Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
  • C12N15/75—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Bacillus
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/14—Hydrolases (3)
  • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
  • C12N9/18—Carboxylic ester hydrolases (3.1.1)
  • C12N9/20—Triglyceride splitting, e.g. by means of lipase
• • C—CHEMISTRY; METALLURGY
  • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
  • C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
  • C11D17/00—Detergent materials or soaps characterised by their shape or physical properties
  • C11D17/06—Powder; Flakes; Free-flowing mixtures; Sheets
• • C—CHEMISTRY; METALLURGY
  • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
  • C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
  • C11D17/00—Detergent materials or soaps characterised by their shape or physical properties
  • C11D17/08—Liquid soap, e.g. for dispensers; capsuled
• • C—CHEMISTRY; METALLURGY
  • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
  • C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
  • C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
  • C11D3/16—Organic compounds
  • C11D3/38—Products with no well-defined composition, e.g. natural products
  • C11D3/386—Preparations containing enzymes, e.g. protease or amylase
  • C11D3/38627—Preparations containing enzymes, e.g. protease or amylase containing lipase
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Y—ENZYMES
  • C12Y301/00—Hydrolases acting on ester bonds (3.1)
  • C12Y301/01—Carboxylic ester hydrolases (3.1.1)

Definitions

• the present invention relates to a novel lipase.
• Lipases are useful in a variety of applications, including laundry detergents, dishwashing detergents, oil and fat processing, pulp processing, feed, and pharmaceutical intermediate synthesis. In cleaning, lipases contribute to the removal of oil-containing soils by hydrolyzing triglycerides to produce fatty acids.
• TLL lipase derived from Thermomyces lanuginosus
• LIPOLASE registered trademark
• Patent Document 1 discloses that lipase Lipr139 derived from Cedecea sp-16640 strain has superior cleaning performance compared to TLL.
• US Pat. No. 5,001,203 discloses variants of lipases from Proteus bacteria (hereinafter PvLip) that have improved cleaning performance when compared to one or more reference lipolytic enzymes.
• PvLip Proteus bacteria
• Patent Document 3 discloses that metagenome-derived lipase Lipr138 has superior cleaning performance compared to TLL.
• Patent Document 1 Japanese Translation of PCT Publication No. 2015-523078 (Patent Document 2) International Publication No. 2020/046613 (Patent Document 3) Japanese Patent Publication No. 2015-525248
• the present invention relates to the following 1) to 6).
• 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.
• a polynucleotide encoding the lipase described in 1).
• a transformed cell containing the vector or DNA fragment of 3).
• a cleaning composition containing the lipase according to 1).
• Lipase activity of each lipase in surfactant solution Lipase activity in model washing solution containing surfactant for each lipase. Detergent power of each lipase in a model cleaning solution containing a surfactant.
• lipase refers to triacylglycerol lipase (EC3.1.1.3), and refers to a group of enzymes that have the activity of hydrolyzing 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 octoate. Specific procedures for measuring lipase activity are detailed in the Examples below.
• the identity of amino acid sequences or nucleotide sequences is calculated by the Lipman-Pearson method (Science, 1985, 227: 1435-1441). Specifically, genetic information processing software GENETYX Ver. It is calculated by performing an analysis using a search homology program of 12 with Unit size to compare (ktup) set to 2.
• operable linkage between a control region such as a promoter and a gene means that the gene and the control region are linked so that the gene can be expressed under the control of the control region. means. Procedures for "operably linking" genes and control regions are well known to those skilled in the art.
• upstream and downstream with respect to a gene refer to upstream and downstream of the gene in the direction of transcription.
• a gene located downstream of a promoter means that the gene is located on the 3' side of the promoter in the DNA sense strand
• upstream of the gene means that the gene is located on the 5' side of the promoter in the DNA sense strand. means the area on the side.
• lipase It is known that the activity of lipase is inhibited by surfactants contained in cleaning compositions. As far as the present inventors actually verified, among the known lipases whose suitability for washing has been disclosed, all of TLL, Lipr139 and PvLip were significantly inhibited in activity by the coexistence of a surfactant. Inhibition of activity by surfactants is a major problem common to cleaning lipases. Furthermore, lipases are often used together with surfactants not only in cleaning applications but also in industrial applications such as pulp processing, and inhibition of activity by surfactants is a problem for all industrial lipases. There is a need for a lipase that is less inhibited by surfactants and exhibits high cleaning effects.
• the lipase of the present invention has significantly high resistance to activity inhibition by surfactants, and exhibits excellent cleaning effects even in the presence of surfactants.
• 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.
• Amino acid sequences having at least 91% identity include amino acid sequences in which one or more amino acids have been deleted, inserted, substituted, or added.
• amino acid sequence in which one or more amino acids are deleted, inserted, substituted, or added refers to 1 to 30, preferably 20 or less, more preferably 10 or less, and even more preferably 5 or less. Examples include amino acid sequences in which amino acids are deleted, inserted, substituted, or added.
• a lipase consisting of an amino acid sequence having at least 91% identity with the amino acid sequence shown in SEQ ID NO: 14 is an artificially produced variant of the lipase consisting of the amino acid sequence shown in SEQ ID NO: 14.
• the mutant can be produced by, for example, introducing a mutation into the gene encoding the amino acid sequence shown by SEQ ID NO: 14 by known mutagenesis methods such as ultraviolet irradiation or site-directed mutagenesis. It can be produced by expressing a gene having the mutation and selecting a protein having the desired lipase activity. Procedures for creating such variants are well known to those skilled in the art.
• the lipase of the present invention has an amino acid sequence different from conventionally isolated or purified lipases and proteins predicted as triacylglycerol lipase in the NCBI protein sequence database.
• Conventionally isolated or purified lipases include, for example, lipase TLL derived from Thermomyces lanuginosus (SEQ ID NO: 18), Cedecea sp.
• Lipase Lipr139 (SEQ ID NO: 2) derived from -16640 strain, lipase derived from a Proteus bacterium (hereinafter referred to as PvLip, SEQ ID NO: 4) disclosed as a parent enzyme of a group of lipase mutants suitable for washing in Patent Document 2.
• metagenome-derived lipase Lipr138 (SEQ ID NO: 16), which is disclosed as a lipase suitable for washing in Patent Document 3, and the like.
• proteins estimated as triacylglycerol lipase in the NCBI protein sequence database include the protein with accession number WP_123598507.1 (hereinafter referred to as PfLip, SEQ ID NO: 6) and the protein with accession number WP_115457195.1 (hereinafter referred to as PfLip).
• the lipase KAL-A8 of the present invention which consists of the amino acid sequence shown in SEQ ID NO: 14, has 72%, 60%, 59%, 68%, 86% and 71% of Lipr139, PvLip, PfLip, EtLip, EspLip and YeLip, respectively. Amino acid sequence identity is shown.
• the lipase of the present invention is a lipase consisting of the amino acid sequence shown by SEQ ID NO: 14.
• the lipase of the present invention can be produced, for example, by expressing the polynucleotide encoding the lipase of the present invention.
• 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.
• a polynucleotide encoding the lipase of the present invention or a vector containing the same is introduced into a host to obtain a transformant, and the transformant is cultured in an appropriate medium, the polynucleotide can be introduced into the transformant.
• the lipase of the present invention is produced from the polynucleotide encoding the lipase of the present invention.
• the lipase of the present invention can be obtained by isolating or purifying the produced lipase from the culture.
• 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. Furthermore, the polynucleotide encoding the lipase of the present invention may be in the form of single-stranded or double-stranded DNA, RNA, or artificial nucleic acid, or may be cDNA or chemically synthesized DNA without introns.
• a polynucleotide encoding the lipase of the present invention can be synthesized chemically or genetically based on the amino acid sequence of the lipase.
• the polynucleotide can be chemically synthesized based on the amino acid sequence of the lipase of the present invention described above.
• a nucleic acid synthesis contract service eg, provided by Medical and Biological Research Institute, Genscript, etc.
• the synthesized polynucleotide can also be amplified by PCR, cloning, etc.
• the polynucleotide encoding the lipase of the present invention can be mutated by a known mutagenesis method such as the ultraviolet irradiation or site-directed mutagenesis described above on the polynucleotide synthesized by the above procedure. It can be produced by introducing.
• a polynucleotide encoding the lipase of the present invention can be obtained by introducing mutations into the polynucleotide of SEQ ID NO: 13 by a known method, expressing the resulting polynucleotide to examine lipase activity, and producing a protein having the desired lipase activity. can be obtained by selecting a polynucleotide encoding.
• Site-specific mutations can be introduced into polynucleotides using any method such as the inverse PCR method or annealing method (edited by Muramatsu et al., "New Genetic Engineering Handbook, Revised 4th Edition,” Yodosha, p. 82-88). This can be done by If necessary, use Stratagene's QuickChange II Site-Directed Mutagenesis Kit or QuickChange Multi Site-Directed Mutagenesis Various commercially available site-directed mutagenesis kits such as Kit can also be used.
• any method such as the inverse PCR method or annealing method (edited by Muramatsu et al., "New Genetic Engineering Handbook, Revised 4th Edition," Yodosha, p. 82-88). This can be done by If necessary, use Stratagene's QuickChange II Site-Directed Mutagenesis Kit or QuickChange Multi Site-Directed Mutagenesis Various commercially available site-directed mutagenesis kits such as Kit can also be used.
• 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 a bacterium, preferably a bacterium of the genus Bacillus (for example, Bacillus subtilis or a mutant strain thereof), and more preferably a vector capable of amplifying the introduced gene in a bacterium of the genus Bacillus.
• shuttle vectors which are vectors that can be replicated in both Bacillus bacteria and other organisms, can be suitably used for recombinant production of the lipase of the present invention.
• preferred vectors include, but are not limited to, pHA3040SP64, pHSP64R or pASP64 (Patent No.
• pHY300PLK expression vector capable of transforming both Escherichia coli and Bacillus subtilis; Jpn J Genet, 1985, 60: Shuttle vectors such as pAC3 (Nucleic Acids Res, 1988, 16:8732); pUB110 (J Bacteriol, 1978, 134:318-329), pTA10607 (Plasmid, 1987, 18:8-1) 5) etc.
• Examples include plasmid vectors that can be used to transform bacteria belonging to the genus Bacillus.
• plasmid vectors derived from E. coli eg, pET22b(+), pBR322, pBR325, pUC57, pUC118, pUC119, pUC18, pUC19, pBluescript, etc.
• E. coli eg, pET22b(+), pBR322, pBR325, pUC57, pUC118, pUC119, pUC18, pUC19, p
• the above-mentioned vector may include a DNA region containing a DNA replication initiation region or an origin of replication.
• a promoter region, a terminator region, or an expressed protein for initiating transcription of the gene is placed upstream of the polynucleotide encoding the lipase of the present invention (i.e., the lipase gene of the present invention).
• a control sequence such as a secretory signal region for secretion to the outside may be operably linked.
• the term "operably linked" between a gene and a control sequence means that the gene and the control region are arranged such that the gene can be expressed under the control of the control region. means.
• control sequences such as the promoter region, terminator region, secretion signal region, etc. 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.
• suitable examples of control sequences that can be incorporated into vectors include Bacillus sp.
• Examples include the cellulase gene promoter and secretion signal sequence of the KSM-S237 strain.
• the vector of the present invention may further incorporate a marker gene (for example, a drug resistance gene for ampicillin, neomycin, kanamycin, chloramphenicol, etc.) for selecting a host into which the vector has been appropriately introduced. You can leave it there.
• a marker gene for example, a drug resistance gene for ampicillin, neomycin, kanamycin, chloramphenicol, etc.
• a gene encoding a required nutrient synthesizing enzyme may be incorporated into the vector as a marker gene.
• a gene related to the metabolism may be incorporated into the vector as a marker gene.
• An example of such a metabolism-related gene is an acetamidase gene for utilizing acetamide as a nitrogen source.
• the above polynucleotide encoding the lipase of the present invention and the control sequence and marker gene can be linked by a method known in the art such as SOE (splicing by overlap extension)-PCR method (Gene, 1989, 77:61-68). This can be done by any method. Procedures for introducing ligated fragments into vectors are well known in the art.
• the transformed cells of the present invention can be transformed by introducing into a host a vector containing a polynucleotide encoding the lipase of the present invention, or by introducing a DNA fragment containing a polynucleotide encoding the lipase of the present invention into the genome of the host. Obtainable.
• Examples of host cells include microorganisms such as bacteria and filamentous fungi.
• bacteria include Escherichia coli, bacteria belonging to the genus Staphylococcus, genus Enterococcus, genus Listeria, and genus Bacillus.
• Bacillus bacteria for example, Bacillus subtilis Marburg No. 168 or mutant strains thereof) are preferred.
• Bacillus subtilis mutants include J. subtilis mutants. Biosci. Bioeng. , 2007, 104(2): 135-143, and the protease 9-deficient strain KA8AX, as well as Biotechnol. Lett.
• the D8PA strain is a protease octad-deficient strain with improved protein folding efficiency.
• filamentous fungi include Trichoderma, Aspergillus, Rhizopus, and the like.
• a method for introducing the vector into the host methods commonly used in the field, such as protoplast method and electroporation method, can be used. By selecting a strain into which the introduction has been properly carried out using marker gene expression, auxotrophy, etc. as indicators, it is possible to obtain the desired transformant into which the vector has been introduced.
• a fragment in which a polynucleotide encoding the lipase of the present invention, a control sequence, and a marker gene are linked can be directly introduced into the host genome.
• a DNA fragment is constructed by adding sequences complementary to the host's genome to both ends of the above-mentioned ligated fragment by SOE-PCR method, etc., and this is introduced into the host to create a gap between the host genome and the DNA fragment.
• a polynucleotide encoding the lipase of the present invention is introduced into the genome of the host.
• the thus obtained transformant into which the polynucleotide encoding the lipase of the present invention or the vector containing the same has been introduced is cultured in an appropriate medium, the gene encoding the protein on the vector will be expressed.
• a lipase of the invention is produced.
• the medium used for culturing the transformant can be appropriately selected by those skilled in the art depending on the type of microorganism of the transformant.
• 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.
• a "cell-free translation system” is an in vitro transcription translation system or an in vitro translation system in which reagents such as amino acids necessary for protein translation are added to a suspension obtained by mechanically disrupting host cells. It is composed of
• the lipase of the present invention produced in the above-mentioned culture or cell-free translation system can be produced by conventional methods used for protein purification, such as centrifugation, ammonium sulfate precipitation, gel chromatography, ion exchange chromatography, affinity chromatography, etc. Isolation or purification can be achieved by using either alone or in appropriate combination. Proteins recovered from the culture may be further purified by known means.
• the thus obtained lipase of the present invention has significantly higher resistance to inhibition of lipase activity by surfactants than known proteins, and has significantly higher detergency even in the presence of surfactants. Show power.
• “high resistance” refers to a known protein, specifically a lipase consisting of the amino acid sequence shown by SEQ ID NO: 2, 4, 6, 8, 10 or 12, or a lipase estimated to be a lipase in the NCBI protein sequence database. It means high resistance compared to proteins. Resistance to inhibition of lipase activity by detergents can be assessed using methods well known in the art.
• a lipase solution and a surfactant solution are mixed, a 4-nitrophenyl octoate solution, which is a substrate for lipase, is added to the mixture, and the absorbance change at 405 nm (OD/min) due to the release of 4-nitrophenol is measured. is measured, and the difference ⁇ OD/min from the blank is determined as the lipase activity value (lipase activity value in the surfactant solution).
• calculate the lipase activity value when a buffer is used instead of the surfactant solution (lipase activity value in the buffer) divide the lipase activity value in the surfactant solution by the lipase activity value in the buffer, and calculate 100.
• the multiplied value is calculated as relative activity (%). It can be determined that the higher the relative activity (%), the higher the resistance to inhibition of lipase activity by surfactants.
• the lipase of the present invention preferably has a relative lipase activity (%) of 20% or more, more preferably 30% in an SDS solution (0.1% (w/v)) under the conditions (3) of Examples below. These are the above lipases.
• the lipase of the present invention has a relative lipase activity (%) in a Triton X-100 solution (0.1% (w/v)) of preferably 50% or more, more preferably is more than 70% lipase.
• high detergency refers to a known protein, specifically a lipase consisting of the amino acid sequence shown by SEQ ID NO: 2, 4, 6, 8, 10, or 12, or a lipase estimated to be a lipase in the NCBI protein sequence database. It refers to the ability to provide high detergency, eg, soil removal, in a laundering or washing process compared to proteins. Detergency can be assessed using methods well known in the art. For example, a cleaning solution containing lipase is added to a model stain containing a predetermined indicator substance (for example, a dye with high fat solubility such as Sudan III), and the stain is washed under predetermined conditions. A portion of the cleaning liquid is taken out, and the concentration of the indicator substance in the model soil solubilized in the cleaning liquid through the cleaning process is measured, for example, by absorbance measurement, and the difference from the blank can be determined as the cleaning power.
• a predetermined indicator substance for example, a dye with high fat solubility such as Sudan
• the lipase of the present invention is useful as an enzyme for blending in various cleaning compositions, and is particularly useful as an enzyme for blending in cleaning compositions suitable for low-temperature cleaning.
• “low temperature” includes 40°C or lower, 35°C or lower, 30°C or lower, and 25°C or lower, and also includes 5°C or higher, 10°C or higher, and 15°C or higher. Further examples include 5 to 40°C, 10 to 35°C, 15 to 30°C, and 15 to 25°C.
• the amount of the lipase of the present invention incorporated into the cleaning composition is not particularly limited as long as the lipase exhibits activity, but for example, preferably 0.1 mg or more, more preferably 1 mg per 1 kg of the cleaning composition. Above, it is more preferably 5 mg or more, and preferably 5000 mg or less, more preferably 1000 mg or less, and even more preferably 500 mg or less. Further, it is preferably 0.1 to 5000 mg, more preferably 1 to 1000 mg, and even more preferably 5 to 500 mg.
• the cleaning composition preferably contains a sulfosuccinate or a salt thereof in addition to the lipase of the present invention.
• Sulfosuccinic acid esters or salts thereof are known as components to be included in cleaning compositions (for example, Japanese Patent Application Publication No. 2019-182911).
• the sulfosuccinic acid ester or its salt is preferably a sulfosuccinic acid branched alkyl ester or its salt having a branched chain alkyl group having 9 to 12 carbon atoms, and a sulfosuccinic acid branched alkyl ester having a branched chain alkyl group having 9 or 10 carbon atoms or Salts thereof are more preferred, and sulfosuccinic acid branched alkyl esters having a branched alkyl group having 10 carbon atoms or salts thereof are even more preferred.
• the sulfosuccinic acid ester or its salt is a sulfosuccinic acid di-branched alkyl ester or its salt
• the two branched-chain alkyl groups are each a branched-chain alkyl group having 9 or more and 12 or less carbon atoms.
• Esters or salts thereof are preferred, and sulfosuccinic acid di-branched alkyl esters or salts thereof, in which the two branched alkyl groups each have 9 or 10 carbon atoms, are more preferred, and the two branched alkyl groups each have 10 carbon atoms.
• sulfosuccinic acid di-branched alkyl esters or salts thereof which are branched chain alkyl groups
• bis-(2-propylheptyl)sulfosuccinic acid or salts thereof are particularly preferred.
• the salt examples include alkali metal salts and alkanolamine salts, preferably alkali metal salts or alkanolamine salts, and salts selected from sodium salts, potassium salts, triethanolamine salts, diethanolamine salts, and monoethanolamine salts. is more preferred, and sodium salt is even more preferred.
• Examples of the sulfosuccinic acid ester or its salt include a compound represented by the following formula 1.
• R 1 and R 2 are each a branched alkyl group having 9 to 12 carbon atoms, and A 1 O and A 2 O are each an alkyleneoxy group having 2 to 4 carbon atoms.
• x1 and x2 are average numbers of added moles, each of which is a number from 0 to 10, and M is a cation.
• R 1 and 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.
• a 1 O and A 2 O are each an alkyleneoxy group having 2 or more and 4 or less carbon atoms, preferably 2 or 3 carbon atoms from the viewpoint of lubricity against water.
• x1 and x2 represent the average number of added moles of A 1 O and A 2 O, respectively, from 0 to 10, preferably from the viewpoint of water lubricity to 6 or less, more preferably 4 or less, and Preferably the number is 2 or less, and 0 is more preferable.
• M is a cation.
• M is preferably a cation other than a hydrogen ion.
• M include alkali metal ions such as lithium ions, sodium ions, and potassium ions, alkaline earth metal ions such as calcium ions and barium ions, triethanolammonium ions, diethanolammonium ions, monoethanolammonium ions, and trimethylammonium ions. , organic ammonium ions such as monomethylammonium ion, and the like.
• M is preferably an alkali metal ion or an alkanol ammonium ion, more preferably a sodium ion, a potassium ion, a triethanol ammonium ion, a diethanol ammonium ion, or a monoethanol ammonium ion, and even more preferably a sodium ion.
• the sulfosuccinic acid ester or its salt 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 in formula 1 are each 0.
• R 1 and R 2 are each a branched alkyl group having 9 or more and 12 or less carbon atoms, and M is a cation.
• Specific examples and preferred examples of R 1 , R 2 and M in Formula 1-1 are the same as in Formula 1.
• the sulfosuccinate or salt thereof is bis-(2-propylheptyl)sulfosuccinic acid or a salt thereof.
• the amount of the sulfosuccinate or its salt added to the cleaning 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 It is 1.0% by mass or less. Further, it is preferably 0.01 to 2.0% by mass, and more preferably 0.1 to 1.0% by mass.
• the cleaning composition can also contain various enzymes in addition to the lipase of the present invention.
• enzymes include hydrolase, oxidase, reductase, transferase, lyase, isomerase, ligase, synthetase, and the like.
• lipases, amylases, proteases, cellulases, keratinases, esterases, cutinases, pullulanase, pectinases, mannanases, glucosidases, glucanases, cholesterol oxidases, peroxidases, laccases, etc. which are different from the lipase of the present invention, are preferable, and particularly proteases, cellulases, and amylases. , lipase is preferred.
• Proteases include commercially available Alcalase, Esperase, Everlase, Savinase, Kannase, Progress Uno (registered trademark; Novozymes), PREFERENZ, EFFECTENZ, EXCELLENZ (registered trademark; DuPont), Lav ergy (registered trademark; BASF), and KAP ( Kao), etc.
• cellulases include Celluclean, Carezyme (registered trademark; Novozymes), KAC, alkaline cellulase produced by Bacillus sp. Examples include alkaline cellulase (Kao).
• amylase examples include Termamyl, Duramyl, Stainzyme, Stainzyme Plus, Amplify Prime (registered trademark; Novozymes), PREFERENZ, EFFECTENZ (registered trademark; DuPont), and KAM (Kao).
• lipases examples include Lipolase and Lipex (registered trademark; Novozymes).
• Known detergent components can be blended into the detergent composition, and examples of the known detergent components include the following.
• the surfactant is blended in the cleaning composition in an amount of 0.5 to 60% by mass, particularly 10 to 45% by mass for powdered cleaning compositions, and 20 to 90% for liquid cleaning compositions. It is preferable to mix % by mass. Furthermore, when the detergent composition of the present invention is a laundry detergent or a detergent for automatic dishwashers, the surfactant is generally blended in an amount of 1 to 10% by mass, preferably 1 to 5% by mass.
• the surfactants used in the cleaning composition include anionic surfactants, nonionic surfactants, amphoteric surfactants, and cationic surfactants other than the above-mentioned sulfosuccinates or their salts.
• anionic surfactants and nonionic surfactants are preferred.
• anionic surfactants include sulfate ester salts of alcohols having 10 to 18 carbon atoms, sulfate ester salts of alkoxylated alcohols having 8 to 20 carbon atoms, alkylbenzene sulfonates, paraffin sulfonates, and ⁇ -olefin sulfones.
• Preferred are acid salts, internal olefin sulfonates, ⁇ -sulfo fatty acid salts, ⁇ -sulfo fatty acid alkyl ester salts, or fatty acid salts.
• a linear alkylbenzene sulfonate having an alkyl chain of 10 to 14 carbon atoms, more preferably 12 to 14 carbon atoms, and an internal olefin having an alkylene chain of 12 to 20 carbon atoms, more preferably 16 to 18 carbon atoms
• One or more anionic surfactants selected from sulfones are preferred, and as counterions, alkali metal salts and amines are preferred, with sodium and/or potassium, monoethanolamine, and diethanolamine being particularly preferred.
• alkali metal salts and amines are preferred, with sodium and/or potassium, monoethanolamine, and diethanolamine being particularly preferred.
• Nonionic surfactants include polyoxyalkylene alkyl (8 to 20 carbon atoms) ether, alkyl polyglycoside, polyoxyalkylene alkyl (8 to 20 carbon atoms) phenyl ether, polyoxyalkylene sorbitan fatty acid (8 to 20 carbon atoms) 22) Ester, polyoxyalkylene glycol fatty acid (carbon number 8 to 22) ester, and polyoxyethylene polyoxypropylene block polymer are preferred.
• 4 to 20 moles of alkylene oxide such as ethylene oxide or propylene oxide are added to an alcohol having 10 to 18 carbon atoms [HLB value (calculated by Griffin method) of 10.5 to 15. 0, preferably 11.0 to 14.5] are preferred.
• the divalent metal ion scavenger is blended in an amount of 0.01 to 50% by mass, preferably 5 to 40% by mass.
• the divalent metal ion scavenger used in the cleaning composition of the present invention includes condensed phosphates such as tripolyphosphate, pyrophosphate, and orthophosphate, aluminosilicates such as zeolite, and synthetic layered crystalline silicates. , nitrilotriacetate, ethylenediaminetetraacetate, citrate, isocitrate, polyacetal carboxylate, and the like.
• crystalline aluminosilicate is particularly preferred, and among A-type, X-type, and P-type zeolites, A-type is particularly preferred.
• the synthetic zeolite preferably has an average primary particle size of 0.1 to 10 ⁇ m, particularly 0.1 to 5 ⁇ m.
• Alkaline agent is blended in an amount of 0.01 to 80% by weight, preferably 1 to 40% by weight.
• examples include alkali metal carbonates such as sodium carbonate, which are collectively referred to as dense ash and light ash, and amorphous alkali metal silicates such as JIS No. 1, No. 2, and No. 3.
• alkali metal carbonates such as sodium carbonate, which are collectively referred to as dense ash and light ash
• amorphous alkali metal silicates such as JIS No. 1, No. 2, and No. 3.
• These inorganic alkaline agents are effective in forming particle skeletons during detergent drying, and can yield detergents that are relatively hard and have excellent fluidity.
• alkalis other than these include sodium sesquicarbonate and sodium hydrogen carbonate, and phosphates such as tripolyphosphate also act as alkaline agents.
• sodium hydroxide and mono-, di-, or triethanolamine can be used as alkaline agents used in liquid detergents, and can also be
• Anti-restaining agent is blended in an amount of 0.001 to 10% by mass, preferably 1 to 5% by mass.
• anti-recontamination agents used in the cleaning composition of the present invention include polyethylene glycol, carboxylic acid polymers, polyvinyl alcohol, and polyvinylpyrrolidone.
• carboxylic acid polymers have the ability to prevent recontamination, as well as the ability to capture metal ions and disperse solid particle stains from clothing into the washing bath.
• the carboxylic acid polymer is a homopolymer or copolymer of acrylic acid, methacrylic acid, itaconic acid, etc.
• the preferred copolymer is a copolymer of the above monomers and maleic acid, and those with a molecular weight of several thousand to 100,000 are suitable. preferable.
• polymers such as polyglycidylate, cellulose derivatives such as carboxymethylcellulose, and aminocarboxylic acid-based polymers such as polyaspartic acid also have metal ion scavenging agents, dispersing agents, and re-fouling prevention abilities. Therefore, it is preferable.
• Bleaching agent Bleaching agents such as hydrogen peroxide and percarbonate are preferably blended in an amount of 1 to 10% by mass.
• a bleaching activator such as tetraacetylethylenediamine (TAED) or the one described in JP-A-6-316700 can be added.
• Fluorescent agent examples include biphenyl type fluorescent agents (eg, Tinopal CBS-X, etc.) and stilbene type fluorescent agents (eg, DM type fluorescent dye, etc.).
• the fluorescent agent is preferably blended in an amount of 0.001 to 2% by mass.
• the detergent composition includes builders, softeners, reducing agents (such as sulfites), foam suppressants (such as silicones), fragrances, and antibacterial and antifungal agents known in the field of laundry detergents. (Proxel [trade name], benzoic acid, etc.) and other additives can be included.
• a cleaning composition can be produced in accordance with a conventional method by combining the lipase of the present invention obtained by the above method and the above-mentioned known cleaning components.
• the form of the detergent can be selected depending on the application, and can be, for example, liquid, powder, granule, paste, solid, etc.
• the cleaning composition thus obtained can be used as a laundry detergent, a dishwashing agent, a bleaching agent, a hard surface cleaning agent, a drain cleaning agent, a denture cleaning agent, a disinfectant cleaning agent for medical instruments, etc.
• laundry detergents and dishwashing agents preferred are laundry detergents and dishwashing agents, and more preferred are laundry detergents (laundry laundry detergents), hand-washing dishwashing agents, and automatic dishwashing detergents.
• the cleaning composition is suitable for use at temperatures of 40°C or lower, 35°C or lower, 30°C or lower, 25°C or lower, and 5°C or higher, 10°C or higher, or 15°C or higher.
• Preferred usage modes include use in low-temperature (15-30°C) washing in laundry facilities and low-temperature (15-30°C) washing in automatic dishwashers.
• the cleaning composition of the present invention By using the cleaning composition of the present invention, objects to be cleaned that require stain removal (e.g., clothes, tableware, hard surfaces, drain pipes, dentures, medical instruments, etc.) can be cleaned, that is, stains can be removed. can do.
• a cleaning method includes bringing the cleaning composition of the present invention into contact with an object to be cleaned that requires stain removal.
• the stain is a sebum stain or a stain containing food-derived oils and fats.
• the object to be cleaned in order to bring the object to be cleaned and the cleaning agent composition into contact, the object to be cleaned may be soaked in water in which the cleaning agent composition is dissolved; The material may be applied directly to the object to be cleaned.
• the object to be cleaned after soaking or applying the detergent composition may be further washed by hand or in a washing machine, but this is not always necessary.
• the present invention further discloses the following aspects.
• ⁇ 1> A lipase consisting of the amino acid sequence shown by SEQ ID NO: 14 or an amino acid sequence having at least 91% identity thereto.
• ⁇ 2> The lipase according to ⁇ 1>, which consists of the amino acid sequence shown by SEQ ID NO: 14.
• ⁇ 3> A polynucleotide encoding the lipase according to ⁇ 1> or ⁇ 2>.
• ⁇ 4> A vector or DNA fragment comprising the polynucleotide according to ⁇ 3>.
• ⁇ 5> A transformed cell containing the vector or DNA fragment according to ⁇ 4>.
• ⁇ 6> The transformed cell according to ⁇ 5>, which is a microorganism.
• ⁇ 7> The transformed cell according to ⁇ 5> or ⁇ 6>, which is Escherichia coli or a bacterium belonging to the genus Bacillus.
• Sulfosuccinic acid ester or salt thereof preferably a sulfosuccinic acid branched alkyl ester or salt thereof having a branched chain alkyl group having 9 to 12 carbon atoms, more preferably a sulfosuccinate having a branched chain alkyl group having 9 or 10 carbon atoms.
• the cleaning composition according to ⁇ 8> further comprising an acid branched alkyl ester or a salt thereof, more preferably a sulfosuccinic acid branched alkyl ester or a salt thereof having a branched alkyl group having 10 carbon atoms.
• a sulfosuccinic acid diester or a salt thereof preferably a sulfosuccinic acid di-branched alkyl ester or a salt thereof, wherein each of the two branched chain alkyl groups is a branched chain alkyl group having 9 to 12 carbon atoms, more preferably two branched chains.
• a di-branched sulfosuccinic acid alkyl ester or salt thereof in which the alkyl groups are each a branched-chain alkyl group having 9 or 10 carbon atoms, more preferably a di-branched sulfosuccinic acid, in which the two branched-chain alkyl groups are each a branched-chain alkyl group having 10 carbon atoms.
• ⁇ 11> The cleaning composition according to any one of ⁇ 8> to ⁇ 10>, which is a clothes cleaning agent or a dishwashing agent.
• ⁇ 12> The cleaning composition according to any one of ⁇ 8> to ⁇ 11>, which is a powder or liquid.
• ⁇ 13> The cleaning composition according to any one of ⁇ 8> to ⁇ 12>, which is used at low temperatures.
• ⁇ 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 from 5 to 40°C, 10 to 35°C, 15°C
• the cleaning composition according to ⁇ 13> which is used at ⁇ 30°C and 15-25°C.
• ⁇ 15> A method for cleaning dirt using the cleaning composition according to any one of ⁇ 8> to ⁇ 14>.
• ⁇ 16> The method according to ⁇ 15>, which comprises contacting the object to be cleaned with the cleaning composition according to any one of ⁇ 8> to ⁇ 14>.
• ⁇ 17> The method according to ⁇ 15> or ⁇ 16>, wherein the stain is a sebum stain or a stain containing food-derived oils and fats.
• ⁇ 18> Use of a lipase consisting of the amino acid sequence shown by SEQ ID NO: 14 or an amino acid sequence having at least 91% identity thereto for the production of a cleaning composition.
• ⁇ 19> The use according to ⁇ 18>, wherein the lipase is a lipase consisting of the amino acid sequence shown by SEQ ID NO: 14.
• ⁇ 20> The use according to ⁇ 18> or ⁇ 19>, wherein the cleaning composition is a laundry detergent or a dishwasher.
• the cleaning composition is a powder or liquid.
• ⁇ 22> The use according to any one of ⁇ 18> to ⁇ 21>, wherein the cleaning composition is used at low temperatures.
• the cleaning composition is used at a temperature of 40°C or lower, 35°C or lower, 30°C or lower, 25°C or lower, and 5°C or higher, 10°C or higher, 15°C or higher, or 5 to 40°C, 10°C or lower.
• ⁇ 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 use according to ⁇ 24>, wherein the lipase is a lipase consisting of the amino acid sequence shown by SEQ ID NO: 14.
• ⁇ 26> The use according to ⁇ 24> or ⁇ 25>, wherein the stain is a sebum stain or a stain containing food-derived oils and fats.
• ⁇ 27> The use according to any one of ⁇ 24> to ⁇ 26>, wherein the washing is washing at a low temperature.
• Washing is at 40°C or lower, 35°C or lower, 30°C or lower, 25°C or lower, and at 5°C or higher, 10°C or higher, 15°C or higher, or 5-40°C, 10-35°C
• lipase expression plasmid was constructed using the plasmid for expression of VHH of SEQ ID NO: 26 containing the Bacillus subtilis spoVG gene-derived promoter described in WO2021/153129 as a template. It was constructed by replacing each lipase gene with the full length ORF containing the VHH gene of the above plasmid by In-Fusion reaction.
• Lipr139 (polynucleotides of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13 and 15, respectively, SEQ ID NO: 2) , 4, 6, 8, 10, 12, 14 and 16) from the plasmids pHY-Lipr139, pHY-PvLip, pHY-PfLip, pHY-EtLip, pHY-EspLip, pHY-YeLip, pHY, respectively.
• -KAL-A8 pHY-Lipr138 were constructed.
• a lipase TLL gene in 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) is linked to the full length ORF containing the Lipr139 gene of plasmid pHY-Lipr139 on the N-terminal side.
• pHY-amyEsig-TLL was constructed by replacing the artificially synthesized gene of polynucleotide number 17 (encoding the amino acid sequence of SEQ ID NO: 18) by In-Fusion reaction.
• a lipase expression plasmid was introduced into a Bacillus subtilis strain by the protoplast method, and a 2 ⁇ L-maltose medium (2% tryptone, 1% yeast extract, 1% NaCl, 7.5% maltose, 7.5 ppm sulfuric acid After culturing at 30°C for 2 days in manganese pentahydrate, 0.04% calcium chloride dihydrate, 15 ppm tetracycline (% is (w/v)%), the culture supernatant containing lipase was collected by centrifugation. Recovered. 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 determined from the band intensity of SDS-PAGE.
• TLL, Lipr139, PvLip and Lipr138 whose suitability for washing has been disclosed in Patent Documents 1, 2 and 3, and the naturally occurring lipase sequences PfLip, EtLip, EspLip and YeLip, were prepared using SDS and Triton X-100. Activity was significantly inhibited in the presence of Compared to these, the novel lipase KAL-A8 showed high resistance to activity inhibition by SDS and Triton X-100.
• TLL, Lipr139, PvLip and Lipr138 whose suitability for washing has been disclosed in Patent Documents 1, 2 and 3, and the naturally occurring lipase sequences PfLip, EtLip, EspLip and YeLip, are significantly active in the model washing solution. inhibited.
• the novel lipase KAL-A8 showed high resistance to activity inhibition in the model wash solution.
• Lipr139 whose suitability for cleaning is disclosed in Patent Document 1, showed high cleaning power in a model cleaning solution compared to TLL, Lipr138, PvLip, PfLip, EtLip, EspLip, and YeLip.
• the novel lipase KAL-A8 showed significantly higher detergency in the model washing solution than other lipases including Lipr139.

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