Novel lipase

Abstract

To provide a lipase whose activity is less likely to be inhibited by surfactants and which exhibits a high cleaning effect.SOLUTION: Provided is a lipase comprising an amino acid sequence represented by SEQ ID NO: 14 or an amino acid sequence having at least 91% identity to the amino acid sequence.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a novel lipase. [Background technology]

[0002] Lipases are useful in a variety of applications, including laundry detergents, dishwashing detergents, oil and fat processing, pulp treatment, animal feed, and pharmaceutical intermediate synthesis. In cleaning, lipases contribute to the removal of oily stains by hydrolyzing triglycerides to produce fatty acids.

[0003] Current cleaning compositions and cleaning environments contain various components that inhibit lipase activity and cleaning effects, and lipases that function under such conditions are needed. As a lipase useful for cleaning, lipase derived from Thermomyces lanuginosus (hereinafter referred to as TLL) is sold under the trade name LIPOLASE®. Patent Document 1 discloses that lipase Lipr139 derived from Cedecea sp.-16640 strain has superior cleaning performance compared to TLL. Patent Document 2 discloses a variant of lipase derived from Proteus genus bacteria (hereinafter referred to as PvLip) that has improved cleaning performance compared to one or more reference lipolytic enzymes. Patent Document 3 discloses that metagenomic-derived lipase Lipr138 has superior cleaning performance compared to TLL. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Special Publication No. 2015-523078 [Patent Document 2] International Publication No. 2020 / 046613 [Patent Document 3] Special Publication No. 2015-525248 Summary of the Invention [Problem to be solved by the invention]

[0005] It is known that the activity of lipases is inhibited by surfactants contained in cleaning compositions. In fact, as far as the inventors have been able to verify, all of the known lipases disclosed as being suitable for cleaning, namely TLL, Lipr139, and PvLip, were significantly inhibited in the presence of surfactants. Activity inhibition by surfactants is a major issue common to all cleaning lipases. Furthermore, lipases are often used in conjunction with surfactants not only for cleaning applications but also for industrial applications such as pulp processing, and activity inhibition by surfactants is an issue for all industrial lipases. There is a demand for lipases that are less susceptible to activity inhibition by surfactants and exhibit high cleaning efficacy. [Means for solving the problem]

[0006] In view of the above problems, the present inventors have conducted extensive research and have discovered a novel lipase, KAL-A8, which has remarkably high resistance to activity inhibition by surfactants and exhibits a dramatic cleaning effect.

[0007] That is, the present invention relates to the following 1) to 6). 1) A lipase consisting of the amino acid sequence shown in SEQ ID NO: 14 or an amino acid sequence having at least 91% identity thereto. 2) A polynucleotide encoding the lipase according to 1). 3) A vector or DNA fragment containing the polynucleotide of 2). 4) A transformed cell containing the vector or DNA fragment of 3). 5) A cleaning composition containing the lipase according to 1). 6) A method for cleaning stains using the cleaning agent composition according to 5). [Effects of the Invention]

[0008] The lipase of the present invention has remarkably high resistance to activity inhibition by surfactants and exhibits excellent cleaning effect even in the presence of surfactants. [Brief explanation of the drawings]

[0009] [Figure 1] Lipase activity of each lipase in surfactant solution. [Figure 2] Lipase activity of each lipase in a model detergent solution containing a surfactant. [Figure 3] Detergency of each lipase in a model detergent solution containing a surfactant. DETAILED DESCRIPTION OF THE INVENTION

[0010] All patents, non-patent documents, and other publications cited herein are hereby incorporated by reference in their entirety.

[0011] As used herein, the term "lipase" refers to triacylglycerol lipase (EC 3.1.1.3), a group of enzymes that hydrolyze triglycerides to produce fatty acids. Lipase activity can be determined by measuring the rate of increase in absorbance associated with the release of 4-nitrophenol by hydrolysis of 4-nitrophenyl octanoate. Specific procedures for measuring lipase activity are described in detail in the Examples below.

[0012] Herein, the identity of an amino acid sequence or a nucleotide sequence is calculated by the Lipman-Pearson method (Science, 1985, 227:1435-1441). Specifically, it is calculated by performing analysis using the homology analysis (Search homology) program of the genetic information processing software GENETYX Ver. 12, with the unit size to compare (ktup) set to 2.

[0013] As used herein, the term "operably linked" between a gene and a regulatory region such as a promoter means that the gene and regulatory region are linked in such a way that the gene can be expressed under the control of the regulatory region. Procedures for "operably linking" a gene to a regulatory region are well known to those skilled in the art.

[0014] As used herein, "upstream" and "downstream" in relation to a gene refer to upstream and downstream in the transcription direction of the gene. For example, "a gene located downstream of a promoter" means that the gene is located on the 3' side of the promoter on the DNA sense strand, and "upstream" of a gene means the 5' region of the gene on the DNA sense strand.

[0015] <1. Lipase> The lipase of the present invention is a lipase consisting of the amino acid sequence shown in SEQ ID NO: 14 or an amino acid sequence having at least 91% identity thereto. The lipase of the present invention has significantly high resistance to inhibition of lipase activity by surfactants and exhibits significantly high detergency even in the presence of surfactants.

[0016] Examples of lipases having an amino acid sequence that is at least 91% identical to the amino acid sequence shown in SEQ ID NO: 14 include lipases having an amino acid sequence that is at least 91% identical to the amino acid sequence shown in SEQ ID NO: 14, specifically, 91% or more, preferably 93% or more, more preferably 95% or more, even more preferably 96% or more, even more preferably 97% or more, even more preferably 98% or more, and even more preferably 99% or more. Amino acid sequences with at least 91% identity include amino acid sequences in which one or more amino acids have been deleted, inserted, substituted, or added. "Amino acid sequences in which one or more amino acids have been deleted, inserted, substituted, or added" include amino acid sequences in which 1 to 30, preferably 20 or less, more preferably 10 or less, and even more preferably 5 or less amino acids have been deleted, inserted, substituted, or added.

[0017] An example of a lipase consisting of an amino acid sequence having at least 91% identity to the amino acid sequence shown in SEQ ID NO: 14 is an artificially created mutant of the lipase consisting of the amino acid sequence shown in SEQ ID NO: 14. The mutant can be prepared, for example, by introducing a mutation into a gene encoding the amino acid sequence shown in SEQ ID NO: 14 by known mutagenesis methods such as ultraviolet irradiation or site-directed mutagenesis, expressing the gene having the mutation, and selecting a protein having the desired lipase activity. Procedures for preparing such mutants are well known to those skilled in the art.

[0018] The lipase of the present invention has an amino acid sequence different from that of conventionally isolated or purified lipases and proteins predicted as triacylglycerol lipases in the NCBI protein sequence database. Examples of conventionally isolated or purified lipases include lipase TLL (SEQ ID NO: 18) derived from Thermomyces lanuginosus, lipase Lipr139 (SEQ ID NO: 2) derived from Cedecea sp.-16640 strain, which is disclosed in Patent Document 1 as a lipase suitable for cleaning, lipase derived from Proteus bacteria (hereinafter referred to as PvLip, SEQ ID NO: 4), which is disclosed in Patent Document 2 as a parent enzyme of a group of lipase variants suitable for cleaning, and metagenomic lipase Lipr138 (SEQ ID NO: 16), which is disclosed in Patent Document 3 as a lipase suitable for cleaning. Furthermore, proteins predicted to be triacylglycerol lipases in the NCBI protein sequence database include the protein with accession number WP_123598507.1 (hereinafter referred to as PfLip, SEQ ID NO: 6), the protein with accession number WP_115457195.1 (hereinafter referred to as EtLip, SEQ ID NO: 8), the protein with accession number WP_135495634.1 (hereinafter referred to as EspLip, SEQ ID NO: 10), and the protein with accession number WP_005161363.1 (hereinafter referred to as YeLip, SEQ ID NO: 12). The enzymatic properties of these proteins registered in the database have not yet been reported. The lipase KAL-A8 of the present invention, consisting of the amino acid sequence shown in SEQ ID NO: 14, shows 72%, 60%, 59%, 68%, 86% and 71% amino acid sequence identity with Lipr139, PvLip, PfLip, EtLip, EspLip and YeLip, respectively.

[0019] In a preferred embodiment, the lipase of the present invention is a lipase consisting of the amino acid sequence shown in SEQ ID NO:14.

[0020] <2. Lipase production method> The lipase of the present invention can be produced, for example, by expressing a polynucleotide encoding the lipase of the present invention. Preferably, the lipase of the present invention can be produced from a transformant into which a polynucleotide encoding the lipase of the present invention has been introduced. For example, a polynucleotide encoding the lipase of the present invention, or a vector containing the polynucleotide, is introduced into a host to obtain a transformant, and the transformant is then cultured in an appropriate medium, whereby the lipase of the present invention is produced from the polynucleotide encoding the lipase of the present invention introduced into the transformant. The lipase of the present invention can be obtained by isolating or purifying the produced lipase from the culture.

[0021] The polynucleotide encoding the lipase of the present invention may be a polynucleotide encoding a lipase consisting of the amino acid sequence shown in SEQ ID NO: 14 or an amino acid sequence having at least 91% identity thereto. The polynucleotide encoding the lipase of the present invention may be in the form of single-stranded or double-stranded DNA, RNA, or an artificial nucleic acid, or may be cDNA or chemically synthesized DNA containing no introns.

[0022] A polynucleotide encoding the lipase of the present invention can be synthesized chemically or by genetic engineering based on the amino acid sequence of the lipase. For example, the polynucleotide can be chemically synthesized based on the amino acid sequence of the lipase of the present invention described above. Chemical synthesis of polynucleotides can be performed using nucleic acid synthesis contract services (such as those provided by Medical & Biological Laboratories, Inc. and Genscript, Inc.). Furthermore, synthesized polynucleotides can be amplified by PCR, cloning, or the like.

[0023] Alternatively, a polynucleotide encoding the lipase of the present invention can be prepared by introducing a mutation into the polynucleotide synthesized by the above procedure using a known mutagenesis method, such as UV irradiation or site-directed mutagenesis. For example, a polynucleotide encoding the lipase of the present invention can be obtained by introducing a mutation into the polynucleotide of SEQ ID NO: 13 using a known method, expressing the resulting polynucleotide, examining its lipase activity, and selecting a polynucleotide encoding a protein having the desired lipase activity.

[0024] Site-directed mutagenesis into a polynucleotide can be carried out by any method, such as inverse PCR or annealing (Muramatsu et al., eds., "New Genetic Engineering Handbook, 4th Revised Edition," Yodosha, pp. 82-88). If necessary, various commercially available site-directed mutagenesis kits, such as Stratagene's QuickChange II Site-Directed Mutagenesis Kit and QuickChange Multi Site-Directed Mutagenesis Kit, can also be used.

[0025] A polynucleotide encoding the lipase of the present invention can be incorporated into a vector. The type of vector containing the polynucleotide is not particularly limited and may be any vector, such as a plasmid, phage, phagemid, cosmid, virus, YAC vector, or shuttle vector. The vector is preferably, but not limited to, a vector that can be amplified in bacteria, preferably in Bacillus bacteria (e.g., Bacillus subtilis or a mutant thereof), and more preferably an expression vector that can induce expression of an introduced gene in Bacillus bacteria. In particular, shuttle vectors, which are vectors that can be replicated in both Bacillus bacteria and other organisms, are suitable for recombinant production of the lipase of the present invention. Examples of preferred vectors include, but are not limited to, shuttle vectors such as pHA3040SP64, pHSP64R, or pASP64 (Japanese 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 plasmid vectors that can be used to transform Bacillus bacteria, such as pUB110 (J Bacteriol, 1978, 134:318-329) and pTA10607 (Plasmid, 1987, 18:8-15). Plasmid vectors derived from E. coli (e.g., pET22b(+), pBR322, pBR325, pUC57, pUC118, pUC119, pUC18, pUC19, pBluescript, etc.) can also be used.

[0026] The vector may contain a DNA replication origin region or a DNA region containing a replication origin. Alternatively, the vector may have a control sequence, such as a promoter region for initiating transcription of the gene, a terminator region, or a secretion signal region for secreting the expressed protein extracellularly, operably linked upstream of the polynucleotide encoding the lipase of the present invention (i.e., the lipase gene of the present invention). As used herein, the phrase "operably linked" between a gene and a control sequence means that the gene and the control region are positioned so that the gene can be expressed under the control of the control region.

[0027] The types of the control sequences such as the promoter region, terminator region, and secretion signal region are not particularly limited, and commonly used promoters and secretion signal sequences can be appropriately selected and used depending on the host to be introduced into. For example, suitable examples of control sequences that can be incorporated into a vector include the promoter and secretion signal sequence of the cellulase gene of Bacillus sp. KSM-S237 strain.

[0028] Alternatively, the vector of the present invention may further incorporate a marker gene (e.g., a resistance gene to a drug such as ampicillin, neomycin, kanamycin, or chloramphenicol) for selecting a host into which the vector has been appropriately introduced. Alternatively, when an auxotrophic strain is used as the host, a gene encoding an enzyme that synthesizes the required nutrient may be incorporated into the vector as a marker gene. Furthermore, when a selective medium requiring a specific metabolism for growth is used, a gene related to that metabolism may be incorporated into the vector as a marker gene. An example of such a metabolism-related gene is the acetamidase gene for utilizing acetamide as a nitrogen source.

[0029] The polynucleotide encoding the lipase of the present invention can be ligated to a regulatory sequence and a marker gene by methods known in the art, such as splicing by overlap extension (SOE)-PCR (Gene, 1989, 77:61-68). Procedures for introducing the ligated fragments into a vector are well known in the art.

[0030] The transformed cell of the present invention can be obtained by introducing a vector containing a polynucleotide encoding the lipase of the present invention into a host, or by introducing a DNA fragment containing a polynucleotide encoding the lipase of the present invention into the genome of the host.

[0031] Host cells include microorganisms such as bacteria and filamentous fungi. Examples of bacteria include Escherichia coli, Staphylococcus, Enterococcus, Listeria, and Bacillus bacteria, among which Escherichia coli and Bacillus bacteria (e.g., Bacillus subtilis Marburg No. 168 (Bacillus subtilis 168 strain) or mutants thereof) are preferred. Examples of Bacillus mutant strains include the KA8AX protease 9-fold deletion strain described in J. Biosci. Bioeng., 2007, 104(2):135-143, and the D8PA strain, an octaprotease deletion strain with improved protein folding efficiency described in Biotechnol. Lett., 2011, 33(9):1847-1852. Examples of filamentous fungi include the genera Trichoderma, Aspergillus, and Rhizopus.

[0032] The vector can be introduced into the host by a method commonly used in the field, such as the protoplast method, electroporation, etc. Strains into which the vector has been appropriately introduced can be selected based on the expression of a marker gene, auxotrophy, etc., to obtain the desired transformant into which the vector has been introduced.

[0033] Alternatively, a fragment comprising a polynucleotide encoding the lipase of the present invention, a regulatory sequence, and a marker gene can be directly introduced into the genome of a host. For example, a DNA fragment having sequences complementary to the host genome added to both ends of the above-mentioned ligated fragment can be constructed by SOE-PCR or other methods, and this can be introduced into a host to induce homologous recombination between the host genome and the DNA fragment, thereby introducing the polynucleotide encoding the lipase of the present invention into the genome of the host.

[0034] When the thus obtained transformant into which the polynucleotide encoding the lipase of the present invention or a vector containing the same has been introduced is cultured in an appropriate medium, the gene encoding the protein on the vector is expressed to produce the lipase of the present invention. The medium used to culture the transformant can be appropriately selected by those skilled in the art depending on the type of microorganism used as the transformant.

[0035] Alternatively, the lipase of the present invention may be expressed from a polynucleotide encoding the lipase of the present invention or a transcription product thereof using a cell-free translation system. The "cell-free translation system" is an in vitro transcription / translation system or an in vitro translation system constructed by adding reagents such as amino acids necessary for protein translation to a suspension obtained by mechanically disrupting host cells.

[0036] The lipase of the present invention produced in the above-mentioned culture or cell-free translation system can be isolated or purified by a general method used for protein purification, such as centrifugation, ammonium sulfate precipitation, gel chromatography, ion exchange chromatography, affinity chromatography, etc., either alone or in appropriate combination. The protein recovered from the culture may be further purified by known means.

[0037] <3. Cleaning composition> The lipase of the present invention thus obtained has significantly higher resistance to inhibition of lipase activity by surfactants than known proteins, and exhibits significantly higher detergency even in the presence of surfactants. Here, "high resistance" refers to a higher resistance than known proteins, specifically lipases consisting of the amino acid sequences set forth in SEQ ID NOs: 2, 4, 6, 8, 10, or 12, or proteins predicted to be lipases in the NCBI protein sequence database. Resistance to surfactant-induced lipase activity inhibition can be evaluated using methods well known in the art. For example, a lipase solution and a surfactant solution are mixed, and a 4-nitrophenyl octanoate solution, which serves as a lipase substrate, is added to the mixture. The change in absorbance at 405 nm (OD / min) associated with the release of 4-nitrophenol is measured, and the difference ΔOD / min from the blank is calculated as the lipase activity (lipase activity in the surfactant solution). Furthermore, the lipase activity (lipase activity in the buffer) is calculated when a buffer is used instead of the surfactant solution, and the lipase activity in the surfactant solution is divided by the lipase activity in the buffer and multiplied by 100 to determine the relative activity (%). A higher relative activity (%) indicates a higher resistance to surfactant-induced lipase activity inhibition. The lipase of the present invention is a lipase whose relative lipase activity (%) in an SDS solution (0.1% (w / v)) under the conditions of (3) in the Examples below is preferably 20% or more, more preferably 30% or more. The lipase of the present invention is a lipase whose relative lipase activity (%) in a Triton X-100 solution (0.1% (w / v)) under the conditions of (3) in the Examples below is preferably 50% or more, more preferably 70% or more. Furthermore, "high detergency" refers to a higher detergency, e.g., the ability to remove stains, during a washing or cleaning process than known proteins, specifically lipases consisting of the amino acid sequences set forth in SEQ ID NOs: 2, 4, 6, 8, 10, or 12, or proteins predicted to be lipases in the NCBI protein sequence database. Detergency can be evaluated using methods well known in the art. For example, a washing solution containing lipase is added to a model stain containing a predetermined indicator substance (e.g., a stain with high fat solubility, such as Sudan III), and the model stain is washed under specified conditions. A portion of the washing solution is sampled, and the concentration of the indicator substance in the model stain solubilized in the washing solution by the washing process is measured, for example, by absorbance measurement. The difference from the blank can be used to determine the detergency.

[0038] The lipase of the present invention is useful as an enzyme to be incorporated into various detergent compositions, and is particularly useful as an enzyme to be incorporated into detergent compositions suitable for low-temperature washing. Here, "low temperature" includes 40°C or lower, 35°C or lower, 30°C or lower, and 25°C or lower, as well as 5°C or higher, 10°C or higher, and 15°C or higher, and also includes 5 to 40°C, 10 to 35°C, 15 to 30°C, and 15 to 25°C.

[0039] The amount of the lipase of the present invention to be blended in the detergent composition is not particularly limited as long as the lipase exhibits activity, but is, for example, preferably 0.1 mg or more, more preferably 1 mg or more, more preferably 5 mg or more, and preferably 5,000 mg or less, more preferably 1,000 mg or less, more preferably 500 mg or less, per 1 kg of the detergent composition. The amount is also preferably 0.1 to 5,000 mg, more preferably 1 to 1,000 mg, and more preferably 5 to 500 mg.

[0040] The detergent composition preferably contains, in addition to the lipase of the present invention, a sulfosuccinate ester or a salt thereof. Sulfosuccinate esters or salts thereof are known as components incorporated into detergent compositions (for example, JP 2019-182911 A). The sulfosuccinate ester or salt thereof is preferably a branched alkyl sulfosuccinate ester having a branched alkyl group of 9 to 12 carbon atoms or a salt thereof, more preferably a branched alkyl sulfosuccinate ester having a branched alkyl group of 9 or 10 carbon atoms or a salt thereof, and even more preferably a branched alkyl sulfosuccinate ester having a branched alkyl group of 10 carbon atoms or a salt thereof. Furthermore, the sulfosuccinate ester or a salt thereof is a dibranched alkyl sulfosuccinate ester or a salt thereof, and is preferably a dibranched alkyl sulfosuccinate ester or a salt thereof in which two branched alkyl groups each have from 9 to 12 carbon atoms, more preferably a dibranched alkyl sulfosuccinate ester or a salt thereof in which two branched alkyl groups each have 9 or 10 carbon atoms, even more preferably a dibranched alkyl sulfosuccinate ester or a salt thereof in which two branched alkyl groups each have 10 carbon atoms, and even more preferably bis-(2-propylheptyl)sulfosuccinate or a salt thereof.

[0041] Examples of the salt include alkali metal salts and alkanolamine salts. Alkali metal salts or alkanolamine salts are preferred, and salts selected from sodium salts, potassium salts, triethanolamine salts, diethanolamine salts, and monoethanolamine salts are more preferred, with sodium salts being even more preferred.

[0042] Examples of sulfosuccinate esters or salts thereof include compounds represented by the following formula 1:

[0043] [ka]

[0044] [In formula 1, R 1 , R 2are branched alkyl groups having 9 to 12 carbon atoms, and A 1 O, A 2 Each O is an alkyleneoxy group having 2 to 4 carbon atoms, x1 and x2 are the average number of moles added and are each a number of 0 to 10, and M is a cation.

[0045] In formula 1, R 1 , R 2 are each preferably a branched alkyl group selected from a branched nonyl group, a branched decyl group, and a branched dodecyl group, and more preferably a branched decyl group. The branched decyl group is preferably a 2-propylheptyl group.

[0046] In formula 1, A 1 O, A 2 O is an alkyleneoxy group having 2 to 4 carbon atoms, preferably 2 or 3 carbon atoms in view of lubricity to water. 1 O, A 2 Each represents the average number of moles of O added, and is from 0 to 10, and from the viewpoint of lubricity in water, is preferably 6 or less, more preferably 4 or less, even more preferably 2 or less, with 0 being even more preferred.

[0047] In formula 1, M is a cation. M is preferably a cation other than a hydrogen ion. Examples of M include alkali metal ions such as lithium ion, sodium ion, and potassium ion, alkaline earth metal ions such as calcium ion and barium ion, and organic ammonium ions such as triethanolammonium ion, diethanolammonium ion, monoethanolammonium ion, trimethylammonium ion, and monomethylammonium ion. From the viewpoint of dispersibility in water, M is preferably an alkali metal ion or an alkanolammonium ion, more preferably a sodium ion, a potassium ion, a triethanolammonium ion, a diethanolammonium ion or a monoethanolammonium ion, and even more preferably a sodium ion.

[0048] The sulfosuccinate ester or a salt thereof is preferably a compound represented by the following formula 1-1: The compound of formula 1-1 is a compound in which x1 and x2 are each 0 in formula 1.

[0049] [ka]

[0050] [In formula 1-1, R 1 , R 2 are each a branched alkyl group having 9 to 12 carbon atoms, and M is a cation. R in Formula 1-1 1 , R 2 Specific examples and preferred examples of M are the same as those of formula 1. In a preferred embodiment, the sulfosuccinate or salt thereof is bis-(2-propylheptyl) sulfosuccinic acid or a salt thereof.

[0051] The amount of the sulfosuccinate or its salt in the detergent composition is preferably 0.01% by mass or more, more preferably 0.1% by mass or more, and preferably 2.0% by mass or less, more preferably 1.0% by mass or less, and is preferably 0.01 to 2.0% by mass, and more preferably 0.1 to 1.0% by mass.

[0052] The detergent composition can also contain various enzymes in addition to the lipase of the present invention. For example, hydrolases, oxidases, reductases, transferases, lyases, isomerases, ligases, synthetases, etc. Among these, lipases other than the lipase of the present invention, amylases, proteases, cellulases, keratinases, esterases, cutinases, pullulanases, pectinases, mannanases, glucosidases, glucanases, cholesterol oxidases, peroxidases, laccases, etc. are preferred, with proteases, cellulases, amylases, and lipases being particularly preferred. Examples of commercially available proteases include Alcalase, Esperase, Everlase, Savinase, Kannase, Progress Uno (registered trademark; Novozymes), PREFERENZ, EFFECTENZ, EXCELLENZ (registered trademark; DuPont), Lavergy (registered trademark; BASF), and KAP (Kao). Examples of cellulases include Celluclean, Carezyme (registered trademark; Novozymes), KAC, alkaline cellulase produced by Bacillus sp. KSM-S237 strain described in JP-A-10-313859, and mutant alkaline cellulase described in JP-A-2003-313592 (all Kao products). Examples of amylases include Termamyl, Duramyl, Stainzyme, Stainzyme Plus, Amplify Prime (registered trademark; Novozymes), PREFERENZ, EFFECTENZ (registered trademark; DuPont), and KAM (Kao). Examples of lipase include Lipolase and Lipex (registered trademark; Novozymes).

[0053] The detergent composition may contain known detergent components, and examples of such known detergent components include the following:

[0054] (1) Surfactants The surfactant is blended in the detergent composition in an amount of 0.5 to 60% by mass, preferably 10 to 45% by mass for powder detergent compositions and 20 to 90% by mass for liquid detergent compositions. When the detergent composition of the present invention is a laundry detergent or an automatic dishwasher detergent, the surfactant is blended in an amount of generally 1 to 10% by mass, preferably 1 to 5% by mass.

[0055] The surfactant used in the detergent composition may be one or a combination of anionic surfactants, nonionic surfactants, amphoteric surfactants, and cationic surfactants other than the above-mentioned sulfosuccinate esters or salts thereof, but anionic surfactants and nonionic surfactants are preferred.

[0056] Preferred anionic surfactants include sulfate salts of alcohols having 10 to 18 carbon atoms, sulfate salts of alkoxylated alcohols having 8 to 20 carbon atoms, alkylbenzenesulfonates, paraffin sulfonates, α-olefinsulfonates, internal olefinsulfonates, α-sulfofatty acid salts, α-sulfofatty acid alkyl ester salts, and fatty acid salts. In the present invention, one or more anionic surfactants selected from linear alkylbenzenesulfonates having an alkyl chain of 10 to 14 carbon atoms, more preferably 12 to 14 carbon atoms, and internal olefinsulfones having an alkylene chain of 12 to 20 carbon atoms, more preferably 16 to 18 carbon atoms, are particularly preferred. The counter ion is preferably an alkali metal salt or an amine, particularly preferably sodium and / or potassium, monoethanolamine, or diethanolamine. For internal olefinsulfonic acids, see, for example, WO2017 / 098637.

[0057] Preferred nonionic surfactants include polyoxyalkylene alkyl (C8-20) ethers, alkyl polyglycosides, polyoxyalkylene alkyl (C8-20) phenyl ethers, polyoxyalkylene sorbitan fatty acid (C8-22) esters, polyoxyalkylene glycol fatty acid (C8-22) esters, and polyoxyethylene polyoxypropylene block polymers. Particularly preferred nonionic surfactants are polyoxyalkylene alkyl ethers in which 4 to 20 moles of alkylene oxide, such as ethylene oxide or propylene oxide, are added to alcohols having 10 to 18 carbon atoms (thus having an HLB value (calculated by the Griffin method) of 10.5 to 15.0, preferably 11.0 to 14.5).

[0058] (2) Divalent metal ion scavenger The divalent metal ion scavenger is blended in an amount of 0.01 to 50 mass%, preferably 5 to 40 mass%. Examples of divalent metal ion scavenger agents used in the detergent composition of the present invention include condensed phosphates such as tripolyphosphates, pyrophosphates, and orthophosphates, aluminosilicates such as zeolites, synthetic layered crystalline silicates, nitrilotriacetates, ethylenediaminetetraacetates, citrates, isocitrates, and polyacetalcarboxylates. Among these, crystalline aluminosilicates (synthetic zeolites) are particularly preferred, with A-type zeolites being particularly preferred among A-, X-, and P-type zeolites. Synthetic zeolites with an average primary particle size of 0.1 to 10 μm, and particularly 0.1 to 5 μm, are preferably used.

[0059] (3) Alkaline agent The alkaline agent is blended in an amount of 0.01 to 80% by mass, preferably 1 to 40% by mass. In the case of powder detergents, examples include alkali metal carbonates such as sodium carbonate, collectively known as dense ash or light ash, and amorphous alkali metal silicates such as JIS No. 1, No. 2, and No. 3. These inorganic alkaline agents are effective in forming the particle skeleton during detergent drying, resulting in a detergent that is relatively hard and has excellent fluidity. Other examples of alkalis include sodium sesquicarbonate and sodium bicarbonate, and phosphates such as tripolyphosphates also function as alkaline agents. In addition to the above alkaline agents, sodium hydroxide and mono-, di-, or triethanolamine can also be used as alkaline agents in liquid detergents, and they can also be used as counterions for the active agent.

[0060] (4) Anti-redeposition agent The anti-redeposition agent is blended in an amount of 0.001 to 10% by mass, preferably 1 to 5% by mass. Examples of anti-redeposition agents used in the detergent composition of the present invention include polyethylene glycol, carboxylic acid polymers, polyvinyl alcohol, and polyvinylpyrrolidone. Among these, carboxylic acid polymers have the ability to prevent redeposition, as well as the ability to capture metal ions and disperse solid particle soils from clothing into the wash bath. The carboxylic acid polymer is a homopolymer or copolymer of acrylic acid, methacrylic acid, itaconic acid, or the like. Suitable copolymers are copolymers of the above monomers with maleic acid, and preferably have a molecular weight of several thousand to 100,000. In addition to the above carboxylic acid polymers, polymers such as polyglycidyl acid salts, cellulose derivatives such as carboxymethylcellulose, and aminocarboxylic acid polymers such as polyaspartic acid are also preferred because they have the ability to capture metal ions, disperse, and prevent redeposition.

[0061] (5) Bleach For example, bleaching agents such as hydrogen peroxide and percarbonate are preferably blended in an amount of 1 to 10% by mass. When using bleaching agents, tetraacetylethylenediamine (TAED) or a bleaching activator such as that described in JP-A-6-316700 can be blended in an amount of 0.01 to 10% by mass.

[0062] (6) Fluorescent agent Fluorescent agents used in the detergent composition include biphenyl-type fluorescent agents (such as Tinopal CBS-X) and stilbene-type fluorescent agents (such as DM-type fluorescent dyes). The fluorescent agent is preferably blended in an amount of 0.001 to 2% by mass.

[0063] (7) Other ingredients The detergent composition may contain builders, softeners, reducing agents (such as sulfites), foam inhibitors (such as silicones), fragrances, antibacterial and antifungal agents (such as Proxel (trade name) and benzoic acid), and other additives known in the field of laundry detergents.

[0064] The detergent composition can be produced by combining the lipase of the present invention obtained by the above method with the above-mentioned known detergent components according to a conventional method. The detergent form can be selected depending on the application, and can be, for example, a liquid, powder, granule, paste, solid, etc.

[0065] The detergent composition thus obtained can be used as a clothing detergent, dish detergent, bleach, detergent for cleaning hard surfaces, drain cleaner, denture cleaner, disinfectant cleaner for medical instruments, etc., but is preferably used as a clothing detergent or dish detergent, and more preferably used as a laundry detergent (laundry laundry detergent), dish detergent for hand washing, or detergent for automatic dishwashers. The detergent composition is suitable for use at temperatures of 40° C. or lower, 35° C. or lower, 30° C. or lower, or 25° C. or lower, and 5° C. or higher, 10° C. or higher, or 15° C. or higher. The detergent composition is also suitable for use at temperatures of 5 to 40° C., 10 to 35° C., 15 to 30° C., or 15 to 25° C. Preferred modes of use include use in low-temperature (15 to 30° C.) washing in a laundry or low-temperature (15 to 30° C.) washing in an automatic dishwasher.

[0066] The detergent composition of the present invention can be used to clean objects requiring stain removal (e.g., clothes, tableware, hard surfaces, drain pipes, dentures, medical instruments, etc.), i.e., to remove stains. Such a cleaning method includes contacting the object requiring stain removal with the detergent composition of the present invention. Preferably, the stain is sebum stain or stain containing oils and fats derived from food.

[0067] In the cleaning method of the present invention, the object to be cleaned may be brought into contact with the detergent composition by immersing the object in water containing the detergent composition, or by directly applying the detergent composition to the object. In the method of the present invention, the object to be cleaned after the immersion or application of the detergent composition may be further washed by hand or in a washing machine, but this is not necessarily required.

[0068] In relation to the above-described embodiment, the present invention further discloses the following aspects. <1> A lipase consisting of the amino acid sequence shown in SEQ ID NO: 14 or an amino acid sequence having at least 91% identity thereto. <2> It consists of the amino acid sequence shown in SEQ ID NO: 14. <1> The lipase described in <3> <1> or <2> A polynucleotide encoding the lipase described in <4> <3> A vector or DNA fragment comprising the polynucleotide described in . <5> <4> A transformed cell containing the vector or DNA fragment described in 1. <6> It is a microorganism, <5> A transformed cell according to claim 1. <7> E. coli or Bacillus bacteria, <5> or <6> A transformed cell according to claim 1.

[0069] <8> <1> or <2> A detergent composition containing the lipase described in 1. <9> The composition further contains a sulfosuccinate ester or a salt thereof, preferably a branched alkyl sulfosuccinate ester having a branched alkyl group having from 9 to 12 carbon atoms or a salt thereof, more preferably a branched alkyl sulfosuccinate ester having a branched alkyl group having 9 or 10 carbon atoms or a salt thereof, and even more preferably a branched alkyl sulfosuccinate ester having a branched alkyl group having 10 carbon atoms or a salt thereof, <8> The cleaning composition according to claim 1. <10> The sulfosuccinic acid diester or a salt thereof, preferably a sulfosuccinic acid di-branched alkyl ester or a salt thereof in which two branched chain alkyl groups each have from 9 to 12 carbon atoms, more preferably a sulfosuccinic acid di-branched alkyl ester or a salt thereof in which two branched chain alkyl groups each have 9 or 10 carbon atoms, even more preferably a sulfosuccinic acid di-branched alkyl ester or a salt thereof in which two branched chain alkyl groups each have 10 carbon atoms, and even more preferably bis-(2-propylheptyl)sulfosuccinic acid or a salt thereof, <8> The cleaning composition according to claim 1. <11> A laundry detergent or dishwashing detergent, <8> ~ <10> The cleaning composition according to any one of the preceding claims. <12> It is a powder or a liquid, <8> ~ <11> The cleaning composition according to any one of the preceding claims. <13> Used at low temperatures, <8> ~ <12> The cleaning composition according to any one of the preceding claims. <14> Used at temperatures below 40°C, below 35°C, below 30°C, below 25°C, and above 5°C, above 10°C, above 15°C, or used at temperatures between 5 and 40°C, 10 and 35°C, 15 and 30°C, or 15 and 25°C. <13> The cleaning composition according to claim 1.

[0070] <15> <8> ~ <14> A method for cleaning stains using the detergent composition according to any one of the above. <16> The item to be washed <8> ~ <14> The cleaning composition according to any one of the preceding claims is contacted with the cleaning composition. <15> The method described below. <17> The stain is a sebum stain or a stain containing oil or fat derived from food. <15> or <16> The method described below.

[0071] <18> Use of a lipase consisting of the amino acid sequence shown in SEQ ID NO: 14 or an amino acid sequence having at least 91% identity thereto for the production of a cleaning composition. <19> The lipase is a lipase consisting of the amino acid sequence shown in SEQ ID NO: 14. <18> Use as described in. <20> The detergent composition is a laundry detergent or a dish detergent; <18> or <19> Use as described in. <21> The cleaning composition is a powder or a liquid. <18> ~ <20> The use according to any one of the preceding claims. <22> The cleaning composition is used at low temperatures. <18> ~ <21> The use according to any one of the preceding claims. <23> The detergent composition is used at 40°C or lower, 35°C or lower, 30°C or lower, or 25°C or lower, and 5°C or higher, 10°C or higher, or 15°C or higher, or at 5 to 40°C, 10 to 35°C, 15 to 30°C, or 15 to 25°C. <22> Use as described in.

[0072] <24> Use of a lipase consisting of the amino acid sequence shown in SEQ ID NO: 14 or an amino acid sequence having at least 91% identity thereto for cleaning stains. <25> The lipase is a lipase consisting of the amino acid sequence shown in SEQ ID NO: 14. <24> Use as described in. <26> The stain is a sebum stain or a stain containing oil or fat derived from food. <24> or <25> Use as described in. <27> The washing is done at low temperatures, <24> ~ <26> The use according to any one of the preceding claims. <28> Washing is performed at a temperature of 40°C or less, 35°C or less, 30°C or less, or 25°C or less, and 5°C or more, 10°C or more, or 15°C or more, or at a temperature of 5 to 40°C, 10 to 35°C, 15 to 30°C, or 15 to 25°C. <27> Use as described in. [Example]

[0073] The present invention will be described in more detail below based on examples, but the present invention is not limited to these examples.

[0074] (1) Construction of lipase expression plasmid The lipase expression plasmid was constructed using as a template the VHH expression plasmid of SEQ ID NO: 26, which contains the Bacillus subtilis spoVG gene-derived promoter described in WO2021 / 153129. Each lipase gene was inserted into the full-length ORF containing the VHH gene of the above plasmid by an in-fusion reaction. The artificially synthesized lipase genes Lipr139, PvLip, PfLip, EtLip, EspLip, YeLip, KAL-A8, and Lipr138 (encoding the polynucleotides of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, and 15, respectively, and the amino acid sequences of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, and 16, respectively) were used to construct the plasmids pHY-Lipr139, pHY-PvLip, pHY-PfLip, pHY-EtLip, pHY-EspLip, pHY-YeLip, pHY-KAL-A8, and pHY-Lipr138, respectively. pHY-amyEsig-TLL was constructed by replacing the full-length ORF containing the Lipr139 gene of the plasmid pHY-Lipr139 with an artificially synthesized gene for the lipase TLL gene (polynucleotide of SEQ ID NO: 17, encoding the amino acid sequence of SEQ ID NO: 18) to which a signal sequence derived from Bacillus subtilis amyE (polynucleotide of SEQ ID NO: 19, encoding the amino acid sequence of SEQ ID NO: 20) was linked at the N-terminus by an In-Fusion reaction.

[0075] (2) Preparation of lipase solution The lipase expression plasmid was introduced into a Bacillus subtilis strain by the protoplast method and cultured in 2x L-maltose medium (2% tryptone, 1% yeast extract, 1% NaCl, 7.5% maltose, 7.5 ppm manganese sulfate pentahydrate, 0.04% calcium chloride dihydrate, 15 ppm tetracycline; % (w / v)%) at 30°C for 2 days. The culture supernatant containing lipase was then collected by centrifugation. The culture supernatant was buffer-exchanged into 10 mM Tris-HCl + 0.01% Triton-X100 (pH 7.0) by dialysis, and the lipase concentration was quantified based on the band intensity on SDS-PAGE.

[0076] (3) Activity measurement in surfactant solution 4-Nitrophenyl octanoate (SIGMA) was used as the substrate. Lipase activity can be determined by measuring the rate of increase in absorbance associated with the release of 4-nitrophenol by lipase. 20 mM 4-nitrophenyl octanoate in 20 mM Tris-HCl (pH 7.0) was used as the substrate solution. 20 mM Tris-HCl (pH 7.0) supplemented with 0.1% (w / v) SDS or Triton X-100 was used as the surfactant solution. 5 μL of lipase solution adjusted to 2 ppm and 100 μL of surfactant solution were mixed in each well of a 96-well assay plate, and 10 μL of substrate solution was added. The change in absorbance (OD / min) at 405 nm was measured at 30°C. The difference ΔOD / min from the blank (sample without enzyme addition) was used as the activity value. For each lipase, the activity in the surfactant solution was divided by the activity in 20 mM Tris-HCl (pH 7.0) instead of the surfactant solution (activity in buffer), and the result was multiplied by 100 to determine the relative activity (%) (Figure 1). The activities of TLL, Lipr139, PvLip, and Lipr138, whose suitability for cleaning has been disclosed in Patent Documents 1, 2, and 3, and naturally occurring lipase sequences PfLip, EtLip, EspLip, and YeLip, were significantly inhibited in the presence of SDS and Triton X-100. In comparison, the novel lipase KAL-A8 showed high resistance to activity inhibition by SDS and Triton X-100.

[0077] (4) Activity measurement in model cleaning solution 20 mM 4-nitrophenyl octanoate in 20 mM Tris-HCl (pH 7.0) was used as the substrate solution. The aqueous media listed in Table 1 were used as model cleaning solutions. 2 μL of lipase solution adjusted to 4 ppm and 100 μL of test solution (model cleaning solution or 20 mM Tris-HCl (pH 7.0)) were mixed in each well of a 96-well assay plate, and 10 μL of substrate solution was added. The absorbance change (OD / min) at 405 nm was measured at 30°C. The difference (ΔOD / min) from the blank (sample without enzyme addition) was calculated. 10 μL of 20 mM Tris-HCl (pH 7.0) containing 31 to 500 μM 4-nitrophenol was mixed with 100 μL of each test solution, and the absorbance at 405 nm was measured to create a calibration curve. The release rate of 4-nitrophenol per minute (μM / min) was calculated as the activity value from the calibration curve and ΔOD / min value of each test solution. For each lipase, the activity value (activity in buffer) when 20 mM Tris-HCl (pH 7.0) was used as the test solution was divided by the activity value when the model cleaning solution was used as the test solution, and the result was multiplied by 100 to obtain the relative activity (%) (Figure 2). The activities of TLL, Lipr139, PvLip, and Lipr138, whose suitability for cleaning has been disclosed in Patent Documents 1, 2, and 3, and naturally occurring lipase sequences PfLip, EtLip, EspLip, and YeLip, were significantly inhibited in the model cleaning solutions. On the other hand, the novel lipase KAL-A8 showed high resistance to activity inhibition in the model cleaning solutions.

[0078] [Table 1]

[0079] (5) Cleaning evaluation in model cleaning solution A model soil was prepared by mixing beef tallow (SIGMA, 03-0660) and rapeseed oil (SIGMA, 23-0450) in a weight ratio of 9:1, dissolving the mixture in three volumes of chloroform, and then coloring it with 0.2 wt% Sudan III. Ten microliters of the model soil was dropped onto the bottom of each well of a 96-well polypropylene deep-well plate, and the chloroform was evaporated and dried to prepare the soil plate. The model cleaning solution listed in Table 1 was prepared by adding 1 / 100th the volume of a 240 ppm lipase solution. 300 μL of the cleaning solution was slowly added to the soiled plate and allowed to stand at room temperature (approximately 22°C) for 15 minutes for immersion cleaning. 100 μL of the cleaning solution was then transferred to a new 96-well plate, avoiding contact with the soil at the bottom. The absorbance at 500 nm (A500) was measured to quantify the amount of Sudan III in the model soil solubilized in the cleaning solution by immersion cleaning. The value ΔA500, calculated by subtracting the A500 before cleaning from the A500 after cleaning, corresponds to the amount of oil released into the cleaning solution and can be used as an indicator of cleaning power. The cleaning power ΔA500 of each lipase is shown in Figure 3. Lipr139, whose suitability for cleaning is disclosed in Patent Document 1, exhibited higher detergency in a model cleaning solution than TLL, Lipr138, PvLip, PfLip, EtLip, EspLip, and YeLip. On the other hand, the novel lipase KAL-A8 exhibited significantly higher detergency in a model cleaning solution than other lipases, including Lipr139.

Claims

1. A lipase consisting of the amino acid sequence shown in SEQ ID NO: 14 or an amino acid sequence having at least 91% identity thereto.

2. A polynucleotide encoding the lipase of claim 1.

3. A vector or DNA fragment comprising the polynucleotide of claim 2.

4. A transformed cell containing the vector or DNA fragment according to claim 3.

5. The transformed cell of claim 4, which is a microorganism.

6. A detergent composition containing the lipase according to claim 1.

7. The detergent composition according to claim 6, which is a laundry detergent or a dish detergent.

8. The cleaning composition of claim 7, which is a powder or a liquid.

9. The cleaning composition according to claim 7 or 8, which is used at low temperatures.

10. The cleaning composition according to claim 9, which is used at a temperature of 5 to 40°C.

11. A method for cleaning stains, comprising using the cleaning composition according to claim 6.

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