WO2022050387A1 - Novel enzyme specific to phosphatidylglycerol - Google Patents

Abstract

The present inventor found a novel PG-specific phospholipase C (PG-PLC) from a microorganism belonging to Actinomycetales. Glycerol phosphate is a compound that has a chiral center and occurs as three positional isomers including a pair of enantiomers (G1P, G2P, and G3P; G1P and G3P being enantiomers). A reagent, which comprises a specific enantiomer of glycerol phosphate having been isolated alone, is expensive. By hydrolyzing PG with the use of the PG-PLC according to the present invention, in contrast thereto, glycerol phosphate in the form of positional isomers such as G1P and G3P can be efficiently produced at low cost.

Description

Phosphatidylglycerol-specific novel enzyme

The present invention relates to a novel protein having a phosphatidylglycerol (PG) hydrolytic decomposing ability and a method for using the same.

Development of substrate-specific enzymes is useful in various fields. By the way, although there are many examples of research reports on phospholipase C (PLC) (Japanese Patent Laid-Open No. 2006-223119), the report of PG-specific PLC (PG-PLC) is not known to the present inventor.

Japanese Unexamined Patent Publication No. 2006-223119

When the inventors searched for phospholipase, they found PG-specific PLC (PG-PLC) from microorganisms belonging to the order Actinomycetales. 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.
(Item 3)
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.
(Item 4)
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. , And the polypeptide of item 3, further comprising an amino acid corresponding to 432S-435D.
(Item 5)
A phospholipase C having a phosphatidylglycerol hydrolysis resolution, which is the following polypeptide (a), (b) or (c).
(A) Polypeptide containing the amino acid sequence represented by SEQ ID NO: 2 (b) Polypeptide containing an amino acid sequence in which one or more amino acids are deleted, substituted or imparted in the amino acid sequence represented by SEQ ID NO: 2. (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 (item 6).
The polypeptide of any of items 1-5, further comprising a label.
(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). A polynucleotide containing a base sequence containing a codon synonymous with the represented base sequence (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 μg-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 (〇), BisTris-HCl (Δ), MES-NaOH (●), or Tris-HCl (□). FIG. 3-2 shows the effect of temperature on PLC activity. Using POPG as a substrate, 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 (Δ), or Tris-HCl (〇). FIG. 4-2 shows the temperature stability of the PLC. Purified enzyme samples were incubated in 0.2 M MES-NaOH (pH 6.0) at each temperature for 30 minutes. Residual activity was measured in 0.2 M MES-NaOH (pH 6.0) at 55 ° C. using POPG as a substrate. 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. 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. FIG. 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.

[Definition of terms]
It should be understood that the terms used herein are used in the meaning commonly used in the art unless otherwise noted. Accordingly, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. In case of conflict, this specification (including definitions) takes precedence.

As used herein, "about" refers to the indicated value ± 10% unless otherwise defined.

As used herein, "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. The simplest is phosphatidylic acid (PA) if the hydrophilic part of the phospholipid is hydrogen, phosphatidylcholine (PC) if it is choline, phosphatidylethanolamine (PE) if it is ethanolamine, phosphatidylserine (PS), inositol if it is serine. If it is, it is called phosphatidylinositol (PI), and if it is glycerol, it is called phosphatidylglycerol (PG). Therefore, in the present specification, "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. Refers to bound phospholipids. In addition, "cardiolipin" (also known as diphosphatidylglycerol; CL) is known as a phospholipid having a special structure. This is a phospholipid in which two PGs are linked while sharing their glycerol, and has a structure showing hydrophobicity derived from four acyl chains and negatively charged polarity of two phosphoric acids. Since the pKa of the above is about 3, it is also considered that 50% or more of CL is negatively charged at pH 3 or higher.
Phosphatidylglycerol:

As used herein, the term "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.

In the present invention, "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, and 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. Refers to an enzyme that produces a phospholipid hydrophilic moiety (phosphodiester compound) and diacylglycerol.
Hydrolysis mode by PLC:

As used herein, the term "identity" of an amino acid sequence refers to the proportion of residues having the same amino acid when comparing a plurality of amino acid sequences. Regarding the homology of amino acid sequences, 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.

In general, an enzyme catalyzes a chemical reaction and has specificity for the type of reaction and specificity for a substrate. As used herein, 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. As used herein, 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.

As used herein, the term "polynucleotide that hybridizes under stringent conditions" refers to a polynucleotide that hybridizes under well-known conditions commonly used in the art. Such 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. Identified by washing the filter under 65 ° C. conditions using a ~ 2x concentration SSC (saline-sodium polynucleotide) solution (the composition of the 1x concentration SSC solution is 150 mM sodium chloride, 15 mM sodium citrate). Means a polynucleotide that can. 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. Yes, for example, J. The conditions described in Sambrook et al., Molecular Cloning, ALaboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989) can be mentioned. Further, for example, 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. Specifically, for example, 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. Sodium chloride (15 mM sodium citrate), preferably conditions for hybridizing at a salt concentration corresponding to 0.1 × SSC.

As used herein, the term "vector" or "recombinant vector" refers to a gene construct capable of transferring a polynucleotide sequence of interest into a cell of interest. Such 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. When a microorganism belonging to the above is used as a host, λgt / λC, λgt / λB 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. for the genus Streptomyces, and pTip series, pCPi series, pNit series, etc. of Hokkaido System Science Co., Ltd. for the genus Rhodococcus, especially Saccharomyces cerevisiae. In this case, 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. Furthermore, various cell-free protein expression systems can also be used. From the viewpoint that the safety of the recombinant vector of the present invention has been confirmed, the Ministerial Ordinance (Heisei 10) stipulates the diffusion prevention measures, etc. that should be taken for industrial use, etc., among the second-class use of genetically modified organisms, etc. Six years Ministry of Finance, Ministry of Health, Labor and Welfare, Ministry of Agriculture, Forestry and Fisheries, Ministry of Economy, Trade and Industry, Ministry of Environment Ordinance No. 1) Vectors listed in Appendix 1 of GILSP transgenic microorganisms specified by the Minister of Economy, Trade and Industry based on the provisions of Appendix 1. 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.

In the present specification, "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. Point to.
sn-glycerol-1-phosphate:

sn-glycerol-3-phosphate:

G1P and G3P are related to enantiomers. These are referred to by various names by nomenclature, G1P is also called L-glycerol-1-phosphate or D-glycerol-3-phosphate, and G3P is L-glycerol-3-phosphate or D-glycerol. Also called -1-phosphate. Further, in the present specification, "diaacylglycerol" (DG) refers to a fatty acid ester of glycerol to which two acyl groups are bonded, which is also referred to as diglyceride. Among them, 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)

As described in the definition of the above term, 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.

(In the formula, R ₁ and R ₂ are arbitrary acyl groups, respectively)

A compound having an enantiomer requires a complicated operation to separate a specific enantiomer, and the compound reagent obtained by the separation is generally expensive. 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.

[Embodiment]
The following embodiments describe preferred embodiments of the present invention. It is understood that the embodiments provided below are provided for a better understanding of the present invention and are not intended to limit the scope of the invention to the following description. sea bream.

(Polypeptide)
In one aspect, the present specification 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.

In one embodiment, 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. Monounsaturated fatty acids such as nervonic acid, n-6 (ω6) series polyunsaturated fatty acids such as linolenic acid, γ-linolenic acid, dihomo-γ-linolenic acid, arachidonic acid, or docosapentaenoic acid, or It may be derived from n-3 (ω3) series polyunsaturated fatty acids such as α-linolenic acid, stearidonic acid, eicosatetraenoic acid, eicosapentaenoic acid, or docosahexaenoic acid, but is not limited to these (Kihara). Akio, "Various Metaunsaturated Fatty Acids, Physiological Functions and Related Diseases," Biochemistry, 82 (7), 591-605, (2010)). In a preferred embodiment, 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. In one embodiment, 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.

In one embodiment, 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. In one embodiment, 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. for 5 minutes is 100%, one of the other substrates (PA, PE, PC, CL, PS, and PI) under this condition. Or, as long as the water resolution for (s) is at most 30% or less, 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. In addition, 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). It has been reported that the substrate specificity and specific activity were maintained, and that the heat resistance was further improved (Nakano S. et. Al., Benchmark Analysis of Native and Artificial NAD + -Dependent Enzymes Generated by with or without Phylogenetic Data. Biochemistry 2018, 372-3732).

In one embodiment, 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 are known to those of skill in the art and 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.

In one embodiment, when the polypeptide of the invention is used, 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. Can be applied. 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. In one embodiment, 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-. 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)). Further, it may be a fusion signal tag in which a plurality of types of signals or tags are freely combined. For example, OpPA and PelB, SKIK and PelB, OpPA and SKIK 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. 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 α-aglutinin and a GPI-anchored signal sequence.

In general, mutating other amino acid residues without altering important amino acid residues involved in enzyme activity or substrate binding keeps the amino acid sequence homology low, similar to or similar to the original enzyme. New enzymes can be created with further improved functions and properties. Methods for producing proteins, peptides and enzymes having such properties are known in the art. Therefore, a polypeptide produced by such a method, which has low homology between amino acid sequences but has similar properties to PG-PLC represented by SEQ ID NO: 2, is also included in the scope of the present application. In one embodiment, the polypeptides of the invention have a substrate binding site. In one embodiment, 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. In one embodiment, 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. In one embodiment, 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. In one embodiment, 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. Here, in the present specification, 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. 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. Furthermore, although it is not desired to be bound by a specific theory, in the polypeptide of the present invention, 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. That is, since a person skilled in the art can easily predict the three-dimensional structure from the primary structure (amino acid sequence) of the protein and predict the spatial position of the constituent amino acids with considerable accuracy, they are arranged at similar positions in the three-dimensional structure. 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.

In one embodiment, 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. In addition, 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. In one embodiment, 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. In one embodiment, 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. In one embodiment, the metal labeling is for electron microscopy and may be a labeling with gold, uranium, tungsten, or vanadium. In one embodiment, the enzymatic labeling may be luciferase, β-galactosidase, peroxidase, alkaline phosphatase, or the like. In one embodiment, 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. In one embodiment, 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. Here, 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.

In one embodiment, the optimum pH of the polypeptide of the invention is in the range of about pH 5.5 to pH 6.5. In one embodiment, 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.

In one embodiment, the optimum temperature for the polypeptide of the invention is about 53-57 ° C. In one embodiment, 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.

In one embodiment, 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.

A method for measuring the water resolution is known to those skilled in the art. In one embodiment, 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. In one embodiment, 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. In one embodiment, a method of measuring the concentration of a decomposition product using an enzyme or a radioisotope can be mentioned. In one embodiment, as a method for measuring the concentration of decomposition products by PLC using an enzyme, 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. Methods using (reagents utilizing color development), molybdenum blue method, malakite green method, and improved methods thereof (BIOMOL (registered trademark) Green, etc.) are known.

In one embodiment, the composition of the solution containing the PLC and substrate can be modified by one of ordinary skill in the art. In one embodiment, 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. In one embodiment, 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 chelator may be, but is not limited to, EDTA, EGTA, BAPTA, DTPA, HEADTA, NTA, DTPA, GLDA, TTHA, HIDA, DHEG and the like. In one embodiment, 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.

In one embodiment, the polypeptide of the invention may be of a microorganism belonging to the order Actinomycetales. As the microorganism, 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, and the microorganism belonging to the genus Amycolatopsis sp. Is preferable. Whether or not the strain isolated from the soil, lakes, sea, the surface of living organisms, the inside of the body cavity, etc. 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. Whether or not this is the case is described in "Checkebrand E., Evers J .: Taxonomy parameters reviewed: tarnished gold standards, Microbiology today, nov, p. 152-155, 2006". That is, if there is 70% or more homology in DNA-DNA hybridization, or if 16s rRNA is 98.5% or more the same, it can be judged to be the same genus and the same species. Preferably, if DNA-DNA hybridization has 70% or more homology, it can be determined to be of the same genus and species.

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.

(Polynucleotide)
In one aspect, 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.

In one embodiment, 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. In one embodiment, 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. Methods of deleting, substituting or imparting a base are known to those skilled in the art. In one embodiment, 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). ), That is, when the substrate specificity is, for example, pH 6.0, 55 ° C., and 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. Well, more preferably 15 or less. Further, the mutation of the base may be an artificial mutation or a mutation occurring in the natural world.

In one embodiment, 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. In one embodiment, 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.

(Recombinant vector and transformant)
In one aspect, 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. In one embodiment, 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.

As a method of introducing into the target cell as a recombinant vector containing the target gene, 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.

(Method for producing polypeptide)
In one aspect, the present specification provides a method for producing a polypeptide having a hydration decomposing ability of PG. In one embodiment, the method for producing a polypeptide comprises culturing a transformant to produce the polypeptide. In one embodiment, 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.

In one embodiment, 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.

In one embodiment, 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. In one embodiment, 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. In one embodiment, 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. In one embodiment, 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. Alternatively, the polypeptide may be extracted by concentrating the required fraction. In one embodiment, 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. In one embodiment, when extracting a polypeptide, the polypeptide may be efficiently extracted by using a precipitation method in which the polypeptide is precipitated with a precipitating agent and centrifuged. Precipitants 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. In one embodiment, when extracting a polypeptide, a surfactant 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.

In one embodiment, 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. In one embodiment, if the purified polypeptide has a label such as a tag, affinity chromatography for that tag may be used. For example, when the pET vector system is used as described above, tag labels such as His tag, T7 tag, and Strep tag are attached to the expressed protein, and the binding affinity for these tags is used. Methods for purifying proteins are known in the art. In one embodiment, the polypeptides of the invention may be purified in combination with reciprocal hydrophobic action chromatography, anion exchange chromatography, and gel filtration chromatography. As the chromatography method, 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 are known to those skilled in the art and 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.

(How to use polypeptide)
In one aspect, the invention provides a method of using a polypeptide having PG water decomposing ability. In one embodiment, 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.

In one embodiment, the PG in the method using the polypeptide of the present invention may be of biological origin or may not be of biological origin. Although we do not want to be bound by a specific theory, 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. In one embodiment, the organism used is assumed to be any organism as long as it contains PG. For example, 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. For example, eukaryotes include molds, yeasts and mushrooms, and prokaryotes such as archaea and eubacteria include actinomycetes, Bacillus subtilis, and Escherichia coli, but Escherichia coli with established treatment is preferable. be. 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. Although not bound by any particular theory, the phospholipid hydrophilic moieties of PG in E. coli cell membranes are predominantly phospho- (1'-sn-glycerol) type and, for example, above 40 ° C. It has been reported that the proportion of phospho- (3'-sn-glycerol) type can be increased by changing the culture conditions such as increasing the temperature (Yunori Fujishima et al., "Phosphatidylglycerol stereoisomer present in marine bacteria". Yutaka Itahashi, 2002 Performance Report "Analysis, Distribution and Physiological Functions of D-Type Phospholipids in Aquatic Organisms", https://kaken.nii.ac.jp/report/KAKENHI-PROJECT-13460088/134600882002jisseki /) .. Therefore, G1P can be efficiently obtained by hydrolyzing PG synthesized by Escherichia coli with the polypeptide of the present invention. Furthermore, 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.

In one embodiment, 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. In one embodiment, the phospholipid may be extracted from the recovered E. coli and then reacted with the polypeptide having the hydration decomposing ability of PG. Generally, 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. On the other hand, in one embodiment, the phospholipid may not be extracted from E. coli, and the polypeptide having the hydration decomposing ability of PG may not be purified. Although we do not want to be bound by a specific theory, 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. In one embodiment, the composition of the solution containing the polypeptide and PG can be modified by one of ordinary skill in the art. In one embodiment, 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. In one embodiment, 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. In one embodiment, 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 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. Examples of 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. Methods for discriminating mirror image isomers of purified glycerol phosphate are known in the art and include optical rotatory dispersion (ORD) measurement methods, circular dichroism (CD) measurement methods, X-ray crystal structure analysis, and nuclear magnetic resonance apparatus. And so on.

(Composition containing polypeptide)
In one aspect, the invention provides a composition for degrading PG containing the polypeptide of the invention. In one embodiment, the composition containing the polypeptide of the invention may be sold as a kit. In one embodiment, 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-α-Phosphatidylgycerol, Egg (PG, Avanti Polar Yeast Co., Ltd.) Model number 46261003), L-α-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) and other reagents not specifically mentioned were all commercially available special grade products.

Screening for bacteria producing the target enzyme (1) Culture 1) 5 ml of ISP2 medium and 3 glass beads (Φ4 mm) were placed in a test tube (Φ18 × 180 mm) and sterilized by autoclave (121 ° C, 15 min).
2) 1% (v / v) of a glycerol stock suspension of the screening strain was inoculated into a sterilized medium, and reciprocating shaking culture was performed at 28 ° C. and 160 spm.

(2) PLC measurement method When PLC acts on PG, DG and two kinds of glycerol phosphates (G1P and G3P) are produced when PG is a racemate. Among them, G3P is oxidized by glycerol-3-phosphate oxidase (GPO) to generate hydrogen peroxide. 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. It is known that 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 μmol of G3P per 1 min was defined as 1 U.

(Results and discussion)
Acquisition and taxonomic identification of PLC-producing bacteria (1) Screening As a result of a total of 72 strains, PG-PLC activity was found in the culture supernatant of the NT115 strain. As a result of simple morphological observation, the NT115 strain showed a colony on a circular central depression on the ISP2 agar medium, and the color tone on the front surface was white and the back surface was brown. In addition, as a result of microscopic observation, aerial hyphae were formed and chained spores were observed. In the molecular phylogenetic tree analyzed based on the BLAST homology test for DB-BA and the international nucleotide sequence database using the Technosuruga Lab Microbial Identification System, 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]
(experimental method)
Amycolatopsis sp. Purification of NT115 strain PLC (1) Culture A 5 ml of ISP2 medium and 3 glass beads (Φ4 mm) were placed in a culture test tube (Φ18 × 180 mm) and sterilized by autoclave (121 ° C., 15 min). After cooling, 50 μl 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. Ten bottles containing 100 ml of ISP2 medium and a stainless coil in a 500 ml Erlenmeyer flask were prepared and sterilized by autoclave. In a clean bench, 1% (v / v) of the preculture solution was inoculated into the medium, and the culture was shaken at 28 ° C. and 160 rpm for 72 hours.

(2) Ammonium sulfate (AS) fraction The culture solution of (1) was centrifuged (18,800 × g, 10 min) to obtain a culture supernatant. A saturated AS aqueous solution (stored at 4 ° C.) was added to the culture supernatant so as to be 20% saturated. After allowing to stand for 1 hour, the resulting precipitate was collected by centrifugation (18,800 × g, 10 min).

(3) PLC activity In the following experiments, the PLC activity value was calculated by an enzymatic reaction in the same manner as in Example 1.

(4) PPG-Toyopearl Column Chromatography The recovered precipitate was dissolved in 1M AS / 20 mM Tris-HCl (pH 7.0) (no filter, no centrifugation).
1) PPG-Toyopearl column chromatography was performed under the conditions shown in Table 1.
A sol. 1M AS / 20m M Tris-HCl (pH 7)
Bsol. : 20 mM Tris-HCl (pH 7)
Volume volume: 15 ml (= Φ2.5 cm, H = 3.0 cm)
Elution1: linear gradient of 1 to 0M AS
with 4CV
Elution2: 100% B sol. with 4CV
Fraction: 6 ml
Table 1: Conditions for PPG-Toyopearl column chromatography

2) The PLC activity (substrate; PG) of each fraction was measured, and fractions showing an activity of 2.5 U / ml or more were recovered (PPG fraction).

(5) 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).

(6) Giga Cap Q-Toyopearl Column Chromatography 1) The dialysis internal solution was collected and loaded into Giga Cap Q-Toyopearl (hereinafter abbreviated as GCQ). The sample was column loaded without filter filtration and centrifugation, and GCQ column chromatography was performed under the conditions shown in Table 2.
Asol. : 20 mM Tris-HCl (pH 9.0)
B sol. : 0.5M NaCl / 20 mM Tris-HCl (pH 9.0)
Volume volume: 15 ml (= Φ2.5 cm, H = 3.0 cm)
Elution: linear gradient of 0 to 0.5M NaCl with 10CV
Fraction: 5 ml
Table 2: Conditions for Giga Cap Q-Toyopeal column chromatography

2) The PG-PLC activity of each fraction was measured, and the active fraction was used as the GCQ fraction.

(7) Gel Filtration Chromatography The GCQ fraction was concentrated to 500 μl using Amicon-30K (15 mL). Concentrated samples were loaded onto Superdex 200 increase 10/300 GL (Spd 200 Inc) after filtration through a 0.45 μm filter.
1) Column chromatography was performed under the following conditions and the conditions shown in Table 3.
A sol. : 0.15M NaCl / Tris-HCl (pH 8.0)
Volume volume: 24 ml (= Φ1.0 cm, H = 30 cm)
Elution: 0.15M NaCl with 1.5CV
Fraction: 0.5 ml
Table 3: Gel filtration chromatography conditions

2) The PG-PLC activity of each fraction was measured, and buffer exchange was performed with 20 mM Tris-HCl (pH 7.0) using Amicon-30K to obtain purified PG-PLC.

Quantification of Protein Concentration Using the BCA Protein Assay Reagent Kit (Thermo Fisher), the absorbance (A ₅₆₂ ) at 562 nm was measured according to the instruction manual, and the protein concentration was quantified.

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.

(Results and discussion)
From the supernatant cultured in ISP2 medium for 2 days (FIG. 1), PG-PLC could be purified up to 2981 times (Table 4), and the specific activity was 6.19 U / mg-protein. SDS-PAGE analysis detected a single band with a molecular weight of approximately 54 kDa (FIG. 2).
Table 4: Purification results of PG-PLC derived from NT115 strain

[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. 2-oleoyl-sn-glycello-3-phopsphoethanolamine (POPE), 1-palmitoyl-2-oleoyl-sn-gycero-3-phopsphoinositol (POPI), 1-palmitoyl-2-oleoyl-sn- POPS), 1-palmitoyl-2-oleoyl-sn-glycello-3-phopsphate (POPA) cardiolipin (CL), L-α-phospatidyglycerol (PG) were used. In addition, BIOMOL Green (registered trademark) Reagent (hereinafter abbreviated as BG) and Alkaline phosphatase (Oriental Yeast Co., Ltd., derived from calf small intestine, hereinafter abbreviated as CIAP) were used. 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%.

1) Effect of pH and temperature on enzyme reaction, pH and temperature stability test Sodium acetate-sodium acetate (pH 4.0-5.5, hereinafter abbreviated as Ac-Na), MES-NaO (pH 5.5-7.0) , Tris-HCl (pH 7.0-9.0) at a temperature adjusted to the effect of pH and temperature (FIGS. 3-1 and 3-2) on the enzyme reaction or enzyme and stability (FIG. 4). -1, Fig. 4-2) was investigated.

2) Effect of metal ion and surfactant on PLC activity Investigate the effect of metal ion (Fig. 5-1) and surfactant (Fig. 5-2) under the conditions of Tris-HCl (pH 7.0) and 37 ° C. rice field.

3) Substrate specificity test of PLC Each substrate was reacted with PLC and the enzyme activity for each substrate was examined. Inorganic phosphoric acid (Pi) liberated by CIAP, which is a dephosphorylating enzyme, was quantified from the corresponding phosphoric acid monoester such as glycerol phosphoric acid generated by the enzymatic reaction. However, POPA was not treated with dephosphorylating enzyme, and the inorganic phosphoric acid (Pi) released by PG-PLC was quantified as it was. The quantification of inorganic phosphoric acid was carried out by measuring the absorbance (A ₆₂₀ ) at 620 nm according to the instruction manual using the BG reagent.

(Results and discussion)
Unless otherwise specified, the data shown below show the results of three tests as mean ± standard deviation (SD).
Effect of pH and temperature on enzyme reaction and enzyme stability When PLC activity (37 ° C) at each pH was examined, it showed high activity in the weakly acidic range pH 5.0 to pH 6.0, and MES-NaOH (pH 6. It showed the highest activity in 0) (Fig. 3-1). When the activity at each temperature was examined at pH 6.0, the maximum activity was shown at 55 ° C, and the relative activity was about 30% at 20 ° C and about 40% at 70 ° C (Fig. 3-2). Since the cloud point of Triton® X-100 used for substrate preparation is 63 ° C, it is considered that the PLC activity is characteristic at 60 ° C or lower.

As shown in FIG. 4-1 the pH was stable in a wide range from pH 4.0 to pH 9.0. In the temperature stability test, as shown in FIG. 4-2, the temperature was stable up to 50 ° C., and the activity was halved at 60 ° C.

Effect of metal ions As shown in Fig. 5-1 it showed activity even in the presence of EDTA, the activity was slightly improved by Ca ²⁺ , and the activity was improved by about 1.3 times in the presence of Al ³⁺ . In addition, Triton® X-100 showed the best results under the conditions tested as shown in FIG. 5-2.

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]
(experimental method)
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. A total of 11 peptide sequences were decoded, and the amino acid sequences of all of these peptides matched the sequences in the Amycolatopsis orientalis-derived metallophosphosterase registered in the public database as WP_037369513.1.

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. By analyzing it with a DNA sequencer, the base sequence of a part of this enzyme gene (SEQ ID NOs: 9 and 10) was decoded. Next, inverse PCR (iPCR) was performed using the primer sets of SEQ ID NOs: 11 and 12 to decode the 5'and 3'undeciphered regions. The iPCR conditions are as follows.
The iPCR reaction solution was column-purified at 94 ° C. for 1 minute → (98 ° C., 10 seconds → 57 ° C., 15 seconds → 68 ° C., 2.5 minutes) for 25 cycles, 68 ° C. for 5 minutes, and sequenced. From the analysis results, 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. Of the amino acid sequences shown in FIG. 8, 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, and 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. The two are mixed and inserted into the HindIII site of pET-24a (+) using the Infusion Cloning Kit (Takara Bio), 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 μg / mL kanamycin, and PG- PLC / pET-24a (+) / BL21 (DE3) was obtained. It also contains kanamycin at a final concentration of 50 μg / 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. Centrifuge the culture broth cultured at 20 ° C to obtain a culture supernatant, and centrifuge the culture cultivated at 30 ° C to obtain PG-PLC / pET-24a (+) / BL21 (DE3) cells. The cells obtained by collecting the culture supernatant were suspended in a buffer solution and crushed by ultrasonic waves, and the cells were centrifuged to obtain a supernatant, which was used as a crude protein solution (CFE).

Recombinant E. cultivated after transformation with an empty pET-24a (+) vector. The colli BL21 (DE3) was recovered by centrifugation and crushed by ultrasonic waves to obtain E.I. A colli crushed solution was obtained. The crushed solution was mixed with a culture supernatant at 20 ° C. or CFE at 30 ° C., and the enzyme activity of PG-PLC was measured by the method for measuring the water resolution described in Example 1. As a result, the concentration of G3P increased in each of the reaction solutions in a time-dependent manner (FIGS. 9-1 and 9-2). As a result, E. It was confirmed that the PG contained in the colli crushed solution was hydrolyzed by the action of the PG-PLC to produce glycerol phosphate. This result indicates that 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]
In order to estimate the catalytic (active) center and substrate binding sites of PG-PLC, 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.

First, the 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. From these results, the three-dimensional (three-dimensional) structure of PG-PLC and the metal ion-requiring phosphoesterase are similar, and therefore the structure of PG-PLC can be estimated using the metal ion-requiring phosphoesterase as a model. I thought.

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. When the active center residue that interacts with phosphoric acid in PG-PLC was estimated from the comparison with the three-dimensional structure of 2xmo, histidine (H) and aspartic acid (H) and aspartic acid of D41 / H43 / D103 / N191 / H364 / H407 / H409 ( It was estimated that D) was the active center residue, and among them, H43 and H409, which are close to the phosphate, were the catalytic residues.

Next, in order to predict the substrate binding site, 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. Was in contact with the substrate and was estimated to hit the substrate binding site (40T to 75T, 100T to 120L, 184P to 197V, 205P to 227K, 266F, 271L, 296Y to 297Y of the amino acid sequence represented by SEQ ID NO: 2). 310L-322S, 363S-366T, 378R-384R, 406G-409H, and 432S-435D). Among them, 56L / 59FVG61 / 204IPGI207 / 209AW210 / 316GGFA320, which are non-polar amino acids, are considered to be amino acid residues that bind to the acyl group of PG.

Furthermore, in order to investigate that these substrate binding sites, active central amino acid residues, and catalytic residues are conserved among actinomycetes, the partial sequence of 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. coli expression. : Sequence confirmed to be highly conserved among species in PG-PLC

Claims (14)

1. Phospholipase C polypeptide having substrate-specific water resolution for phosphatidylglycerol.
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 claim 1, which has an amino acid corresponding to.
3. The polypeptide according to claim 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.
4. 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. , And the polypeptide of claim 3, further comprising an amino acid corresponding to 432S-435D.
5. A phospholipase C having a phosphatidylglycerol hydrolysis resolution, which is the following polypeptide (a), (b) or (c).
   (A) Polypeptide containing the amino acid sequence represented by SEQ ID NO: 2 (b) Polypeptide containing an amino acid sequence in which one or more amino acids are deleted, substituted or imparted in the amino acid sequence represented by SEQ ID NO: 2. (C) A polypeptide containing an amino acid sequence having at least about 80% identity with the amino acid sequence represented by SEQ ID NO: 2.
6. The polypeptide according to any one of claims 1 to 5, further comprising a label.
7. The polypeptide according to any one of claims 1 to 6, which is derived from a microorganism belonging to the order Actinomycetales.
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). A polynucleotide containing a base sequence having a codon synonymous with the represented base sequence (f) A polynucleotide containing a base sequence having at least about 80% identity with the base sequence represented by SEQ ID NO: 1.
9. A recombinant vector containing the polynucleotide according to claim 8.
10. A transformant containing the recombinant vector according to claim 9.
11. A method for producing a polypeptide having a substrate-specific hydrolysis resolution with respect to phosphatidylglycerol, comprising a step of producing the polypeptide from a transformant containing the polynucleotide according to claim 8.
12. A method for decomposing phosphatidylglycerol using the polypeptide according to any one of claims 1 to 7.
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 claims 1 to 7.
14. A composition for decomposing phosphatidylglycerol containing the polypeptide according to any one of claims 1 to 7.

Images