WO2022050387A1 - Nouvelle enzyme spécifique du phosphatidylglycérol - Google Patents

Nouvelle enzyme spécifique du phosphatidylglycérol Download PDF

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WO2022050387A1
WO2022050387A1 PCT/JP2021/032497 JP2021032497W WO2022050387A1 WO 2022050387 A1 WO2022050387 A1 WO 2022050387A1 JP 2021032497 W JP2021032497 W JP 2021032497W WO 2022050387 A1 WO2022050387 A1 WO 2022050387A1
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polypeptide
amino acid
seq
plc
phosphatidylglycerol
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Japanese (ja)
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大助 杉森
聖人 梶山
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大助 杉森
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
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    • 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)
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    • 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
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
    • C12P7/18—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic polyhydric
    • C12P7/20—Glycerol
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    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/62—Carboxylic acid esters

Definitions

  • the present invention relates to a novel protein having a phosphatidylglycerol (PG) hydrolytic decomposing ability and a method for using the same.
  • PG phosphatidylglycerol
  • PLC phospholipase C
  • PG-PLC PG-specific PLC
  • PG-PLC PG-specific PLC
  • the PG-PLC of the present invention has also made it possible to efficiently obtain a specific glycerol phosphoric acid.
  • the present invention provides, for example, the following items.
  • (Item 1) A phospholipase C polypeptide having substrate-specific hydrolysis resolution for phosphatidylglycerol.
  • (Item 2) (I) Amino acids corresponding to 56L, 59FVG61, 204IPGI207, 209AW210, and 316GGFA320 of the amino acid sequence represented by SEQ ID NO: 2 and / or (ii) H43 and H409 of the amino acid sequence represented by SEQ ID NO: 2.
  • the polypeptide according to item 1 which has an amino acid corresponding to.
  • the polypeptide according to item 2 further comprising an amino acid corresponding to D41, D103, N191, H364, and H407 of the amino acid sequence represented by SEQ ID NO: 2.
  • the amino acid sequences represented by SEQ ID NO: 2 are 40T to 75T, 100T to 120L, 184P to 197V, 205P to 227K, 266F, 271L, 296Y to 297Y, 310L to 322S, 363S to 366T, 378R to 384R, 406G to 409H.
  • the polypeptide of item 3 further comprising an amino acid corresponding to 432S-435D.
  • a phospholipase C having a phosphatidylglycerol hydrolysis resolution which is the following polypeptide (a), (b) or (c).
  • (Item 7) The polypeptide according to any one of items 1 to 6, which is derived from a microorganism belonging to the order Actinomycetales.
  • (Item 8) A polynucleotide according to any one of (a) to (f) below, which encodes a phospholipase C polypeptide having substrate-specific hydrolytic resolution with respect to phosphatidylglycerol.
  • A Nucleotide containing the base sequence represented by SEQ ID NO: 1
  • B Polynucleotide containing a base sequence complementary to the base sequence represented by SEQ ID NO: 1
  • c Polynucleotides and stringents of (b) Nucleotide (d) that hybridizes under various conditions In the nucleotide sequence represented by SEQ ID NO: 1, one or more bases are deleted, substituted, or imparted with the nucleotide sequence (e).
  • f A polynucleotide containing a base sequence having at least about 80% identity with the base sequence represented by SEQ ID NO: 1 (item 9).
  • a recombinant vector containing the polynucleotide according to item 8. (Item 10) A transformant containing the recombinant vector according to item 9. (Item 11) A method for producing a polypeptide having substrate-specific hydrolysis resolution with respect to phosphatidylglycerol, comprising a step of producing the polypeptide from a transformant containing the polynucleotide according to item 8. (Item 12) A method for degrading phosphatidylglycerol using the polypeptide according to any one of items 1 to 7.
  • (Item 13) A method for producing sn-glycerol-1-phosphate, sn-glycerol-3-phosphate, and / or diacylglyceride from phosphatidylglycerol using the polypeptide according to any one of items 1 to 7.
  • (Item 14) A composition for degrading phosphatidylglycerol, which comprises the polypeptide according to any one of items 1 to 7.
  • FIG. 1 shows the relationship between the type of medium and the activity of PG-PLC.
  • the PG-PLC activity when cultured using ISP2 medium was normalized as 100%.
  • FIG. 2 shows the results of SDS-PAGE (separation gel concentration 12%) analysis of purified PG-PLC.
  • Lane M marker
  • lane 1 purified PG-PLC (0.03 mg-protein / ml, 0.3 ⁇ g-protein).
  • FIG. 3-1 shows the effect of pH on PLC activity. Using POPG as a substrate, enzyme activity was measured at 37 ° C. in various 0.16 M buffers. The buffer used is Ac-Na ( ⁇ ), BisTris-HCl ( ⁇ ), MES-NaOH ( ⁇ ), or Tris-HCl ( ⁇ ).
  • FIG. 1 shows the relationship between the type of medium and the activity of PG-PLC.
  • the PG-PLC activity when cultured using ISP2 medium was normalized as 100%.
  • FIG. 2 shows the results of SDS-P
  • FIG. 3-2 shows the effect of temperature on PLC activity.
  • enzyme activity was measured at 0.16 M MES-NaOH (pH 6.0) at each temperature.
  • FIG. 4-1 shows the pH stability of PLC. Purified enzyme samples were incubated at 4 ° C. for 4 hours in each 50 mM buffer. Residual activity was measured in 0.16 M MES-NaOH (pH 6.0) at 55 ° C. using POPG as a substrate. The buffer used for incubation is Ac-Na ( ⁇ ), MES-NaOH ( ⁇ ), or Tris-HCl ( ⁇ ).
  • FIG. 4-2 shows the temperature stability of the PLC.
  • FIG. 5-1 shows the effect of metal ions on PG-PLC activity when POPG is used as a substrate. Enzyme activity was measured at 55 ° C. in 0.2 M MES-NaOH (pH 6.0).
  • FIG. 5-2 shows the effect of the surfactant on the PG-PLC activity when POPG is used as a substrate. Enzyme activity was measured at 55 ° C. in 0.2 M MES-NaOH (pH 6.0).
  • FIG. 5-2 shows the effect of the surfactant on the PG-PLC activity when POPG is used as a substrate. Enzyme activity was measured at 55 ° C. in 0.2 M MES-NaOH (pH 6.0).
  • FIG. 6 shows the substrate specificity of the purified enzyme sample. Enzyme (PLC) activity was measured at 55 ° C. in 0.2M MES-NaOH (pH 6.0).
  • FIG. 7 shows the peptide sequences obtained by LC-MS analysis and the protein sequences obtained from the database matching these peptide sequences.
  • FIG. 8 shows the base sequence of the PG-PLC gene obtained by DNA sequencing.
  • FIG. 9-1 shows an enzymatic reaction (substrate POPG, reaction temperature 45 ° C., buffer: 0.16M MES-NaOH (pH 6.0). The horizontal axis is the enzyme reaction time, vertical axis) using the culture supernatant of the 20 ° C. culture. The axis shows the amount of G3P produced.
  • 9-2 shows an enzymatic reaction (substrate POPG, reaction temperature 45 ° C., buffer: 0.16M MES-NaOH (pH 6.0)) using a crude protein solution (CFE) cultured at 30 ° C., and the horizontal axis is an enzymatic reaction. The time and the vertical axis indicate the amount of G3P produced.
  • the amino acid residues presumed to be involved in substrate binding based on the predicted three-dimensional structure of PG-PLC using SwissModel are shown. The amino acid residue is surrounded by a frame.
  • phospholipid is a general term for lipids having a phosphoric acid ester and a phosphonic acid ester.
  • Glycerophospholipid which is the main component of the cell membrane composed of a lipid bilayer and has a glycerol as a skeleton, is one of them.
  • Glycerophospholipids In glycerophospholipids, fatty acids are bound to the sn-1 and sn-2 positions of glycerol, and phosphoric acid is bound to the sn-3 position. Glycerophospholipids are further classified by the phospholipid hydrophilic moiety (X in the above structural formula) that binds to this phosphoric acid.
  • PA phosphatidylic acid
  • PC phosphatidylcholine
  • PE phosphatidylethanolamine
  • PS phosphatidylserine
  • inositol if it is serine. If it is, it is called phosphatidylinositol (PI), and if it is glycerol, it is called phosphatidylglycerol (PG).
  • PI phosphatidylinositol
  • PG phosphatidylglycerol
  • phosphatidylglycerol means that a fatty acid is ester-bonded to the sn-1 position and the sn-2 position of glycerol, phosphoric acid is ester-bonded to the sn-3 position, and glycerol is further added to the phosphoric acid.
  • cardiacolipin also known as diphosphatidylglycerol; CL
  • CL diphosphatidylglycerol
  • hydrolysis refers to a mode of bond cleavage by the reaction of AB + H 2 O ⁇ A—OH + B—H.
  • An enzyme that hydrolyzes is called a “hydrolase”, its ability is called “hydrolytic resolution”, and its strength (height) is called “activity”, “hydrolyzing activity”, or “enzyme activity”.
  • Hydrolysis resolution / activity can be examined, for example, by measuring a decrease in the concentration of the substrate to be hydrolyzed or by measuring an increase in the concentration of a product produced by hydrolysis.
  • phospholipase refers to an enzyme that hydrolyzes glycerophospholipids. Phospholipases are classified according to the site that hydrolyzes glycerophospholipids, phospholipase A1 degrades the ester bond at the sn-1 position of glycerol, phospholipase A2 degrades the ester bond at the sn-2 position of glycerol, and phospholipase B degrades the ester bond at the sn-2 position of glycerol. The ester bonds at both the sn-1 and sn-2 positions of the above are decomposed, and both release fatty acids.
  • Phospholipase C decomposes the ester bond on the glycerol skeleton side of the phosphodiester bond connecting glycerol and X
  • phospholipase D decomposes the ester bond on the opposite side of the glycerol skeleton of the phosphodiester bond. Therefore, in the present specification, "phospholipase C (PLC)" decomposes the ester bond on the glycerol skeleton side of the phosphodiester bond bound to the glycerol skeleton among the phospholipases that hydrolyze glycerophospholipids, and the phosphate is bound.
  • PLC phospholipase C
  • identity refers to the proportion of residues having the same amino acid when comparing a plurality of amino acid sequences.
  • homology when comparing multiple amino acid sequences, if the amino acids are the same, or if the amino acids are not the same but have the same properties, they are treated as homologous and are homologous. Refers to the percentage of residues.
  • an enzyme catalyzes a chemical reaction and has specificity for the type of reaction and specificity for a substrate.
  • an enzyme is "substrate-specific” when it selectively catalyzes a chemical reaction to a particular chemical, that is, to a chemical other than the particular chemical. It means that the chemical reaction is not catalyzed or the degree of catalysis is weak enough.
  • an enzyme or polypeptide is "substrate-specific with respect to phosphatidylglycerol", “phosphatidylglycerol-specific” or "PG-specific” as the pH of the enzyme or polypeptide is 6.0, 55 ° C. When the water resolution of PG under the condition of 5 minutes is 100%, the water resolution for substrates other than PG selected from the group consisting of PA, PE, PC, CL, PS, and PI is at most about 30% or less. It means that it is.
  • polynucleotide that hybridizes under stringent conditions refers to a polynucleotide that hybridizes under well-known conditions commonly used in the art.
  • a polynucleotide can be obtained by using a polynucleotide selected from the polynucleotides of the present invention as a probe and using a colony hybridization method, a plaque hybridization method, a Southern blot hybridization method, or the like. Specifically, using a filter on which DNA derived from the genome or its digest, colonies or plaques is immobilized, hybridization is performed at 65 ° C. in the presence of 0.7 to 1.0 M NaCl, and then 0.1.
  • Stringent conditions are usually about 5 ° C. to about 30 ° C., preferably about 10 ° C. to about 25 ° C. lower than the melting temperature (Tm) of the complete hybrid, under conditions where a specific hybrid is formed.
  • Tm melting temperature
  • the condition may be such that DNAs having 90% or more homology hybridize with each other and DNAs having lower homology than that do not hybridize with each other.
  • a complete hybrid in the temperature range of Tm to (Tm-30) ° C., preferably Tm to (Tm-20) ° C., and the composition of 1 ⁇ SSC (1-fold concentration SSC solution) is 150 mM.
  • vector refers to a gene construct capable of transferring a polynucleotide sequence of interest into a cell of interest.
  • vectors can be self-sustainingly replicated in host cells such as prokaryotic cells, yeast, animal cells, plant cells, insect cells, animal individuals and plant individuals, or can be integrated into chromosomes, and are the poly of the present invention. Examples include those containing a promoter at a position suitable for transcription of a nucleotide.
  • the vector into which the gene is incorporated is not particularly limited, but a phage, plasmid, or cosmid that can grow autonomously in the host microorganism and constructed for gene recombination is suitable, and the phage vector is, for example, Escherichia coli.
  • a microorganism belonging to the above is used as a host, ⁇ gt / ⁇ C, ⁇ gt / ⁇ B and the like can be used.
  • Examples of the plasmid vector include Novagen's pET vector, Takarabio's pCold I to IV, pCold TF, Promega's pFN18A HaloTag T7Flexi Vector, pFN18K HaloTag T7FlexR , PACYC184, pUC12, pUC13, pUC18, pUC19, pUC118, pIN I, BluescriptKS +, etc., pWH1520, pUB110, pKH300PLK, etc. Hosts such as HisT, pIJ680, pIJ702, pTONA, etc.
  • an expression system using the methanol-utilizing yeast Pichia plasmid as a host an expression system using the filamentous fungus Aspergillus oryzae or Aspergillus niger, or the like, such as YRp7, pYC1, and YEp13, can also be used.
  • various cell-free protein expression systems can also be used.
  • the Ministerial Ordinance stipulates the diffusion prevention measures, etc. that should be taken for industrial use, etc., among the second-class use of genetically modified organisms, etc.
  • a recombinant vector into which the above-mentioned gene of the present invention is inserted is preferable.
  • the promoter is not particularly limited as long as it can be expressed in the host.
  • the recombinant vector of the present invention can be prepared by a method known to those skilled in the art using, for example, the gene of the present invention and the above-mentioned vector.
  • sn-glycerol-1-phosphate (G1P) and “sn-glycerol-3-phosphate (G3P)” are compounds represented by the following chemical formulas in which phosphoric acid is bound to glycerol, respectively.
  • diaacylglycerol refers to a fatty acid ester of glycerol to which two acyl groups are bonded, which is also referred to as diglyceride.
  • 1,2-diacylglycerol represented by the following chemical formula is known to be important as an intermediate for biosynthesis of phospholipids or triglycerides.
  • Diacylglycerol (In the formula, R 1 and R 2 are arbitrary acyl groups, respectively)
  • PLC decomposes the ester bond on the glycerol skeleton side of the phosphodiester bond bound to the glycerol skeleton to produce a phospholipid hydrophilic moiety to which phosphoric acid is bound, and DG.
  • Hydrolysis of PG with PLC produces DG and glycerol phosphate, for example, as shown in the reaction formula below. If the phospholipid hydrophilic moiety is phospho- (3'-sn-glycerol) as glycerol phosphate, G3P Is generated, and if it is phospho- (1'-sn-glycerol), G1P is generated.
  • R 1 and R 2 are arbitrary acyl groups, respectively
  • Glycerol phosphate has a chiral center and contains three positional isomers containing a set of mirror isomers (G1P, sn-glycerol-2-phosphate (G2P), and G3P; G1P and G3P are mirror isomers). It is a compound having. Even in this glycerol phosphate, a reagent from which only a specific enantiomer is separated is expensive. On the other hand, by using the PG-PLC of the present invention, it becomes possible to inexpensively and efficiently produce glycerol phosphate, which is a positional isomer such as G1P and G3P, by hydrolyzing PG.
  • polypeptide in one aspect, provides a polypeptide having a hydration decomposing ability of PG.
  • the polypeptide may be an artificially synthesized polypeptide or a biologically derived polypeptide.
  • the polypeptide may contain non-naturally occurring amino acids.
  • the two acyl groups contained in the PG of the present invention may be the same acyl group or different acyl groups. Further, it may be saturated or unsaturated. In one embodiment, the acyl group contained in the PG of the present invention may be a saturated fatty acid such as palmitic acid, stearic acid, arachidic acid, behenic acid, or linolenic acid, palmitoleic acid, oleic acid, gondonic acid, erucic acid, or.
  • a saturated fatty acid such as palmitic acid, stearic acid, arachidic acid, behenic acid, or linolenic acid, palmitoleic acid, oleic acid, gondonic acid, erucic acid, or.
  • Monounsaturated fatty acids such as nervonic acid, n-6 ( ⁇ 6) series polyunsaturated fatty acids such as linolenic acid, ⁇ -linolenic acid, dihomo- ⁇ -linolenic acid, arachidonic acid, or docosapentaenoic acid, or It may be derived from n-3 ( ⁇ 3) series polyunsaturated fatty acids such as ⁇ -linolenic acid, stearidonic acid, eicosatetraenoic acid, eicosapentaenoic acid, or docosahexaenoic acid, but is not limited to these (Kihara).
  • the acyl group may be derived from palmitoleic acid, stearic acid, palmitoleic acid, oleic acid, linoleic acid, arachidonic acid, docosapentaenoic acid, or docosapentaenoic acid, and in a particularly preferred embodiment, the acyl group. May be derived from oleic acid, linoleic acid, arachidonic acid, docosapentaenoic acid, or docosapentaenoic acid.
  • the polypeptide of the invention may have hydrolytic resolution regardless of the acyl group contained in the PG. This is because the polypeptide of the invention is a PLC, which degrades the phosphodiester bond at the sn-3 position to which the phosphate group is attached, rather than the sn-1 or sn-2 position to which the acyl group is attached. Because.
  • the polypeptide of the invention lacks one or more amino acids in (a) the amino acid sequence represented by SEQ ID NO: 2 or (b) the amino acid sequence represented by SEQ ID NO: 2.
  • Methods of deleting, substituting or imparting an amino acid, comprising a polypeptide comprising a substituted or imparted amino acid sequence are known to those of skill in the art.
  • the number of deleted, substituted or conferred amino acids that the polypeptide of the invention may contain is not significantly altered in function (if substantially equivalent), i.e., substrate specificity. However, for example, when the water resolution of PG under the condition of pH 6.0, 55 ° C.
  • the number may be 100 or more, 100 or less, 50 or less, 25 or less, and preferably 10 or less. , More preferably 5 or less.
  • the amino acid mutation may be an artificial mutation or a naturally occurring mutation. In fact, in the field of enzyme engineering focusing on the function of the enzyme and its amino acid sequence, for example, even if 100 or more amino acid mutations are added to the short-chain L-threonine dehydrogenase (about 350 amino acid length).
  • the polypeptide of the invention comprises (c) a polypeptide comprising an amino acid sequence having at least about 80% identity with the amino acid sequence represented by SEQ ID NO: 2.
  • the identity may be at least 80%, preferably at least 90%, and even more preferably at least 95%, as long as it has the substrate specificity intended in the present invention.
  • Methods for searching for protein identity and homology include integrated databases such as NCBI, EBI, SIB, or Genomenet, base sequence databases such as GenBank, EMBL, or DDBJ, PIR, Swiss-Prot. , UniProt, Entrez Protein, or a protein database such as PRF, which can be done via the Internet using software such as BLAST or FASTA.
  • amino acid deletions, substitutions, or additions to the polypeptide of the invention are made without impairing the function and properties of the polypeptide of the invention.
  • Deletions, substitutions, and additions of these amino acids may be at the N-terminus, C-terminus, or anywhere in the amino acid sequence.
  • Amino acid deletions, substitutions, and additions are, for example, the result of ligation of the tag and restriction enzyme site of the vector when introducing the polypeptide encoding the polypeptide of the invention into the multicloning site of the vector. It may be an amino acid imparted to the N-terminal and / or C-terminal of the peptide.
  • a signal sequence may be imparted to the polypeptide of the present invention, and a method of imparting a signal sequence to a polypeptide to manipulate the localization of the polypeptide is known in the art.
  • the signal sequences used include signal peptides such as endoplasmic reticulum transport signal, retention signal in the endoplasmic reticulum cavity, mitochondrial transport signal, nuclear translocation signal, membrane mooring signal, secretory signal, or cell membrane permeation peptide, myristoylation, etc. Examples thereof include, but are not limited to, a signal sequence for undergoing a lipid modification of, or a signal sequence for undergoing a sugar chain modification such as glycosylation.
  • Cell membrane permeation peptides include Tat signal peptides and Sec signal peptides, and periplasm transition signals include alkaline phosphatase signals, PelB signals and OpPA signals (Sigma, pFLAG-ATS), and other Penestratin peptides, Polyarginin peptides, Transportin peptides, Pep-.
  • periplasm transition signals include alkaline phosphatase signals, PelB signals and OpPA signals (Sigma, pFLAG-ATS), and other Penestratin peptides, Polyarginin peptides, Transportin peptides, Pep-.
  • One peptide, LL-37 peptide, or Pep-7 peptide, SKIK tag is known, but is not limited to these (Naoki Kajiwara and Tadashi Shibasaki, "Cell Membrane Permeable Peptide", Nikkei Journal, 141,220-221 ( 2013)).
  • OpPA and PelB may be fused, such as OpPA / PelB and SKIK / PelB, or vice versa.
  • the polypeptide of the present invention comprises a plurality of domains, and it is possible to prepare a chimeric protein in which one or a plurality of specific domains that are a part of the polypeptide of the present invention are fused with a label.
  • a chimeric protein in which one or a plurality of specific domains that are a part of the polypeptide of the present invention are fused with a label.
  • How to make chimeric proteins is known in the art. For example, it can be presented on the cell surface such as a cell membrane or a cell wall by fusion with GFP or fusion with the C-terminal region of ⁇ -aglutinin and a GPI-anchored signal sequence.
  • polypeptides of the invention have a substrate binding site.
  • the substrate binding site may comprise, for example, the amino acids corresponding to 56L, 59FVG61, 204IPGI207, 209AW210, and 316GGFA320 of the amino acid sequence represented by SEQ ID NO: 2, or selected from these amino acids. May be done. Since these amino acid residues are hydrophobic, they are considered to interact with the acyl group of PG.
  • the polypeptides of the invention have catalytic residues that interact with the substrate.
  • the catalytic residue may contain, for example, the amino acids corresponding to H43 and H409 of the amino acid sequence represented by SEQ ID NO: 2, or may be selected from these amino acids.
  • the polypeptides of the invention have active central amino acid residues that interact with the substrate.
  • the active central amino acid residue may include, for example, the amino acid residues corresponding to D41, D103, N191, H364, and H407 of the amino acid sequence represented by SEQ ID NO: 2, or may be selected from these amino acids. It may contain catalytic residues.
  • the substrate binding site is, for example, 40T to 75T, 100T to 120L, 184P to 197V, 205P to 227K, 266F, 271L, 296Y to 297Y, 310L to 322S, 363S to 366T of the amino acid sequence represented by SEQ ID NO: 2. It may contain amino acids corresponding to 378R to 384R, 406G to 409H, and 432S to 435D, or may be selected from these amino acids.
  • the polypeptides of the invention are zinc-dependent phospholipase C signatures derived from one or more prokaryotic organisms (HYx- [GT] -D- [LIVMAF]-[DNSH] -x-. P-x-H- [PA] -x-N; Kim, Y.G. et al., Structural and Functional Analysis of the Lmo2642 Cyclic Nucleotide Phosphodiesterase.
  • the "amino acid corresponding to a certain amino acid residue” means that the amino acid residue A'in the polypeptide A is used as a substrate when comparing a certain polypeptide A with another polypeptide B.
  • amino acid residue B' When it is an important residue in the interaction of, it means an amino acid residue B'that interacts with a substrate similar to the amino acid residue A'in polypeptide B. Therefore, for example, since polypeptide A contains a signal sequence but does not contain polypeptide B, the primary structural position of amino acid residue A'in polypeptide A (that is, the amino acid residue number when counted from the N-terminal). And, even if the position of the amino acid residue B'in the corresponding polypeptide B is different, if the role in the interaction with the substrate is the same, the amino acid residue B'is the amino acid residue A'. It can be said that it is an amino acid corresponding to.
  • the amino acid sequence (SEQ ID NO: 16) of a part of the mature protein (SEQ ID NO: 2) is referred to by various actinomycetes. It has been found to be conserved among the proteins it has, and the substrate binding site, active central amino acid residue, and catalytic residue are included in this conserved sequence. Therefore, for example, for the residue A'belonging to the substrate binding site of the polypeptide of the present invention, this amino acid residue is conserved, and in the polypeptides of other actinomycetes, the primary structural position is different, but the role in substrate binding is different. It is easily inferred that a similar amino acid residue B'is present.
  • amino acid residues can be identified to some extent. Therefore, for amino acid residues contained in conserved sequences containing substrate binding sites, active center residues, and catalytic centers, there are significant or similar amino acid residues between prokaryotes, or at least between actinomycetes. , This can be found by sequence comparison. Furthermore, even among all living organisms, amino acid residues including substrate binding sites, active center residues, and catalytic centers are conserved according to the closeness of the species, and amino acids corresponding to or similar to certain amino acid residues are found. would be possible.
  • Methods for predicting active central amino acid residues and substrate binding sites are known in the art, and protein databases (such as SwissProt and SwissModel) are used to obtain structural and / or functionally similar protein conformational information. Based on the structure, the structure of the protein can be predicted. Since the three-dimensional structure information of the structural and / or functionally similar proteins obtained from these databases also includes the positional information of the substrate or the substrate-like compound and the ligand such as the metal ion and the catalytic residue information, the predicted structure of the target protein Can also estimate the site of interaction with the substrate. Further, there is also known a method of estimating an interaction site with a substrate by docking simulation analysis of a substrate or a substrate-like compound using software such as AutoDock.
  • the label may be a label with a fluorescent molecule, a label with a radioactive substance, a label with a metal, a label with an enzyme, or a label with a tag, a compound that binds to an enzyme such as an inhibitor, or PEG. It may be a compound to be modified such as (polyethylene glycol), or these may be used in combination.
  • the label may be bound, adsorbed or coated, and its principle includes adhesion utilizing any physicochemical interaction or reaction including covalent bonds and hydrophobic interactions.
  • the labeling with a fluorescent molecule is for fluorescence microscopy and may be a fluorescent molecule such as rhodamine or FM dye or a fluorescent protein such as GFP.
  • the label with a fluorescent molecule may change its brightness due to binding to various biomolecules such as phospholipids and proteins, or may reversibly fade.
  • the labeling with radioactive material is 3 H, 14 C, 32 P, 33 P, 35 S, or 125 I, preferably 3 H, 14 C, or 35 S.
  • the metal labeling is for electron microscopy and may be a labeling with gold, uranium, tungsten, or vanadium.
  • the enzymatic labeling may be luciferase, ⁇ -galactosidase, peroxidase, alkaline phosphatase, or the like.
  • tag labeling has the effect of increasing the solubility of recombinantly expressed proteins: GST tag, Halo tag, TF tag, FLAG tag, HA tag, His tag, Myc tag, V5 tag, S tag, E. It may be a tag, a T7 tag, a VSV-G tag, a Glu-Glu tag, a Strept-tagII, a CBD tag, a CBP tag, an Fc tag, a GST tag, an MBP tag, a Trx tag, a biotin-streptavidin tag, or the like.
  • the polypeptide of the present invention may have substrate-specific water resolution with respect to PG.
  • the substrate-specific water resolution is 30% for other substrates under the condition of pH 6.0, 55 ° C., and 5 minutes, where the water resolution of PG is 100%. 20% or less, 15% or less, 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, 1% or less, Alternatively, it may be determined by 0% or less.
  • the other substrate is a phospholipid, for example, may be selected from the group consisting of PA, PE, PC, CL, PS, and PI, preferably CL. In a more preferred embodiment, other substrates may include PA, PE, PC, CL, PS, and PI.
  • the optimum pH of the polypeptide of the invention is in the range of about pH 5.5 to pH 6.5.
  • the lower limit of pH at which the polypeptide of the invention is allowed to act is pH 4, preferably pH 5.0, at pH 5.5, which exhibits an action of 90% or more compared to maximum activity. Is particularly preferable.
  • the upper limit is pH 10, preferably pH 8.5, and is particularly preferably pH 6.5, which exhibits an action of 90% or more as compared with the maximum activity.
  • the optimum temperature for the polypeptide of the invention is about 53-57 ° C.
  • the lower limit of the temperature at which the polypeptide of the invention is allowed to act is 30 ° C, preferably 37 ° C, at 50 ° C, which exhibits an action of 80% or more compared to the maximum activity. Is particularly preferable.
  • the upper limit is 67 ° C., preferably 65 ° C., and 60 ° C., which exhibits an action of 80% or more as compared with the maximum activity, is particularly preferable.
  • the reaction time of the polypeptide of the present invention may be changed as long as the intended degradation of PG can be achieved.
  • the reaction time is about 15 seconds or longer, preferably about 1 minute or longer, and more preferably about 3 minutes or longer.
  • the upper limit of the reaction time is not particularly limited, but it may be preferably about 30 minutes or less, more preferably about 15 minutes or less, and particularly preferably about 10 minutes or less.
  • the method for measuring the water resolution by the polypeptide of the present invention includes a method for measuring a decrease in the concentration of a substrate due to hydrolysis and a method for measuring an increase in the concentration of a decomposition product due to hydrolysis.
  • a method for measuring an increase in the concentration of a decomposition product due to hydrolysis includes a method for measuring the concentration of the decomposition product and a method for further decomposing the decomposition product to measure the concentration.
  • a method of measuring the concentration of a decomposition product using an enzyme or a radioisotope can be mentioned.
  • an enzyme and a Trinder reagent for example, an enzyme and a Trinder reagent (TODB, TOOS, phenol, and a halogenated phenol derivative are condensed in the presence of peroxidase) are condensed.
  • Trinder reagent TODB, TOOS, phenol, and a halogenated phenol derivative are condensed in the presence of peroxidase
  • Methods using (reagents utilizing color development), molybdenum blue method, malakite green method, and improved methods thereof (BIOMOL (registered trademark) Green, etc.) are known.
  • the composition of the solution containing the PLC and substrate can be modified by one of ordinary skill in the art.
  • the solution may comprise a buffer, wherein the buffer is acetate buffer (Ac-Na), phosphate buffer, citrate buffer, citrate phosphate buffer, borate buffer, tartrate buffer, Tris buffer (Tris). -HCl), Bis-Tris buffer, MES buffer (MES-NaOH), HEEPS buffer, or phosphate buffer, but is not limited thereto.
  • the solution may contain metal ions or metal ion chelators, such as Al 3+ , Ca 2+ , Mg 2+ , Li 2+ , Na + , Mn 2+ , Fe 3+ , Li + , Cu.
  • the metal ion chelator may be, but is not limited to, EDTA, EGTA, BAPTA, DTPA, HEADTA, NTA, DTPA, GLDA, TTHA, HIDA, DHEG and the like.
  • the solution may contain a surfactant, which includes Triton® X-100, Triton® X-114, Nonidet P40, Tween® 20. , Sodium deoxycholate, Tween® 80, Briji35, and sodium cholate, but are not limited thereto.
  • the polypeptide of the invention may be of a microorganism belonging to the order Actinomycetales.
  • the microorganism belonging to the family Pseudonocardiace is preferable among the microorganisms belonging to the order Actinomycetales
  • the microorganism belonging to the genus Amycolatopsis is particularly preferable among the microorganisms belonging to the family Pseudonocardiace
  • Amycolatopsis is a microorganism belonging to the genus Amycolatopsis is, for example, "Bergey's Manual 2nd Edition (2001)", “Classification of microorganisms. Identification experiment method-Focusing on molecular genetics and molecular biology methods (Springer Lab Manual) Springer Fairlark Tokyo, September 2001 ”, etc., and commercially available identification test products (for example, BIOMERIEUX) It may be confirmed by the method of use, the method of outsourcing to "Technosuruga Lab Co., Ltd. (Shizuoka City, Shizuoka Prefecture)", etc. Furthermore, those strains are found in Amycolatopsis sp.
  • Methods for measuring the molecular weight of the polypeptide of the present invention are known to those skilled in the art, for example, electrophoresis using Native-PAGE or SDS-PAGE, gel filtration chromatography, HPLC, mass analysis, ultracentrifugation. It can be measured by a precipitation equilibrium method using a separation device, a light scattering method using dynamic light scattering (DLS), or the like.
  • the molecular weight of the polypeptide of the present invention can be from about 49,000 Da to about 59,000 Da, preferably from about 52,000 Da to about 56,000 Da, as measured by electrophoresis using SDS-PAGE. , More preferably about 54,000 Da.
  • the molecular weight of the polypeptide of the present invention can be about 51,000 Da to about 62,000 Da, preferably about 54,000 Da to about 58,000 Da, when estimated from SEQ ID NO: 2, which is an amino acid sequence excluding the signal sequence. It can be, more preferably about 56,000 Da.
  • the present specification provides a polynucleotide encoding the above-mentioned polypeptide.
  • the polynucleotide may be DNA or RNA. In the case of RNA, T may be changed to U from the described sequence.
  • the polynucleotide may be single-stranded or double-stranded.
  • the polynucleotide of the present invention is a polynucleotide containing (a) a base sequence represented by SEQ ID NO: 1 and (b) a base sequence complementary to the base sequence represented by SEQ ID NO: 1. c) Containing a polynucleotide that hybridizes with the polynucleotide of (b) under stringent conditions, stringent conditions are defined herein.
  • the polynucleotide of the invention comprises (d) a polynucleotide comprising a base sequence in which one or more bases have been deleted, substituted or added in the base sequence represented by SEQ ID NO: 1.
  • the number of deletions, substitutions or imparted bases that the polynucleotides of the invention may contain will not significantly change the function of the expressed polypeptide (provided they are substantially equivalent).
  • the substrate specificity is, for example, pH 6.0, 55 ° C.
  • the water resolution of PG under the condition of 5 minutes is 100%
  • other substrates PA, PE, PC, CL, PS, And if the water resolution for one or more of PIs) is at most 30% or less
  • the number may be 300 or more, 300 or less, 200 or less, and preferably 100 or less.
  • the mutation of the base may be an artificial mutation or a mutation occurring in the natural world.
  • the polynucleotide of the invention comprises (e) a polynucleothio comprising a base sequence containing a base sequence synonymous with the base sequence represented by SEQ ID NO: 1, and a species of organism expressing this polynucleotide.
  • the base sequence can be changed according to the codon frequency without changing the amino acid sequence. Methods of changing the codon frequency and base sequence in each species are known to those of skill in the art.
  • the polynucleotide of the invention comprises (f) a polynucleotide comprising a base sequence having at least about 80% identity with the base sequence represented by SEQ ID NO: 1.
  • the identity may be at least 80%, preferably at least 90%, more preferably at least 95%, as long as the encoded polypeptide has the substrate specificity intended in the present invention. May be.
  • the present specification provides a recombinant vector containing a polynucleotide encoding a polypeptide having a hydration resolution of PG, and a transformant containing the recombinant vector.
  • the vectors that can be used include, but are not limited to, plasmid vectors, cosmid vectors, phage vectors, viral vectors, or transposon vectors. In the present invention, any vector can be used as long as the polynucleotide sequence of interest can be transferred into the cell of interest.
  • the vector is introduced by the lipofection method, the electric perforation method, the calcium phosphate method, etc., the gene knock-in using the CRISPR-Cas9 system, and the DNA injection into the fertilized egg nucleus.
  • Examples include, but are not limited to, gene knock-in, gene knock-in using homologous recombination, and the like.
  • the host organism to which the gene can be introduced as a transformant is not limited as long as it is an organism capable of introducing a recombinant vector and expressing a target protein.
  • Host organisms that can be used include, for example, germs such as Bacillus subtilis, Bacillus subtilis, or fungi such as Escherichia coli, fungi such as yeast or mold, cultured cells derived from plants such as Chinese hamster ovaria, or HEK cells, HeLa cells, or CHO cells. Examples include, but are not limited to, cultured cells derived from mammals such as.
  • the present specification provides a method for producing a polypeptide having a hydration decomposing ability of PG.
  • the method for producing a polypeptide comprises culturing a transformant to produce the polypeptide.
  • the method for producing a polypeptide further comprises the step of purifying the polypeptide of interest.
  • Experimental methods using polypeptides are known to those skilled in the art and are described, for example, in Tairo Oshima et al. (Editor), "Post-Sequence Protein Experimental Method", Tokyo Kagaku Dojin, Volumes 1 to 4.
  • the transformant used to produce the polypeptide is E. coli.
  • Methods for culturing Escherichia coli and expressing proteins are known to those skilled in the art, and are described in, for example, Hiroki Nakayama and Takahito Nishikata, "Bio-Experiment Illustrated 1 Basics of Molecular Biology Experiments", Shujunsha, etc. There is.
  • the step of purifying a polypeptide from a cultured transformant may include a step of extracting the polypeptide from cultured cells, and may further include a step of purifying the target polypeptide.
  • the polypeptide produced by the cells may be secreted into the culture medium, distributed in the cytoplasm, or distributed on the cell membrane or periplasm.
  • the polypeptide produced by the cell may degrade the PG contained in the cell membrane of the cultured cell and be secreted extracellularly or periplasm.
  • the cells when the polypeptide is distributed in the cytoplasm, periplasm, or cell membrane, the cells are collected by centrifugation or the like, and then mechanically disrupted using a homogenizer or the like, or ultrasonic waves are used.
  • the cells are destroyed by a crushing method, a crushing method using a dairy pot and a dairy stick, or a chemical cell lysis method using lysozyme or a surfactant, and unnecessary fractions are removed by centrifugation or the like.
  • the polypeptide may be extracted by concentrating the required fraction.
  • the protein when the polypeptide is secreted into the culture medium, the protein may be extracted by collecting the culture medium and removing unnecessary cell debris and the like by centrifugation.
  • the polypeptide when extracting a polypeptide, may be efficiently extracted by using a precipitation method in which the polypeptide is precipitated with a precipitating agent and centrifuged.
  • a precipitating agent include, but are not limited to, chaotropic salts such as ammonium sulfate, water-soluble polymers such as polyethylene glycol or dextran, acids such as trichloroacetic acid or hydrochloric acid, and organic solvents such as acetone or alcohol.
  • the preferred precipitant is ammonium sulphate, ethylene glycol, or dextran, which is less likely to denature the polypeptide, more preferably ammonium sulphate.
  • a surfactant when extracting a polypeptide, may be added to solubilize the polypeptide, if necessary.
  • Surfactants include Triton® X-100, Triton® X-114, Nonidet P40, Tween® 20, Sodium Deoxycholate, Tween® 80, Briji35, and Cole. Examples include, but are not limited to, sodium acid.
  • the extracted polypeptide may be purified by a chromatographic method.
  • the procedure of the chromatography method is known to those skilled in the art, and gel filtration chromatography using the size of the molecule, cation exchange chromatography and anion exchange chromatography using charge, and hydrophobic interaction chromatography using hydrophobicity. Examples include imaging and reverse phase chromatography, as well as affinity chromatography for purification utilizing binding affinity. These chromatographic methods may be used alone or in combination.
  • affinity chromatography for that tag may be used if the purified polypeptide has a label such as a tag.
  • the polypeptides of the invention may be purified in combination with reciprocal hydrophobic action chromatography, anion exchange chromatography, and gel filtration chromatography.
  • a column may be used, or a batch method using a chromatography carrier may be used.
  • the concentration of the purified polypeptide may be measured and combined with the above-mentioned method for measuring the water resolution of the polypeptide to measure the specific activity of the polypeptide.
  • Methods for measuring the concentration of a polypeptide include absorptiometry (absorptiometry, Bradford, WST, Biuret, Lowry, and BCA), fluorescence, and electrophoresis. However, it is not limited to these (Yoshio Suzuki, “Method for quantifying total protein", Bunseki, 1, 2-9 (2018)).
  • the absorptiometry method is preferable, and the BCA method is more preferable.
  • the invention provides a method of using a polypeptide having PG water decomposing ability.
  • this method of use provides a method of degrading PG with a polypeptide and a method of producing G1P and G3P, and DG from PG.
  • the PG in the method using the polypeptide of the present invention may be of biological origin or may not be of biological origin.
  • the phospholipids that make up the cell membrane of bacteria contain about 10 to 25% of PG, which is higher than that of eukaryotic cell membranes. .. Therefore, it is conceivable to react the polypeptide of the present invention with the cell membrane of a bacterium to produce glycerol phosphate.
  • the organism used is assumed to be any organism as long as it contains PG.
  • it may be a prokaryote, a eukaryote, or an organism contained in excess sewage sludge, waste agricultural products, or waste plants, and is not particularly limited.
  • eukaryotes include molds, yeasts and mushrooms
  • prokaryotes such as archaea and eubacteria include actinomycetes, Bacillus subtilis, and Escherichia coli, but Escherichia coli with established treatment is preferable.
  • As a method for obtaining a large amount of E. coli not only a laboratory method using a culture solution but also a method for culturing E. coli using biomass, which is a resource derived from a renewable organism, is assumed.
  • Biomass includes waste paper, livestock manure, food waste, construction waste, sawmill residue, black liquor, and waste-based biomass such as sewage sludge, rice straw, wheat straw, rice husks, non-food parts of agricultural products, and forest land. Examples include unused biomass such as residual material, and resource crops such as corn and sugar cane cultivated for the purpose of using as biomass.
  • waste-based biomass such as sewage sludge, rice straw, wheat straw, rice husks, non-food parts of agricultural products, and forest land.
  • examples include unused biomass such as residual material, and resource crops such as corn and sugar cane cultivated for the purpose of using as biomass.
  • the phospholipid hydrophilic moieties of PG in E. coli cell membranes are predominantly phospho- (1'-sn-glycerol) type and, for example, above 40 ° C.
  • E. coli when E. coli is cultured in an environment that increases the rate at which E. coli synthesizes phospho- (3'-sn-glycerol) type PG, for example, under high temperature conditions of 50 ° C., about 25% of the total PG is phospho- (3'-sn-glycerol). Since it becomes a 3'-sn-glycerol) type PG, G3P can be efficiently obtained by hydrolyzing the PG with the polypeptide of the present invention.
  • a method for recovering E. coli from biomass is known to those skilled in the art, and examples thereof include recovery of E. coli from a liquid component in biomass, washing of biomass with water, and recovery of E. coli from a washing solution.
  • the phospholipid may be extracted from the recovered E. coli and then reacted with the polypeptide having the hydration decomposing ability of PG.
  • a method for extracting a phospholipid from Escherichia coli is known to those skilled in the art, and examples thereof include a Brich-Dyer method using an organic solvent such as chloroform and methanol, and a Folch method. If necessary, thin layer chromatography, solid phase extraction, high performance liquid chromatography and the like may be performed.
  • the phospholipid may not be extracted from E. coli, and the polypeptide having the hydration decomposing ability of PG may not be purified.
  • PG-PLC when PG-PLC is expressed in E. coli cells, it decomposes PG contained in the cell membrane of E. coli to produce glycerol phosphate and PG. -PLC is secreted extracellularly. Then, the PG-PLC secreted extracellularly can decompose PG contained in the cell membrane of other Escherichia coli to produce glycerol phosphate. Therefore, in one embodiment, E.
  • coli expressing the polypeptide is grown in the biomass without purifying the polypeptide and extracting phospholipids, which is then added to the cell membrane of the same or heterologous E. coli in the biomass.
  • the PG may be cleaved and the obtained glycerol phosphate may be recovered.
  • the method using the polypeptide of the present invention is preferably carried out in a liquid, and an aqueous phase and an organic solvent phase are assumed as the liquid, and the method is preferably carried out in the aqueous phase.
  • the composition of the solution containing the polypeptide and PG can be modified by one of ordinary skill in the art.
  • the solution may comprise a buffer, wherein the buffer is acetate buffer (Ac-Na), phosphate buffer, citrate buffer, citrate phosphate buffer, borate buffer, tartrate buffer, Tris buffer (Tris). -HCl), Bis-Tris buffer, MES buffer (MES-NaOH), HEEPS buffer, or phosphate buffer, but is not limited thereto.
  • the solution may contain metal ions or metal ion chelators, such as Al 3+ , Ca 2+ , Mg 2+ , Li 2+ , Na + , Mn 2+ , Fe 3+ , Li + , Cu. It may be, but is not limited to, 2+ , Co 2+ , Zn 2+ , and the like.
  • the metal ion killer may be, but is not limited to, EDTA, EGTA, BAPTA, DTPA, HEADTA, NTA, DTPA, GLDA, TTHA, HIDA, DHEG and the like.
  • the solution may contain a surfactant, which includes Triton® X-100, Triton® X-114, Nonidet P40, Tween® 20. , Sodium deoxycholate, Tween® 80, Briji35, and sodium cholate, but are not limited thereto.
  • a surfactant which includes Triton® X-100, Triton® X-114, Nonidet P40, Tween® 20. , Sodium deoxycholate, Tween® 80, Briji35, and sodium cholate, but are not limited thereto.
  • the glycerol phosphoric acid produced by using the polypeptide of the present invention may remain mixed with raw materials or other components, but may be purified so as not to contain impurities according to the purpose and use.
  • Methods for purifying glycerol phosphate are well known in the art, and include thin layer chromatography, recrystallization of glycerol phosphate as a salt with calcium or sodium (Japanese Patent Laid-Open No. 2014-189552; Ryo Fujimori). Toshi and Yoshito Takatsu, "Material Cycle Using Used CaO Catalysts in Biodiesel Production", Proceedings of the Biomass Science Conference, 13 (0), 121-122, 2018).
  • Glycerol phosphate produced using the polypeptide of the present invention is expected to contain a large amount of specific enantiomers of G1P or G3P, but the purity of the enantiomers is further increased by a method known in the art. You may.
  • the optical resolution method for the enantiomer include a crystallization method (priority crystallization method, diastereomer method, inclusion complex method, method using preferential enrichment, etc.), HPLC method using a chiral column, and the like. Will be.
  • composition containing polypeptide provides a composition for degrading PG containing the polypeptide of the invention.
  • the composition containing the polypeptide of the invention may be sold as a kit.
  • the composition containing the polypeptide of the invention may be a solution, a frozen solution of the solution, or a dried solution (by vacuum drying, lyophilization, spray drying, etc.). Includes, for example, pH buffers, salts, surfactants, reducing agents, metal chelators, freeze protectants (such as glycerol or ethylene glycol), preservatives (such as sodium azide or thimerosal), and protease inhibitors. But it may be.
  • a glycerol solution containing the polypeptide or a lyophilized product containing the polypeptide is preferred.
  • Example 1 Screening for acquisition of PG-PLC-producing bacteria and taxonomic identification for genus determination of PG-PLC-producing bacteria] (experimental method)
  • the reagents used in the experiment are as follows. Bacto-Malt extract (Becton Dickinson, model number 218630), Yeast Extract BSP-B (Oriental Yeast Co., Ltd.), L- ⁇ -Phosphatidylgycerol, Egg (PG, Avanti Polar Yeast Co., Ltd.) Model number 46261003), L- ⁇ -glycellophothsite oxidase (GPO, Toyobo Co., Ltd., model number G3O-321), 4-Aminoantipyrine (4-AA, Nacalai Tesque, model number 01907), N, N-Bis (4-sulfobutyl) -3.
  • -Methylaniline, disposable salt TODB, Dojin Chemical Research Institute, model number OC22
  • G3P is oxidized by glycerol-3-phosphate oxidase (GPO) to generate hydrogen peroxide.
  • GPO glycerol-3-phosphate oxidase
  • the generated hydrogen peroxide quantitatively oxidatively condenses 4-aminoantipyrine (4-AA) with N, N-Bis (4-sulfovutyl) -3-methylaniline and disodium salt (TODB) by the action of peroxidase (POD). It produces a reddish-purple quinoimine dye.
  • the water resolution of PLC can be measured by measuring the intensity of this color development at an absorbance (A 550 ) of 500 to 630 nm, particularly 550 nm. Using the measurement method, it was examined whether or not PLC that hydrolyzes PG was present in the supernatant of the culture solution of the screening strain. The amount of enzyme that produces 1 ⁇ mol of G3P per 1 min was defined as 1 U.
  • the NT115 strain was included in the cluster composed of the genus Amycolatopsis. It showed a molecular phylogenetic position different from that of known species. Therefore, from the result of this 16SrDNA partial base sequence analysis, the NT115 strain is Amycoloptissis sp. Attributed. This result was also in agreement with the result of simple morphological observation.
  • Example 2 Purification of PLC secreted and produced by NT115 strain outside the cells
  • Amycolatopsis sp Purification of NT115 strain PLC
  • ISP2 medium and 3 glass beads were placed in a culture test tube ( ⁇ 18 ⁇ 180 mm) and sterilized by autoclave (121 ° C., 15 min). After cooling, 50 ⁇ l of a 10% (v / v) glycerol stock suspension of NT115 strain was inoculated in a clean bench and cultured with reciprocating shaking at 28 ° C. and 160 spm for 2 days to prepare a preculture solution.
  • Dialysis PPG fraction is transferred to a dialysis membrane (Sanko Pure Medicine, cellulose tube 36/32) and dialyzed twice with 20 mM Tris-HCl (pH 9.0), which is 10 times the sample volume (buf exchange; 1h, 12h).
  • GCQ Giga Cap Q-Toyopearl Column Chromatography
  • SDS-PAGE analysis The SDS-PAGE method was performed according to the method of Laemmli. A silver staining kit (Aproscience) was used to detect protein bands, and the procedure was performed according to the instruction manual.
  • Example 3 Analysis of various characteristics of PG-PLC] (experimental method) Except for the substrate specificity test, the enzyme activity was measured by the method described in Example 1. In the substrate specificity test, 1-palmitoyl-2-oleoyl-sn-glycello-3-phopsphoglycerol (POPG), 1-palmitoyl-2-oleoyl-sn-glycello-3-phopsphocholine (POPC), 1-palmit as substrates.
  • POPG 1-palmitoyl-2-oleoyl-sn-glycello-3-phopsphocholine
  • POPC 1-palmit as substrates.
  • POPE 2-oleoyl-sn-glycello-3-phopsphoethanolamine
  • POPI 1-palmitoyl-2-oleoyl-sn-gycero-3-phopsphoinositol
  • POPS 1-palmitoyl-2-oleoyl-sn- POPS
  • POPA 1-palmitoyl-2-oleoyl-sn-glycello-3-phopsphate
  • CL L- ⁇ -phospatidyglycerol
  • BIOMOL Green (registered trademark) Reagent hereinafter abbreviated as BG
  • Alkaline phosphatase Oriental Yeast Co., Ltd., derived from calf small intestine, hereinafter abbreviated as CIAP
  • the enzyme solution (purified PG-PLC sample) to be added to the enzyme reaction solution was 0.05 U / ml, 1.50 U / mg-protein or more, and was appropriately diluted so that the substrate consumption was about 10%.
  • the pH was stable in a wide range from pH 4.0 to pH 9.0.
  • the temperature was stable up to 50 ° C., and the activity was halved at 60 ° C.
  • Substrate specificity As shown in Fig. 6, it showed no activity on PC, PE, and CL, showed no activity on PA (data not shown), and had almost no effect on anything other than PG. It can be determined that the enzyme is a PG-specific PLC.
  • Example 4 PG-PLC sequence
  • Example 4 Peptide sequence determination After electrophoresis of PG-PLC by SDS-PAGE, a band was excised, and trypsin treatment and LC-MS analysis were performed at Antegral Co., Ltd. to analyze the amino acid sequence.
  • the peptide sequences obtained by LC-MS analysis and the protein sequences obtained from the database matching these peptide sequences are shown in FIG.
  • the gray highlight part is the sequence that matches the peptide sequence of this enzyme.
  • Forward primers of amino acid SEQ ID NO: 6 to SEQ ID NO: 5 and reverse primers of amino acid SEQ ID NO: 8 to SEQ ID NO: 7 were designed and PCR was performed.
  • the PCR conditions are as follows.
  • the amplified fragment obtained by PCR at 94 ° C. for 1 minute ⁇ (98 ° C., 10 seconds ⁇ 55 ° C., 15 seconds ⁇ 68 ° C., 45 seconds) for 25 cycles and 68 ° C. for 5 minutes was used as a cloning vector pMD20 (Takara Bio). After ligation, Escherichia coli DH5alpha was transformed and cultured, and the recombinant plasmid was recovered and purified.
  • iPCR inverse PCR
  • the 5'and 3'undeciphered regions were decoded, and the full-length sequence of this enzyme gene (the sequence of SEQ ID NOs: 1 and 2 with the signal sequences of SEQ ID NOs: 3 and 4 added) was decoded.
  • the amino acid sequence shown in FIG. 8 was obtained.
  • the signal sequence consisting of 25 amino acids is shown as SEQ ID NO: 4
  • the base sequence thereof is shown as SEQ ID NO: 3
  • the amino acid sequence of the mature protein consisting of 514 amino acids is shown in SEQ ID NO: 2
  • the base sequence thereof is shown in SEQ ID NO: 2. Shown as 1.
  • Example 5 Production of heterologous recombinant PG-PLC and decomposition of Escherichia coli PG
  • the DNA encoding PG-PLC set forth in SEQ ID NO: 1 obtained in Example 4 was PCR amplified using the primers of SEQ ID NOs: 13 and 14, gel electrophoresis was performed, and the amplified band was excised and purified.
  • the PCR conditions are as follows. 94 ° C, 1 minute ⁇ (98 ° C, 10 seconds ⁇ 64 ° C, 15 seconds ⁇ 68 ° C, 1 minute 15 seconds) for 25 cycles, 68 ° C, 5 minutes, pET-24a (+) after HindIII digestion, gel electrophoresis It was run, cut out and purified.
  • PG-PLC / pET-24a (+) containing the polynucleotide encoding the polypeptide of the invention. It was created. Therefore, 21 amino acids consisting of MASMTGGGQQMGRGSQFQLRRQ containing the T7 epitope derived from the multicloning site (MCS) are added to the N-terminal side of the PG-PLC mature sequence.
  • PG-PLC / pET-24a (+) was transformed into Zip Competent Cell BL21 (DE3) (biodynamics, product number: DS255), cultured on LB agar medium containing a final concentration of 50 ⁇ g / mL kanamycin, and PG- PLC / pET-24a (+) / BL21 (DE3) was obtained. It also contains kanamycin at a final concentration of 50 ⁇ g / mL Overnight express instant TB medium. The cells were inoculated into 5 mL (liquid medium) and cultured at 20 ° C. or 30 ° C. for 1 day to express PG-PLC.
  • glycerol phosphate can be obtained from Escherichia coli, a biomass that can grow in large quantities.
  • Example 6 Estimation of catalyst (active) central site and substrate binding site.
  • the structures and sequences on the database predict the three-dimensional (stereo) structure of PG-PLC based on existing proteins. Based on the above, the amino acids that serve as the catalytic (active) central site and substrate binding site of the enzyme were estimated.
  • PG-PLC protein family to which PG-PLC belongs was investigated using Pfam, which is a database of protein domain motifs, and CATH, which is a database of protein conformations and families. Then, using any of the databases, PG-PLC was presumed to belong to the metal ion-requiring phospholipase, and the prokaryotic zinc-dependent (requiring) phospholipase C signature (HYx- [GT]. -D- [LIVMAF]-[DNSH] -x-P-x-H- [PA] -x-N) had five sequences. Furthermore, when the three-dimensional (three-dimensional) structures of the proteins belonging to this family were compared, the structures were similar.
  • PG-PLC In order to predict the structure of PG-PLC, a protein having a similar three-dimensional (three-dimensional) structure in amino acid sequence was investigated using SwissModel, which is a database of protein three-dimensional structure. Then, a metal ion-requiring phosphoesterase called 2xmo was found, and a three-dimensional structure model of PG-PLC was obtained based on the three-dimensional structure of 2xmo.
  • the three-dimensional structure model of PG-PLC was compared with four types of PLCs with known structures. As a result, it was considered that each PLC had a pocket rich in acidic amino acids near the center of the protein, and the substrate was bound to this pocket. Furthermore, when the position of the substrate in a PG non-specific PLC (for example, PC-specific PLC) having a known structure was applied to PG-PLC and the amino acid residues in contact with the substrate were estimated, the amino acid residues obtained in FIG. 10 were obtained.
  • a PG non-specific PLC for example, PC-specific PLC
  • SEQ ID NO: 2 was sequenced among various actinomycetes using BLAST. Compared. As a result, in the amino acids at positions 35 to 488 of SEQ ID NO: 2 (AFV ... SYN; SEQ ID NO: 16), high sequence homology and similarity were observed among more than 25 types of actinomycetes, and the substrate binding site, It was shown that the active central amino acid residue, and the catalytic residue, are conserved across species at least among the actinomycetes. In addition, in the analysis by Pfam, the amino acids at positions 36 to 409 of SEQ ID NO: 2 are the Metallophos motif (Pfam). It was assigned as an accession ID: PF00149).
  • SEQ ID NO: 1 Amycolatopsis sp. Nucleobase sequence of PG-PLC from which it was derived
  • SEQ ID NO: 2 Amycolatopsis sp. Derived PG-PLC mature protein sequence
  • SEQ ID NO: 3 Amycolatopsis sp. Nucleobase sequence of the signal sequence of PG-PLC in FIG.
  • Signal sequence of PG-PLC in 5 Forward primer for sequence identification of PG-PLC SEQ ID NO: 6: Amino acid sequence corresponding to the primer of SEQ ID NO: 5
  • SEQ ID NO: 7 Reverse for sequence identification of PG-PLC Primer
  • SEQ ID NO: 8 Amino acid sequence corresponding to the primer of SEQ ID NO: 7
  • SEQ ID NO: 9 Partial base sequence of the identified PG-PLC SEQ ID NO: 10: Partial amino acid sequence of the identified PG-PLC SEQ ID NO: 11: PG-PLC Primer for iPCR 1 for sequence identification of SEQ ID NO: 12: Primer 2 for iPCR for sequence identification of PG-PLC SEQ ID NO: 13: Forward primer for subcloning SEQ ID NO: 14: Reverse primer for subcloning SEQ ID NO: 15: Amino acid SEQ ID NO: 16 containing the MCS-derived T7 epitope imparted to the N-terminal of PG-PLC during E.

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Abstract

La présente invention concerne une nouvelle phospholipase C spécifique du PG (PG-PLC) provenant d'un microorganisme appartenant au genre Actinomycetales. Le phosphate de glycérol est un composé doté d'un centre chiral qui se présente sous la forme de trois isomères positionnels, dont une paire d'énantiomères (G1P, G2P et G3P, G1P et G3P étant des énantiomères). Un réactif, comprenant un énantiomère spécifique du phosphate de glycérol ayant été isolé seul, présente un coût élevé. En hydrolysant le PG avec l'utilisation de la PG-PLC selon la présente invention, contrairement à l'utilisation de réactif susmentionnée, le phosphate de glycérol sous forme d'isomères positionnels tels que G1P et G3P peut être produit efficacement à faible coût.
PCT/JP2021/032497 2020-09-04 2021-09-03 Nouvelle enzyme spécifique du phosphatidylglycérol WO2022050387A1 (fr)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS4955893A (fr) * 1972-10-07 1974-05-30
JP2005328781A (ja) * 2004-05-21 2005-12-02 Nagase Chemtex Corp 新規ホスホリパーゼｃ
JP2006223119A (ja) * 2005-02-15 2006-08-31 Sankyo Lifetech Co Ltd 新規ホスホリパーゼｃ
JP2011010608A (ja) * 2009-07-02 2011-01-20 Fukushima Univ ホスホリパーゼｃおよびそれを用いたリン脂質の分解方法

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS4955893A (fr) * 1972-10-07 1974-05-30
JP2005328781A (ja) * 2004-05-21 2005-12-02 Nagase Chemtex Corp 新規ホスホリパーゼｃ
JP2006223119A (ja) * 2005-02-15 2006-08-31 Sankyo Lifetech Co Ltd 新規ホスホリパーゼｃ
JP2011010608A (ja) * 2009-07-02 2011-01-20 Fukushima Univ ホスホリパーゼｃおよびそれを用いたリン脂質の分解方法

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
DATABASE PROTEIN 27 July 2019 (2019-07-27), ANONYMOUS: "TIGR03767 family metallophosphoesterase [Amycolatopsis orientalis]", XP055907608, retrieved from GENBANK Database accession no. WP_037369513 *
S̆IMOC̆KOVÁ MÁRIA, HOLIC̆ ROMAN, TAHOTNÁ DANA, PATTON-VOGT JANA, GRIAC̆ PETER: "Yeast Pgc1p (YPL206c) Controls the Amount of Phosphatidylglycerol via a Phospholipase C-type Degradation Mechanism", JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 283, no. 25, 1 June 2008 (2008-06-01), US , pages 17107 - 17115, XP055907607, ISSN: 0021-9258, DOI: 10.1074/jbc.M800868200 *

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