WO2018021324A1 - Polypeptide having lipase activity with excellent stability - Google Patents

Polypeptide having lipase activity with excellent stability Download PDF

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Publication number
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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French (fr)
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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Priority to JP2018529920A priority Critical patent/JP7063807B2/en
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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).

Abstract

The purpose of the present invention is to provide a polypeptide having lipase activity with improved stability (heat, pH, solvent). The polypeptide at least includes, from the N-terminal side, β sheet (A), α helix (A), α helix (B), α helix (C), β sheet (B), and α helix (D), and has a lipase activity. The region between the β sheet (A) and the α helix (A) includes any of the amino acid residues shown in No. 1 - 125 of table I, and/or the region between the β sheet (B) and the α helix (D) includes any of the amino acid residues shown in No. 1 - 150 of table II.

Description

[規則37.2に基づきISAが決定した発明の名称]　安定性に優れたリパーゼ活性を有するポリペプチド[Name of invention determined by ISA based on Rule 37.2] Polypeptide having excellent lipase activity
　本発明は、リパーゼ活性を有するポリペプチドに関する。具体的には、安定性に優れたリパーゼ活性を有するポリペプチド、そのポリペプチドをコードするＤＮＡ、組換えベクター、形質転換体、組成物、酵素剤、ポリペプチドの製造方法、そのポリペプチドを用いた油脂処理方法、排水処理方法、医薬中間体の製造方法、及びファインケミカル素材の製造方法に関する。 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.
　バークホルデリア・セパシア（Ｂｕｒｋｈｏｌｄｅｒｉａ．ｃｅｐａｃｉａ）由来のリパーゼは、医薬中間体製造など幅広く商業利用されている酵素の一つである。しかしながら、溶媒存在下や高温条件下など酵素の使用条件によっては酵素の触媒活性が失われてしまうため、使用用途が限定されることが問題点として挙げられる。 A lipase derived from Burkholderia cepacia is one of the enzymes that are widely used commercially such as pharmaceutical intermediate production. However, since 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.
　酵素の安定性（熱、ｐＨ、溶媒など）を向上させる方法として、蛋白質工学的手法が用いられている。蛋白質工学を用いた酵素の安定性向上の具体的な手法として、ループ領域への変異導入がこれまでに報告されており、例えば、非特許文献１では、枯草菌由来α－アミラーゼについて表面ループのヒンジ領域になる７残基に変異を導入することにより、標的残基のうち５残基の変異でデンプン分解活性が増大し、かつ安定な好冷性酵素を得られたことが報告されている。 Protein engineering techniques are used as methods for improving enzyme stability (heat, pH, solvent, etc.). As a specific technique for improving the stability of an enzyme using protein engineering, mutation introduction into a loop region has been reported so far. For example, in Non-Patent Document 1, a surface loop of Bacillus subtilis-derived α-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. .
　また例えば、非特許文献２では、動的表面ループを除去することで、ホスファチジルイノシトール合成型ストレプトマイセス（Ｓｔｒｅｐｔｍｙｃｅｓ）属由来ホスホリパーゼＤの熱安定性を向上し得たことが報告されている。 For example, Non-Patent Document 2 reports that the thermal stability of phosphatidylinositol-synthesizing Streptomyces-derived phospholipase D could be improved by removing dynamic surface loops.
　しかしながら、バークホルデリア（Ｂｕｒｋｈｏｌｄｅｒｉａ）属、シュードモナス（Ｐｓｅｕｄｏｍｏｎａｓ）属、及び類縁菌由来のリパーゼに関しては当該手法を用いた報告は未だされていない。ループ領域への変異導入は、１アミノ酸置換やアミノ酸挿入、及びアミノ酸の欠損によりなされるが、変異導入点の選定など容易に実施できるものではない。 However, no report has been made on the lipases derived from the genus Burkholderia, the genus Pseudomonas, and related bacteria using this technique. Mutation is introduced into the loop region by single amino acid substitution, amino acid insertion, and amino acid deletion, but it cannot be easily performed such as selecting a mutation introduction point.
　本発明は、前記現状に鑑みて、従来と比べて、安定性が向上したリパーゼ活性を有するポリペプチドを提供することを目的とする。 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.
　本発明者は、上記の課題に鑑み鋭意研究を重ねた結果、バークホルデリア・セパシア（Ｂｕｒｋｈｏｌｄｅｒｉａ　ｃｅｐａｃｉａ）由来のリパーゼ活性を有するポリペプチドにおいて、特定のループ領域の３つのアミノ酸残基を特定のアミノ酸残基に置換することにより、リパーゼ活性の安定性、特に熱安定性が格段に向上することを見出した。本発明は、これらの知見に基づいて更に検討を重ねることにより完成したものである。 As a result of intensive studies in view of the above problems, the present inventor has determined that three amino acid residues in a specific loop region are specific amino acids in a polypeptide having lipase activity derived from Burkholderia cepacia. It has been found that substitution with a residue significantly improves the stability of lipase activity, particularly thermal stability. The present invention has been completed by further studies based on these findings.
　すなわち、本発明は、下記に掲げる態様の発明を提供する。
項１．　少なくとも、Ｎ末端側から、βシート（Ａ）、αへリックス（Ａ）、αへリックス（Ｂ）、αへリックス（Ｃ）、βシート（Ｂ）、及びαへリックス（Ｄ）を備え、リパーゼ活性を有しており、
　前記βシート（Ａ）は、そのＮ末端側のアミノ酸残基がポリペプチドのＮ末端側から７～１３位に存在し、アミノ酸残基数が２～６で構成されており、
　前記αへリックス（Ａ）は、そのＮ末端側のアミノ酸残基が前記βシート（Ａ）のＣ末端側のアミノ酸残基を０位とした場合に１５～２５位に存在し、アミノ酸残基数が３～９で構成されており、
　前記αへリックス（Ｂ）は、そのＮ末端側のアミノ酸残基が前記αへリックス（Ａ）のＣ末端側のアミノ酸残基を０位とした場合に７０～９２位に存在し、アミノ酸残基数が４～１５で構成されており
　前記αへリックス（Ｃ）は、そのＮ末端側のアミノ酸残基が前記αへリックス（Ｂ）のＣ末端側のアミノ酸残基を０位とした場合に３～１１位に存在し、アミノ酸残基数が１１～２０で構成されており、
　前記βシート（Ｂ）は、そのＮ末端側のアミノ酸残基が前記αへリックス（Ｃ）のＣ末端側のアミノ酸残基を０位とした場合に６５～８１位に存在し、アミノ酸残基数が２～８で構成されており、
　前記αへリックス（Ｄ）は、そのＮ末端側のアミノ酸残基が前記αへリックス（Ｃ）のＣ末端側のアミノ酸残基を０位とした場合に６～１５位に存在し、アミノ酸残基数が２～２４で構成されている、ポリペプチドであって
　前記βシート（Ａ）とαへリックス（Ａ）の間の領域が、表ＩのＮｏ．１～１２５に示すいずれかのアミノ酸残基を含んでおり、及び／又は、
　前記βシート（Ｂ）とαへリックス（Ｄ）の間の領域が、表ＩＩのＮｏ．１～１５０に示すいずれかのアミノ酸残基を含んでいる、
ポリペプチド。
Figure JPOXMLDOC01-appb-T000003
Figure JPOXMLDOC01-appb-T000004
 項２．　更に、αへリックス（Ｅ）、βシート（Ｃ）、βシート（Ｄ）、αへリックス（Ｇ）、及びβシート（Ｅ）を備え、
　前記αへリックス（Ｅ）は、そのＮ末端側のアミノ酸残基が前記αへリックス（Ａ）のＣ末端側のアミノ酸残基を０位とした場合に１７～２８位に存在し、アミノ酸残基数が１３～１９で構成されており、
　前記βシート（Ｃ）は、そのＮ末端側のアミノ酸残基が前記αへリックス（Ｅ）のＣ末端側のアミノ酸残基を０位とした場合に２～８位に存在し、アミノ酸残基数が４～８で構成されており、
　前記βシート（Ｄ）は、そのＮ末端側のアミノ酸残基が前記βシート（Ｃ）のＣ末端側のアミノ酸残基を０位とした場合に１４～２２位に存在し、アミノ酸残基数が３～１１で構成されており、
　前記αへリックス（Ｇ）は、そのＮ末端側のアミノ酸残基が前記αへリックス（Ｃ）のＣ末端側のアミノ酸残基を０位とした場合に３～１３位に存在し、アミノ酸残基数が４～１３で構成されており、
　前記βシート（Ｅ）は、そのＮ末端側のアミノ酸残基が前記αへリックス（Ｇ）のＣ末端側のアミノ酸残基を０位とした場合に２４～３６位に存在し、アミノ酸残基数が２～６で構成されている、
項１に記載のポリペプチド。
項３．　更に、αへリックス（Ｆ）、αへリックス（Ｈ）、及びβシート（Ｆ）を備え、
　前記αへリックス（Ｆ）は、そのＮ末端側のアミノ酸残基が前記βシート（Ｃ）のＣ末端側のアミノ酸残基を０位とした場合に１～５位に存在し、アミノ酸残基数が３～１５で構成されており、
　前記αへリックス（Ｈ）は、そのＮ末端側のアミノ酸残基が前記αへリックス（Ｇ）のＣ末端側のアミノ酸残基を０位とした場合に１～１０位に存在し、アミノ酸残基数が２～１３で構成されており、
　前記βシート（Ｆ）は、そのＮ末端側のアミノ酸残基が前記βシート（Ｅ）のＣ末端側のアミノ酸残基を０位とした場合に１～５位に存在し、アミノ酸残基数が１５～２３で構成されている、
項２に記載のポリペプチド。
項４．　以下の（１）から（１２）のいずれかに示すポリペプチド。
（１）配列番号１に示すアミノ酸配列において、２５～２７位のアミノ酸残基が表ＩのＮｏ．１～１２５に示すいずれかのアミノ酸残基に置換されている、及び／又は２３３～２３５位のアミノ酸残基が表ＩＩのＮｏ．１～１５０に示すいずれかのアミノ酸残基に置換されているアミノ酸配列からなるポリペプチド、
（２）配列番号２に示すアミノ酸配列において、２４～２６位のアミノ酸残基が表ＩのＮｏ．１～１２５に示すいずれかのアミノ酸残基に置換されている、及び／又は２３２～２３４位のアミノ酸残基が表ＩＩのＮｏ．１～１５０に示すいずれかのアミノ酸残基に置換されているアミノ酸配列からなるポリペプチド、
（３）配列番号３に示すアミノ酸配列において、２５～２７位のアミノ酸残基が表ＩのＮｏ．１～１２５に示すいずれかのアミノ酸残基に置換されている、及び／又は２３３～２３５位のアミノ酸残基が表ＩＩのＮｏ．１～１５０に示すいずれかのアミノ酸残基に置換されているアミノ酸配列からなるポリペプチド、
（４）配列番号４に示すアミノ酸配列において、２５～２７位のアミノ酸残基が表ＩのＮｏ．１～１２５に示すいずれかのアミノ酸残基に置換されている、及び／又は２３３～２３５位のアミノ酸残基が表ＩＩのＮｏ．１～１５０に示すいずれかのアミノ酸残基に置換されているアミノ酸配列からなるポリペプチド、
（５）配列番号１に示すアミノ酸配列における２５～２７位のアミノ酸残基が表ＩのＮｏ．１～１２５に示すいずれかのアミノ酸残基に置換されている、及び／又は２３３～２３５位のアミノ酸残基が表ＩＩのＮｏ．１～１５０に示すいずれかのアミノ酸残基に置換されているアミノ酸配列において、前記置換が導入されたアミノ酸残基以外の１個又は数個のアミノ酸残基が置換、付加、挿入又は欠失されてなり、且つ、リパーゼ活性を有し、配列番号１に示すアミノ酸配列からなるポリペプチドに比して熱安定性が向上しているポリペプチド、
（６）配列番号２に示すアミノ酸配列における２４～２６位のアミノ酸残基が表ＩのＮｏ．１～１２５に示すいずれかのアミノ酸残基に置換されている、及び／又は２３２～２３４位のアミノ酸残基が表ＩＩのＮｏ．１～１５０に示すいずれかのアミノ酸残基に置換されているアミノ酸配列において、前記置換が導入されたアミノ酸残基以外の１個又は数個のアミノ酸残基が置換、付加、挿入又は欠失されてなり、且つ、リパーゼ活性を有し、配列番号２に示すアミノ酸配列からなるポリペプチドに比して熱安定性が向上しているポリペプチド、
（７）配列番号３に示すアミノ酸配列における２５～２７位のアミノ酸残基が表ＩのＮｏ．１～１２５に示すいずれかのアミノ酸残基に置換されている、及び／又は２３３～２３５位のアミノ酸残基が表ＩＩのＮｏ．１～１５０に示すいずれかのアミノ酸残基に置換されているアミノ酸配列において、前記置換が導入されたアミノ酸残基以外の１個又は数個のアミノ酸残基が置換、付加、挿入又は欠失されてなり、且つ、リパーゼ活性を有し、配列番号３に示すアミノ酸配列からなるポリペプチドに比して熱安定性が向上しているポリペプチド、
（８）配列番号４に示すアミノ酸配列における２５～２７位のアミノ酸残基が表ＩのＮｏ．１～１２５に示すいずれかのアミノ酸残基に置換されている、及び／又は２３３～２３５位のアミノ酸残基が表ＩＩのＮｏ．１～１５０に示すいずれかのアミノ酸残基に置換されているアミノ酸配列において、前記置換が導入されたアミノ酸残基以外の１個又は数個のアミノ酸残基が置換、付加、挿入又は欠失されてなり、且つ、リパーゼ活性を有し、配列番号４に示すアミノ酸配列からなるポリペプチドに比して熱安定性が向上しているポリペプチド、
（９）配列番号１に示すアミノ酸配列における２５～２７位のアミノ酸残基が表ＩのＮｏ．１～１２５に示すいずれかのアミノ酸残基に置換されている、及び／又は２３３～２３５位のアミノ酸残基が表ＩＩのＮｏ．１～１５０に示すいずれかのアミノ酸残基に置換されているアミノ酸配列において、配列番号１に示すアミノ酸配列に対する前記置換が導入されたアミノ酸残基を除いた配列同一性が８０％以上であり、且つ、リパーゼ活性を有し、配列番号１に示すアミノ酸配列からなるポリペプチドに比して熱安定性が向上しているポリペプチド、
（１０）配列番号２に示すアミノ酸配列における２４～２６位のアミノ酸残基が表ＩのＮｏ．１～１２５に示すいずれかのアミノ酸残基に置換されている、及び／又は２３２～２３４位のアミノ酸残基が表ＩＩのＮｏ．１～１５０に示すいずれかのアミノ酸残基に置換されているアミノ酸配列において、配列番号２に示すアミノ酸配列に対する前記置換が導入されたアミノ酸残基を除いた配列同一性が８０％以上であり、且つ、リパーゼ活性を有し、配列番号２に示すアミノ酸配列からなるポリペプチドに比して熱安定性が向上しているポリペプチド、
（１１）配列番号３に示すアミノ酸配列における２５～２７位のアミノ酸残基が表ＩのＮｏ．１～１２５に示すいずれかのアミノ酸残基に置換されている、及び／又は２３３～２３５位のアミノ酸残基が表ＩＩのＮｏ．１～１５０に示すいずれかのアミノ酸残基に置換されているアミノ酸配列において、配列番号３に示すアミノ酸配列に対する前記置換が導入されたアミノ酸残基を除いた配列同一性が８０％以上であり、且つ、リパーゼ活性を有し、配列番号３に示すアミノ酸配列からなるポリペプチドに比して熱安定性が向上しているポリペプチド、
（１２）配列番号４に示すアミノ酸配列における２５～２７位のアミノ酸残基が表ＩのＮｏ．１～１２５に示すいずれかのアミノ酸残基に置換されている、及び／又は２３３～２３５位のアミノ酸残基が表ＩＩのＮｏ．１～１５０に示すいずれかのアミノ酸残基に置換されているアミノ酸配列において、配列番号４に示すアミノ酸配列に対する前記置換が導入されたアミノ酸残基を除いた配列同一性が８０％以上であり、且つ、リパーゼ活性を有し、配列番号４に示すアミノ酸配列からなるポリペプチドに比して熱安定性が向上しているポリペプチド。
項５．　項１～４に記載のポリペプチドをコードしているＤＮＡ。
項６．　項５に記載のＤＮＡを含む組換えベクター。
項７．　項６に記載の組換えベクターにより宿主を形質転換して得られる形質転換体。
項８．　項７に記載の形質転換体を培養する工程を含む、項１～５のいずれかに記載のポリペプチドの製造方法。
項９．　項１～４のいずれかに記載のポリペプチドを含む組成物。
項１０．　項１～４のいずれかに記載のポリペプチド、又は項９に記載の組成物を含む酵素剤。
項１１．　項１～４のいずれかに記載のポリペプチド、項９に記載の組成物、又は項１０に記載の酵素剤を、油脂に作用させる、油脂処理方法。
項１２．
　項１～４のいずれかに記載のポリペプチド、項９に記載の組成物、又は項１０に記載の酵素剤を排水に作用させる、排水処理方法。
項１３．　項１～４のいずれかに記載のポリペプチド、項９に記載の組成物、又は項１０に記載の酵素剤を医薬中間体原料に作用させる、医薬中間体の製造方法。
項１４．　項１～４のいずれかに記載のポリペプチド、項９に記載の組成物、又は項１０に記載の酵素剤をファインケミカル素材原料に作用させる、ファインケミカル素材の製造方法。 That is, this invention provides the invention of the aspect hung up below.
Item 1. At least, from the N-terminal side, β sheet (A), α helix (A), α helix (B), α helix (C), β sheet (B), and α helix (D), Has lipase activity,
In the β sheet (A), 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 α-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 β-sheet (A). The number consists of 3-9,
The α 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 α helix (A). When the α-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 α-helix (B). 3 to 11 and is composed of 11 to 20 amino acid residues,
The β 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 α helix (C). The number consists of 2-8,
The α 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 α helix (C). A polypeptide having a radix number of 2 to 24, wherein the region between the β sheet (A) and the α helix (A) is represented by No. 1 in Table I. Contains any amino acid residue shown in 1-125, and / or
The region between the β sheet (B) and the α helix (D) is shown in Table II. Any one of the amino acid residues shown in 1-150,
Polypeptide.
Figure JPOXMLDOC01-appb-T000003
Figure JPOXMLDOC01-appb-T000004
 Item 2. Furthermore, an α helix (E), a β sheet (C), a β sheet (D), an α helix (G), and a β sheet (E) are provided,
The α 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 α helix (A). The radix consists of 13-19,
The β 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 α helix (E). The number consists of 4-8,
The β 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 β sheet (C). Consists of 3 to 11,
The α-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 α-helix (C). The radix is composed of 4-13,
The β 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 α helix (G). The number consists of 2-6,
Item 2. The polypeptide according to Item 1.
Item 3. Furthermore, an α helix (F), an α helix (H), and a β sheet (F) are provided,
The α-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 β-sheet (C), and the amino acid residue The number consists of 3 to 15,
The α helix (H) is present at the 1 to 10 position when the amino acid residue on the N-terminal side of the α helix (G) is 0 position on the C-terminal side of the α helix (G). The radix is composed of 2 to 13,
The β 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 β-sheet (E). Consists of 15-23,
Item 3. The polypeptide according to Item 2.
Item 4. The polypeptide shown in any one of (1) to (12) below.
(1) In the amino acid sequence shown in SEQ ID NO: 1, the amino acid residues at positions 25 to 27 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. A polypeptide comprising an amino acid sequence substituted with any one of the amino acid residues shown in 1-150,
(2) In the amino acid sequence shown in SEQ ID NO: 2, the amino acid residues at positions 24 to 26 are Nos. Nos. 1 to 125 are substituted with amino acid residues and / or amino acid residues at positions 232 to 234 A polypeptide comprising an amino acid sequence substituted with any one of the amino acid residues shown in 1-150,
(3) In the amino acid sequence shown in SEQ ID NO: 3, the amino acid residues at positions 25 to 27 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. A polypeptide comprising an amino acid sequence substituted with any one of the amino acid residues shown in 1-150,
(4) In the amino acid sequence shown in SEQ ID NO: 4, the amino acid residues at positions 25 to 27 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. A polypeptide comprising an amino acid sequence substituted with any one of the amino acid residues shown in 1-150,
(5) 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. 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. A polypeptide having a lipase activity and having improved thermal stability compared to a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1,
(6) 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. A polypeptide that has lipase activity and has improved thermal stability compared to a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2,
(7) 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. 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. A polypeptide having a lipase activity and having improved thermal stability compared to a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 3,
(8) 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. 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. A polypeptide having a lipase activity and having improved thermal stability compared to a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 4,
(9) 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. In the amino acid sequence substituted with any one of the amino acid residues shown in 1-150, 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, And a polypeptide having lipase activity and having improved thermal stability compared to a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1,
(10) 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, 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, And a polypeptide having lipase activity and improved thermal stability compared to a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2.
(11) 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. In the amino acid sequence substituted with any one of the amino acid residues shown in 1-150, 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, And a polypeptide having lipase activity and improved thermal stability compared to a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 3.
(12) 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. In the amino acid sequence substituted with any one of the amino acid residues shown in 1-150, 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. 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. Item 7. A transformant obtained by transforming a host with the recombinant vector according to Item 6.
Item 8. 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.
Item 10. Item 10. 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. A method for treating fats and oils, 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 fats and oils.
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.
　本発明によれば、ｐＨ、熱、溶媒等の安定性、特に熱安定性が向上したリパーゼ活性を有するポリペプチドを取得することができる。従って、本発明は、安定性に優れたリパーゼを必要とする用途等に利用することができる。また、本発明のポリペプチドは、高い安定性が求められる用途に好適に用いることができ、排水処理、医薬中間体製造、ファインケミカル素材製造、機能代替油脂製造、洗剤、食品加工、キラル合成、バイオエタノール等の分野で好適に利用することができる。 According to 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. In addition, 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.
バークホルデリア・セパシア由来リパーゼの立体構造を示す図である。It is a figure which shows the three-dimensional structure of lipase derived from Burkholderia cepacia. シュードモナス・グルマエ由来リパーゼの立体構造を示す図である。It is a figure which shows the three-dimensional structure of a lipase derived from Pseudomonas grumae. シュードモナス・フルオレセンス由来リパーゼの立体構造を示す図である。It is a figure which shows the three-dimensional structure of Pseudomonas fluorescens origin lipase. シュードモナス・エルギノーサ由来リパーゼの立体構造を示す図である。It is a figure which shows the three-dimensional structure of Pseudomonas aeruginosa origin lipase. バークホルデリア・セパシア由来リパーゼの立体構造とシュードモナス・グルマエ由来リパーゼの立体構造を重ね合わせた図である。黒がバークホルデリア・セパシア由来リパーゼで、灰色がシュードモナス・グルマエ由来リパーゼを示す。It is the figure which piled up the three-dimensional structure of the lipase derived from Burkholderia cepacia and the three-dimensional structure of Pseudomonas grumae lipase. Black indicates a lipase derived from Burkholderia cepacia, and gray indicates a lipase derived from Pseudomonas grumae. バークホルデリア・セパシア由来リパーゼの立体構造とシュードモナス・フルオレセンス由来リパーゼの立体構造を重ね合わせた図である。黒がバークホルデリア・セパシア由来リパーゼで、灰色がシュードモナス・フルオレセンス由来リパーゼを示す。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. バークホルデリア・セパシア由来リパーゼの立体構造とシュードモナス・エルギノーサ由来リパーゼの立体構造を重ね合わせた図である。黒がバークホルデリア・セパシア由来リパーゼで、灰色がシュードモナス・エルギノーサ由来リパーゼを示す。It is the figure which superimposed the three-dimensional structure of the lipase derived from Burkholderia cepacia and the three-dimensional structure of Pseudomonas aeruginosa lipase. Black indicates lipase derived from Burkholderia cepacia, and gray indicates lipase derived from Pseudomonas aeruginosa. 三重変異体（Ｐ２３３Ｇ/Ｌ２３４Ｅ/Ｖ２３５Ｍ）のｐＨ安定性の評価結果の図である。It is a figure of the evaluation result of pH stability of a triple mutant (P233G / L234E / V235M). 三重変異体（Ｐ２３３Ｇ/Ｌ２３４Ｅ/Ｖ２３５Ｍ）の有機溶媒安定性の評価結果の図である。It is a figure of the evaluation result of the organic-solvent stability of a triple mutant (P233G / L234E / V235M).
　以下、本発明を詳細に説明する。なお、配列表以外では、アミノ酸配列における２０種類のアミノ酸残基は、一文字略記で表現している。即ち、グリシン（Ｇｌｙ）はＧ、アラニン（Ａｌａ）はＡ、バリン（Ｖａｌ）はＶ、ロイシン（Ｌｅｕ）はＬ、イソロイシン（Ｉｌｅ）はＩ、フェニルアラニン（Ｐｈｅ）はＦ、チロシン（Ｔｙｒ）はＹ、トリプトファン（Ｔｒｐ）はＷ、セリン（Ｓｅｒ）はＳ、スレオニン（Ｔｈｒ）はＴ、システイン（Ｃｙｓ）はＣ、メチオニン（Ｍｅｔ）はＭ、アスパラギン酸（Ａｓｐ）はＤ、グルタミン酸（Ｇｌｕ）はＥ、アスパラギン（Ａｓｎ）はＮ、グルタミン（Ｇｌｎ）はＱ、リジン（Ｌｙｓ）はＫ、アルギニン（Ａｒｇ）はＲ、ヒスチジン（Ｈｉｓ）はＨ、プロリン（Ｐｒｏ）はＰである。 Hereinafter, the present invention will be described in detail. In addition to the sequence listing, the 20 types of amino acid residues in the amino acid sequence are expressed by single letter abbreviations. That is, glycine (Gly) is G, alanine (Ala) is A, valine (Val) is V, leucine (Leu) is L, isoleucine (Ile) is I, phenylalanine (Phe) is F, tyrosine (Tyr) is Y , Tryptophan (Trp) is W, serine (Ser) is S, threonine (Thr) is T, cysteine (Cys) is C, methionine (Met) is M, aspartic acid (Asp) is D, glutamic acid (Glu) is E Asparagine (Asn) is N, glutamine (Gln) is Q, lysine (Lys) is K, arginine (Arg) is R, histidine (His) is H, and proline (Pro) is P.
　本明細書における「Ｆ４５Ｖ」等の表現は、アミノ酸置換の表記法である。例えば、「Ｆ４５Ｖ」とは、特定のアミノ酸配列におけるＮ末端側から４５番目のアミノ酸Ｆが、アミノ酸Ｖに置換されていることを意味する。また、本明細書における「Ｖ２７２Ａ／Ｈ２７３Ｇ」等の表現は、多重変異を意味している。例えば、「Ｖ２７２Ａ／Ｈ２７３Ｇ」とは、Ｖ２７２Ａ及びＨ２７３Ｇのアミノ酸置換を同時に導入していることを意味する。 In the present specification, expressions such as “F45V” are amino acid substitution notations. For example, “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. In addition, expressions such as “V272A / H273G” in the present specification mean multiple mutations. For example, “V272A / H273G” means that amino acid substitutions of V272A and H273G are introduced simultaneously.
　また、本明細書において、「非極性アミノ酸」には、アラニン、バリン、ロイシン、イソロイシン、プロリン、メチオニン、フェニルアラニン、及びトリプチファンが含まれる。また、「非電荷アミノ酸」には、グリシン、セリン、トレオニン、システイン、チロシン、アスパラギン、及びグルタミンが含まれる。また、「酸性アミノ酸」には、アスパラギン酸及びグルタミン酸が含まれる。また、「塩基性アミノ酸」には、リジン、アルギニン、及びヒスチジンが含まれる。 In the present specification, “nonpolar amino acids” include alanine, valine, leucine, isoleucine, proline, methionine, phenylalanine, and tryptophan. In addition, “uncharged amino acid” includes glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. The “acidic amino acid” includes aspartic acid and glutamic acid. The “basic amino acid” includes lysine, arginine, and histidine.
　通常は翻訳開始点に対応するメチオニンをＮ末端の１番目の位置とするが、プロペプチド、プレペプチド、シグナルペプチドを含んだ状態で翻訳されて、これらのペプチドが切断された成熟体では、これらのペプチドが切断された後の成熟体のＮ末端を１番目の位置とする。例えば、本明細書における配列番号１は配列番号５から４４アミノ酸が切断された成熟体の配列であり、Ｎ末端アミノ酸残基はアラニン（Ａ）となっている。配列番号２は配列番号６から４０アミノ酸が切断された成熟体の配列であり、Ｎ末端アミノ酸残基はアスパラギン酸（Ｄ）となっている。配列番号７は配列番号Ｃから４４アミノ酸が切断された成熟体の配列であり、Ｎ末端アミノ酸残基はアラニン（Ａ）となっている。配列番号４は配列番号８から２６アミノ酸が切断された成熟体の配列であり、Ｎ末端アミノ酸残基はセリン（Ｓ）となっている。 Normally, 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. For example, 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).
　また、特に明示しない限り、ペプチドおよびタンパク質のアミノ酸残基の配列は、左端から右端にかけてＮ末端からＣ末端となるように表される。 Unless otherwise specified, the 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.
　また、本明細書において、「置換」とは、人為的にアミノ酸残基の置換を導入した場合のみならず、自然にアミノ酸残基の置換が導入された場合、すなわち、もともとアミノ酸残基が相違していた場合も含まれる。本明細書においては、アミノ酸残基の置換は、人為的な置換であってもよく、自然な置換であってもよいが、人為的な置換が好ましい。 In this specification, the term “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. In the present specification, substitution of amino acid residues may be artificial substitution or natural substitution, but artificial substitution is preferred.
ポリペプチド
　本発明のポリペプチドは、少なくとも、Ｎ末端側から、βシート（Ａ）、αへリックス（Ａ）、αへリックス（Ｂ）、αへリックス（Ｃ）、βシート（Ｂ）、及びαへリックス（Ｄ）を備え、リパーゼ活性を有しており、
　前記βシート（Ａ）は、そのＮ末端側のアミノ酸残基がポリペプチドのＮ末端側から７～１３位に存在し、アミノ酸残基数が２～６で構成されており、
　前記αへリックス（Ａ）は、そのＮ末端側のアミノ酸残基が前記βシート（Ａ）のＣ末端側のアミノ酸残基を０位とした場合に１５～２５位に存在し、アミノ酸残基数が３～９で構成されており、
　前記αへリックス（Ｂ）は、そのＮ末端側のアミノ酸残基が前記αへリックス（Ａ）のＣ末端側のアミノ酸残基を０位とした場合に７０～９２位に存在し、アミノ酸残基数が４～１５で構成されており、
　前記αへリックス（Ｃ）は、そのＮ末端側のアミノ酸残基が前記αへリックス（Ｂ）のＣ末端側のアミノ酸残基を０位とした場合に３～１１位に存在し、アミノ酸残基数が１１～２０で構成されており、
　前記βシート（Ｂ）は、そのＮ末端側のアミノ酸残基が前記αへリックス（Ｃ）のＣ末端側のアミノ酸残基を０位とした場合に６５～８１位に存在し、アミノ酸残基数が２～８で構成されており、
　前記αへリックス（Ｄ）は、そのＮ末端側のアミノ酸残基が前記αへリックス（Ｃ）のＣ末端側のアミノ酸残基を０位とした場合に６～１５位に存在し、アミノ酸残基数が２～２４で構成されている、ポリペプチドであって
　前記βシート（Ａ）とαへリックス（Ａ）の間の領域が、表ＩのＮｏ．１～１２５に示すいずれかのアミノ酸残基を含んでおり、及び／又は、
　前記βシート（Ｂ）とαへリックス（Ｄ）の間の領域が、表ＩＩのＮｏ．１～１５０に示すいずれかのアミノ酸残基を含んでいる、ポリペプチドである。 Polypeptide The polypeptide of the present invention comprises, at least from the N-terminal side, a β sheet (A), an α helix (A), an α helix (B), an α helix (C), a β sheet (B), and It has α helix (D) and has lipase activity,
In the β sheet (A), 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 α-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 β-sheet (A). The number consists of 3-9,
The α 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 α helix (A). The radix is composed of 4-15,
The α 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 α-helix (B). The radix is composed of 11-20,
The β 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 α helix (C). The number consists of 2-8,
The α 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 α helix (C). A polypeptide having a radix number of 2 to 24, wherein the region between the β sheet (A) and the α helix (A) is represented by No. 1 in Table I. Contains any amino acid residue shown in 1-125, and / or
The region between the β sheet (B) and the α helix (D) is shown in Table II. A polypeptide comprising any one of the amino acid residues shown in 1-150.
Figure JPOXMLDOC01-appb-T000005
Figure JPOXMLDOC01-appb-T000005
Figure JPOXMLDOC01-appb-T000006
Figure JPOXMLDOC01-appb-T000006
基本構造
　本発明は、少なくとも、Ｎ末端側から、βシート（Ａ）、αへリックス（Ａ）、αへリックス（Ｂ）、αへリックス（Ｃ）、βシート（Ｂ）、及びαへリックス（Ｄ）を順に備え、リパーゼ活性を有しており、
　前記βシート（Ａ）は、そのＮ末端側のアミノ酸残基がポリペプチドのＮ末端側から７～１３位に存在し、アミノ酸残基数が２～６で構成されており、
　前記αへリックス（Ａ）は、そのＮ末端側のアミノ酸残基が前記βシート（Ａ）のＣ末端側のアミノ酸残基を０位とした場合に１５～２５位に存在し、アミノ酸残基数が３～９で構成されており、
　前記αへリックス（Ｂ）は、そのＮ末端側のアミノ酸残基が前記αへリックス（Ａ）のＣ末端側のアミノ酸残基を０位とした場合に７０～９２位に存在し、アミノ酸残基数が４～１５で構成されており、
　前記αへリックス（Ｃ）は、そのＮ末端側のアミノ酸残基が前記αへリックス（Ｂ）のＣ末端側のアミノ酸残基を０位とした場合に３～１１位に存在し、アミノ酸残基数が１１～２０で構成されており、
　前記βシート（Ｂ）は、そのＮ末端側のアミノ酸残基が前記αへリックス（Ｃ）のＣ末端側のアミノ酸残基を０位とした場合に６５～８１位に存在し、アミノ酸残基数が２～８で構成されており、
　前記αへリックス（Ｄ）は、そのＮ末端側のアミノ酸残基が前αへリックス（Ｃ）のＣ末端側のアミノ酸残基を０位とした場合に６～１５位に存在し、アミノ酸残基数２～２４で構成されている、ポリペプチドである。 Basic Structure In the present invention, at least from the N-terminal side, β sheet (A), α helix (A), α helix (B), α helix (C), β sheet (B), and α helix (D) in order, having lipase activity,
In the β sheet (A), 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 α-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 β-sheet (A). The number consists of 3-9,
The α 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 α helix (A). The radix is composed of 4-15,
The α 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 α-helix (B). The radix is composed of 11-20,
The β 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 α helix (C). The number consists of 2-8,
The α-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 α-helix (C). A polypeptide composed of 2 to 24 bases.
　Ｎ末端からβシート（Ａ）間の領域、βシート（Ａ）とαへリックス（Ａ）間の領域、αへリックス（Ａ）とαへリックス（Ｂ）間の領域、αへリックス（Ｂ）とβシート（Ｂ）間の領域、βシート（Ｂ）とαへリックス（Ｄ）間の領域、及びαへリックス（Ｄ）からＣ末端までの領域には、それぞれ１又は複数のアミノ酸残基を含む。これらの各領域には、１又は複数のループ、シート、又はヘリックスを含んでいてもよい。 Region from N-terminal to β sheet (A), region between β sheet (A) and α helix (A), region between α helix (A) and α helix (B), α helix (B ) And β-sheet (B), β-sheet (B) and α-helix (D), and α-helix (D) to C-terminal region, respectively. Contains groups. Each of these regions may include one or more loops, sheets, or helices.
　なお、本発明において、ポリペプチドのＮ末端からβシート（Ａ）Ｎ末端側アミノ酸残基までの領域を領域（Ｉ）、βシート（Ａ）Ｃ末端側アミノ酸残基からαへリックス（Ａ）Ｎ末端側アミノ酸残基まで領域を領域（ＩＩ）、αへリックス（Ａ）Ｃ末端側アミノ酸残基からαへリックス（Ｂ）Ｎ末端側アミノ酸残基までの領域を領域（ＩＩＩ）、αへリックス（Ｂ）Ｃ末端側アミノ酸残基からαへリックス（Ｃ）Ｎ末端側アミノ酸残基までの領域を領域（ＩＶ）、αへリックス（Ｃ）Ｃ末端側アミノ酸残基からβシート（Ｂ）Ｎ末端側アミノ酸残基までの領域を領域（Ｖ）、βシート（Ｂ）Ｃ末端側アミノ酸残基からαへリックス（Ｄ）Ｎ末端側アミノ酸残基までの領域を領域（ＶＩ）、αへリックス（Ｄ）Ｃ末端側アミノ酸残基からポリペプチドのＣ末端までの領域を領域（ＶＩＩ）とする。 In the present invention, the region from the N-terminus of the polypeptide to the β-sheet (A) N-terminal amino acid residue is defined as region (I), and β-sheet (A) from the C-terminal amino acid residue to α-helix (A) Region to region (II) up to N-terminal amino acid residue, α helix (A) Region from C-terminal amino acid residue to α-helix (B) N-terminal amino acid residue to region (III), α Region (IV) from Helix (B) C-terminal side amino acid residue to α-helix (C) N-terminal side amino acid residue, α-helix (C) β-sheet (B) from C-terminal side amino acid residue The region from the N-terminal amino acid residue to the region (V), β sheet (B) from the C-terminal amino acid residue to the α helix (D) the region from the N-terminal amino acid residue to the region (VI), α Lix (D) Polypeptide from C-terminal amino acid residue A region up to the C-terminus of the region (VII).
　このようなポリペプチドは、分子量が３０～３３ｋＤａ程度のリパーゼで認められる構造である。 Such a polypeptide has a structure recognized by a lipase having a molecular weight of about 30 to 33 kDa.
　具体的には、このような構造を有するポリペプチドは、バークホルデリア・セパシア（Ｂｕｒｋｈｏｌｄｅｒｉａ　ｃｅｐａｃｉａ）、バークホルデリア・テリトリ（Ｂｕｒｋｈｏｌｄｅｒｉａ　ｔｅｒｒｉｔｏｒｉｉ）、バークホルデリア・セノセパシア（Ｂｕｒｋｈｏｌｄｅｒｉａ　ｃｅｎｏｃｅｐａｃｉａ）、バークホルデリア・アンビファリア（Ｂｕｒｋｈｏｌｄｅｒｉａ　ａｍｂｉｆａｒｉａ）、バークホルデリア・コンタミナンス（Ｂｕｒｋｈｏｌｄｅｒｉａ　ｃｏｎｔａｍｉｎａｎｓ）、バークホルデリア・ラタ（Ｂｕｒｋｈｏｌｄｅｒｉａ　ｌａｔａ）、バークホルデリア・セミナリス（Ｂｕｒｋｈｏｌｄｅｒｉａ　ｓｅｍｉｎａｌｉｓ）、バークホルデリア・アンセナ（Ｂｕｒｋｈｏｌｄｅｒｉａ　ａｎｔｈｉｎａ）等のバークホルデリア（Ｂｕｒｋｈｏｌｄｅｒｉａ）属；シュードモナス・グルマエ（Ｐｓｅｕｄｏｍｏｎａｓ　ｇｌｕｍａｅ）、シュードモナス・フルオレセンス（Ｐｓｅｕｄｏｍｏｎａｓ　ｆｌｕｏｒｅｓｃｅｎｃｅ）、シュードモナス・エルギノーサ（Ｐｓｅｕｄｏｍｏｎａｓ　ａｅｒｕｇｉｎｏｓａ）等のシュードモナス属；クロモバクテリウム・ヴィスコスム（Ｃｈｒｏｍｏｂａｃｔｅｒｉｕｍ ｖｉｓｃｏｓｕｍ）等のクロモバクテリウム属等が産生するリパーゼで認められている。 Specifically, 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 The genus Burkholderia; Pseudomonas glumae, Pseudomonas fluorescens, Pseudomonas ummo somomoscos, etc .; It is recognized as a lipase produced by bacteria.
　このような構造を有するポリペプチドとしては、具体的には、後述するタイプＩとタイプＩＩが挙げられる。タイプＩに含まれるポリペプチドとしては、例えば、バークホルデリア・セパシア由来リパーゼ、シュードモナス・グルマエ由来リパーゼ、シュードモナス・フルオレセンス由来リパーゼが挙げられる。これらの立体構造を図１～３に示す。また、タイプＩＩに含まれるポリペプチドとしては、例えば、シュードモナス・エルギノーサ由来リパーゼが挙げられる。シュードモナス・エルギノーサ由来のリパーゼの立体構造を図４に示す。なかでも、タイプＩのポリペプチドが好ましい。なお、ポリペプチドのタイプＩ、タイプＩＩについては、ＭＯＥ（Ｍｏｌｅｃｕｌａｒ Ｏｐｅｒａｔｉｎｇ Ｅｎｖｉｒｏｎｍｅｎｔ）（バージョンＭＯＥ２０１３）、ＰｙＭＯＬ、ＲａｓＭＯＬ、ＷｉｎＣｏｏｔ等のタンパク質の立体構造を解析するソフトウェアを用いて分析することにより、確認することができる。 Specific examples of the polypeptide having such a structure 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. In addition, 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.
　前記立体構造において、αへリックス、βシートは、前記ソフトウェアのＭＯＥを用いて規定することができる。ループもまた前記ソフトウェアのＭＯＥを用いて規定することができる。なお、本発明においては、前記ソフトウェアで規定されるループに限定されず、αへリックスとβシート以外の部分を全てループと定義する。 In the above three-dimensional structure, the α helix and β 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 α helix and the β sheet are defined as a loop.
　以下に、タイプＩとタイプＩＩのポリペプチドについてそれぞれ詳細に説明する。なお、タイプＩ、タイプＩＩ以外のポリペプチドであっても、本発明のポリペプチドの基本構造を有するものは、本発明に含まれる。 Hereinafter, each of the type I and type II polypeptides will be described in detail. In addition, even if it is polypeptide except type I and type II, what has the basic structure of the polypeptide of this invention is contained in this invention.
＜タイプＩ＞
　タイプＩのポリペプチドとしては、例えば、配列番号１～３のアミノ酸配列を有するポリペプチド、及びこれらと類似の立体構造を有するポリペプチドが挙げられる。タイプＩのポリペプチドの構成についてＮ末端からＣ末端まで順に説明する。 <Type I>
Examples of 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末端からβシート（Ａ）Ｎ末端側のアミノ酸残基までの領域（Ｉ）
　領域（Ｉ）のアミノ酸残基数としては、６～１３、好ましくは８～１１である。領域（Ｉ）のアミノ酸配列としては、具体的には、バークホルデリア・セパシア由来のものであれば、配列番号１に示す１～１０位のアミノ酸残基、シュードモナス・グルマエ由来のものであれば、配列番号２に示す１～９位のアミノ酸残基、シュードモナス・フルオレセンス由来のものであれば、配列番号３に示す１～１０位のアミノ酸残基が挙げられる。 Region from N-terminal to β-sheet (A) 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.
βシート（Ａ）
　βシート（Ａ）は、そのＮ末端側のアミノ酸残基がポリペプチドのＮ末端側から７～１３位に存在し、好ましくは８～１１位に存在し、さらに好ましくは９～１０位に存在する。βシート（Ａ）を構成するアミノ酸残基数としては、２～６、好ましくは３～５、さらに好ましくは３～４が挙げられる。
　βシート（Ａ）のアミノ酸配列としては、特に限定されないが、例えばｘ1ＩＬＶ（ｘ1は、いずれのアミノ酸残基であってもよく、好ましくはＩ又はＶである）が挙げられ、好ましくはＩＩＬＶが挙げられる。βシート（Ａ）のアミノ酸配列としては、具体的には、バークホルデリア・セパシア由来のものであれば、配列番号１に示す１１～１４位のアミノ酸残基、シュードモナス・グルマエ由来のものであれば、配列番号２に示す１０～１３位のアミノ酸残基、シュードモナス・フルオレセンス由来のものであれば、配列番号３に示す１１～１４位のアミノ酸残基が挙げられる。 β sheet (A)
In the β sheet (A), 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. To do. Examples of the number of amino acid residues constituting the β sheet (A) include 2 to 6, preferably 3 to 5, and more preferably 3 to 4.
The amino acid sequence of the β 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. Specifically, the amino acid sequence of the β 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. For example, 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.
βシート（Ａ）Ｃ末端側アミノ酸残基からαへリックス（Ａ）Ｎ末端側アミノ酸残基までの領域（ＩＩ）
　領域（ＩＩ）のアミノ酸残基数としては、１４～２３、好ましくは１６～２２、さらに好ましくは１７～２１である。領域（ＩＩ）のアミノ酸配列としては、具体的には、バークホルデリア・セパシア由来のものであれば、配列番号１に示す１５～３２位のアミノ酸残基、シュードモナス・グルマエ由来のものであれば、配列番号２に示す１４～３１位のアミノ酸残基、シュードモナス・フルオレセンス由来のものであれば、配列番号３に示す１５～３２位のアミノ酸残基が挙げられる。 β sheet (A) Region from C-terminal amino acid residue to α-helix (A) N-terminal amino acid residue (II)
The number of amino acid residues in the region (II) is 14 to 23, preferably 16 to 22, and more preferably 17 to 21. Specifically, as 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.
αへリックス（Ａ）
　αへリックス（Ａ）は、そのＮ末端側のアミノ酸残基が前記βシート（Ａ）のＣ末端側のアミノ酸残基を０位とした場合に１５～２５位に存在し、好ましくは１７～２３位に存在し、さらに好ましくは１９～２１に存在する。αへリックス（Ａ）を構成するアミノ酸残基数としては、３～９、好ましくは４～８、さらに好ましくは５～７が挙げられる。
　αへリックス（Ａ）のアミノ酸配列としては、特に限定されないが、例えば、ＩＱｘ2ＤＬＱｘ3（ｘ2及びｘ3はそれぞれいずれのアミノ酸残基であってもよく、好ましくは、ｘ2はＥ又はＳ、ｘ3はＱ又はＳである。）が挙げられ、好ましくはＩＱＥＤＬＱＱ、ＩＱＳＤＬＱＳが挙げられる。αへリックス（Ａ）のアミノ酸配列としては、具体的には、バークホルデリア・セパシア由来のものであれば、配列番号１に示す３３～３９位のアミノ酸残基、シュードモナス・グルマエ由来のものであれば、配列番号２に示す３２～３８位のアミノ酸残基、シュードモナス・フルオレセンス由来のものであれば、配列番号３に示す３３～３９位のアミノ酸残基が挙げられる。 α helix (A)
The α-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 β sheet (A), preferably 17- Present at the 23rd position, more preferably at 19-21. Examples of the number of amino acid residues constituting the α helix (A) include 3 to 9, preferably 4 to 8, and more preferably 5 to 7.
The amino acid sequence of the α helix (A) is not particularly limited. For example, 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. Specifically, the amino acid sequence of α-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.
αへリックス（Ａ）Ｃ末端側アミノ酸残基からαへリックス（Ｂ）Ｎ末端側アミノ酸残基までの領域（ＩＩＩ）
　領域（ＩＩＩ）のアミノ酸残基数としては、６９～９２、好ましくは７４～８７、さらに好ましくは７８～８３である。領域（ＩＩＩ）のアミノ酸配列としては、具体的には、バークホルデリア・セパシア由来のものであれば、配列番号１に示す４０～１１７位のアミノ酸残基、シュードモナス・グルマエ由来のものであれば、配列番号２に示す３９～１１６位のアミノ酸残基、シュードモナス・フルオレセンス由来のものであれば、配列番号３に示す４０～１１９位のアミノ酸残基が挙げられる。 α-helix (A) region from C-terminal amino acid residue to α-helix (B) N-terminal amino acid residue (III)
The number of amino acid residues in region (III) is 69 to 92, preferably 74 to 87, and more preferably 78 to 83. Specifically, as 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.
αへリックス（Ｂ）
　αへリックス（Ｂ）は、そのＮ末端側のアミノ酸残基が前記αへリックス（Ａ）のＣ末端側のアミノ酸残基を０位とした場合に７０～９２位に存在し、好ましくは７５～８７位に存在し、さらに好ましくは７９～８３位に存在する。αへリックス（Ｂ）を構成するアミノ酸残基数としては、４～１５、好ましくは５～１４、さらに好ましくは７～１２が挙げられる。
　αへリックス（Ｂ）のアミノ酸配列としては、特に限定されないが、例えば、ＥＦＡＤＦＶＱＧＶＬ、ＥＦＡＤＦＶＱＤＶＬＫＴ、ＡＤＦＶＱＧＶＬ等が挙げられ、好ましくはＥＦＡＤＦＶＱＧＶＬが挙げられる。αへリックス（Ｂ）のアミノ酸配列としては、具体的には、バークホルデリア・セパシア由来のものであれば、配列番号１に示す１１８～１２７位のアミノ酸残基、シュードモナス・グルマエ由来のものであれば、配列番号２に示す１１７～１２８位のアミノ酸残基、シュードモナス・フルオレセンス由来のものであれば、配列番号３に示す１２０～１２８位のアミノ酸残基が挙げられる。 α helix (B)
The α-helix (B) is present at the 70-92 position when the amino acid residue on the N-terminal side of the α-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 α helix (B) include 4 to 15, preferably 5 to 14, and more preferably 7 to 12.
The amino acid sequence of α-helix (B) is not particularly limited, and examples thereof include EFADFVQGVL, EFADFVQDVLKT, ADFVQGVL, and preferably EFADFVQGVL. Specifically, the amino acid sequence of α-helix (B), if it is derived from Burkholderia cepacia, is the amino acid residue at positions 118 to 127 shown in SEQ ID NO: 1, derived from Pseudomonas grumae. If there are, amino acid residues at positions 117 to 128 shown in SEQ ID NO: 2 and amino acid residues at positions 120 to 128 shown in SEQ ID NO: 3 if derived from Pseudomonas fluorescens.
αへリックス（Ｂ）Ｃ末端側アミノ酸残基からαへリックス（Ｃ）Ｎ末端側アミノ酸残基までの領域（ＩＶ）
　領域（ＩＶ）のアミノ酸残基数としては、２～１０、好ましくは４～９、さらに好ましくは５～８が挙げられる。領域（ＩＶ）のアミノ酸配列としては、具体的には、バークホルデリア・セパシア由来のものであれば、配列番号１に示す１２８～１３３位のアミノ酸残基、シュードモナス・グルマエ由来のものであれば、配列番号２に示す１２９～１３５位のアミノ酸残基、シュードモナス・フルオレセンス由来のものであれば、配列番号３に示す１２９～１３３位のアミノ酸残基が挙げられる。 α-helix (B) region from C-terminal amino acid residue to α-helix (C) N-terminal amino acid residue (IV)
Examples of the number of amino acid residues in region (IV) include 2 to 10, preferably 4 to 9, and more preferably 5 to 8. Specifically, as 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. Examples of the 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.
αへリックス（Ｃ）
　αへリックス（Ｃ）は、そのＮ末端側のアミノ酸残基が前記αへリックス（Ｂ）のＣ末端側のアミノ酸残基を０位とした場合に３～１１位に存在し、好ましくは５～１０位に存在し、さらに好ましくは６～９位に存在する。αへリックス（Ｃ）を構成するアミノ酸残基数としては、１１～２０、好ましくは１３～１８、さらに好ましくは１４～１７が挙げられる。
　αへリックス（Ｃ）のアミノ酸配列としては、特に限定されないが、例えば、ＬＳＳＴＶＩＡＡＦＶＮＶｘ4Ｇｘ5ＬＴ（ｘ4及びｘ5はそれぞれいずれのアミノ酸残基であってもよく、好ましくは、ｘ4はＦ又はＩ、ｘ5はＩ又はＡである。）、ＴＶＩＡＡＦＶＮＶＦＧＴＬＶ等が挙げられ、好ましくはＬＳＳＴＶＩＡＡＦＶＮＶｘ4Ｇｘ5ＬＴ（ｘ4及びｘ5は、それぞれいずれのアミノ酸残基であってもよく、好ましくは、ｘ4はＦ又はＩ、ｘ5はＩ又はＡである。）が挙げられ、より好ましくはＬＳＳＴＶＩＡＡＦＶＮＶＦＧＩＬＴ、ＬＳＳＴＶＩＡＡＦＶＮＶＩＧＡＬＴが挙げられる。αへリックス（Ｃ）のアミノ酸配列としては、具体的には、バークホルデリア・セパシア由来のものであれば、配列番号１に示す１３４～１５０位、シュードモナス・グルマエ由来のものであれば、配列番号２では１３６～１４９位、シュードモナス・フルオレセンス由来のものであれば、配列番号３に示す１３４～１５０位のアミノ酸残基が挙げられる。 α helix (C)
The α helix (C) is present at the 3 to 11 position when the amino acid residue on the N-terminal side of the α 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 α helix (C) include 11 to 20, preferably 13 to 18, and more preferably 14 to 17.
The amino acid sequence of the α helix (C) is not particularly limited. For example, 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. As the amino acid sequence of α-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.
αへリックス（Ｃ）Ｃ末端側アミノ酸残基からβシート（Ｂ）Ｎ末端側アミノ酸残基までの領域（Ｖ）
　領域（Ｖ）のアミノ酸残基数としては、６４～８０、好ましくは６８～７６、さらに好ましくは７０～７５が挙げられる。領域（Ｖ）のアミノ酸配列としては、具体的には、バークホルデリア・セパシア由来のものであれば、配列番号１に示す１５１～２２２位のアミノ酸残基、シュードモナス・グルマエ由来のものであれば、配列番号２に示す１５０～２２１位のアミノ酸残基、シュードモナス・フルオレセンス由来のものであれば、配列番号３に示す１５１～２２２位のアミノ酸残基が挙げられる。 α-helix (C) region from amino acid residue on the C-terminal side to β-sheet (B) amino acid residue on the N-terminal side (V)
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. Specifically, as 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. Examples of 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.
βシート（Ｂ）
　βシート（Ｂ）は、そのＮ末端側のアミノ酸残基が前記αへリックス（Ｃ）のＣ末端側のアミノ酸残基を０位とした場合に６５～８１位に存在し、好ましくは６９～７７位に存在し、さらに好ましくは７１～７５位に存在する。βシート（Ｂ）を構成するアミノ酸残基数としては、２～８、好ましくは３～７、さらに好ましくは４～６が挙げられる。
　βシート（Ｂ）のアミノ酸配列としては、特に限定されないが、例えば、ＶＴＧＡｘ6Ｄ（ｘ6は、いずれのアミノ酸残基であってもよく、好ましくはＴ又はＲである。）等が挙げられ、好ましくはＶＴＧＡＴＤが挙げられる。βシート（Ｂ）のアミノ酸配列としては、具体的には、バークホルデリア・セパシア由来のものであれば、配列番号１に示す２２３～２２８位のアミノ酸残基、シュードモナス・グルマエ由来のものであれば、配列番号２に示す２２２～２２７位のアミノ酸残基、シュードモナス・フルオレセンス由来のものであれば、配列番号３に示す２２３～２２８位のアミノ酸残基が挙げられる。 β sheet (B)
The β 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 α 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 β sheet (B) include 2 to 8, preferably 3 to 7, and more preferably 4 to 6.
The amino acid sequence of the β 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. Specifically, the amino acid sequence of the β 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. For example, 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.
βシート（Ｂ）Ｃ末端側アミノ酸残基からαへリックス（Ｄ）Ｎ末端側アミノ酸残基までの領域（ＶＩ）
　領域（ＶＩ）のアミノ酸残基数としては、５～１４、好ましくは７～１２位、さらに好ましくは８～１１が挙げられる。領域（ＶＩ）のアミノ酸配列としては、具体的には、バークホルデリア・セパシア由来のものであれば、配列番号１に示す２２９～２３７位のアミノ酸残基、シュードモナス・グルマエ由来のものであれば、配列番号２に示す２２８～２３５位のアミノ酸残基、シュードモナス・フルオレセンス由来のものであれば、配列番号３に示す２２９～２３６位のアミノ酸残基が挙げられる。 β sheet (B) Region from C-terminal amino acid residue to α 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. Specifically, as the 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. Examples of the 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.
αへリックス（Ｄ）
　αへリックス（Ｄ）は、そのＮ末端側のアミノ酸残基が前記αへリックス（Ｃ）のＣ末端側のアミノ酸残基を０位とした場合に６～１５位に存在し、好ましくは８～１３位に存在し、さらに好ましくは９～１２位に存在する。αへリックス（Ｄ）を構成するアミノ酸残基数としては、２～２４、好ましくは３～２３、さらに好ましくは４～２２が挙げられる。
　αへリックス（Ｄ）のアミノ酸配列としては、特に限定されないが、例えば、ＡＮｘ7ｘ8（ｘ7及びｘ8はそれぞれいずれのアミノ酸残基であってもよく、好ましくは、ｘ7はＡ又はＶ、ｘ8はＬ又はＴである。）、ＰＡＮＶＴ、ＳＴＬＡＬｘ9ｘ10ＴＧｘ11ＶＭｘ12（ｘ9、ｘ10、ｘ11、及びｘ12はそれぞれいずれのアミノ酸残基であってもよく、好ましくは、ｘ9はＦ又はＬ、ｘ10はＧ又はＡ、ｘ11はＴ又はＡ、ｘ12はＶ又はＩである。）、ＳＴＬＡＬＬＧＳＧＴＶＭＶＮ等が挙げられ、好ましくはＡＮｘ7ｘ8（ｘ7及びｘ8は、それぞれいずれのアミノ酸残基であってもよく、好ましくは、ｘ7はＡ又はＶ、ｘ8はＬ又はＴである。）、ＳＴＬＡＬＦＧＴＧＴＶＭＶ、ＳＴＬＡＬＬＡＴＧＡＶＭＩ、ＳＴＬＡＬＬＧＳＧＴＶＭＶＮ、が挙げられ、より好ましくはＡＮＡＬ、ＡＮＶＴ、ＳＴＬＡＬＦＧＴＧＴＶＭＶ、ＳＴＬＡＬＬＡＴＧＡＶＭＩ、更に好ましくはＡＮＡＬ、ＳＴＬＡＬＦＧＴＧＴＶＭＶが挙げられる。αへリックス（Ｄ）領域には、前記で例示した２種以上の配列が、直接又は数個のアミノ酸残基を介して連結した配列であることが好ましく、ＡＮＡＬ、ＳＴＬＡＬＦＧＴＧＴＶＭＶを含む配列であることが好ましい。αへリックス（Ｄ）のアミノ酸配列としては、具体的には、バークホルデリア・セパシア由来のものであれば、配列番号１に示す２３８～２５６位のアミノ酸残基、シュードモナス・グルマエ由来のものであれば、配列番号２に示す２３６～２５４位のアミノ酸残基、シュードモナス・フルオレセンス由来のものであれば、配列番号３に示す２３７～２５７位のアミノ酸残基が挙げられる。 α helix (D)
The α-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 α-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 α helix (D) include 2 to 24, preferably 3 to 23, and more preferably 4 to 22.
The amino acid sequence of the α helix (D) is not particularly limited. For example, 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 α 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. As the amino acid sequence of α-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.
αへリックス（Ｄ）Ｃ末端側アミノ酸残基からポリペプチドのＣ末端までの領域（ＶＩＩ）
　領域（ＶＩＩ）のアミノ酸配列の残基数としては、５５～８８、好ましくは５８～８４、さらに好ましくは６１～８０が挙げられる。領域（ＶＩＩ）のアミノ酸配列としては、具体的には、バークホルデリア・セパシア由来のものであれば、配列番号１に示す２５７～３２０位のアミノ酸残基、シュードモナス・グルマエ由来のものであれば、配列番号２に示す２５５～３１８位のアミノ酸残基、シュードモナス・フルオレセンス由来のものであれば、配列番号３に示す２５８～３２０位のアミノ酸残基が挙げられる。 α-helix (D) Region from the C-terminal amino acid residue to the C-terminus of the polypeptide (VII)
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. Specifically, as the 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. And 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.
　また、本発明のポリペプチドは、更に、領域（ＩＩＩ）において、Ｎ末端側から、αへリックス（Ｅ）、βシート（Ｃ）、αへリックス（Ｆ）、又はβシート（Ｄ）を備えていてもよい。これらのαへリックス、βシートの間の領域には、１又は複数のループ、αへリックス、又はβシートを含んでもよい。 The polypeptide of the present invention further comprises an α helix (E), β sheet (C), α helix (F), or β sheet (D) from the N-terminal side in the region (III). It may be. The region between these α-helix and β-sheets may include one or more loops, α-helix, or β-sheet.
αへリックス（Ｅ）
　αへリックス（Ｅ）は、そのＮ末端側のアミノ酸残基が前記αへリックス（Ａ）のＣ末端側のアミノ酸残基を０位とした場合に１７～２８位、好ましくは１９～２６位、さらに好ましくは２１～２４に存在し、アミノ酸残基数が１３～１９、好ましくは１４～１８、さらに好ましくは１５～１７で構成される。
　αへリックス（Ｅ）のアミノ酸配列としては、特に限定されないが、例えば、ＲＧＥＱＬＬＡＹＶＫｘ13ＶＬＡｘ14Ｔ（ｘ13及びｘ14は、それぞれいずれのアミノ酸残基であってもよく、好ましくは、ｘ13はＴ又はＱ、ｘ14はＡ又はＱである。）等が挙げられ、好ましくはＲＧＥＱＬＬＡＹＶＫＴＶＬＡＡＴ、ＲＧＥＱＬＬＡＹＶＫＱＶＬＡＡＴ、ＲＧＥＱＬＬＡＹＶＫＱＶＬＡＱＴ、より好ましくはＲＧＥＱＬＬＡＹＶＫＴＶＬＡＡＴが挙げられる。αへリックス（Ｅ）のアミノ酸配列としては、具体的には、バークホルデリア・セパシア由来のものであれば、配列番号１に示す６１～７６位のアミノ酸残基、シュードモナス・グルマエ由来のものであれば、配列番号２に示す６０～７５位のアミノ酸残基、シュードモナス・フルオレセンス由来のものであれば、配列番号３に示す６１～７６位のアミノ酸残基が挙げられる。 α helix (E)
The α helix (E) has 17 to 28 positions, preferably 19 to 26 positions, when the amino acid residue on the N-terminal side of the α 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, but 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. Specifically, the amino acid sequence of α-helix (E), if derived from Burkholderia cepacia, is the amino acid residue at positions 61 to 76 shown in SEQ ID NO: 1, derived from Pseudomonas grumae. If there are amino acid residues at positions 60 to 75 shown in SEQ ID NO: 2, and amino acid residues at positions 61 to 76 shown in SEQ ID NO: 3 if they are derived from Pseudomonas fluorescens.
βシート（Ｃ）
　βシート（Ｃ）は、そのＮ末端側のアミノ酸残基が前記αへリックス（Ｅ）のＣ末端側のアミノ酸残基を０位とした場合に２～８位、好ましくは３～７位、さらに好ましくは４～６に存在し、アミノ酸残基数が４～８、好ましくは５～７で構成される。
　βシート（Ｃ）のアミノ酸配列としては、特に限定されないが、例えば、ＶＮＬｘ15ＧＨ（ｘ15はいずれのアミノ酸残基であってもよく、好ましくはＶ又はＩである。）等が挙げられ、好ましくはＶＮＬＶＧＨが挙げられる。βシート（Ｃ）のアミノ酸配列としては、具体的には、バークホルデリア・セパシア由来のものであれば、配列番号１に示す８１～８６位のアミノ酸残基、シュードモナス・グルマエ由来のものであれば、配列番号２に示す８０～８５位のアミノ酸残基、シュードモナス・フルオレセンス由来のものであれば、配列番号３に示す８１～８６位のアミノ酸残基が挙げられる。 β sheet (C)
The β 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 α 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 β 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. Specifically, the amino acid sequence of the β 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. For example, 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.
αへリックス（Ｆ）
　αへリックス（Ｆ）は、そのＮ末端側のアミノ酸残基が前記βシート（Ｃ）のＣ末端側のアミノ酸残基を０位とした場合に１～５位、好ましくは２～４位に存在し、アミノ酸残基数が３～１５、好ましくは５～１４、さらに好ましくは１０～１３で構成される。
　αへリックス（Ｆ）のアミノ酸配列としては、特に限定されないが、例えば、ＧＧＬＴＳＲＹＶＡＡＶ、ＱＧＧＬＴＳＲＹＶＡＡＶ等が挙げられ、好ましくはＧＧＬＴＳＲＹＶＡＡＶが挙げられる。αへリックス（Ｆ）のアミノ酸配列としては、具体的には、バークホルデリア・セパシア由来のものであれば、配列番号１に示す８９～９９位のアミノ酸残基、シュードモナス・グルマエ由来のものであれば、配列番号２に示す８７～９８位のアミノ酸残基、シュードモナス・フルオレセンス由来のものであれば、配列番号３に示す８８～９９位のアミノ酸残基が挙げられる。 α helix (F)
α-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 β-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 α-helix (F) is not particularly limited, and examples thereof include GGLTSRYVAAV, QGGLTSRYVAAV, and preferably GGLTSRYVAAV. As the amino acid sequence of α-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.
βシート（Ｄ）
　βシート（Ｄ）は、そのＮ末端側のアミノ酸残基が前記βシート（Ｃ）のＣ末端側のアミノ酸残基を０位とした場合に１４～２２位、好ましくは１６～２０位、さらに好ましくは１７～１９位に存在する。また、βシート（Ｄ）は、そのＮ末端側のアミノ酸残基が前記αへリックス（Ｆ）のＣ末端側のアミノ酸残基を０位とした場合に３～１３位、好ましくは４～１２位、さらに好ましくは４～６位に存在する。βシート（Ｄ）は、アミノ酸残基数が３～１１、好ましくは５～９、さらに好ましくは、６～８で構成される。
　βシート（Ｄ）のアミノ酸配列としては、特に限定されないが、例えば、ＶＡＳＶＴＴＩ等が挙げられる。βシート（Ｄ）のアミノ酸配列としては、具体的には、バークホルデリア・セパシア由来のものであれば、配列番号１に示す１０４～１１０位のアミノ酸残基、シュードモナス・グルマエ由来のものであれば、配列番号２に示す１０３～１０９位、シュードモナス・フルオレセンス由来のものであれば、配列番号３に示す１０４～１１０位のアミノ酸残基が挙げられる。 β sheet (D)
The β-sheet (D) has 14 to 22 positions, preferably 16 to 20 positions when the amino acid residue on the N-terminal side of the β-sheet (C) is 0-position. Preferably it is located at positions 17-19. The β 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 α helix (F) is 0 position. , More preferably 4-6. The β 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 | seat (D), For example, VASVTTI etc. are mentioned. Specifically, the amino acid sequence of the β sheet (D), if it is derived from Burkholderia cepacia, is the amino acid residue at positions 104 to 110 shown in SEQ ID NO: 1, derived from Pseudomonas grumae. For example, the 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.
　また、本発明のポリペプチドは、更に、領域（Ｖ）において、Ｎ末端側から、αへリックス（Ｇ）、αへリックス（Ｈ）、βシート（Ｅ）、又はβシート（Ｆ）を備えていてもよい。これらのαへリックス、βシートの間の領域には、１又は複数のループ、αへリックス、又はβシートを含んでもよい。 The polypeptide of the present invention further comprises an α helix (G), α helix (H), β sheet (E), or β sheet (F) from the N-terminal side in the region (V). It may be. The region between these α-helix and β-sheets may include one or more loops, α-helix, or β-sheet.
αへリックス（Ｇ）
　αへリックス（Ｇ）は、そのＮ末端側のアミノ酸残基が前記αへリックス（Ｃ）のＣ末端側のアミノ酸残基を０位とした場合に３～１３位、好ましくは４～１２位、さらに好ましくは５～１１に存在し、アミノ酸残基数が４～１３、好ましくは５～１２、さらに好ましくは６～１１で構成される。
　αへリックス（Ｇ）のアミノ酸配列としては、特に限定されないが、例えば、ＡＬＡＡＬＫＴ、ＴＤＱＤＡＬＡＡＬＲ、ＡＬＡＡＬＱＴＬ等が挙げられ、好ましくはＡＬＡＡＬＫＴが挙げられる。αへリックス（Ｇ）のアミノ酸配列としては、具体的には、バークホルデリア・セパシア由来のものであれば、配列番号１に示す１６０～１６６位のアミノ酸残基、シュードモナス・グルマエ由来のものであれば、配列番号２に示す１５５～１６４位のアミノ酸残基、シュードモナス・フルオレセンス由来のものであれば、配列番号３に示す１６０～１６７位のアミノ酸残基が挙げられる。 α helix (G)
α-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 α-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.
Although it does not specifically limit as an amino acid sequence of (alpha) helix (G), For example, ALAALKT, TDQDALAALR, ALAALQTL etc. are mentioned, Preferably ALAALKT is mentioned. Specifically, the amino acid sequence of α-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.
αへリックス（Ｈ）
　αへリックス（Ｈ）は、そのＮ末端側のアミノ酸残基が前記αへリックス（Ｇ）のＣ末端側のアミノ酸残基を０位とした場合に１～１０位、好ましくは２～８位、さらに好ましくは２～５位に存在し、アミノ酸残基数が２～１３、好ましくは４～１２、さらに好ましくは９～１１で構成される。
　αへリックス（Ｈ）のアミノ酸配列としては、特に限定されないが、例えば、ＴＡｘ16ｘ17ＡＴＹＮｘ18Ｎ（ｘ16、ｘ17、及びｘ18はそれぞれいずれのアミノ酸残基であってもよく、好ましくは、ｘ16はＱ又はＲ、ｘ17はＡ又はＴ、ｘ18はＱ又はＲである。）等が挙げられ、好ましくはＴＡＱＡＡＴＹＮＱＮ、ＴＡＱＴＡＴＹＮＲＮ、又はＴＡＲＡＡＴＹＮＱＮが挙げられ、より好ましくはＴＡＱＡＡＴＹＮＱＮが挙げられる。αへリックス（Ｈ）のアミノ酸配列としては、具体的には、バークホルデリア・セパシア由来のものであれば、配列番号１に示す１６９～１７８位のアミノ酸残基、シュードモナス・グルマエ由来のものであれば、配列番号２に示す１６８～１７７位のアミノ酸残基、シュードモナス・フルオレセンス由来のものであれば、配列番号３に示す１６９～１７８位のアミノ酸残基が挙げられる。 α helix (H)
α-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 α-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 α helix (H) is not particularly limited. For example, TAx 16 x 17 ATYNx 18 N (x 16 , x 17 , and x 18 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 . As the amino acid sequence of α-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.
βシート（Ｅ）
　βシート（Ｅ）は、そのＮ末端側のアミノ酸残基が前記αへリックス（Ｇ）のＣ末端側のアミノ酸残基を０位とした場合に２４～３６位、好ましくは２７～３４位、さらに好ましくは２９～３２に存在し、アミノ酸残基数が２～６、好ましくは３～５で構成される。また、βシート（Ｅ）は、そのＮ末端側のアミノ酸残基が前記αへリックス（Ｈ）のＣ末端側のアミノ酸残基を０位とした場合に１３～２４位、好ましくは１５～２２位、さらに好ましくは１７～２０に存在する。βシート（Ｅ）は、アミノ酸残基数が２～６、好ましくは３～５で構成される。
　βシート（Ｅ）のアミノ酸配列としては、特に限定されないが、例えば、ＴＥＴＶ等が挙げられる。βシート（Ｅ）のアミノ酸配列としては、具体的には、例えば、バークホルデリア・セパシア由来のものであれば、配列番号１に示す１９６～１９９位のアミノ酸残基、シュードモナス・グルマエ由来のものであれば、配列番号２に示す１９５～１９８位のアミノ酸残基、シュードモナス・フルオレセンス由来のものであれば、配列番号３に示す１９６～１９９位のアミノ酸残基が挙げられる。 β sheet (E)
The β 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 α 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. In the β sheet (E), 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 α helix (H) is 0 position. , More preferably 17-20. The β sheet (E) is composed of 2 to 6, preferably 3 to 5, amino acid residues.
The amino acid sequence of the β sheet (E) is not particularly limited, and examples thereof include TETV. Specifically, the amino acid sequence of the β 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.
βシート（Ｆ）
　βシート（Ｆ）は、そのＮ末端側のアミノ酸残基が前記βシート（Ｅ）のＣ末端側のアミノ酸残基を０位とした場合に１～５位、好ましくは２～４位に存在し、アミノ酸残基数が１５～２３、好ましくは１７～２１、さらに好ましくは１８～２０で構成される。
　βシート（Ｆ）のアミノ酸配列としては、特に限定されないが、例えば、ＮＴＨＬＬＹＳＷＡＧ、ＳＱＨＬＬＹＳＷ、ＩＱＰＴｘ19ｘ20Ｖ（ｘ19、及びｘ20はそれぞれいずれのアミノ酸残基であってもよく、好ましくは、ｘ19はＩ、Ｓ又はＦ、ｘ20はＳ又はＴである。）等が挙げられ、好ましくはＮＴＨＬＬＹＳＷＡＧ、ＩＱＰＴｘ19ｘ20Ｖ（ｘ19、及びｘ20はそれぞれいずれのアミノ酸残基であってもよく、好ましくは、ｘ19はＩ、Ｓ又はＦ、ｘ20はＳ又はＴである。）が挙げられ、より好ましくはＮＴＨＬＬＹＳＷＡＧ、ＩＱＰＴＩＳＶが挙げられる。βシート（Ｆ）領域には、前記で例示した２種以上の配列が、直接又は数個のアミノ酸残基を介して連結した配列であることが好ましく、ＮＴＨＬＬＹＳＷＡＧとＩＱＰＴＩＳＶを含む配列であることが好ましい。βシート（Ｆ）のアミノ酸配列としては、具体的には、バークホルデリア・セパシア由来のものであれば、配列番号１に示す２０２～２２０位のアミノ酸残基、シュードモナス・グルマエ由来のものであれば、配列番号２に示す２０１～２１９位のアミノ酸残基、シュードモナス・フルオレセンス由来のものであれば、配列番号３に示す２０２～２２０位のアミノ酸残基が挙げられる。 β sheet (F)
The β 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 β-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 β sheet (F) is not particularly limited. For example, NTHLLYSWAG, SQHLLYSW, IQPTx 19 x 20 V (x 19 , and x 20 may be any amino acid residue, preferably, x 19 is I, S or F, and x 20 is S or T.), etc., preferably 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 β 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. Specifically, the amino acid sequence of the β 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. For example, 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.
　本発明のポリペプチドは、前述のβシート（Ａ）、αへリックス（Ａ）、αへリックス（Ｂ）、αへリックス（Ｃ）、βシート（Ｂ）、及びαへリックス（Ｄ）を基本的に有するものであるが、好ましくは、更にαへリックス（Ｅ）、βシート（Ｃ）、βシート（Ｄ）、αへリックス（Ｇ）、及びβシート（Ｅ）を備え、より好ましくは、更にαへリックス（Ｅ）、βシート（Ｃ）、αへリックス（Ｆ）、βシート（Ｄ）、αへリックス（Ｇ）、αへリックス（Ｈ）、βシート（Ｅ）、及びβシート（Ｆ）を備える。 The polypeptide of the present invention comprises the aforementioned β sheet (A), α helix (A), α helix (B), α helix (C), β sheet (B), and α helix (D). Basically, it preferably has an α helix (E), β sheet (C), β sheet (D), α helix (G), and β sheet (E), more preferably Are the α helix (E), β sheet (C), α helix (F), β sheet (D), α helix (G), α helix (H), β sheet (E), and A β sheet (F) is provided.
＜タイプＩＩ＞
　タイプＩＩのポリペプチドとしては、例えば、配列番号４のアミノ酸配列を有するポリペプチド、及びこれと類似の立体構造を有するポリペプチドが挙げられる。タイプＩＩのポリペプチドの構成についてＮ末端からＣ末端まで順に説明する。 <Type II>
Examples of the type II polypeptide 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.
Ｎ末端からβシート（Ａ）Ｎ末端側のアミノ酸残基までの領域（Ｉ）
　領域（Ｉ）のアミノ酸残基数としては、６～１３、好ましくは８～１１である。領域（Ｉ）のアミノ酸配列としては、具体的には、シュードモナス・エルギノーサ由来のものであれば、配列番号４に示す１～１０位のアミノ酸残基が挙げられる。 Region from N-terminal to β-sheet (A) 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.
βシート（Ａ）
　βシート（Ａ）は、そのＮ末端側のアミノ酸残基がポリペプチドのＮ末端側７～１３位に存在し、好ましくは８～１１位に存在し、さらに好ましくは９～１０位に存在する。βシート（Ａ）を構成するアミノ酸残基数としては、２～６、好ましくは３～５、さらに好ましくは３～４が挙げられる。
　βシート（Ａ）のアミノ酸配列としては、特に限定されないが、例えば、ＶＬＡ等が挙げられる。βシート（Ａ）のアミノ酸配列としては、具体的には、シュードモナス・エルギノーサ由来のものであれば、配列番号４に示す１１～１３位のアミノ酸残基が挙げられる。 β sheet (A)
In the β sheet (A), 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 β sheet (A) include 2 to 6, preferably 3 to 5, and more preferably 3 to 4.
The amino acid sequence of the β sheet (A) is not particularly limited, and examples thereof include VLA. Specific examples of the amino acid sequence of the β sheet (A) include amino acid residues at positions 11 to 13 shown in SEQ ID NO: 4 if they are derived from Pseudomonas aeruginosa.
βシート（Ａ）Ｃ末端側アミノ酸残基からαへリックス（Ａ）Ｎ末端側アミノ酸残基までの領域（ＩＩ）
　領域（ＩＩ）のアミノ酸残基数としては、１４～２３、好ましくは１６～２２、さらに好ましくは１７～２１である。領域（ＩＩ）のアミノ酸配列としては、具体的には、シュードモナス・エルギノーサ由来のものであれば、配列番号４に示す１４～３３位のアミノ酸残基が挙げられる。 β sheet (A) Region from C-terminal amino acid residue to α-helix (A) N-terminal amino acid residue (II)
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.
αへリックス（Ａ）
　αへリックス（Ａ）は、そのＮ末端側のアミノ酸残基が前記βシート（Ａ）のＣ末端側のアミノ酸残基を０位とした場合に１５～２５位に存在し、好ましくは１７～２３位に存在し、さらに好ましくは１９～２１に存在する。αへリックス（Ａ）を構成するアミノ酸残基数としては、３～９、好ましくは４～８、さらに好ましくは５～７が挙げられる。
　αへリックス（Ａ）のアミノ酸配列としては、特に限定されないが、例えば、ＡＬＲＲＤ等が挙げられる。αへリックス（Ａ）のアミノ酸配列としては、具体的には、シュードモナス・エルギノーサ由来のものであれば、配列番号４では３４～３８位のアミノ酸残基が挙げられる。 α helix (A)
The α-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 β sheet (A), preferably 17- Present at the 23rd position, more preferably at 19-21. Examples of the number of amino acid residues constituting the α helix (A) include 3 to 9, preferably 4 to 8, and more preferably 5 to 7.
Although it does not specifically limit as an amino acid sequence of alpha helix (A), For example, ALRRD etc. are mentioned. Specifically, the amino acid sequence of α-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.
αへリックス（Ａ）Ｃ末端側アミノ酸残基からαへリックス（Ｂ）Ｎ末端側アミノ酸残基までの領域（ＩＩＩ）
　領域（ＩＩＩ）のアミノ酸残基数としては、６９～９２、好ましくは７４～８７、さらに好ましくは７８～８３である。領域（ＩＩＩ）のアミノ酸配列としては、具体的には、シュードモナス・エルギノーサ由来のものであれば、配列番号４に示す３９～１２０位のアミノ酸残基が挙げられる。 α-helix (A) region from C-terminal amino acid residue to α-helix (B) N-terminal amino acid residue (III)
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.
αへリックス（Ｂ）
　αへリックス（Ｂ）は、そのＮ末端側のアミノ酸残基が前記αへリックス（Ａ）のＣ末端側のアミノ酸残基を０位とした場合に７０～９２位に存在し、好ましくは７５～８７位に存在し、さらに好ましくは７９～８３位に存在する。αへリックス（Ｂ）を構成するアミノ酸残基数としては、４～１５、好ましくは５～１４、さらに好ましくは７～１２が挙げられる。
　αへリックス（Ｂ）のアミノ酸配列としては、特に限定されないが、例えば、ＩＰＰＧＳＡＧ等が挙げられる。αへリックス（Ｂ）のアミノ酸配列としては、具体的には、シュードモナス・エルギノーサ由来のものであれば、配列番号４に示す１２１～１２７位のアミノ酸残基が挙げられる。 α helix (B)
The α-helix (B) is present at the 70-92 position when the amino acid residue on the N-terminal side of the α-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 α 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 α-helix (B) include amino acid residues 121 to 127 shown in SEQ ID NO: 4 as long as they are derived from Pseudomonas aeruginosa.
αへリックス（Ｂ）Ｃ末端側アミノ酸残基からαへリックス（Ｃ）Ｎ末端側アミノ酸残基までの領域（ＩＶ）
　領域（ＩＶ）のアミノ酸残基数としては、２～１０、好ましくは４～９、さらに好ましくは５～８が挙げられる。領域（ＩＶ）のアミノ酸配列としては、具体的には、シュードモナス・エルギノーサ由来のものであれば、配列番号４に示す１２８～１３３位のアミノ酸残基が挙げられる。 α-helix (B) region from C-terminal amino acid residue to α-helix (C) N-terminal amino acid residue (IV)
Examples of the number 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.
αへリックス（Ｃ）
　αへリックス（Ｃ）は、そのＮ末端側のアミノ酸残基が前記αへリックス（Ｂ）のＣ末端側のアミノ酸残基を０位とした場合に３～１１位に存在し、好ましくは５～１０位に存在し、さらに好ましくは６～９位に存在する。αへリックス（Ｃ）を構成するアミノ酸残基数としては、１１～２０、好ましくは１３～１８、さらに好ましくは１４～１７が挙げられる。
　αへリックス（Ｃ）のアミノ酸配列としては、特に限定されないが、例えば、ＬＶＮＳＬＧＡＬＩＳＦＬＳＳＧＳＴ等が挙げられる。αへリックス（Ｃ）のアミノ酸配列としては、具体的には、シュードモナス・エルギノーサ由来のものであれば、配列番号４に示す１３４～１５０位のアミノ酸残基が挙げられる。 α helix (C)
The α helix (C) is present at the 3 to 11 position when the amino acid residue on the N-terminal side of the α 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 α helix (C) include 11 to 20, preferably 13 to 18, and more preferably 14 to 17.
Although it does not specifically limit as an amino acid sequence of (alpha) helix (C), For example, LVNSLGALISFLSSGST etc. are mentioned. Specifically, the amino acid sequence of α-helix (C) includes amino acid residues at positions 134 to 150 shown in SEQ ID NO: 4 if it is derived from Pseudomonas aeruginosa.
αへリックス（Ｃ）Ｃ末端側アミノ酸残基からβシート（Ｂ）Ｎ末端側アミノ酸残基までの領域（Ｖ）
　領域（Ｖ）のアミノ酸残基数としては、６４～８０、好ましくは６８～７６、さらに好ましくは７０～７５が挙げられる。領域（Ｖ）のアミノ酸配列としては、具体的には、シュードモナス・エルギノーサ由来のものであれば、配列番号４に示す１５１～２２２位のアミノ酸残基が挙げられる。 α-helix (C) region from amino acid residue on the C-terminal side to β-sheet (B) amino acid residue on the N-terminal side (V)
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.
βシート（Ｂ）
　βシート（Ｂ）は、そのＮ末端側のアミノ酸残基が前記αへリックス（Ｃ）のＣ末端側のアミノ酸残基を０位とした場合に６５～８１位に存在し、好ましくは６９～７７位に存在し、さらに好ましくは７１～７５位に存在する。βシート（Ｂ）を構成するアミノ酸残基数としては、２～８、好ましくは３～７、さらに好ましくは４～６が挙げられる。
　βシート（Ｂ）のアミノ酸配列としては、特に限定されないが、例えば、ＫＮＧＴ等が挙げられる。βシート（Ｂ）のアミノ酸配列としては、具体的には、シュードモナス・エルギノーサ由来のものであれば、配列番号４に示す２２３～２２６のアミノ酸残基が挙げられる。 β sheet (B)
The β 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 α 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 β sheet (B) include 2 to 8, preferably 3 to 7, and more preferably 4 to 6.
The amino acid sequence of the β sheet (B) is not particularly limited, and examples thereof include KNGT. Specific examples of the amino acid sequence of the β sheet (B) include the amino acid residues 223 to 226 shown in SEQ ID NO: 4 if they are derived from Pseudomonas aeruginosa.
βシート（Ｂ）Ｃ末端側アミノ酸残基からαへリックス（Ｄ）Ｎ末端側アミノ酸残基までの領域（ＶＩ）
　領域（ＶＩ）のアミノ酸残基数としては、５～１４、好ましくは７～１２位、さらに好ましくは８～１１が挙げられる。領域（ＶＩ）のアミノ酸配列としては、具体的には、シュードモナス・エルギノーサ由来のものであれば、配列番号４に示す２２７～２３７位のアミノ酸残基が挙げられる。 β sheet (B) Region from C-terminal amino acid residue to α 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.
αへリックス（Ｄ）
　αへリックス（Ｄ）は、そのＮ末端側のアミノ酸残基が前記βシート（Ｂ）のＣ末端側のアミノ酸残基を０位とした場合に６～１５位に存在し、好ましくは８～１３位に存在し、さらに好ましくは９～１２位に存在する。αへリックス（Ｄ）を構成するアミノ酸残基数としては、２～２４、好ましくは３～２３、さらに好ましくは４～２２が挙げられる。
　αへリックス（Ｄ）のアミノ酸配列としては、特に限定されないが、例えば、ＨＬＧＭ、ＩＲＤＮＹＲＭＮＨＬＤＥＶＮＱ等が挙げられる。αへリックス（Ｄ）領域には、前記で例示した２種以上の配列が、直接又は数個のアミノ酸残基を介して連結した配列であることが好ましく、ＨＬＧＭ、ＩＲＤＮＹＲＭＮＨＬＤＥＶＮＱを含む配列であることが好ましい。２種以上の配列が含まれる場合、その間にループが存在していてもよい。αへリックス（Ｄ）のアミノ酸配列としては、具体的には、シュードモナス・エルギノーサ由来のものであれば、配列番号４に示す２３８～２５７位のアミノ酸残基が挙げられる。 α helix (D)
The α 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 β 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 α helix (D) include 2 to 24, preferably 3 to 23, and more preferably 4 to 22.
The amino acid sequence of the α helix (D) is not particularly limited, and examples thereof include HLGM, IRDNYRMNHLDEVNQ, and the like. The α 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 α-helix (D) include amino acid residues at positions 238 to 257 shown in SEQ ID NO: 4 if they are derived from Pseudomonas aeruginosa.
αへリックス（Ｄ）のＣ末端側アミノ酸残基からポリペプチドのＣ末端までの領域（ＶＩＩ）
　領域（ＶＩＩ）のアミノ酸配列の残基数としては、２１～５１、好ましくは２４～４９、さらに好ましくは２６～４７が挙げられる。領域（ＶＩＩ）のアミノ酸配列としては、具体的には、シュードモナス・エルギノーサ由来のものであれば、配列番号４に示す２５８～２８５位のアミノ酸残基が挙げられる。 Region from the C-terminal amino acid residue of α-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.
　また、本発明のポリペプチドは、更に、領域（ＩＩＩ）において、Ｎ末端側から、αへリックス（Ｅ）、βシート（Ｃ）、及びβシート（Ｄ）を備えていることが好ましい。これらのαへリックス、βシートとの間の領域には、ループ、αへリックス、又はβシートを含んでもよい。 The polypeptide of the present invention preferably further comprises an α helix (E), a β sheet (C), and a β sheet (D) from the N-terminal side in the region (III). The region between these α helix and β sheet may include a loop, α helix, or β sheet.
αへリックス（Ｅ）
　αへリックス（Ｅ）は、そのＮ末端側のアミノ酸残基が前記αへリックス（Ａ）のＣ末端側のアミノ酸残基を０位とした場合に１７～２８位、好ましくは１９～２６位、さらに好ましくは２１～２４に存在し、アミノ酸残基数が１３～１９、好ましくは１４～１８、さらに好ましくは１５～１７で構成される。
　αへリックス（Ｅ）のアミノ酸配列としては、特に限定されないが、例えば、ＬＱＱＶＥＥＩＶＡＬＳＧＱＰＬＶ等が挙げられる。αへリックス（Ｅ）のアミノ酸配列としては、具体的には、シュードモナス・エルギノーサ由来のものであれば、配列番号４に示す６１～７６位のアミノ酸残基が挙げられる。 α helix (E)
The α helix (E) has 17 to 28 positions, preferably 19 to 26 positions, when the amino acid residue on the N-terminal side of the α 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 α helix (E) is not particularly limited, and examples thereof include LQQVEEIVALSGQPLV. Specifically, the amino acid sequence of α-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.
βシート（Ｃ）
　βシート（Ｃ）は、そのＮ末端側のアミノ酸残基が前記αへリックス（Ｅ）のＣ末端側のアミノ酸残基を０位とした場合に２～８位、好ましくは３～７位、さらに好ましくは４～６に存在し、アミノ酸残基数が４～８、好ましくは５～７で構成される。
　βシート（Ｃ）のアミノ酸配列としては、特に限定されないが、例えば、ＨＳＨＧＧＰ等が挙げられる。βシート（Ｃ）のアミノ酸配列としては、具体的には、シュードモナス・エルギノーサ由来のものであれば、配列番号４に示す８１～８６位のアミノ酸残基が挙げられる。 β sheet (C)
The β 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 α 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 | seat (C), For example, HSHGPGP etc. are mentioned. Specific examples of the amino acid sequence of the β sheet (C) include amino acid residues at positions 81 to 86 shown in SEQ ID NO: 4 as long as they are derived from Pseudomonas aeruginosa.
βシート（Ｄ）
　βシート（Ｄ）は、そのＮ末端側のアミノ酸残基が前記βシート（Ｃ）のＣ末端側のアミノ酸残基を０位とした場合に１４～２２位、好ましくは１６～２０位、さらに好ましくは１７～１９位に存在し、アミノ酸残基数が３～１１、好ましくは５～９、さらに好ましくは６～８で構成される。
　βシート（Ｄ）のアミノ酸配列としては、特に限定されないが、例えば、ＳＶＧＡＰＨＫ等が挙げられる。βシート（Ｄ）のアミノ酸配列としては、具体的には、シュードモナス・エルギノーサ由来のものであれば、配列番号４に示す１０４～１１０位のアミノ酸残基が挙げられる。 β sheet (D)
The β-sheet (D) has 14 to 22 positions, preferably 16 to 20 positions when the amino acid residue on the N-terminal side of the β-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 | seat (D), For example, SVGAPHK etc. are mentioned. Specific examples of the amino acid sequence of the β sheet (D) include amino acid residues at positions 104 to 110 shown in SEQ ID NO: 4 if they are derived from Pseudomonas aeruginosa.
　また、本発明のポリペプチドは、更に、領域（ＶＩ）において、Ｎ末端側から、αへリックス（Ｇ）、及びβシート（Ｅ）を備えていることが好ましい。これらのαへリックス、βシートの間の領域には、ループ、αへリックス、又はβシートを含んでもよい。 The polypeptide of the present invention preferably further comprises an α helix (G) and a β sheet (E) from the N-terminal side in the region (VI). The region between these α-helix and β-sheets may include loops, α-helixes, or β-sheets.
αへリックス（Ｇ）
　αへリックス（Ｇ）は、そのＮ末端側のアミノ酸残基が前記αへリックス（Ｃ）のＣ末端側のアミノ酸残基を０位とした場合に３～１３位、好ましくは４～１２位、さらに好ましくは５～１１に存在し、アミノ酸残基数が４～１３、好ましくは５～１２、さらに好ましくは６～１１で構成される。
　αへリックス（Ｇ）のアミノ酸配列としては、特に限定されないが、例えば、ＥＳＬＮＳＥＧ等が挙げられる。αへリックス（Ｇ）のアミノ酸配列としては、具体的には、シュードモナス・エルギノーサ由来のものであれば、配列番号４に示す１６０～１６６位のアミノ酸残基が挙げられる。 α helix (G)
α-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 α-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 α helix (G) is not particularly limited, and examples thereof include ESLNSEG. Specifically, the amino acid sequence of α-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.
βシート（Ｅ）
　βシート（Ｅ）は、そのＮ末端側のアミノ酸残基が前記αへリックス（Ｇ）のＣ末端側のアミノ酸残基を０位とした場合に２４～３６位、好ましくは２７～３４位、さらに好ましくは２９～３２位に存在し、アミノ酸残基数が２～６、好ましくは３～５で構成される。
　βシート（Ｅ）のアミノ酸配列としては、特に限定されないが、例えば、ＹＳＷＳ等が挙げられる。βシート（Ｅ）のアミノ酸配列としては、具体的には、シュードモナス・エルギノーサ由来のものであれば、配列番号４に示す１９６～１９９位のアミノ酸残基が挙げられる。 β sheet (E)
The β 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 α 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 | seat (E), For example, YSWS etc. are mentioned. Specific examples of the amino acid sequence of the β sheet (E) include amino acid residues at positions 196 to 199 shown in SEQ ID NO: 4 as long as they are derived from Pseudomonas aeruginosa.
　本発明のポリペプチドは、前述のβシート（Ａ）、αへリックス（Ａ）、αへリックス（Ｂ）、αへリックス（Ｃ）、βシート（Ｂ）、及びαへリックス（Ｄ）を基本的に有するものであるが、好ましくは、更にαへリックス（Ｅ）、βシート（Ｃ）、βシート（Ｄ）、αへリックス（Ｇ）、及びβシート（Ｅ）を備える。 The polypeptide of the present invention comprises the aforementioned β sheet (A), α helix (A), α helix (B), α helix (C), β sheet (B), and α helix (D). Basically, it preferably has an α helix (E), a β sheet (C), a β sheet (D), an α helix (G), and a β sheet (E).
アミノ酸置換領域
　本発明のポリペプチドでは、前記βシート（Ａ）とαへリックス（Ａ）の間の領域が、表ＩのＮｏ．１～１２５に示すいずれかのアミノ酸残基を含み、及び／又は、前記βシート（Ｂ）とαへリックス（Ｄ）の間の領域が、表ＩＩのＮｏ．１～１５０に示すいずれかのアミノ酸残基を含む。
　本発明のポリペプチドにおいて、前記βシート（Ａ）とαへリックス（Ａ）の間の領域（ＩＩ）に存在するループを含む領域のアミノ酸配列、好ましくはそのＮ末端側のアミノ酸残基が前記βシート（Ａ）のＣ末端側のアミノ酸残基を０位とした場合に８～１５位、好ましくは１０～１３位、さらに好ましくは１１～１２位に存在するアミノ酸配列は、表ＩのＮｏ．１～１２５に示すいずれかのアミノ酸残基（３つのアミノ酸残基からなる配列）を含むように設定され、前記シート（Ｂ）とαへリックス（Ｄ）の間の領域（ＶＩ）に存在するループを含む領域のアミノ酸配列、好ましくはそのＮ末端側のアミノ酸残基が前記βシート（Ｂ）のＣ末端側のアミノ酸残基を０位とした場合に３～９位、好ましくは４～８位、さらに好ましくは５～７位に存在するアミノ酸配列は、表ＩＩのＮｏ．１～１５０に示すいずれかのアミノ酸残基（３つのアミノ酸残基からなる配列）を含むように設定される。領域（ＩＩ）と領域（ＶＩ）の少なくとも一方に、このような変異を導入することにより、ポリペプチドのリパーゼ活性の熱安定性が向上し得る。 Amino acid substitution region In the polypeptide of the present invention, the region between the β sheet (A) and the α 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 β sheet (B) and the α helix (D) is represented by No. 1 in Table II. Any amino acid residue shown in 1-150 is included.
In the polypeptide of the present invention, the amino acid sequence of a region containing a loop existing in the region (II) between the β sheet (A) and the α helix (A), preferably the N-terminal amino acid residue is When the amino acid residue on the C-terminal side of the β sheet (A) is 0 position, the amino acid sequence present at positions 8 to 15, preferably positions 10 to 13, more preferably positions 11 to 12, . It is set to include any of the amino acid residues shown in 1-125 (sequence consisting of three amino acid residues) and exists in the region (VI) between the sheet (B) and the α helix (D). 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 β 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). By introducing such a mutation into at least one of region (II) and region (VI), the thermal stability of the lipase activity of the polypeptide can be improved.
　リパーゼ活性の熱安定性がより一層向上し得る点で、領域（ＩＩ）に含まれる前記アミノ酸配列としては、好ましくは、表ＩのＮｏ．１～１２５、より好ましくはＮｏ．１～９６、より一層好ましくはＮｏ．１～６８、更に好ましくはＮｏ．１～５０、特に好ましくはＮｏ．１～４０、特に一層好ましくはＮｏ．１～３３に示すいずれかのアミノ酸残基が挙げられ、領域（ＶＩ）に含まれる前記アミノ酸配列としては、好ましくは表ＩＩのＮｏ．１～１５０、より好ましくはＮｏ．１～１２９、より一層好ましくはＮｏ．１～８８、更に好ましくはＮｏ．１～６８、特に好ましくはＮｏ．１～５５、特に一層好ましくはＮｏ．１～４０に示すいずれかのアミノ酸残基が挙げられる。 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.
　前記βシート（Ａ）とαへリックス（Ａ）の間の領域（ＩＩ）に存在するアミノ酸置換領域となるアミノ酸配列は、少なくとも１のアミノ酸残基がループ（Ａ）領域に含まれ、好ましくは１～３、より好ましくは２又は３のアミノ酸残基がループ（Ａ）領域に含まれる。 The amino acid sequence to be an amino acid substitution region present in the region (II) between the β sheet (A) and the α 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.
　ループ（Ａ）領域は、前記βシート（Ａ）とαへリックス（Ａ）の間の領域（ＩＩ）に存在する。ループ（Ａ）は、そのＮ末端側のアミノ酸残基がβシート（Ａ）のＣ末端側のアミノ酸残基を０位とした場合に１～１２位、好ましくは１～１１、より好ましくは８～１１に存在する。
　ループ（Ａ）を構成するアミノ酸残基数は、１～１３、好ましくは２～１２である。ループ（Ａ）のアミノ酸配列としては、特に限定されず、例えば、ｘ21ｘ22Ｖ（ｘ21は及びｘ22はそれぞれいずれのアミノ酸残基であってもよく、好ましくは、ｘ21はＡ又はＶであり、ｘ22はＧ又はＮである。）、ＧＶＤ、ＨＧＬＡＧＴＤＫＦＡＮＶ等が挙げられ、好ましくはＡＧＶ、ＨＧＬＡＧＴＤＫＦＡＮＶ、ＶＧＶ、ＧＶＤ等が挙げられるが、ループ（Ａ）のアミノ酸配列に、領域（ＩＩ）の前記アミノ酸配列の少なくとも１のアミノ酸残基が含まれることが好ましい。 The loop (A) region exists in the region (II) between the β sheet (A) and the α 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 β 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. For example, x 21 x 22 V (x 21 and x 22 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.
　前記βシート（Ｂ）とαへリックス（Ｄ）の間の領域（ＶＩ）に存在するアミノ酸置換領域となるアミノ酸配列は、少なくとも１のアミノ酸残基がループ（Ｂ）領域にあり、好ましくは１～３、より好ましくは２又は３のアミノ酸残基がループ（Ｂ）領域にある。 The amino acid sequence to be an amino acid substitution region existing in the region (VI) between the β sheet (B) and the α 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.
　ループ（Ｂ）領域は、前記βシート（Ｂ）とαへリックス（Ｄ）の間の領域（ＶＩ）に存在する。ループ（Ｂ）は、そのＮ末端側のアミノ酸残基がβシート（Ｂ）のＣ末端側のアミノ酸残基を０位とした場合に１～７位、好ましくは１～６位、更に好ましくは１～５位に存在する。ループ（Ｂ）を構成するアミノ酸残基数は、１～１２、好ましくは２～１１である。
　ループ（Ｂ）のアミノ酸配列としては、特に限定されず、例えばＴＳＴｘ23ｘ24ｘ25ＶＤ（ｘ23、ｘ24、及びｘ25はそれぞれいずれのアミノ酸残基であってもよく、好ましくは。ｘ23はＩ又はＧであり、ｘ24はＰ又はＴであり、ｘ25はＶ又はＬである。）、ＴＳＴＧＴＬＤＶ、ＡＮＤＧＬＶＧＴＣＳＳ等が挙げられ、好ましくは、ＴＳＴＩＰＬＶＤ、ＴＳＴＩＰＬＶＤ、ＴＳＴＧＴＬＤＶ、ＡＮＤＧＬＶＧＴＣＳＳが挙げられるが、ループ（Ｂ）のアミノ酸配列に、領域（ＶＩ）の前記アミノ酸配列の少なくとも１のアミノ酸残基が含まれることが好ましい。 The loop (B) region exists in the region (VI) between the β sheet (B) and the α helix (D). In the loop (B), 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 β-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. 23 is I or G, x 24 is P or T, and x 25 is V or L.), TSTGTLDV, ANDGLVGTCSS, etc. 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).
　本発明のポリペプチドの総アミノ酸残基数としては、２７０～３４０、好ましくは２８０～３３０、より好ましくは２８５～３２５、より一層好ましくは３００～３２５、特に好ましくは３１０～３２５である。 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.
本発明のポリペプチドの好適な一態様
　本発明のポリペプチドの好適な一態様として、具体的には、下記（１）～（４）に示すポリペプチドが挙げられる。
（１）配列番号１に示すアミノ酸配列において、２５～２７位のアミノ酸残基が表ＩのＮｏ．１～１２５に示すいずれかのアミノ酸残基に置換されている、及び／又は２３３～２３５位のアミノ酸残基が表ＩＩのＮｏ．１～１５０に示すいずれかのアミノ酸残基に置換されているアミノ酸配列からなるポリペプチド、
（２）配列番号２に示すアミノ酸配列において、２４～２６位のアミノ酸残基が表ＩのＮｏ．１～１２５に示すいずれかのアミノ酸残基に置換されている、及び／又は２３２～２３４位のアミノ酸残基が表ＩＩのＮｏ．１～１５０に示すいずれかのアミノ酸残基に置換されているアミノ酸配列からなるポリペプチド、
（３）配列番号３に示すアミノ酸配列において、２５～２７位のアミノ酸残基が表ＩのＮｏ．１～１２５に示すいずれかのアミノ酸残基に置換されている、及び／又は２３３～２３５位のアミノ酸残基が表ＩＩのＮｏ．１～１５０に示すいずれかのアミノ酸残基に置換されているアミノ酸配列からなるポリペプチド、
（４）配列番号４に示すアミノ酸配列において、２５～２７位のアミノ酸残基が表ＩのＮｏ．１～１２５に示すいずれかのアミノ酸残基に置換されている、及び／又は２３３～２３５位のアミノ酸残基が表ＩＩのＮｏ．１～１５０に示すいずれかのアミノ酸残基に置換されているアミノ酸配列からなるポリペプチド。 Preferred Embodiment of Polypeptide of the Present Invention As a preferred embodiment of the polypeptide of the present invention , specifically, polypeptides shown in the following (1) to (4) can be mentioned.
(1) In the amino acid sequence shown in SEQ ID NO: 1, the amino acid residues at positions 25 to 27 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. A polypeptide comprising an amino acid sequence substituted with any one of the amino acid residues shown in 1-150,
(2) In the amino acid sequence shown in SEQ ID NO: 2, the amino acid residues at positions 24 to 26 are Nos. Nos. 1 to 125 are substituted with amino acid residues and / or amino acid residues at positions 232 to 234 A polypeptide comprising an amino acid sequence substituted with any one of the amino acid residues shown in 1-150,
(3) In the amino acid sequence shown in SEQ ID NO: 3, the amino acid residues at positions 25 to 27 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. A polypeptide comprising an amino acid sequence substituted with any one of the amino acid residues shown in 1-150,
(4) In the amino acid sequence shown in SEQ ID NO: 4, the amino acid residues at positions 25 to 27 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. A polypeptide comprising an amino acid sequence substituted with any one of amino acid residues shown in 1-150.
　配列番号１に示すアミノ酸配列は、バークホルデリア・セパシア由来の野生型リパーゼのアミノ酸配列である。配列番号２に示すアミノ酸配列は、シュードモナス・グルマエ由来の野生型リパーゼのアミノ酸配列である。配列番号３に示すアミノ酸配列は、シュードモナス・フルオレセンス由来の野生型リパーゼのアミノ酸配列である。配列番号４に示すアミノ酸配列は、シュードモナス・エルギノーサの野生型リパーゼのアミノ酸配列である。なお、本発明においては、前記（１）～（４）のポリペプチドには、人為的に置換して得られるポリペプチドのみならず、そのようなアミノ酸配列を元々有するポリペプチドも含まれる。 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. In the present invention, the polypeptides of (1) to (4) include not only polypeptides obtained by artificial substitution, but also polypeptides originally having such amino acid sequences.
　前記（１）～（４）のポリペプチドにおいて、所定部位に置換される表Ｉ及びＩＩに示すアミノ酸残基の内、好ましいものについては、前述する通りである。 Of the amino acid residues shown in Tables I and II that are substituted at predetermined sites in the polypeptides (1) to (4), preferred ones are as described above.
　また、本発明のポリペプチドの他の好適な態様として、下記（５）～（１２）に示すポリペプチドが挙げられる。
（５）配列番号１に示すアミノ酸配列における２５～２７位のアミノ酸残基が表ＩのＮｏ．１～１２５に示すいずれかのアミノ酸残基に置換されている、及び／又は２３３～２３５位のアミノ酸残基が表ＩＩのＮｏ．１～１５０に示すいずれかのアミノ酸残基に置換されているアミノ酸配列において、前記置換が導入されたアミノ酸残基以外の１個又は数個のアミノ酸残基が置換、付加、挿入又は欠失されてなり、且つ、リパーゼ活性を有し、配列番号１に示すアミノ酸配列からなるポリペプチドに比して熱安定性が向上しているポリペプチド、
（６）配列番号２に示すアミノ酸配列における２４～２６位のアミノ酸残基が表ＩのＮｏ．１～１２５に示すいずれかのアミノ酸残基に置換されている、及び／又は２３２～２３４位のアミノ酸残基が表ＩＩのＮｏ．１～１５０に示すいずれかのアミノ酸残基に置換されているアミノ酸配列において、前記置換が導入されたアミノ酸残基以外の１個又は数個のアミノ酸残基が置換、付加、挿入又は欠失されてなり、且つ、リパーゼ活性を有し、配列番号２に示すアミノ酸配列からなるポリペプチドに比して熱安定性が向上しているポリペプチド、
（７）配列番号３に示すアミノ酸配列における２５～２７位のアミノ酸残基が表ＩのＮｏ．１～１２５に示すいずれかのアミノ酸残基に置換されている、及び／又は２３３～２３５位のアミノ酸残基が表ＩＩのＮｏ．１～１５０に示すいずれかのアミノ酸残基に置換されているアミノ酸配列において、前記置換が導入されたアミノ酸残基以外の１個又は数個のアミノ酸残基が置換、付加、挿入又は欠失されてなり、且つ、リパーゼ活性を有し、配列番号３に示すアミノ酸配列からなるポリペプチドに比して熱安定性が向上しているポリペプチド、
（８）配列番号４に示すアミノ酸配列における２５～２７位のアミノ酸残基が表ＩのＮｏ．１～１２５に示すいずれかのアミノ酸残基に置換されている、及び／又は２３３～２３５位のアミノ酸残基が表ＩＩのＮｏ．１～１５０に示すいずれかのアミノ酸残基に置換されているアミノ酸配列において、前記置換が導入されたアミノ酸残基以外の１個又は数個のアミノ酸残基が置換、付加、挿入又は欠失されてなり、且つ、リパーゼ活性を有し、配列番号４に示すアミノ酸配列からなるポリペプチドに比して熱安定性が向上しているポリペプチド、
（９）配列番号１に示すアミノ酸配列における２５～２７位のアミノ酸残基が表ＩのＮｏ．１～１２５に示すいずれかのアミノ酸残基に置換されている、及び／又は２３３～２３５位のアミノ酸残基が表ＩＩのＮｏ．１～１５０に示すいずれかのアミノ酸残基に置換されているアミノ酸配列において、配列番号１に示すアミノ酸配列に対する前記置換が導入されたアミノ酸残基を除いた配列同一性が８０％以上であり、且つ、リパーゼ活性を有し、配列番号１に示すアミノ酸配列からなるポリペプチドに比して熱安定性が向上しているポリペプチド、
（１０）配列番号２に示すアミノ酸配列における２４～２６位のアミノ酸残基が表ＩのＮｏ．１～１２５に示すいずれかのアミノ酸残基に置換されている、及び／又は２３２～２３４位のアミノ酸残基が表ＩＩのＮｏ．１～１５０に示すいずれかのアミノ酸残基に置換されているアミノ酸配列において、配列番号２に示すアミノ酸配列に対する前記置換が導入されたアミノ酸残基を除いた配列同一性が８０％以上であり、且つ、リパーゼ活性を有し、配列番号２に示すアミノ酸配列からなるポリペプチドに比して熱安定性が向上しているポリペプチド、
（１１）配列番号３に示すアミノ酸配列における２５～２７位のアミノ酸残基が表ＩのＮｏ．１～１２５に示すいずれかのアミノ酸残基に置換されている、及び／又は２３３～２３５位のアミノ酸残基が表ＩＩのＮｏ．１～１５０に示すいずれかのアミノ酸残基に置換されているアミノ酸配列において、配列番号３に示すアミノ酸配列に対する前記置換が導入されたアミノ酸残基を除いた配列同一性が８０％以上であり、且つ、リパーゼ活性を有し、配列番号３に示すアミノ酸配列からなるポリペプチドに比して熱安定性が向上しているポリペプチド、
（１２）配列番号４に示すアミノ酸配列における２５～２７位のアミノ酸残基が表ＩのＮｏ．１～１２５に示すいずれかのアミノ酸残基に置換されている、及び／又は２３３～２３５位のアミノ酸残基が表ＩＩのＮｏ．１～１５０に示すいずれかのアミノ酸残基に置換されているアミノ酸配列において、配列番号４に示すアミノ酸配列に対する前記置換が導入されたアミノ酸残基を除いた配列同一性が８０％以上であり、且つ、リパーゼ活性を有し、配列番号４に示すアミノ酸配列からなるポリペプチドに比して熱安定性が向上しているポリペプチド。 Other preferred embodiments of the polypeptide of the present invention include the polypeptides shown in the following (5) to (12).
(5) 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. 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. A polypeptide having a lipase activity and having improved thermal stability compared to a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1,
(6) 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. A polypeptide that has lipase activity and has improved thermal stability compared to a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2,
(7) 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. 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. A polypeptide having a lipase activity and having improved thermal stability compared to a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 3,
(8) 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. 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. A polypeptide having a lipase activity and having improved thermal stability compared to a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 4,
(9) 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. In the amino acid sequence substituted with any one of the amino acid residues shown in 1-150, 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, And a polypeptide having lipase activity and having improved thermal stability compared to a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1,
(10) 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, 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, And a polypeptide having lipase activity and improved thermal stability compared to a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2.
(11) 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. In the amino acid sequence substituted with any one of the amino acid residues shown in 1-150, 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, And a polypeptide having lipase activity and improved thermal stability compared to a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 3.
(12) 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. In the amino acid sequence substituted with any one of the amino acid residues shown in 1-150, 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.
　なお、本発明において、前記（５）～（１２）のポリペプチドには、人為的に置換して得られるポリペプチドのみならず、そのようなアミノ酸配列を元々有するポリペプチドも含まれる。 In the present invention, the polypeptides (5) to (12) include not only polypeptides obtained by artificial substitution, but also polypeptides having such an amino acid sequence.
　以下、前記（５）～（１２）のポリペプチドにおいて、前記表I及びＩＩに示すアミノ酸残基による置換がされているアミノ酸残基以外を「任意相違部位」と表記することもある。本明細書において、用語「任意相違部位」とは、ポリペプチドの特性に大きく影響しない限り、相違が許容される部位である。また、本明細書において、前記（１）～（４）のポリペプチドと比較して、任意相違部位においてアミノ酸配列に相違がみられるものの、前記（１）～（４）のポリペプチドと同程度又はそれ以上のリパーゼ活性を有し、かつ、前記（１）～（４）のポリペプチドと同程度又はそれ以上の熱安定性を有しているものを、前記（１）～（４）のポリペプチドの相違体という。また、前記ポリペプチドの相違体は、前記（１）～（４）のポリペプチドと比較して、任意相違部位においてアミノ酸配列に相違がみられるものの、当該ポリペプチドの特性が実質的に同一であることが好ましい。なお、「実質的に同一」とは、前記（１）～（４）のポリペプチドと同程度のリパーゼ活性及び熱安定性を有しているものをいう。 Hereinafter, in the polypeptides (5) to (12) above, 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”. In the present specification, 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. Further, in the present specification, although 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). Or 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. In addition, the 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).
　前記（５）及び（９）のポリペプチドは、前記（１）のポリペプチドの相違体である。前記（６）及び（１０）のポリペプチドは、前記（２）のポリペプチドの相違体である。前記（７）及び（１１）のポリペプチドは、前記（３）のポリペプチドの相違体である。前記（８）及び（１２）のポリペプチドは、前記（４）のポリペプチドの相違体である。 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).
　前記（５）～（８）のポリペプチドにおけるアミノ酸の相違は、置換、付加、挿入、および欠失の中から１種類の相違（例えば置換）のみを含むものであってもよく、２種以上の相違（例えば、置換と挿入）を含んでいても良い。前記（５）～（８）のポリペプチドにおいて、任意相違部位におけるアミノ酸の相違は、１個若しくは数個であればよく、例えば１～５０個、好ましくは１～２０個、１～１０個、１～８個、１～７個、１～６個、１～５個、又は１～４個、更に好ましくは１～３個、特に好ましくは１又は２個或いは１個が挙げられる。 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. In the polypeptides (5) to (8), 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.
　また、前記（９）～（１２）のポリペプチドにおいて、配列番号１～４に示す各アミノ酸配列に対する前記アミノ酸置換がされた部位を除いた配列同一性は、８０％以上であればよいが、好ましくは８５％以上又は９０％以上、更に好ましくは９５％以上、９６％以上、９７％以上、又は９８％以上、特に好ましくは９９％以上が挙げられる。 In the polypeptides (9) to (12), the 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.
　ここで、前記（９）～（１２）のポリペプチドにおいて、配列番号１～４に示す各アミノ酸配列に対する前記アミノ酸置換がされた部位を除いた配列同一性とは、配列番号１～４に示す各アミノ酸配列から前記任意相違部位のみを抜き出して、当該任意相違部位のみを比較して算出される配列同一性である。また、「配列同一性」とは、ＢＬＡＳＴ　ＰＡＣＫＡＧＥ［ｓｇｉ３２　ｂｉｔ　ｅｄｉｔｉｏｎ，Ｖｅｒｓｉｏｎ　２．０．１２；ａｖａｉｌａｂｌｅ　ｆｒｏｍ　Ｎａｔｉｏｎａｌ　Ｃｅｎｔｅｒ　ｆｏｒ　Ｂｉｏｔｅｃｈｎｏｌｏｇｙ　Ｉｎｆｏｒｍａｔｉｏｎ（ＮＣＢＩ）］のｂｌ２ｓｅｑ　ｐｒｏｇｒａｍ（Ｔａｔｉａｎａ　Ａ．Ｔａｔｓｕｓｏｖａ，Ｔｈｏｍａｓ　Ｌ．Ｍａｄｄｅｎ，ＦＥＭＳ　Ｍｉｃｒｏｂｉｏｌ．Ｌｅｔｔ．，Ｖｏｌ．１７４，ｐ２４７－２５０，１９９９）により得られるアミノ酸配列の同一性の値を示す。パラメーターは、Ｇａｐ　ｉｎｓｅｒｔｉｏｎ　Ｃｏｓｔ　ｖａｌｕｅ：１１、Ｇａｐ　ｅｘｔｅｎｓｉｏｎ　Ｃｏｓｔ　ｖａｌｕｅ：１に設定すればよい。 Here, in the polypeptides of (9) to (12) above, the 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 is shown in SEQ ID NOs: 1 to 4 This is sequence identity calculated by extracting only the arbitrary different sites from each amino acid sequence and comparing only the arbitrary different sites. “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.
　また、前記（５）～（１２）のポリペプチドの任意相違部位に導入されるアミノ酸置換の他の態様として、保存的置換が挙げられる。即ち、前記任意相違部位における置換としては、例えば、置換前のアミノ酸が非極性アミノ酸であれば他の非極性アミノ酸への置換、置換前のアミノ酸が非荷電性アミノ酸であれば他の非荷電性アミノ酸への置換、置換前のアミノ酸が酸性アミノ酸であれば他の酸性アミノ酸への置換、及び置換前のアミノ酸が塩基性アミノ酸であれば他の塩基性アミノ酸への置換が挙げられる。 Further, 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.
　前記（５）のポリペプチドにおいて、「リパーゼ活性を有し、配列番号１に示すアミノ酸配列からなるポリペプチドに比して熱安定性が向上しているポリペプチド」とは、リパーゼ活性があり、且つ下記条件で測定した残存活性が、同条件で測定した配列番号１に示すアミノ酸配列からなるポリペプチドの残存活性よりも高いことを意味する。具体的には、下記条件で測定したポリペプチドの残存活性が、同条件で測定した配列番号１に示すアミノ酸配列からなるポリペプチドの残存活性に比べて１．５倍以上、好ましくは２倍以上、更に好ましくは３倍以上高いことを意味する。なお、前記（６）～（１２）のポリペプチドも、同様である。なお、「残存活性」とは、熱処理後に残存するリパーゼ活性であり、ポリペプチドの熱処理前のリパーゼ活性値に対する熱処理後のリパーゼ活性値を百分率で示したものである。
（測定条件）
　熱処理：６０℃、３０分
　活性値の測定：Ｌｉｐａｓｅ　Ｋｉｔ　Ｓ（ＤＳ　ファーマバイオメディカル）を使用して、３７℃、２０分反応させた後、ＰｏｗｅｒＳｃａｎＨＴ（ＤＳ　ファーマバイオメディカル）にて測定した吸光値（４１２ｎｍ）から算出する。
　残存活性（残存率）（％）：［活性値（熱処理サンプル）／活性値（未処理サンプル）×１００］ In the polypeptide of (5) above, “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.
(Measurement condition)
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]
前記ポリペプチドをコードしているＤＮＡ
　本発明のポリペプチドをコードしているＤＮＡ（以下、「本発明のＤＮＡ」と表記することもある）は、例えば、野生型リパーゼのアミノ酸配列（配列番号１～４）をコードしているＤＮＡに前記アミノ酸変異を導入することにより得ることができる。また、本発明のＤＮＡは、遺伝子の全合成法によって人工合成することもできる。 DNA encoding the polypeptide
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.
　配列番号１に示すアミノ酸配列からなるポリペプチドをコードしているＤＮＡは、例えば、配列番号９に示される塩基配列として知られており、バークホルデリア・セパシア（Ｂｕｒｋｈｏｌｄｅｒｉａ　ｃｅｐａｃｉａ）のＭ－１２－３３株のゲノムＤＮＡからＰＣＲを用いた定法により単離することができる。 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.
　配列番号２に示すアミノ酸配列からなるポリペプチドをコードしているＤＮＡは、例えば、配列番号１０に示される塩基配列として知られており、シュードモナス・グルマエ（Ｐｓｅｕｄｏｍｏｎａｓ　ｇｌｕｍａｅ）のＰＧ１株のゲノムＤＮＡからＰＣＲを用いた定法により単離することができる。 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
　配列番号３に示すアミノ酸配列からなるポリペプチドをコードしているＤＮＡは、例えば、配列番号１１に示される塩基配列として知られており、シュードモナス・フルオレセンス（Ｐｓｅｕｄｏｍｏｎａｓ　ｆｌｕｏｒｅｓｃｅｎｃｅ）のＡＫ１０２株のゲノムＤＮＡからＰＣＲを用いた定法により単離することができる。 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.
　配列番号４に示すアミノ酸配列からなるポリペプチドをコードしているＤＮＡは、例えば、配列番号１２に示される塩基配列として知られており、シュードモナス・エルギノーサ（Ｐｓｅｕｄｏｍｏｎａｓ　ａｅｒｕｇｉｎｏｓａ）のＴＥ３２８５株のゲノムＤＮＡからＰＣＲを用いた定法により単離することができる。 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
　塩基配列の特定の部位に特定の変異を導入する方法は公知であり、例えばＤＮＡの部位特異的変異導入法等が利用できる。ＤＮＡ中の塩基を変換する具体的な方法としては、例えば、市販のキット（ＱｕｉｃｋＣｈａｎｇｅ　Ｌｉｇｈｔｎｉｎｇ　Ｓｉｔｅ－Ｄｉｒｅｃｔｅｄ　Ｍｕｔａｇｅｎｅｓｉｓ　ｋｉｔ：Ｓｔｒａｔａｇｅｎｅ製、ＫＯＤ－Ｐｌｕｓ－Ｍｕｔａｇｅｎｅｓｉｓ　ｋｉｔ：東洋紡製など）の利用等が挙げられる。 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.).
　塩基配列に変異を導入したＤＮＡは、ＤＮＡシーケンサーを用いて塩基配列を確認することができる。一旦、塩基配列が確定されると、その後は化学合成、クローニングされたプローブを鋳型としたＰＣＲ、又は当該塩基配列を有するＤＮＡ断片をプローブとするハイブリダイゼーションによって、前記ポリペプチドをコードするＤＮＡを得ることができる。 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.
　また、部位特異的突然変異誘発法等によって前記ペプチドをコードするＤＮＡの変異型であって変異前と同等の機能を有するものを合成することができる。なお、前記ペプチドをコードするＤＮＡに変異を導入するには、Ｋｕｎｋｅｌ法、Ｇａｐｐｅｄ　ｄｕｐｌｅｘ法、メガプライマーＰＣＲ法等の公知の手法によって行うことができる。 In addition, 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. In order to introduce a mutation into the DNA encoding the peptide, a known method such as Kunkel method, Gapped duplex method, or megaprimer PCR method can be used.
　本発明のＤＮＡは、コドン利用頻度を宿主に最適化したものが好ましく、コドン利用頻度を大腸菌に最適化させたＤＮＡがより好ましい。 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.
　コドン利用頻度を表す指標として、各コドンの宿主最適コドン利用頻度の総計を採択すればよい。最適コドンとは、同じアミノ酸に対応するコドンのうち利用頻度が最も高いコドンと定義される。コドン利用頻度は、宿主に最適化したものであれば特に限定されないが、例えば、大腸菌の最適コドンの一例として以下のものが挙げられる。
Ｆ：フェニルアラニン（ｔｔｔ）、Ｌ：ロイシン（ｃｔｇ）、Ｉ：イソロイシン（ａｔｔ）、Ｍ：メチオニン（ａｔｇ）、Ｖ：バリン（ｇｔｇ）、Ｙ：チロシン（ｔａｔ）、終止コドン（ｔａａ）、Ｈ：ヒスチジン（ｃａｔ）、Ｑ：グルタミン（ｃａｇ）、Ｎ：アスパラギン（ａａｔ）、Ｋ：リジン（ａａａ）、Ｄ：アスパラギン酸（ｇａｔ）、Ｅ：グルタミン酸（ｇａａ）、Ｓ：セリン（ａｇｃ）、Ｐ：プロリン（ｃｃｇ）、Ｔ：スレオニン（ａｃｃ）、Ａ：アラニン（ｇｃｇ）、Ｃ：システイン（ｔｇｃ）、Ｗ：トリプトファン（ｔｇｇ）、Ｒ：アルギニン（ｃｇｃ）、Ｇ：グリシン（ｇｇｃ）。 What is necessary is just to employ | adopt the total of the host optimal codon usage frequency of each codon as a parameter | index showing codon usage frequency. 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).
　本発明のＤＮＡの具体的態様として、配列番号９～１３に示す塩基配列を含むＤＮＡが挙げられる。配列番号９又は１３に示される塩基配列からなるＤＮＡは、前記（１）のポリペプチドであって、２５～２７位に表Ｉに示されるアミノ酸残基、２３３～２３５位に表ＩＩに示されるアミノ酸残基が導入されたポリペプチドをコードしている。配列番号１０に示される塩基配列からなるＤＮＡは、前記（２）のポリペプチドであって、２４～２６位に表Ｉに示されるアミノ酸残基、２３２～２３４位に表ＩＩに示されるアミノ酸残基が導入されたポリペプチドをコードしている。配列番号１１に示される塩基配列からなるＤＮＡは、前記（３）のポリペプチドであって、２５～２７位に表Ｉに示されるアミノ酸残基、２３３～２３５位に表ＩＩに示されるアミノ酸残基が導入されたポリペプチドをコードしている。配列番号１２に示される塩基配列からなるＤＮＡは、前記（４）のポリペプチドであって、２５～２７位に表Ｉに示されるアミノ酸残基、２３３～２３５位に表ＩＩに示されるアミノ酸残基が導入されたポリペプチドをコードしている。 Specific examples of the DNA of the present invention 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.
　また、本発明のＤＮＡには、（ｉ）リパーゼ活性を有し、配列番号１に示すアミノ酸配列からなるポリペプチドに比して熱安定性が向上しているポリペプチドをコードし、配列番号９又は１３に示す塩基配列からなるＤＮＡと相補的な塩基配列を含むＤＮＡと、ストリンジェントな条件下でハイブリダイズするＤＮＡ、（ｉｉ）リパーゼ活性を有し、配列番号２に示すアミノ酸配列からなるポリペプチドに比して熱安定性が向上しているポリペプチドをコードし、配列番号１０に示す塩基配列からなるＤＮＡと相補的な塩基配列を含むＤＮＡと、ストリンジェントな条件下でハイブリダイズするＤＮＡ、（ｉｉｉ）リパーゼ活性を有し、配列番号３に示すアミノ酸配列からなるポリペプチドに比して熱安定性が向上しているポリペプチドをコードし、配列番号１１に示す塩基配列からなるＤＮＡと相補的な塩基配列を含むＤＮＡと、ストリンジェントな条件下でハイブリダイズするＤＮＡ、（ｉｖ）リパーゼ活性を有し、配列番号４に示すアミノ酸配列からなるポリペプチドに比して熱安定性が向上しているポリペプチドをコードし、配列番号１２に示す塩基配列からなるＤＮＡと相補的な塩基配列を含むＤＮＡと、ストリンジェントな条件下でハイブリダイズするＤＮＡ、が包含される。 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; (iv) having lipase activity and shown in SEQ ID NO: 4 A DNA encoding a polypeptide having improved thermal stability as compared to a polypeptide comprising an amino acid sequence, and comprising a DNA comprising a base sequence complementary to the DNA comprising the base sequence represented by SEQ ID NO: 12, and stringent conditions DNA that hybridizes with is included.
　ここで、「ストリンジェントな条件下」とは、０．５％ＳＤＳ、５×デンハルツ〔Ｄｅｎｈａｒｔｚ’ｓ、０．１％ウシ血清アルブミン（ＢＳＡ）、０．１％ポリビニルピロリドン、０．１％フィコール４００〕および１００μｇ／ｍｌサケ精子ＤＮＡを含む６×ＳＳＣ（１×ＳＳＣは、０．１５Ｍ　ＮａＣｌ、０．０１５Ｍ　クエン酸ナトリウム、ｐＨ７．０）中で、５０℃～６５℃で４時間～一晩保温する条件をいう。 Here, “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 μg / 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.
　ストリンジェントな条件下でのハイブリダイゼーションは、具体的には、以下の手法によって行われる。即ち、ＤＮＡライブラリー又はｃＤＮＡライブラリーを固定化したナイロン膜を作成し、６×ＳＳＣ、０．５％　ＳＤＳ、５×デンハルツ、１００μｇ／ｍｌサケ精子ＤＮＡを含むプレハイブリダイゼーション溶液中、６５℃でナイロン膜をブロッキングする。その後、32Ｐでラベルした各プローブを加えて、６５℃で一晩保温する。このナイロン膜を６×ＳＳＣ中、室温で１０分間、０．１％ＳＤＳを含む２×ＳＳＣ中、室温で１０分間、０．１％ＳＤＳを含む０．２×ＳＳＣ中、４５℃で３０分間洗浄した後、オートラジオグラフィーをとり、プローブと特異的にハイブリダイズしているＤＮＡを検出することができる。 Specifically, 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 μg / 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.
　更に、本発明のＤＮＡには、（ｖ）リパーゼ活性を有し、配列番号１に示すアミノ酸配列からなるポリペプチドに比して熱安定性が向上しているポリペプチドをコードし、且つ配列番号９に示す塩基配列からなるＤＮＡに８０％以上の相同性を有するＤＮＡ、（ｖｉ）リパーゼ活性を有し、配列番号２に示すアミノ酸配列からなるポリペプチドに比して熱安定性が向上しているポリペプチドをコードし、且つ配列番号１０に示す塩基配列からなるＤＮＡに８０％以上の相同性を有するＤＮＡ、（ｖｉｉ）リパーゼ活性を有し、配列番号３に示すアミノ酸配列からなるポリペプチドに比して熱安定性が向上しているポリペプチドをコードし、且つ配列番号１１に示す塩基配列からなるＤＮＡに８０％以上の相同性を有するＤＮＡ、並びに（ｖｉｉｉ）リパーゼ活性を有し、配列番号４に示すアミノ酸配列からなるポリペプチドに比して熱安定性が向上しているポリペプチドをコードし、且つ配列番号１２に示す塩基配列からなるＤＮＡに８０％以上の相同性を有するＤＮＡも包含される。当該相同性として、好ましくは８５％以上又は９０％以上、更に好ましくは９５％以上、９６％以上、又は９７％以上、特に好ましくは９８％以上又は９９％以上が挙げられる。 Furthermore, 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 A DNA encoding a polypeptide having improved thermal stability as compared to the DNA having a nucleotide sequence of SEQ ID NO: 11 and having a homology of 80% or more, and (v ii) It encodes a polypeptide having lipase activity and having improved thermal stability as compared with the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 4 and has a DNA sequence consisting of 80% of the DNA shown in SEQ ID NO: 12 Also included are DNAs with% homology or greater. The homology is preferably 85% or more or 90% or more, more preferably 95% or more, 96% or more, or 97% or more, and particularly preferably 98% or more or 99% or more.
　ここで、ＤＮＡの「相同性」は、基準配列を照会配列として比較するアルゴリズムをもった公開又は市販されているソフトウェアを用いて計算される。具体的には、ＢＬＡＳＴ、ＦＡＳＴＡ、又はＧＥＮＥＴＹＸ（ソフトウエア開発株式会社製）等を用いることができ、これらはデフォルトパラメーターに設定して使用すればよい。 Here, 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. Specifically, BLAST, FASTA, GENETYX (manufactured by Software Development Co., Ltd.) or the like can be used, and these may be used by setting default parameters.
組換えベクター
　本発明のペプチドをコードするＤＮＡを含む組換えベクター（以下、「本発明の組換えベクター」と表記することもある）は、発現ベクターに本発明のＤＮＡを挿入することにより得ることができる。 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.
　本発明の組換えベクターには、本発明のＤＮＡに作動可能に連結されたプロモーター等の制御因子が含まれる。制御因子としては、代表的にはプロモーターが挙げられるが、更に必要に応じてエンハンサー、ＣＣＡＡＴボックス、ＴＡＴＡボックス、ＳＰＩ部位等の転写要素が含まれていてもよい。また、作動可能に連結とは、本発明のＤＮＡを調節するプロモーター、エンハンサー等の種々の制御因子と本発明のＤＮＡが、宿主細胞中で作動し得る状態で連結されることをいう。 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 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. The term “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.
　発現ベクターとしては、宿主内で自律的に増殖し得るファージ、プラスミド、又はウイルスから遺伝子組換え用として構築されたものが好適である。このような発現ベクターは公知であり、例えば、商業的に入手可能な発現ベクターとしては、ｐＱＥ系ベクター（株式会社キアゲン）、ｐＤＲ５４０、ｐＲＩＴ２Ｔ（ＧＥヘルスケアバイオサイエンス株式会社）、ｐＥＴ系ベクター（メルク株式会社）等が挙げられる。発現ベクターは、宿主細胞との適切な組み合わせを選んで使用すればよく、例えば、大腸菌を宿主細胞とする場合には、ｐＥＴ系ベクターとＤＨ５α大腸菌株の組み合わせ、ｐＥＴ系ベクターとＢＬ２１（ＤＥ３）大腸菌株の組み合わせ、又はｐＤＲ５４０ベクターとＪＭ１０９大腸菌株の組み合わせ等が好ましく挙げられる。 As the expression vector, 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. For example, commercially available expression vectors include pQE vectors (Qiagen), pDR540, pRIT2T (GE Healthcare Biosciences), pET vectors (Merck). Etc.). For the expression vector, an appropriate combination with a host cell may be selected and used. For example, when E. coli is used as a host cell, a combination of a pET vector and a DH5α 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.
形質転換体
　本発明の組換えベクターを用いて宿主を形質転換することによって形質転換体（以下、「本発明の形質転換体」と表記することもある）が得られる。 Transformant A transformant (hereinafter sometimes referred to as “transformant of the present invention”) is obtained by transforming a host using the recombinant vector of the present invention.
　形質転換体の製造に使用される宿主としては、組換えベクターが安定であり、且つ自律増殖可能で外来性遺伝子の形質を発現できるのであれば特に制限されないが、例えば、大腸菌（Ｅｓｃｈｅｒｉｃｈｉａ　ｃｏｌｉ）等のエッシェリヒア属、バチルス・ズブチリス（Ｂａｃｉｌｌｕｓ　ｓｕｂｔｉｌｉｓ）等のバチルス属、シュードモナス・プチダ（Ｐｓｅｕｄｏｍｏｎａｓ　ｐｕｔｉｄａ）等のシュードモナス属等に属する細菌；酵母等が好適な例として挙げられるが、その他、動物細胞、昆虫細胞、植物等であってもよい。これらの中でも大腸菌が特に好ましい。 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. For example, 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. Among these, Escherichia coli is particularly preferable.
　本発明の形質転換体は、宿主に本発明の組換えベクターを導入することによって得ることができ、宿主に組換えベクターを導入する条件は、宿主の種類等に応じて適宜設定すればよい。宿主が細菌の場合であれば、例えば、カルシウムイオン処理によるコンピテントセルを用いる方法及びエレクトロポレーション法等が挙げられる。宿主が酵母の場合であれば、例えば、電気穿孔法（エレクトロポレーション法）、スフェロプラスト法及び酢酸リチウム法等が挙げられる。宿主が動物細胞の場合であれば、例えば、エレクトロポレーション法、リン酸カルシウム法及びリポフェクション法等が挙げられる。宿主が昆虫細胞の場合であれば、例えば、リン酸カルシウム法、リポフェクション法及びエレクトロポレーション法等が挙げられる。宿主が植物胞の場合であれば、例えば、エレクトロポレーション法、アグロバクテリウム法、パーティクルガン法、及びＰＥＧ法等が挙げられる。 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. When 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. When the host is yeast, for example, electroporation method (electroporation method), spheroplast method, lithium acetate method and the like can be mentioned. When the host is an animal cell, examples thereof include an electroporation method, a calcium phosphate method, and a lipofection method. When the host is an insect cell, examples include calcium phosphate method, lipofection method, electroporation method and the like. When the host is a plant vesicle, examples thereof include an electroporation method, an Agrobacterium method, a particle gun method, and a PEG method.
　本発明の組換えベクターが宿主に組み込まれたか否かの確認は、ＰＣＲ法、サザンハイブリダイゼーション法、及びノーザンハイブリダイゼーション法等により行うことができる。 Whether or not the recombinant vector of the present invention has been incorporated into the host can be confirmed by PCR, Southern hybridization, Northern hybridization, or the like.
　ＰＣＲ法よって本発明の組換えベクターが宿主に組み込まれたか否かを確認する場合、例えば、形質転換体から組換えベクターを分離・精製すればよい。 When confirming whether or not the recombinant vector of the present invention has been incorporated into the host by PCR, for example, the recombinant vector may be separated and purified from the transformant.
　組換えベクターの分離・精製は、例えば、宿主が細菌の場合、細菌を溶菌して得られる溶菌物に基づいて行われる。溶菌の方法としては、例えばリゾチームなどの溶菌酵素により処理が施され、必要に応じてプロテアーゼ、及び他の酵素並びにラウリル硫酸ナトリウム（ＳＤＳ）等の界面活性剤が併用される。 For example, when the host is a bacterium, the recombinant vector is separated and purified based on a lysate obtained by lysing the bacterium. As a method of lysis, for example, treatment is performed with a lytic enzyme such as lysozyme, and a protease, other enzyme, and a surfactant such as sodium lauryl sulfate (SDS) are used in combination as necessary.
　更に、凍結融解およびフレンチプレス処理のような物理的破砕方法を組み合わせてもよい。溶菌物からのＤＮＡの分離・精製は、例えば、フェノール処理およびプロテアーゼ処理による除蛋白処理、リボヌクレアーゼ処理、アルコール沈殿処理並びに市販のキットを適宜組み合わせることにより行うことができる。 Furthermore, physical crushing methods such as freezing and thawing and French press treatment may be combined. 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.
　ＤＮＡの切断は、常法に従い、例えば制限酵素処理を用いて行うことができる。制限酵素としては、例えば特定のヌクレオチド配列に作用するＩＩ型制限酵素を用いる。ＤＮＡと発現ベクターとの結合は、例えばＤＮＡリガーゼを用いて行う。 The DNA can be cleaved according to a conventional method, for example, using a restriction enzyme treatment. As 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.
　その後、分離・精製したＤＮＡを鋳型として、本発明のＤＮＡに特異的なプライマーを設計してＰＣＲを行う。ＰＣＲにより得られた増幅産物についてアガロースゲル電気泳動、ポリアクリルアミドゲル電気泳動、キャピラリー電気泳動等を行い、臭化エチジウムおよびＳＹＢＲ　Ｇｒｅｅｎ液等により染色し、そして増幅産物をバンドとして検出することにより、形質転換されたことを確認することができる。 Thereafter, 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.
　また、予め蛍光色素等により標識したプライマーを用いてＰＣＲを行い、増幅産物を検出することもできる。さらに、マイクロプレート等の固相に増幅産物を結合させ、蛍光および酵素反応等により増幅産物を確認する方法も採用してもよい。 In addition, PCR can be performed using a primer previously labeled with a fluorescent dye or the like to detect an amplification product. Furthermore, 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.
ポリペプチドの製造
　本発明のポリペプチドは、本発明の形質転換体を培養することによって製造することができる。 Production of Polypeptide The polypeptide of the present invention can be produced by culturing the transformant of the present invention.
　形質転換体の培養条件は、宿主の栄養生理的性質を考慮して適宜設定すればよいが、好ましくは液体培養が挙げられる。また、工業的製造を行う場合であれば、通気攪拌培養が好ましい。 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.
　培地の栄養源としては、形質転換体の生育に必要とされるものが使用され得る。炭素源としては、資化可能な炭素化合物であればよく、例えば、グルコース、シュークロース、ラクトース、マルトース、糖蜜、ピルビン酸等が挙げられる。 As the nutrient source of the medium, those required for the growth of the transformant can be used. The carbon source may be any carbon compound that can be assimilated, and examples thereof include glucose, sucrose, lactose, maltose, molasses, and pyruvic acid.
　窒素源としては、資化可能な窒素化合物であればよく、例えば、ペプトン、肉エキス、酵母エキス、カゼイン加水分解物、大豆粕アルカリ抽出物が挙げられる。 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.
　炭素源及び窒素源の他に、例えば、リン酸塩、炭酸塩、硫酸塩、マグネシウム、カルシウム、カリウム、鉄、マンガンおよび亜鉛などの塩類、特定のアミノ酸並びに特定のビタミンなどを必要に応じて使用してもよい。 In addition to the carbon source and nitrogen source, for example, 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.
　培養温度は、本発明の形質転換体が生育可能であり、且つ本発明の形質転換体が本発明のポリペプチドを産生する範囲で適宜設定し得るが、好ましくは１５～３７℃程度である。培養は、本発明のポリペプチドが最高収量に達する時期を見計らって適当時期に完了すればよく、通常は培養時間が１２～４８時間程度である。 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.
　本発明の形質転換体を培養し、培養液を遠心分離などの方法により培養上清または菌体を回収し、菌体は超音波およびフレンチプレスといった機械的方法又はリゾチーム等の溶菌酵素により処理を施し、必要に応じてプロテアーゼ等の酵素やラウリル硫酸ナトリウム（ＳＤＳ）等の界面活性剤を使用することにより可溶化し、本発明のポリペプチドを含む水溶性画分を得ることができる。 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.
　また、適当な発現ベクターと宿主を選択することにより、発現した本発明のポリペプチドを培養液中に分泌させることもできる。 The expressed polypeptide of the present invention can also be secreted into the culture medium by selecting an appropriate expression vector and host.
　上記のようにして得られた本発明のポリペプチドを含む水溶性画分は、そのまま精製処理に供してもよいが、該水溶性画分中の本発明のポリペプチドを濃縮した後に精製処理に供してもよい。 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.
　濃縮は、例えば、減圧濃縮、膜濃縮、塩析処理、親水性有機溶媒（例えば、メタノール、エタノールおよびアセトン）による分別沈殿法等により行うことができる。 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).
　本発明のポリペプチドの精製処理は、例えば、ゲルろ過、吸着クロマトグラフィー、イオン交換クロマトグラフィー、アフィニティクロマトグラフィー等の方法を適宜組み合わせることによって行うことができる。 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.
　前記精製処理は既に公知であり、適当な文献、雑誌および教科書等を参照することで進めることができる。このようにして精製された本発明のポリペプチドは、必要に応じて、凍結乾燥、真空乾燥、スプレードライ等により粉末化して市場に流通させることができる。 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.
組成物
　本発明のポリペプチドは、例えば組成物の形態で提供されてもよい。前記組成物は、本発明のポリペプチドを有効成分として含む。前記組成物の精製の程度は特に限定されないが、前記組成物は、本発明の効果に影響を与えない程度に、他の成分を含んでいてもよい。他の成分としては、培地由来の成分や夾雑タンパク質等が挙げられる。 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.
　前記組成物はまた、他の酵素を含んでいてもよい。他の酵素としては、例えば、アミラーゼ（α－アミラーゼ、β－アミラーゼ、グルコアミラーゼ）、グルコシダーゼ（α－グルコシダーゼ、β－グルコシダーゼ）、ガラクトシダーゼ（α－ガラクトシダーゼ、β－ガラクトシダーゼ）、プロテアーゼ（酸性プロテアーゼ、中性プロテアーゼ、アルカリプロテアーゼ）、ペプチダーゼ（ロイシンペプチダーゼ、アミノペプチダーゼ）、リパーゼ、エステラーゼ、セルラーゼ、ホスファターゼ（酸性ホスファターゼ、アルカリホスファターゼ）、ヌクレアーゼ、デアミナーゼ、オキシダーゼ、デヒドロゲナーゼ、グルタミナーゼ、ペクチナーゼ、カタラーゼ、デキストラナーゼ、トランスグルタミナーゼ、蛋白質脱アミド酵素、プルラナーゼ等が挙げられる。 The composition may also contain other enzymes. Examples of other enzymes include amylase (α-amylase, β-amylase, glucoamylase), glucosidase (α-glucosidase, β-glucosidase), galactosidase (α-galactosidase, β-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, pullulanase, and the like.
　前記組成物における本発明のポリペプチドの含有量としては、特に限定されないが、好ましくは前記組成物の全タンパク質中１０質量％以上、より好ましくは３０質量％以上が挙げられる。前記組成物の形態は、特に限定されないが、例えば、液体、粉末、顆粒等が挙げられる。前記組成物は、一般的に公知の方法で調製することができる。 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. Although 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.
酵素剤
　本発明のポリペプチド、又は本発明のポリペプチドを含む組成物は、例えば酵素剤の形態で提供されてもよい。酵素剤は、本発明のポリペプチド、又は前記組成物の他、賦形剤、緩衝剤、懸濁剤、安定剤、保存剤、防腐剤、生理食塩水などを含有していてもよい。賦形剤としてはデンプン、デキストリン、マルトース、トレハロース、乳糖、Ｄ－グルコース、ソルビトール、Ｄ－マンニトール、白糖、グリセロール等を用いることができる。緩衝剤としてはリン酸塩、クエン酸塩、酢酸塩等を用いることができる。安定剤としてはプロピレングリコール、アスコルビン酸等を用いることができる。保存剤としてはフェノール、塩化ベンザルコニウム、ベンジルアルコール、クロロブタノール、メチルパラベン等を用いることができる。防腐剤としてはエタノール、塩化ベンザルコニウム、パラオキシ安息香酸、クロロブタノール等と用いることができる。前記酵素剤における前記ポリペプチドの含有量としては、前記ポリペプチドの効果が発揮される範囲で適宜設定される。 Enzyme Agent 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. As the 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. As the stabilizer, propylene glycol, ascorbic acid or the like can be used. As preservatives, phenol, benzalkonium chloride, benzyl alcohol, chlorobutanol, methylparaben, and the like can be used. As 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.
用途
本発明のポリペプチドは、リパーゼ活性を有し熱安定性に優れる。そのため、本発明のポリペプチドは、リパーゼによる酵素処理が必要とされる用途、例えば、油脂（トリグリセリド）の処理（分解、エステル交換）が要求される用途に利用することができる。リパーゼによる酵素処理が必要とされる用途が要求される用途としては、例えば、油脂の精製、食品又は食品素材の製造、消化酵素剤等の医薬品、化粧品添加剤、食品工場等の排水処理、医薬中間体製造、ファインケミカル素材製造、機能代替油脂製造、等の用途が挙げられ、好ましくは排水処理用途が挙げられる。本発明のポリペプチド、又は本発明のポリペプチドを含む前述の酵素剤は、油脂に作用させることにより、油脂処理することができる。そのような油脂処理方法も本発明の一つである。 Use 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.
　また、本発明のポリペプチド又は本発明のポリペプチドを含む前述の酵素剤を、油脂を含む排水に作用させることにより、排水を処理することができる。排水としては、特に限定されず、家庭用排水、工業用排水、農業用排水等が挙げられるが、なかでも油脂を多く含む排水の処理に適用することが好ましい。そのような排水処理方法も本発明の一つである。 Moreover, 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.
　また、本発明のポリペプチド又は本発明のポリペプチドを含む前述の酵素剤を医薬中間体原料に作用させることにより、医薬中間体を製造することができる。医薬中間体原料としては、例えばコレステロール脂肪酸エステル、モノアシルグリセロール、グリシッド酸エステル類、ベンゾチアゼピン類化合物等が挙げられる。そのような医薬中間体の製造方法も本発明の一つである。 Alternatively, 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. Examples of 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.
　更に、本発明のポリペプチド又は本発明のポリペプチドを含む前述の酵素剤をファインケミカル素材原料に作用させることにより、ファインケミカル素材を製造することができる。ファインケミカル素材としては、例えば香料（乳フレーバー、大環状ラクトン、フェネチルアルコール配糖体等）、化粧品原料、乳化剤等が挙げられる。そのようなファインケミカル素材の製造方法も本発明の一つである。 Furthermore, 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. Examples of 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.
以下に、実施例を挙げて本発明を更に詳しく説明するが、本発明は下記の実施例に何ら限定されるものではない。 Hereinafter, the present invention will be described in more detail with reference to examples. However, the present invention is not limited to the following examples.
＜実験例１＞
Ｅ．ｃｏｌｉ　発現用プラスミドの構築
　Ｅ．ｃｏｌｉ発現系を構築するにあたり、Ｂ．ｃｅｐａｃｉａ　Ｍ１２－３３　の各遺伝子（Ｌｉｐ　Ａ（配列番号９）、Ｌｉｐ　Ｘ（配列番号１４））をＥ．ｃｏｌｉ　発現用にコドン最適化した遺伝子を全合成した。全合成した構造遺伝子（Ｌｉｐ　Ａ；配列番号１３）を鋳型としたＰＣＲ増幅（ＰｒｉｍｅＳＴＡＲ　ＧＸＬ　ＤＮＡ　Ｐｏｌｙｍｅｒａｓｅ　（ＴａＫａＲａ））、プライマー（フォワードプライマー：５’－ＴＴＴＴＣＣＡＴＧＧＣＴＣＧＴＴＣＴＡＴＧＣＧＴＴＣＴＣＧ－３’、リバースプライマー：５’－ＡＡＡＡＡＡＧＣＴＴＡＡＡＣＡＣＣＣＧＣＣＡＧＴＴＴＣＡＧＡＣＧＧ－３’））にてリンカー配列（Ｎｃｏ　Ｉ，　Ｈｉｎｄ　ＩＩＩ）を付加した後、精製（ＮｕｃｌｅｏＳｐｉｎ　Ｇｅｌ　ａｎｄ　ＰＣＲ　Ｃｌｅａｎ－ｕｐ　（ＭＡＣＨＥＲＥＹ－ＮＡＧＥＬ））して遺伝子断片（ＢＣＬ－ＬｉｐＡ）を取得した。 <Experimental example 1>
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. PCR amplification (PrimeSTAR GXL DNA Polymerase (TaKaRa)), primer (forward primer: 5'-TTTCCATGGCTCGTTCTATGCGTTCTGCGAGAGTATAGCAGAGTAGTAGAC) 3 ′)), a linker sequence (Nco I, Hind III) was added and purified (NucleoSpin Gel and PCR Clean-up (MACHEREY-NAGEL)) to obtain a gene fragment (BCL-LipA).
　遺伝子断片（ＢＣＬ－ＬｉｐＡ）、及びｐＥＴＤｕｅｔ－１（Ｎｏｖａｇｅｎ）は、制限酵素（Ｎｃｏ　Ｉ（ＴａＫａＲａ），Ｈｉｎｄ　ＩＩＩ（ＴａＫａＲａ））で処理した後、ライゲーション（ＤＮＡ　Ｌｉｇａｔｉｏｎ　Ｋｉｔ　＜Ｍｉｇｈｔｙ　Ｍｉｘ＞（ＴａＫａＲａ））して、Ｅ．ｃｏｌｉ　ＤＨ５α　（ＴａＫａＲａ）に形質転換することで、Ｅ．ｃｏｌｉ　ＢＣＬ－ＬｉｐＡを取得した。Ｅ．ｃｏｌｉ　ＢＣＬ－ＬｉｐＡからのプラスミド抽出は、ＬＢ　Ｂｒｏｔｈ　Ｂａｓｅ（ｉｎｖｉｔｒｏｇｅｎ）＋　Ａｍｐ　：　１００μｇ／ｍＬ：５ｍＬに植菌して振とう培養（３７℃、１６ｈ、１４０ｒｐｍ）した後、ＮｕｃｌｅｏＳｐｉｎ　Ｐｌａｓｍｉｄ　ＥａｓｙＰｕｒｅ（ＭＡＣＨＥＲＥＹ－ＮＡＧＥＬ）を用いて抽出して、プラスミド（ｐＥＴＢＣＬ－ＬｉｐＡ）を取得した。 The gene fragment (BCL-LipA) and pETDuet-1 (Novagen) were treated with restriction enzymes (Nco I (TaKaRa), Hind III (TaKaRa)) and then ligated (DNA Ligation Kit <Mighty Mix> (TaKaRa)). E. by transformation into E. coli DH5α (TaKaRa). E. coli BCL-LipA was obtained. E. Plasmid extraction from E. coli BCL-LipA was carried out by inoculating LB Broth Base (invitrogen) + Amp: 100 μg / 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).
　シャペロン遺伝子（Ｌｉｐ　Ｘ）についても、同様の操作を行った。すなわち、全合成したシャペロン遺伝子（Ｌｉｐ　Ｘ；配列番号１５）を鋳型としてＰＣＲ増幅（プライマー（フォワードプライマー：５’－ＴＴＴＴＣＡＴＡＴＧＡＣＣＧＣＡＣＧＴＧＡＡＧＧＴＣＧＣＧＣ－３’、リバースプライマー：５’－ＡＡＡＡＣＴＣＧＡＧＴＴＡＣＴＧＴＧＣＡＧＡＡＣＣＣＧＣＡＣＣＧ－３’）にてリンカー配列（Ｎｄｅ　Ｉ，Ｘｈｏ　Ｉ）を付加した後、精製（ＮｕｃｌｅｏＳｐｉｎ　Ｇｅｌ　ａｎｄ　ＰＣＲ　Ｃｌｅａｎ－ｕｐ　（ＭＡＣＨＥＲＥＹ－ＮＡＧＥＬ））して遺伝子断片（ＢＣＬ－ＬｉｐＸ）を取得した。 The same operation was performed for the chaperone gene (Lip X). Specifically, a linker sequence (PCR primer (forward primer: 5′-TTTTCATATGACCGCACGTGAAGGTCGGCGC-3 ′, reverse primer: 5′-AAAAACTCGAGTTTACTGGCAGACACCGCGCCG-3 ′) using the fully synthesized chaperone gene (Lip X; SEQ ID NO: 15) as a template. After adding Nde I, Xho I), purification (NucleoSpin Gel and PCR Clean-up (MACHEREY-NAGEL)) was performed to obtain a gene fragment (BCL-LipX).
　遺伝子断片（ＢＣＬ－ＬｉｐＸ）、及びｐＥＴＢＣＬ－ＬｉｐＡを制限酵素（ＮｄｅＩ（ＴａＫａＲａ），Ｘｈｏ　Ｉ（ＴａＫａＲａ））で処理した後、ライゲーションしてＥ．ｃｏｌｉ　ＤＨ５αに形質転換することで、Ｅ．ｃｏｌｉ　ＢＣＬ－ＬｉｐＡＸを取得した。Ｅ．ｃｏｌｉ　ＢＣＬ－ＬｉｐＡＸからのプラスミド抽出は、ＬＢ　Ｂｒｏｔｈ　Ｂａｓｅ　＋　Ａｍｐ　：　１００μｇ／ｍＬ：５ｍＬに植菌して振とう培養（３７℃、１６ｈ、１４０ｒｐｍ）した後、ＮｕｃｌｅｏＳｐｉｎ　Ｐｌａｓｍｉｄ　ＥａｓｙＰｕｒｅ（ＭＡＣＨＥＲＥＹ－ＮＡＧＥＬ）を用いて抽出して、Ｅ．ｃｏｌｉ発現プラスミド（ｐＥＴＢＣＬ－ＬｉｐＡＸ）を取得した。 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 DH5α. E. coli BCL-LipAX was obtained. E. Plasmid extraction from E. coli BCL-LipAX was inoculated into LB Broth Base + Amp: 100 μg / 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．ｃｏｌｉ　発現系の構築
　取得したＥ．ｃｏｌｉ発現プラスミド（ｐＥＴＢＣＬ－ＬｉｐＡＸ）をＥ．ｃｏｌｉ　ＢＬ２１（ＤＥ３）　（Ｎｉｐｐｏｎｇｅｎｅ）に形質転換して、Ｅ．ｃｏｌｉ　発現菌株：Ｅ．ｃｏｌｉ　ＢＬ２１（ＢＣＬ－ＬｉｐＡＸ）を取得した。 E. E. coli expression system constructed E. coli expression plasmid (pETBCL-LipAX) was transformed into E. coli. E. coli BL21 (DE3) (Nippongene) E. coli expression strain: E. coli E. coli BL21 (BCL-LipAX) was obtained.
変異株のシークエンス確認
　構築したプラスミド（ｐＥＴＢＣＬ－ＬｉｐＡ、ｐＥＴＢＣＬ－ＬｉｐＡＸ）のシークエンス確認は、Ｓｅｑｕｅｎｃｅ　Ｐｒｉｍｅｒ（ｐＥＴ　Ｕｐｓｔｒｅａｍ　Ｐｒｉｍｅｒ：５’－ＡＴＧＣＧＴＣＣＧＧＣＧＴＡＧＡ－３’、ＤｕｅｔＤＯＷＮ１　Ｐｒｉｍｅｒ：５’－ＧＡＴＴＡＴＧＣＧＧＣＣＧＴＧＴＡＣＡＡ－３’、ＤｕｅｔＵＰ２　Ｐｒｉｍｅｒ：５’－ＴＴＧＴＡＣＡＣＧＧＣＣＧＣＡＴＡＡＴＣ－３’、Ｔ７　Ｔｅｒｍｉｎａｔｏｒ　Ｐｒｉｍｅｒ：５’－ＧＣＴＡＧＴＴＡＴＴＧＣＴＣＡＧＣＧＧ－３’）を用いて行った。 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').
＜実験例２＞
Ｌｉｐａｓｅ　（ＢＣＬ）の変異導入点の選定、及びランダム変異ライブラリーの作製
（Ｌｉｐａｓｅ（ＢＣＬ）の変異導入点の選定）
　Ｂｕｒｋｈｏｌｄｅｒｉａ　ｃｅｐａｃｉａ由来Ｌｉｐａｓｅの立体構造情報〔１〕、〔２〕を参考にして、１２箇所の変異導入点（Ｌ１～Ｌ１２）を選定した。
＊〔１〕Ｋｉｍ，Ｋ．Ｋ．；Ｓｏｎｇ，Ｈ．Ｋ．；Ｓｈｉｎ，Ｄ．Ｈ．；Ｓｕｈ，Ｓ．Ｗ．Ｓｔｒｕｃｔｕｒｅ．１９９７，５，１７３－１８５、
＊〔２〕Ｌａｎｇ，Ｄ．Ａ．；Ｍａｎｎｅｓｓｅ，Ｍ．Ｌ．Ｍ．；Ｄｅ　Ｈａ　ａｓ，Ｇ．；Ｖｅｒｈｅｉｊ，　Ｈ．Ｍ．；Ｄｉｊｋｓｔｒａ，Ｂ．Ｗ．Ｅ　ｕｒ　Ｊ　Ｂｉｏｃｈｅｍ．１９９８，２５４，３３３－３４０ <Experimental example 2>
Selection of mutation introduction point of Lipase (BCL) and preparation of random mutation library (selection of mutation introduction point of Lipase (BCL))
Twelve mutation introduction points (L1 to L12) were selected with reference to the three-dimensional structure information [1] and [2] of Lipase derived from Burkholderia cepacia.
* [1] Kim, K .; K. Song, H .; K. Shin, D .; H. Suh, S .; W. Structure. 1997, 5, 173-185,
* [2] Lang, D .; A. Mannesse, M .; L. M.M. De Ha as, G .; Verheij, H .; M.M. Dijkstra, B .; W. Eur J Biochem. 1998,254,333-340
（ランダム変異株の作製）
　選定した１２箇所の変異導入点についてランダム変異ライブラリーを作製するため、ランダムＰｒｉｍｅｒを設計した。 (Production of random mutants)
Random Primers were designed to create a random mutation library for the 12 selected mutation introduction points.
・Ｌ１（Ａ７４Ｘ／Ａ７５Ｘ／Ｔ７６Ｘ）：
（フォワードプライマー：　５’－ＮＮＫＮＮＫＮＮＫＧＧＣＧＣＡＡＣＣＡＡＡＧＴＴＡＡＣＣＴＧＧＴＴＧ－３’、
リバースプライマー：　５’－ＣＡＧＣＡＣＡＧＴＴＴＴＣＡＣＡＴＡＣＧＣＣＡＧＣ－３’）
・Ｌ２（Ｖ１９９Ｘ／Ｇ２００Ｘ／Ｇ２０Ｘ）：
（フォワードプライマー：　５’－ＮＮＫＮＮＫＮＮＫＡＡＣＡＣＴＣＡＣＣＴＧＣＴＧＴＡＣＴＣＴＴＧＧＧＣ－３’、
リバースプライマー：　５’－ＡＧＴＣＴＣＧＧＴＣＧＧＣＧＣＡＣＣ－３’）
・Ｌ３（Ｌ１２７Ｘ／Ａ１２８Ｘ／Ｙ１２９Ｘ）：
（フォワードプライマー：　５’－ＮＮＫＮＮＫＮＮＫＧＡＣＣＣＧＡＣＴＧＧＣＣＴＧＴＣＴＴＣＴＡＣＣ－３’、
リバースプライマー：　５’－ＡＡＣＧＣＣＣＴＧＡＡＣＧＡＡＡＴＣＣＧＣＧ－３’）
・Ｌ４（Ｐ２１６Ｘ／Ｔ２１７Ｘ／Ｉ２１８Ｘ）：
（フォワードプライマー：　５’－ＮＮＫＮＮＫＮＮＫＴＣＴＧＴＴＴＴＣＧＧＴＧＴＴＡＣＴＧＧＴＧＣＧＡＣ－３’、
リバースプライマー：　５’－ＣＴＧＧＡＴＣＧＣＡＧＴＡＣＣＣＧＣＣＣＡＡＧ－３’）
・Ｌ５（Ｓ２１９Ｘ／Ｖ２２０Ｘ／Ｆ２２１Ｘ）：
（フォワードプライマー：　５’－ＮＮＫＮＮＫＮＮＫＧＧＴＧＴＴＡＣＴＧＧＴＧＣＧＡＣＴＧＡＴＡＣＣＴＣＴＡＣ－３’、
リバースプライマー：　５’－ＧＡＴＧＧＴＣＧＧＣＴＧＧＡＴＣＧＣＡＧＴＡＣ－３’）
・Ｌ６（Ｇ２２２Ｘ／Ｖ２２３Ｘ／Ｔ２２４Ｘ）：
（フォワードプライマー：　５’－ＮＮＫＮＮＫＮＮＫＧＧＴＧＣＧＡＣＴＧＡＴＡＣＣＴＣＴＡＣＴＡＴＣＣＣＧＣ－３’、
リバースプライマー：　５’－ＧＡＡＡＡＣＡＧＡＧＡＴＧＧＴＣＧＧＣＴＧＧＡＴＣＧＣ－３’）
・Ｌ７（Ｐ２３３Ｘ／Ｌ２３４Ｘ／Ｖ２３５Ｘ）：
（フォワードプライマー：　５’－ＮＮＫＮＮＫＮＮＫＧＡＴＣＣＧＧＣＡＡＡＣＧＣＡＣＴＧＧＡＣＣ－３’、
リバースプライマー：　５’－ＧＡＴＡＧＴＡＧＡＧＧＴＡＴＣＡＧＴＣＧＣＡＣＣＡＧＴＡＡＣ－３’）
・Ｌ８（Ｒ２５８Ｘ／Ｇ２５９Ｘ／Ｓ２６０Ｘ）：
（フォワードプライマー：　５’－ＮＮＫＮＮＫＮＮＫＧＧＴＣＡＧＡＡＣＧＡＴＧＧＴＧＴＧＧＴＧＴＣＴＡＡＧＴＧ－３’、
リバースプライマー：　５’－ＧＴＴＣＡＣＣＡＴＡＡＣＧＧＴＧＣＣＧＧＴＡＣＣＡＡＡＣ－３’）
・Ｌ９（Ｑ２９２Ｘ／Ｌ２９３Ｘ／Ｌ２９４Ｘ）：
（フォワードプライマー：　５’－ＮＮＫＮＮＫＮＮＫＧＧＴＧＴＴＣＧＴＧＧＴＧＣＴＡＡＣＧＣＧＧＡＡＧＡＴＣ－３’、
リバースプライマー：　５’－ＧＴＴＧＡＴＣＴＣＧＴＣＣＡＧＧＴＧＧＴＴＣＣＡＴＴＴＧＴＡＡＧＡＧ－３’）
・Ｌ１０（Ｇ２５Ｘ／Ｖ２６Ｘ／Ｌ２７Ｘ）：
（フォワードプライマー：　５’－ＮＮＫＮＮＫＮＮＫＧＡＧＴＡＣＴＧＧＴＡＣＧＧＴＡＴＴＣＡＧＧＡＡＧＡＣＣＴＧＣ－３’、
リバースプライマー：　５’－ＡＧＣＡＴＡＣＴＴＡＴＣＧＧＴＧＣＣＡＧＴＣＡＧＡＣＣＡＴＧ－３’）
・Ｌ１１（Ｐ５８Ｘ／Ｎ５９Ｘ／Ｇ６０Ｘ）：
（フォワードプライマー：　５’－ＮＮＫＮＮＫＮＮＫＣＧＣＧＧＣＧＡＡＣＡＧＣＴＧＣＴＧＧＣＧＴＡＴＧＴＧＡＡＡＡＣ－３’、
リバースプライマー：　５’－ＧＣＣＧＴＣＧＴＣＧＧＡＣＴＧＧＡＡＡＣＣＡＧＡＣＡＧＧＴＴＣ－３’）
・Ｌ１２（Ｑ３９Ｘ／Ｒ４０Ｘ／Ｇ４１Ｘ）：
（フォワードプライマー：　５’－ＮＮＫＮＮＫＮＮＫＧＣＧＡＣＴＧＴＴＴＡＣＧＴＴＧＣＧＡＡＣＣＴＧＴＣＴＧＧＴＴＴＣ－３’、
リバースプライマー：　５’－ＣＴＧＣＡＧＧＴＣＴＴＣＣＴＧＡＡＴＡＣＣＧＴＡＣＣＡＧＴＡＣＴＣ－３’） 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′-NNKNNKNNKTCTGTTTTCGGTGTTACTGGTGCAC-3 ′,
Reverse primer: 5'-CTGGATCCGAGTACCCCGCCCAAG-3 ')
L5 (S219X / V220X / F221X):
(Forward primer: 5′-NNKNNKNNKGGGTGTACTGGGTCGACTACTATACCTCTAC-3 ′,
Reverse primer: 5'-GATGGTCGGCTGGATCGCAGTAC-3 ')
L6 (G222X / V223X / T224X):
(Forward primer: 5′-NNKNNKNNKGGGTCGCGACTGATACCTCTACTATCCCCGC-3 ′,
Reverse primer: 5'-GAAAACAGAGATGGTCCGCTGGATCGC-3 ')
L7 (P233X / L234X / V235X):
(Forward primer: 5′-NNKNNKNNKGATCCCGGCAAACGCCACTGGACC-3 ′,
Reverse primer: 5′-GATAGTAGAGGTATCAGGTCGCACCAGTAAC-3 ′)
L8 (R258X / G259X / S260X):
(Forward primer: 5′-NNKNNKNNKGGGTCAGAACGATGGGTGTGGTTCTAAGTG-3 ′,
Reverse primer: 5'-GTTCACCATAACGGTGGCCGTACCAAAC-3 ')
L9 (Q292X / L293X / L294X):
(Forward primer: 5′-NNKNNKNNKGGGTGTTCGTGGTGCTAACGCGGAAGATC-3 ′,
Reverse primer: 5'-GTTGATCTCGTCCCAGGTGGTCTCTTTGTAAGAG-3 ')
L10 (G25X / V26X / L27X):
(Forward primer: 5′-NNKNNKNNKGAGTACTGGTACGGTATTCAGGGAAGACCCTGC-3 ′,
Reverse primer: 5'-AGCATACTTATCGGTGCCCATCCAGACCATG-3 ')
L11 (P58X / N59X / G60X):
(Forward primer: 5′-NNKNNKNNKCGCGCGGCAACACGCTGCTGGCGTATGTGAAAAC-3 ′,
Reverse primer: 5′-GCCGTCGTCGGAACTGGGAAAACCAGACAGGTTC-3 ′)
L12 (Q39X / R40X / G41X):
(Forward primer: 5'-NNKNNKNNKGCCGACTGTTTACGTTGGAACCTGTCTGGTTTC-3 ',
Reverse primer: 5′-CTGCAGGTCTTCCTGAATACCGTACCAGTACTC-3 ′)
　各変異導入点へのランダム変異導入は、プラスミド（ｐＥＴＢＣＬ－ＬｉｐＡＸ）を鋳型として、ランダムＰｒｉｍｅｒを用いたＰＣＲ増幅（ＰｒｉｍｅＳＴＡＲ　ＧＸＬ　ＤＮＡ　Ｐｏｌｙｍｅｒａｓｅ　（ＴａＫａＲａ））にて行った。ＰＣＲ増幅後、Ｄｐｎ　Ｉ（ＴａＫａＲａ）を用いた鋳型プラスミドの処理（３７℃、１６ｈ）、及びＴ４　ｐｏｌｙｍｅｒａｓｅ　（Ｔｏｙｏｂｏ）、Ｌｉｇａｔｉｏｎ　Ｈｉｇｈ　（Ｔｏｙｏｂｏ）を用いたライゲーション反応（１６℃、ｏ／ｎ）をした後、Ｅ．ｃｏｌｉ　ＢＬ２１（ＤＥ３）に形質転換して、各変異導入点にランダム変異導入されたランダム変異株（　Ｅ．ｃｏｌｉ　ＢＬ２１（ＢＣＬ－Ｒａｎ　Ｌ１　～　ＢＣＬ－Ｒａｎ　Ｌ１２））を取得した。 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. After PCR amplification, 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. Later, 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.
　（ランダム変異ライブラリーの作製）
　ランダム変異株（Ｅ．ｃｏｌｉ　ＢＬ２１（ＢＣＬ－Ｒａｎ　Ｌ１　～　ＢＣＬ－Ｒａｎ　Ｌ１２））を用いたランダム変異ライブラリーの作製は、２段階にて行った。 (Preparation of random mutation library)
Preparation of a random mutation library using a random mutant strain (E. coli BL21 (BCL-Ran L1 to BCL-Ran L12)) was performed in two steps.
１．ＬＢ　Ａｇａｒ　＋　０．１％　Ｔｒｉｂｕｔｙｒｉｎ（Ａｍｐ：１００μｇ／ｍＬ）プレートを用いたプレートアッセイ
　ランダム変異株から加水分解活性を有した変異株を選抜するため、ＬＢ　Ａｇａｒ（ｉｎｖｉｔｒｏｇｅｎ）　＋　０．１％　Ｔｒｉｂｕｔｙｒｉｎ（ｗａｋｏ）（Ａｍｐ　：　１００　μｇ／ｍＬ）を用いたプレートアッセイを行った。ランダム変異株を上記のプレート培地に植菌して培養（３７℃、２４ｈ）した後、クリアハロー形成が認められた変異株を選抜した。 1. Plate assay using LB Agar + 0.1% Tributyrin (Amp: 100 μg / mL) plate In order to select mutants having hydrolytic activity from random mutants, LB Agar (invitrogen) + 0.1% Tributyrin ( plate assay using Wako) (Amp: 100 μg / mL). A random mutant was inoculated into the above plate medium and cultured (37 ° C., 24 h), and then a mutant having clear halo formation was selected.
２．Ｔｅｒｉｆｆｉｃ　Ｂｒｏｔｈ（Ａｍｐ：１００μｇ／ｍＬ）を用いたランダム変異ライブラリーの作製
　各変異導入点についてランダム変異ライブラリーを作製するため、上記で選抜した変異株を９６穴Ｄｅｅｐ　Ｗｅｌｌ　Ｐｌａｔｅ（Ｃｏａｓｔａｒ）に分注したＴｅｒｉｆｆｉｃ　Ｂｒｏｔｈ（ｉｎｖｉｔｒｏｇｅｎ）（Ａｍｐ　：　１００　μｇ／ｍＬ）：１ｍＬ　に植菌（１８０株／変異導入点）後、振とう培養機（Ｔａｉｔｅｃ）にて培養（３３℃、４８ｈ、１，０００ｒｐｍ）した。酵素発現の誘導は、培養：２４ｈ時点で終濃度０．１　ｍＭとなるようにＩＰＴＧを培養液に添加して行った。培養後、遠心分離（３，３００　ｇ　×１５ｍｉｎ、４℃）にて菌体を回収した後、Ｂ－ＰＥＲ（ＴｈｅｒｍｏＦｉｓｈｅｒ）を用いた溶菌処理（２５℃、１，０００ｒｐｍ）にて酵素抽出液を取得した。酵素抽出液を遠心分離（３，３００ｇ×１５ｍｉｎ、４℃）した後、上清を回収して、各変異導入点のランダム変異ライブラリーとした。
※ランダム変異ライブラリーを用いて、検討した菌株数
１つの変異導入点について、１８０株を培養・評価（概算合計：２１６０株（内訳：１８０株／変異点×１２箇所＝２１６０株））
　ＢＣＬのランダム変異ライブラリーの評価にて、Ｌ７（Ｐ２３３／Ｌ２３４／Ｖ２３５）、及びＬ１０（Ｇ２５／Ｖ２６／Ｌ２７）への変異導入により、安定性の向上が確認された。 2. Preparation of random mutation library using Terific Broth (Amp: 100 μg / mL) In order to prepare a random mutation library for each mutation introduction point, the mutant strain selected above was dispensed into 96-well Deep Well Plate (Coastar). Terific Broth (invitrogen) (Amp: 100 μg / mL): Inoculated into 1 mL (180 strains / mutation introduction point) and then cultured in a shaker (Taitec) (33 ° C., 48 h, 1,000 rpm) . Induction of enzyme expression was performed by adding IPTG to the culture solution so that the final concentration was 0.1 mM after 24 hours of culture. After culturing, 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.
* Using a random mutation library, 180 strains were cultivated and evaluated for one mutation introduction point (approximate total: 2160 strains (breakdown: 180 strains / mutation points x 12 locations = 2160 strains))
In the evaluation of the random mutation library of BCL, improvement in stability was confirmed by introducing mutations into L7 (P233 / L234 / V235) and L10 (G25 / V26 / L27).
＜実験例３＞
ＢＣＬ（Ｇ２５、Ｖ２６、Ｌ２７、Ｐ２３３、Ｌ２３４、Ｖ２３５）－飽和変異ライブラリーの作製
　（ＢＣＬ（Ｇ２５、Ｖ２６、Ｌ２７、Ｐ２３３、Ｌ２３４、Ｖ２３５）－飽和変異Ｐｒｉｍｅｒの設計）
　各変異点を構成するアミノ酸の飽和変異ライブラリーを作製して、安定性の向上に寄与する置換アミノ酸を検討した。
　各変異点の飽和変異ライブラリーを作製するため、飽和変異プライマーを設計した。表５～１０にプライマーを示す。 <Experimental example 3>
BCL (G25, V26, L27, P233, L234, V235)-Construction of saturation mutation library (BCL (G25, V26, L27, P233, L234, V235)-Designation of saturation mutation Primer)
A saturation mutation library of amino acids constituting each mutation point was prepared, and substitution amino acids contributing to the improvement of stability were examined.
Saturation mutation primers were designed to create a saturation mutation library at each mutation point. Tables 5 to 10 show the primers.
Figure JPOXMLDOC01-appb-T000007
Figure JPOXMLDOC01-appb-T000007
Figure JPOXMLDOC01-appb-T000008
Figure JPOXMLDOC01-appb-T000008
Figure JPOXMLDOC01-appb-T000009
Figure JPOXMLDOC01-appb-T000009
Figure JPOXMLDOC01-appb-T000010
Figure JPOXMLDOC01-appb-T000010
Figure JPOXMLDOC01-appb-T000011
Figure JPOXMLDOC01-appb-T000011
Figure JPOXMLDOC01-appb-T000012
Figure JPOXMLDOC01-appb-T000012
ＢＣＬ（Ｇ２５、Ｖ２６、Ｌ２７、Ｐ２３３、Ｌ２３４、Ｖ２３５）－飽和変異プラスミドの作製
　各変異点への飽和変異導入は、プラスミド（ｐＥＴＢＣＬ－ＬｉｐＡＸ）を鋳型として、飽和変異Ｐｒｉｍｅｒを用いたＰＣＲ増幅（ＰｒｉｍｅＳＴＡＲ　ＧＸＬ　ＤＮＡ　Ｐｏｌｙｍｅｒａｓｅ　（ＴａＫａＲａ））にて行った。
　ＰＣＲ増幅後、Ｄｐｎ　Ｉ（ＴａＫａＲａ）を用いた鋳型プラスミドの処理（３７℃、１６ｈ）、及びＴ４　ｐｏｌｙｍｅｒａｓｅ　（Ｔｏｙｏｂｏ）、Ｌｉｇａｔｉｏｎ　Ｈｉｇｈ　（Ｔｏｙｏｂｏ）を用いたライゲーション反応（１６℃、ｏ／ｎ）をした後、Ｅ．ｃｏｌｉ　ＤＨ５αに形質転換して、各変異点について飽和変異株（　Ｅ．ｃｏｌｉ　ＤＨ５α（ＢＣＬ－Ｇ２５Ａ～Ｇ２５Ｙ、ＢＣＬ－Ｖ２６Ａ～Ｖ２６Ｙ、ＢＣＬ－Ｌ２７Ａ～Ｌ２７Ｙ、ＢＣＬ－Ｐ２３３Ａ～Ｐ２３３Ｙ、　ＢＣＬ－Ｌ２３４Ａ～Ｌ２３４Ｙ、ＢＣＬ－Ｖ２３５Ａ～Ｖ２３５Ｙ））を取得した。
　各飽和変異株からのプラスミド抽出は、ＬＢ　Ｂｒｏｔｈ　Ｂａｓｅ（ｉｎｖｉｔｒｏｇｅｎ）＋Ａｍｐ：１００μｇ／ｍＬで振とう培養（３７℃、１６ｈ、１４０ｒｐｍ）した後、ＮｕｃｌｅｏＳｐｉｎ　Ｐｌａｓｍｉｄ　ＥａｓｙＰｕｒｅ（ＭＡＣＨＥＲＥＹ－ＮＡＧＥＬ）を用いて抽出して、飽和変異プラスミド（ｐＥＴＢＣＬ－Ｇ２５Ａ～Ｇ２５Ｙ、ｐＥＴＢＣＬ－Ｖ２６Ａ～Ｖ２６Ｙ、ｐＥＴＢＣＬ－Ｌ２７Ａ～Ｌ２７Ｙ、ｐＥＴＢＣＬ－Ｐ２３３Ａ～Ｐ２３３Ｙ、ｐＥＴＢＣＬ－Ｌ２３４Ａ～Ｌ２３４Ｙ、ｐＥＴＢＣＬ－Ｖ２３５Ａ～Ｖ２３５Ｙ）を取得した。
　取得したプラスミドのシークエンス確認は、Ｓｅｑｕｅｎｃｅ　Ｐｒｉｍｅｒ（ｐＥＴ　Ｕｐｓｔｒｅａｍ　Ｐｒｉｍｅｒ：５’－ＡＴＧＣＧＴＣＣＧＧＣＧＴＡＧＡ－３’、　ＤｕｅｔＤＯＷＮ１　Ｐｒｉｍｅｒ：５’－ＧＡＴＴＡＴＧＣＧＧＣＣＧＴＧＴＡＣＡＡ－３’）を用いて行った。 Preparation of BCL (G25, V26, L27, P233, L234, V235) -saturation mutation plasmid Saturation mutation introduction into each mutation point was carried out by PCR amplification using a plasmid (pETBCL-LipAX) as a template (PrimeSTAR) GXL DNA Polymerase (TaKaRa)).
After PCR amplification, template plasmid treatment with Dpn I (TaKaRa) (37 ° C., 16 h) and ligation reaction (16 ° C., o / n) with T4 polymerase (Toyobo) and Ligation High (Toyobo) were performed. Later, E.E. E. coli DH5α (BCL-G25A to G25Y, BCL-V26A to V26Y, BCL-L27A to L27Y, BCL-P233A to P233Y, BCL-L234A to L234Y) , BCL-V235A to V235Y)).
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 μg / 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 ′).
　（ＢＣＬ（Ｇ２５、Ｖ２６、Ｌ２７、Ｐ２３３、Ｌ２３４、Ｖ２３５）－　Ｅ．ｃｏｌｉ　発現菌株の作製）
　取得した飽和変異プラスミドをＥ．ｃｏｌｉ　ＢＬ２１（ＤＥ３）　（Ｎｉｐｐｏｎｇｅｎｅ）に形質転換して、Ｅ．ｃｏｌｉ　発現菌株：Ｅ．ｃｏｌｉ　ＢＬ２１（ＢＣＬ－Ｇ２５Ａ～Ｇ２５Ｙ、ＢＣＬ－Ｖ２６Ａ～Ｖ２６Ｙ、ＢＣＬ－Ｌ２７Ａ～Ｌ２７Ｙ、ＢＣＬ－Ｐ２３３Ａ～Ｐ２３３Ｙ、　ＢＣＬ－Ｌ２３４Ａ～Ｌ２３４Ｙ、ＢＣＬ－Ｖ２３５Ａ～Ｖ２３５Ｙ））を取得した。 (BCL (G25, V26, L27, P233, L234, V235)-Production of E. coli expression strain)
The obtained saturation mutant plasmid was designated as E. coli. 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.
　（ＢＣＬ（Ｇ２５、Ｖ２６、Ｌ２７、Ｐ２３３、Ｌ２３４、Ｖ２３５）－飽和変異ライブラリーの作製）
　各変異点の飽和変異ライブラリーを作製するため、上記で作製したＥ．ｃｏｌｉ発現菌株を９６穴Ｄｅｅｐ　Ｗｅｌｌ　Ｐｌａｔｅ（Ｃｏａｓｔａｒ）に分注したＴｅｒｉｆｆｉｃ　Ｂｒｏｔｈ（ｉｎｖｉｔｒｏｇｅｎ）（Ａｍｐ：１００μｇ／ｍＬ）：　１ｍＬ　に植菌（１８０株／変異導入点）後、振とう培養機（ＴＡＩＴＥＣ）にて培養（３３℃、４８ｈ、１，０００ｒｐｍ）した。酵素発現の誘導は、培養：２４ｈ時点で終濃度０．１　ｍＭとなるようにＩＰＴＧを培養液に添加して行った。培養後、遠心分離（３，３００ｇ×１５ｍｉｎ、４℃）にて菌体を回収した後、Ｂ－ＰＥＲ（ＴｈｅｒｍｏＦｉｓｈｅｒ）を用いた溶菌処理（２５℃、１，０００ｒｐｍ）にて酵素抽出液を取得した。酵素抽出液を遠心分離（３，３００ｇ×１５ｍｉｎ、４℃）した後、上清を回収して、各変異点の飽和変異ライブラリーとした。 (BCL (G25, V26, L27, P233, L234, V235)-Preparation of a saturation mutation library)
In order to prepare a saturation mutation library of each mutation point, the E. coli prepared above was used. E. coli expression strain was dispensed into 96-well Deep Well Plate (Coastar). (33 ° C., 48 h, 1,000 rpm). Induction of enzyme expression was performed by adding IPTG to the culture solution so that the final concentration was 0.1 mM after 24 hours of culture. After culturing, 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.
＜実験例４＞
活性測定法、及び各ポリペプチド変異ライブラリーの評価（熱安定性）
（活性測定法）
　リパーゼ（ＢＣＬ）、実験例２で調製したランダム変異ライブラリー（Ｌ７、Ｌ１０）及び実験例３で調製した各変異点の飽和変異ライブラリーの活性値は、Ｌｉｐａｓｅ　Ｋｉｔ　Ｓ（ＤＳ　ファーマバイオメディカル）を使用して、３７℃、２０分反応させた後、ＰｏｗｅｒＳｃａｎＨＴ（ＤＳ　ファーマバイオメディカル）にて測定した吸光値（４１２ｎｍ）から算出した。測定サンプルの希釈は、２０ｍＭ　Ｐｏｔａｓｓｉｕｍ　Ｐｈｏｓｐｈａｔｅ　Ｂｕｆｆｅｒ　ｐＨ７．０を用いた。 <Experimental example 4>
Activity measurement method and evaluation of each polypeptide mutation library (thermal stability)
(Activity measurement method)
The activity values of the lipase (BCL), the random mutation library (L7, L10) prepared in Experimental Example 2 and the saturation mutation library of each mutation point prepared in Experimental Example 3 were determined using Lipase Kit S (DS Pharma Biomedical). It was calculated from the absorbance value (412 nm) measured by PowerScanHT (DS Pharma Biomedical) after reacting at 37 ° C. for 20 minutes. Dilution of the measurement sample was performed using 20 mM Potassium Phosphate Buffer pH 7.0.
（各変異ライブラリーの評価（熱安定性））
　各変異ライブラリーの熱安定性の評価は、熱処理（６０℃、３０分）したサンプルの活性の残存率（活性値（熱処理サンプル）／活性値（未処理サンプル）×　１００）で比較した。活性測定は、Ｌｉｐａｓｅ　Ｋｉｔ　Ｓ（ＤＳ　ファーマバイオメディカル）を使用した。 (Evaluation of each mutation library (thermal stability))
Evaluation of the thermal stability of each mutation library was made by comparing the activity remaining ratio (activity value (heat-treated sample) / activity value (untreated sample) × 100) of the heat-treated (60 ° C., 30 minutes) sample. For the activity measurement, Lipase Kit S (DS Pharma Biomedical) was used.
（ＰＣＲ条件）
　なお、ＢＣＬのＥ．ｃｏｌｉ発現系の構築、及び変異導入株の作製は、全ての検討において　ＰｒｉｍｅＳＴＡＲ　ＧＸＬ　ＤＮＡ　Ｐｏｌｙｍｅｒａｓｅ　（ＴａＫａＲａ）を使用した。反応組成は、５×ＰｒｉｍｅＳＴＡＲ　ＧＸＬ　Ｂｕｆｆｅｒ：１０μｌ、ｄＮＴＰ　Ｍｉｘｔｕｒｅ（２．５　ｍＭ　ｅａｃｈ）：４μｌ、フォワードプライマー：１０ｐｍｏｌ、リバースプライマー：１０　ｐｍｏｌ、Ｔｅｍｐｌａｔｅ：１０ｎｇ、ＰｒｉｍｅＳＴＡＲ　ＧＸＬ　ＤＮＡ　Ｐｏｌｙｍｅｒａｓｅ：１μｌ、滅菌蒸留水：ｕｐ　ｔｏ　５０μｌ　である。反応条件は、９８℃：１０ｓｅｃ、６０℃：３０ｓｅｃ、６８℃：１．５ｍｉｎ　を３０ｃｙｃｌｅ　である。 (PCR conditions)
BCL E.I. In all studies, PrimeSTAR GXL DNA Polymerase (TaKaRa) was used for the construction of an E. coli expression system and the production of a mutation-introduced strain. Reaction composition: 5 × PrimeStar GXL Buffer: 10 μl, dNTP Mixture (2.5 mMeach): 4 μl, Forward primer: 10 pmol, Reverse primer: 10 pmol, Template: 10 ng, PrimeSTAR GXL DNA Polymerase: 1 μl, Distilled water: 1 μl up to 50 μl. The reaction conditions are 98 ° C .: 10 sec, 60 ° C .: 30 sec, 68 ° C .: 1.5 min, 30 cycles.
各ポリペプチド変異ライブラリーの熱安定性の評価結果を表１１、及び表１２に示す。 Tables 11 and 12 show the evaluation results of the thermal stability of each polypeptide mutation library.
Figure JPOXMLDOC01-appb-T000013
Figure JPOXMLDOC01-appb-I000014
Figure JPOXMLDOC01-appb-I000015
Figure JPOXMLDOC01-appb-I000016
Figure JPOXMLDOC01-appb-T000013
Figure JPOXMLDOC01-appb-I000014
Figure JPOXMLDOC01-appb-I000015
Figure JPOXMLDOC01-appb-I000016
Figure JPOXMLDOC01-appb-T000017
Figure JPOXMLDOC01-appb-I000018
Figure JPOXMLDOC01-appb-I000019
Figure JPOXMLDOC01-appb-I000020
Figure JPOXMLDOC01-appb-I000021
Figure JPOXMLDOC01-appb-T000017
Figure JPOXMLDOC01-appb-I000018
Figure JPOXMLDOC01-appb-I000019
Figure JPOXMLDOC01-appb-I000020
Figure JPOXMLDOC01-appb-I000021
　表１１によれば、表１１に示すＮｏ．１～１２５のいずれかのアミノ酸配列の変異が導入されたポリペプチドは、熱処理後のリパーゼ活性の残存率が、野生型よりも高いことが示された。また、表１１に示すＮｏ．１～６８のいずれかのアミノ酸配列の変異が導入されたポリペプチドは、熱処理後のリパーゼ活性の残存率が、野生型よりも２倍以上高く、更に、Ｎｏ．１～４０のいずれかのアミノ酸配列の変異が導入されたポリペプチドは、野生型よりも３倍以上高いことが示された。また、表１２によれば、表１２に示すＮｏ．１～１５０のいずれかのアミノ酸配列の変異が導入されたポリペプチドは、熱処理後のリパーゼ活性の残存率が、野生型よりも高いことが示された。また、表１２に示すＮｏ．１～８８のいずれかのアミノ酸配列の変異が導入されたポリペプチドは、熱処理後のリパーゼ活性の存存率が、野生型よりも４倍以上高く、更に、Ｎｏ．１～５５のいずれかのアミノ酸配列の変異が導入されたポリペプチドは、野生型よりも６倍以上高いことが示された。 According to Table 11, No. It was shown that the polypeptide into which any amino acid sequence of 1-125 was introduced has a higher lipase activity remaining rate after heat treatment than that of the wild type. In addition, No. A polypeptide into which a mutation in any one of amino acid sequences 1 to 68 has been introduced has a residual rate of lipase activity after heat treatment more than twice that of the wild type. It was shown that a polypeptide into which a mutation of any one of amino acid sequences 1 to 40 was introduced was three times higher than the wild type. Further, according to Table 12, No. 1 shown in Table 12 was obtained. It was shown that the polypeptide introduced with a mutation of any amino acid sequence of 1-150 has a higher lipase activity remaining rate after heat treatment than the wild type. In addition, No. 1 shown in Table 12 is used. 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.
＜実験例５＞
（三重変異体の評価（ｐＨ安定性））
　三重変異体（Ｐ２３３Ｇ／Ｌ２３４Ｅ／Ｖ２３５Ｍ、表１２のＮｏ．８１）のｐＨ安定性を評価した。サンプルは実験例２で調製したランダム変異ライブラリーを用いた。ｐＨ安定性の評価は、ｐＨ２～１２の各ｐＨで処理した野生型（表１２のＮｏ．１５１）と三重変異体の活性の残存率（活性値（ｐＨ処理サンプル）／活性値（未処理サンプル）×１００）で比較した。ｐＨ２～４処理は０．１ｍｏｌ／Ｌ　グリシン緩衝液、ｐＨ５～７処理は０．１ｍｏｌ／Ｌ　リン酸カリウム緩衝液、ｐＨ８～９処理は０．１ｍｏｌ／Ｌ　Ｔｒｉｓ緩衝液、ｐＨ１０～１２処理は０．１ｍｏｌ／Ｌ　グリシン緩衝液を用いた。サンプルの酵素液１０μＬを前述の緩衝液９０μＬと混合後、３７℃、１時間静置することにより処理した。処理後、１ｍｏｌ／Ｌリン酸カリウム緩衝液（ｐＨ７．０）を等量加えてｐＨ７．０に戻してから活性測定した。活性測定は、Ｌｉｐａｓｅ　Ｋｉｔ　Ｓ（ＤＳファーマバイオメディカル）を使用して行った。 <Experimental example 5>
(Evaluation of triple mutant (pH stability))
The pH stability of the triple mutant (P233G / L234E / V235M, No. 81 in Table 12) was evaluated. As a sample, the random mutation library prepared in Experimental Example 2 was used. The evaluation of pH stability is based on the residual rate of activity (activity value (pH-treated sample) / activity value (untreated sample) of wild-type (No. 151 in Table 12) and triple mutant treated at each pH of pH 2-12. ) × 100). 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 μL of the sample enzyme solution was mixed with 90 μL 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).
　三重変異体（Ｐ２３３Ｇ／Ｌ２３４Ｅ／Ｖ２３５Ｍ）と野生型のｐＨ安定性の評価結果を表１３及び図８に示す。 The evaluation results of the pH stability of the triple mutant (P233G / L234E / V235M) and the wild type are shown in Table 13 and FIG.
Figure JPOXMLDOC01-appb-T000022
Figure JPOXMLDOC01-appb-T000022
　表１３及び図８によれば、三重変異体（Ｐ２３３Ｇ／Ｌ２３４Ｅ／Ｖ２３５Ｍ）は、ｐＨ３及び１２におけるｐＨ処理後のリパーゼ活性の残存率が、野生型よりも高いことが示された。 According to Table 13 and FIG. 8, 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.
（三重変異体の評価（有機溶媒安定性））
　三重変異体（Ｐ２３３Ｇ／Ｌ２３４Ｅ／Ｖ２３５Ｍ）の有機溶媒安定性を評価した。サンプルは実験例２で調製したランダム変異ライブラリーを用いた。有機溶媒安定性の評価は、野生型と三重変異体のサンプルをそれぞれ水とアセトンで０～５０ｖ／ｖ％アセトン濃度になるように希釈し、３７℃、１時間処理したときの水処理サンプルに対する各濃度のアセトン処理サンプルの相対活性（活性値（水処理サンプル）／活性値（アセトン処理サンプル）×　１００）で比較した。活性測定は、Ｌｉｐａｓｅ　Ｋｉｔ　Ｓ（ＤＳ　ファーマバイオメディカル）を使用して行った。 (Evaluation of triple mutants (organic solvent stability))
The organic solvent stability of the triple mutant (P233G / L234E / V235M) was evaluated. As a sample, 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).
　三重変異体（Ｐ２３３Ｇ／Ｌ２３４Ｅ／Ｖ２３５Ｍ）の溶媒安定性の評価結果を表１４及び図９に示す。 The evaluation results of the solvent stability of the triple mutant (P233G / L234E / V235M) are shown in Table 14 and FIG.
Figure JPOXMLDOC01-appb-T000023
Figure JPOXMLDOC01-appb-T000023
　表１４及び図９によれば、三重変異体（Ｐ２３３Ｇ／Ｌ２３４Ｅ／Ｖ２３５Ｍ）は、１０～５０ｖ／ｖ％アセトン溶液中におけるリパーゼ活性の残存率が、野生型よりも高いことが示された。 According to Table 14 and FIG. 9, it was shown that 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.
　以上の結果から、本発明における熱安定性が向上したポリペプチドは、ｐＨ安定性や有機溶媒安定性も向上することが示唆された。 From the above results, it was suggested that the polypeptide having improved thermal stability in the present invention also has improved pH stability and organic solvent stability.
配列番号１は、バークホルデリア・セパシア由来リパーゼ（成熟体）のアミノ酸配列である。
配列番号２は、シュードモナス・グルマエ由来リパーゼ（成熟体）のアミノ酸配列である。
配列番号３は、シュードモナス・フルオレセンス由来リパーゼ（成熟体）のアミノ酸配列である。
配列番号４は、シュードモナス・エルギノーサ由来リパーゼ（成熟体）のアミノ酸配列である。
配列番号５は、バークホルデリア・セパシア由来リパーゼ（全長）のアミノ酸配列である。
配列番号６は、シュードモナス・グルマエ由来リパーゼ（全長）のアミノ酸配列である。配列番号７は、シュードモナス・フルオレセンス由来リパーゼ（全長）のアミノ酸配列である。
配列番号８は、シュードモナス・エルギノーサ由来リパーゼ（全長）のアミノ酸配列である。
配列番号９は、バークホルデリア・セパシア由来リパーゼ（野生型）の塩基配列である。配列番号１０は、シュードモナス・グルマエ由来リパーゼ（野生型）の塩基配列である。配列番号１１は、シュードモナス・フルオレセンス由来リパーゼ（野生型）の塩基配列である。
配列番号１２は、シュードモナス・エルギノーサ由来リパーゼ（野生型）の塩基配列である。
配列番号１３は、バークホルデリア・セパシア由来リパーゼＬｉｐＡ（Ｅ．ｃｏｌｉコドン最適化）の塩基配列である。
配列番号１４は、バークホルデリア・セパシア由来のシャペロン遺伝子（ＬｉｐＸ）野生型の塩基配列である。
配列番号１５は、バークホルデリア・セパシア由来シャペロン遺伝子ＬｉｐＸ（Ｅ．ｃｏｌｉコドン最適化）の塩基配列である。 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. aeruginosa-derived lipase (full length). 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).

Claims (14)

  1. 　少なくとも、Ｎ末端側から、βシート（Ａ）、αへリックス（Ａ）、αへリックス（Ｂ）、αへリックス（Ｃ）、βシート（Ｂ）、及びαへリックス（Ｄ）を備え、リパーゼ活性を有しており、
    　前記βシート（Ａ）は、そのＮ末端側のアミノ酸残基がポリペプチドのＮ末端側から７～１３位に存在し、アミノ酸残基数が２～６で構成されており、
    　前記αへリックス（Ａ）は、そのＮ末端側のアミノ酸残基が前記βシート（Ａ）のＣ末端側のアミノ酸残基を０位とした場合に１５～２５位に存在し、アミノ酸残基数が３～９で構成されており、
    　前記αへリックス（Ｂ）は、そのＮ末端側のアミノ酸残基が前記αへリックス（Ａ）のＣ末端側のアミノ酸残基を０位とした場合に７０～９２位に存在し、アミノ酸残基数が４～１５で構成されており
    　前記αへリックス（Ｃ）は、そのＮ末端側のアミノ酸残基が前記αへリックス（Ｂ）のＣ末端側のアミノ酸残基を０位とした場合に３～１１位に存在し、アミノ酸残基数が１１～２０で構成されており、
    　前記βシート（Ｂ）は、そのＮ末端側のアミノ酸残基が前記αへリックス（Ｃ）のＣ末端側のアミノ酸残基を０位とした場合に６５～８１位に存在し、アミノ酸残基数が２～８で構成されており、
    　前記αへリックス（Ｄ）は、そのＮ末端側のアミノ酸残基が前記αへリックス（Ｃ）のＣ末端側のアミノ酸残基を０位とした場合に６～１５位に存在し、アミノ酸残基数が２～２４で構成されている、ポリペプチドであって
    　前記βシート（Ａ）とαへリックス（Ａ）の間の領域が、表ＩのＮｏ．１～１２５に示すいずれかのアミノ酸残基を含んでおり、及び／又は、
    　前記βシート（Ｂ）とαへリックス（Ｄ）の間の領域が、表ＩＩのＮｏ．１～１５０に示すいずれかのアミノ酸残基を含んでいる、
    ポリペプチド。
    Figure JPOXMLDOC01-appb-T000001
    Figure JPOXMLDOC01-appb-T000002
         At least, from the N-terminal side, β sheet (A), α helix (A), α helix (B), α helix (C), β sheet (B), and α helix (D), Has lipase activity,
    In the β sheet (A), 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 α-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 β-sheet (A). The number consists of 3-9,
    The α 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 α helix (A). When the α-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 α-helix (B). 3 to 11 and is composed of 11 to 20 amino acid residues,
    The β 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 α helix (C). The number consists of 2-8,
    The α 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 α helix (C). A polypeptide having a radix number of 2 to 24, wherein the region between the β sheet (A) and the α helix (A) is represented by No. 1 in Table I. Contains any amino acid residue shown in 1-125, and / or
    The region between the β sheet (B) and the α helix (D) is shown in Table II. Any one of the amino acid residues shown in 1-150,
    Polypeptide.
    Figure JPOXMLDOC01-appb-T000001
    Figure JPOXMLDOC01-appb-T000002
  2. 　更に、αへリックス（Ｅ）、βシート（Ｃ）、βシート（Ｄ）、αへリックス（Ｇ）、及びβシート（Ｅ）を備え、
    　前記αへリックス（Ｅ）は、そのＮ末端側のアミノ酸残基が前記αへリックス（Ａ）のＣ末端側のアミノ酸残基を０位とした場合に１７～２８位に存在し、アミノ酸残基数が１３～１９で構成されており、
    　前記βシート（Ｃ）は、そのＮ末端側のアミノ酸残基が前記αへリックス（Ｅ）のＣ末端側のアミノ酸残基を０位とした場合に２～８位に存在し、アミノ酸残基数が４～８で構成されており、
    　前記βシート（Ｄ）は、そのＮ末端側のアミノ酸残基が前記βシート（Ｃ）のＣ末端側のアミノ酸残基を０位とした場合に１４～２２位に存在し、アミノ酸残基数が３～１１で構成されており、
    　前記αへリックス（Ｇ）は、そのＮ末端側のアミノ酸残基が前記αへリックス（Ｃ）のＣ末端側のアミノ酸残基を０位とした場合に３～１３位に存在し、アミノ酸残基数が４～１３で構成されており、
    　前記βシート（Ｅ）は、そのＮ末端側のアミノ酸残基が前記αへリックス（Ｇ）のＣ末端側のアミノ酸残基を０位とした場合に２４～３６位に存在し、アミノ酸残基数が２～６で構成されている、
    請求項１に記載のポリペプチド。     Furthermore, an α helix (E), a β sheet (C), a β sheet (D), an α helix (G), and a β sheet (E) are provided,
    The α 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 α helix (A). The radix consists of 13-19,
    The β 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 α helix (E). The number consists of 4-8,
    The β 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 β sheet (C). Consists of 3 to 11,
    The α-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 α-helix (C). The radix is composed of 4-13,
    The β 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 α helix (G). The number consists of 2-6,
    The polypeptide of claim 1.
  3. 　更に、αへリックス（Ｆ）、αへリックス（Ｈ）、及びβシート（Ｆ）を備え、
    　前記αへリックス（Ｆ）は、そのＮ末端側のアミノ酸残基が前記βシート（Ｃ）のＣ末端側のアミノ酸残基を０位とした場合に１～５位に存在し、アミノ酸残基数が３～１５で構成されており、
    　前記αへリックス（Ｈ）は、そのＮ末端側のアミノ酸残基が前記αへリックス（Ｇ）のＣ末端側のアミノ酸残基を０位とした場合に１～１０位に存在し、アミノ酸残基数が２～１３で構成されており、
    　前記βシート（Ｆ）は、そのＮ末端側のアミノ酸残基が前記βシート（Ｅ）のＣ末端側のアミノ酸残基を０位とした場合に１～５位に存在し、アミノ酸残基数が１５～２３で構成されている、
    請求項２に記載のポリペプチド。     Furthermore, an α helix (F), an α helix (H), and a β sheet (F) are provided,
    The α-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 β-sheet (C), and the amino acid residue The number consists of 3 to 15,
    The α helix (H) is present at the 1 to 10 position when the amino acid residue on the N-terminal side of the α helix (G) is 0 position on the C-terminal side of the α helix (G). The radix is composed of 2 to 13,
    The β 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 β-sheet (E). Consists of 15-23,
    The polypeptide of claim 2.
  4. 　以下の（１）から（１２）のいずれかに示すポリペプチド。
    （１）配列番号１に示すアミノ酸配列において、２５～２７位のアミノ酸残基が表ＩのＮｏ．１～１２５に示すいずれかのアミノ酸残基に置換されている、及び／又は２３３～２３５位のアミノ酸残基が表ＩＩのＮｏ．１～１５０に示すいずれかのアミノ酸残基に置換されているアミノ酸配列からなるポリペプチド、
    （２）配列番号２に示すアミノ酸配列において、２４～２６位のアミノ酸残基が表ＩのＮｏ．１～１２５に示すいずれかのアミノ酸残基に置換されている、及び／又は２３２～２３４位のアミノ酸残基が表ＩＩのＮｏ．１～１５０に示すいずれかのアミノ酸残基に置換されているアミノ酸配列からなるポリペプチド、
    （３）配列番号３に示すアミノ酸配列において、２５～２７位のアミノ酸残基が表ＩのＮｏ．１～１２５に示すいずれかのアミノ酸残基に置換されている、及び／又は２３３～２３５位のアミノ酸残基が表ＩＩのＮｏ．１～１５０に示すいずれかのアミノ酸残基に置換されているアミノ酸配列からなるポリペプチド、
    （４）配列番号４に示すアミノ酸配列において、２５～２７位のアミノ酸残基が表ＩのＮｏ．１～１２５に示すいずれかのアミノ酸残基に置換されている、及び／又は２３３～２３５位のアミノ酸残基が表ＩＩのＮｏ．１～１５０に示すいずれかのアミノ酸残基に置換されているアミノ酸配列からなるポリペプチド、
    （５）配列番号１に示すアミノ酸配列における２５～２７位のアミノ酸残基が表ＩのＮｏ．１～１２５に示すいずれかのアミノ酸残基に置換されている、及び／又は２３３～２３５位のアミノ酸残基が表ＩＩのＮｏ．１～１５０に示すいずれかのアミノ酸残基に置換されているアミノ酸配列において、前記置換が導入されたアミノ酸残基以外の１個又は数個のアミノ酸残基が置換、付加、挿入又は欠失されてなり、且つ、リパーゼ活性を有し、配列番号１に示すアミノ酸配列からなるポリペプチドに比して熱安定性が向上しているポリペプチド、
    （６）配列番号２に示すアミノ酸配列における２４～２６位のアミノ酸残基が表ＩのＮｏ．１～１２５に示すいずれかのアミノ酸残基に置換されている、及び／又は２３２～２３４位のアミノ酸残基が表ＩＩのＮｏ．１～１５０に示すいずれかのアミノ酸残基に置換されているアミノ酸配列において、前記置換が導入されたアミノ酸残基以外の１個又は数個のアミノ酸残基が置換、付加、挿入又は欠失されてなり、且つ、リパーゼ活性を有し、配列番号２に示すアミノ酸配列からなるポリペプチドに比して熱安定性が向上しているポリペプチド、
    （７）配列番号３に示すアミノ酸配列における２５～２７位のアミノ酸残基が表ＩのＮｏ．１～１２５に示すいずれかのアミノ酸残基に置換されている、及び／又は２３３～２３５位のアミノ酸残基が表ＩＩのＮｏ．１～１５０に示すいずれかのアミノ酸残基に置換されているアミノ酸配列において、前記置換が導入されたアミノ酸残基以外の１個又は数個のアミノ酸残基が置換、付加、挿入又は欠失されてなり、且つ、リパーゼ活性を有し、配列番号３に示すアミノ酸配列からなるポリペプチドに比して熱安定性が向上しているポリペプチド、
    （８）配列番号４に示すアミノ酸配列における２５～２７位のアミノ酸残基が表ＩのＮｏ．１～１２５に示すいずれかのアミノ酸残基に置換されている、及び／又は２３３～２３５位のアミノ酸残基が表ＩＩのＮｏ．１～１５０に示すいずれかのアミノ酸残基に置換されているアミノ酸配列において、前記置換が導入されたアミノ酸残基以外の１個又は数個のアミノ酸残基が置換、付加、挿入又は欠失されてなり、且つ、リパーゼ活性を有し、配列番号４に示すアミノ酸配列からなるポリペプチドに比して熱安定性が向上しているポリペプチド、
    （９）配列番号１に示すアミノ酸配列における２５～２７位のアミノ酸残基が表ＩのＮｏ．１～１２５に示すいずれかのアミノ酸残基に置換されている、及び／又は２３３～２３５位のアミノ酸残基が表ＩＩのＮｏ．１～１５０に示すいずれかのアミノ酸残基に置換されているアミノ酸配列において、配列番号１に示すアミノ酸配列に対する前記置換が導入されたアミノ酸残基を除いた配列同一性が８０％以上であり、且つ、リパーゼ活性を有し、配列番号１に示すアミノ酸配列からなるポリペプチドに比して熱安定性が向上しているポリペプチド、
    （１０）配列番号２に示すアミノ酸配列における２４～２６位のアミノ酸残基が表ＩのＮｏ．１～１２５に示すいずれかのアミノ酸残基に置換されている、及び／又は２３２～２３４位のアミノ酸残基が表ＩＩのＮｏ．１～１５０に示すいずれかのアミノ酸残基に置換されているアミノ酸配列において、配列番号２に示すアミノ酸配列に対する前記置換が導入されたアミノ酸残基を除いた配列同一性が８０％以上であり、且つ、リパーゼ活性を有し、配列番号２に示すアミノ酸配列からなるポリペプチドに比して熱安定性が向上しているポリペプチド、
    （１１）配列番号３に示すアミノ酸配列における２５～２７位のアミノ酸残基が表ＩのＮｏ．１～１２５に示すいずれかのアミノ酸残基に置換されている、及び／又は２３３～２３５位のアミノ酸残基が表ＩＩのＮｏ．１～１５０に示すいずれかのアミノ酸残基に置換されているアミノ酸配列において、配列番号３に示すアミノ酸配列に対する前記置換が導入されたアミノ酸残基を除いた配列同一性が８０％以上であり、且つ、リパーゼ活性を有し、配列番号３に示すアミノ酸配列からなるポリペプチドに比して熱安定性が向上しているポリペプチド、
    （１２）配列番号４に示すアミノ酸配列における２５～２７位のアミノ酸残基が表ＩのＮｏ．１～１２５に示すいずれかのアミノ酸残基に置換されている、及び／又は２３３～２３５位のアミノ酸残基が表ＩＩのＮｏ．１～１５０に示すいずれかのアミノ酸残基に置換されているアミノ酸配列において、配列番号４に示すアミノ酸配列に対する前記置換が導入されたアミノ酸残基を除いた配列同一性が８０％以上であり、且つ、リパーゼ活性を有し、配列番号４に示すアミノ酸配列からなるポリペプチドに比して熱安定性が向上しているポリペプチド。     The polypeptide shown in any one of (1) to (12) below.
    (1) In the amino acid sequence shown in SEQ ID NO: 1, the amino acid residues at positions 25 to 27 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. A polypeptide comprising an amino acid sequence substituted with any one of the amino acid residues shown in 1-150,
    (2) In the amino acid sequence shown in SEQ ID NO: 2, the amino acid residues at positions 24 to 26 are Nos. Nos. 1 to 125 are substituted with amino acid residues and / or amino acid residues at positions 232 to 234 A polypeptide comprising an amino acid sequence substituted with any one of the amino acid residues shown in 1-150,
    (3) In the amino acid sequence shown in SEQ ID NO: 3, the amino acid residues at positions 25 to 27 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. A polypeptide comprising an amino acid sequence substituted with any one of the amino acid residues shown in 1-150,
    (4) In the amino acid sequence shown in SEQ ID NO: 4, the amino acid residues at positions 25 to 27 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. A polypeptide comprising an amino acid sequence substituted with any one of the amino acid residues shown in 1-150,
    (5) 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. 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. A polypeptide having a lipase activity and having improved thermal stability compared to a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1,
    (6) 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. A polypeptide that has lipase activity and has improved thermal stability compared to a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2,
    (7) 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. 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. A polypeptide having a lipase activity and having improved thermal stability compared to a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 3,
    (8) 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. 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. A polypeptide having a lipase activity and having improved thermal stability compared to a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 4,
    (9) 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. In the amino acid sequence substituted with any one of the amino acid residues shown in 1-150, 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, And a polypeptide having lipase activity and having improved thermal stability compared to a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1,
    (10) 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, 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, And a polypeptide having lipase activity and improved thermal stability compared to a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2.
    (11) 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. In the amino acid sequence substituted with any one of the amino acid residues shown in 1-150, 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, And a polypeptide having lipase activity and improved thermal stability compared to a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 3.
    (12) 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. In the amino acid sequence substituted with any one of the amino acid residues shown in 1-150, 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.
  5. 　請求項１～４に記載のポリペプチドをコードしているＤＮＡ。 DNA encoding the polypeptide according to claims 1 to 4.
  6. 　請求項５に記載のＤＮＡを含む組換えベクター。 A recombinant vector comprising the DNA according to claim 5.
  7. 　請求項６に記載の組換えベクターにより宿主を形質転換して得られる形質転換体。 A transformant obtained by transforming a host with the recombinant vector according to claim 6.
  8. 　請求項７に記載の形質転換体を培養する工程を含む、請求項１～５のいずれかに記載のポリペプチドの製造方法。 The method for producing a polypeptide according to any one of claims 1 to 5, comprising a step of culturing the transformant according to claim 7.
  9. 　請求項１～４のいずれかに記載のポリペプチドを含む組成物。 A composition comprising the polypeptide according to any one of claims 1 to 4.
  10. 　請求項１～４のいずれかに記載のポリペプチド、又は請求項９に記載の組成物を含む酵素剤。 An enzyme agent comprising the polypeptide according to any one of claims 1 to 4 or the composition according to claim 9.
  11. 　請求項１～４のいずれかに記載のポリペプチド、請求項９に記載の組成物、又は請求項１０に記載の酵素剤を、油脂に作用させる、油脂処理方法。 A method for treating fats and oils, wherein the polypeptide according to any one of claims 1 to 4, the composition according to claim 9, or the enzyme agent according to claim 10 is allowed to act on fats and oils.
  12. 　請求項１～４のいずれかに記載のポリペプチド、請求項９に記載の組成物、又は請求項１０に記載の酵素剤を排水に作用させる、排水処理方法。 A wastewater treatment method in which the polypeptide according to any one of claims 1 to 4, the composition according to claim 9 or the enzyme agent according to claim 10 is allowed to act on wastewater.
  13. 　請求項１～４のいずれかに記載のポリペプチド、請求項９に記載の組成物、又は請求項１０に記載の酵素剤を医薬中間体原料に作用させる、医薬中間体の製造方法。 A method for producing a pharmaceutical intermediate, wherein the polypeptide according to any one of claims 1 to 4, the composition according to claim 9 or the enzyme agent according to claim 10 is allowed to act on a raw material for the pharmaceutical intermediate.
  14. 　請求項１～４のいずれかに記載のポリペプチド、請求項９に記載の組成物、又は請求項１０に記載の酵素剤をファインケミカル素材原料に作用させる、ファインケミカル素材の製造方法。 A method for producing a fine chemical material, wherein the polypeptide according to any one of claims 1 to 4, the composition according to claim 9 or the enzyme agent according to claim 10 is allowed to act on the raw material of the fine chemical material.
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