Protein having peptidoglycan-decomposing activity, dna encoding the protein, microbial decomposition preparation, and microbial decomposition method

Abstract

Provided herein is a protein from Tumebacillus sp. NITE BP-02779 having a peptidoglycan-decomposing activity. Provided herein is a protein comprising the amino acid sequence of SEQ ID NO: 2 (1-164) or the amino acid sequence of SEQ ID NO: 4 (1-493), or a protein comprising an amino acid sequence in which 1 to 10 amino acid residues of these amino acid sequences are substituted, deleted, inserted, or added, or an amino acid sequence having 90% or more identity to these amino acid sequences, and having a peptidoglycan-decomposing activity.

Description

Technical Field

[0001] This invention relates to proteins with peptidoglycan-degrading activity, the DNA encoding such proteins, microbial degrading agents, and microbial degrading methods. Background Technology

[0002] When wastewater is purified using the activated sludge process, the removed organic matter becomes flocculent material containing microorganisms (bacteria), producing sludge known as residual sludge. Residual sludge discharged from wastewater treatment plants accounts for as much as 20% or more of industrial waste. Typically, residual sludge is dewatered, dried, and then incinerated (Non-Patent Literature 1: Fiscal Year 30 Survey Report on the Discharge and Treatment of Industrial and Commercial Waste, Fiscal Year 28 (Summary Version); Non-Patent Literature 2: Masayuki Yamamoto, “On Wastewater Combustion Technology,” Journal of the Japan Society for Combustion, Japan Society for Combustion, 2011, Vol. 53, No. 164, pp. 91-96).

[0003] Existing technical documents

[0004] Non-patent literature

[0005] Non-patent document 1: "Investigation Report on the Discharge and Treatment of Industrial and Commercial Waste in Fiscal Year 30 (Heisei 30), Performance in Fiscal Year 28 (Summary Version)" [Online], March 31 (Heisei 31), Waste Regulation Division, Environmental Regeneration and Resource Recycling Bureau, Ministry of the Environment [Searched on March 17, Reiwa 2], Internet <URL:https: / / www.e-stat.go.jp / stat-search / file-download?statInfId=000031887949&fileKind=2>

[0006] Non-Patent Literature 2: Masayuki Yamamoto, “On Combustion Technology for Wastewater”, Journal of the Japan Society for Combustion, 2011, Vol. 53, No. 164, pp. 91-96 Summary of the Invention

[0007] The problem that the invention aims to solve

[0008] However, incinerating waste sludge produces greenhouse gases, therefore, for environmental reasons, it is necessary to reduce the volume of waste sludge. As a method for reducing the volume of waste sludge, a method for decomposing the microorganisms that constitute the waste sludge has been proposed.

[0009] The purpose of this invention is to provide a novel protein capable of decomposing peptidoglycan, the DNA encoding the protein, a microbial decomposition agent, and a microbial decomposition method.

[0010] Methods for solving problems

[0011] The present invention relates to the following examples [1] to

[11] .

[0012] [1] A protein derived from a species of the genus *Tumebacillus*, NITE BP-02779, and possessing peptidoglycan-degrading activity.

[0013] [2] According to the protein described in [1], it is a secretory protein.

[0014] [3] A protein, which is any one of the following (A1) to (A3),

[0015] (A1) A protein containing the amino acid sequence from position 1 to 164 of sequence number 2 and possessing peptidoglycan-degrading activity.

[0016] (A2) A protein containing an amino acid sequence in which 1 to 10 amino acid residues in positions 1 to 164 of sequence number 2 are substituted, deleted, inserted, or added, and which exhibits peptidoglycan-degrading activity.

[0017] (A3) A protein containing an amino acid sequence that is more than 90% identical to the amino acid sequence of positions 1-164 relative to sequence number 2 and has peptidoglycan decomposition activity.

[0018] [4] The protein according to any one of [1] to [3], wherein,

[0019] The aforementioned peptidoglycan decomposition activity refers to the activity of breaking down the glycosidic bond between N-acetylmuramic acid and N-acetylglucosamine.

[0020] [5] A protein, which is any one of the following (a1) to (a4),

[0021] (a1) A protein composed of amino acid sequences 1-164 of sequence number 2.

[0022] (a2) A protein composed of amino acids in sequence number 2, where amino acid 1 (Ala) is replaced by Met.

[0023] (a3) A protein composed of the amino acid sequence of sequence number 2, where position 1 (Ala) is replaced by Met, and the amino acid sequence of sequence number 21 is added to the N-terminus of the Met.

[0024] (a4) A protein consisting of an amino acid sequence consisting of the amino acid sequence of positions 1-164 of sequence number 2, with the amino acid sequence of position 1 having ...

[0025] [6] A protein, which is any one of the following (B1) to (B3),

[0026] (B1) A protein containing the amino acid sequence 1-493 of sequence number 4 (SEQ ID No: 4) and possessing peptidoglycan-degrading activity.

[0027] (B2) A protein containing an amino acid sequence in which 1 to 10 amino acid residues in positions 1 to 493 of sequence number 4 are substituted, deleted, inserted, or added, and which has peptidoglycan-degrading activity.

[0028] (B3) A protein containing an amino acid sequence that is more than 90% identical to the amino acid sequence of positions 1 to 493 relative to sequence number 4, and having peptidoglycan decomposition activity.

[0029] [7] The protein according to [1], [2] or [6], wherein,

[0030] The above-mentioned peptidoglycan degradation activity is N-acetylmuracil-L-alanine amidase activity.

[0031] [8] A protein, which is any one of the following (b1) to (b4),

[0032] (b1) A protein composed of amino acid sequences 1-493 of sequence number 4.

[0033] (b2) A protein composed of amino acids in sequence number 4, where Glu at position 1 is replaced by Met.

[0034] (b3) A protein composed of Glu at position 1 of amino acid sequence 4 (1-493) being replaced by Met, and the amino acid sequence of sequence number 21 being added to the N-terminus of the Met.

[0035] (b4) A protein consisting of an amino acid sequence consisting of amino acid sequence 1-493 of sequence number 4 with the amino acid sequence of Met added to the N-terminus of Glu at position 1.

[0036] [9] A DNA that encodes any one of the proteins described in [1] to [8].

[0037]

[10] A vector comprising the DNA described in [9].

[0038]

[11] A transformant comprising the DNA described in [9] or the vector described in

[10] .

[0039]

[12] A microbial decomposition agent comprising at least one protein selected from any one of [1] to [8], and the transformant and its culture described in

[11] .

[0040]

[13] A method for microbial decomposition, comprising a step of acting on a target microorganism with at least one of the proteins selected from any one of [1] to [8] and the transformants and cultures of

[11] .

[0041] Invention Effects

[0042] According to the present invention, it is possible to provide a novel protein capable of decomposing peptidoglycan, the DNA encoding the protein, a microbial decomposition agent, and a microbial decomposition method.

[0043] Brief description of the attached diagram

[0044] [ Figure 1 This diagram illustrates the structure of a representative peptidoglycan and the sites that can be cleaved by peptidoglycan-degrading enzymes.

[0045] [ Figure 2 The figure shows the dry weight of the remaining sludge after adding Tumebacillus sp. NITEBP-02779 in Experiment 4.

[0046] [ Figure 3 [Graph showing the fractionation of the culture supernatant of Tumebacillus sp. NITE BP-02779 in Experiment 6 using column chromatography and the examination of peptidoglycan degradation activity.]

[0047] [ Figure 4 [Image showing the fractionation of the culture supernatant of Bacillus sp. NITE BP-02779 in Experiment 6 using column chromatography and the detection of proteins in the elution fractions by electrophoresis.]

[0048] Detailed Implementation of the Invention

[0049] The following provides a detailed description of the methods for implementing the present invention. It should be noted that the present invention is not limited to the following embodiments.

[0050] [Protein from Tumebacillus sp. NITE BP-02779]

[0051] One embodiment of the present invention relates to a protein derived from a species of *Tumebacillus* sp., NITE BP-02779 (hereinafter sometimes referred to as "NITE BP-02779"), and possessing peptidoglycan-degrading activity. The protein can be synthetic, purified, or isolated. Whether a protein is the protein involved in this invention can be determined based on the results of the protein identification method and the peptidoglycan-degrading activity determination method described below. Proteins derived from NITE BP-02779 and possessing peptidoglycan-degrading activity can reduce the volume of excess sludge because they can decompose target microorganisms.

[0052] NITE BP-02779 is a bacterium that was internationally deposited on September 11, 2018, at the Patent Microbial Collection Center of the National Institute for Technical Evaluation (Room 122, 2-5-8, Kazusa-kama-zu, Kisarazu City, Chiba Prefecture, Japan 292-0818), and a certificate of deposit and a certificate of viability were issued on September 26, 2018. NITE BP-02779 is considered a new species of *Tumebacillus*. NITE BP-02779 can decompose various target microorganisms such as *Bacillus*, *Micrococcus*, and *Staphylococcus*, as well as residual sludge, and is therefore useful for treating residual sludge. The bacteriological properties of NITE BP-02779 are shown in Tables 1-4 of the examples described below. NITEBP-02779 is a bacterium that possesses a 16S rRNA gene containing the base sequence described in sequence number 10.

[0053] "From NITE BP-02779" can refer to a substance synthesized or generated from NITE BP-02779, or it can refer to a substance synthesized or generated artificially or within the cells of other organisms based on the genome sequence of NITE BP-02779. Examples of substances derived from NITE BP-02779 include biological molecules and metabolites such as proteins, nucleic acids, and sugars. These substances can exist locally within the cell or be secreted extracellularly.

[0054] One method for checking whether a protein originates from NITE BP-02779 is as follows: First, the genome sequence of NITE BP-02779 is interpreted using a next-generation sequencer. Adapter sequences are trimmed from the obtained sequence, and de novo sequencing assembly is performed. The scaffold sequence obtained through de novo sequencing is analyzed to predict and annotate gene regions, creating a protein database for quality analysis. Next, the target protein undergoes quality analysis and is compared with the aforementioned protein database. This allows determination of whether the target protein originates from NITE BP-02779.

[0055] As another method to check whether a protein is from NITE BP-02779, the amino acid sequence of the target protein can be interpreted using an amino acid sequencer and compared with the aforementioned protein database.

[0056] Proteins derived from NITE BP-02779 and possessing peptidoglycan-degrading activity are preferred, especially secretory proteins. Secretory proteins are capable of functioning extracellularly. For reducing the volume of excess sludge, it is useful for proteins with peptidoglycan-degrading activity to exhibit this activity in an extracellular environment. Secretory proteins with peptidoglycan-degrading activity can be recovered from the culture supernatant of the cells that produce the protein, or by destroying the cells that produce the protein.

[0057] Peptidoglycans are molecules composed of polysaccharides with alternating β-1,4 bonds of N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc) linked by oligopeptides. Figure 1 The diagram shows the structure of a representative peptidoglycan. "Peptidoglycan degradative activity" refers to the state of a reaction that promotes the conversion of peptidoglycan into two or more substances. "Proteins with peptidoglycan degradative activity" (hereinafter, sometimes referred to as "peptidoglycan degradative enzymes") are proteins that promote the above-mentioned reactions.

[0058] Peptidoglycans are effectively broken down through the glycosidic bonds between sugars that make up polysaccharides and the peptide bonds within oligopeptides. Figure 1 Arrows indicate the sites that can be broken down by peptidoglycan-degrading enzymes. Examples of proteins with peptidoglycan-degrading activity include: (1) proteins with activity in breaking down the glycosidic bond between N-acetylglucosamine and N-acetylmuramic acid; (2) proteins with activity in breaking down the glycosidic bond between N-acetylmuramic acid and N-acetylglucosamine; (3) proteins with activity in breaking down the amide bond between N-acetylmuramic acid and L-alanine; and (4) proteins with activity in breaking down various peptide bonds.

[0059] Specifically, as (1), N-acetylglucosidase can be cited; as (2), N-acetylmuraminase and lysozyme can be cited; as (3), N-acetylmuramin-L-alanine amidase can be cited; and as (4), L,D-endopeptidase, D,L-endopeptidase, carboxypeptidase, and D,D-endopeptidase can be cited.

[0060] One method for checking whether a protein exhibits peptidoglycan-degrading activity is to react the target protein with peptidoglycan in a buffer solution for a certain period of time and then measure the degradation of the peptidoglycan. There are no particular limitations on the method for checking degradation; examples include methods for measuring the turbidity of peptidoglycan, methods for detecting peptidoglycan using SLP reagents, and methods for detecting degradation products from peptidoglycan using high-performance liquid chromatography (HPLC), mass spectrometry (MS), thin-layer chromatography (TLC), nuclear magnetic resonance (NMR), gas chromatography (GC), etc.

[0061] As a method for checking whether a protein possesses peptidoglycan-degrading activity (2) involving the breakdown of the glycosidic bond between N-acetylmuramic acid and N-acetylglucosamine, one example is the use of *Micrococcus lysodeikticus* as a substrate. Specifically, *Micrococcus lysodeikticus* culture cells can be used as the substrate, or lysozyme activity kits (Sigma-Aldrich) and EnzChek lysozyme assay kits (ThermoFisher Scientific) can be used.

[0062] As a method for examining the amidase activity of (3) hydrolysis of the amide bond between N-acetylmuramic acid and L-alanine in peptidoglycan degradation activity, an example is the use of L-aniline-p-nitroaniline hydrochloride (manufactured by Sigma-Aldrich) as a matrix for N-acetylmuramic acid-L-alanine amidase, as described in Appl Microbiol Biotechnol. 2015, Oct, 99(20):8563-73.

[0063] [The protein described by sequence number 2 or 4]

[0064] The protein involved in one embodiment of the present invention is any of the following proteins.

[0065] (A1) A protein containing the amino acid sequence from position 1 to 164 of sequence number 2 and possessing peptidoglycan-degrading activity.

[0066] (B1) A protein containing the amino acid sequence from position 1 to 493 of sequence number 4 and possessing peptidoglycan-degrading activity.

[0067] The amino acid sequences 1-164 of sequence number 2 and 1-493 of sequence number 4 are the amino acid sequences of the mature protein predicted from the genomic sequence of NITE BP-02779. Whether a protein possesses peptidoglycan-degrading activity can be determined using methods such as those described above. Proteins with peptidoglycan-degrading activity can reduce the volume of excess sludge because they can decompose target microorganisms.

[0068] One embodiment of the present invention relates to a protein that, as long as it maintains peptidoglycan-degrading activity, can be a variant of a protein having amino acid sequences 1-164 of sequence number 2 or amino acid sequences 1-493 of sequence number 4. Variants that maintain peptidoglycan-degrading activity are sometimes referred to as "conserved variants." In the present invention, proteins determined to have peptidoglycan-degrading activity include, in addition to the proteins described above, their conserved variants and fusion proteins of proteins with other peptides.

[0069] The following are examples of conserved variants of peptidoglycan-degrading enzymes.

[0070] One embodiment of the present invention relates to a protein that, as long as it maintains peptidoglycan degradation activity, can be a protein having an amino acid sequence in which one or more amino acid residues in amino acid sequences 1-164 of sequence number 2 or 1-493 of sequence number 4 are substituted, deleted, inserted, or added. "One or more" varies depending on the position and type of amino acid residues in the protein's three-dimensional structure, and can be, for example, 1-10, 1-8, 1-5, or 1-3.

[0071] One embodiment of the present invention relates to a protein that only needs to maintain peptidoglycan degradation activity. It may be a protein having the following amino acid sequence, wherein the amino acid sequence has high identity with the amino acid sequence of sequence number 2 (positions 1-164) or sequence number 4 (positions 1-493), for example, more than 90%, more than 93%, more than 95%, more than 97%, or more than 99% identity.

[0072] The aforementioned variations in amino acid residues are conserved variations that maintain the normal function of proteins. Representative conserved variations are conserved substitutions. Conserved substitutions refer to the following substitution variations: for aromatic amino acids, between Phe, Trp, and Tyr; for hydrophobic amino acids, between Leu, Ile, and Val; for polar amino acids, between Gln and Asn; for basic amino acids, between Lys, Arg, and His; for acidic amino acids, between Asp and Glu; and for amino acids with a hydroxyl group, between Ser and Thr. As substitutions considered conservative, examples include substitutions from Ala to Ser or Thr, from Arg to Gln, His, or Lys, from Asn to Glu, Gln, Lys, His, or Asp, from Asp to Asn, Glu, or Gln, from Cys to Ser or Ala, from Gln to Asn, Glu, Lys, His, Asp, or Arg, from Glu to Gly, Asn, Gln, Lys, or Asp, from Gly to Pro, and from His to Asn, Lys, Gln, Arg, or Tyr. Substitutions include substitutions from Ile to Leu, Met, Val, or Phe; substitutions from Leu to Ile, Met, Val, or Phe; substitutions from Lys to Asn, Glu, Gln, His, or Arg; substitutions from Met to Ile, Leu, Val, or Phe; substitutions from Phe to Trp, Tyr, Met, Ile, or Leu; substitutions from Ser to Thr or Ala; substitutions from Thr to Ser or Ala; substitutions from Trp to Phe or Tyr; substitutions from Tyr to His, Phe, or Trp; and substitutions from Val to Met, Ile, or Leu. Variations in amino acid residues also include variations arising from naturally occurring variations (mutants or variants) based on individual differences, species differences, etc., in the organism from which the protein originates.

[0073] One embodiment of the present invention relates to a protein that can be a fusion protein with other peptides. Examples of other peptides include labeled peptides (labeled proteins), peptide tags, and pro or prepro sequences, such as secretory signal peptides. The peptide linked to the peptidoglycanase can be a single peptide or two or more peptides. When the peptidoglycanase is a fusion protein with a signal peptide, after expression in a secretory host cell, the signal peptide is cleaved, and only the peptidoglycanase portion (mature protein) can be secreted extracellularly. When the peptidoglycanase is a fusion protein, the aforementioned identity refers to the identity of the mature protein portion other than the other peptide portions.

[0074] As a labeling peptide, any peptide that can function as a label is acceptable, without particular limitations. Examples include alkaline phosphatase, the Fc region of an antibody, HRP, and GFP. As a peptide tag, there are no particular limitations; previously known peptide tags such as Myc tags, His tags, FLAG tags, and GST tags can be used. As a secretion signal peptide, in addition to the secretion signal peptide of the peptidoglycan-degrading enzyme itself present in NITE BP-02779, secretion signal peptides that function in hosts expressing peptidoglycan-degrading enzymes can also be used. The fusion protein can be prepared using conventional methods.

[0075] The protein involved in one embodiment of the present invention may be any one of the following proteins (a1) to (a4).

[0076] (a1) A protein composed of amino acid sequences 1-164 of sequence number 2.

[0077] (a2) A protein composed of amino acids in sequence number 2, where amino acid 1 (Ala) is replaced by Met.

[0078] (a3) A protein composed of the amino acid sequence of MetGlySerSerHisHisHisHisHisHisHisHisHisGlyLeuValProArgGlySerHis (Sequence No. 21) consisting of the amino acid sequence of the first amino acid in sequence No. 1 to 164 of sequence No. 2 being replaced by Met, and the amino acid sequence of MetGlySerHisHisHisHisHisHisHisHisHisHisGlyLeuValProArg ...

[0079] (a4) A protein consisting of an amino acid sequence consisting of the amino acid sequence of positions 1-164 of sequence number 2, with the amino acid sequence of position 1 having ...

[0080] The protein involved in one embodiment of the present invention may be any one of the following proteins (b1) to (b4).

[0081] (b1) A protein composed of amino acid sequences 1-493 of sequence number 4.

[0082] (b2) A protein composed of amino acids in sequence number 4, where Glu at position 1 is replaced by Met.

[0083] (b3) A protein composed of Glu at position 1 of amino acid sequence 4 (1-493) being replaced by Met, and the amino acid sequence of sequence number 21 being added to the N-terminus of the Met.

[0084] (b4) A protein consisting of an amino acid sequence consisting of amino acid sequence 1-493 of sequence number 4 with the amino acid sequence of Met added to the N-terminus of Glu at position 1.

[0085] For a protein composed of amino acid sequences 1 to 164 of sequence number 2, it is considered to have the activity of breaking the glycosidic bond between N-acetylmuramic acid and N-acetylglucosamine in the above-mentioned peptidoglycan decomposition activity (2). (A1) A protein containing amino acid sequences 1 to 164 of sequence number 2 and having peptidoglycan decomposition activity, (A2) A protein containing an amino acid sequence in which 1 to 10 amino acid residues in amino acid sequences 1 to 164 of sequence number 2 are substituted, deleted, inserted or added, and having peptidoglycan decomposition activity, (A3) A protein containing an amino acid sequence that has more than 90% identity with the amino acid sequence in sequences 1 to 164 of sequence number 2 and having peptidoglycan decomposition activity, preferably having the activity of breaking the glycosidic bond between N-acetylmuramic acid and N-acetylglucosamine in the above-mentioned peptidoglycan decomposition activity (2).

[0086] For a protein composed of amino acid sequences 1 to 493 of sequence number 4, it is considered to have the (3) N-acetylmurayl-L-alanine amidase activity in the peptidoglycan decomposition activity described above. (B1) A protein containing amino acid sequences 1 to 493 of sequence number 4 and having peptidoglycan decomposition activity; (B2) A protein containing amino acid sequences in which 1 to 10 amino acid residues in amino acid sequences 1 to 493 of sequence number 4 are substituted, deleted, inserted, or added, and having peptidoglycan decomposition activity; (B3) A protein containing amino acid sequences that are more than 90% identical to amino acid sequences 1 to 493 of sequence number 4 and having peptidoglycan decomposition activity, preferably having the (3) N-acetylmurayl-L-alanine amidase activity in the peptidoglycan decomposition activity described above.

[0087] The proteins involved in (A1)~(A3) and (B1)~(B3) may be synthetic proteins or purified or isolated proteins. The proteins involved in (A1)~(A3) and (B1)~(B3) may be proteins derived from NITE BP-02779 or proteins that are artificially synthesized or generated in the cells of NITE BP-02779 or other organisms.

[0088] DNA encoding peptidoglycan-degrading enzymes

[0089] The DNA involved in this invention is DNA encoding the aforementioned proteins. Specifically, examples include DNA encoding amino acid sequences 1-164 of sequence number 2, DNA encoding amino acid sequences 1-493 of sequence number 4, and DNA encoding conserved variants of proteins having these amino acid sequences. In one embodiment of this invention, the DNA may be DNA having a base sequence of 205-696 of sequence number 1 or a base sequence of 85-1563 of sequence number 3. Sequence numbers 1 and 3 are base sequences based on the NITEBP-02779 genome sequence, encoding mature proteins with peptidoglycan-degrading activity. The DNA involved in this invention may contain a start codon and / or a base sequence encoding other peptides at the 5' end of the aforementioned base sequences, and may also contain a stop codon at the 3' end of the aforementioned base sequences. The DNA may be recombinant DNA. The DNA may be synthetic DNA or complementary DNA (cDNA). The DNA may be purified or isolated DNA.

[0090] The DNA involved in this invention is not limited to the DNA consisting of the sequence present in NITE BP-02779, but can be DNA containing base sequences in the coding region in which codons encoding each amino acid are replaced with other equivalent codons encoding the same amino acid. In one embodiment of the invention, the DNA may also be DNA containing base sequences in which the codon usage is altered in a manner that enhances the expression of proteins with peptidoglycan-degrading activity.

[0091] One embodiment of the present invention relates to DNA that further includes DNA encoding a protein with peptidoglycan-degrading activity, which can hybridize under stringent conditions with a probe having a base sequence complementary to the aforementioned base sequence or a probe that can be prepared from the aforementioned complementary base sequence. The stringent conditions refer to conditions under which so-called specific hybridization occurs, and non-specific hybridization does not occur. As an example, it is possible to exemplify conditions where DNAs with high identity have, for example, 50% or more, 65% or more, 80% or more, preferably 90% or more, more preferably 95% or more, further preferably 97% or more, particularly preferably 99% or more identity with each other, while DNAs with lower identity do not hybridize with each other. Alternatively, it can be conditions where washing is performed once, preferably two to three times, at a salt concentration and temperature equivalent to that of typical Southern hybridization: 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. Additionally, when using DNA fragments of approximately 300 bp as probes, washing conditions for hybridization could include 50°C, 2×SSC, and 0.1% SDS.

[0092] The proportion of identity between two sequences can be determined using, for example, mathematical algorithms. 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 sequence alignment algorithm described in Needleman and Wunsch (1970) J. Mol. Biol. 48:443-453; the similarity retrieval method described in Pearson and Lipman (1988) Proc. Natl. Acad. Sci. 85:2444-2448; and the improved algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA, 90:587-5877.

[0093] Using programs based on these mathematical algorithms, sequence alignment can be performed to determine sequence identity. These programs can be executed by a computer. Examples of such programs are not particularly limited, but include the PC / Gene program CLUSTAL (available from Intelligenetics, Mountain View, Calif.), the ALIGN program (Version 2.0), and GAP, BESTFIT, BLAST, FASTA, and TFASTA from the Wisconsin Genetics Software Package, Version 8 (available from GeneticsComputer Group (GCG), 575 Science Drive, Madison, Wis., USA). Sequence alignment using these programs can be performed using, for example, initial parameters. The CLUSTAL procedure is well documented 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.

[0094] To obtain a base sequence identical to the base sequence encoding the target protein, a BLAST nucleotide search can be performed using the BLASTN program with a score of 100 and a word length of 12. To obtain an amino acid sequence identical to the target protein, a BLAST protein search can be performed using the BLASTX program with a score of 50 and a word length of 3. For information on BLAST nucleotide and BLAST protein searches, refer to http: / / www.ncbi.nlm.nih.gov. To obtain sequence alignments with gaps added for comparison purposes, Gapped BLAST (BLAST 2.0) can be used. To perform repetitive searches to detect distant relationships between sequences, PSI-BLAST (BLAST 2.0) can be used. For information on 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 for each program (e.g., BLASTN for base sequences, BLASTX for amino acid sequences) can be used. Sequence alignment can be performed manually.

[0095] The identity between two sequences is calculated as the ratio of identical residues between the two sequences when they are arranged in the most consistent manner.

[0096] The DNA involved in this invention can be obtained through chemical synthesis or by using PCR or other methods with NITE BP-02779.

[0097] "Carrier"

[0098] The vector involved in this invention contains the aforementioned DNA. The vector is a nucleic acid molecule capable of amplifying and maintaining DNA, and can be an expression vector or a cloning vector. The vector involved in this invention can be constructed by integrating the aforementioned DNA into a basic vector based on conventional genetic engineering methods. The basic vector can replicate autonomously, for example, in a host cell, can be isolated and purified from the host cell, and has a detectable marker. The DNA involved in this invention is inserted into an expression vector, for example, that can be used in a host cell into which the gene is introduced. By being introduced into the host cell, the vector is able to express a protein with peptidoglycan-degrading activity in the host cell.

[0099] Basic vectors can be, for example, vectors derived from bacterial plasmids, vectors derived from yeast plasmids, viral vectors, granular vectors, phage vectors, and artificial chromosome vectors. Examples of basic vectors include pBR322, pUC plasmid vectors, and pET lineage plasmid vectors. Specifically, when using *E. coli* as the host cell, examples include vectors pUC19, pUC18, and pUC119 (manufactured by Takara Shuzo), phage vector pBluescriptII (manufactured by Stratagene), pET28a(+) vector, and pET22b(+) vector (Merck Millipore). When using budding yeast as the host cell, examples include vectors pGBT9, pGAD424, and pACT2 (manufactured by Clontech). When using mammalian cells as host cells, examples include vectors such as pRc / RSV and pRc / CMV (manufactured by Invitrogen), bovine papillomavirus vector pBPV (manufactured by Amersham Pharmacia Biotech), Epstein-Barr virus vector pCEP4 (manufactured by Invitrogen), vaccinia virus vectors, retroviral vectors, lentiviral vectors, adenovirus vectors, and adeno-associated virus vectors. When using insect cells as host cells, examples include baculovirus vectors.

[0100] When the vector involved in this invention is constructed using a basic vector having an autonomous origin of replication (ori), the vector remains in the cell as a free organism upon introduction into the host cell. When a vector integrating SV40 and ori is introduced into, for example, COS cells transformed with an SV40 genome lacking ori, the copy number of the vector can be significantly increased within the cell.

[0101] Expression vectors can contain promoter and terminator sequences for expressing the integrated gene. There are no particular restrictions as long as the promoter functions in the host cell. A "functional promoter" refers to a promoter that has promoter activity in the host and can control the expression of the integrated gene. Typically, the DNA sequence integrated into the basic vector is inserted downstream of the promoter in a functional state. For example, a cloning site may be located downstream of the promoter sequence in the basic vector. The basic vector may contain a selection marker sequence.

[0102] Promoters can be derived from the host or from a different species. They can be intrinsic promoters of peptidoglycan-degrading enzyme genes or promoters of other genes. When the host cell is *E. coli*, examples of promoters include the *lacP* promoter of the *E. coli* lactose operon, the *trpP* promoter of the *tryptophan* operon, the *argP* promoter of the *arginine* operon, the *galP* promoter of the *galactose* operon, the *tac* promoter, the T7 promoter, the T3 promoter, and the promoters of *λ* phage (λ-pL, λ-pR). When the host cell is animal cell or dividing yeast, examples of promoters include the *Rouse sarcoma virus* (RSV) promoter, the *CMV* promoter, the early or late promoters of *simian virus* (SV40), and the *MMTV* promoter. When the host cell is budding yeast, examples of promoters include the ADH1 promoter. The ADH1 promoter can be prepared from, for example, the yeast expression vector pAAH5, which retains both the ADH1 promoter and the same terminator [available from the Washington Research Fund, Ammerer et al., Method in Enzymology, 10 1 part (p. 192-201)] using conventional genetic engineering methods. The vector pACT2, which contains the ADH1 promoter, can express the target gene in large quantities in budding yeasts such as CG1945 (manufactured by Clontech) by inserting the target gene downstream of the ADH1 promoter.

[0103] [Transformation]

[0104] The transformants of this invention comprise the DNA or vector described above. Transformants comprising the DNA or vector described above are capable of expressing proteins with peptidoglycan-degrading activity.

[0105] The host cell for introducing the DNA or vector involved in this invention can be a eukaryotic or prokaryotic cell, such as bacterial, fungal, plant, animal, or insect cells. Escherichia coli is preferred as the host cell. By introducing the DNA or vector into the host cell, the host cell can be transformed, and a transformant can be produced.

[0106] The DNA or vector involved in this invention can be maintained outside the chromosome within the host cell or integrated into the chromosome. In a transformant, the DNA or vector involved in this invention is preferably maintained in a gene-expressing state under the control of a promoter that functions within the host cell.

[0107] For the method of introducing the DNA or vector involved in this invention into a host cell, any commonly used introduction method suitable for the host cell can be applied. When using *E. coli* as the host cell, gene introduction methods such as the calcium chloride method and electroporation method described in *Molecular Cloning 2nd Edition*, published by Cold Spring Harbor Laboratory (1989), can be cited. When using mammalian or insect cells as the host cell, gene introduction methods such as the calcium phosphate method, DEAE dextran method, electroporation method, and lipid transfection method can be cited. When using yeast as the host cell, gene introduction methods such as the lithium method used in yeast transformation kits (manufactured by Clontech) can be cited. When a virus is used as a vector, in addition to using the above-mentioned gene introduction method to introduce the genome of a virus containing the DNA of the present invention into a host cell, the DNA of the present invention can also be introduced into a host cell by infecting the host cell with viral particles containing the genome of the virus.

[0108] When screening transformants introduced with the DNA or vector of the present invention, a selection marker can be used. For example, the DNA or vector of the present invention and a selection marker gene can be simultaneously introduced into host cells, and the cells can be cultured using a method that conforms to the properties of the selection marker. For example, if the selection marker gene is a gene that confers resistance to a screening agent that exhibits lethal activity against the host cell, host cells introduced with the DNA or vector of the present invention can be cultured using a medium supplemented with the screening agent. Examples of combinations of the gene conferring resistance to the screening agent include, for example, a combination of a neomycin resistance-conferring gene and neomycin, a combination of a hygromycin resistance-conferring gene and hygromycin, and a combination of a blast fungicide S resistance-conferring gene and blast fungicide S. Furthermore, if the marker gene is a gene that supplements the nutritional requirements of the host cell, host cells introduced with the DNA or vector of the present invention can be cultured using a minimum medium that does not contain nutrients that meet those nutritional requirements. Transformants can also be screened based on the activity of the enzyme expressed by the DNA or vector of the present invention.

[0109] To obtain a transformant in which the DNA of the present invention is located within the chromosome of a host cell, the following method is employed: First, the vector of the present invention and a vector containing a marker gene are digested with a restriction enzyme to form a linear structure. Then, the vector is introduced into a host cell using the gene introduction method described above. After culturing the introduced cells for several weeks, the target transformant is obtained based on the expression level of the marker gene. When using a gene conferring resistance to a screening agent as the marker gene, the vector of the present invention and a vector containing a marker gene are introduced into a host cell using the gene introduction method described above. The cells are then passaged in a culture medium containing a screening agent for several weeks or more, and the screening agent-resistant clones that survive as colonies are purified. This process yields a transformant in which the DNA of the present invention is introduced into the chromosome of the host cell. To confirm the integration of the DNA of the present invention into the chromosome of the host cell, genomic DNA of the cell is prepared using conventional genetic engineering methods. PCR, Southern hybridization, or similar procedures are then performed using the introduced DNA containing a portion of the base sequence of the DNA of the present invention as a primer or probe to detect the presence of the DNA of the present invention. The present invention relates to a transformant in which DNA is integrated into the chromosome of a host cell. It can be cryopreserved and can be thawed and used as needed, thus saving the time of transformant preparation for each experiment. In addition, experiments can be carried out using transformants whose properties and processing conditions have been confirmed in advance.

[0110] [The present invention relates to the manufacture of proteins]

[0111] The protein involved in this invention can be obtained from, for example, NITE BP-02779. Obtaining from NITE BP-02779 means obtaining it from the cells or culture supernatant of NITE BP-02779. For example, when obtaining the protein involved in this invention from the cells, the protein can be recovered by appropriately disrupting, dissolving, or extracting the cells. The cells can be recovered from the culture using centrifugation or the like. Cell disruption, dissolution, or extraction can be performed using known methods. Examples of such methods include, for instance, ultrasonic disruption, Dyno grinding, bead disruption, Freund's crushing, and lysozyme treatment. One of these methods can be used alone, or two or more can be appropriately combined. When the protein involved in this invention accumulates in the culture supernatant, the protein can be recovered from the culture supernatant by obtaining the culture supernatant using centrifugation or the like.

[0112] The protein purification involved in this invention can be performed using known methods employed in 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 can be used individually or in combination of two or more as appropriate. The protein purification involved in this invention can be performed to the desired extent.

[0113] The proteins of this invention can also be manufactured using transformants in which the DNA encoding them is introduced into a suitable host. The DNA is preferably integrated into a vector for transformation. The proteins of this invention can be synthesized artificially in cell-free lines.

[0114] When the protein of the present invention is manufactured using a transformant, the transformant can be cultured using a method for culturing host cells. When the transformant is a microorganism, it can be cultured using various culture media that are commonly used in microbial culture and appropriately contain carbon sources, nitrogen sources, organic or inorganic salts, etc.

[0115] 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 culture medium is typically around 0.1% to 30% (w / v) for the culture medium.

[0116] 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, dried yeast, and casein amino acids; amino acids; 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 also be used as carbon sources in various situations. The amount of these nitrogen sources added to the culture medium is typically around 0.1% to 30% (w / v) for the culture medium.

[0117] Examples of organic or inorganic salts include chlorides, sulfates, acetates, carbonates, and phosphates of potassium, sodium, magnesium, iron, manganese, cobalt, and zinc. Specifically, 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 and / or inorganic salts added to the culture medium is typically around 0.0001–5% (w / v) for the culture medium.

[0118] When a gene is expressed by linking a promoter such as the tac promoter, trc promoter, or lac promoter induced by isolaxose to the DNA involved in this invention, a small amount of, for example, isopropyl thio-β-D-galactoside (IPTG) may be added to the culture medium as an inducer for producing the protein involved in this invention.

[0119] The cultivation of the transformants involved in this invention can be carried out using methods commonly used in host cell culture, such as liquid and solid cultures including test tube shaking culture, reciprocating shaking culture, Jar Fermenter culture, and tank culture. The culture temperature can be appropriately varied within the range where the transformants can grow, typically from about 15°C to about 40°C. The pH of the culture medium is preferably in the range of about 6.0 to about 8.0. The culture time varies depending on the culture conditions and can be from about 1 day to 5 days.

[0120] By culturing the transformant of the present invention, a culture containing the protein of the present invention can be obtained. The protein of the present invention can accumulate, for example, in the cells of the transformant and / or in the culture supernatant. The production of the protein of the present invention can be confirmed by measuring the peptidoglycan-degrading activity of appropriate fractions, such as culture supernatant and cell extracts, according to the method described above.

[0121] The protein of this invention can be obtained by appropriately disrupting, dissolving, extracting, and purifying the transformant using the same method as described in NITE BP-02779. The protein of this invention is not limited to the purified protein of this invention; any fraction containing the protein of this invention can be used as "the protein of this invention" for applications such as peptidoglycan hydrolysis. There are no particular limitations as long as the fraction containing the protein of this invention contains it in a manner capable of acting on peptidoglycan. Examples of such fractions include, for instance, transformants, culture supernatants of transformants, disrupted products, dissolved products, extracts (cell-free extracts, etc.), partially purified products of these (crude purified products), and combinations thereof. These fractions can be used individually in applications such as peptidoglycan hydrolysis, or together with the purified protein of this invention. In the culture, other enzymes different from the protein of this invention can also be generated and accumulated together with the protein of this invention. The protein of this invention can be recovered as a mixture with such other enzymes, or it can be recovered separately from such other enzymes.

[0122] [Microbial decomposition agents]

[0123] The microbial degradation agent comprises at least one protein selected from the group consisting of the proteins involved in this invention and the transformants and their cultures involved in this invention. The culture of the transformant comprises the cultured transformant, the culture supernatant of the transformant, fragments, lysates, cell extracts (cell-free extracts, etc.), partially purified forms thereof (crude purified forms), and combinations thereof.

[0124] The microbial degradation agent preferably comprises a protein consisting of amino acid sequences 1-164 of Serial No. 2 and / or a protein consisting of amino acid sequences 1-493 of Serial No. 4. The microbial degradation agent may contain one enzyme with peptidoglycan degradation activity, or a combination of two or more enzymes. Other components may also be included, provided they do not impede peptidoglycan degradation activity. The transformants that may be included in the microbial degradation agent may be one or two or more.

[0125] There are no particular limitations on the content of the proteins involved in this invention in the microbial decomposition preparation, as well as the content of the transformants and their cultures involved in this invention. The content can be appropriately set according to the type and concentration of the target microorganism and the usage environment of the microbial decomposition preparation (volume of the reaction system, temperature, etc.).

[0126] Microbial decomposition agents, containing proteins with peptidoglycan-decomposing activity or transformants expressing such proteins, are capable of decomposing peptidoglycan. These agents can decompose target microorganisms containing peptidoglycan. The target microorganisms are preferably bacteria that constitute excess sludge. These target microorganisms can be dead or live. Microbial decomposition agents can contribute to the volume reduction of excess sludge.

[0127] Whether or not microorganisms have been decomposed can be confirmed using methods commonly used by those skilled in the art. Examples of such confirmation methods include reacting a microbial decomposition agent with the target microorganism in a suitable culture medium or buffer for a certain period of time, and then detecting the decomposition of the target microorganism in the culture medium or buffer. There are no particular limitations on the methods for detecting the decomposition of the target microorganism; examples include measuring the turbidity of the target microorganism, detecting the target microorganism using SLP reagents, detecting the DNA of the target microorganism using PCR, measuring the dried cell weight of the target microorganism, and detecting decomposition products 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. Using the methods described above for checking peptidoglycan decomposition activity, it is also possible to check whether the target microorganism has been decomposed.

[0128] When using turbidity to examine the decomposition of target microorganisms, decomposition of 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 less than 80%, preferably less than 50%, and more preferably less than 30%. When using the weight of dried bacterial cells to examine the decomposition of target microorganisms, decomposition of target microorganisms means, for example, that the weight of dried bacterial cells after the reaction is significantly lower than the weight before the reaction, for example, the weight of dried bacterial cells after the reaction is less than 95%, preferably less than 90%.

[0129] The dosage form of the microbial decomposition preparation is not particularly limited as long as it does not lose the function of the protein and the transformant and its culture involved in this invention. Examples of dosage forms include liquids, suspensions, powders, solids, capsules, or frozen or lyophilized versions of these. During formulation, additives such as excipients, binders, disintegrants, lubricants, stabilizers, diluents, and surfactants can be used.

[0130] Microbial decomposition agents can be used in sludge treatment plants and wastewater treatment plants that generate excess sludge. They can also be used in tanks that accumulate excess sludge or target microorganisms.

[0131] One aspect of the invention is the use of at least one of the proteins and transformants and their cultures as described in the manufacture of a microbial decomposition agent.

[0132] [Microbial Decomposition Methods]

[0133] The microbial degradation method includes a step of applying at least one protein selected from the group consisting of the proteins and transformants and their cultures involved in this invention to a target microorganism. The transformant culture includes a cultured transformant, a culture supernatant of the transformant, fragments, a lysate, cell extracts (cell-free extracts, etc.), partially purified forms thereof (crude purified forms), and combinations thereof. The step of applying at least one protein selected from the proteins and transformants and their cultures involved in this invention to the target microorganism can be performed using the aforementioned microbial degradation agent.

[0134] Applying the protein of the present invention to a target microorganism means, for example, contacting the protein of the present invention with the target microorganism. Applying the transformant of the present invention to a target microorganism means, for example, culturing the transformant of the present invention in the presence of the target microorganism. Applying a culture of the transformant of the present invention to a target microorganism means, for example, contacting the culture of the transformant, preferably containing a protein in the culture and having peptidoglycan-degrading activity, with the target microorganism.

[0135] According to the microbial decomposition method of the present invention, target microorganisms containing peptidoglycan can be decomposed. The target microorganisms are preferably bacteria constituting excess sludge. The target microorganisms can be dead or live bacteria. The microbial decomposition method can facilitate the reduction of the volume of excess sludge.

[0136] The microbial degradation method preferably includes a step of applying a protein consisting of amino acid sequences 1-164 of Serial No. 2 and / or a protein consisting of amino acid sequences 1-493 of Serial No. 4 to a target microorganism. In this step, one protein with peptidoglycan degradation activity may be used, or two or more may be used in combination. In the microbial degradation method, the transformant applied to the target microorganism may be one or two or more.

[0137] The process of applying the protein, the transformant, and its culture to the target microorganisms involved in this invention is not particularly limited, as long as the conditions for synthesizing the protein are not lost or the peptidoglycan-degrading activity of the protein are not lost, or the transformant is not killed. This process can be carried out, for example, at a temperature of 20–35°C or 25–30°C. It can also be carried out at a pH of 5.5–8.0 or 6.0–7.5.

[0138] There are no particular limitations on the amount of the protein, the transformant and its culture used in the microbial decomposition method, as well as the amount of the transformant and its culture, which can be appropriately set considering the type and concentration of the target microorganism, the volume of the reaction system, the reaction temperature, etc.

[0139] Whether the target microorganism has been decomposed can be confirmed using the methods described in the section on microbial decomposition agents.

[0140] One aspect of the invention is the use of a microbial decomposition agent comprising at least one of the proteins involved in the invention and the transformants and cultures thereof involved in the invention, for decomposing target microorganisms. Example

[0141] The present invention will be described in more detail below with reference to specific embodiments, but the present invention is not limited to these embodiments.

[0142] [Experiment 1. Isolation of Microbial Degrading Bacteria]

[0143] (method)

[0144] A culture medium using Micrococcus bacteria as a carbon source was used to cultivate a microbial community present in the environment (water), thereby enriching microorganisms that decompose Micrococcus bacteria. Subsequently, several well-proliferating strains were isolated from the enriched microbial community.

[0145] (result)

[0146] The micrococcal decomposition ability of each isolated strain was examined, and micrococcal decomposition ability was confirmed in one strain. Hereinafter, the strain in which micrococcal decomposition ability was confirmed will sometimes be referred to as "strain A".

[0147] [Experiment 2. Identification of strain A]

[0148] (Material)

[0149] • Forward primer for cloning (27F: sequence number 5)

[0150] • Reverse primer for cloning (1492R: sequence number 6)

[0151] • Primers used for sequencing analysis (339F: sequence number 7, 536R: sequence number 8, 907F: sequence number 9)

[0152] (method)

[0153] The identification of strain A was carried out through 16S rRNA gene analysis, morphological observation, and physiological and biochemical trait tests.

[0154] 16S rRNA gene analysis was performed according to the following steps. Genomic DNA was extracted from strain A. Using the obtained genomic DNA as a template, PCR amplification of the 16S rRNA gene was performed using forward primer (27F) and reverse primer (1492R) for cloning. PCR amplification was performed using a KOD FX (manufactured by Toyobo Co., Ltd.), and the amplified products were purified.

[0155] Cyclic sequencing was performed using purified PCR amplification products. The cyclic sequencing reaction was performed using the BigDyeTerminator v3.1 cyclic sequencing kit. The resulting reaction solution was purified, and DNA sequencing analysis (3730xl DNA analyzer) was performed to determine the nucleotide sequence of the 16S rRNA gene from the template DNA extracted from strain A.

[0156] Morphological observation and physiological and biochemical tests were conducted using optical microscopy, BARROW and other methods (Cowan and Steel's Manual for the Identification of Medical Bacteria 3rd Edition 1993, Cambridge University Press.), and API50CHB (bioMerieux, Lyon, France).

[0157] (result)

[0158] The obtained 16S rRNA gene sequence (accession number 10) was compared with the international base sequence databases (DDBJ / ENA(EMBL) / GenBank) for homology analysis. In the reference strain, the 16S rRNA gene sequence of *Tumebacillus permanentifrigoris* Eurl_9.5 showed 98.1% identity. However, no microorganism with a 16S rRNA gene completely identical to the obtained sequence was found. Furthermore, strain A did not grow at 10°C, but this characteristic was not observed in *Tumebacillus permanentifrigoris* Eurl_9.5, which showed the highest 16S rRNA gene identity. Therefore, strain A is a novel species different from previously known *Tumebacillus* species. Strain A was deposited internationally as species NITE BP-02779 of the *Tumebacillus* genus.

[0159] The morphological observation and physiological and biochemical trait test results of NITE BP-02779 are shown in Tables 1 to 4.

[0160] [Table 1]

[0161]

[0162] +: Positive, -: Negative, +w: Weak reaction

[0163] [Table 2]

[0164]

[0165] +: positive, -: negative

[0166] [Table 3]

[0167]

[0168] +: positive, -: negative

[0169] [Table 4]

[0170]

[0171] +: positive, -: negative

[0172] [Experiment 3. Evaluation of NITE BP-02779's ability to decompose excess sludge (dead bacteria) - 1]

[0173] (Material)

[0174] • R2A medium: R2A broth medium DAIGO (manufactured by Nippon Pharmaceutical Co., Ltd.) was dissolved in 3.2g of ultrapure water at a ratio of 1000mL and then autoclaved.

[0175] • Inorganic culture medium containing residual sludge (dead bacteria): A culture medium consisting of 986 mL of matrix 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.

[0176] The following were used as matrix solutions and solutions A through D.

[0177] Matrix solution: A solution prepared by washing residual sludge, mixing it with 986 mL of ultrapure water to achieve a turbidity (OD660) of 0.2, and then autoclaving it.

[0178] Solution A: 4.35g dipotassium hydrogen phosphate, 1.70g potassium dihydrogen phosphate, 8.92g disodium hydrogen phosphate 1 / 2 hydrate, and 0.34g ammonium chloride were dissolved in ultrapure water to prepare a 200mL solution, which was then autoclaved.

[0179] Solution B: 4.50 g of magnesium sulfate hexahydrate was dissolved in ultrapure water to prepare a 200 mL solution, which was then autoclaved.

[0180] Solution C: 5.50 g of anhydrous calcium chloride was dissolved in ultrapure water to prepare a 200 mL solution, which was then autoclaved.

[0181] Solution D: 0.05 g of ferric chloride hexahydrate was dissolved in ultrapure water to prepare a 200 mL solution, which was then filtered and sterilized using a 0.2 μm syringe filter.

[0182] (method)

[0183] NITE BP-02779 was inoculated into R2A medium and incubated at 25°C for 24–48 hours. After incubation, 50 μL of NITE BP-02779 culture medium and 5.0 mL of inorganic medium containing residual sludge (dead bacteria) were added to test tubes, and the reaction was carried out at 25°C and 200 rpm. The turbidity (OD660) of the test tubes was measured over time using a simple turbidimeter (simple OD monitor miniphoto 518R, TAITEC). The number of days elapsed when the turbidity (OD660) of the inorganic medium containing residual sludge (dead bacteria) showed 50% of the turbidity (OD660) of the negative control was calculated. The negative control used the turbidity (OD660) of inorganic medium containing target bacteria (dead bacteria) without the addition of NITE BP-02779.

[0184] (result)

[0185] It was confirmed that the addition of NITE BP-02779 reduced the turbidity of the residual sludge (dead bacteria), reaching 50% of the turbidity of the negative control at 3.2 days. Therefore, NITE BP-02779 can decompose residual sludge (dead bacteria).

[0186] [Experiment 4. Evaluation of NITE BP-02779's ability to decompose residual sludge (dead bacteria) - 2]

[0187] (Material)

[0188] The same materials as in Experiment 3 were used.

[0189] (method)

[0190] The reaction was conducted using the same method as in Experiment 3. The reaction was performed in five consecutive replicates, and on day 4 after the start of the reaction, the residual sludge in the test tubes was recovered in total. The recovered residual sludge was dried and weighed, and a significance test (t-test, one-sided) was performed using the negative control group and the NITE BP-02779 addition group.

[0191] (result)

[0192] The results are shown in Figure 2 The study confirmed a significant reduction in the dry weight of residual sludge in the NITE BP-02779-added group compared to the negative control group (p<0.01). Therefore, the addition of NITE BP-02779 can reduce residual sludge.

[0193] [Experiment 5. Determination of the genome sequence of NITE BP-02779]

[0194] (method)

[0195] The genome sequence of NITE BP-02779 was analyzed using a next-generation sequencer following these steps. 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 bacterial cells were centrifuged, collected, and genomic DNA was extracted using a genomic DNA extraction kit (QIAamp DNA Mini Kit (250), QIAGEN). The extracted DNA was used as a template and sequenced using a HiSeq 2500 next-generation sequencer (Illumina) with paired-end sequencing. Next, adapter sequences were trimmed from the obtained sequence, and de novo assembly was performed using Velvet. The scaffold sequence obtained from the de novo sequencing assembly was analyzed for gene region prediction and annotation. RAST (Rapid Annotations using Subsystems Technology) was used for gene region prediction and annotation.

[0196] (result)

[0197] The de novo sequencing assembly yielded 149 scaffolds with a total scaffold sequence length of 4.44 Mbp, predicting the presence of 4446 genes.

[0198] [Experiment 6. Identification of a protein with peptidoglycan-degrading activity (ID2839)]

[0199] (1) Preparation of NITE BP-02779 culture supernatant

[0200] (method)

[0201] 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 then inoculated into 1.5 L of R2A medium and cultured with shaking at 25 °C and 130 rpm. After 48 hours, the culture was centrifuged (4 °C, 8000 × g, 10 min), and the resulting supernatant was filtered (0.20 μm). The filtered supernatant was concentrated using a centrifuge filter unit (Merck Millipore) and used as the starting material for enzyme purification.

[0202] (2) Fractionation using column chromatography

[0203] Enzyme fractionation and purification were performed using column chromatography as shown in (a)–(c) below. The peptidoglycan-degrading activity of each fraction was determined using a lysozyme activity kit (Sigma-Aldrich). Proteins in the fractions were identified by electrophoresis on 4–20% mini-Protean TGX prepreg gels (BioRad) followed by CBB staining.

[0204] (a) Cation exchange chromatography

[0205] (Material)

[0206] • Sample: NITE BP-02779 culture supernatant concentrate

[0207] (method)

[0208] Using a centrifugal filter unit (Merck Millipore), the buffer in the sample was replaced with 50 mM phosphate buffer (pH 5.8). The sample was then applied to a HiTrap QHP, 5 mL column (GE Healthcare) equilibrated with the same buffer. After washing the column with the same buffer, the adsorbed proteins were eluted using a linear sodium chloride concentration gradient from 0 to 1.0 M.

[0209] (result)

[0210] The peptidoglycan degradation activity was examined, and the activity was confirmed in the unadsorbed fraction. Therefore, the unadsorbed fraction was provided for hydrophobic interaction chromatography.

[0211] (b) Hydrophobic interaction chromatography

[0212] • Sample: Unadsorbed fraction from cation exchange chromatography

[0213] (method)

[0214] Using a centrifugal filter unit (Merck Millipore), the buffer in the unadsorbed fraction of (a) cation exchange chromatography was replaced with 50 mM phosphate buffer (pH 5.8) containing 1 M ammonium sulfate. This sample was then applied to a Hitrap HIC, 5 mL column (GE Healthcare) equilibrated with the same buffer. After washing the column with the same buffer, adsorbed proteins were eluted using a linear concentration gradient of 1.0–0 M ammonium sulfate.

[0215] (result)

[0216] The peptidoglycan degradation activity was examined, and the activity was confirmed in the adsorption fraction. Therefore, the fraction with confirmed activity was used in gel filtration chromatography.

[0217] (c) Gel filtration chromatography

[0218] • Sample: Adsorption fraction of hydrophobic interaction chromatography

[0219] (method)

[0220] Using a centrifugal filter unit (Merck Millipore), the buffer in the adsorption fraction of (b) hydrophobic interaction chromatography was replaced with 66 mM potassium phosphate (pH 6.24). The sample was then eluted in a Superdex™ 75 10 / 300 GL (GE Healthcare) buffer equilibrated with the same buffer.

[0221] (result)

[0222] The peptidoglycan degradation activity was examined, and the results are as follows: Figure 3 The activity was confirmed in the elution fraction, as shown.

[0223] (3) Identification of proteins with peptidoglycan-degrading activity using mass analysis

[0224] (Material)

[0225] • Sample: Elution fraction of gel filtration chromatography

[0226] (method)

[0227] The elution fractions with confirmed activity were concentrated using a centrifugal filter unit (Merck Millipore) and electrophoretically analyzed using 4–20% mini-Protean TGX pre-prepared gels (BioRad). The results were as follows: Figure 4As shown, the protein bands from the elution fraction were confirmed to be single bands. For the purified fractions from gel filtration chromatography (C) and the bands separated by electrophoresis, trypsin was used for liquid-phase digestion or in-gel digestion, and the digestion solutions were desalted. Data-dependent MS / MS acquisition (DDA) was performed on the desalted samples using nano-LC-MS (Thermo Scientific, Q Exactive HF, nanocolumn: Acclaim PepMap RSLC C18 (Thermo Scientific)). Following the determination, a Masco search (MS / MS ion search) using ProteomeDiscoverer 2.1 was performed on the protein sequence database based on the genomic information of NITEBP-02779.

[0228] (result)

[0229] A search was performed on trypsin-cleaved peptides detected by quality analysis, and amino acid sequences 11-17 of a partial sequence of ID2839 were detected. These partial sequences correspond to amino acid sequences 15-164 of sequence number 2. Based on this, it is suggested that the protein ID2839 expressed by NITE BP-02779 possesses peptidoglycan-degrading activity.

[0230] (4) Identification of the N-terminal cut site of ID2839

[0231] (method)

[0232] Furthermore, using the DDA assay data file that identified ID2839 as the target, we searched whether the N-terminal side contained any cleaved peptide at positions -11 to 14 of sequence number 2.

[0233] (result)

[0234] The peptide with the highest signal strength was identified as a partial sequence of SEQ ID NO: 18. This partial sequence corresponds to positions 1-10 of SEQ ID NO: 2. Therefore, the overall amino acid sequence of SEQ ID NO: 2 is considered to be the amino acid sequence of the precursor (pro sequence) of ID2839. The mature amino acid sequence of ID2839 is considered to be positions 1-164 of SEQ ID NO: 2, encoded by bases 205-696 of SEQ ID NO: 1. Positions -68 to -1 of SEQ ID NO: 2 are considered to be the signal peptide. Hereinafter, the protein composed of amino acid positions 1-164 of SEQ ID NO: 2 is sometimes referred to as the "mature ID2939," and the protein composed of the overall amino acid sequence of SEQ ID NO: 2 (positions -68-164) is sometimes referred to as the "precursor ID2839." It is believed that cleavage of the signal peptide from the precursor ID2839 results in the mature ID2839, which is secreted extracellularly by bacteria and can decompose peptidoglycan. The molecular weight of the ID2839 precursor is approximately 25 kDa, and the molecular weight of the mature ID2839 is approximately 18 kDa.

[0235] [Experiment 7. Specific activity determination of mature ID2839 organisms]

[0236] (Material)

[0237] • Mature ID2839 fraction (elution fraction obtained by gel filtration chromatography)

[0238] (method)

[0239] Activity (units / mL) and protein quantification (mg / mL) of mature ID2839 were performed to calculate specific activity (units / mg). Two commercially available lysozymes (Sigma-Aldrich) were used as controls. Peptidoglycan degradation activity was evaluated using a lysozyme activity kit (Sigma-Aldrich) based on the activity in breaking down the glycosidic bond between N-acetylmuramic acid and N-acetylglucosamine.

[0240] (result)

[0241] The results are shown in Table 5. It demonstrates that the mature ID2839 has the activity to break the glycosidic bond between N-acetylmuramic acid and N-acetylglucosamine, with a higher specific activity than commercially available lysozyme and glucocorticoid lysozyme.

[0242] [Table 5]

[0243]

[0244] [Experiment 8. Preparation of mature ID2839 in E. coli]

[0245] (1) Construction of mature expression line of ID2839

[0246] (Material)

[0247] Primers F and R (serial numbers 19 and 20) were used for cloning ID2839.

[0248] • LB agar medium: Dissolve LB Ager (Sigma-Aldrich) at a ratio of 10 to 500 mL of ultrapure water, autoclave, and then pour the agar medium into the dish.

[0249] (method)

[0250] Using the genomic DNA of NITE BP-02779 as a template, PCR amplification was performed using primers F and R for ID2839 cloning to a region containing the base sequence encoding the mature ID2839 (where Ala at position 1 in sequence number 2 is replaced by Met). PCR amplification was performed using a KOD FX (Toyobo Co., Ltd.). The reaction solution was prepared according to the contents of the kit and amplified 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, with steps (2) to (4) repeated for 25 cycles. The obtained PCR fragment and the pET-28a(+) vector (Merck Millipore Co., Ltd.) were digested with NdeI and XhoI, respectively, and ligated using a high-efficiency ligation reagent (Ligation High Ver.2, Toyobo Co., Ltd.). In this ligation reaction solution, E. coli DH5α was transformed, and the target plasmid was extracted from the kanamycin-resistant strain. Using this plasmid, E. coli BL21(DE3) was transformed to obtain an expression strain of the mature ID2839 (sometimes denoted as "BL21(DE3) / pET28a-ID2839 mature strain"). In the BL21(DE3) / pET28a-ID2839 mature strain, a protein containing a His tag and an amino acid sequence with a thrombin cleavage site (MetGlySerSerHisHisHisHisHisHisHisHisGlyLeuValProArgGlySerHis: Serial No. 21) was expressed. As a control strain, a strain that did not express the mature ID2839 (sometimes denoted as "BL21(DE3) / pET28a") was obtained.

[0251] (2) Expression induction of mature ID2839 and preparation of soluble fractions

[0252] (Material)

[0253] • LB liquid medium: 1.1g PER TABLET (Sigma-Aldrich) of LB broth was dissolved in 10 parts of 500mL of ultrapure water and then autoclaved.

[0254] Transformed form: BL21(DE3) / pET28a-ID2839 mature form

[0255] • Transformant (control): BL21(DE3) / pET28a

[0256] (method)

[0257] 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 in a flask. When the OD660 reached 0.6, IPTG (final concentration 0.1 mM) was added, and the culture was further incubated at 18°C ​​for 16 hours. After incubation, the cells were collected from the culture medium by centrifugation, and a specific amount of BugBuster (Novagen) was added per wet cell volume to disrupt the cells. Cell residue was removed from the disruption liquid by centrifugation, and the resulting supernatant was used as the soluble fraction. A soluble fraction containing mature ID2839 was obtained from the BL21(DE3) / pET28a-ID2839 mature strain. A soluble fraction excluding ID2839 was obtained from the BL21(DE3) / pET28a strain.

[0258] (3) Activity evaluation of soluble fractions

[0259] (Material)

[0260] • Soluble fraction from mature BL21(DE3) / pET28a-ID2839

[0261] • Soluble fraction from BL21(DE3) / pET28a

[0262] (method)

[0263] The activity (in units / mL) of lysozyme that breaks down the glycosidic bond between N-acetylmuramic acid and N-acetylglucosamine was calculated using a lysozyme activity kit (Sigma-Aldrich). The activity (in units / mL) was calculated using the following formula.

[0264] The activity of breaking the glycosidic bond between N-acetylmuramic acid and N-acetylglucosamine (unit / mL) = {ΔABS450nm / min (phosphate buffer containing target bacteria with added soluble fraction) - ΔABS450nm / min (phosphate buffer containing target bacteria without added soluble fraction)} / (0.001 × 0.1)

[0265] (result)

[0266] The results are shown in Table 6. In the soluble fraction from the mature BL21(DE3) / pET28a-ID2839, activity was confirmed to break the glycosidic bond between N-acetylmuramic acid and N-acetylglucosamine. However, in the soluble fraction from the control BL21(DE3) / pET28a, almost no activity was confirmed to break the glycosidic bond between N-acetylmuramic acid and N-acetylglucosamine. This indicates that the mature ID2839 possesses peptidoglycan-degrading activity, specifically the activity to break the glycosidic bond between N-acetylmuramic acid and N-acetylglucosamine.

[0267] [Table 6]

[0268]

[0269] [Experiment 9. Identification of a protein with peptidoglycan-degrading activity (ID1644)]

[0270] (1) Identification based on genomic information

[0271] The genome sequence of NITE BP-02779 revealed a gene for an enzyme with high identity to N-acetylmurayl-L-alanine amidase. This enzyme was named ID1644. ID1644 is secreted in the culture supernatant of NITE BP-02779. The amino acid sequence of sequence number 4 is considered to be the overall amino acid sequence of the precursor (pro sequence). The amino acid sequence of the mature protein is considered to be positions 1-493 of sequence number 4, encoded by positions 85-1563 of sequence number 3. Positions -28 to -1 of sequence number 4 are considered to be the signal peptide. Hereinafter, the protein composed of amino acid sequences 1-493 of sequence number 4 will sometimes be referred to as the "mature ID1644", and the protein composed of the overall amino acid sequence of sequence number 4 (positions -28-493) will be referred to as the "precursor ID1644". It is believed that when the signal peptide is cleaved from the ID1644 precursor, it becomes the mature ID1644. The mature ID1644 is secreted outside the bacterial cell and can decompose peptidoglycan. The molecular weight of the ID1644 precursor is approximately 56 kDa, and the molecular weight of the mature ID1644 is approximately 53 kDa.

[0272] [Experiment 10. Preparation of mature ID1644 in E. coli]

[0273] (1) Construction of mature expression line of ID1644

[0274] (Material)

[0275] Primers F and R (serial numbers 22 and 23) were used for cloning ID1644.

[0276] LB agar medium

[0277] (method)

[0278] Using the genomic DNA of NITE BP-02779 as a template, PCR amplification of the region encoding the base sequence of the mature ID1644 was performed using primers F and R for ID1644 cloning. PCR amplification was performed using KOD FX (manufactured by Toyobo). The reaction solution was prepared according to the contents of the kit and amplified 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, with steps (2) to (4) repeated for 25 cycles. The obtained PCR fragment and pET-22b(+) vector (manufactured by Merck Millipore) were digested with NdeI and XhoI, respectively, and ligated using a high-efficiency ligation reagent (Ligation High Ver.2, manufactured by Toyobo). In this ligation reaction solution, E. coli DH5α was transformed, and the target plasmid was extracted from the ampicillin-resistant strain. Using this plasmid, E. coli BL21(DE3) was transformed to obtain an expression strain of the mature ID1644 (sometimes denoted as "BL21(DE3) / pET22b-ID1644 mature strain"). In the BL21(DE3) / pET22b-ID1644 mature strain, a protein with a methionine residue attached to the N-terminus of the mature protein (serial number 4, positions 1-493) was expressed. As a control strain, a strain that did not express the mature ID1644 was obtained (sometimes denoted as "BL21(DE3) / pET22b").

[0279] (2) Expression induction of mature ID1644 and preparation of soluble fractions

[0280] (Material)

[0281] LB liquid culture medium

[0282] Transformed form: BL21(DE3) / pET22b-ID1644 mature form

[0283] • Transformant (control): BL21(DE3) / pET22b

[0284] (method)

[0285] 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 in a flask. When the OD660 reached 0.6, IPTG (final concentration 0.1 mM) was added, and the culture was further incubated at 18°C ​​for 16 hours. After incubation, the bacterial cells were collected from the culture medium by centrifugation, and a specific amount of BugBuster (Novagen) was added per wet cell volume to disrupt the cells. The bacterial residue was removed from the disruption liquid by centrifugation, and the resulting supernatant was used as the soluble fraction. A soluble fraction containing mature ID1644 cells was obtained from the BL21(DE3) / pET22b-ID1644 mature strain. A soluble fraction not containing mature ID1644 cells was obtained from the BL21(DE3) / pET22b strain.

[0286] (3) Activity evaluation of soluble fractions - 1

[0287] (Material)

[0288] • Soluble fraction from mature BL21(DE3) / pET22b-ID1644

[0289] • Soluble fraction from BL21(DE3) / pET22b

[0290] • Peptidoglycan solution: A solution of peptidoglycan (manufactured by Sigma-Aldrich) dissolved at a ratio of 10 mg to 50 mL of phosphate buffer (pH 7.0).

[0291] (method)

[0292] Mix 5 mL of peptidoglycan solution with 50 μL of soluble fraction, and measure the OD450 over time to calculate the peptidoglycan decomposition activity (units / mL). The peptidoglycan decomposition activity (units / mL) is calculated using the following formula.

[0293] Peptidoglycan degradation activity (unit / mL) = {ΔABS450nm / min (phosphate buffer containing peptidoglycan with added soluble fraction) - ΔABS450nm / min (phosphate buffer containing peptidoglycan without added soluble fraction)} / (0.001 × 0.05)

[0294] (result)

[0295] The results are shown in Table 7. Peptidoglycan-degrading activity was confirmed in the soluble fraction from the mature BL21(DE3) / pET22b-ID1644, while almost no peptidoglycan-degrading activity was confirmed in the soluble fraction from the control BL21(DE3) / pET22b. This indicates that the mature ID1644 possesses peptidoglycan-degrading activity.

[0296] [Table 7]

[0297]

[0298] (4) Activity evaluation of soluble fractions - 2

[0299] (Material)

[0300] • Soluble fraction from mature BL21(DE3) / pET22b-ID1644

[0301] • Soluble fraction from BL21(DE3) / pET22b

[0302] • Substrate for N-acetylmurayl-L-alanine amidase: L-aniline-p-nitroaniline hydrochloride (Sigma-Aldrich)

[0303] (method)

[0304] 10 μL of soluble fraction, 10 μL of 1 mg / mL matrix solution, and 80 μL of 100 mM tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl, pH 7.6) were incubated at 37 °C for 10 minutes, and the absorbance (405 nm) was measured using an ELISA reader (Molecular Devices).

[0305] (result)

[0306] The results are shown in Table 8. The soluble fraction from the mature BL21(DE3) / pET22b-ID1644 showed a higher ABS405nm value than the soluble fraction from BL21(DE3) / pET22b. This indicates that the mature ID1644 possesses N-acetylmurayl-L-alanine amidase activity.

[0307] [Table 8]

[0308]

[0309] [Experiment 11. Preparation of mature ID2839-2 in E. coli]

[0310] (1) Construction of ID2839 mature-2 expression line

[0311] (Material)

[0312] Primers F and R (serial numbers 24 and 25) were used for cloning ID2839.

[0313] LB agar medium

[0314] (method)

[0315] Using the genomic DNA of NITE BP-02779 as a template, PCR amplification was performed using primers for ID2839 cloning (serial numbers 24 and 25) to a region encoding the base sequence of mature ID2839-2 (a protein composed of amino acids from amino acid sequence 1 to 164 of sequence number 2 with a Met amino acid sequence added to the N-terminus of Ala at position 1). PCR amplification was performed using KODplus NEO (manufactured by Toyobo). The reaction solution was prepared according to the contents of the kit and the reaction was performed 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 for 30 cycles. The obtained PCR fragment was ligated to a fragment prepared by digesting the pET-28a(+) vector (manufactured by Merck Millipore) with NcoI and XhoI using the In-fusion HD Cloning Kit (manufactured by Takara Bio). In this ligation reaction solution, E. coli DH5α was transformed, and the target plasmid was extracted from the kanamycin-resistant strain. Using this plasmid, E. coli BL21(DE3) was transformed to obtain the expression strain of ID2839 mature-2 (sometimes referred to as "BL21(DE3) / pET28a-ID2839 mature-2"). In BL21(DE3) / pET28a-ID2839 mature-2, the mature protein (a protein composed of amino acids consisting of amino acids 1-164 of sequence number 2 with Met added to the N-terminus of Ala at position 1) was expressed.

[0316] (2) Expression induction of ID2839 mature-2 and preparation of soluble fraction

[0317] (Material)

[0318] LB liquid culture medium

[0319] Transformed form: BL21(DE3) / pET28a-ID2839 mature form-2

[0320] • Transformant (control): BL21(DE3) / pET28a

[0321] (method)

[0322] 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 in a flask. When the OD660 reached 0.6, IPTG (final concentration 1 mM) was added, and the culture was further incubated at 24°C for 20 hours. After the culture was completed, the soluble fraction of the cells was obtained using the same method as in Experiment 8.

[0323] (3) Activity evaluation of soluble fractions

[0324] (Material)

[0325] • Soluble fraction from mature BL21(DE3) / pET28a-ID2839-2

[0326] • Soluble fraction from BL21(DE3) / pET28a

[0327] (method)

[0328] The same method as in Experiment 8 was used.

[0329] (result)

[0330] The results are shown in Table 9. Peptidoglycan-degrading activity was confirmed in the soluble fraction of BL21(DE3) / pET28a-ID2839 mature form-2.

[0331] [Table 9]

[0332]

[0333] [Experiment 12. Preparation of mature ID2839-3 in E. coli]

[0334] (1) Construction of ID2839 mature-3 expression line

[0335] (Material)

[0336] Primers F and R (serial numbers 26 and 25) were used for cloning ID2839.

[0337] LB agar medium

[0338] (method)

[0339] In addition to using the cloning primers described in Serial Numbers 26 and 25, the expression strain of ID2839 mature-3 (sometimes referred to as "BL21(DE3) / pET28a-ID2839 mature-3") was obtained using the same method as in Experiment 11. In BL21(DE3) / pET28a-ID2839 mature-3, the mature protein (a protein composed of amino acids consisting of the amino acid sequence in Serial Number 2 where Ala is replaced by Met at position 1) was expressed.

[0340] (2) Expression induction of ID2839 mature-3 and preparation of soluble fractions

[0341] (Material)

[0342] LB liquid culture medium

[0343] Transformed form: BL21(DE3) / pET28a-ID2839 mature form-3

[0344] • Transformant (control): BL21(DE3) / pET28a

[0345] (method)

[0346] The soluble fractions of the bacterial cells were obtained using the same method as in Experiment 11.

[0347] (3) Activity evaluation of soluble fractions

[0348] (Material)

[0349] • Soluble fraction from BL21(DE3) / pET28a-ID2839 mature form-3

[0350] • Soluble fraction from BL21(DE3) / pET28a

[0351] (method)

[0352] The same method as in Experiment 8 was used.

[0353] (result)

[0354] The results are shown in Table 10. Peptidoglycan degradation activity was confirmed in the soluble fraction from BL21(DE3) / pET28a-ID2839 mature-3.

[0355] [Table 10]

[0356]

[0357]

Claims

1. A protein comprising the amino acid sequence from position 1 to position 164 of SEQ ID NO: 2 and having an activity of decomposing a glycosidic bond between N-acetylmuramic acid and N-acetylglucosamine.

2. A protein which is any one of the following (al) to (a4), (al) a protein consisting of the amino acid sequence from position 1 to position 164 of SEQ ID NO: 2, (a2) a protein consisting of the amino acid sequence in which Ala at position 1 of the amino acid sequence from position 1 to position 164 of SEQ ID NO: 2 is substituted with Met, (a3) a protein consisting of the amino acid sequence in which Ala at position 1 of the amino acid sequence from position 1 to position 164 of SEQ ID NO: 2 is substituted with Met, and the amino acid sequence of the amino acid sequence recited in SEQ ID NO: 21 is added to the N terminus of the Met, and (a4) a protein consisting of the amino acid sequence in which Met is added to the N terminus of Ala at position 1 of the amino acid sequence from position 1 to position 164 of SEQ ID NO:

2.

3. A protein comprising the amino acid sequence from position 1 to position 493 of SEQ ID NO: 4 and having N-acetylmuramoyl-L-alanine amidase activity.

4. A protein which is any one of the following (bl) to (b4), (bl) a protein consisting of the amino acid sequence from position 1 to position 493 of SEQ ID NO: 4, (b2) a protein consisting of the amino acid sequence in which Glu at position 1 of the amino acid sequence from position 1 to position 493 of SEQ ID NO: 4 is substituted with Met, (b3) a protein consisting of the amino acid sequence in which Glu at position 1 of the amino acid sequence from position 1 to position 493 of SEQ ID NO: 4 is substituted with Met, and the amino acid sequence of the amino acid sequence recited in SEQ ID NO: 21 is added to the N terminus of the Met, and (b4) a protein consisting of the amino acid sequence in which Met is added to the N terminus of Glu at position 1 of the amino acid sequence from position 1 to position 493 of SEQ ID NO:

4.

5. A DNA encoding the protein according to any one of claims 1 to 4.

6. A vector comprising the DNA according to claim 5.

7. A transformant comprising the DNA according to claim 5 or the vector according to claim 6.

8. A microbial decomposition preparation comprising at least one selected from the group consisting of the protein according to any one of claims 1 to 4 and the transformant according to claim 7 and a culture thereof.

9. Use of at least one selected from the group consisting of the protein according to any one of claims 1 to 4 and the transformant according to claim 7 and a culture thereof for producing a microbial decomposition preparation. ​ ​ ​ ​ ​ ​ ​ ​

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