WO2018021324A1 - Polypeptide à activité lipase présentant une excellente stabilité - Google Patents

Polypeptide à activité lipase présentant une excellente stabilité Download PDF

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WO2018021324A1
WO2018021324A1 PCT/JP2017/026895 JP2017026895W WO2018021324A1 WO 2018021324 A1 WO2018021324 A1 WO 2018021324A1 JP 2017026895 W JP2017026895 W JP 2017026895W WO 2018021324 A1 WO2018021324 A1 WO 2018021324A1
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amino acid
acid residues
positions
helix
polypeptide
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PCT/JP2017/026895
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Japanese (ja)
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和典 吉田
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天野エンザイム株式会社
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    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F1/00—Treatment of water, waste water, or sewage
    • 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
    • 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
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • 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
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats

Definitions

  • the present invention relates to a polypeptide having lipase activity. Specifically, a polypeptide having excellent lipase activity, DNA encoding the polypeptide, recombinant vector, transformant, composition, enzyme agent, method for producing the polypeptide, and use of the polypeptide
  • the present invention relates to a method for treating fats and oils, a method for treating wastewater, a method for producing a pharmaceutical intermediate, and a method for producing a fine chemical material.
  • a lipase derived from Burkholderia cepacia is one of the enzymes that are widely used commercially such as pharmaceutical intermediate production.
  • the catalytic activity of the enzyme is lost depending on the use conditions of the enzyme, such as in the presence of a solvent or under high temperature conditions, there is a problem that the usage is limited.
  • Protein engineering techniques are used as methods for improving enzyme stability (heat, pH, solvent, etc.).
  • mutation introduction into a loop region has been reported so far.
  • Non-Patent Document 1 a surface loop of Bacillus subtilis-derived ⁇ -amylase is reported. It has been reported that by introducing mutations into 7 residues that become the hinge region, amylolytic activity was increased by mutation of 5 residues among the target residues, and a stable psychrophilic enzyme was obtained. .
  • Non-Patent Document 2 reports that the thermal stability of phosphatidylinositol-synthesizing Streptomyces-derived phospholipase D could be improved by removing dynamic surface loops.
  • thermotolerant psychroenzyme by loop design, Takao Nibi, Takafumi Ito, Yoshiaki Nishiya, Grant-in-Aid for Scientific Research Funds, https: // kaken. nii. ac. jp / d / p / 23658095. ja. html
  • Deletion of a dynamic surface loop improves stability and changes Kinetic behavior of phosphatidylinositol-synthesizing properties. , Damnjanovic J, Nakano H. , And Iwasaki Y. , Biotechnol Bioeng. , 2014, Apr; 111 (4): 674-82. Doi: 10.1002 / bit. 25149. Epub 2013 Nov 30.
  • the present invention has been made in view of the above situation, and an object of the present invention is to provide a polypeptide having a lipase activity with improved stability as compared with the prior art.
  • this invention provides the invention of the aspect hung up below.
  • Item 1 At least, from the N-terminal side, ⁇ sheet (A), ⁇ helix (A), ⁇ helix (B), ⁇ helix (C), ⁇ sheet (B), and ⁇ helix (D), Has lipase activity,
  • the amino acid residue on the N-terminal side is present at positions 7 to 13 from the N-terminal side of the polypeptide, and the number of amino acid residues is 2 to 6.
  • the ⁇ -helix (A) is present at positions 15 to 25 when the amino acid residue on the N-terminal side is 0-position from the amino acid residue on the C-terminal side of the ⁇ -sheet (A).
  • the number consists of 3-9
  • the ⁇ helix (B) is present at positions 70 to 92 when the amino acid residue on the N-terminal side is 0-position of the amino acid residue on the C-terminal side of the ⁇ helix (A).
  • the ⁇ -helix (C) is composed of 4 to 15 radicals
  • the N-terminal amino acid residue is the 0-position of the C-terminal amino acid residue of the ⁇ -helix (B).
  • the ⁇ sheet (B) is present at positions 65 to 81 when the amino acid residue on the N-terminal side is defined as 0-position of the amino acid residue on the C-terminal side of the ⁇ helix (C).
  • the number consists of 2-8,
  • the ⁇ helix (D) is present at positions 6 to 15 when the amino acid residue on the N-terminal side is 0-position of the amino acid residue on the C-terminal side of the ⁇ helix (C).
  • an ⁇ helix (E), a ⁇ sheet (C), a ⁇ sheet (D), an ⁇ helix (G), and a ⁇ sheet (E) are provided,
  • the ⁇ helix (E) is present at positions 17 to 28 when the amino acid residue on the N-terminal side is 0-position of the amino acid residue on the C-terminal side of the ⁇ helix (A).
  • the radix consists of 13-19
  • the ⁇ sheet (C) is present at the 2nd to 8th positions when the amino acid residue on the N-terminal side is 0-position of the amino acid residue on the C-terminal side of the ⁇ helix (E).
  • the number consists of 4-8,
  • the ⁇ sheet (D) is present at positions 14 to 22 when the amino acid residue on the N-terminal side is 0-position of the amino acid residue on the C-terminal side of the ⁇ sheet (C). Consists of 3 to 11,
  • the ⁇ -helix (G) is present in the 3rd to 13th positions when the amino acid residue on the N-terminal side is 0-position of the amino acid residue on the C-terminal side of the ⁇ -helix (C).
  • the radix is composed of 4-13,
  • the ⁇ sheet (E) is present at positions 24 to 36 when the amino acid residue on the N-terminal side is defined as 0-position of the amino acid residue on the C-terminal side of the ⁇ helix (G).
  • the number consists of 2-6, Item 2.
  • the polypeptide according to Item 1. Item 3. Furthermore, an ⁇ helix (F), an ⁇ helix (H), and a ⁇ sheet (F) are provided,
  • the ⁇ -helix (F) is present in the 1st to 5th positions when the amino acid residue on the N-terminal side is 0-position of the amino acid residue on the C-terminal side of the ⁇ -sheet (C), and the amino acid residue
  • the number consists of 3 to 15,
  • the ⁇ helix (H) is present at the 1 to 10 position when the amino acid residue on the N-terminal side of the ⁇ helix (G) is 0 position on the C-terminal side of the ⁇ helix (G).
  • the radix is composed of 2 to 13
  • the ⁇ sheet (F) is present at the 1st to 5th positions when the amino acid residue on the N-terminal side is 0-position of the amino acid residue on the C-terminal side of the ⁇ -sheet (E). Consists of 15-23, Item 3.
  • the amino acid residues substituted with any of the amino acid residues shown in 1-125 and / or the amino acid residues at positions 233-235 are shown in Table II.
  • amino acid sequence substituted with any one of the amino acid residues shown in 1-150 one or several amino acid residues other than the amino acid residue into which the substitution is introduced are substituted, added, inserted or deleted.
  • the amino acid residues at positions 24 to 26 in the amino acid sequence shown in SEQ ID NO: 2 are Nos. Nos.
  • amino acid residues 1 to 125 are substituted with amino acid residues and / or amino acid residues at positions 232 to 234
  • amino acid residues substituted with any one of the amino acid residues shown in 1-150 one or several amino acid residues other than the amino acid residue into which the substitution is introduced are substituted, added, inserted or deleted.
  • the amino acid residues at positions 25 to 27 in the amino acid sequence shown in SEQ ID NO: 3 are Nos.
  • the amino acid residues substituted with any of the amino acid residues shown in 1-125 and / or the amino acid residues at positions 233-235 are shown in Table II.
  • amino acid sequence substituted with any one of the amino acid residues shown in 1-150 one or several amino acid residues other than the amino acid residue into which the substitution is introduced are substituted, added, inserted or deleted.
  • the amino acid residues at positions 25 to 27 in the amino acid sequence shown in SEQ ID NO: 4 are Nos.
  • the amino acid residues substituted with any of the amino acid residues shown in 1-125 and / or the amino acid residues at positions 233-235 are shown in Table II.
  • amino acid sequence substituted with any one of the amino acid residues shown in 1-150 one or several amino acid residues other than the amino acid residue into which the substitution is introduced are substituted, added, inserted or deleted.
  • the amino acid residues at positions 25 to 27 in the amino acid sequence shown in SEQ ID NO: 1 are No.
  • the amino acid residues substituted with any of the amino acid residues shown in 1-125 and / or the amino acid residues at positions 233-235 are shown in Table II.
  • the sequence identity excluding the amino acid residue introduced with the substitution with respect to the amino acid sequence shown in SEQ ID NO: 1 is 80% or more
  • the amino acid residues at positions 24 to 26 in the amino acid sequence shown in SEQ ID NO: 2 are Nos. Nos.
  • 1 to 125 are substituted with amino acid residues and / or amino acid residues at positions 232 to 234
  • the sequence identity excluding the amino acid residue introduced with the substitution with respect to the amino acid sequence shown in SEQ ID NO: 2 is 80% or more
  • the amino acid residues at positions 25 to 27 in the amino acid sequence shown in SEQ ID NO: 3 are Nos.
  • the amino acid residues substituted with any of the amino acid residues shown in 1-125 and / or the amino acid residues at positions 233-235 are shown in Table II.
  • the sequence identity excluding the amino acid residue introduced with the substitution with respect to the amino acid sequence shown in SEQ ID NO: 3 is 80% or more
  • the amino acid residues at positions 25 to 27 in the amino acid sequence shown in SEQ ID NO: 4 are Nos.
  • the amino acid residues substituted with any of the amino acid residues shown in 1-125 and / or the amino acid residues at positions 233-235 are shown in Table II.
  • the sequence identity excluding the amino acid residue into which the substitution is introduced with respect to the amino acid sequence shown in SEQ ID NO: 4 is 80% or more, A polypeptide having lipase activity and improved thermal stability as compared to the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 4.
  • Item 5. A DNA encoding the polypeptide according to Item 1-4.
  • Item 6. A recombinant vector comprising the DNA of Item 5.
  • Item 7. A transformant obtained by transforming a host with the recombinant vector according to Item 6.
  • the method for producing a polypeptide according to any one of Items 1 to 5, comprising a step of culturing the transformant according to Item 7.
  • Item 9. Item 5.
  • a composition comprising the polypeptide according to any one of Items 1 to 4.
  • An enzyme agent comprising the polypeptide according to any one of Items 1 to 4 or the composition according to Item 9.
  • Item 11. Item 11.
  • Item 12. Item 11.
  • a wastewater treatment method wherein the polypeptide according to any one of Items 1 to 4, the composition according to Item 9, or the enzyme agent according to Item 10 is allowed to act on wastewater.
  • Item 13. Item 11. A method for producing a pharmaceutical intermediate, comprising causing the polypeptide according to any one of Items 1 to 4, the composition according to Item 9, or the enzyme agent according to Item 10 to act on a raw material for the pharmaceutical intermediate.
  • Item 14. Item 11.
  • a method for producing a fine chemical material wherein the polypeptide according to any one of Items 1 to 4, the composition according to Item 9, or the enzyme agent according to Item 10 is allowed to act on a raw material for the fine chemical material.
  • the present invention it is possible to obtain a polypeptide having a lipase activity with improved stability such as pH, heat, solvent, etc., particularly thermal stability. Therefore, the present invention can be used for applications that require a lipase with excellent stability.
  • the polypeptide of the present invention can be suitably used for applications that require high stability, such as wastewater treatment, pharmaceutical intermediate production, fine chemical material production, functional substitute oil production, detergent, food processing, chiral synthesis, biosynthesis. It can be suitably used in fields such as ethanol.
  • Black indicates a lipase derived from Burkholderia cepacia, and gray indicates a lipase derived from Pseudomonas grumae. It is the figure which piled up the three-dimensional structure of lipase derived from Burkholderia cepacia and the three-dimensional structure of Pseudomonas fluorescens lipase. Black indicates lipase derived from Burkholderia cepacia and gray indicates lipase derived from Pseudomonas fluorescens.
  • expressions such as “F45V” are amino acid substitution notations.
  • F45V means that the 45th amino acid F from the N-terminal side in the specific amino acid sequence is substituted with the amino acid V.
  • expressions such as “V272A / H273G” in the present specification mean multiple mutations.
  • V272A / H273G means that amino acid substitutions of V272A and H273G are introduced simultaneously.
  • nonpolar amino acids include alanine, valine, leucine, isoleucine, proline, methionine, phenylalanine, and tryptophan.
  • uncharged amino acid includes glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine.
  • acidic amino acid includes aspartic acid and glutamic acid.
  • basic amino acid includes lysine, arginine, and histidine.
  • the methionine corresponding to the translation start point is the first position at the N-terminus, but in mature forms in which these peptides have been cleaved after being translated including propeptides, prepeptides and signal peptides, these
  • the N-terminus of the matured body after cleaving the peptide is taken as the first position.
  • SEQ ID NO: 1 in the present specification is a mature sequence obtained by cleaving 44 amino acids from SEQ ID NO: 5, and the N-terminal amino acid residue is alanine (A).
  • SEQ ID NO: 2 is a matured sequence having 40 amino acids cleaved from SEQ ID NO: 6, and the N-terminal amino acid residue is aspartic acid (D).
  • SEQ ID NO: 7 is a mature sequence obtained by cleaving 44 amino acids from SEQ ID NO: C, and the N-terminal amino acid residue is alanine (A).
  • SEQ ID NO: 4 is a matured sequence obtained by cleaving 26 amino acids from SEQ ID NO: 8, and the N-terminal amino acid residue is serine (S).
  • amino acid residue sequences of peptides and proteins are expressed from the N-terminus to the C-terminus from the left end to the right end.
  • substitution refers not only to the case where amino acid residue substitution is artificially introduced, but also to the case where amino acid residue substitution is naturally introduced, that is, the amino acid residue is originally different. It also includes the case where it was.
  • substitution of amino acid residues may be artificial substitution or natural substitution, but artificial substitution is preferred.
  • polypeptide of the present invention comprises, at least from the N-terminal side, a ⁇ sheet (A), an ⁇ helix (A), an ⁇ helix (B), an ⁇ helix (C), a ⁇ sheet (B), and It has ⁇ helix (D) and has lipase activity
  • the amino acid residue on the N-terminal side is present at positions 7 to 13 from the N-terminal side of the polypeptide, and the number of amino acid residues is 2 to 6.
  • the ⁇ -helix (A) is present at positions 15 to 25 when the amino acid residue on the N-terminal side is 0-position from the amino acid residue on the C-terminal side of the ⁇ -sheet (A).
  • the number consists of 3-9
  • the ⁇ helix (B) is present at positions 70 to 92 when the amino acid residue on the N-terminal side is 0-position of the amino acid residue on the C-terminal side of the ⁇ helix (A).
  • the radix is composed of 4-15
  • the ⁇ helix (C) is present in the 3rd to 11th positions when the amino acid residue on the N-terminal side is 0-position of the amino acid residue on the C-terminal side of the ⁇ -helix (B).
  • the radix is composed of 11-20
  • the ⁇ sheet (B) is present at positions 65 to 81 when the amino acid residue on the N-terminal side is defined as 0-position of the amino acid residue on the C-terminal side of the ⁇ helix (C).
  • the number consists of 2-8
  • the ⁇ helix (D) is present at positions 6 to 15 when the amino acid residue on the N-terminal side is 0-position of the amino acid residue on the C-terminal side of the ⁇ helix (C).
  • ⁇ sheet (A), ⁇ helix (A), ⁇ helix (B), ⁇ helix (C), ⁇ sheet (B), and ⁇ helix (D) in order, having lipase activity
  • the amino acid residue on the N-terminal side is present at positions 7 to 13 from the N-terminal side of the polypeptide, and the number of amino acid residues is 2 to 6.
  • the ⁇ -helix (A) is present at positions 15 to 25 when the amino acid residue on the N-terminal side is 0-position from the amino acid residue on the C-terminal side of the ⁇ -sheet (A).
  • the number consists of 3-9
  • the ⁇ helix (B) is present at positions 70 to 92 when the amino acid residue on the N-terminal side is 0-position of the amino acid residue on the C-terminal side of the ⁇ helix (A).
  • the radix is composed of 4-15
  • the ⁇ helix (C) is present in the 3rd to 11th positions when the amino acid residue on the N-terminal side is 0-position of the amino acid residue on the C-terminal side of the ⁇ -helix (B).
  • the radix is composed of 11-20
  • the ⁇ sheet (B) is present at positions 65 to 81 when the amino acid residue on the N-terminal side is defined as 0-position of the amino acid residue on the C-terminal side of the ⁇ helix (C).
  • the number consists of 2-8
  • the ⁇ -helix (D) is present at positions 6 to 15 when the amino acid residue on the N-terminal side is 0-position of the amino acid residue on the C-terminal side of the previous ⁇ -helix (C).
  • a polypeptide composed of 2 to 24 bases.
  • Contains groups. Each of these regions may include one or more loops, sheets, or helices.
  • the region from the N-terminus of the polypeptide to the ⁇ -sheet (A) N-terminal amino acid residue is defined as region (I), and ⁇ -sheet (A) from the C-terminal amino acid residue to ⁇ -helix (A) Region to region (II) up to N-terminal amino acid residue, ⁇ helix (A) Region from C-terminal amino acid residue to ⁇ -helix (B) N-terminal amino acid residue to region (III), ⁇ Region (IV) from Helix (B) C-terminal side amino acid residue to ⁇ -helix (C) N-terminal side amino acid residue, ⁇ -helix (C) ⁇ -sheet (B) from C-terminal side amino acid residue
  • Such a polypeptide has a structure recognized by a lipase having a molecular weight of about 30 to 33 kDa.
  • polypeptides having such a structure include Burkholderia cepacia, Burkholderia territory, Burkholderia cenocepaceria, Burkholderia cenocepaceria, Faria (Burkholderia ambaria), Burkholderia contaminans, Burkholderia lata (Burkholderia lath), Burkholderia Baholderina
  • Burkholderia Pseudomonas glumae, Pseudomonas fluorescens, Pseudomonas ummo somomoscos, etc .
  • polypeptide having such a structure examples include type I and type II described later.
  • examples of the polypeptide included in type I include Burkholderia cepacia-derived lipase, Pseudomonas glumae-derived lipase, and Pseudomonas fluorescens-derived lipase. These three-dimensional structures are shown in FIGS.
  • Examples of the polypeptide included in type II include Pseudomonas aeruginosa-derived lipase.
  • FIG. 4 shows the three-dimensional structure of a lipase derived from Pseudomonas aeruginosa. Of these, type I polypeptides are preferred.
  • type I and type II of polypeptides should be confirmed by analyzing using a software for analyzing the three-dimensional structure of proteins such as MOE (Molecular Operating Environment) (version MOE 2013), PyMOL, RasMOL, WinCoot, etc. Can do.
  • MOE Molecular Operating Environment
  • the ⁇ helix and ⁇ sheet can be defined using the MOE of the software. Loops can also be defined using the MOE of the software. In the present invention, it is not limited to the loop defined by the software, and all parts other than the ⁇ helix and the ⁇ sheet are defined as a loop.
  • type I polypeptides include polypeptides having the amino acid sequences of SEQ ID NOs: 1 to 3, and polypeptides having a similar three-dimensional structure. The structure of the type I polypeptide will be described in order from the N-terminal to the C-terminal.
  • N-terminal amino acid residue (I) The number of amino acid residues in region (I) is 6 to 13, preferably 8 to 11. Specifically, as the amino acid sequence of region (I), if it is derived from Burkholderia cepacia, it is an amino acid residue at positions 1 to 10 shown in SEQ ID NO: 1, if it is derived from Pseudomonas grumae. Examples of the amino acid residues at positions 1 to 9 shown in SEQ ID NO: 2 and those derived from P. fluorescens include the amino acid residues at positions 1 to 10 shown in SEQ ID NO: 3.
  • the N-terminal amino acid residue is present at the 7th to 13th positions from the N-terminal side of the polypeptide, preferably at the 8th to 11th positions, more preferably at the 9th to 10th positions.
  • Examples of the number of amino acid residues constituting the ⁇ sheet (A) include 2 to 6, preferably 3 to 5, and more preferably 3 to 4.
  • the amino acid sequence of the ⁇ sheet (A) is not particularly limited, and examples thereof include x 1 ILV (x 1 may be any amino acid residue, preferably I or V), preferably IILV is mentioned.
  • amino acid sequence of the ⁇ sheet (A) if it is derived from Burkholderia cepacia, is the amino acid residue at positions 11 to 14 shown in SEQ ID NO: 1, derived from Pseudomonas grumae.
  • amino acid residues at positions 10 to 13 shown in SEQ ID NO: 2 and those derived from P. fluorescens include amino acid residues at positions 11 to 14 shown in SEQ ID NO: 3.
  • ⁇ sheet Region from C-terminal amino acid residue to ⁇ -helix
  • A N-terminal amino acid residue
  • the number of amino acid residues in the region (II) is 14 to 23, preferably 16 to 22, and more preferably 17 to 21.
  • the amino acid sequence of region (II) if it is derived from Burkholderia cepacia, it is an amino acid residue at positions 15 to 32 shown in SEQ ID NO: 1 and is derived from Pseudomonas grumae. Examples of the amino acid residues at positions 14 to 31 shown in SEQ ID NO: 2 and those derived from P. fluorescens include the amino acid residues at positions 15 to 32 shown in SEQ ID NO: 3.
  • ⁇ helix (A) The ⁇ -helix (A) is present at positions 15 to 25 when the amino acid residue on the N-terminal side is 0-position from the amino acid residue on the C-terminal side of the ⁇ sheet (A), preferably 17- Present at the 23rd position, more preferably at 19-21.
  • Examples of the number of amino acid residues constituting the ⁇ helix (A) include 3 to 9, preferably 4 to 8, and more preferably 5 to 7.
  • the amino acid sequence of the ⁇ helix (A) is not particularly limited.
  • IQx 2 DLQx 3 (x 2 and x 3 may each be any amino acid residue, and preferably x 2 is E or S and x 3 are Q or S.), preferably IQEDLQQ and IQDLDLQS.
  • amino acid sequence of ⁇ -helix (A) if it is derived from Burkholderia cepacia, is the amino acid residue at positions 33 to 39 shown in SEQ ID NO: 1, derived from Pseudomonas grumae. If present, amino acid residues at positions 32 to 38 shown in SEQ ID NO: 2 and those derived from P. fluorescens include amino acid residues at positions 33 to 39 shown in SEQ ID NO: 3.
  • the number of amino acid residues in region (III) is 69 to 92, preferably 74 to 87, and more preferably 78 to 83.
  • the amino acid sequence of region (III) if it is derived from Burkholderia cepacia, it is an amino acid residue at positions 40 to 117 shown in SEQ ID NO: 1 and is derived from Pseudomonas grumae. Examples of the amino acid residues at positions 39 to 116 shown in SEQ ID NO: 2 and those derived from P. fluorescens include amino acid residues at positions 40 to 119 shown in SEQ ID NO: 3.
  • ⁇ helix (B) The ⁇ -helix (B) is present at the 70-92 position when the amino acid residue on the N-terminal side of the ⁇ -helix (A) is 0-position, preferably 75. It is present at the ⁇ 87th position, more preferably at the 79th to 83rd position. Examples of the number of amino acid residues constituting the ⁇ helix (B) include 4 to 15, preferably 5 to 14, and more preferably 7 to 12.
  • the amino acid sequence of ⁇ -helix (B) is not particularly limited, and examples thereof include EFADFVQGVL, EFADFVQDVLKT, ADFVQGVL, and preferably EFADFVQGVL.
  • the number of amino acid residues in region (IV) include 2 to 10, preferably 4 to 9, and more preferably 5 to 8.
  • the amino acid sequence of region (IV) if it is derived from Burkholderia cepacia, it is the amino acid residue at positions 128 to 133 shown in SEQ ID NO: 1, and it is derived from Pseudomonas grumae.
  • amino acid residues at positions 129 to 135 shown in SEQ ID NO: 2 and those derived from P. fluorescens include the amino acid residues at positions 129 to 133 shown in SEQ ID NO: 3.
  • the ⁇ helix (C) is present at the 3 to 11 position when the amino acid residue on the N-terminal side of the ⁇ helix (B) is 0-position, preferably 5 Present at the 10th position, more preferably at the 6th to 9th position.
  • Examples of the number of amino acid residues constituting the ⁇ helix (C) include 11 to 20, preferably 13 to 18, and more preferably 14 to 17.
  • the amino acid sequence of the ⁇ helix (C) is not particularly limited.
  • LSSTVIAAFVNVx 4 Gx 5 LT (x 4 and x 5 may be any amino acid residue, and preferably x 4 is F Or I, x 5 is I or A.), TVIAAFVNVFGTLV and the like, preferably LSSTVIAAFVNVx 4 Gx 5 LT (x 4 and x 5 may each be any amino acid residue, preferably x 4 is F or I, and x 5 is I or A. More preferably, LSSTVIAAFVNVFGILT and LSSTVIAAFVNVIGALT are mentioned.
  • amino acid sequence of ⁇ -helix (C) specifically, if it is derived from Burkholderia cepacia, positions 134 to 150 shown in SEQ ID NO: 1, if it is derived from Pseudomonas grumae, the sequence In No. 2, amino acid residues at positions 136 to 149 and those derived from Pseudomonas fluorescens at positions 134 to 150 shown in SEQ ID NO: 3 can be mentioned.
  • Examples of the number of amino acid residues in region (V) include 64 to 80, preferably 68 to 76, and more preferably 70 to 75.
  • the amino acid sequence of region (V) if it is derived from Burkholderia cepacia, it is an amino acid residue at positions 151 to 222 shown in SEQ ID NO: 1 and is derived from Pseudomonas grumae.
  • amino acid residues at positions 150 to 221 shown in SEQ ID NO: 2 and those derived from P. fluorescens include amino acid residues at positions 151 to 222 shown in SEQ ID NO: 3.
  • the ⁇ sheet (B) is present at the 65th to 81st positions when the amino acid residue on the N-terminal side is 0-position of the amino acid residue on the C-terminal side of the ⁇ helix (C), preferably 69- It exists at the 77th position, more preferably at the 71st to 75th positions.
  • Examples of the number of amino acid residues constituting the ⁇ sheet (B) include 2 to 8, preferably 3 to 7, and more preferably 4 to 6.
  • the amino acid sequence of the ⁇ sheet (B) is not particularly limited, and examples thereof include VTGAx 6 D (x 6 may be any amino acid residue, preferably T or R). Preferably, VTGATD is mentioned.
  • amino acid sequence of the ⁇ sheet (B), if it is derived from Burkholderia cepacia, is the amino acid residue at positions 223 to 228 shown in SEQ ID NO: 1, derived from Pseudomonas grumae.
  • amino acid residues 222 to 227 shown in SEQ ID NO: 2 and those derived from P. fluorescens include amino acid residues 223 to 228 shown in SEQ ID NO: 3.
  • ⁇ sheet Region from C-terminal amino acid residue to ⁇ helix
  • D N-terminal amino acid residue
  • VI Examples of the number of amino acid residues in region (VI) include 5 to 14, preferably 7 to 12, and more preferably 8 to 11.
  • amino acid sequence of region (VI) if it is derived from Burkholderia cepacia, it is an amino acid residue at positions 229 to 237 shown in SEQ ID NO: 1 and is derived from Pseudomonas grumae.
  • amino acid residues at positions 228 to 235 shown in SEQ ID NO: 2 and those derived from P. fluorescens include amino acid residues at positions 229 to 236 shown in SEQ ID NO: 3.
  • ⁇ helix (D) The ⁇ -helix (D) is present at positions 6 to 15 when the amino acid residue on the N-terminal side is 0-position of the amino acid residue on the C-terminal side of the ⁇ -helix (C), preferably 8 It exists in the ⁇ 13th position, more preferably in the 9th-12th position.
  • Examples of the number of amino acid residues constituting the ⁇ helix (D) include 2 to 24, preferably 3 to 23, and more preferably 4 to 22.
  • the amino acid sequence of the ⁇ helix (D) is not particularly limited.
  • ANx 7 x 8 (x 7 and x 8 may be any amino acid residue, and preferably x 7 is A or V, x 8 is L or T.), PANVT, STALL x 9 x 10 TG x 11 VM x 12 (x 9 , x 10 , x 11 , and x 12 may each be any amino acid residue, preferably X 9 is F or L, x 10 is G or A, x 11 is T or A, x 12 is V or I.), STALLLGSGTVMVN, etc., preferably ANx 7 x 8 (x 7 and x 8 may be any amino acid residue, and preferably x 7 is A or V, and x 8 is L or T.), STLALFTGTVMVMV, STLLLATGGAVMI, STALLLGSGTVMVN, More preferably, ANAL, ANVT, STLALFTGTVMVMV, STALLATGGAMVMI, and more preferably ANAL, STLALFTGTVMVMV.
  • the ⁇ helix (D) region is preferably a sequence in which two or more of the sequences exemplified above are linked directly or via several amino acid residues, and includes sequences such as ANAL and STALLFGTGTTVMV Is preferred.
  • amino acid sequence of ⁇ -helix (D) specifically, if it is derived from Burkholderia cepacia, the amino acid residue at positions 238 to 256 shown in SEQ ID NO: 1, derived from Pseudomonas grumae If there are, amino acid residues at positions 236 to 254 shown in SEQ ID NO: 2 and amino acid residues at positions 237 to 257 shown in SEQ ID NO: 3 if derived from Pseudomonas fluorescens.
  • Examples of the number of residues in the amino acid sequence of region (VII) include 55 to 88, preferably 58 to 84, and more preferably 61 to 80.
  • amino acid sequence of region (VII) if it is derived from Burkholderia cepacia, it is the amino acid residue at positions 257 to 320 shown in SEQ ID NO: 1, and it is derived from Pseudomonas grumae.
  • amino acid residues at positions 258 to 318 shown in SEQ ID NO: 2 and those derived from P. fluorescens include amino acid residues at positions 258 to 320 shown in SEQ ID NO: 3.
  • the polypeptide of the present invention further comprises an ⁇ helix (E), ⁇ sheet (C), ⁇ helix (F), or ⁇ sheet (D) from the N-terminal side in the region (III). It may be.
  • the region between these ⁇ -helix and ⁇ -sheets may include one or more loops, ⁇ -helix, or ⁇ -sheet.
  • the ⁇ helix (E) has 17 to 28 positions, preferably 19 to 26 positions, when the amino acid residue on the N-terminal side of the ⁇ helix (A) is 0-position. More preferably, it is present in 21 to 24, and the number of amino acid residues is 13 to 19, preferably 14 to 18, and more preferably 15 to 17.
  • the amino acid sequence of the helix (E) to alpha are not limited to, for example, RGEQLLAYVKx 13 VLAx 14 T (x 13 and x 14, respectively may be any amino acid residue, preferably, x 13 is T or Q, x 14 is A or Q.), etc., and preferably RGEQLLAYVKTVLAAT, RGEQLLAYVKQVLAAT, RGEQLLAYVKQVLAQT, more preferably RGEQLLAYVKTVLAAT.
  • the ⁇ sheet (C) has an N-terminal amino acid residue of 2 to 8 positions, preferably 3 to 7 positions when the C-terminal amino acid residue of the ⁇ helix (E) is 0 position. More preferably, it is present in 4-6, and the number of amino acid residues is 4-8, preferably 5-7.
  • the amino acid sequence of the ⁇ sheet (C) is not particularly limited, and examples thereof include VNLx 15 GH (x 15 may be any amino acid residue, preferably V or I). VNLVGGH is preferable.
  • the amino acid sequence of the ⁇ sheet (C) may be an amino acid residue at positions 81 to 86 shown in SEQ ID NO: 1, Pseudomonas grumae, as long as it is derived from Burkholderia cepacia.
  • the amino acid residues at positions 80 to 85 shown in SEQ ID NO: 2 and those derived from P. fluorescens include the amino acid residues at positions 81 to 86 shown in SEQ ID NO: 3.
  • ⁇ helix (F) ⁇ -helix (F) has 1 to 5 positions, preferably 2 to 4 positions when the amino acid residue on the N-terminal side is 0-position on the C-terminal side of ⁇ -sheet (C). Is present and comprises 3 to 15, preferably 5 to 14, more preferably 10 to 13 amino acid residues.
  • the amino acid sequence of ⁇ -helix (F) is not particularly limited, and examples thereof include GGLTSRYVAAV, QGGLTSRYVAAV, and preferably GGLTSRYVAAV.
  • amino acid sequence of ⁇ -helix (F) specifically, if it is derived from Burkholderia cepacia, it is the amino acid residue at positions 89 to 99 shown in SEQ ID NO: 1, derived from Pseudomonas grumae. If there are amino acid residues at positions 87 to 98 shown in SEQ ID NO: 2, and amino acid residues at positions 88 to 99 shown in SEQ ID NO: 3 if derived from Pseudomonas fluorescens.
  • the ⁇ -sheet (D) has 14 to 22 positions, preferably 16 to 20 positions when the amino acid residue on the N-terminal side of the ⁇ -sheet (C) is 0-position. Preferably it is located at positions 17-19.
  • the ⁇ sheet (D) has an N-terminal amino acid residue of 3 to 13 positions, preferably 4 to 12 when the C-terminal amino acid residue of the ⁇ helix (F) is 0 position. , More preferably 4-6.
  • the ⁇ sheet (D) is composed of 3 to 11, preferably 5 to 9, more preferably 6 to 8 amino acid residues. Although it does not specifically limit as an amino acid sequence of (beta) sheet
  • SEQ ID NO: 1 derived from Pseudomonas grumae.
  • amino acid residues at positions 103 to 109 shown in SEQ ID NO: 2 and those derived from P. fluorescens at positions 104 to 110 shown in SEQ ID NO: 3 can be mentioned.
  • the polypeptide of the present invention further comprises an ⁇ helix (G), ⁇ helix (H), ⁇ sheet (E), or ⁇ sheet (F) from the N-terminal side in the region (V). It may be.
  • the region between these ⁇ -helix and ⁇ -sheets may include one or more loops, ⁇ -helix, or ⁇ -sheet.
  • ⁇ helix (G) ⁇ -helix (G) has 3 to 13 positions, preferably 4 to 12 positions, when the amino acid residue on the N-terminal side is 0-position of the amino acid residue on the C-terminal side of ⁇ -helix (C). More preferably, it is present in 5 to 11, and comprises 4 to 13, preferably 5 to 12, more preferably 6 to 11 amino acid residues.
  • an amino acid sequence of (alpha) helix (G) For example, ALAALKT, TDQDALAALR, ALAALQTL etc. are mentioned, Preferably ALAALKT is mentioned.
  • amino acid sequence of ⁇ -helix (G) if it is derived from Burkholderia cepacia, is the amino acid residue at positions 160 to 166 shown in SEQ ID NO: 1, derived from Pseudomonas grumae. If there are, amino acid residues at positions 155 to 164 shown in SEQ ID NO: 2 and those derived from P. fluorescens include amino acid residues at positions 160 to 167 shown in SEQ ID NO: 3.
  • ⁇ helix (H) ⁇ -helix (H) has 1 to 10 positions, preferably 2 to 8 positions when the amino acid residue on the N-terminal side is 0-position on the C-terminal side of ⁇ -helix (G). More preferably, it is present at positions 2 to 5, and the number of amino acid residues is 2 to 13, preferably 4 to 12, and more preferably 9 to 11.
  • the amino acid sequence of the ⁇ helix (H) is not particularly limited.
  • TAx 16 x 17 ATYNx 18 N may be any amino acid residue, preferably it is, x 16 is Q or R, x 17 a or T, x 18 is Q or R.) and the like, preferably TAQAATYNQN, TAQTATYNRN, or TARAATYNQN the like, and more preferably include TAQAATYNQN .
  • amino acid sequence of ⁇ -helix (H) specifically, if it is derived from Burkholderia cepacia, it is an amino acid residue at positions 169 to 178 shown in SEQ ID NO: 1, derived from Pseudomonas grumae. If there are amino acid residues at positions 168 to 177 shown in SEQ ID NO: 2, and amino acid residues at positions 169 to 178 shown in SEQ ID NO: 3 if they are derived from Pseudomonas fluorescens.
  • the ⁇ sheet (E) has an amino acid residue on the N-terminal side in positions 24 to 36, preferably 27 to 34, when the amino acid residue on the C-terminal side of the ⁇ helix (G) is 0 position. More preferably, it exists in 29 to 32, and the number of amino acid residues is 2 to 6, preferably 3 to 5.
  • the amino acid residue on the N-terminal side is 13 to 24, preferably 15 to 22, when the C-terminal amino acid residue of the ⁇ helix (H) is 0 position. , More preferably 17-20.
  • the ⁇ sheet (E) is composed of 2 to 6, preferably 3 to 5, amino acid residues.
  • the amino acid sequence of the ⁇ sheet (E) is not particularly limited, and examples thereof include TETV.
  • the amino acid sequence of the ⁇ sheet (E) is, for example, an amino acid residue at positions 196 to 199 shown in SEQ ID NO: 1, derived from Pseudomonas grumae, if it is derived from Burkholderia cepacia Then, amino acid residues at positions 195 to 198 shown in SEQ ID NO: 2 and amino acid residues at positions 196 to 199 shown in SEQ ID NO: 3 can be mentioned if they are derived from Pseudomonas fluorescens.
  • the ⁇ sheet (F) is present at the 1st to 5th positions, preferably the 2nd to 4th positions when the amino acid residue on the N-terminal side is 0-position of the amino acid residue on the C-terminal side of the ⁇ -sheet (E).
  • the number of amino acid residues is 15 to 23, preferably 17 to 21, and more preferably 18 to 20.
  • the amino acid sequence of the ⁇ sheet (F) is not particularly limited.
  • NTHLLYSWAG, SQHLLYSW, IQPTx 19 x 20 V may be any amino acid residue, preferably, x 19 is I, S or F, and x 20 is S or T.
  • NTHLLYSWAG, IQPTx 19 x 20 V x 19 and x 20 are any amino acid residues, respectively
  • X 19 is preferably I, S or F, and x 20 is S or T. More preferably, NTHLLYSWAG and IQPTISV are mentioned.
  • the ⁇ sheet (F) region is preferably a sequence in which two or more of the sequences exemplified above are linked directly or via several amino acid residues, and may be a sequence containing NTHLLYSWAG and IQPTISV. preferable.
  • the amino acid sequence of the ⁇ sheet (F) if it is derived from Burkholderia cepacia, is the amino acid residue at positions 202 to 220 shown in SEQ ID NO: 1, derived from Pseudomonas grumae.
  • the amino acid residues at positions 201 to 219 shown in SEQ ID NO: 2 and those derived from P. fluorescens include the amino acid residues at positions 202 to 220 shown in SEQ ID NO: 3.
  • the polypeptide of the present invention comprises the aforementioned ⁇ sheet (A), ⁇ helix (A), ⁇ helix (B), ⁇ helix (C), ⁇ sheet (B), and ⁇ helix (D).
  • it preferably has an ⁇ helix (E), ⁇ sheet (C), ⁇ sheet (D), ⁇ helix (G), and ⁇ sheet (E), more preferably Are the ⁇ helix (E), ⁇ sheet (C), ⁇ helix (F), ⁇ sheet (D), ⁇ helix (G), ⁇ helix (H), ⁇ sheet (E), and A ⁇ sheet (F) is provided.
  • Type II polypeptide examples include a polypeptide having the amino acid sequence of SEQ ID NO: 4 and a polypeptide having a similar three-dimensional structure. The structure of the type II polypeptide will be described in order from the N-terminal to the C-terminal.
  • Region from N-terminal to ⁇ -sheet N-terminal amino acid residue (I)
  • the number of amino acid residues in region (I) is 6 to 13, preferably 8 to 11.
  • Specific examples of the amino acid sequence of region (I) include the amino acid residues at positions 1 to 10 shown in SEQ ID NO: 4 if they are derived from Pseudomonas aeruginosa.
  • the N-terminal amino acid residue is present at the 7th to 13th position of the polypeptide, preferably at the 8th to 11th position, and more preferably at the 9th to 10th position.
  • Examples of the number of amino acid residues constituting the ⁇ sheet (A) include 2 to 6, preferably 3 to 5, and more preferably 3 to 4.
  • the amino acid sequence of the ⁇ sheet (A) is not particularly limited, and examples thereof include VLA. Specific examples of the amino acid sequence of the ⁇ sheet (A) include amino acid residues at positions 11 to 13 shown in SEQ ID NO: 4 if they are derived from Pseudomonas aeruginosa.
  • ⁇ sheet Region from C-terminal amino acid residue to ⁇ -helix
  • A N-terminal amino acid residue
  • the number of amino acid residues in the region (II) is 14 to 23, preferably 16 to 22, and more preferably 17 to 21.
  • Specific examples of the amino acid sequence of region (II) include the amino acid residues at positions 14 to 33 shown in SEQ ID NO: 4 if they are derived from Pseudomonas aeruginosa.
  • ⁇ helix (A) The ⁇ -helix (A) is present at positions 15 to 25 when the amino acid residue on the N-terminal side is 0-position from the amino acid residue on the C-terminal side of the ⁇ sheet (A), preferably 17- Present at the 23rd position, more preferably at 19-21.
  • Examples of the number of amino acid residues constituting the ⁇ helix (A) include 3 to 9, preferably 4 to 8, and more preferably 5 to 7.
  • an amino acid sequence of alpha helix (A) For example, ALRRD etc. are mentioned.
  • the amino acid sequence of ⁇ -helix (A) includes amino acid residues at positions 34 to 38 in SEQ ID NO: 4 as long as they are derived from Pseudomonas aeruginosa.
  • the number of amino acid residues in region (III) is 69 to 92, preferably 74 to 87, and more preferably 78 to 83.
  • Specific examples of the amino acid sequence of region (III) include the amino acid residues at positions 39 to 120 shown in SEQ ID NO: 4 if they are derived from Pseudomonas aeruginosa.
  • ⁇ helix (B) The ⁇ -helix (B) is present at the 70-92 position when the amino acid residue on the N-terminal side of the ⁇ -helix (A) is 0-position, preferably 75. It is present at the ⁇ 87th position, more preferably at the 79th to 83rd position. Examples of the number of amino acid residues constituting the ⁇ helix (B) include 4 to 15, preferably 5 to 14, and more preferably 7 to 12. Although it does not specifically limit as an amino acid sequence of alpha helix (B), For example, IPPGGSAG etc. are mentioned. Specific examples of the amino acid sequence of ⁇ -helix (B) include amino acid residues 121 to 127 shown in SEQ ID NO: 4 as long as they are derived from Pseudomonas aeruginosa.
  • numbers of amino acid residues in region (IV) include 2 to 10, preferably 4 to 9, and more preferably 5 to 8.
  • Specific examples of the amino acid sequence of region (IV) include amino acid residues at positions 128 to 133 shown in SEQ ID NO: 4 if they are derived from Pseudomonas aeruginosa.
  • ⁇ helix (C) The ⁇ helix (C) is present at the 3 to 11 position when the amino acid residue on the N-terminal side of the ⁇ helix (B) is 0-position, preferably 5 Present at the 10th position, more preferably at the 6th to 9th position.
  • Examples of the number of amino acid residues constituting the ⁇ helix (C) include 11 to 20, preferably 13 to 18, and more preferably 14 to 17.
  • an amino acid sequence of (alpha) helix (C) For example, LVNSLGALISFLSSGST etc. are mentioned.
  • the amino acid sequence of ⁇ -helix (C) includes amino acid residues at positions 134 to 150 shown in SEQ ID NO: 4 if it is derived from Pseudomonas aeruginosa.
  • Examples of the number of amino acid residues in region (V) include 64 to 80, preferably 68 to 76, and more preferably 70 to 75.
  • Specific examples of the amino acid sequence of the region (V) include amino acid residues 151 to 222 shown in SEQ ID NO: 4 if it is derived from Pseudomonas aeruginosa.
  • the ⁇ sheet (B) is present at the 65th to 81st positions when the amino acid residue on the N-terminal side is 0-position of the amino acid residue on the C-terminal side of the ⁇ helix (C), preferably 69- It exists at the 77th position, more preferably at the 71st to 75th positions.
  • Examples of the number of amino acid residues constituting the ⁇ sheet (B) include 2 to 8, preferably 3 to 7, and more preferably 4 to 6.
  • the amino acid sequence of the ⁇ sheet (B) is not particularly limited, and examples thereof include KNGT. Specific examples of the amino acid sequence of the ⁇ sheet (B) include the amino acid residues 223 to 226 shown in SEQ ID NO: 4 if they are derived from Pseudomonas aeruginosa.
  • ⁇ sheet Region from C-terminal amino acid residue to ⁇ helix
  • D N-terminal amino acid residue
  • VI Examples of the number of amino acid residues in region (VI) include 5 to 14, preferably 7 to 12, and more preferably 8 to 11.
  • Specific examples of the amino acid sequence of region (VI) include the amino acid residues at positions 227 to 237 shown in SEQ ID NO: 4 if they are derived from Pseudomonas aeruginosa.
  • the ⁇ helix (D) is present at the 6th to 15th positions when the amino acid residue on the N-terminal side is 0-position of the amino acid residue on the C-terminal side of the ⁇ sheet (B), preferably 8 to Present at the 13th position, more preferably at the 9th to 12th positions.
  • Examples of the number of amino acid residues constituting the ⁇ helix (D) include 2 to 24, preferably 3 to 23, and more preferably 4 to 22.
  • the amino acid sequence of the ⁇ helix (D) is not particularly limited, and examples thereof include HLGM, IRDNYRMNHLDEVNQ, and the like.
  • the ⁇ helix (D) region is preferably a sequence in which two or more of the sequences exemplified above are linked directly or via several amino acid residues, and includes HLGM and IRDNYRMNHLDEVNQ. Is preferred. When two or more kinds of sequences are included, a loop may exist between them.
  • Specific examples of the amino acid sequence of ⁇ -helix (D) include amino acid residues at positions 238 to 257 shown in SEQ ID NO: 4 if they are derived from Pseudomonas aeruginosa.
  • Region from the C-terminal amino acid residue of ⁇ -helix (D) to the C-terminus of polypeptide (VII) The number of residues in the amino acid sequence of region (VII) is 21 to 51, preferably 24 to 49, and more preferably 26 to 47.
  • Specific examples of the amino acid sequence in the region (VII) include amino acid residues at positions 258 to 285 shown in SEQ ID NO: 4 if it is derived from Pseudomonas aeruginosa.
  • the polypeptide of the present invention preferably further comprises an ⁇ helix (E), a ⁇ sheet (C), and a ⁇ sheet (D) from the N-terminal side in the region (III).
  • the region between these ⁇ helix and ⁇ sheet may include a loop, ⁇ helix, or ⁇ sheet.
  • the ⁇ helix (E) has 17 to 28 positions, preferably 19 to 26 positions, when the amino acid residue on the N-terminal side of the ⁇ helix (A) is 0-position. More preferably, it is present in 21 to 24, and the number of amino acid residues is 13 to 19, preferably 14 to 18, and more preferably 15 to 17.
  • the amino acid sequence of the ⁇ helix (E) is not particularly limited, and examples thereof include LQQVEEIVALSGQPLV.
  • the amino acid sequence of ⁇ -helix (E) includes the amino acid residues at positions 61 to 76 shown in SEQ ID NO: 4 if it is derived from Pseudomonas aeruginosa.
  • the ⁇ sheet (C) has an N-terminal amino acid residue of 2 to 8 positions, preferably 3 to 7 positions when the C-terminal amino acid residue of the ⁇ helix (E) is 0 position. More preferably, it is present in 4-6, and the number of amino acid residues is 4-8, preferably 5-7. Although it does not specifically limit as an amino acid sequence of (beta) sheet
  • the ⁇ -sheet (D) has 14 to 22 positions, preferably 16 to 20 positions when the amino acid residue on the N-terminal side of the ⁇ -sheet (C) is 0-position. It is preferably located at positions 17 to 19, and is composed of 3 to 11, preferably 5 to 9, more preferably 6 to 8 amino acid residues. Although it does not specifically limit as an amino acid sequence of (beta) sheet
  • the polypeptide of the present invention preferably further comprises an ⁇ helix (G) and a ⁇ sheet (E) from the N-terminal side in the region (VI).
  • the region between these ⁇ -helix and ⁇ -sheets may include loops, ⁇ -helixes, or ⁇ -sheets.
  • ⁇ helix (G) ⁇ -helix (G) has 3 to 13 positions, preferably 4 to 12 positions, when the amino acid residue on the N-terminal side is 0-position of the amino acid residue on the C-terminal side of ⁇ -helix (C). More preferably, it is present in 5 to 11, and comprises 4 to 13, preferably 5 to 12, more preferably 6 to 11 amino acid residues.
  • the amino acid sequence of the ⁇ helix (G) is not particularly limited, and examples thereof include ESLNSEG. Specifically, the amino acid sequence of ⁇ -helix (G) includes the amino acid residues at positions 160 to 166 shown in SEQ ID NO: 4 if it is derived from Pseudomonas aeruginosa.
  • the ⁇ sheet (E) has an amino acid residue on the N-terminal side in positions 24 to 36, preferably 27 to 34, when the amino acid residue on the C-terminal side of the ⁇ helix (G) is 0 position. More preferably, it is located at positions 29 to 32, and the number of amino acid residues is 2 to 6, preferably 3 to 5. Although it does not specifically limit as an amino acid sequence of (beta) sheet
  • the polypeptide of the present invention comprises the aforementioned ⁇ sheet (A), ⁇ helix (A), ⁇ helix (B), ⁇ helix (C), ⁇ sheet (B), and ⁇ helix (D). Basically, it preferably has an ⁇ helix (E), a ⁇ sheet (C), a ⁇ sheet (D), an ⁇ helix (G), and a ⁇ sheet (E).
  • Amino acid substitution region In the polypeptide of the present invention, the region between the ⁇ sheet (A) and the ⁇ helix (A) is represented by No. 1 in Table I. The region between any of the amino acid residues shown in 1-125 and / or between the ⁇ sheet (B) and the ⁇ helix (D) is represented by No. 1 in Table II. Any amino acid residue shown in 1-150 is included.
  • the amino acid sequence of a region containing a loop existing in the region (II) between the ⁇ sheet (A) and the ⁇ helix (A), preferably the N-terminal amino acid residue is
  • the amino acid sequence of the region including the loop, preferably the N-terminal amino acid residue is 3 to 9, preferably 4 to 8 when the amino acid residue on the C-terminal side of the ⁇ sheet (B) is 0-position.
  • the amino acid sequence present in the position, more preferably in positions 5 to 7, is represented by No. It is set so as to include any one of amino acid residues shown in 1-150 (sequence consisting of three amino acid residues).
  • the amino acid sequence contained in region (II) is preferably No. 1 in Table I in that the thermal stability of lipase activity can be further improved. 1-125, more preferably no. 1-96, even more preferably no. 1 to 68, more preferably No. 1-50, particularly preferably no. 1 to 40, particularly preferably No.1. Any of the amino acid residues shown in 1-33 can be mentioned, and the amino acid sequence contained in the region (VI) is preferably No. 1 in Table II. 1-150, more preferably no. 1 to 129, more preferably No. 1 to 88, more preferably No. 1 to 68, particularly preferably no. 1 to 55, more preferably no. Any amino acid residue shown in 1 to 40 can be mentioned.
  • the amino acid sequence to be an amino acid substitution region present in the region (II) between the ⁇ sheet (A) and the ⁇ helix (A) includes at least one amino acid residue in the loop (A) region, preferably 1 to 3, more preferably 2 or 3 amino acid residues are included in the loop (A) region.
  • the loop (A) region exists in the region (II) between the ⁇ sheet (A) and the ⁇ helix (A).
  • the N-terminal amino acid residue of the loop (A) is 1 to 12 positions, preferably 1 to 11, more preferably 8 when the C-terminal amino acid residue of the ⁇ sheet (A) is 0 position. Present in ⁇ 11.
  • the number of amino acid residues constituting the loop (A) is 1 to 13, preferably 2 to 12.
  • the amino acid sequence of the loop (A) is not particularly limited.
  • x 21 x 22 V may be any amino acid residues, and preferably x 21 is A or V, x 22 is G or N.), GVD, HGLAGTDDKFANV, and the like, and preferably AGV, HGLAGTDDKFANV, VGV, GVD, and the like. It is preferable that at least one amino acid residue of the amino acid sequence is included.
  • the amino acid sequence to be an amino acid substitution region existing in the region (VI) between the ⁇ sheet (B) and the ⁇ helix (D) has at least one amino acid residue in the loop (B) region, preferably 1 ⁇ 3, more preferably 2 or 3 amino acid residues are in the loop (B) region.
  • the loop (B) region exists in the region (VI) between the ⁇ sheet (B) and the ⁇ helix (D).
  • the amino acid residue on the N-terminal side is 1 to 7, preferably 1 to 6, more preferably when the amino acid residue on the C-terminal side of ⁇ -sheet (B) is 0-position. Present in the 1st to 5th positions.
  • the number of amino acid residues constituting the loop (B) is 1 to 12, preferably 2 to 11.
  • the amino acid sequence of the loop (B) is not particularly limited, and for example, TSTx 23 x 24 x 25 VD (x 23 , x 24 , and x 25 may be any amino acid residue, preferably x.
  • the amino acid sequence of the loop (B) preferably contains at least one amino acid residue of the amino acid sequence of the region (VI).
  • the total number of amino acid residues of the polypeptide of the present invention is 270 to 340, preferably 280 to 330, more preferably 285 to 325, still more preferably 300 to 325, and particularly preferably 310 to 325.
  • polypeptides shown in the following (1) to (4) can be mentioned.
  • polypeptides shown in the following (1) to (4) are shown in the following (1) to (4).
  • the amino acid residues substituted with any of the amino acid residues shown in 1-125 and / or the amino acid residues at positions 233-235 are shown in Table II.
  • the amino acid residues at positions 24 to 26 are Nos. Nos.
  • amino acid residues substituted with any of the amino acid residues shown in 1-125 and / or the amino acid residues at positions 233-235 are shown in Table II.
  • the amino acid sequence shown in SEQ ID NO: 1 is the amino acid sequence of wild-type lipase derived from Burkholderia cepacia.
  • the amino acid sequence shown in SEQ ID NO: 2 is the amino acid sequence of wild-type lipase derived from Pseudomonas grumae.
  • the amino acid sequence shown in SEQ ID NO: 3 is the amino acid sequence of wild-type lipase derived from Pseudomonas fluorescens.
  • the amino acid sequence shown in SEQ ID NO: 4 is the amino acid sequence of Pseudomonas aeruginosa wild-type lipase.
  • the polypeptides of (1) to (4) include not only polypeptides obtained by artificial substitution, but also polypeptides originally having such amino acid sequences.
  • polypeptide of the present invention include the polypeptides shown in the following (5) to (12).
  • the amino acid residues at positions 25 to 27 in the amino acid sequence shown in SEQ ID NO: 1 are No.
  • the amino acid residues substituted with any of the amino acid residues shown in 1-125 and / or the amino acid residues at positions 233-235 are shown in Table II.
  • the amino acid sequence substituted with any one of the amino acid residues shown in 1-150 one or several amino acid residues other than the amino acid residue into which the substitution is introduced are substituted, added, inserted or deleted.
  • the amino acid residues at positions 24 to 26 in the amino acid sequence shown in SEQ ID NO: 2 are Nos. Nos. 1 to 125 are substituted with amino acid residues and / or amino acid residues at positions 232 to 234
  • In the amino acid sequence substituted with any one of the amino acid residues shown in 1-150 one or several amino acid residues other than the amino acid residue into which the substitution is introduced are substituted, added, inserted or deleted.
  • the amino acid residues at positions 25 to 27 in the amino acid sequence shown in SEQ ID NO: 3 are Nos.
  • the amino acid residues substituted with any of the amino acid residues shown in 1-125 and / or the amino acid residues at positions 233-235 are shown in Table II.
  • the amino acid sequence substituted with any one of the amino acid residues shown in 1-150 one or several amino acid residues other than the amino acid residue into which the substitution is introduced are substituted, added, inserted or deleted.
  • the amino acid residues at positions 25 to 27 in the amino acid sequence shown in SEQ ID NO: 4 are Nos.
  • the amino acid residues substituted with any of the amino acid residues shown in 1-125 and / or the amino acid residues at positions 233-235 are shown in Table II.
  • the amino acid sequence substituted with any one of the amino acid residues shown in 1-150 one or several amino acid residues other than the amino acid residue into which the substitution is introduced are substituted, added, inserted or deleted.
  • the amino acid residues at positions 25 to 27 in the amino acid sequence shown in SEQ ID NO: 1 are No.
  • the amino acid residues substituted with any of the amino acid residues shown in 1-125 and / or the amino acid residues at positions 233-235 are shown in Table II.
  • the sequence identity excluding the amino acid residue introduced with the substitution with respect to the amino acid sequence shown in SEQ ID NO: 1 is 80% or more
  • the amino acid residues at positions 24 to 26 in the amino acid sequence shown in SEQ ID NO: 2 are Nos. Nos.
  • 1 to 125 are substituted with amino acid residues and / or amino acid residues at positions 232 to 234
  • the sequence identity excluding the amino acid residue introduced with the substitution with respect to the amino acid sequence shown in SEQ ID NO: 2 is 80% or more
  • the amino acid residues at positions 25 to 27 in the amino acid sequence shown in SEQ ID NO: 3 are Nos.
  • the amino acid residues substituted with any of the amino acid residues shown in 1-125 and / or the amino acid residues at positions 233-235 are shown in Table II.
  • the sequence identity excluding the amino acid residue introduced with the substitution with respect to the amino acid sequence shown in SEQ ID NO: 3 is 80% or more
  • the amino acid residues at positions 25 to 27 in the amino acid sequence shown in SEQ ID NO: 4 are Nos.
  • the amino acid residues substituted with any of the amino acid residues shown in 1-125 and / or the amino acid residues at positions 233-235 are shown in Table II.
  • the sequence identity excluding the amino acid residue into which the substitution is introduced with respect to the amino acid sequence shown in SEQ ID NO: 4 is 80% or more, A polypeptide having lipase activity and improved thermal stability as compared to the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 4.
  • the polypeptides (5) to (12) include not only polypeptides obtained by artificial substitution, but also polypeptides having such an amino acid sequence.
  • amino acid residues other than the amino acid residues substituted with the amino acid residues shown in Tables I and II may be referred to as “arbitrarily different sites”.
  • the term “arbitrary difference site” is a site where a difference is allowed as long as it does not greatly affect the properties of the polypeptide.
  • the amino acid sequence is different at an arbitrarily different site as compared with the polypeptides (1) to (4), it is the same as the polypeptides (1) to (4).
  • having a lipase activity higher than that and having a thermal stability equivalent to or higher than that of the polypeptide of (1) to (4) above It is called a polypeptide variant.
  • polypeptide variants have substantially the same characteristics as the polypeptides of the above (1) to (4), although the amino acid sequences are different at arbitrary different sites. Preferably there is.
  • the term “substantially the same” refers to those having the same lipase activity and thermal stability as the polypeptides (1) to (4).
  • the polypeptides (5) and (9) are different from the polypeptide (1).
  • the polypeptides (6) and (10) are different from the polypeptide (2).
  • the polypeptides (7) and (11) are different from the polypeptide (3).
  • the polypeptides (8) and (12) are different from the polypeptide (4).
  • the amino acid difference in the polypeptides (5) to (8) may include only one type of difference (for example, substitution) from substitution, addition, insertion, and deletion, or two or more types (For example, substitution and insertion) may be included.
  • the number of amino acid differences at arbitrary different sites may be one or several, for example 1 to 50, preferably 1 to 20, 1 to 10, 1 to 8, 1 to 7, 1 to 6, 1 to 5, or 1 to 4, more preferably 1 to 3, particularly preferably 1 or 2, or 1.
  • sequence identity excluding the site where the amino acid substitution is made for each amino acid sequence shown in SEQ ID NOs: 1 to 4 may be 80% or more, Preferably 85% or more or 90% or more, more preferably 95% or more, 96% or more, 97% or more, or 98% or more, and particularly preferably 99% or more.
  • sequence identity refers to BLAST PACKAGE [sgi32 bit edition, Version 2.0.12; available from National Center for Biotechnology Information (NCBI), Bl2seqpromTam. Microbiol.Lett., Vol.174, p247-250, 1999) shows the identity value of amino acid sequences.
  • the parameters may be set to Gap insertion Cost value: 11 and Gap extension Cost value: 1.
  • conservative substitution may be mentioned as another embodiment of the amino acid substitution introduced at an arbitrarily different site in the polypeptides (5) to (12). That is, as the substitution at the arbitrarily different site, for example, when the amino acid before substitution is a nonpolar amino acid, substitution with another nonpolar amino acid, and when the amino acid before substitution is an uncharged amino acid, Examples include substitution with an amino acid, substitution with another acidic amino acid if the amino acid before substitution is an acidic amino acid, and substitution with another basic amino acid if the amino acid before substitution is a basic amino acid.
  • a polypeptide having lipase activity and having improved thermal stability compared to the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1 has lipase activity, And it means that the residual activity measured under the following conditions is higher than the residual activity of the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1 measured under the same conditions. Specifically, the remaining activity of the polypeptide measured under the following conditions is 1.5 times or more, preferably 2 times or more compared to the remaining activity of the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1 measured under the same conditions. More preferably, it means 3 times higher. The same applies to the polypeptides (6) to (12).
  • “Residual activity” is lipase activity remaining after heat treatment, and is a percentage of the lipase activity value after heat treatment with respect to the lipase activity value before heat treatment of the polypeptide.
  • Heat treatment 60 ° C., 30 minutes
  • Measurement of activity value Using Lipase Kit S (DS Pharma Biomedical), the reaction was carried out at 37 ° C. for 20 minutes, and then the absorbance value measured with PowerScanHT (DS Pharma Biomedical) ( 412 nm).
  • Residual activity (residual rate) (%): [activity value (heat treated sample) / activity value (untreated sample) ⁇ 100]
  • the DNA encoding the polypeptide of the present invention (hereinafter sometimes referred to as “the DNA of the present invention”) is, for example, a DNA encoding the amino acid sequence of wild-type lipase (SEQ ID NOs: 1 to 4). It can be obtained by introducing the amino acid mutation into.
  • the DNA of the present invention can also be artificially synthesized by a total gene synthesis method.
  • the DNA encoding the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1 is known, for example, as the base sequence shown in SEQ ID NO: 9, and is M-12-33 of Burkholderia cepacia. It can be isolated from the genomic DNA of the strain by a conventional method using PCR.
  • DNA encoding a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2 is known, for example, as the base sequence shown in SEQ ID NO: 10, and PCR is performed from the genomic DNA of Pseudomonas glumae PG1 strain. It can be isolated by a conventional method using
  • DNA encoding a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 3 is known, for example, as the base sequence shown in SEQ ID NO: 11, and is genomic DNA of the AK102 strain of Pseudomonas fluorescence. Can be isolated by a conventional method using PCR.
  • the DNA encoding the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 4 is known as, for example, the base sequence shown in SEQ ID NO: 12, and PCR is performed from the genomic DNA of the Pseudomonas aeruginosa TE3285 strain. It can be isolated by a conventional method using
  • a method for introducing a specific mutation into a specific site in a base sequence is known, and for example, a site-specific mutagenesis method for DNA can be used.
  • Specific examples of the method for converting the base in the DNA include use of a commercially available kit (QuickChange Lightning Site-Directed Mutagenesis kit: manufactured by Stratagene, KOD-Plus-Mutageness kit: manufactured by Toyobo, etc.).
  • the base sequence of DNA having a mutation introduced into the base sequence can be confirmed using a DNA sequencer. Once the base sequence is determined, DNA encoding the polypeptide is obtained by chemical synthesis, PCR using the cloned probe as a template, or hybridization using a DNA fragment having the base sequence as a probe. be able to.
  • a mutant form of DNA encoding the peptide having a function equivalent to that before mutation can be synthesized by site-directed mutagenesis or the like.
  • a known method such as Kunkel method, Gapped duplex method, or megaprimer PCR method can be used.
  • the DNA of the present invention is preferably a DNA whose codon usage frequency is optimized for the host, and more preferably a DNA whose codon usage frequency is optimized for E. coli.
  • the optimal codon is defined as the codon that is most frequently used among codons corresponding to the same amino acid.
  • the codon usage frequency is not particularly limited as long as it is optimized for the host. Examples of the optimal codon for Escherichia coli include the following.
  • F phenylalanine (ttt), L: leucine (ctg), I: isoleucine (att), M: methionine (atg), V: valine (gtg), Y: tyrosine (tat), stop codon (taa), H: Histidine (cat), Q: glutamine (cag), N: asparagine (aat), K: lysine (aaa), D: aspartic acid (gat), E: glutamic acid (gaa), S: serine (agc), P: Proline (ccg), T: threonine (acc), A: alanine (gcg), C: cysteine (tgc), W: tryptophan (tgg), R: arginine (cgg), G: glycine (ggc).
  • DNAs containing the base sequences shown in SEQ ID NOs: 9 to 13 include DNAs containing the base sequences shown in SEQ ID NOs: 9 to 13.
  • the DNA consisting of the base sequence shown in SEQ ID NO: 9 or 13 is the polypeptide of (1) above, and is shown in Table II at amino acid residues shown in Table I at positions 25-27 and Table II at positions 233-235 It encodes a polypeptide into which an amino acid residue has been introduced.
  • the DNA consisting of the base sequence shown in SEQ ID NO: 10 is the polypeptide of (2) described above, and the amino acid residues shown in Table I at positions 24 to 26 and amino acid residues shown in Table II at positions 232 to 234. It encodes a polypeptide into which a group has been introduced.
  • the DNA consisting of the base sequence shown in SEQ ID NO: 11 is the polypeptide of (3) above, and the amino acid residues shown in Table I at positions 25 to 27 and amino acid residues shown in Table II at positions 233 to 235, respectively. It encodes a polypeptide into which a group has been introduced.
  • the DNA consisting of the base sequence shown in SEQ ID NO: 12 is the polypeptide of the above (4), the amino acid residues shown in Table I at positions 25 to 27, and the amino acid residues shown in Table II at positions 233 to 235. It encodes a polypeptide into which a group has been introduced.
  • the DNA of the present invention encodes a polypeptide having (i) lipase activity and improved thermal stability as compared with the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1, Or a DNA comprising a base sequence complementary to the DNA consisting of the base sequence shown in 13, and a DNA that hybridizes under stringent conditions; (ii) having a lipase activity and consisting of an amino acid sequence shown in SEQ ID NO: 2 A DNA that encodes a polypeptide having improved thermal stability compared to a peptide, and that hybridizes under stringent conditions with a DNA comprising a base sequence complementary to the DNA consisting of the base sequence shown in SEQ ID NO: 10 (Iii) a polypeptide having lipase activity and improved thermal stability as compared to the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 3 A DNA comprising a base sequence complementary to the DNA comprising the base sequence shown in SEQ ID NO: 11, and a DNA hybridizing under stringent conditions; (i
  • stringent conditions means 0.5% SDS, 5 ⁇ Denhartz [Denhartz's, 0.1% bovine serum albumin (BSA), 0.1% polyvinylpyrrolidone, 0.1% Ficoll. 400] and 100 ⁇ g / ml salmon sperm DNA (1 ⁇ SSC is 0.15 M NaCl, 0.015 M sodium citrate, pH 7.0) at 50 ° C. to 65 ° C. for 4 hours to overnight This refers to the conditions for keeping warm.
  • BSA bovine serum albumin
  • polyvinylpyrrolidone 0.1% Ficoll. 400
  • hybridization under stringent conditions is performed by the following method. That is, a nylon membrane on which a DNA library or cDNA library is immobilized is prepared, and a prehybridization solution containing 6 ⁇ SSC, 0.5% SDS, 5 ⁇ Denharz, 100 ⁇ g / ml salmon sperm DNA at 65 ° C. Block nylon membrane. Then add each probe labeled with 32 P and incubate at 65 ° C. overnight. This nylon membrane was placed in 6 ⁇ SSC for 10 minutes at room temperature, in 2 ⁇ SSC containing 0.1% SDS, for 10 minutes at room temperature, in 0.2 ⁇ SSC containing 0.1% SDS for 30 minutes at 45 ° C. After washing, autoradiography can be taken to detect DNA specifically hybridized with the probe.
  • the DNA of the present invention encodes a polypeptide having (v) lipase activity and improved thermal stability as compared to the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1, and SEQ ID NO: DNA having 80% homology or more to DNA consisting of the base sequence shown in 9; (vi) having lipase activity and improved thermal stability compared to the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2 (Vii) a polypeptide having the lipase activity and having the lipase activity and comprising the amino acid sequence shown in SEQ ID NO: 3
  • the “homology” of DNA is calculated using publicly available or commercially available software having an algorithm for comparing a reference sequence as a reference sequence.
  • BLAST, FASTA, GENETYX manufactured by Software Development Co., Ltd.
  • these may be used by setting default parameters.
  • Recombinant vector A recombinant vector containing DNA encoding the peptide of the present invention (hereinafter sometimes referred to as "the recombinant vector of the present invention") is obtained by inserting the DNA of the present invention into an expression vector. Can do.
  • the recombinant vector of the present invention includes a control factor such as a promoter operably linked to the DNA of the present invention.
  • a control factor such as a promoter operably linked to the DNA of the present invention.
  • a typical example of a control factor is a promoter, but a transcription element such as an enhancer, a CCAAT box, a TATA box, or an SPI site may be further included as necessary.
  • operably linked means that various regulatory factors such as promoters and enhancers that regulate the DNA of the present invention and the DNA of the present invention are linked in a state in which they can operate in a host cell.
  • a vector constructed for gene recombination from a phage, plasmid, or virus capable of autonomously growing in a host is suitable.
  • Such expression vectors are known.
  • commercially available expression vectors include pQE vectors (Qiagen), pDR540, pRIT2T (GE Healthcare Biosciences), pET vectors (Merck). Etc.).
  • an appropriate combination with a host cell may be selected and used.
  • E. coli when E. coli is used as a host cell, a combination of a pET vector and a DH5 ⁇ E. coli strain, a pET vector and BL21 (DE3) E. coli A combination of strains or a combination of pDR540 vector and JM109 E. coli strain is preferable.
  • Transformant of the present invention is obtained by transforming a host using the recombinant vector of the present invention.
  • the host used for the production of the transformant is not particularly limited as long as the recombinant vector is stable, can autonomously proliferate, and can express a foreign gene trait.
  • Escherichia coli etc.
  • Bacteria belonging to the genus Escherichia, Bacillus subtilis, such as Bacillus subtilis, Pseudomonas putida, Pseudomonas genus, etc .; yeast and the like are preferred examples, but other animal cells, insect cells It may be a plant or the like.
  • Escherichia coli is particularly preferable.
  • the transformant of the present invention can be obtained by introducing the recombinant vector of the present invention into a host, and the conditions for introducing the recombinant vector into the host may be appropriately set according to the type of the host.
  • the host is a bacterium, for example, a method using competent cells by calcium ion treatment, an electroporation method and the like can be mentioned.
  • the host is yeast, for example, electroporation method (electroporation method), spheroplast method, lithium acetate method and the like can be mentioned.
  • the host is an animal cell, examples thereof include an electroporation method, a calcium phosphate method, and a lipofection method.
  • examples include calcium phosphate method, lipofection method, electroporation method and the like.
  • examples thereof include an electroporation method, an Agrobacterium method, a particle gun method, and a PEG method.
  • the recombinant vector may be separated and purified from the transformant.
  • the recombinant vector is separated and purified based on a lysate obtained by lysing the bacterium.
  • a lytic enzyme such as lysozyme
  • a protease other enzyme
  • a surfactant such as sodium lauryl sulfate (SDS) are used in combination as necessary.
  • Separation and purification of DNA from the lysate can be performed by, for example, appropriately combining a deproteinization treatment by a phenol treatment and a protease treatment, a ribonuclease treatment, an alcohol precipitation treatment, and a commercially available kit.
  • the DNA can be cleaved according to a conventional method, for example, using a restriction enzyme treatment.
  • a restriction enzyme for example, a type II restriction enzyme that acts on a specific nucleotide sequence is used.
  • the DNA and the expression vector are bound using, for example, DNA ligase.
  • PCR is performed by designing primers specific to the DNA of the present invention using the separated and purified DNA as a template.
  • the amplified product obtained by PCR is subjected to agarose gel electrophoresis, polyacrylamide gel electrophoresis, capillary electrophoresis, etc., stained with ethidium bromide and SYBR Green solution, etc., and the amplified product is detected as a band to You can confirm that it has been converted.
  • PCR can be performed using a primer previously labeled with a fluorescent dye or the like to detect an amplification product.
  • a method of binding an amplification product to a solid phase such as a microplate and confirming the amplification product by fluorescence, enzyme reaction, or the like may be employed.
  • polypeptide of the present invention can be produced by culturing the transformant of the present invention.
  • the culture conditions for the transformant may be appropriately set in consideration of the nutritional physiological properties of the host, and liquid culture is preferable. In addition, when industrial production is performed, aeration stirring culture is preferable.
  • the carbon source may be any carbon compound that can be assimilated, and examples thereof include glucose, sucrose, lactose, maltose, molasses, and pyruvic acid.
  • the nitrogen source may be any assimitable nitrogen compound, and examples thereof include peptone, meat extract, yeast extract, casein hydrolyzate, and soybean meal alkaline extract.
  • salts such as phosphate, carbonate, sulfate, magnesium, calcium, potassium, iron, manganese and zinc, specific amino acids and specific vitamins are used as necessary May be.
  • the culture temperature can be appropriately set within the range in which the transformant of the present invention can grow and the transformant of the present invention produces the polypeptide of the present invention, but is preferably about 15 to 37 ° C.
  • the culture may be completed at an appropriate time in consideration of the time when the polypeptide of the present invention reaches the maximum yield, and the culture time is usually about 12 to 48 hours.
  • the transformant of the present invention is cultured, and the culture supernatant or cells are collected by a method such as centrifugation, and the cells are treated with a mechanical method such as ultrasonic and French press or a lytic enzyme such as lysozyme. It is solubilized by using an enzyme such as protease or a surfactant such as sodium lauryl sulfate (SDS) as necessary, and a water-soluble fraction containing the polypeptide of the present invention can be obtained.
  • a mechanical method such as ultrasonic and French press or a lytic enzyme such as lysozyme.
  • the expressed polypeptide of the present invention can also be secreted into the culture medium by selecting an appropriate expression vector and host.
  • the water-soluble fraction containing the polypeptide of the present invention obtained as described above may be subjected to purification treatment as it is. However, after the polypeptide of the present invention in the water-soluble fraction is concentrated, the water-soluble fraction is subjected to purification treatment. May be provided.
  • Concentration can be performed by, for example, vacuum concentration, membrane concentration, salting-out treatment, fractional precipitation with a hydrophilic organic solvent (for example, methanol, ethanol and acetone).
  • a hydrophilic organic solvent for example, methanol, ethanol and acetone.
  • the purification treatment of the polypeptide of the present invention can be performed by appropriately combining methods such as gel filtration, adsorption chromatography, ion exchange chromatography, affinity chromatography and the like.
  • the purification process is already known and can be carried out by referring to appropriate documents, magazines, textbooks, and the like.
  • the polypeptide of the present invention thus purified can be pulverized by lyophilization, vacuum drying, spray drying or the like and distributed to the market as necessary.
  • compositions The polypeptides of the invention may be provided, for example, in the form of a composition.
  • the composition contains the polypeptide of the present invention as an active ingredient.
  • the degree of purification of the composition is not particularly limited, but the composition may contain other components to the extent that the effect of the present invention is not affected. Examples of other components include media-derived components and contaminating proteins.
  • the composition may also contain other enzymes.
  • other enzymes include amylase ( ⁇ -amylase, ⁇ -amylase, glucoamylase), glucosidase ( ⁇ -glucosidase, ⁇ -glucosidase), galactosidase ( ⁇ -galactosidase, ⁇ -galactosidase), protease (acidic protease, medium Sex protease, alkaline protease), peptidase (leucine peptidase, aminopeptidase), lipase, esterase, cellulase, phosphatase (acid phosphatase, alkaline phosphatase), nuclease, deaminase, oxidase, dehydrogenase, glutaminase, pectinase, catalase, dextranase, trans Examples include glutaminase, protein deamidase, pullulanas
  • the content of the polypeptide of the present invention in the composition is not particularly limited, but preferably 10% by mass or more, more preferably 30% by mass or more, based on the total protein of the composition.
  • the form of the said composition is not specifically limited, For example, a liquid, a powder, a granule etc. are mentioned.
  • the composition can be prepared by a generally known method.
  • the polypeptide of the present invention or the composition containing the polypeptide of the present invention may be provided, for example, in the form of an enzyme agent.
  • the enzyme agent may contain an excipient, a buffer, a suspension, a stabilizer, a preservative, a preservative, a physiological saline and the like in addition to the polypeptide of the present invention or the composition.
  • an excipient starch, dextrin, maltose, trehalose, lactose, D-glucose, sorbitol, D-mannitol, sucrose, glycerol and the like can be used.
  • Phosphate, citrate, acetate, etc. can be used as the buffer.
  • propylene glycol, ascorbic acid or the like can be used.
  • preservatives phenol, benzalkonium chloride, benzyl alcohol, chlorobutanol, methylparaben, and the like can be used.
  • preservatives ethanol, benzalkonium chloride, paraoxybenzoic acid, chlorobutanol and the like can be used.
  • the content of the polypeptide in the enzyme agent is appropriately set within a range in which the effect of the polypeptide is exhibited.
  • the polypeptide of the present invention has lipase activity and is excellent in thermal stability. Therefore, the polypeptide of the present invention can be used in applications that require enzyme treatment with lipase, for example, applications that require treatment of fats and oils (triglycerides) (decomposition, transesterification).
  • Applications that require enzyme treatment with lipase include, for example, purification of oils and fats, production of food or food materials, pharmaceuticals such as digestive enzymes, cosmetic additives, wastewater treatment of food factories, etc.
  • Uses such as intermediate production, fine chemical material production, functional substitute oil production and the like can be mentioned, preferably wastewater treatment use.
  • the polypeptide of the present invention or the above-mentioned enzyme agent containing the polypeptide of the present invention can be treated with fats and oils by acting on the fats and oils. Such an oil treatment method is also one aspect of the present invention.
  • wastewater can be treated by allowing the polypeptide of the present invention or the above-mentioned enzyme agent containing the polypeptide of the present invention to act on wastewater containing fats and oils.
  • the wastewater is not particularly limited, and examples thereof include household wastewater, industrial wastewater, agricultural wastewater, and the like, and it is preferable to apply to wastewater containing a lot of oils and fats.
  • Such a wastewater treatment method is also one aspect of the present invention.
  • a pharmaceutical intermediate can be produced by allowing the above-mentioned enzyme agent containing the polypeptide of the present invention or the polypeptide of the present invention to act on the raw material for the pharmaceutical intermediate.
  • the raw material for pharmaceutical intermediates include cholesterol fatty acid esters, monoacylglycerols, glycidic acid esters, and benzothiazepine compounds.
  • a method for producing such a pharmaceutical intermediate is also one aspect of the present invention.
  • the fine chemical material can be produced by allowing the above-mentioned enzyme agent containing the polypeptide of the present invention or the polypeptide of the present invention to act on the fine chemical material raw material.
  • fine chemical materials include fragrances (milk flavors, macrocyclic lactones, phenethyl alcohol glycosides, etc.), cosmetic raw materials, emulsifiers, and the like. Such a method for producing a fine chemical material is also one aspect of the present invention.
  • E. Construction of E. coli expression plasmid In constructing the E. coli expression system, C. cepacia M12-33 genes (Lip A (SEQ ID NO: 9), Lip X (SEQ ID NO: 14)) A gene codon optimized for E. coli expression was fully synthesized.
  • coli BCL-LipA was carried out by inoculating LB Broth Base (invitrogen) + Amp: 100 ⁇ g / mL: 5 mL and shaking culture (37 ° C., 16 h, 140 rpm), and then NucleoSpin Plasmid EasyPure (N. ) was used to obtain a plasmid (pETBCL-LipA).
  • PCR primer forward primer: 5′-TTTTCATATGACCGCACGTGAAGGTCGGCGC-3 ′
  • reverse primer 5′-AAAAACTCGAGTTTACTGGCAGACACCGCGCCG-3 ′
  • E. coli BCL-LipAX The gene fragment (BCL-LipX) and pETBCL-LipA were treated with restriction enzymes (NdeI (TaKaRa), Xho I (TaKaRa)), then ligated and treated with E. coli. by transformation into E. coli DH5 ⁇ .
  • E. coli BCL-LipAX was obtained.
  • Plasmid extraction from E. coli BCL-LipAX was inoculated into LB Broth Base + Amp: 100 ⁇ g / mL: 5 mL and shake-cultured (37 ° C., 16 h, 140 rpm), and then NucleoSpin Plasmid EasyPure (MACHEREY-NAGEL) was used.
  • E. An E. coli expression plasmid (pETBCL-LipAX) was obtained.
  • E. E. coli expression system constructed E. coli expression plasmid (pETBCL-LipAX) was transformed into E. coli.
  • Sequence Confirmation of Mutant Strains Sequence confirmation of the constructed plasmids (pETBCL-LipA, pETBCL-LipAX) was performed using Sequence Primer (pET Upstream Primer: 5'-ATGCGTCCGGCGTAGGATGAGTGAGTATGG : 5'-TTGTACACGGCCGCCATAATC-3 ', T7 Terminator Primer: 5'-GCTAGTTATTGCTCAGCGGG-3').
  • Random Primers were designed to create a random mutation library for the 12 selected mutation introduction points.
  • L1 (A74X / A75X / T76X): (Forward primer: 5′-NNKNNKNNKGGCGCGACACAAAGTTTAACCTGGTTG-3 ′, Reverse primer: 5'-CAGCACAGTTTTCACATACCGCCAGC-3 ') L2 (V199X / G200X / G20X): (Forward primer: 5′-NNKNNKNNKAAACACTCACCTGCTGTTACCTTGGGC-3 ′, Reverse primer: 5′-AGTCTCGGTCGGCGCCACC-3 ′) L3 (L127X / A128X / Y129X): (Forward primer: 5′-NNKNNKNNKGACCCGAACTGGCCTGTCTCTCACC-3 ′, Reverse primer: 5'-AACGCCCTGAACGAAATCCGCG-3 ') L4 (P216X / T217X / I218X): (Forward primer: 5′-NNKNNKNNKTCTGTTTTCGGTGTTACT
  • Random mutagenesis at each mutagenesis point was performed by PCR amplification (PrimSTAR GXL DNA Polymerase (TaKaRa)) using a random primer using a plasmid (pETBCL-LipAX) as a template.
  • template plasmid treatment using Dpn I (TaKaRa) (37 ° C., 16 h) and ligation reaction (16 ° C., o / n) using T4 polymerase (Toyobo) and Ligation High (Toyobo) were performed.
  • E.E. E. coli BL21 (DE3) was transformed to obtain a random mutant strain (E. coli BL21 (BCL-Ran L1 to BCL-Ran L12)) in which random mutations were introduced at each mutation introduction point.
  • the cells were collected by centrifugation (3,300 g ⁇ 15 min, 4 ° C.), and then the enzyme extract was subjected to lysis using B-PER (ThermoFisher) (25 ° C., 1,000 rpm). I got it. After the enzyme extract was centrifuged (3,300 g ⁇ 15 min, 4 ° C.), the supernatant was collected to obtain a random mutation library at each mutation introduction point.
  • B-PER ThermoFisher
  • Plasmid extraction from each saturated mutant was carried out by shaking culture (37 ° C., 16 h, 140 rpm) at LB Broth Base (invitrogen) + Amp: 100 ⁇ g / mL, followed by extraction using NucleoSpin Plasmid EasyPure (MACHEREY-NAGEL).
  • Saturated mutant plasmids (pETBCL-G25A to G25Y, pETBCL-V26A to V26Y, pETBCL-L27A to L27Y, pETBCL-P233A to P233Y, pETBCL-L234A to L234Y, pETBCL-V235A to V235Y) were obtained. Sequence confirmation of the obtained plasmid was performed using Sequence Primer (pET Upstream Primer: 5′-ATGCGTCCGGCGTAGA-3 ′, DuetDOWN1 Primer: 5′-GATTATGCGCGCGTGCAA-3 ′).
  • E. coli BL21 (DE3) (Nippongene)
  • E. coli expression strain E. coli E. coli BL21 (BCL-G25A to G25Y, BCL-V26A to V26Y, BCL-L27A to L27Y, BCL-P233A to P233Y, BCL-L234A to L234Y, BCL-V235A to V235Y)) were obtained.
  • the cells are collected by centrifugation (3,300 g ⁇ 15 min, 4 ° C.), and then the enzyme extract is obtained by lysis treatment (25 ° C., 1,000 rpm) using B-PER (ThermoFisher). did.
  • the enzyme extract was centrifuged (3,300 g ⁇ 15 min, 4 ° C.), and the supernatant was collected to obtain a saturated mutation library at each mutation point.
  • Tables 11 and 12 show the evaluation results of the thermal stability of each polypeptide mutation library.
  • Polypeptides into which any one of the amino acid sequences 1 to 88 has been introduced have a lipase activity survival rate of 4 times or more higher than that of the wild type after heat treatment. It was shown that a polypeptide into which a mutation of any one of amino acid sequences 1 to 55 was introduced was 6 times or more higher than the wild type.
  • Treatment with pH 2-4 is 0.1 mol / L glycine buffer
  • treatment with pH 5-7 is 0.1 mol / L potassium phosphate buffer
  • treatment with pH 8-9 is 0.1 mol / L Tris buffer
  • treatment with pH 10-12 is 0 .1 mol / L glycine buffer was used.
  • 10 ⁇ L of the sample enzyme solution was mixed with 90 ⁇ L of the aforementioned buffer solution, and then treated by allowing to stand at 37 ° C. for 1 hour. After the treatment, an equal amount of 1 mol / L potassium phosphate buffer (pH 7.0) was added to return to pH 7.0, and the activity was measured. The activity was measured using Lipase Kit S (DS Pharma Biomedical).
  • the triple mutant (P233G / L234E / V235M) showed that the residual rate of lipase activity after pH treatment at pH 3 and 12 was higher than that of the wild type.
  • the organic solvent stability of the triple mutant (P233G / L234E / V235M) was evaluated.
  • the random mutation library prepared in Experimental Example 2 was used.
  • the organic solvent stability was evaluated by comparing the wild-type and triple mutant samples with water and acetone to a concentration of 0 to 50 v / v% acetone and treating them at 37 ° C for 1 hour, respectively. Comparison was made based on the relative activity (activity value (water-treated sample) / activity value (acetone-treated sample) ⁇ 100) of each concentration-treated acetone sample. The activity was measured using Lipase Kit S (DS Pharma Biomedical).
  • the triple mutant (P233G / L234E / V235M) has a higher residual rate of lipase activity in 10-50 v / v% acetone solution than the wild type.
  • the polypeptide having improved thermal stability in the present invention also has improved pH stability and organic solvent stability.
  • SEQ ID NO: 1 is the amino acid sequence of a lipase derived from Burkholderia cepacia (mature).
  • SEQ ID NO: 2 is the amino acid sequence of Pseudomonas gulmae lipase (mature).
  • SEQ ID NO: 3 is an amino acid sequence of Pseudomonas fluorescens-derived lipase (mature).
  • SEQ ID NO: 4 is an amino acid sequence of Pseudomonas aeruginosa-derived lipase (mature).
  • SEQ ID NO: 5 is the amino acid sequence of lipase (full length) derived from Burkholderia cepacia.
  • SEQ ID NO: 6 is the amino acid sequence of P.
  • SEQ ID NO: 7 is the amino acid sequence of Pseudomonas fluorescens-derived lipase (full length).
  • SEQ ID NO: 8 is the amino acid sequence of Pseudomonas aeruginosa-derived lipase (full length).
  • SEQ ID NO: 9 is the base sequence of Burkholderia cepacia derived lipase (wild type).
  • SEQ ID NO: 10 is the base sequence of Pseudomonas glumae-derived lipase (wild type).
  • SEQ ID NO: 11 is the base sequence of Pseudomonas fluorescens lipase (wild type).
  • SEQ ID NO: 12 is the base sequence of Pseudomonas aeruginosa-derived lipase (wild type).
  • SEQ ID NO: 13 is the base sequence of Burkholderia cepacia-derived lipase LipA (E. coli codon optimized).
  • SEQ ID NO: 14 is a wild-type base sequence of chaperone gene (LipX) derived from Burkholderia cepacia.
  • SEQ ID NO: 15 is the base sequence of the Burkholderia cepacia chaperone gene LipX (E. coli codon optimization).

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Abstract

L'objectif de la présente invention est de fournir un polypeptide à activité lipase présentant une stabilité améliorée (chaleur, pH, solvant). Le polypeptide comprend au moins, à partir de l'extrémité N-terminale, un feuillet β (A), une hélice α (A), une hélice α (B), une hélice α (C), un feuillet β (B) et une hélice α (D), et possède une activité lipase. La région entre le feuillet β (A) et l'hélice α (A) comprend n'importe lesquels des résidus acides aminés montrés sous les No 1 à 125 de la table I, et/ou la région entre le feuillet β (B) et l'hélice α (D) comprend n'importe lesquels des résidus acides aminés montrés sous les No 1 à 150 de la table II.
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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021020458A1 (fr) * 2019-08-01 2021-02-04 天野エンザイム株式会社 Nouvelle lipase et son utilisation
CN115279908A (zh) * 2020-03-13 2022-11-01 天野酶制品株式会社 具有对l-薄荷醇的酯化活性和/或对l-薄荷醇酯的水解活性的多肽
WO2023203080A1 (fr) 2022-04-20 2023-10-26 Novozymes A/S Procede de production d'acides gras libres

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995030744A2 (fr) * 1994-05-04 1995-11-16 Genencor International Inc. Lipases a resistance aux tensioactifs amelioree
WO2000032758A1 (fr) * 1998-11-27 2000-06-08 Novozymes A/S Variants d'enzyme lipolytique
WO2003035878A2 (fr) * 2001-10-22 2003-05-01 Basf Aktiengesellschaft Variantes de lipase

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995030744A2 (fr) * 1994-05-04 1995-11-16 Genencor International Inc. Lipases a resistance aux tensioactifs amelioree
WO2000032758A1 (fr) * 1998-11-27 2000-06-08 Novozymes A/S Variants d'enzyme lipolytique
WO2003035878A2 (fr) * 2001-10-22 2003-05-01 Basf Aktiengesellschaft Variantes de lipase

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
KIM, K.K. ET AL.: "The crystal structure of a triacylglcerol lipase from Pseudomonas cepacia reveals a highly open conformation in the absence of a bound inhibitor", STRUCTURE, vol. 5, 15 February 1997 (1997-02-15), pages 173 - 185, XP055459223, ISSN: 0969-2126 *
KOGA, Y. ET AL.: "In vitro construction and screening of a Burkholderia cepacia lipase library using single-molecular PCR and cell-free protein synthesis", JOURNAL OF BIOSCIENCE AND BIOENGINEERING, vol. 94, no. 1, 2002, pages 84 - 86, XP008159635, ISSN: 1389-1723 *
YANG, J. ET AL.: "In vitro analysis of roles of a disulfide bridge and calcium binding site in activation of Pseudomonas sp. strain KWI-56 lipase", JOURNAL OF BACTERIOLOGY, vol. 182, no. 2, January 2000 (2000-01-01), pages 295 - 302, XP001085342, ISSN: 0021-9193 *
YEDAVALLI, P. ET AL.: "Engineering the loops in a lipase for stability in DMSO", PROTEIN ENGINEERING, DESIGN & SELECTION, vol. 26, no. 4, 2013, pages 317 - 324, XP055459217, ISSN: 1741-0126 *

Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021020458A1 (fr) * 2019-08-01 2021-02-04 天野エンザイム株式会社 Nouvelle lipase et son utilisation
JP6846577B1 (ja) * 2019-08-01 2021-03-24 天野エンザイム株式会社 新規リパーゼ及びその用途
CN112639091A (zh) * 2019-08-01 2021-04-09 天野酶制品株式会社 新型脂肪酶和其用途
JP2021090440A (ja) * 2019-08-01 2021-06-17 天野エンザイム株式会社 新規リパーゼ及びその用途
JP7148659B2 (ja) 2019-08-01 2022-10-05 天野エンザイム株式会社 新規リパーゼ及びその用途
JP2022180554A (ja) * 2019-08-01 2022-12-06 天野エンザイム株式会社 新規リパーゼ及びその用途
US11718837B2 (en) 2019-08-01 2023-08-08 Amano Enzyme Inc. Lipase and uses of the same
JP7358592B2 (ja) 2019-08-01 2023-10-10 天野エンザイム株式会社 新規リパーゼ及びその用途
CN115279908A (zh) * 2020-03-13 2022-11-01 天野酶制品株式会社 具有对l-薄荷醇的酯化活性和/或对l-薄荷醇酯的水解活性的多肽
EP4119673A4 (fr) * 2020-03-13 2024-05-29 Amano Enzyme Inc. Polypeptide presentant une action d'estherification sur l-menthol et/ou une action d'hydrolyse sur un ester de l-menthol
WO2023203080A1 (fr) 2022-04-20 2023-10-26 Novozymes A/S Procede de production d'acides gras libres

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