Hard surface treatment agent

Abstract

To provide a hard surface treatment agent that can make a hard surface anti-bacterial, and a hard surface treatment method using the same.SOLUTION: A hard surface treatment agent contains a polypeptide comprising an amino acid sequence of SEQ ID NO: 2, and a polypeptide comprising an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 2, and having the activity of decomposing glycine-glycine bond in the peptide sequence as active ingredients.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a hard surface treatment agent and its use.

Background Art

[0002] Microbial contamination on environmental surfaces has become an important source of infection for pathogenic bacteria (Non-Patent Document 1). In addition to pathogenic bacteria, microbial contamination in sinks, drains, washing machines, etc. causes bad odors and stickiness. Such surface microbial contamination is typically removed using ethanol, hypochlorous acid, hot water, surfactants, etc., but frequent cleaning is required to maintain a surface free of microbial contamination. Therefore, there is a need for a technology to continuously control microorganisms on the surfaces of sinks, drains, washing machines, medical devices, hospital environments, personal items, livestock products, etc.

[0003] As an example of such technology, a well-known technique involves hygiene management through antibacterial processing that inhibits bacterial growth on hard surfaces. This technology is widely used not only on hard surfaces that are frequently touched by many people, such as public doorknobs, handrails, and straps, but also on all kinds of equipment and tools used in nursing and care facilities and hospitals, as well as on general household goods, and is increasingly being used on personal belongings. These technologies impart antibacterial properties by kneading antibacterial agents into the plastic that makes up the hard surface, or by applying or spraying antibacterial agents onto the hard surface. Common antibacterial agents include organic compounds with well-known antibacterial properties, and metal particles or metal ions of silver and copper, but some technologies claim antibacterial properties based on the residual effect of disinfectants. Patent Document 1 describes a hard surface antibacterial cleaner containing a specific structure of silicone, a quaternary ammonium compound, phenolic resin, guanide derivative, alkyl alcohol, and other disinfectants. Patent Document 2 describes an antibacterial agent using nanoparticle-sized silver particles, and Patent Documents 3 and 4 disclose antifouling agents containing cationic polymers. Patent Document 5 describes an antibacterial composition for hard surfaces containing a cationic antibacterial active substance, and Patent Document 6 discloses an antibacterial cleaning agent composition containing catechin and a cationic surfactant.

[0004] One technique using enzymes is to immobilize lysostafin, which has lytic activity against Staphylococcus aureus, onto a plastic surface, thereby imparting bactericidal activity against Staphylococcus aureus to the surface (Non-Patent Literature 2). This effect is achieved simply by bringing a lysostafin solution into contact with the plastic. However, lysostafin is known to be effective only against Staphylococcus species (Non-Patent Literature 3), and a technique to control a wider range of microorganisms is needed. On the other hand, a hard surface antimicrobial treatment technique using lysozyme, which exhibits antimicrobial activity against a wider range of microorganisms, has been reported (Non-Patent Literature 4 and 5). However, these techniques require covalent crosslinking and / or surface pretreatment for immobilization on the surface, limiting their applicability. Furthermore, Staphylococcus aureus, a serious hazardous bacterium, is known to be resistant to lysozyme due to its cell wall structure (Non-Patent Literature 6).

[0005] Beta-lytic metallopeptidases (BLPs), belonging to the M23A subfamily of proteases, have been reported to have strong lytic activity against Gram-positive bacteria such as Staphylococcus aureus and Bacillus subtilis (Non-Patent Documents 7 and 8). Furthermore, it has been found that M23A subfamily proteases can be efficiently produced from cultures by introducing the M23A family protease gene into a Bacillus host and culturing it (Patent Document 7).

[0006] However, the use of M23A family proteases for antibacterial treatment of hard surfaces has not been reported to date. [Prior art documents] [Patent Documents]

[0007] [Patent Document 1] Special Publication No. 2004-532300 [Patent Document 2] Japanese Patent Publication No. 2000-178595 [Patent Document 3] Special Announcement No. 2002-60786 [License 4] Special Announcement No. 2020-152856 [Patent Document 5] Special Announcement No. 2003-510450 [License 6] Special Announcement No. 2008-195917 [License 7] International Publication No. 2019 / 142773 [Non-licensed literature]

[0008] [Non-licensed Document 1] Donskey, Curtis J. American journal of infection control, 2013, 41(5): S12-S19 [Non-licensed Document 2] Shah, Anjali et al. Antimicrobial agents and chemotherapy, 2004, 48(7): 2704-2707 [Non-licensed Document 3] Schindler, Ch A. and VT Schuhardt, Proceedings of the National Academy of Sciences of the United States of America, 1964, 51(3): 414-421 [Non-licensed Document 4] Yuan, Shaojun, et al. Langmuir, 2011, 27.6: 2761-2774 [Non-licensed Document 5] Yu, Wu-Zhong, et al. Materials & Design, 2018, 139: 351-362 [Non-licensed Document 6] Bera, Agnieszka et al. Journal of Bacteriology, 2007, 189(1): 280-283 [Non-licensed Document 7] Li, Shaoliang et al. The Journal of Biochemistry, 1998, 124(2): 332-339 [Non-Patent Document 8] Ahmed, Kashfia et al. Journal of Bioscience and Bioengineering, 2003, 95(1): 27-34 [Overview of the project] [Problems that the invention aims to solve]

[0009] The present invention relates to a hard surface treatment agent that can impart antibacterial properties to a hard surface, and a hard surface treatment method using the same. [Means for solving the problem]

[0010] The inventors have discovered that antibacterial properties can be imparted to a hard surface simply by bringing BLP into contact with the surface.

[0011] In other words, the present invention relates to the following 1) to 3). 1) A hard surface treatment agent comprising as an active ingredient a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2, or a polypeptide consisting of an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 2, and having glycine-glycine bond degrading activity in the peptide sequence. 2) A hard surface treatment method comprising the step of contacting a hard surface with a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2, or a polypeptide consisting of an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 2 and having glycine-glycine bond degrading activity in the peptide sequence, or an enzyme composition containing the same. 3) A method for imparting antibacterial properties to a hard surface, comprising a step of bringing into contact with the hard surface a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2, or a polypeptide consisting of an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 2 and having the activity of decomposing the glycine-glycine bond in the peptide sequence, or an enzyme composition containing the same.

Advantages of the Invention

[0012] The enzyme polypeptide provided by the present invention can impart antibacterial properties to a hard surface simply by bringing it into contact with the hard surface. The enzyme can impart antibacterial properties even in the coexistence of a surfactant. The imparted antibacterial properties can be maintained even when the hard surface is subjected to washing or drying after contact with the enzyme.

Brief Description of the Drawings

[0013] [Figure 1] Bactericidal activity of BLP and lysostaphin in solution. [Figure 2] Antibacterial activity of BLP against stainless steel. [Figure 3] Antibacterial activity of BLP against stainless steel. [Figure 4] Antibacterial activity of BLP against stainless steel. [Figure 5] Antibacterial activity of BLP against stainless steel. [Figure 6] Antibacterial activity of BLP against stainless steel. [Figure 7] Antibacterial activity of BLP, lysostaphin, and lysozyme against stainless steel. [Figure 8] Antibacterial activity of BLP and lysostaphin against plastic.

Modes for Carrying Out the Invention

[0014] In this specification, "at least 80% identity" with respect to a nucleotide sequence or amino acid sequence means identity of 80% or more, preferably 85% or more, more preferably 90% or more, even more preferably 95% or more, even more preferably 97% or more, even more preferably 98% or more, and even more preferably 99% or more.

[0015] In this specification, the identity between nucleotide sequences or amino acid sequences can be calculated using the Lipman-Pearson method (Science, 1985, 227:1435-41). Specifically, it can be calculated by performing the homology analysis (Search homology) using the genetic information processing software Genetyx-Win (Ver. 5.1.1; software development) with a Unit size to compare (ktup) of 2.

[0016] In this specification, the "corresponding positions" on amino acid sequences and nucleotide sequences can be determined by aligning the target sequence and the reference sequence (for example, the amino acid sequence shown in SEQ ID NO: 2) to give maximum homology to the conserved amino acid residues or nucleotides present in each amino acid sequence or nucleotide sequence. Alignment can be performed using known algorithms, and the procedures are known to those skilled in the art. For example, alignment can be performed using the Clustal W multiple alignment program (Thompson, JD et al, 1994, Nucleic Acids Res., 22:4673-4680) with default settings. Alternatively, revised versions of Clustal W, such as Clustal W2 or Clustal Omega, can be used. Clustal W, Clustal W2, and Clustal omega can be used, for example, on the websites of the European Bioinformatics Institute (EBI [www.ebi.ac.uk / index.html]) and the DNA Databank of Japan (DDBJ [www.ddbj.nig.ac.jp / Welcome-j.html]) operated by the National Institute of Genetics. The positions of amino acid residues or nucleotides in the target sequence aligned to any position in the reference sequence by the above alignment are considered to be "corresponding positions" to those arbitrary positions.

[0017] In this specification, "operable linkage" between a regulatory region and a gene means that the gene and the regulatory region are linked in such a way that the gene can be expressed under the control of the regulatory region. Procedures for "operable linkage" between a gene and a regulatory region are well known to those skilled in the art.

[0018] M23A subfamily proteases are proteases that possess the activity to degrade glycine-glycine bonds in peptide sequences and, according to the classification method of the MEROPS database (Rawlings, Neil D., et al. "MEROPS: the database of proteolytic enzymes, their substrates and inhibitors." Nucleic acids research 42.D1 (2013): D503-D509), are classified as the M23A subfamily, which is a subfamily of metalloproteases belonging to the M23 family.

[0019] "Beta-lytic metallopeptidase (BLP)" (MEROPS ID: M23.001) is an enzyme also known as a β-lytic protease, and is a type of protease belonging to the M23A subfamily.

[0020] The polypeptides of the present invention include BLP and polypeptides having equivalent functions, and it is preferable to appropriately select and use one of these. BLP is a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2, encoded by the nucleotide sequence from positions 595 to 1134 of SEQ ID NO: 1. BLP has the activity to degrade glycine-glycine bonds in peptide sequences. Furthermore, polypeptides having equivalent functionality to BLP include polypeptides that consist of an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 2 and that have glycine-glycine bond degrading activity in the peptide sequence. Preferred examples of polypeptides having equivalent functionality to BLP include polypeptides that consist of an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 2, preferably having His at positions corresponding to positions 22, 121, and 123 of the amino acid sequence of SEQ ID NO: 2 and Asp at position corresponding to position 36, and that have glycine-glycine bond degrading activity in the peptide sequence. Furthermore, the presence or absence of glycine-glycine bond degradation activity can be determined by testing the degradation ability of, for example, oligoglycine peptides or Fret-GGGGG substrates. However, this method is not the only way to determine this.

[0021] The polypeptide of the present invention can be extracted or prepared from the microorganism that produces it or from its culture. For example, BLP can be extracted or prepared from Lysobacter sp. (NBRC 12725 or NBRC 12726), Achromobacter lyticus M497-1, Lysobacter sp. IB-9374, Lysobacter gummosus DSMZ 6980, etc., or from their cultures. The above microorganisms can be purchased from public microorganism storage institutions.

[0022] Microorganisms that produce the polypeptide of the present invention can be cultured under appropriate conditions using a culture medium containing assimilated carbon sources, nitrogen sources, metal salts, vitamins, etc. From the microorganisms or culture medium thus obtained, enzymes can be collected and prepared by general methods, and the required enzyme form can be obtained by freeze-drying, spray-drying, crystallization, etc. For example, the recovery and preparation of enzymes from the culture can be carried out using conventional methods such as separation of microorganisms by centrifugation or filtration, precipitation of the enzyme in the supernatant or filtrate by adding a salt such as ammonium sulfate or by adding an organic solvent such as ethanol, concentration and desalting using an ultrafiltration membrane, purification using various chromatography methods such as ion exchange or gel filtration.

[0023] Alternatively, the polypeptide of the present invention can be produced by chemical synthesis or biological methods using the above-described amino acid sequence. For example, the polypeptide of the present invention can be obtained by extracting genomic DNA from a microorganism that naturally produces the polypeptide of the present invention by conventional methods, or by extracting RNA and synthesizing cDNA by reverse transcription, and then culturing a Bacillus bacterium transformed to express a polynucleotide encoding a protein prepared by introducing mutations as necessary, and preparing the target enzyme from the culture. Examples of the transformed Bacillus bacterium prepared here include, for example, a Bacillus bacterium obtained by introducing a gene encoding the polypeptide of the present invention, which is operably linked to a regulatory region, into the genome or plasmid of a host cell, or a Bacillus bacterium into which an expression vector in which the target gene is incorporated at an appropriate position has been introduced.

[0024] Here, the "regulatory region" of a gene is a region that has the function of controlling the intracellular expression of downstream genes, and preferably has the function of constitutively expressing or hyperexpressing downstream genes. Specifically, it can be defined as a region located upstream of the coding region of the gene, which has the function of controlling the transcription of the gene through interaction with RNA polymerase. Preferably, the regulatory region of a gene refers to a region of about 200 to 600 nucleotides upstream of the coding region of the gene. The regulatory region includes the transcription initiation regulatory region and / or the translation initiation regulatory region of the gene, or the region from the transcription initiation regulatory region to the translation initiation regulatory region. The transcription initiation regulatory region is the region containing the promoter and the transcription start site, and the translation initiation regulatory region is the region corresponding to the Shine-Dalgarno (SD) sequence that forms a ribosome binding site together with the start codon (Shine, J., Dalgarno, L., Proc. Natl. Acad. Sci. USA., 1974, 71:1342-1346).

[0025] An expression vector containing the gene encoding the polypeptide of the present invention can be produced by incorporating the gene encoding the polypeptide of the present invention into a vector that can stably retain the gene, replicate and maintain it within a host microorganism, and stably express the polypeptide. Examples of such vectors include shuttle vectors such as pHA3040SP64, pHSP64R, or pASP64 (Patent No. 3492935), pHY300PLK (an expression vector capable of transforming both Escherichia coli and Bacillus subtilis; Jpn J Genet, 1985, 60:235-243), and pAC3 (Nucleic Acids Res, 1988, 16:8732); and plasmids usable for the transformation of Bacillus bacteria such as pUB110 (J Bacteriol, 1978, 134:318-329) and pTA10607 (Plasmid, 1987, 18:8-15). Plasmids derived from E. coli (e.g., pET22b(+), pBR322, pBR325, pUC57, pUC118, pUC119, pUC18, pUC19, pBluescript, etc.) can also be used.

[0026] Transformation of host Bacillus species can be carried out using methods such as the protoplast method, competent cell method, and electroporation. Preferably, the host Bacillus species is Bacillus subtilis or a mutant thereof. For example, a Bacillus subtilis strain in which extracellular protease production has been reduced within a range that allows for sufficient M23A maturation is a suitable host.

[0027] The resulting transformants can be cultured under appropriate conditions using a medium containing assimilated carbon sources, nitrogen sources, metal salts, vitamins, etc. From the culture thus obtained, enzymes can be collected and prepared by general methods, and the required enzyme form can be obtained by freeze-drying, spray-drying, crystallization, etc. For example, the recovery and preparation of enzymes from the culture can be carried out using conventional methods such as separation of recombinant microorganisms by centrifugation or filtration, precipitation of the enzyme in the supernatant or filtrate by adding a salt such as ammonium sulfate or by adding an organic solvent such as ethanol, concentration and desalting using an ultrafiltration membrane, purification using various chromatography methods such as ion exchange or gel filtration.

[0028] Alternatively, the polypeptide of the present invention can be prepared from an enzyme composition containing it. For example, BLP can be prepared from achromopeptidase. Achromopeptidase is a lytic enzyme derived from Lysobacter enzymogenes and contains BLP. Achromopeptidase is commercially available from Wako Pure Chemical Industries, Ltd. and others.

[0029] As shown in the examples described later, the polypeptide of the present invention, for example, BLP, has an antibacterial effect on hard surfaces, and by contacting a hard surface, it can impart antibacterial properties to the hard surface. The bactericidal effect of BLP on Staphylococcus aureus in solution is equivalent to that of lysostafin described in Non-Patent Literature 2, while the antibacterial effect achieved by contacting BLP with a hard surface was unexpectedly superior to that achieved by contacting lysostafin with a hard surface. Furthermore, surprisingly, the antibacterial effect of BLP on hard surfaces was highly maintained even when the hard surface was washed after contact with BLP, that is, even when the hard surface was in contact with water or dried. Moreover, the antibacterial effect of BLP on hard surfaces was observed regardless of the material of the hard surface, whether it was a stainless steel surface or a plastic surface, and was also observed in the presence of a surfactant. Therefore, the polypeptide of the present invention is useful as an enzyme for hard surface treatment to impart antibacterial properties to hard surfaces, and can become a hard surface treatment agent, preferably a hard surface treatment agent for imparting antibacterial properties. Alternatively, the polypeptide of the present invention can be used to produce a hard surface treatment agent, preferably a hard surface treatment agent for imparting antibacterial properties. Furthermore, the polypeptide of the present invention can be used for hard surface treatment, preferably for hard surface treatment to impart antibacterial properties. For example, by bringing the polypeptide of the present invention into contact with a hard surface, antibacterial properties can be imparted to the hard surface.

[0030] In this invention, "antibacterial" is a term that includes any of the following concepts: inhibiting the adhesion of bacteria on hard surfaces, inhibiting the residue of bacteria on hard surfaces, "sterilization" and "disinfection" which kill bacteria on hard surfaces, and "antimicrobial," "bacteriostatic," and "bacteriostatic" which suppress the occurrence, growth, and proliferation of bacteria on hard surfaces. The inventors have found that BLP is particularly excellent in inhibiting the residue or adhesion of bacteria on hard surfaces. Antimicrobial activity can be evaluated using methods well known in the art. For example, it can be evaluated by immersing a test piece having a hard surface in a solution containing the target polypeptide for a predetermined time to bring it into contact with the polypeptide, then immersing the test piece in a test solution containing the test bacteria for a predetermined time to bring it into contact with the test bacteria, then extracting the test bacteria from the test piece, culturing them in a suitable solid medium, and measuring the number of colonies produced to calculate the number of viable bacteria attached to the test piece.

[0031] In the present invention, the bacteria that can be targeted for "antibacterial treatment" are not particularly limited, but Gram-positive bacteria are preferred. Gram-positive bacteria include, for example, Staphylococcus aureus and Staphylococcus epidermidis; Micrococcus species such as Micrococcus luteus; Streptococcus species such as Streptococcus pneumoniae, Streptococcus viridans, Group A β-hemolytic streptococcus, and Group B β-hemolytic streptococcus agalactiae; Enterococcus species such as Enterococcus faecalis; Bacillus species such as Bacillus anthracis; Clostridium tetani, Clostridium perfringens, and Clostridium botulinum. Examples include Clostridium bacteria such as *Clostridium botulinum*; Corynebacterium bacteria such as *Corynebacterium diphtheriae*; and Listeria bacteria such as *Listeria monocytogenes*. Of these, Staphylococcus and Micrococcus bacteria are more preferred, and Staphylococcus aureus and *Luteus* are even more preferred.

[0032] The hard surface treatment agent of the present invention may be in the form of using the polypeptide of the present invention alone, or in the form of an enzyme composition containing it. The enzyme composition may be a solid composition such as a powder or a liquid composition. Furthermore, the enzyme composition may be in an undiluted form or a diluted form. The undiluted form is used for hard surface treatment without dilution. The diluted form is used for hard surface treatment after being diluted with a suitable medium such as water so that the content of the polypeptide of the present invention after dilution is within the following range. Preferably, the hard surface treatment agent is a product or formulation used for antibacterial treatment of hard surfaces of animate objects in which bacteria are present, may be present, or may be attached, or is used as an antibacterial material in such products or formulations.

[0033] Examples of hard surfaces include hard surfaces of inanimate objects on which bacteria are present, potentially present, or potentially attached. Examples include hard surfaces of counters, sinks, restrooms, toilets, washing machines, bathtubs, showers, floors, windows, doors, doorknobs, walls, drains, and pipes in homes and business facilities; hard surfaces of various utensils and equipment such as kitchenware, furniture, telephones, toys, medical devices, livestock equipment, and food processing equipment, as well as food processing devices, tools, and miscellaneous goods; and hard surfaces of water-cooling towers in building air conditioning systems that come into contact with water. Examples of materials for hard surfaces include plastics (including silicone resins), metals, ceramics, wood, glass, or combinations thereof. Preferably, they are plastics, metals, or combinations thereof, and more preferably, they are plastics, stainless steel, or combinations thereof. Furthermore, as hard surfaces, hard surfaces that are in an environment where they come into contact with water periodically or irregularly, or hard surfaces that are in an environment where they dry periodically or irregularly, are preferred. The antibacterial effect of the hard surface treatment agent of the present invention on hard surfaces is maintained even under such conditions. Here, "environment" refers to the external conditions surrounding the hard surface, and includes not only naturally occurring environments but also environments created by intentional actions. Therefore, "hard surfaces in an environment where they come into contact with water" can be hard surfaces that may come into contact with water in their natural state of existence, or hard surfaces that may come into contact with water due to the user's intention, and "hard surfaces in a dry environment" can be hard surfaces that may dry in their natural state of existence, or hard surfaces that may dry due to the user's intention.

[0034] The above-mentioned products and formulations may take the form of liquid, emulsion, cream, lotion, paste, gel, sheet (with base material), aerosol, spray, oil, or gel, but are not limited to these forms.

[0035] The above-mentioned products and formulations may, in addition to the polypeptide of the present invention, optionally contain antimicrobial substances such as hypochlorous acid, hydrogen peroxide, and silver ion compounds, as well as cationic antimicrobial agents (such as benzethonium chloride), disinfectants (such as triclosan and isopropylmethylphenol), ethanol, surfactants, etc. They can be prepared according to conventional methods by appropriately blending additives such as chelating agents, humectants, lubricants, builders, buffers, abrasives, electrolytes, bleaches, fragrances, dyes, foaming control agents, corrosion inhibitors, essential oils, thickeners, pigments, gloss enhancers, enzymes other than the polypeptide of the present invention, detergents, solvents, dispersants, polymers, silicones, and hydrophobic substances.

[0036] The content of the polypeptide of the present invention in the hard surface treatment agent of the present invention can be appropriately determined depending on the form of the enzyme composition. For example, the content of the polypeptide of the present invention is preferably 0.00001% by mass or more, more preferably 0.0002% by mass or more, even more preferably 0.0005% by mass or more, even more preferably 0.001% by mass or more, even more preferably 0.005% by mass or more, even more preferably 0.01% by mass or more, and preferably 20% by mass or less, more preferably 5% by mass or less, and even more preferably 2% by mass or less, based on the total mass of the composition. Furthermore, the numerical range of the content of the polypeptide of the present invention is preferably 0.00001 to 20% by mass, more preferably 0.0002 to 5% by mass, even more preferably 0.0005 to 2% by mass, even more preferably 0.001 to 2% by mass, even more preferably 0.005 to 2% by mass, and even more preferably 0.01 to 2% by mass.

[0037] The hard surface treatment agent of the present invention is used by contacting a hard surface and can impart antibacterial properties to the hard surface. From the viewpoint of imparting antibacterial properties, the contact time is preferably 10 seconds or more, more preferably 1 minute or more, even more preferably 5 minutes or more, and even more preferably 10 minutes or more. There is no particular upper limit to the contact time, and it may be left as is after contact, but if washing or the like is performed after contact, from the viewpoint of workload, it is preferably 3 hours or less, more preferably 1 hour or less, and even more preferably 30 minutes or less. Furthermore, the contact time is preferably 10 seconds or more and 3 hours or less, more preferably 1 minute or more and 1 hour or less, even more preferably 5 to 30 minutes, and even more preferably 10 to 30 minutes. The means of contact are not particularly limited and may include applying the hard surface treatment agent to a hard surface, immersing the hard surface in the hard surface treatment agent, spraying or scattering the hard surface treatment agent onto the hard surface in an atomized state using an atomizing device such as a pump spray, aerosol, pressurized liquid spray, or pressurized air atomizing spray device, wiping the hard surface with a sheet, gauze, towel, wet wipe, tissue, or wet wipe impregnated with the hard surface treatment agent, or slowly releasing the hard surface treatment agent from an upstream location using flowing water to bring it into contact with a downstream hard surface.

[0038] The concentration of the polypeptide of the present invention when the hard surface treatment agent of the present invention is brought into contact with a hard surface is preferably 1 ppm or more, more preferably 5 ppm or more, and even more preferably 10 ppm or more, from the viewpoint of imparting antibacterial properties. The upper limit of the polypeptide concentration is not particularly limited, but is preferably 1000 ppm or less, more preferably 500 ppm or less, and even more preferably 100 ppm or less. Furthermore, the numerical range of the concentration is preferably 1 to 1000 ppm, more preferably 5 to 500 ppm, and even more preferably 10 to 100 ppm.

[0039] In another embodiment, the present invention provides a method for treating a hard surface using the polypeptide of the present invention. In yet another embodiment, the present invention provides a method for imparting antibacterial properties to a hard surface using the polypeptide of the present invention. The method comprises contacting the hard surface with the polypeptide of the present invention or an enzyme composition containing it. The mode of contact between the polypeptide of the present invention and the hard surface can be appropriately selected depending on the shape and type of material of the hard surface, and the processing time and the amount of enzyme used can also be arbitrarily set according to the mode of processing. For example, the hard surface may be coated, sprayed, or sprayed with a solution containing the polypeptide of the present invention and left for a certain period of time (e.g., 10 seconds to 3 hours), or the hard surface may be immersed in the solution and left for a certain period of time (e.g., 10 seconds to 3 hours). After contact, the hard surface may be washed or rinsed with a medium such as water, and may also be dried. The number of washings is not particularly limited, but for example, it is at least once, preferably four times or less, and more preferably two times or less. The mode of drying is not particularly limited and may be natural drying or heat drying.

[0040] With regard to the embodiments described above, the present invention further discloses the following embodiments. <1> A hard surface treatment agent comprising as an active ingredient a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2, or a polypeptide consisting of an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 2, and having glycine-glycine bond degrading activity in the peptide sequence. <2> It is a hard surface treatment agent for providing antibacterial properties. <1> The hard surface treatment agent described. <3> The hard surface is preferably a plastic surface, a metal surface, or a combination thereof, more preferably a plastic surface, a stainless steel surface, or a combination thereof. <1> or <2> The hard surface treatment agent described. <4> The aforementioned antibacterial effect is antibacterial effect against Gram-positive bacteria. <1> ~ <3> A hard surface treatment agent as described in any one of the following items. <5> The hard surface is a hard surface that is in an environment where it comes into contact with water periodically or irregularly. <1> ~ <4> A hard surface treatment agent as described in any one of the following items. <6> The hard surface is a hard surface that is in an environment where it dries regularly or irregularly. <1> ~ <4> A hard surface treatment agent as described in any one of the following items. <7> A hard surface treatment method comprising the step of contacting a hard surface with a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2, or a polypeptide consisting of an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 2 and having glycine-glycine bond degrading activity in the peptide sequence, or an enzyme composition containing the same. <8> A method for imparting antibacterial properties to a hard surface, comprising the step of contacting a hard surface with a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2, or a polypeptide consisting of an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 2 and having glycine-glycine bond degrading activity in the peptide sequence, or an enzyme composition containing the same. <9> The hard surface is preferably a plastic surface, a metal surface, or a combination thereof, more preferably a plastic surface, a stainless steel surface, or a combination thereof. <7> or <8> Method of description. <10> The process includes bringing the polypeptide or an enzyme composition containing the polypeptide into contact with the hard surface for preferably 10 seconds or more, more preferably 1 minute or more, even more preferably 5 minutes or more, and even more preferably 10 minutes or more. <7> ~ <9> The method described in any one of the items. <11> The process includes contacting the aforementioned polypeptide or an enzyme composition containing the same with the hard surface at a concentration of the polypeptide preferably 1 ppm or more, more preferably 5 ppm or more, and even more preferably 10 ppm or more. <7> ~ <10> The method described in any one of the items. <12> The process includes applying, spraying, or scattering a solution containing the polypeptide or an enzyme composition containing the polypeptide onto the hard surface. <7> ~ <11> The method described in any one of the items. <13> The process includes immersing the hard surface in a solution containing the polypeptide or an enzyme composition containing the polypeptide. <7> ~ <11> The method described in any one of the items. <14> The aforementioned antibacterial effect is antibacterial effect against Gram-positive bacteria. <8> ~ <13> The method described in any one of the items. <15> The hard surface is a hard surface that is in an environment where it comes into contact with water periodically or irregularly. <7> ~ <14> The method described in any one of the items. <16> The hard surface is a hard surface that is in an environment where it dries regularly or irregularly. <7> ~ <14> The method described in any one of the items. <17> The use of a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2, or a polypeptide consisting of an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 2 and having glycine-glycine bond degrading activity in the peptide sequence, for the production of a hard surface treatment agent, preferably for the production of a hard surface treatment agent that imparts antibacterial properties. <18> Use of a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2, or a polypeptide consisting of an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 2, and having glycine-glycine bond degrading activity in the peptide sequence, for hard surface treatment, preferably for imparting antibacterial properties to a hard surface. <19> The hard surface is a hard surface that is in an environment where it comes into contact with water periodically or irregularly. <17> or <18> Use as described. <20> The hard surface is a hard surface that is in an environment where it dries regularly or irregularly. <17> or <18> Use as described. [Examples]

[0041] Example 1 (1) Enzyme preparation BLP consisting of the amino acid sequence of Sequence ID No. 2 was prepared by culturing and purifying it according to the method described in Example 1 of Japanese Patent Application No. 2020-182945. Lysozyme (Fujifilm Wako Pure Chemical Industries, 129-06723) and lysostafin (Fujifilm Wako Pure Chemical Industries, 120-06611) were dissolved in 20 mM Tris-HCl (pH 7.5). A DC Protein Assay Kit (Bio-Rad) was used to measure the concentration of the enzyme solution. BSA Standard Solution (Fujifilm Wako Pure Chemical Industries) was used as the standard solution for calculating the protein amount.

[0042] (2) Sterilization test Staphylococcus aureus NCTC8325 was used as the test bacterium. SCD liquid medium was SCD medium "Daigo", for general bacterial testing (Fujifilm Wako Pure Chemical Industries), SCD agar medium was SCD agar medium "Daigo", for general bacterial testing (Fujifilm Wako Pure Chemical Industries), LP diluent was LP diluent "Daigo" (Fujifilm Wako Pure Chemical Industries), and 20 mM Tris-HCl (pH 7.5) was used as the buffer. Buffers containing each enzyme (BLP, lysostafine) at a final concentration of 1 ppm were used as the test solution. Test bacteria cultured overnight in SCD liquid medium at 37°C with shaking were collected, washed with buffer, and resuspended. 8-9 The samples were prepared to CFU / mL. 5 μL of bacterial suspension was added to 500 μL of each test solution and incubated at 30°C for 30 minutes. The test solutions were serially diluted with LP diluent and 100 μL each was spread onto SCD agar plates. After incubation at 37°C for 24 hours, the number of viable cells (CFU / mL) in 1 mL of the test solution was calculated by counting the colonies. BLP and lysostafin showed similar levels of Staphylococcus aureus bactericidal activity in the buffer (Figure 1).

[0043] (3) Bacterial adhesion inhibition test on stainless steel (Antibacterial treatment of stainless steel by BLP) Micrococcus luteus was used as the test bacterium. 20 mM Tris-HCl (pH 7.5) was used as the buffer. 1 mL of BLP solution, diluted to 10 ppm with buffer, was dispensed into each well of a 12-well polystyrene plate (CORNING, 351143). One 1 × 15 × 15 mm SUS430 test piece (Engineering Test Service) was placed in each well and immersed at room temperature for 10 minutes. The water was drained from the test pieces, and they were transferred to a new 12-well plate with 2 mL of buffer dispensed into each well and gently shaken for 2 minutes. Again, the water was drained from the test pieces, and they were transferred to a new 12-well plate with 2 mL of buffer dispensed into each well and gently shaken for 2 minutes before the test pieces were collected. The test bacteria were cultured overnight on SCD agar at 37°C with shaking, then collected, washed with buffer, and resuspended.8 The bacterial suspension was prepared to CFU / mL. 1.5 mL of the above bacterial suspension was dispensed into new 12-well plates, and one enzyme-soaked test piece was placed in each. After standing at room temperature for 15 minutes, the water was drained from the test pieces, and they were transferred to new 12-well plates containing 2 mL of buffer each, and gently shaken for 1 minute. Again, the water was drained from the test pieces, and they were transferred to new 12-well plates containing 2 mL of buffer each, and gently shaken for 1 minute. One test piece was placed in a 50 mL tube containing 7 mL of LP diluent, and bacteria were extracted by sonication for 30 minutes. The extract was serially diluted with LP diluent, and 100 μL was spread onto SCD agar plates. After incubation at 37°C for 24 hours, the number of viable bacteria (CFU / piece) attached to each test piece was calculated by counting the colonies. Test pieces pre-soaked in BLP showed a significantly reduced number of attached viable bacteria compared to test pieces soaked in buffer only (Figure 2).

[0044] (4) Test to inhibit bacterial adhesion to stainless steel (effect of BLP immersion time) The bacterial adhesion inhibition test was performed in the same manner as in (3), except that the immersion time of the test pieces in the enzyme solution was changed to 1, 5, 10, and 30 minutes. Even a one-minute immersion in BLP reduced the number of attached viable bacteria by approximately 90% compared to immersion in buffer alone (Figure 3).

[0045] (5) Bacterial adhesion inhibition test on stainless steel (resistance to residual enzymes on the surface) The bacterial adhesion inhibition test was performed in the same manner as in (3), except that the immersion enzyme concentration was changed to 20 ppm and test pieces treated under the following four conditions were used as test pieces after enzyme immersion. Condition 1: Rinse twice (same conditions as (3)) Condition 2: Rinse 4 times Condition 3: After rinsing twice, allow the test piece to dry at room temperature for 3 hours. Condition 4: After two rinses, dry the test piece at room temperature for 21 hours. Test pieces immersed in BLP maintained their reduction in the number of attached viable bacteria even after four rinses and 21 hours of drying (Figure 4).

[0046] (6) Test to inhibit bacterial adhesion to stainless steel (effect on Staphylococcus aureus) The bacterial adhesion inhibition test was performed in the same manner as in (3), except that the test bacterium was changed to Staphylococcus aureus NCTC8325 and the immersion enzyme concentration was changed to 20 ppm. Test pieces immersed in BLP also showed a reduction in the number of attached Staphylococcus aureus bacteria (Figure 5).

[0047] (7) Test to inhibit bacterial adhesion to stainless steel (effect on Staphylococcus aureus) The bacterial adhesion inhibition test was conducted in the same manner as in (3), except that the buffer was changed to a solution of commercially available laundry detergent (Attack ZERO, Kao) diluted 3000 times with tap water. The effect of BLP in reducing the number of attached viable bacteria was maintained even in aqueous solutions containing surfactants (Figure 6).

[0048] (8) Test to inhibit bacterial adhesion to stainless steel (comparison of performance with other lytic enzymes) The bacterial adhesion inhibition test was performed in the same manner as in (3), except that the test bacterium was changed to Staphylococcus aureus NCTC8325, the immersion enzyme concentration was changed to 20 ppm, and BLP, lysostafin, or lysozyme was used as the enzyme. BLP showed the greatest effect in reducing the number of attached viable bacteria (Figure 7).

[0049] (9) Test to inhibit bacterial adhesion to plastics Staphylococcus aureus NCTC8325 was used as the test bacterium. 20 mM Tris-HCl (pH 7.5) was used as the buffer. 1 mL of the enzyme solution, diluted to 10 ppm with buffer, was dispensed into each well of a 12-well polystyrene plate (CORNING, 351143) and immersed at room temperature for 10 minutes. The entire solution in each well was removed with a pipette, and 2 mL of buffer was dispensed into each well and gently shaken for 2 minutes. The entire solution in each well was again removed with a pipette, and 2 mL of buffer was dispensed into each well and gently shaken for 2 minutes, after which the entire solution in each well was removed with a pipette. The test bacteria were cultured overnight on SCD agar at 37°C with shaking, then harvested, washed with buffer, and resuspended. 8 The bacterial suspension was prepared to CFU / mL. 1.5 mL of the above bacterial suspension was dispensed into each of the enzyme-treated 12-well plates. After standing at room temperature for 15 minutes, all the solution was removed from the wells using a pipette, and 2 mL of buffer was dispensed into each well and gently shaken for 2 minutes. Again, all the solution was removed from the wells using a pipette, and 2 mL of buffer was dispensed into each well and gently shaken for 2 minutes. All the solution was removed from the wells using a pipette, and 2 mL of LP diluent was dispensed into each well, sealed with a seal, and the bacteria were extracted by sonication for 30 minutes. The extract was serially diluted with LP diluent and 100 μL was spread onto SCD agar plates. After incubation at 37°C for 24 hours, the number of viable bacteria (CFU / well) attached to each well was calculated by counting the colonies. BLP also showed an effect of reducing the number of viable bacteria attached to polystyrene, and this effect was greater than that of lysostafin as shown in Non-Patent Document 2 (Figure 8).

Claims

1. An inhibitor of Gram-positive bacteria adhesion on hard surfaces, comprising as an active ingredient a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2, or a polypeptide consisting of an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 2 and having glycine-glycine bond degrading activity in the peptide sequence.

2. A method for inhibiting the adhesion of Gram-positive bacteria to a hard surface, comprising the step of contacting a hard surface with a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2, or a polypeptide consisting of an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 2 and having glycine-glycine bond degrading activity in the peptide sequence, or an enzyme composition containing the same.

3. The method according to claim 2, wherein the hard surface is a hard surface that is in an environment in which it comes into contact with water periodically or irregularly.

4. The method according to claim 2, wherein the hard surface is a hard surface that is in an environment where it dries regularly or irregularly.

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