Microorganisms having biodegradable plastic decomposition activity, compositions for use in the decomposition of biodegradable plastics, and methods for processing biodegradable plastics.

Abstract

This invention provides a novel microorganism capable of decomposing biodegradable plastics, particularly polylactic acid (PLA). [Solution] The microorganism described herein belongs to the family Bacillaceae and is deposited under accession number NITE BP-04272.

Description

Technical Field

[0001] The present disclosure relates to a microorganism having biodegradable plastic-degrading activity, a composition for use in degrading a biodegradable plastic, and a method for treating a biodegradable plastic. Background Art

[0002] In recent years, in order to solve the marine plastic problem and the global warming problem, research and development of biodegradable plastics that are ultimately decomposed into CO2 and water by microorganisms and the like have been advanced.

[0003] Furthermore, along with the expansion of the use of said biodegradable plastics, research into microorganisms capable of degrading said biodegradable plastics and the like has been progressing (Non-Patent Documents 1-2). Prior Art Documents Non-Patent Documents

[0004] Non-Patent Document 1 Arena, Maria et al. “Degradation of poly (lactic acid) and nanocomposites by Bacillus licheniformis.” Environmental science and pollution research international vol. 18,6 (2011): 865-70. doi:10.1007 / s11356-011-0443-2 Non-Patent Document 2 Bonifer, Kyle S et al. “Bacillus pumilus B12 Degrades Polylactic Acid and Degradation Is Affected by Changing Nutrient Conditions.” Frontiers in microbiology vol. 10 2548. 22 Nov. 2019, doi:10.3389 / fmicb.2019.02548 [Overview of the project] [Problems that the invention aims to solve]

[0005] Therefore, this disclosure aims to provide, for example, a novel microorganism capable of degrading biodegradable plastics, particularly polylactic acid (PLA). [Means for solving the problem]

[0006] To achieve the aforementioned objective, the microorganisms described herein belong to the family Bacillaceae and are deposited under accession number NITE BP-04272.

[0007] The microorganisms disclosed herein are mutant strains of the microorganisms disclosed herein.

[0008] The microorganisms disclosed herein belong to the family Bacillaceae. The 16S rRNA gene includes a gene consisting of the following (R) nucleotide sequence: (R) The base sequence of (R1) or (R2) below (R1) The base sequence shown in Sequence ID No. 1; (R2) A nucleotide sequence that has 90% or more identity with the nucleotide sequence shown in Sequence ID No. 1.

[0009] The microorganisms disclosed herein belong to the family Bacillaceae. It has the following characteristics (1) to (10): (1) A rod-shaped bacterium that is Gram-stain positive and forms spores; (2) The growing temperature is 35-65°C; (3) The optimal pH for growth is 7-10; (4) The growth salt concentration is 0-4%; (5) The catalase reaction is positive; (6) The oxidase reaction is positive; (7) Having metabolic activity of D-arabinose, L-arabinose, D-ribose, D-xylose, L-xylose, D-fructose, L-sorbose, N-acetylglucosamine, arbutin, ferric esculincote, D-cellobiose, D-maltose, D-lactose, D-melibiose, D-turanose, D-lyxose, D-tagatose, potassium 2-ketogluconate, and / or potassium 5-ketogluconate; (8) Having L-arginine, sodium citrate, gelatin, and / or esculin metabolic activity; (9) Having esterase activity, esterase lipase activity, and / or naphthol-AS-BI-phosphohydrolase activity; (10) Has the ability to decompose polylactic acid (PLA).

[0010] The composition for use in the decomposition of biodegradable plastics of this disclosure comprises the microorganisms of this disclosure.

[0011] The method for processing biodegradable plastics according to the present disclosure includes the step of bringing the biodegradable plastics into contact with the microorganisms according to the present disclosure, thereby decomposing the biodegradable plastics with the microorganisms. [Effects of the Invention]

[0012] According to this disclosure, for example, it is possible to provide novel microorganisms capable of degrading biodegradable plastics, particularly polylactic acid (PLA). [Brief explanation of the drawing]

[0013] [Figure 1] Figure 1 shows the phylogenetic tree of komham206 based on the partial nucleotide sequence of 16S rRNA in Example 1. [Figure 2] Figure 2 is a photograph showing the results of morphological observation of Komham206 using an optical microscope in Example 1. [Figure 3] Figure 3 is a photograph showing the Gram staining results of Komham 206 in Example 1. [Figure 4]Figure 4 is a photograph showing the results of the second-stage bacterial test using API (registered trademark) 50CH in Example 1. [Figure 5] Figure 5 is a photograph showing the results of the second-stage bacterial test using API (registered trademark) 20E and API (registered trademark) 20NE in Example 1. [Figure 6] Figure 6 is a photograph showing the results of the second-stage bacterial test using API (registered trademark) ZYM in Example 1. [Figure 7] Figure 7 shows the results of dDDH analysis in Example 1. [Figure 8] Figure 8 is a photograph showing the results of polylactic acid degradation in Example 1. MODE FOR CARRYING OUT THE INVENTION

[0014] Hereinafter, the present disclosure will be specifically described with reference to examples. Unless otherwise specified, each description herein may incorporate the descriptions of other disclosures.

[0015] As a result of intensive research, the present inventors found a novel microorganism having degradation activity against biodegradable plastics such as PLA, and established the present disclosure by isolating and identifying the microorganism. Specifically, as a result of morphological observation, physiological and biochemical property tests, partial nucleotide sequence analysis of 16S rRNA, and digital DNA-DNA hybridization (dDDH) analysis of genomic nucleotide sequences, although it was confirmed that the microorganism of the present disclosure belongs to the family Bacillaceae, it does not belong to any known genera within Bacillaceae, and was found to be a microorganism of a novel genus. Furthermore, as a result of intensive research, the present inventors found that the microorganism exhibits the ability to degrade biodegradable plastics, and thus established the present disclosure. Therefore, the microorganism of the present disclosure is expected to be used, for example, in the degradation treatment of biodegradable plastics.

[0016] The microorganisms disclosed herein are novel species belonging to the family Bacillaceae. The microorganisms disclosed herein include, for example, the strain or subsequent lineage deposited under accession number NITE BP-04272 (hereinafter also referred to as "komham206").

[0017] <komham206> The Komham206 or its successor lines as disclosed herein have, for example, the following characteristics (1) to (9). (1) A rod-shaped bacterium that is Gram-stain positive and forms spores; (2) The growing temperature is 35-65°C; (3) The optimal pH for growth is 7-10; (4) The growth salt concentration is 0-4%; (5) The catalase reaction is positive; (6) The oxidase reaction is positive; (7) Having metabolic activity of D-arabinose, L-arabinose, D-ribose, D-xylose, L-xylose, D-fructose, L-sorbose, N-acetylglucosamine, arbutin, ferric esculincote, D-cellobiose, D-maltose, D-lactose, D-melibiose, D-turanose, D-lyxose, D-tagatose, potassium 2-ketogluconate, and / or potassium 5-ketogluconate; (8) Having L-arginine, sodium citrate, gelatin, and / or esculin metabolic activity; (9) Possessing esterase activity, esterase lipase activity, and / or naphthol-AS-BI-phosphohydrolase activity.

[0018] The komham206 of this disclosure may have one of the characteristics (1) to (9) above, or it may have more than one of the characteristics, or it may have all of the characteristics. For example, the komham206 of this disclosure may have one or more of the metabolic activities in (7) above, preferably all of the metabolic activities. For example, the komham206 of this disclosure may have one or more of the metabolic activities in (8) above, preferably all of the metabolic activities. For example, the komham206 of this disclosure may have one or more of the activities in (9) above, preferably all of the activities.

[0019] The characteristics described in (1) to (9) above can be measured in accordance with Examples 1(5) to (6) described later.

[0020] The komham206 of this disclosure may further have, for example, the following characteristics (10). (10) Has the ability to decompose polylactic acid.

[0021] The komham206 disclosed herein may, for example, be a strain (deposited lineage) or a descendant lineage deposited under accession number NITE BP-04272. The deposit information is shown below. Type of deposit: International deposit Depository name: National Institute of Technology and Evaluation (NITE), Patent Microbial Depository Center Address: Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture 292-0818, Japan (komham206) Accession Number: NITE BP-04272 Identification mark: komham206 Entrustment date: January 22, 2025

[0022] The komham206 of this disclosure includes, for example, a gene consisting of the following base sequence (R) as a 16S rRNA gene, and preferably a gene consisting of the following base sequence (R1), i.e., a 16S rRNA gene consisting of the base sequence shown in Sequence ID No. 1.

[0023] (R) The base sequence of (R1) or (R2) below (R1) The base sequence shown in Sequence ID No. 1; (R2) A nucleotide sequence that has 90% or more identity with the nucleotide sequence shown in Sequence ID No. 1.

[0024] In (R1) above, the base sequence of Sequence ID No. 1 is a base sequence that encodes 16S RNA. The base sequence of Sequence ID No. 1 can be isolated, for example, from komham206.

[0025] The base sequence of the 16S rRNA gene of komham206 (SEQ ID NO: 1)

[0026] In (R2) above, "identity" means, for example, that a microorganism having a 16S rRNA gene containing the base sequence of (R2) maintains the characteristics of komham206. The "identity" of (R2) means, for example, that the base sequence of (R1) is 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.94% or more, 99.95% or more, 99.96% or more, 99.97% or more, 99.98% or more, or 99.99% or more. The "identity" can be determined by aligning two base sequences or amino acid sequences (the same applies hereinafter). The alignment can be calculated using default parameters, for example, with BLAST, FASTA, etc.

[0027] The 16S rRNA of komham206 in this disclosure preferably has an identity of, for example, less than 98.7%, 98.6% or less, 98.5% or less, or 98.4% or less compared to the base sequence of a known microorganism's 16S rRNA gene.

[0028] Examples of closely related species to komham206 in this disclosure include bacteria of the genus Bacillus.

[0029] The komham206 of this disclosure has, for example, genomic DNA consisting of the nucleotide sequence shown in Sequence ID No. 2.

[0030] The identification of bacterial species using the genomic DNA of a bacterial strain may be performed, for example, by digital DNA-DNA hybridization (dDDH) analysis. The dDDH analysis is a classification method based on the sequence distance between genomes. In the dDDH analysis, if the homology value is 70% or more, it can be determined that the genomes belong to the same bacterial species, and if the homology value is 70-80%, it can be determined that they are subspecies of the same species. The dDDH analysis can be performed using, for example, a Type (Strain) Genome Server (TYGS, https: / / tygs.dsmz.de / ).

[0031] Preferably, the dDDH values ​​of the komham206 genome sequence (SEQ ID NO: 2) of this disclosure and the genome sequences of known microorganisms are less than 70%, less than 65%, less than 60%, less than 55%, less than 50%, less than 45%, and less than 40%. The known microorganisms include, for example, Compostibacillus humi, Ornithinibacillus hominis, Caldalkalibacillus thermarum, Planococcus salinus, Halalkalibacter akibai, Ureibacillus thermosphaericus, Planococcus mcmeekinii, Halobacillus naozhouensis, Lysinibacillus alkalisoli, Insulibacter Examples include thermoxylanivorax, Savagea serpentis, Robertmurraya mangrovi, Planococcus koreensis, Ornithinibacillus bavariensis, Anoxybacillus ayderensis, Anoxybacillus thermarum, Geobacillus proteiniphilus, Ectobacillus ponti, Oceanobacillus limi, Anoxybacillus kamchatkensis, and the like.

[0032] The komham206 disclosed herein preferably contains genomic DNA having dDDH values ​​of 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, and 99% or more with respect to the base sequence shown in SEQ ID NO: 2.

[0033] The komham206 of this disclosure includes, for example, a gene consisting of the base sequence (G) below as genomic DNA, and preferably a gene consisting of the base sequence (G1) below, i.e., genomic DNA consisting of the base sequence shown in Sequence ID No. 2.

[0034] (G) The base sequence of (G1), (G2), or (G3) below (G1) The base sequence shown in Sequence ID No. 2; (G2) A nucleotide sequence in which one or more nucleotides are deleted, substituted, inserted, and / or added in the nucleotide sequence shown in Sequence ID No. 2; (G3) A nucleotide sequence that has 80% or more identity with the nucleotide sequence shown in Sequence ID No. 2.

[0035] In (G1) above, the base sequence of Sequence ID No. 2 is a base sequence that encodes genomic DNA. The base sequence of Sequence ID No. 2 can be isolated from, for example, Komham206.

[0036] In (G2) above, "one or several" means, for example, that the microorganisms containing genomic DNA having the base sequence of (G2) above maintain the characteristics of komham206. The characteristics of komham206 include, for example, the characteristics of (1) to (10) above, preferably the characteristic of (10) above (the same applies hereinafter). The "one or several" in (G2) above refers to, for example, 1 to 525,852, 1 to 394,389, 1 to 262,926, 1 to 131,463, 1 to 105,170, 1 to 78,877, 1 to 52,585, 1 to 38,438, 1 to 26,292, 1 to 13,146, 1 to 10,517, 1 to 7,887, 1 to 5,258, 1 to 1,314, or 1 to 1,051 in the base sequence of (G1).

[0037] In (G3) above, "identity" means, for example, that a microorganism containing genomic DNA having the base sequence of (G3) maintains the characteristics of komham206. The "identity" in (G3) means, for example, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, and 99% or more in the base sequence of (G1).

[0038] The komham206 of this disclosure may include, for example, genes expressing hypothetical protein, isochorismatase family protein, YaaR family protein, DUF4367 domain-containing protein, acyl-CoA dehydrogenase, and / or YitT family protein. These genes are presumed to contribute to the biodegradable plastic degradation activity characteristics of the komham206 of this disclosure.

[0039] The komham206 of this disclosure includes, for example, the following (Pa) polynucleotides. (Pa) Any of the following polynucleotides (Pa1) to (Pa3): (Pa1) A polynucleotide encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO: 6; (Pa2) A polynucleotide encoding a polypeptide consisting of an amino acid sequence in which one or more amino acids are deleted, substituted, inserted, and / or added in the amino acid sequence of SEQ ID NO: 6; (Pa3) A polynucleotide encoding a polypeptide consisting of an amino acid sequence that has more than 80% identity with the amino acid sequence of SEQ ID NO: 6.

[0040] The base sequence of the polynucleotide (Pa1) can be designed, for example, by replacing the corresponding codons based on the amino acid sequence of Sequence ID No. 6. The amino acid sequence of Sequence ID No. 6 can be obtained, for example, from komham206 deposited under accession number NITE BP-04272.

[0041] Amino acid sequence of hypothetical protein (SEQ ID NO: 6) MIVQIKGNVKFPITLDPSVWIFDDRKIRLEDAFANKTSENVESEETDAKKMAEMFEKDVQSGVIPPGKQPVKRIDKEKVLSESYVMPLKPFLKSAEINEGAKSARLIADDEEIIISIEQLMDSLARFSDSGKPLKDDGPIHIYFGDGSNREQPFKGIKQIIIE

[0042] The "one or several" in (Pa2) above refers to, for example, 1 to 32, 1 to 24, 1 to 16, 1 to 8, 1 to 6, 1 to 4, 1 to 3, 1 or 2, or 1 in the amino acid sequence of Sequence ID No. 6.

[0043] The "identity" in (Pa3) above is, for example, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, and 99% or more with respect to the amino acid sequence of Sequence ID No. 6.

[0044] The komham206 of this disclosure includes, for example, the following polynucleotide (Pb). (Pb) Any of the following polynucleotides (Pb1) to (Pb3): (Pb1) A polynucleotide encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO: 7; (Pb2) A polynucleotide encoding a polypeptide consisting of an amino acid sequence in which one or more amino acids are deleted, substituted, inserted, and / or added to the amino acid sequence of Sequence ID No. 7; (Pb3) A polynucleotide encoding a polypeptide consisting of an amino acid sequence that has more than 80% identity with the amino acid sequence of Sequence ID No. 7.

[0045] The base sequence of the polynucleotide (Pb1) can be designed, for example, by replacing the corresponding codons based on the amino acid sequence of Sequence ID No. 7. The amino acid sequence of Sequence ID No. 7 can be obtained, for example, from komham206 deposited under accession number NITE BP-04272.

[0046] Amino acid sequence of isochorismatase family protein (SEQ ID NO: 7) MITVEDTALIVVDVQGKLAEIVHESEFVLGQIEKLIRGAQILDVPIIWMEQYPEGLGRTNDRLLKHLENERYVTKRTFSACLEASFLEELQNLKRKSYLVAGIEAHVCVYQTVRDLLKRDYEVEVVLDAVSSRTELNRTIGIEKMKKLGASITSVEMALFELMQTSKHPKFKEVLEIIK

[0047] The "one or several" in (Pb2) above refers to, for example, 1 to 35, 1 to 26, 1 to 17, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 3, 1 or 2, or 1 in the amino acid sequence of Sequence ID No. 7.

[0048] The "identity" of (Pb3) above is, for example, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, and 99% or more with respect to the amino acid sequence of Sequence ID No. 7.

[0049] The komham206 of this disclosure includes, for example, the following polynucleotides (Pc). (Pc) Any of the following polynucleotides (Pc1) to (Pc3): (Pc1) A polynucleotide encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO: 8; (Pc2) A polynucleotide encoding a polypeptide consisting of an amino acid sequence in which one or more amino acids are deleted, substituted, inserted, and / or added to the amino acid sequence of Sequence ID No. 8; (Pc3) A polynucleotide encoding a polypeptide consisting of an amino acid sequence that has more than 80% identity with the amino acid sequence of Sequence ID No. 8.

[0050] The base sequence of the polynucleotide (Pc1) can be designed, for example, by replacing the corresponding codons based on the amino acid sequence of Sequence ID No. 8. The amino acid sequence of Sequence ID No. 8 can be obtained, for example, from komham206 deposited under accession number NITE BP-04272.

[0051] Amino acid sequence of YaaR family protein (SEQ ID NO: 8) MMKIDRELRTNVEQKPIHKKTASKQNFEQIVRTKATHMKQQDLEKLLQDITEQGKKVARFRSFKDLARYKRLIQQFLEEAVFDGLSVKETRNFNPSNFSHKLITVEKIDEKLIQLTDDLLDQEKKTVDLLALIGEIEGLLVNLYM

[0052] The "one or several" in (Pc2) above refers to, for example, 1 to 29, 1 to 21, 1 to 14, 1 to 7, 1 to 5, 1 to 4, 1 to 3, 1 or 2, or 1 in the amino acid sequence of Sequence ID No. 8.

[0053] The "identity" of (Pc3) above is, for example, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, and 99% or more with respect to the amino acid sequence of Sequence ID No. 8.

[0054] The komham206 of this disclosure includes, for example, the following polynucleotide (Gd). (Pd) Any of the following polynucleotides (Pd1) to (Pd3): (Pd1) A polynucleotide encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO: 9; (Pd2) A polynucleotide encoding a polypeptide consisting of an amino acid sequence in which one or more amino acids are deleted, substituted, inserted, and / or added to the amino acid sequence of Sequence ID No. 9; (Pd3) A polynucleotide encoding a polypeptide consisting of an amino acid sequence that has more than 80% identity with the amino acid sequence of Sequence ID No. 9.

[0055] The base sequence of the polynucleotide (Pd1) can be designed, for example, by replacing the corresponding codons based on the amino acid sequence of Sequence ID No. 9. The amino acid sequence of Sequence ID No. 9 can be obtained, for example, from komham206 deposited under accession number NITE BP-04272.

[0056] Amino acid sequence of DUF4367 domain-containing protein (SEQ ID NO: 9) MKQLKLVVFLFVVVPLFFGCRSLKSFSPEQIIENAIMAEVQGDVSYYAEIDMKITSKNESLNEVATIKEWRRNGLVRNELISDKEGEVIITANENDIHMYFVDKKKIVKTTMEDVGQYVLSPKEQFHELLNLLRKTHDIETVGKATIADRPAFHLKATTREGQNSIYGNIDLWIDVEYWLP LKMIMKSGSLELVMEFTEIDYDATFDDALFVLGEVEDAEIEVINSAPETLELTLEEVPEHFGKPVYVLEENDRWKISSIHLTKADSIQEHDLLQIDYTFNGVPSLSLLISQFQESDPTVDVFDDVTKKVTVRNQEGYLIDSAEIVMLSWRENGLEYAVQLINPKIDINELFKLAESMMTIK

[0057] The "one or several" in (Pd2) above refers to, for example, 1 to 72, 1 to 54, 1 to 36, 1 to 18, 1 to 14, 1 to 10, 1 to 7, 1 to 4, 1 to 3, 1 or 2, or 1 in the amino acid sequence of Sequence ID No. 9.

[0058] The "identity" of (Pd3) above is, for example, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, and 99% or more with respect to the amino acid sequence of Sequence ID No. 9.

[0059] The komham206 of this disclosure includes, for example, the following polynucleotides (Pe). (Pe) Any of the following polynucleotides (Pe1) to (Pe3): (Pe1) A polynucleotide encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO: 10; (Pe2) A polynucleotide encoding a polypeptide consisting of an amino acid sequence in which one or more amino acids are deleted, substituted, inserted, and / or added in the amino acid sequence of SEQ ID NO: 10; (Pe3) A polynucleotide encoding a polypeptide consisting of an amino acid sequence that has more than 80% identity with the amino acid sequence of SEQ ID NO: 10.

[0060] The base sequence of the polynucleotide (Pe1) can be designed, for example, by replacing the corresponding codons based on the amino acid sequence of Sequence ID No. 10. The amino acid sequence of Sequence ID No. 10 can be obtained, for example, from komham206 deposited under accession number NITE BP-04272.

[0061] Amino acid sequence of Acyl-CoA dehydrogenase (SEQ ID NO: 10) MHVDENTYSFDEFLKARDEYNDFLDNEFLQLAAKHYIKDEWEQLYEKIKTLSEETSFHYRHIANEISKIENQPKIEHYNAYNKRVDRIIRTKQQADMEEDLFKRALFSKETSKWEQVLSRFLFHHNGEAGIMCP VACTDGLVDILRTFEHELNDELKEILRHVTEGIDGDFGIGAQFMTEIQGGSNIPANVLKAVKAGDHWRLYGSKFFCSAIHADYSVVTARVDNTEHVGTFIVPLWKTRERKERNHLVINRLKSKLGTTELASAEV IFNGAKAYQIGPLEKGVAIAVGIVLTKSRLDIGAASSAFMLRAVREALQYSQFREVFGRRIEDFPLAKGQLLDMEHTAKRTTAAVFKIYDEFFKQENWYHKQLSEEEKKRQFVLRELILLQKIKAAHDTVDTVR TAISIFGGNGVIEDFSSLPRLFRDAMVNELWEEGPKNVLLAQIHRDIKRASKWYEPKAFIKDLLEGVDGQIVERFQERMQSLMDIDLYAEPNEQTIRDAKEWARFCDELFYEYQEQAWKEIGDAPIVKTYSFKKQL

[0062] The "one or several" in (Pe2) above refers to, for example, 1 to 107, 1 to 80, 1 to 53, 1 to 26, 1 to 21, 1 to 16, 1 to 10, 1 to 7, 1 to 5, 1 to 4, 1 to 3, 1 or 2, or 1 in the amino acid sequence of Sequence ID No. 10.

[0063] The "identity" of (Pe3) above is, for example, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, and 99% or more with respect to the amino acid sequence of Sequence ID No. 10.

[0064] The komham206 of this disclosure includes, for example, the polynucleotide (Pf) described below. (Pf) Any of the following polynucleotides (Pf1) to (Pf3): (Pf1) A polynucleotide encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO: 11; (Pf2) A polynucleotide encoding a polypeptide consisting of an amino acid sequence in which one or more amino acids are deleted, substituted, inserted, and / or added to the amino acid sequence of SEQ ID NO: 11; (Gf3) A polynucleotide encoding a polypeptide consisting of an amino acid sequence that has more than 80% identity with the amino acid sequence of SEQ ID NO: 11.

[0065] The base sequence of the polynucleotide (Pf1) can be designed, for example, by replacing the corresponding codons based on the amino acid sequence of Sequence ID No. 11. The amino acid sequence of Sequence ID No. 11 can be obtained, for example, from komham206 deposited under accession number NITE BP-04272.

[0066] Amino acid sequence of YitT family protein (SEQ ID NO: 11) MFGLKLKNIFFILVGSAIFSFGVVHFNMQNDLGEGGFTGITLLLYFLFKWDPSITYLLLNIPVFFIGWKVLGRIITFIYTLIGTFAVSGFLNLFQIKPFDINLESDMTLAALFAGAFVGVGLGVIFRYGGTTGGVDIIARIVNKY FGWSMGKTLFVFDFFVIGTSIFTYLDLVQGMYTLLAVYVGVRVIDLIQEGAYSARGATIISKKYAEIADKINHEMERGVTVFNAKGHYTQEIQKVLYCVVGKNEIVKLKNIILEVDPHAFVSVSSVHEVMGEGFTLDEKKNPIYP

[0067] The "one or several" in (Pf2) above refers to, for example, 1 to 57, 1 to 43, 1 to 28, 1 to 14, 1 to 11, 1 to 8, 1 to 5, 1 to 4, 1 to 3, 1 or 2, or 1 in the amino acid sequence of Sequence ID No. 11.

[0068] The "identity" of (Pf3) above is, for example, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, and 99% or more with respect to the amino acid sequence of Sequence ID No. 11.

[0069] The komham206 of this disclosure may be identified as belonging to a taxonomic group by, for example, bacterial morphological observation, first-stage bacterial testing, and second-stage bacterial testing. Examples of bacterial morphological observation include observation of colony color, cell morphology, Gram staining ability, and spore formation ability. Examples of first-stage bacterial testing include observation of colony characteristics, cell morphology, and motility, as well as physiological and biochemical characterization tests such as catalase, oxidase, and O / F tests. Examples of second-stage bacterial testing include tests for assimilation of carbon sources, oxidation / fermentation ability, and enzyme activity using physiological and biochemical characterization test kits. The komham206 of this disclosure exhibits the morphological characteristics and physiological and biochemical properties shown in the results of bacterial morphological observation, first-stage bacterial testing, and second-stage bacterial testing in Example 1 described below.

[0070] The komham206 of this disclosure can be subcultured, for example, by culturing it according to the culture conditions of Example 1 described below. This makes it possible to obtain progeny lines of the komham206.

[0071] <mutant strain> The microorganisms of this disclosure may be, for example, komham206 or a mutant strain of its progeny.

[0072] The mutant strains of this disclosure are, for example, fungal cells that maintain the taxonomic properties of a new species of komham206. These properties include, for example, the characteristics described in (1) to (10) above. The mutant strains may have, for example, one of the characteristics described in (1) to (10), multiple characteristics, or all of them. The description of komham206 in this disclosure can be applied to the mutant strains of this disclosure.

[0073] The mutant strains described herein can be obtained, for example, by mutation into komham206 or by introduction of exogenous genes. The mutation can be induced, for example, by introducing a mutation using a conventional method. The method for introducing the mutation can be, for example, homologous recombination; genome editing technology using ZFN, TALEN, CRISPR-CAS9, CRISPR-CPF1, etc. The method for introducing the mutation may also be carried out by a mutagenesis method such as site-directed mutagenesis. Alternatively, the method for introducing the mutation may be carried out by a random mutagenesis method. Examples of random mutagenesis methods include irradiation with alpha rays, beta rays, gamma rays, X-rays, etc.; chemical treatment with mutagenic agents such as ethyl methanesulfonate (EMS) and ethynylnitrosourea (ENU); heavy ion beam treatment, etc.

[0074] The aforementioned mutant strain contains a gene consisting of the following (R) nucleotide sequence as the 16S rRNA gene: (R) The base sequence of (R1) or (R2) below (R1) The base sequence shown in Sequence ID No. 1; (R2) A nucleotide sequence that has 98.72% or more identity with the nucleotide sequence shown in Sequence ID No. 1.

[0075] The above (R1) to (R2) can be explained by reference to the explanation in komham206 of this disclosure.

[0076] The mutant strain, for example, includes a gene consisting of the base sequence (G) below as genomic DNA, and preferably has a gene consisting of the base sequence (G1) below, i.e., genomic DNA consisting of the base sequence shown in Sequence ID No. 2.

[0077] (G) The base sequence of (G1), (G2), or (G3) below (G1) The base sequence shown in Sequence ID No. 2; (G2) A nucleotide sequence in which one or more nucleotides are deleted, substituted, inserted, and / or added in the nucleotide sequence shown in Sequence ID No. 2; (G3) A nucleotide sequence that has 80% or more identity with the nucleotide sequence shown in Sequence ID No. 2.

[0078] The explanations in (G1) to (G3) above can be found by reference to those in komham206 of this disclosure.

[0079] The aforementioned mutant strain may have, for example, one of the polynucleotides (Pa) to (Pf) described above, multiple polynucleotides, or all of the polynucleotides. The polynucleotides (Pa) to (Pf) can be described by reference to the explanation in komham206 of this disclosure.

[0080] <Methods for disposing of biodegradable plastics> In another embodiment, the Disclosure provides a method for processing biodegradable plastics. The Method for Processing Biodegradable Plastics of the Disclosure (hereinafter also referred to as the "Processing Method") uses the microorganisms of the Disclosure. The Processing Method of the Disclosure includes a step of bringing the microorganisms of the Disclosure into contact with the biodegradable plastic, thereby degrading the biodegradable plastic by the microorganisms (hereinafter also referred to as the "Degradation Step"). The Processing Method of the Disclosure is characterized by the use of the microorganisms of the Disclosure, and other steps and conditions are not particularly limited.

[0081] In this disclosure, "biodegradable plastic" means a plastic that is ultimately decomposed into CO2 or water by microorganisms or the like. Examples of biodegradable plastics include polylactic acid (PLA), polybutylene adipate terephthalate (PBAT), polyhydroxybutyrate (PHB), polycaprolactone (PCL), polybutylene succinate (PBS), polyethylene terephthalate (PET), etc., and polylactic acid is preferred. Examples of polylactic acid include homopolymers of lactic acid such as poly-L-lactic acid (PLLA) and / or poly-D-lactic acid (PDLA), copolymers containing constituent units derived from lactic acid, etc.

[0082] In the decomposition step, the object to be decomposed may include the biodegradable plastic, may consist solely of the biodegradable plastic, or may include the biodegradable plastic and other objects to be processed (e.g., food waste, metals, etc.). The biodegradable plastic may be, for example, a substance formed solely from biodegradable plastic, or a substance that includes other substances such as non-biodegradable plastic in addition to the biodegradable plastic. Examples of substances formed from the biodegradable plastic include tableware, cutlery, packaging containers, trays, bags, mulch films, and packaging materials.

[0083] In the decomposition process, contact between the microorganisms and the biodegradable plastic can be carried out, for example, by mixing. The mixing can be carried out mechanically, for example, using a stirrer or the like.

[0084] In the decomposition step, the decomposition temperature is, for example, a temperature at which the microorganisms of this disclosure can process the biodegradable plastic. The decomposition temperature is, for example, 35 to 65°C, and preferably 50 to 65°C.

[0085] In the decomposition step, the decomposition pH (decomposition pH) is, for example, a pH at which the microorganisms of this disclosure can process the biodegradable plastic. The decomposition pH is, for example, pH 7 to 10, and preferably pH 9 to 10.

[0086] In the decomposition step, for example, oxygen may be supplied. This supply can be carried out, for example, by stirring a mixture of the biodegradable plastic and the microorganisms of the Disclosure. The time interval for the supply is, for example, 1 to 10 hours, preferably 5 to 8 hours, and more preferably 8 hours. The supply time is, for example, 1 to 30 minutes, preferably 1 to 10 minutes, and more preferably 5 minutes.

[0087] In the decomposition step, the moisture content of the mixture of the biodegradable plastic and the microorganisms of this disclosure is, for example, 40 to 70%, preferably 50 to 70%, and more preferably 55 to 65%. [Examples]

[0088] Next, examples of the present invention will be described. However, the present invention is not limited to the following examples. Commercial reagents were used according to their respective protocols unless otherwise specified. Note that "mol / l" may also be denoted as "M".

[0089] [Example 1] We identified a novel microorganism in this disclosure and confirmed that it can decompose biodegradable plastics.

[0090] (1) Isolation of komham206 Compost from a waste disposal site in Hokkaido, Japan, was obtained as a sample. 8 g of hyperpeptone, 3 g of yeast extract, 1 g of dipotassium phosphate, and 0.25 g of ammonium chloride were dissolved in 900 ml of distilled water. After dissolution, 100 ml of alkaline buffer (sodium carbonate, pH 9.0) was added to prepare PYA medium (pH 9.0). After preparation, the sample was diluted with distilled water and inoculated into the PYA medium. After inoculation, it was cultured under conditions of 50°C. After culturing, the colonies that grew on the medium were isolated. After isolation, the colonies were inoculated into the PYA medium and culture was repeated. Subsequently, isolation and purification were performed to isolate a novel microorganism, komham206, which was deposited. The presence of this novel microorganism was also confirmed in several other compost samples in Hokkaido.

[0091] (2) Determination of the nucleotide sequence of komha206 16S rRNA To identify the microorganism komham206 isolated in Example 1(1), the nucleotide sequence of 16S rRNA was analyzed. Specifically, DNA was extracted from the isolated komham206. After extraction, the following primer set (Fasmac, synthesized by request) and KOD One® PCR Master Mix (TOYOBO) were added to the DNA to prepare a reaction solution. After preparation, the 16S rRNA gene of the DNA was amplified using a thermal cycler (T100 thermal cycler, BIO-RAD). After amplification, DNA sequencing was performed using the obtained PCR product to obtain the nucleotide sequence of 16S rRNA from komham206 isolated in Example 1(1). The nucleotide sequence was the nucleotide sequence represented by Sequence ID No. 1.

[0092] • 27F primer (SEQ ID NO: 3) 5'-AGAGTTTGATCMTGGCTCAG-3' • 1492R primer (SEQ ID NO: 4) 5'-GGYTACCTTGTTACGACTT-3'

[0093] (3) Comparison with the 16S rRNA base sequence of existing microorganisms Using the 16S rRNA sequences obtained in Example 1(2) above, a comparison was made between the 16S rRNA of komham206 and the 16S rRNA of existing microorganisms. Specifically, TYGS was used for the comparison. By performing a homology search of TYGS, microorganisms having sequences identical or similar to the 16S rRNA sequence of komham206 were searched for. As a result of the search, microorganisms having the sequence represented by Sequence ID No. 5, belonging to the genus Bacillus ( Bacillus It was found that the Bacillus thermocloacae (Z26939) strain (sp.) has a 16S rRNA sequence that is closest to the 16S rRNA sequence of komham206.

[0094] Base sequence of the 16S rRNA gene of Bacillus thermocloacae (Z26939) strain (SEQ ID NO: 5)

[0095] (4) Construction of a phylogenetic tree Furthermore, based on the homology search results of Example 1(3) above, a phylogenetic tree was constructed based on the partial nucleotide sequence of 16S rRNA, and the phylogenetic position of komham206 was analyzed. TYGS was used to construct the phylogenetic tree. These results are shown in Figure 1.

[0096] Figure 1 shows the phylogenetic tree of komham206 based on partial nucleotide sequences of 16S rRNA. In Figure 1, the scale bar indicates the number of nucleotide substitutions.

[0097] (5) Examination of taxonomic properties 1 Morphological observation and physiological characterization tests (hereinafter also referred to as "first-stage bacterial testing") were performed on komham206 to investigate its taxonomic properties. Specifically, komham206 was cultured aerobically at 50°C for 24 hours. After the culture, morphological observation was performed using a light microscope, and tests were conducted on catalase reaction, oxidase reaction, acid / gas production from glucose, and oxidation / fermentation (O / F) of glucose based on the method described in Reference 4. The light microscope used was an Olympus BX50F4. These results are shown in Figures 2-3 and Table 1 below. Reference 4: Barrow GI, Feltham RKA. Cowan and Steel's Manual for the Identification of Medical Bacteria. 3rd edition. Cambridge: University Press; 1993.

[0098] [Table 1]

[0099] Figure 2 is a photograph showing the morphological results of komham206 observed with an optical microscope. The morphological observation with an optical microscope revealed that komham206 forms colonies as shown in Figure 2.

[0100] Figure 3 is a photograph showing the Gram staining results of komham206. In Figure 3, the scale bar represents 10 μm. In Figure 3, (A) shows the Gram staining results of komham206 cultured on PYA agar, and (B) shows the Gram staining results of komham206 cultured on SCD agar. As shown in Figure 3, komham206 was found to be a Gram-positive bacterium.

[0101] (6) Examination of taxonomic properties 2 Biochemical characterization tests (hereinafter also referred to as "second-stage bacterial testing") were conducted on komham206 to examine its taxonomic properties. Specifically, API® 50CH, API® 20E, API® 20NE, and API® ZYM (all manufactured by Biomerieux) were used for the second-stage bacterial testing. The results are shown in Figures 4 to 6 and Tables 2 to 3 below (+: activity present).

[0102] [Table 2]

[0103] Figure 4 is a photograph showing the results of the second bacterial test using API(registered trademark) 50CH. Table 2 above is a table showing the results of the second bacterial test using API(registered trademark) 50CH. As shown in Figure 4 and Table 2 above, komham206 was found to have metabolic activity for D-arabinose, L-arabinose, D-ribose, D-xylose, L-xylose, D-fructose, L-sorbose, N-acetylglucosamine, arbutin, ferric esculincote, D-cellobiose, D-maltose, D-lactose, D-melibiose, D-turanose, D-lyxose, D-tagatose, potassium 2-ketogluconate, and potassium 5-ketogluconate.

[0104] Figure 5 is a photograph showing the results of the second-stage bacterial test using API® 20E and API® 20NE. As shown in Figure 5, komham206 was found to have metabolic activity for L-arginine (ADH), sodium citrate (CIT), and gelatin (GEL), and to possess esculin hydrolysis activity and gelatinase activity.

[0105] [Table 3]

[0106] Figure 6 is a photograph showing the results of the second-stage bacterial test using API® ZYM. Table 3 above shows the results of the second-stage bacterial test using API® ZYM. As shown in Figure 6 and Table 3 above, komham206 was found to possess esterase activity, esterase lipase activity, and naphthol-AS-BI-phosphohydrolase activity.

[0107] (7) Determination of the base sequence of genomic DNA The genomic DNA sequence of komham206 was determined. Specifically, first, the genomic DNA of komham206 was extracted. Genomictip 20G (QIAGEN) was used for the extraction. After the extraction, a library was prepared using the SMRTbell® gDNA Sample Amplification Kit (PacBio) and the SMRTbell® Express Template Prep Kit 20 (PacBio) according to the procedure described in Procedure & Checklist - Preparing HiFi SMRTbell® Libraries from Ultra-Low DNA Input. After the preparation, polymerase complexes of the library were formed using the Revio® Polymerase kit (PacBio). After the formation, sequencing was performed using Revio® (PacBio). Subsequently, overhang adapter sequences were removed from the obtained sequences using SMRT® Link (ver. 13.0.0.207600) to produce subreads. After the above production, consensus sequences were prepared by aligning the subreads. After the above production, consensus sequences with an average quality value of less than 20 per read were removed to produce HiFi reads. After the above production, Ultra-Low PCR adapters were removed from the HiFi reads using lima (ver. 2.7.1). After the above removal, PCR duplicate reads were removed using pbmarkdup (ver. 1.0.3). After the above removal, reads of 1000 bases or less were deleted using Filtlong (ver. 0.2.1). After the above deletion, HiFi reads of 1000 bases or more were assembled using the default conditions of Flye (ver. 2.9.2-b1786). As a result, it was found that the genomic DNA of komham206 consists of the base sequence shown in Sequence ID No. 2.

[0108] (8) dDDH analysis of genome base sequences Further dDDH analysis was performed on komham206. Specifically, the dDDH values ​​were determined using TYGS for the genome sequence obtained in Example 1(7) above. These results are shown in Figure 7.

[0109] Figure 7 shows the results of the dDDH analysis. As shown in Figure 7, even the genomic DNA with the highest degree of identicality to the genomic DNA of komham206 had an identicality rate of 54.6%, which is less than 70%. From these results, it was found that komham206 is not a microorganism belonging to a known bacterial species, but rather a new species.

[0110] (9) Evaluation of the decomposition rate of biodegradable plastics (polylactic acid) The ability of komham206 to break down biodegradable plastics (polylactic acid) was investigated. Specifically, komham206 obtained in Example 1(1) was cultured in PYA liquid medium (pH 9) until full growth. After the culture, the cells were collected and inoculated into 10 ml of PYA liquid medium (pH 10). After inoculation, fragments of a straw containing biodegradable plastic (polylactic acid) (product name: Naturally Returning Straw, distributor: Daiso Industries Co., Ltd.) were added, and shaking culture was performed at 65°C and 160 rpm. Observations were made on the start day of the shaking culture (day 0), and on days 7, 13, 16, and 24 from the start of the shaking culture. These results are shown in Figure 8.

[0111] Figure 8 shows photographs illustrating the results of polylactic acid degradation. From left to right in Figure 8, the photographs are from day 0, day 7, day 13, day 16, and day 24. As shown in Figure 8, polylactic acid degradation was observed in komham206 compared to the control at days 13, 16, and 24 from the start of shaking culture. These results demonstrate that komham206 of this disclosure can decompose biodegradable plastics. Generally, biodegradable plastics are considered to have low biodegradability by microorganisms in aquatic environments. However, komham206 of this disclosure exhibits excellent biodegradability characteristics, including the ability to decompose polylactic acid even in aquatic environments. Furthermore, komham206 of this disclosure is expected to be able to decompose biodegradable plastics in compost.

[0112] (10) Proteome analysis Proteomic analysis was performed on komham206. Specifically, first, protein extraction was performed from the bacterial cells using the EasyPep® Magnetic MS Sample Prep Kit (Thermo Scientific, Cat. No. A57866). After extraction, the entire extracted sample was subjected to acetone precipitation. Following acetone precipitation, digestion and purification were performed using the EasyPep® Magnetic MS Sample Prep Kit (Thermo Scientific, Cat. No. A57866). Subsequently, LC-MS / MS analysis was performed on the recovered sample solution using Orbitrap Exploris® 480 (Thermo Fisher Scientific). A database search was performed using DIA-NN (Ver. 1.9) on the product ion measurement data of all MS / MS spectra obtained from the above analysis. The database used was the amino acid sequence estimated from the determined genome. Next, in order to identify the PLA-degrading enzyme, two patterns of intracellular protein profiles were compared: one with PLA added and one without. In the two sample patterns described above, 1200 unique proteins were detected in each. In addition, six proteins were identified as being expressed only upon the addition of PLA: hypothetical protein, isochorismatase family protein, YaaR family protein, DUF4367 domain-containing protein, acyl-CoA dehydrogenase, and YitT family protein.

[0113] Hypothetical protein (SEQ ID NO: 6) MIVQIKGNVKFPITLDPSVWIFDDRKIRLEDAFANKTSENVESEETDAKKMAEMFEKDVQSGVIPPGKQPVKRIDKEKVLSESYVMPLKPFLKSAEINEGAKSARLIADDEEIIISIEKLMDSLARFSDSGKPLKDDGPIHIYFGDGSNREQPFKGIKQIIIE

[0114] Isochorismatase family protein MITVEDTALIVVDVQGKLAEIVHESEFVLGQIEKLIRGAQILDVPIIWMEQYPEGLGRTNDRLLKHLENERYVTKRTFSACLEASFLEELQNLKRKSYLVAGIEAHVCVYQTVRDLLKRDYEVEVVLDAVSSRTELNRTIGIEKMKKLGASITSVEMALFELMQTSKHPKFKEVLEIIK

[0115] YaaR family protein (SEQ ID NO:8) MMKIDRELRTNVEQKPIHKKTASKQNFEQIVRTKATHMKQQDLEKLLQDITEQGKKVARFRSFKDLARYKRLIQQFLEEAVFDGLSVKETRNFNPSNFSHKLITVEKIDEKLIQLTDDLLDQEKKTVDLLALIGEIEGLLVNLYM

[0116] DUF4367 domain-containing protein MKQLKLVVFLFVVVPLFFGCRSLKSFSPEQIIENAIMAEVQGDVSYYAEIDMKITSKNESLNEVATIKEWRRNGLVRNELISDKEGEVIITANENDIHMYFVDKKKIVKTTMEDVGQYVLSPKEQFHELLNLLRKTHDIETVGKATIADRPAFHLKATTREGQNSIYGNIDLWIDVEYWLPLKMIMKSGSLELVMEFTEIDYDATFDDALFVLGEVEDAEIEVINSAPETLELTLEEVPEHFGKPVYVLEENDRWKISSIHLTKADSIQEHDLLQIDYTFNGVPSLSLLISQFQESDPTVDVFDDVTKKVTVRNQEGYLIDSAEIVMLSWRENGLEYAVQLINPKIDINELFKLAESMMTIK

[0117] Acyl-CoA dehydrogenase MHVDENTYSFDEFLKARDEYNDFLDNEFLQLAAKHYIKDEWEQLYEKITLSEETSFHYRHIANEISKIENQPKIEHYNAYNKRVDRIIRTKQQADMEEDLFKRALFSKETSKWEQVLSRFLFHNHNEAGIMCP VACTDGLVDILRTFEHLENDELKEILRHVTEGIDGDFGIGAQFMTEIQGGSNIPANVLKAVKAGDHWRLYGSKFFCSAIHADYSVVTARVDNTEHVGTFIVPLWKTRERKERNHLVINRLKSKLGTTELASAEV IFNGAKAYQIGPLEKGVAIAVGIVLTKSRLDIGAASSAFMLRAVREALQYSQFREVFGRRIEDFPLAKGQLLDMEHTAKRTTAAVFKIYDEFFKQENWYHKQLSEEEKKRQFVLRELILLQKIKAAHDTVDTVR TAISIFGGNGVIEDFSSLPRLFRDAMVNELWEGPKNVLLAQIHRDIKRASKWYEPKAFIKDLLEGVDGQIVERFQERMQSLMDIDLYAEPNEQTIRDAKEWARFCDELFYEYQEQAWKEIGDAPIVKTYSFKKQL

[0118] YitT family protein (SEQ ID NO: 11) MFGLKLKNIFFILVGSAIFSFGVVHFNMQNDLGEGGFTGITLLLYFLFKWDPSITYLLLNIPVFFIGWKVLGRIITFIYTLIGTFAVSGFLNLFQIKPFDINLESDMTLAALFAGAFVGVGLGVIFRYGGTTGGVDIIARIVNKY FGWSMGKTLFVFDFFVIGTSIFTYLDLVQGMYTLLAVYVGVRVIDLIQEGAYSARGATIISKKYAEIADKINHEMERGVTVFNAKGHYTQEIQKVLYCVVGKNEIVKLKNIILEVDPHAFVSVSSVHEVMGEGFTLDEKKNPIYP

[0119] While the present disclosure has been described above with reference to embodiments and examples, the present disclosure is not limited to the above embodiments and examples. Various modifications to the structure and details of the present disclosure are possible, as can be understood by those skilled in the art within the scope of the present disclosure.

[0120] <Note> Some or all of the above embodiments and examples may be described as follows, but are not limited to the following. <Deposited microorganisms> (Note 1) It belongs to the family Bacillaceae. Microorganisms deposited under accession number NITE BP-04272. (Note 2) A microorganism that is a mutant strain of the microorganism described in Appendix 1. (Note 3) The aforementioned mutant strain contains a gene consisting of the following (R) nucleotide sequence as the 16S rRNA gene, as described in Appendix 2 of the microorganism: (R) The base sequence of (R1) or (R2) below (R1) The base sequence shown in Sequence ID No. 1; (R2) A nucleotide sequence that has 90% or more identity with the nucleotide sequence shown in Sequence ID No. 1. (Note 4) The aforementioned mutant strain is a microorganism described in Appendix 2 or 3, having at least one characteristic selected from the group consisting of (1) to (8) and (9) below: (1) A rod-shaped bacterium that is Gram-stain positive and forms spores; (2) The growing temperature is 35-65°C; (3) The optimal pH for growth is 7-10; (4) The growth salt concentration is 0-4%; (5) The catalase reaction is positive; (6) The oxidase reaction is positive; (7) Having metabolic activity of D-arabinose, L-arabinose, D-ribose, D-xylose, L-xylose, D-fructose, L-sorbose, N-acetylglucosamine, arbutin, ferric esculincote, D-cellobiose, D-maltose, D-lactose, D-melibiose, D-turanose, D-lyxose, D-tagatose, potassium 2-ketogluconate, and / or potassium 5-ketogluconate; (8) Having L-arginine, sodium citrate, gelatin, and / or esculin metabolic activity; (9) Possessing esterase activity, esterase lipase activity, and / or naphthol-AS-BI-phosphohydrolase activity. (Note 5) Furthermore, microorganisms described in Appendix 4 that have the following characteristics (10): (10) Has the ability to decompose polylactic acid (PLA). (Note 6) The aforementioned mutant strain is a microorganism described in any of Appendix 2 to 5, which contains genomic DNA having a dDDH value of 70% or more relative to the base sequence shown in Sequence ID No. 2. <Microorganisms> (Note 7) It belongs to the family Bacillaceae. Microorganisms containing a gene consisting of the following (R) nucleotide sequence as a 16S rRNA gene: (R) The base sequence of (R1) or (R2) below (R1) The base sequence shown in Sequence ID No. 1; (R2) A nucleotide sequence that has 90% or more identity with the nucleotide sequence shown in Sequence ID No. 1. (Note 8) Microorganisms described in Appendix 7 having at least one characteristic selected from the group consisting of (1) to (9) and (10) below: (1) A rod-shaped bacterium that is Gram-stain positive and forms spores; (2) The growing temperature is 35-65°C; (3) The optimal pH for growth is 7-10; (4) The growth salt concentration is 0-4%; (5) The catalase reaction is positive; (6) The oxidase reaction is positive; (7) Having metabolic activity of D-arabinose, L-arabinose, D-ribose, D-xylose, L-xylose, D-fructose, L-sorbose, N-acetylglucosamine, arbutin, ferric esculincote, D-cellobiose, D-maltose, D-lactose, D-melibiose, D-turanose, D-lyxose, D-tagatose, potassium 2-ketogluconate, and / or potassium 5-ketogluconate; (8) Having L-arginine, sodium citrate, gelatin, and / or esculin metabolic activity; (9) Having esterase activity, esterase lipase activity, and / or naphthol-AS-BI-phosphohydrolase activity; (10) Has the ability to decompose polylactic acid (PLA). (Note 9) A microorganism as described in Appendix 7 or 8, containing genomic DNA having a dDDH value of 70% or more relative to the base sequence shown in Sequence ID No. 2. <Characteristics> (Note 10) It belongs to the family Bacillaceae. Microorganisms having the following characteristics (1) to (10): (1) A rod-shaped bacterium that is Gram-stain positive and forms spores; (2) The growing temperature is 35-65°C; (3) The optimal pH for growth is 7-10; (4) The growth salt concentration is 0-4%; (5) The catalase reaction is positive; (6) The oxidase reaction is positive; (7) Having metabolic activity of D-arabinose, L-arabinose, D-ribose, D-xylose, L-xylose, D-fructose, L-sorbose, N-acetylglucosamine, arbutin, ferric esculincote, D-cellobiose, D-maltose, D-lactose, D-melibiose, D-turanose, D-lyxose, D-tagatose, potassium 2-ketogluconate, and / or potassium 5-ketogluconate; (8) Having L-arginine, sodium citrate, gelatin, and / or esculin metabolic activity; (9) Having esterase activity, esterase lipase activity, and / or naphthol-AS-BI-phosphohydrolase activity; (10) Has the ability to decompose polylactic acid (PLA). (Note 11) It belongs to the family Bacillaceae. Microorganisms listed in Appendix 10 that contain a gene consisting of the following (R) nucleotide sequence as the 16S rRNA gene: (R) The base sequence of (R1) or (R2) below (R1) The base sequence shown in Sequence ID No. 1; (R2) A nucleotide sequence that has 90% or more identity with the nucleotide sequence shown in Sequence ID No. 1. (Note 12) It belongs to the family Bacillaceae. A microorganism as described in Appendix 10 or 11, containing genomic DNA having a dDDH value of 70% or more relative to the base sequence shown in Sequence ID No. 2. <Composition> (Note 13) A composition for use in the decomposition of biodegradable plastics, comprising any of the microorganisms described in Appendix 1 to 12. (Note 14) The biodegradable plastic is the composition described in Appendix 13, which includes polylactic acid. <Method> (Note 15) A method for treating biodegradable plastics, comprising the step of bringing a microorganism described in any of Appendix 1 to 12 into contact with the biodegradable plastic, thereby decomposing the biodegradable plastic with the microorganism. (Note 16) The biodegradable plastic is a polylactic acid-containing plastic, as described in Appendix 15. (Note 17) The processing method according to Appendix 15 or 16, wherein the decomposition is carried out at a temperature of 35 to 65°C. (Note 18) The treatment method according to any one of appendices 15 to 17, wherein the decomposition is carried out under conditions of pH 7 to 10. [Industrial applicability]

[0121] As described above, this disclosure provides a novel microorganism capable of decomposing biodegradable plastics, particularly polylactic acid (PLA). Therefore, the present invention is extremely useful, for example, in fields such as waste treatment.

Claims

1. It belongs to the family Bacillaceae. Microorganisms deposited under accession number NITE BP-04272.

2. A microorganism that is a mutant strain of the microorganism described in claim 1.

3. The mutant strain comprises a gene consisting of the following (R) nucleotide sequence as the 16S rRNA gene, according to claim 2: (R) The base sequence of (R1) or (R2) below (R1) The base sequence shown in Sequence ID No. 1; (R2) A nucleotide sequence that has 99.94% or more identity with the nucleotide sequence shown in Sequence ID No.

1.

4. The mutant strain has at least one characteristic selected from the group consisting of (1) to (8) and (9) below, according to claim 2 or 3: (1) A rod-shaped bacterium that is Gram-stain positive and forms spores; (2) The growing temperature is 35 to 65°C; (3) The optimal pH for growth is 7 to 10; (4) The growth salt concentration is 0-4%; (5) The catalase reaction is positive; (6) The oxidase reaction is positive; (7) Having metabolic activity of D-arabinose, L-arabinose, D-ribose, D-xylose, L-xylose, D-fructose, L-sorbose, N-acetylglucosamine, arbutin, ferric esculincote, D-cellobiose, D-maltose, D-lactose, D-melibiose, D-turanose, D-lyxose, D-tagatose, potassium 2-ketogluconate, and / or potassium 5-ketogluconate; (8) Having L-arginine, sodium citrate, gelatin, and / or esculin metabolic activity; (9) Having esterase activity, esterase lipase activity, and / or naphthol-AS-BI-phosphohydrolase activity.

5. Furthermore, the microorganism according to claim 4 having the following characteristics (10): (10) Has the ability to decompose polylactic acid (PLA).

6. The mutant strain comprises genomic DNA having a dDDH value of 70% or more with respect to the base sequence shown in Sequence ID No. 2, according to claim 2 or 3.

7. It belongs to the family Bacillaceae. Microorganisms containing a gene consisting of the following (R) nucleotide sequence as a 16S rRNA gene: (R) The base sequence of (R1) or (R2) below (R1) The base sequence shown in Sequence ID No. 1; (R2) A nucleotide sequence that has 99.94% or more identity with the nucleotide sequence shown in Sequence ID No.

1.

8. A microorganism according to claim 7 having at least one characteristic selected from the group consisting of (1) to (9) and (10) below: (1) A rod-shaped bacterium that is Gram-stain positive and forms spores; (2) The growing temperature is 35 to 65°C; (3) The optimal pH for growth is 7 to 10; (4) The growth salt concentration is 0-4%; (5) The catalase reaction is positive; (6) The oxidase reaction is positive; (7) Having metabolic activity of D-arabinose, L-arabinose, D-ribose, D-xylose, L-xylose, D-fructose, L-sorbose, N-acetylglucosamine, arbutin, ferric esculincote, D-cellobiose, D-maltose, D-lactose, D-melibiose, D-turanose, D-lyxose, D-tagatose, potassium 2-ketogluconate, and / or potassium 5-ketogluconate; (8) Having L-arginine, sodium citrate, gelatin, and / or esculin metabolic activity; (9) Having esterase activity, esterase lipase activity, and / or naphthol-AS-BI-phosphohydrolase activity; (10) Has the ability to decompose polylactic acid (PLA).

9. The microorganism according to claim 7 or 8, comprising genomic DNA having a dDDH value of 70% or more with respect to the base sequence shown in Sequence ID No.

2.

10. It belongs to the family Bacillaceae. Microorganisms having the following characteristics (1) to (10): (1) A rod-shaped bacterium that is Gram-stain positive and forms spores; (2) The growing temperature is 35 to 65°C; (3) The optimal pH for growth is 7 to 10; (4) The growth salt concentration is 0-4%; (5) The catalase reaction is positive; (6) The oxidase reaction is positive; (7) Having metabolic activity of D-arabinose, L-arabinose, D-ribose, D-xylose, L-xylose, D-fructose, L-sorbose, N-acetylglucosamine, arbutin, ferric esculincote, D-cellobiose, D-maltose, D-lactose, D-melibiose, D-turanose, D-lyxose, D-tagatose, potassium 2-ketogluconate, and / or potassium 5-ketogluconate; (8) Having L-arginine, sodium citrate, gelatin, and / or esculin metabolic activity; (9) Having esterase activity, esterase lipase activity, and / or naphthol-AS-BI-phosphohydrolase activity; (10) Has the ability to decompose polylactic acid (PLA).

11. It belongs to the family Bacillaceae. The microorganism according to claim 10, comprising a gene consisting of the following base sequence (R) as the 16S rRNA gene: (R) The base sequence of (R1) or (R2) below (R1) The base sequence shown in Sequence ID No. 1; (R2) A nucleotide sequence that has 99.94% or more identity with the nucleotide sequence shown in Sequence ID No.

1.

12. It belongs to the family Bacillaceae. The microorganism according to claim 10 or 11, comprising genomic DNA having a dDDH value of 70% or more with respect to the base sequence shown in Sequence ID No.

2.

13. A composition for use in the decomposition of biodegradable plastics, comprising the microorganism described in claim 1 or 2.

14. The composition according to claim 13, wherein the biodegradable plastic comprises polylactic acid.

15. A method for treating biodegradable plastics, comprising the step of contacting a microorganism described in claim 1 or 2 with a biodegradable plastic, thereby decomposing the biodegradable plastic with the microorganism.

16. The treatment method according to claim 15, wherein the biodegradable plastic includes polylactic acid.

17. The processing method according to claim 15, wherein the decomposition is carried out at a temperature of 35 to 65°C.

18. The processing method according to claim 15, wherein the decomposition is carried out under conditions of pH 7 to 10.

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