peptidoglycan structure and sites that can be cleaved by peptidoglycan-degrading enzymes. Figure 2 shows the dry weight of excess sludge after the addition of Tumebacillus sp. NITE BP-02779 in Experiment 4. Figure 3 shows the results of fractionating the culture supernatant of Tumebacillus sp. NITE BP-02779 by column chromatography and examining the peptidoglycan-degrading activity in Experiment 6. Figure 4 shows the results of fractionating the culture supernatant of Tumebacillus sp. NITE BP-02779 by column chromatography and detecting proteins contained in the eluted fraction by electrophoresis in Experiment 6.

[0009] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The following describes in detail the embodiments of the present invention, but the present invention is not limited to the following embodiments.

[0010] [Protein Derived from Tumebacillus sp. NITE BP-02779] A protein according to one embodiment of the present invention is derived from Tumebacillus sp. NITE BP-02779 (hereinafter, sometimes referred to as "NITE BP-02779") and has peptidoglycan decomposition 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 method for measuring peptidoglycan decomposition activity described below. Proteins derived from NITE BP-02779 and having peptidoglycan decomposition activity can decompose target microorganisms, and therefore can reduce the volume of excess sludge.

[0011] NITE BP-02779 is a bacterium that was internationally deposited at the Patent Microorganism Depositary of the National Institute of Technology and Evaluation (Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan 292-0818) on September 11, 2018, and a certificate of deposit 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 Tumebacillus bacteria. NITE BP-02779 is capable of degrading various target microorganisms such as Bacillus, Micrococcus, and Staphylococcus, as well as excess sludge, making it useful for excess sludge treatment. The bacteriological 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 base 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 that it is based on the genome sequence of NITE BP-02779. For example, it may mean that a substance is artificially synthesized or produced in the cells of another organism based on the genome sequence of NITE BP-02779. Substances derived from NITE BP-02779 include, for example, biomolecules such as proteins, nucleic acids, and sugars, as well as metabolites. These may be localized intracellularly after production, or may be secreted extracellularly.

[0013] The following method can be used to determine whether a protein is derived from NITE BP-02779. First, the genome sequence of NITE BP-02779 is decoded using a next-generation sequencer. The adapter sequence is trimmed from the resulting base sequence, and de novo assembly is performed. The scaffold sequence obtained by de novo assembly is analyzed, and gene region prediction and annotation are performed, creating a protein database for mass spectrometry. Next, the target protein is subjected to mass spectrometry and compared with the protein database, making it possible to determine whether the target protein is derived from NITE BP-02779.

[0014] Another method for determining whether a protein is derived from NITE BP-02779 is to decode the amino acid sequence of the target protein using an amino acid sequencer and compare it with the above-mentioned protein database.

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

[0016] Peptidoglycan is a molecule in which polysaccharides composed of alternating β1,4-linked N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc) units 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 that converts peptidoglycan into two or more substances is promoted, and "proteins with peptidoglycan degrading activity" (hereinafter sometimes referred to as "peptidoglycan degrading enzymes") refer to proteins that promote the above reaction.

[0017] Peptidoglycan is efficiently degraded by cleavage of glycosidic bonds between sugars constituting polysaccharides and peptide bonds within oligopeptides. The sites that can be degraded by peptidoglycan-degrading enzymes are indicated by arrows in Figure 1. Proteins with the activity of degrading peptidoglycan include (1) proteins with the activity of degrading the glycosidic bond between N-acetylglucosamine and N-acetylmuramic acid, (2) proteins with the activity of degrading the glycosidic bond between N-acetylmuramic acid and N-acetylglucosamine, (3) proteins with the activity of degrading the amide bond between N-acetylmuramic acid and L-alanine, and (4) proteins with the activity of degrading each peptide bond.

[0018] Specific examples include (1) N-acetylglucosaminidase, (2) N-acetylmuramidase and lytic transglycosylase, (3) N-acetylmuramoyl-L-alanine amidase, and (4) L,D-endopeptidase, D,L-endopeptidase, carboxypeptidase, and D,D-endopeptidase.

[0019] A method for determining whether a protein has peptidoglycan degrading activity includes, for example, reacting a target protein with peptidoglycan in a buffer solution for a certain period of time and then measuring the degradation of peptidoglycan. Methods for determining degradation include, but are not limited to, measuring the turbidity of peptidoglycan, detecting peptidoglycan with an SLP reagent, and detecting peptidoglycan-derived degradation products using high-performance liquid chromatography (HPLC), mass spectrometry (MS), thin-layer chromatography (TLC), nuclear magnetic resonance (NMR), gas chromatography (GC), etc.

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

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

[0022] [Protein Represented by SEQ ID NO: 2 or 4] A 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 decomposition activity (B1) A protein comprising the amino acid sequence of positions 1 to 493 of SEQ ID NO: 4 and having peptidoglycan decomposition 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 mature proteins predicted from the genome sequence of NITE BP-02779. Whether a protein has peptidoglycan decomposition activity can be determined, for example, by the method described above. Proteins with peptidoglycan decomposition activity can decompose target microorganisms and therefore can reduce the volume of excess sludge.

[0024] A 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, so long as it maintains peptidoglycan degrading activity. Variants that maintain peptidoglycan degrading activity may be referred to as "conservative variants." In the present invention, proteins identified as having peptidoglycan degrading activity include, in addition to the above-mentioned proteins, their conservative variants, and fusion proteins of these proteins with other peptides. Examples of conservative variants of peptidoglycan degrading enzymes are provided below.

[0025] As long as the protein according to one embodiment of the present invention maintains peptidoglycan decomposition activity, it may be a protein having an amino acid sequence in which one or several amino acids have been 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. The term "one or several" varies depending on the position and type of amino acid residue in the three-dimensional structure of the protein, and may be, for example, 1 to 10, 1 to 8, 1 to 5, or 1 to 3.

[0026] A protein according to one embodiment of the present invention may be a protein having an amino acid sequence that is highly identical, for example, 90% or more, 93% or more, 95% or more, 97% or more, or 99% or more, 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 it maintains peptidoglycan decomposition activity.

[0027] The above-mentioned mutations of amino acid residues are conservative mutations that maintain normal protein function. A typical conservative mutation is conservative substitution. Conservative substitutions are those that substitute between Phe, Trp, and Tyr when the substitution site is an aromatic amino acid, between Leu, Ile, and Val when the substitution site is a hydrophobic amino acid, between Gln and Asn when the substitution site is a polar amino acid, between Lys, Arg, and His when the substitution site is a basic amino acid, between Asp and Glu when the substitution site is an acidic amino acid, and between Ser and Thr when the substitution site is an amino acid having a hydroxyl group. Specific examples of substitutions that are considered to be conservative substitutions include substitutions of Ala with Ser or Thr, substitutions of Arg with Gln, His, or Lys, substitutions of Asn with Glu, Gln, Lys, His, or Asp, substitutions of Asp with Asn, Glu, or Gln, substitutions of Cys with Ser or Ala, substitutions of Gln with Asn, Glu, Lys, His, Asp, or Arg, substitutions of Glu with Gly, Asn, Gln, Lys, or Asp, substitutions of Gly with Pro, substitutions of His with Asn, Lys, Gln, Arg, or Tyr, and substitutions of Ile with Pro. Examples of such mutations include substitutions of Lys with Leu, Met, Val, or Phe, substitutions of Leu with Ile, Met, Val, or Phe, substitutions of Lys with Asn, Glu, Gln, His, or Arg, substitutions of Met with Ile, Leu, Val, or Phe, substitutions of Phe with Trp, Tyr, Met, Ile, or Leu, substitutions of Ser with Thr or Ala, substitutions of Thr with Ser or Ala, substitutions of Trp with Phe or Tyr, substitutions of Tyr with His, Phe, or Trp, and substitutions of Val with Met, Ile, or Leu. Mutations of amino acid residues also include mutations that occur due to naturally occurring mutations (mutants or variants) based on individual differences, species differences, etc. in the organisms from which the proteins are derived.

[0028] A protein according to one embodiment of the present invention may be a fusion protein with another peptide. Examples of other peptides include a marker peptide (marker protein), a peptide tag, and a pro- or pre-pro-sequence, such as a secretory signal peptide. The peptide linked to the peptidoglycan-degrading enzyme may be one type, or two or more peptides. When the peptidoglycan-degrading enzyme is a fusion protein with a signal peptide, after expression in a secretory host cell, the signal peptide is cleaved and only the peptidoglycan-degrading enzyme portion (mature protein) can be secreted outside the cell. When the peptidoglycan-degrading enzyme is a fusion protein, the above-mentioned identity refers to the identity of the mature protein portion excluding other peptide portions.

[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, and GFP. 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. The secretory signal peptide may be the secretory signal peptide of the peptidoglycan degrading enzyme itself contained in NITE BP-02779, or a secretory signal peptide that can function in a host that expresses the peptidoglycan degrading enzyme. The fusion protein can be produced by a conventional method.

[0030] A protein according to one 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 the amino acid sequence of positions 1 to 164 of SEQ ID NO: 2, in which Ala at position 1 is substituted with Met, (a3) ​​a protein consisting of the amino acid sequence of positions 1 to 164 of SEQ ID NO: 2, in which Ala at position 1 is substituted with Met and the amino acid sequence of MetGlySerSerHisHisHisHisHisHisHisSerSerGlyLeuValProArgGlySerHis (SEQ ID NO: 21) is added to the N-terminus of the Met, and (a4) a protein consisting of the amino acid sequence of positions 1 to 164 of SEQ ID NO: 2, in which Met is added to the N-terminus of Ala at position 1.

[0031] A protein according to one embodiment of the present invention may be any of the following proteins (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 the amino acid sequence of positions 1 to 493 of SEQ ID NO: 4, in which Glu at position 1 is substituted with Met, (b3) a protein consisting of the amino acid sequence of positions 1 to 493 of SEQ ID NO: 4, in which Glu at position 1 is substituted with Met and the amino acid sequence of SEQ ID NO: 21 is added to the N-terminus of the Met, (b4) a protein consisting of the amino acid sequence of positions 1 to 493 of SEQ ID NO: 4, in which Met is added to the N-terminus of Glu at position 1.

[0032] A protein consisting of the amino acid sequence of positions 1 to 164 of SEQ ID NO: 2 is considered to have (2) the activity of degrading the glycosidic bond between N-acetylmuramic acid and N-acetylglucosamine, among the above-mentioned peptidoglycan degrading activities. (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, and (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 preferably has (2) the activity of degrading the glycosidic bond between N-acetylmuramic acid and N-acetylglucosamine, among the above-mentioned peptidoglycan degrading activities.

[0033] A protein consisting of the amino acid sequence of positions 1 to 493 of SEQ ID NO: 4 is considered to have (3) N-acetylmuramoyl-L-alanine amidase activity among the above-mentioned peptidoglycan decomposition activities. (B1) A protein comprising the amino acid sequence of positions 1 to 493 of SEQ ID NO: 4 and having peptidoglycan decomposition 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 decomposition activity, and (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 decomposition activity preferably has (3) N-acetylmuramoyl-L-alanine amidase activity among the above-mentioned peptidoglycan decomposition activities.

[0034] The proteins according to (A1) to (A3) and (B1) to (B3) may be synthesized proteins or purified or isolated proteins. The proteins according to (A1) to (A3) and (B1) to (B3) may be proteins derived from NITE BP-02779, or may be proteins artificially synthesized or produced in cells of NITE BP-02779 or other organisms.

[0035] [DNA Encoding Peptidoglycan-Degrading Enzyme] The DNA of the present invention is DNA encoding the above-described protein. Specific examples include 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. A DNA according to one embodiment of the present invention may be DNA having the nucleotide sequence of positions 205 to 696 of SEQ ID NO: 1 or the nucleotide sequence of positions 85 to 1563 of SEQ ID NO: 3. SEQ ID NOs: 1 and 3 are nucleotide sequences based on the genome sequence of NITE BP-02779 and encode a mature protein having peptidoglycan-degrading activity. The DNA of the present invention may include a nucleotide sequence encoding an initiation codon and / or another peptide at the 5' end of the nucleotide sequence, or a stop codon at the 3' end of the nucleotide sequence. The DNA may be recombinant DNA. The DNA may be synthetic DNA or complementary DNA (cDNA). The DNA may be purified or isolated DNA.

[0036] The DNA according to the present invention is not limited to the DNA consisting of the sequence of NITE BP-02779, but may also be DNA containing a nucleotide sequence in which a codon encoding each amino acid in the coding region is replaced with another equivalent codon encoding the same amino acid. The DNA according to one embodiment of the present invention may also be DNA containing a nucleotide sequence in which the codon usage has been altered to improve the expression of a protein having peptidoglycan degrading activity.

[0037] The DNA according to one embodiment of the present invention also includes a DNA that hybridizes under stringent conditions with a probe having a base sequence complementary to the above base sequence or with a probe that can be prepared from the above complementary base sequence, and encodes a protein having peptidoglycan decomposition activity. Stringent conditions refer to conditions under which so-called specific hybrids are formed but non-specific hybrids are not formed. Examples of conditions include conditions under which DNAs with high identity, for example, DNAs with identity of 50% or more, 65% or more, 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 97% or more, and particularly preferably 99% or more, hybridize with each other, but DNAs with lower identity do not hybridize with each other, or conditions such as washing once, preferably two to three times, at a salt concentration and temperature equivalent to the washing conditions for conventional Southern hybridization, i.e., 60°C, 1×SSC, 0.1% SDS, preferably 60°C, 0.1×SSC, 0.1% SDS, more preferably 68°C, 0.1×SSC, 0.1% SDS. Furthermore, when a DNA fragment of about 300 bp in length is used as the probe, washing conditions for hybridization include 50°C, 2×SSC, 0.1% SDS.

[0038] The percent identity between two sequences can be determined, for example, using a mathematical algorithm, such as the algorithm described by Myers and Miller (1988) CABIOS, 4:11-17, the local homology algorithm described by Smith et al. (1981) Adv. Appl. Math. 2:482, the homology alignment algorithm described by Needleman and Wunsch (1970) J. Mol. Biol. 48:443-453, the similarity search method described by Pearson and Lipman (1988) Proc. Natl. Acad. Sci. 85:2444-2448, or the similarity search method described by Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA, 90:587-5877, and an improved version of the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA, 872264.

[0039] Sequence comparison (alignment) for determining sequence identity can be performed using programs based on these mathematical algorithms. The programs can be suitably executed by a computer. Such programs include, but are not limited to, the PC / Gene program CLUSTAL (available from Intelligenetics, Mountain View, Calif.), and the ALIGN program (Version 2.0), GAP, BESTFIT, BLAST, FASTA and TFASTA from 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 default 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. 16:1088-190, Huang et al. (1992) CABIOS 8:155-65, and Pearson et al. (1994) Meth. Mol. Biol. 24:307-331.

[0040] To obtain nucleotide sequences identical to a nucleotide sequence encoding a protein of interest, for example, a BLAST nucleotide search can be performed using the BLASTN program, score = 100, word length = 12. To obtain amino acid sequences identical to a protein of interest, a BLAST protein search can be performed using the BLASTX program, score = 50, word length = 3. For details on BLAST nucleotide searches and BLAST protein searches, see http: / / www.ncbi.nlm.nih.gov. Gapped BLAST (BLAST 2.0) can be used to obtain gapped alignments for comparison purposes. PSI-BLAST (BLAST 2.0) can be used to perform an iterated search that detects distant relationships between sequences. Gapped BLAST and PSI-BLAST are described in detail in Altschul et al. (1997) Nucleic Acids Res. 25:3389. When using BLAST, Gapped BLAST, or PSI-BLAST, the default parameters of each program (e.g., BLASTN for nucleotide sequences, BLASTX for amino acid sequences) can be used. Alignment can also be performed manually.

[0041] The identity between two sequences is calculated as the percentage of residues that match between the two sequences when the two sequences are aligned for maximum correspondence.

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

[0043] [Vector] The vector of the present invention contains the above-described 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 of the present invention can be constructed by incorporating the above-described DNA into a basic vector in accordance with standard genetic engineering techniques. The basic vector is, for example, autonomously replicable in host cells, can be isolated and purified from host cells, and has a detectable marker. The DNA of the present invention is, for example, inserted into an expression vector that can be used in the host cell to be transfected, and the vector can be introduced into the host cell to express a protein having peptidoglycan degrading activity in the host cell.

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

[0045] When the vector of the present invention is constructed using a basic vector having an autonomous replication origin (ori), the vector is maintained in the cell as an episome when introduced into a host cell. A vector incorporating an SV40 ori can significantly increase the copy number of the vector in the cell when introduced into, for example, COS cells transformed with an SV40 genome lacking the ori.

[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 functions in the host cell. A "promoter that functions in a 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 to be incorporated into the base vector is inserted downstream of the promoter in a state in which the promoter is functional. For example, the base vector has a cloning site downstream of the promoter sequence. The base vector may have a selection marker sequence.

[0047] The promoter may be a promoter derived from the host or a heterologous promoter. 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, examples of the promoter include the E. coli lactose operon promoter (lacP), tryptophan operon promoter (trpP), arginine operon promoter (argP), galactose operon promoter (galP), tac promoter, T7 promoter, T3 promoter, and λ phage promoters (λ-pL, λ-pR). When the host cell is an animal cell or a fission yeast, examples of the promoter include the Rous sarcoma virus (RSV) promoter, cytomegalovirus (CMV) promoter, simian virus (SV40) early or late promoter, and mouse papilloma virus (MMTV) promoter. When the host cell is a budding yeast, examples of the promoter include the ADH1 promoter. The ADH1 promoter can be prepared by conventional genetic engineering methods from the yeast expression vector pAAH5 (available from the Washington Research Foundation; see Ammerer et al., Methods in Enzymology, 10, part 1 (pp. 192-201)) containing the ADH1 promoter and its terminator. The vector pACT2 containing the ADH1 promoter can express a gene of interest in large quantities in budding yeast such as CG1945 (Clontech) by inserting the gene of interest downstream of the ADH1 promoter.

[0048] [Transformant] The transformant according to the present invention comprises the DNA according to the present invention or the vector according to the present invention. The transformant comprising the DNA according to the present invention or the vector according to the present invention can express a protein having peptidoglycan decomposition activity.

[0049] The host cells into which the DNA of the present invention or the vector of the present invention is introduced can be, for example, eukaryotic or prokaryotic cells, including bacteria, fungi, plant cells, animal cells, and insect cells. The host cells are preferably Escherichia coli. By introducing the DNA or vector into the host cells, the host cells can be transformed to produce transformants.

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

[0051] The DNA or vector of the present invention can be introduced into host cells by a conventional introduction method suitable for the host cell. When Escherichia coli is used as the host cell, examples of the gene introduction methods include the calcium chloride method and electroporation method described in J. Sambrook, E. F. Frisch, and T. Maniatis, "Molecular Cloning 2nd Edition," Cold Spring Harbor Laboratory (1989). When mammalian or insect cells are used as the host cell, examples of the gene introduction methods include the calcium phosphate method, DEAE-dextran method, electroporation, and lipofection. When yeast is used as the host cell, examples of the gene introduction methods include the lithium method used in the Yeast transformation kit (Clontech). When a virus is used as a vector, the viral genome into which the DNA of the present invention has been inserted can be introduced into a host cell by the above-mentioned gene transfer method, or the DNA of the present invention can be introduced into a host cell by infecting the host cell with viral particles containing the viral genome.

[0052] A selectable marker can be used to select transformants into which the DNA or vector of the present invention has been introduced. For example, a selectable marker gene is introduced into host cells together with the DNA or vector of the present invention, and the cells are cultured using a method appropriate to the properties of the selectable marker. For example, if the selectable marker gene confers drug resistance to a selective drug that is lethal to the host cell, the host cells into which the DNA or vector of the present invention has been introduced can be cultured in a medium supplemented with the selective drug. Examples of combinations of a gene conferring drug resistance and a selective drug include a neomycin resistance-conferring gene and neomycin, a hygromycin resistance-conferring gene and hygromycin, and a blasticidin S resistance-conferring gene and blasticidin S. Furthermore, if the marker gene complements an auxotrophy of the host cell, the host cells into which the DNA or vector of the present invention has been introduced can be cultured in a minimal medium that does not contain the nutrient corresponding to the auxotrophy. Transformants can also be selected based on the activity of an enzyme expressed by the DNA or vector of the present invention.

[0053] To obtain a transformant in which the DNA of the present invention is located in the chromosome of a host cell, for example, first linearize the vector of the present invention and a vector carrying a marker gene by digestion with a restriction enzyme or the like, and then introduce them into the host cell by the gene transfer method described above. The introduced cells are typically cultured for several weeks, and then the desired transformant is selected based on the expression level of the marker gene. When a gene conferring resistance to a selective drug is used as the marker gene, the vector of the present invention and a vector carrying the marker gene are introduced into the host cell by the gene transfer method described above, and the cells are then subcultured for several weeks or more in a medium supplemented with the selective drug. The selective drug-resistant clones that survive in colony form are then purified and cultured to obtain a transformant in which the DNA of the present invention has been introduced into the chromosome of the host cell. To confirm that the DNA of the present invention has been integrated into the chromosome of the host cell, the genomic DNA of the cell can be prepared according to conventional genetic engineering methods, and the presence of the DNA of the present invention can be detected by PCR, Southern hybridization, or the like using DNA containing a partial nucleotide sequence of the introduced DNA of the present invention as a primer or probe. A transformant in which the DNA of the present invention has been incorporated into the chromosome of a host cell can be stored frozen and can be awakened and used as needed, thereby eliminating the need to prepare a transformant for each experiment and enabling tests to be conducted using a transformant whose properties and handling conditions have been confirmed in advance.

[0054] [Production of the Protein of the Present Invention] The protein of the present invention can be obtained, for example, from NITE BP-02779. Obtaining from NITE BP-02779 means obtaining it from the cells or culture supernatant of NITE-BP02779. For example, when obtaining the protein of the present invention from the cells, the cells can be disrupted, lysed, extracted, or the like as appropriate, and the protein of the present invention can be recovered. The cells can be recovered from the culture by centrifugation or the like. Cell disruption, lysis, extraction, or the like can be performed by known methods. Examples of such methods include ultrasonic disruption, the Dynomill method, bead disruption, French press disruption, and lysozyme treatment. These methods may be used alone, or two or more may be used in appropriate combination. When the protein of the present invention accumulates in the culture supernatant, the culture supernatant can be obtained by centrifugation or the like, and the protein of the present invention can be recovered from the culture supernatant.

[0055] The protein of the present invention can be purified by known methods used for enzyme purification. Examples of such methods include ammonium sulfate fractionation, ion exchange chromatography, hydrophobic chromatography, affinity chromatography, gel filtration chromatography, and isoelectric precipitation. These methods may be used alone or in appropriate combination of two or more. The protein of the present invention can be purified to the desired extent.

[0056] The proteins of the present invention can also be produced using a transformant obtained by introducing DNA encoding the proteins into a suitable host. The DNA is preferably incorporated into a vector and used for transformation. The proteins of the present invention may also be artificially synthesized in a cell-free system.

[0057] When the protein of the present invention is produced using a transformant, the transformant may be cultured by the same method as for culturing host cells. When the transformant is a microorganism, it can be cultured using various media appropriately containing, for example, a carbon source, a nitrogen source, organic or inorganic salts, etc., which are commonly used for culturing microorganisms.

[0058] Examples of carbon sources include sugars 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 amount of these carbon sources added to the medium is usually about 0.1 to 30% (w / v) of the culture solution.

[0059] Examples of nitrogen sources include natural organic nitrogen sources such as meat extract, peptone, yeast extract, malt extract, soybean flour, corn steep liquor, cottonseed flour, dry yeast, and casamino acids; amino acids; ammonium salts of inorganic acids such as sodium nitrate; 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, and amino acids can often be used as carbon sources as well. The amount of these nitrogen sources added to the medium is usually about 0.1 to 30% (w / v) of 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, and the like. Specific examples include 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. The amount of these organic salts and / or inorganic salts added to the medium is usually about 0.0001 to 5% (w / v) of the culture solution.

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

[0062] The transformant of the present invention may be cultured according to a method commonly used for culturing host cells, including 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 varied as appropriate within a range that allows the growth of the transformant, 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 to about 5 days, depending on the culture conditions.

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

[0064] The protein of the present invention may be obtained by appropriately disrupting, lysing, extracting, and purifying the transformant using the same method as in the above-mentioned NITE BP-02779. The protein of the present invention is not limited to the purified protein of the present invention; any fraction containing the protein of the present invention may be used as the "protein of the present invention" for applications such as peptidoglycan degradation. The fraction containing the protein of the present invention is not particularly limited, as long as it contains the protein of the present invention in a manner that allows it to act on peptidoglycan. Examples of such fractions include transformants, culture supernatants of transformants, disruptants, lysates, extracts (cell-free extracts), etc.), partially purified products (crudely purified products) thereof, and combinations thereof. These fractions may be used alone for applications such as peptidoglycan degradation, or may be used together with the purified protein of the present invention. In addition to the protein of the present invention, other enzymes different from the protein of the present invention may also be produced and accumulated in the culture. The protein of the present invention may be recovered as a mixture with such other enzymes, or may be recovered separately from such other enzymes.

[0065] [Microbial Degradation Preparation] The microbial degradative preparation contains at least one selected from the group consisting of the protein of the present invention and the transformant of the present invention and a culture thereof. The culture of the transformant includes the cultured transformant, a culture supernatant of the transformant, a disrupted transformant, a lysate, an extract of cells (cell-free extract), etc.), a partially purified product thereof (crudely purified product), and a combination thereof.

[0066] The microbial degradation 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 degradation preparation may contain one type of enzyme having peptidoglycan decomposition activity, or a combination of two or more types. Other components may also be contained as long as they do not inhibit the peptidoglycan decomposition activity. The microbial degradation preparation may contain one type or two or more types of transformants.

[0067] The content of the protein of the present invention and the transformant of the present invention and its culture in the microbial degradation preparation is not particularly limited, and may be set appropriately depending on the type and concentration of the target microorganism and the environment in which the microbial degradation preparation is used (volume of the reaction system, temperature, etc.).

[0068] The microbial degradation preparation contains a protein having peptidoglycan decomposition activity or a transformant capable of expressing a protein having peptidoglycan decomposition activity, and is therefore capable of decomposing peptidoglycan. The microbial degradation preparation can decompose target microorganisms having peptidoglycan. The target microorganisms are preferably bacteria that constitute excess sludge. The target microorganisms may be dead or live bacteria. The microbial degradation preparation can contribute to reducing the volume of excess sludge.

[0069] Whether or not microorganisms have been degraded can be confirmed by methods commonly used by those skilled in the art. Such confirmation methods include, for example, reacting a microbial degradation preparation with a target microorganism in an appropriate medium or buffer solution for a certain period of time, and then detecting the degradation of the target microorganism in the medium or buffer solution. The method for detecting the degradation of the target microorganism is not particularly limited, and examples thereof include measuring the turbidity of the target microorganism, detecting the target microorganism with an SLP reagent, detecting the DNA of the target microorganism by PCR, measuring the dry cell weight of the target microorganism, and detecting degradation 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. Whether or not the target microorganism has been degraded can also be determined by the above-mentioned method for examining peptidoglycan degradation activity.

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

[0071] The dosage form of the microbially degraded preparation is not particularly limited, as long as the functions of the protein of the present invention and the transformant and its culture of the present invention are not lost. Examples of dosage forms include liquids, suspensions, powders, solids, encapsulated forms, and frozen or lyophilized forms of these. Additives such as excipients, binders, disintegrants, lubricants, stabilizers, diluents, and surfactants can be used in the formulation.

[0072] The microbial decomposition preparation can be used by being added to sludge treatment equipment, wastewater treatment equipment, etc., where excess sludge is generated. The microbial decomposition preparation can also be used by being added to a tank, etc., in which excess sludge or target microorganisms have accumulated.

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

[0074] [Microbial Degradation Method] The microbial degradation method includes a step of allowing at least one selected from the group consisting of the protein of the present invention and the transformant of the present invention and a culture thereof to act on a target microorganism. The culture of the transformant includes a cultured transformant, a culture supernatant of the transformant, a disrupted product, a lysate, a cell extract (cell-free extract), etc.), a partially purified product thereof (crudely purified product), or a combination thereof. The step of allowing at least one selected from the protein of the present invention and the transformant of the present invention and a culture thereof to act on a target microorganism may be carried out using the above-mentioned microbial degradation preparation.

[0075] "Allowing the protein of the present invention to act on a target microorganism" refers to, for example, contacting the protein of the present invention with the target microorganism. "Allowing the transformant of the present invention to act on a target microorganism" refers to, for example, culturing the transformant of the present invention in the presence of the target microorganism. "Allowing a culture of the transformant of the present invention to act on a target microorganism" refers to, for example, contacting a culture of the transformant, preferably a protein having peptidoglycan degrading activity contained in the culture, with the target microorganism.

[0076] The microbial decomposition method of the present invention can decompose target microorganisms having peptidoglycan. The target microorganisms are preferably bacteria that constitute excess sludge. The target microorganisms may be dead or live. The microbial decomposition method can contribute to reducing the volume of excess sludge.

[0077] The microbial degradation method preferably comprises a step of allowing a target microorganism to react with 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. In this step, one type of protein having peptidoglycan decomposition activity may be used, or two or more types may be used in combination. In the microbial degradation method, one type of transformant may be allowed to act on the target microorganism, or two or more types of transformants may be allowed to act on the target microorganism.

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

[0079] The amounts of the protein of the present invention and the transformant of the present invention and its culture used in the microbial degradation method can be appropriately set taking into consideration the type and concentration of the target microorganism, the volume of the reaction system, the reaction temperature, etc.

[0080] Whether or not the target microorganisms have been decomposed can be confirmed by the method described in the section on microorganism-decomposing preparations.

[0081] One aspect of the present invention is use of at least one selected from the group consisting of the protein of the present invention and the transformant of the present invention and a culture thereof for degrading a target microorganism. One aspect of the present invention is use of a microbial degradation preparation comprising at least one selected from the group consisting of the protein of the present invention and the transformant of the present invention and a culture thereof for degrading a target microorganism.

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

[0083] [Experiment 1. Isolation of Microbial Degrading Bacteria] (Method) Microbial communities present in the environment (water) were cultured in a medium containing Micrococcus bacteria as a carbon source, and microorganisms capable of decomposing Micrococcus bacteria were enriched and cultured. Next, several strains that showed good growth were isolated from the enriched microbial community.

[0084] (Results) The ability of each isolated strain to degrade Micrococcus bacteria was examined, and one strain was found to be capable of degrading Micrococcus bacteria. Hereinafter, the strain that was found to be capable of degrading Micrococcus bacteria will 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] (Method) Strain A was identified by 16S rRNA gene analysis, morphological observation, and physiological and biochemical property tests.

[0087] 16S rRNA gene analysis was performed as follows. Genomic DNA was extracted from strain A, and the resulting genomic DNA was used as a template to perform PCR amplification of the 16S rRNA gene using a cloning forward primer (27F) and a cloning reverse primer (1492R). PCR amplification was performed using KOD FX (manufactured by Toyobo Co., Ltd.), and the amplified product after PCR was purified.

[0088] The purified PCR amplification product was used for cycle sequencing. The cycle sequencing reaction was performed using the BigDye Terminator v3.1 Cycle Sequencing Kit. The resulting reaction solution was purified, and the purified solution was subjected to DNA sequence analysis (3730xl DNA Analyzer) to determine the base 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 (bioMérieux, Lyon, France).

[0090] (Results) The obtained 16S rRNA gene base sequence (SEQ ID NO: 10) was subjected to homology analysis against the international base sequence database (DDBJ / ENA (EMBL) / GenBank). Among the type strains, it showed 98.1% identity to the 16S rRNA gene base sequence of Tumebacillus permanentifrigoris Eurl_9.5. However, no microorganisms possessing a 16S rRNA gene that completely matched the obtained base sequence existed. Furthermore, strain A did not grow at 10°C, whereas Tumebacillus permanentifrigoris Eurl_9.5, which had the highest 16S rRNA gene identity, did not exhibit this characteristic. Therefore, it was suggested that strain A is a new species distinct from conventional Tumebacillus species. Strain A was deposited internationally 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]

[0093]

[0094]

[0095]

[0096] [Experiment 3. Evaluation of the decomposition ability of excess sludge (dead bacteria) of NITE BP-02779 - 1] (Materials) - R2A medium: A medium prepared by dissolving 3.2 g of R2A Broth, DAIGO (manufactured by Nippon Pharmaceutical Co., Ltd.) in 1000 mL of ultrapure water and sterilizing with high-pressure steam - 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 excess sludge, the solution was mixed with 986 mL of ultrapure water to a turbidity (OD660) of 0.2 and sterilized by autoclaving. Solution A: 4.35 g dipotassium hydrogen phosphate, 1.70 g monopotassium dihydrogen phosphate, 8.92 g disodium hydrogen phosphate dodecahydrate, and 0.34 g ammonium chloride were dissolved in ultrapure water, the volume was adjusted to 200 mL, and the solution was sterilized by autoclaving. Solution B: 4.50 g magnesium sulfate heptahydrate was dissolved in ultrapure water, the volume was adjusted to 200 mL, and the solution was sterilized by autoclaving. Solution C: 5.50 g calcium chloride anhydrous was dissolved in ultrapure water, the volume was adjusted to 200 mL, and the solution was sterilized by autoclaving. Solution D: 0.05 g iron chloride hexahydrate was dissolved in ultrapure water, the volume was adjusted to 200 mL, and the solution was sterilized by filtration using 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 was completed, 50 μL of NITE BP-02779 culture solution and 5.0 mL of inorganic medium containing excess sludge (killed 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 using a simple turbidity meter (simple OD monitor miniphoto 518R, manufactured by TAITEC). The number of days until the turbidity (OD660) of the inorganic medium containing excess sludge (killed bacteria) was 50% of the turbidity (OD660) of the negative control was calculated. The negative control was the turbidity (OD660) of inorganic medium containing target bacteria (killed bacteria) without the addition of NITE BP-02779.

[0098] (Results) Addition of NITE BP-02779 reduced the turbidity of the excess sludge (dead bacteria), reaching 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 decomposition ability of excess sludge (dead bacteria) of NITE BP-02779 - 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 carried out in quintuple, and all excess sludge remaining in the test tube was collected on the fourth day after the start of the reaction. The collected excess sludge was dried and then weighed, and a significance test (one-tailed t-test) was carried out between the negative control group and the NITE BP-02779-added group.

[0101] (Results) The results are shown in Figure 2. A significant decrease (p<0.01) in the dry weight of excess sludge was observed in the NITE BP-02779-added group compared to 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 genome sequence of NITE BP-02779 was analyzed using a next-generation sequencer according to the following procedure. NITE BP-02779 was inoculated into 50 mL of R2A medium and cultured at 25°C and 130 rpm for 2 days with shaking. The resulting cells were collected by centrifugation, and genomic DNA was extracted using a genomic DNA extraction kit (QIAamp DNA Mini Kit (250), manufactured by QIAGEN). The extracted DNA was used as a template for paired-end sequencing using a next-generation sequencer, HiSeq 2500 (manufactured by Illumina). Next, adapter sequences were trimmed from the obtained base sequence, and de novo assembly was performed using Velvet. The scaffold sequences obtained by de novo assembly were analyzed, and gene region prediction and annotation were performed using Rapid Annotations using Subsystems Technology (RAST).

[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 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 resulting pre-culture was inoculated into 1.5 L of R2A medium and cultured at 25°C with shaking at 130 rpm. After 48 hours, the culture was centrifuged (4°C, 8000 × g, 10 minutes), and the resulting culture supernatant was filtered (0.20 μm). The filtered culture supernatant was concentrated using a centrifugal filter unit (Merck Millipore) and used as the starting material for enzyme purification.

[0105] (2) Fractionation by column chromatography The enzyme was fractionated and purified by column chromatography as shown in (a) to (c) below. The peptidoglycan decomposition activity of each fraction was measured using a Lysozyme activity kit (Sigma-Aldrich). The proteins contained in the obtained fractions were electrophoresed using a 4-20% Mini-Protean TGX precast gel (BioRad) and confirmed by CBB staining.

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

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

[0108] (Results) When the peptidoglycan decomposition activity was examined, the activity was found in the unadsorbed fraction, and 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 (Merck Millipore), the buffer in the unadsorbed fraction from (a) cation exchange chromatography was exchanged for 50 mM phosphate buffer (pH 5.8) containing 1 M ammonium sulfate. This sample was applied to a Hitrap HIC, 5 mL column (GE Healthcare) equilibrated with the same buffer. After washing the column with the same buffer, the adsorbed protein was eluted with a linear gradient of 1.0 to 0 M ammonium sulfate.

[0111] (Results) When the peptidoglycan decomposition activity was examined, the activity was found in the adsorbed fraction, and the fraction in which the activity was found was subjected to gel filtration chromatography.

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

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

[0114] (Results) When the peptidoglycan decomposition activity was examined, the activity was found in the eluted fraction, as shown in FIG.

[0115] (3) Identification of proteins with peptidoglycan degrading activity by mass spectrometry (Materials) Sample: Elution fraction of gel filtration chromatography

[0116] (Method) Elution fractions showing activity were concentrated using a centrifugal filter unit (Merck Millipore) and subjected to electrophoresis using a 4-20% Mini-Protean TGX precast gel (BioRad). As a result, as shown in Figure 4, the protein band contained in the elution fraction was confirmed to be a single band. (c) The purified fractions from gel filtration chromatography and the bands separated by electrophoresis were each subjected to liquid-phase or in-gel digestion using trypsin, and the digested solution was desalted. The desalted samples were subjected to Data Dependent MS / MS Acquisition (DDA) measurement using a nano LC-MS (Thermo Scientific, Q Exactive HF, nanocolumn: Acclaim PepMap RSLC C18 (Thermo Scientific)). After the measurement, a Mascot search (MS / MS ion search) was performed using Proteome Discoverer 2.1 against a protein sequence database created from the genome information of NITE BP-02779.

[0117] (Results) A search for tryptic peptides detected by mass spectrometry revealed the amino acid sequences of SEQ ID NOs: 11 to 17, which are partial sequences of ID2839. These partial sequences correspond to a part of the amino acid sequence from positions 15 to 164 of SEQ ID NO: 2. These findings suggest that protein ID2839 expressed by NITE BP-02779 has peptidoglycan decomposition 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 cleaved at any site between positions −11 and 14 of SEQ ID NO: 2 on the N-terminal side.

[0119] (Results) The partial sequence of SEQ ID NO: 18 was identified as the peptide with the highest signal intensity. The partial sequence 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 ID2839 precursor (pro-sequence). The mature amino acid sequence of ID2839 was considered to be positions 1 to 164 of SEQ ID NO: 2, 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 a signal peptide. Hereinafter, a protein consisting of the amino acid sequence of positions 1 to 164 of SEQ ID NO: 2 will be referred to as the "ID2839 mature protein," and a protein consisting of the entire amino acid sequence of SEQ ID NO: 2 (positions -68 to 164) will be referred to as the "ID2839 precursor." Cleavage of the signal peptide from the ID2839 precursor results in the ID2839 mature protein, which is thought to be secreted extracellularly and capable of degrading peptidoglycan. The molecular weight of the ID2839 precursor was approximately 25 kDa, and the molecular weight of the mature ID2839 was approximately 18 kDa.

[0120] [Experiment 7. Measurement of specific activity of ID2839 matured product] (Materials) ID2839 matured product (elution fraction of gel filtration chromatography)

[0121] (Method) The activity (units / mL) and protein content (mg / mL) of the ID2839 mature form were measured, and the specific activity (units / mg) was calculated. Two commercially available lytic enzymes (Sigma-Aldrich) were used as controls. Peptidoglycan decomposition activity was assessed by the activity of decomposing the glycosidic bond between N-acetylmuramic acid and N-acetylglucosamine using a Lysozyme activity kit (Sigma-Aldrich).

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

[0123]

[0124] [Experiment 8. Preparation of ID2839 mature form in E. coli] (1) Construction of strain expressing ID2839 mature form (Materials) Primers F and R for ID2839 cloning (SEQ ID NOs: 19 and 20) LB agar medium: LB Ager (Sigma-Aldrich) dissolved in 500 mL of ultrapure water at a ratio of 10 pieces, sterilized by high-pressure steam, and dispensed into Petri dishes

[0125] (Method) Using the genomic DNA of NITE BP-02779 as a template, PCR amplification of a region containing the nucleotide sequence encoding the ID2839 mature form (Ala at position 1 in SEQ ID NO: 2 was replaced with Met) was performed using ID2839 cloning primers F and R. PCR amplification was performed using KOD FX (Toyobo Co., Ltd.). The reaction solution was prepared according to the composition provided with the kit and the following conditions were performed: (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. Steps (2) to (4) were repeated 25 times. The resulting PCR fragment and pET-28a(+) vector (Merck Millipore) were digested with NdeI and XhoI, respectively, and ligated using Ligation High Ver. 2 (Toyobo Co., Ltd.). This ligation reaction mixture was used to transform E. E. coli DH5α was transformed with this plasmid, and the desired plasmid was extracted from a kanamycin-resistant strain. E. coli BL21(DE3) was transformed with this plasmid to obtain an expression strain for the ID2839 mature form (sometimes referred to as "BL21(DE3) / pET28a-ID2839 mature form"). The BL21(DE3) / pET28a-ID2839 mature form expresses a protein in which an amino acid sequence containing a His tag and a thrombin cleavage site (MetGlySerSerHisHisHisHisHisHisSerSerGlyLeuValProArgGlySerHis: SEQ ID NO: 21) is added to the N-terminus of the mature protein (Ala at position 1 in SEQ ID NO: 2 is replaced with Met). As a control strain, a strain that does not express the mature ID2839 (sometimes referred to as "BL21(DE3) / pET28a") was also obtained.

[0126] (2) Induction of expression of ID2839 matured body and preparation of soluble fraction (Materials) LB liquid medium: A medium prepared by dissolving 10 LB Broth 1.1 g per tablet (Sigma-Aldrich) in 500 mL of ultrapure water and sterilizing the medium with high-pressure steam. Transformant: BL21 (DE3) / pET28a - ID2839 matured body. Transformant (control): BL21 (DE3) / pET28a.

[0127] (Method) Each transformant was inoculated into 5 mL of LB medium containing 50 mg / L kanamycin and pre-cultured at 37°C. 1.0 mL of the resulting pre-culture was inoculated into 100 mL of LB medium containing 50 mg / L kanamycin, and cultured with shaking using a baffled flask. When the OD660 reached 0.6, IPTG was added (final concentration: 0.1 mM), and the culture was further cultured at 18°C ​​for 16 hours. After culture, the resulting culture was centrifuged to collect the cells, and a fixed amount of BugBuster (Novagen) was added per wet cell mass to disrupt the cells. Cell debris was removed from the disrupted solution by centrifugation, and the resulting supernatant was used as the soluble fraction. A soluble fraction containing the ID2839 matured form was obtained from the BL21(DE3) / pET28a-ID2839 matured form. A soluble fraction free of ID2839 was obtained from BL21(DE3) / pET28a.

[0128] (3) Activity evaluation of soluble fractions (Materials) Soluble fraction derived from BL21(DE3) / pET28a-ID2839 matured body Soluble fraction derived from BL21(DE3) / pET28a

[0129] (Method) The activity (units / mL) of degrading the glycosidic bond between N-acetylmuramic acid and N-acetylglucosamine was calculated using a Lysozyme activity kit (Sigma-Aldrich). The activity (units / mL) was calculated using the following formula: Activity (units / mL) of degrading the glycosidic bond between N-acetylmuramic acid and N-acetylglucosamine = {ΔABS 450 nm / min (phosphate buffer solution containing target bacteria with soluble fraction added) - ΔABS 450 nm / min (phosphate buffer solution containing target bacteria without soluble fraction added)} / (0.001 x 0.1)

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

[0131]

[0132] [Experiment 9. Identification of a Protein (ID1644) with Peptidoglycan Degradation Activity] (1) Identification from Genomic Information From the results of deciphering the genome sequence of NITE BP-02779, a gene encoding an enzyme highly identical 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 thought to be the amino acid sequence of a precursor (pro-sequence). The amino acid sequence of the mature protein is from positions 1 to 493 of SEQ ID NO: 4, and is thought to be encoded by the base sequence from positions 85 to 1563 of SEQ ID NO: 3. Positions -28 to -1 of SEQ ID NO: 4 were thought to be a signal peptide. Hereinafter, a protein consisting of the amino acid sequence of positions 1 to 493 of SEQ ID NO: 4 will be referred to as the "ID1644 mature protein," and a protein consisting of the entire amino acid sequence of SEQ ID NO: 4 (positions -28 to 493) will be referred to as the "ID1644 precursor." Cleavage of the signal peptide from the ID1644 precursor results in the ID1644 mature protein, which is thought to be secreted outside the bacterial cell and capable of degrading peptidoglycan. The molecular weight of the ID1644 precursor was approximately 56 kDa, and the molecular weight of the ID1644 mature protein was approximately 53 kDa.

[0133] [Experiment 10. Preparation of ID1644 mature form in E. coli] (1) Construction of strain expressing ID1644 mature form (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 the ID1644 mature form was performed using ID1644 cloning primers F and R. PCR amplification was performed using KOD FX (Toyobo Co., Ltd.). The reaction solution was prepared according to the composition provided with the kit and the following conditions were performed: (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. Steps (2) to (4) were repeated 25 times. The resulting PCR fragment and pET-22b(+) vector (Merck Millipore) were digested with NdeI and XhoI, respectively, and ligated using Ligation High Ver. 2 (Toyobo Co., Ltd.). This ligation reaction mixture was used to transform E. E. coli DH5α was transformed with the plasmid, and the desired plasmid was extracted from an ampicillin-resistant strain. E. coli BL21(DE3) was transformed with the plasmid to obtain a strain expressing the ID1644 mature form (sometimes referred to as "BL21(DE3) / pET22b-ID1644 mature form"). In the BL21(DE3) / pET22b-ID1644 mature form, a protein with a 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 the ID1644 mature form (sometimes referred to as "BL21(DE3) / pET22b") was also obtained.

[0135] (2) Induction of expression of ID1644 matured strain and preparation of soluble fraction (Materials) LB liquid medium Transformant: BL21(DE3) / pET22b-ID1644 matured strain Transformant (control): BL21(DE3) / pET22b

[0136] (Method) Each transformant was inoculated into 5 mL of LB medium containing 100 mg / L ampicillin and pre-cultured at 37°C. 1.0 mL of the resulting pre-culture was inoculated into 100 mL of LB medium containing 100 mg / L ampicillin, and cultured with shaking using a baffled flask. When the OD660 reached 0.6, IPTG was added (final concentration: 0.1 mM), and the culture was further cultured at 18°C ​​for 16 hours. After culture, the resulting culture was centrifuged to collect the bacterial cells, and a fixed amount of BugBuster (Novagen) was added per wet bacterial cell mass to disrupt the bacterial cells. The bacterial cell debris was removed from the disrupted solution by centrifugation, and the resulting supernatant was used as the soluble fraction. A soluble fraction containing the ID1644 matured form was obtained from the BL21(DE3) / pET22b-ID1644 matured form. A soluble fraction containing no ID1644 matured form was obtained from BL21(DE3) / pET22b.

[0137] (3) Activity Evaluation of Soluble Fractions-1 (Materials) Soluble fraction derived from BL21(DE3) / pET22b-ID1644 matured body Soluble fraction derived from BL21(DE3) / pET22b Peptidoglycan solution: a solution prepared by dissolving 10 mg of peptidoglycan (Sigma-Aldrich) 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 the OD450 was measured over time to calculate the peptidoglycan degradation activity (Units / mL). The peptidoglycan degradation activity (UNITS / mL) was calculated using the following formula: Peptidoglycan degradation activity (UNITS / mL) = {ΔABS450 nm / min (peptidoglycan-containing phosphate buffer solution with soluble fraction added) - ΔABS450 nm / min (peptidoglycan-containing phosphate buffer solution without soluble fraction added)} / (0.001 × 0.05)

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

[0140]

[0141] (4) Activity Evaluation of Soluble Fractions-2 (Materials) Soluble fraction derived from BL21(DE3) / pET22b-ID1644 matured cell Soluble fraction derived from BL21(DE3) / pET22b Substrate for N-acetylmuramoyl-L-alanine amidase: L-aniline-p-nitroanilide hydrochloride (Sigma-Aldrich)

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

[0143] (Results) The results are shown in Table 8. The soluble fraction derived from BL21(DE3) / pET22b-ID1644 matured strain showed a higher ABS 405 nm value than the soluble fraction derived from BL21(DE3) / pET22b. It was found that the ID1644 matured strain had N-acetylmuramoyl-L-alanine amidase activity.

[0144]

[0145] [Experiment 11. Preparation of ID2839 matured body-2 in E. coli] (1) Construction of ID2839 matured body-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 a region containing the nucleotide sequence encoding ID2839 mature form-2 (a protein consisting of the amino acid sequence of positions 1 to 164 of SEQ ID NO: 2, with Met added to the N-terminus of Ala at position 1) was performed using ID2839 cloning primers (SEQ ID NOs: 24 and 25). PCR amplification was performed using a KOD plus NEO (manufactured by Toyobo Co., Ltd.). The reaction solution was prepared according to the composition provided with the kit, and the reaction was carried out at (1) 94°C for 2 minutes, (2) 98°C for 10 seconds, and (3) 68°C for 30 seconds, with steps (2) and (3) repeated 30 cycles. The resulting PCR fragment was ligated to a fragment obtained by digesting pET-28a(+) vector (Merck Millipore) with NcoI and XhoI using an In-Fusion HD Cloning Kit (Takara Bio). E. coli DH5α was transformed with this ligation reaction solution, and the target plasmid was extracted from a kanamycin-resistant strain. E. coli BL21(DE3) was transformed with this plasmid to obtain an expression strain of ID2839 mature form-2 (sometimes referred to as "BL21(DE3) / pET28a-ID2839 mature form-2"). BL21(DE3) / pET28a-ID2839 mature form-2 expresses a mature protein (a protein consisting of an amino acid sequence consisting of positions 1 to 164 of SEQ ID NO: 2, with Met added to the N-terminus of Ala at position 1).

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

[0148] (Method) Each transformed 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 resulting pre-culture solution was inoculated into 100 mL of LB medium containing 50 mg / L of kanamycin and cultured with shaking using a baffled flask. When the OD660 reached 0.6, IPTG was added (final concentration: 1 mM), and the culture was further continued at 24°C for 20 hours. After completion of the culture, the soluble fraction of the bacterial cells was obtained using the same method as in Experiment 8.

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

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

[0151] (Results) The results are shown in Table 9. The soluble fraction of BL21(DE3) / pET28a-ID2839 matured form-2 was found to have peptidoglycan decomposition activity.

[0152]

[0153] [Experiment 12. Preparation of ID2839 matured form-3 in E. coli] (1) Construction of ID2839 matured form-3 expression strain (Materials) ID2839 cloning primers F and R (SEQ ID NOs: 26 and 25) LB agar medium

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

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

[0156] (Method) A soluble fraction of the cells was obtained in the same manner as in Experiment 11.

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

[0158] (Method) The experiment was carried out in the same manner as in Experiment 8.

[0159] (Results) The results are shown in Table 10. The soluble fraction derived from BL21(DE3) / pET28a-ID2839 matured form-3 was found to have peptidoglycan decomposition activity.

[0160]

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. 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 claims 1 to 3, wherein the peptidoglycan-degrading activity is an activity of degrading the 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 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. Any of the following proteins (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. Any of the following proteins (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 of 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 its culture.

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 its culture to act on a target microorganism.