JP6063207B2 - Enzyme having hydrolysis action on glycerol-3-phosphodiester such as glycerol-3-phosphocholine (GPC) and method for producing the same - Google Patents

Description

  The present invention relates to a novel enzyme having a hydrolysis action on glycerol-3-phosphodiester and a method for producing the same.

  In recent years, it has been found that phospholipids, lysophospholipids and glycerol-3-phosphodiester have various physiological actions, and these compounds and enzymes related to these compounds have attracted attention. Glycerol-3-phosphodiester is a compound produced from phospholipids by the action of enzymes such as phospholipases A1, A2, and B, and is thought to be deeply involved in metabolism in cells. For example, glycerol-3-phosphocholine (hereinafter, also referred to as “GPC”) is a safe component contained in breast milk and can pass through the portal vein, and therefore has a much higher body absorption than phosphatidylcholine. GPC also has a central action and a peripheral action. It has been found that the central action has the effect of improving cognitive function, learning ability (rat discrimination learning), and stress hormone secretion in Alzheimer-type dementia patients. Peripheral effects are known to have growth hormone secretion promoting effects, liver dysfunction improving effects, and blood pressure lowering effects. In addition, GPC is known to dilate blood vessels and lower blood pressure by vasorelaxant action, like acetylcholine. Furthermore, after strenuous exercise, GPC can suppress the decrease in serum choline concentration, which can be expected to improve the performance decline. Further, GPC is expected to improve the repair ability at the time of injury, and it is known that GPC exhibits an excellent effect of promoting repair of muscle cells damaged by intense exercise (Non-patent Document 1). In addition, glycerol-3-phosphocholine is expected to prevent dementia (Patent Document 1). Thus, glycerol-3-phosphocholine is a substance having many physiological activities. In addition to glycerol-3-phosphocholine, glycerol-3-phosphodiester is expected to have many physiological activities, including glycerol-3-phosphocholine.

International Publication No. 2007/010892

BIO INDUSTRY, 29, 2, 2012 Glycerylphosphocholine phosphocholine phosphodiesterase activity is reduced in multiple sclerosisplaques, Janzen, L .; Tourtellotte, W.W .; Kanfer J.N .; Exp. Neurol. 109, 243-246 (1990) Properties of aZn (2 +)-glycerophosphocholine cholinephosphodiesterase from bovine brain membranes, Sok D.E .; Neurochem.Res. 21, 1193-1199 (1996) Somecharacteristics of sn-glycero 3-phosphocholine diesterases from rat brain, Abra, R.M .; Quinn, P.J .; Biochim. Biophys. Acta 431, 631-639 (1976) Aphosphodiesterase in rat kidney cortex that hydrolysesglycerylphosphorylinositol, Dawson, R.M.C .; Hemington, N .; Biochem. J. 162,241-245 (1977)

  Among glycerol-3-phosphodiesters, GPC (glycerol-3-phosphocholine) and glycero-3-phosphoethanolamine (hereinafter also referred to as “GPE”) are important analytes in relation to physiological activities and diseases. It is. Therefore, if these substances can be selectively and easily and quickly quantitatively measured, it will be a very effective measurement method. However, in the conventional technique, there is only an analysis method using HPLC, LC-MS or the like with complicated pretreatment, and analysis requires time and cost. Furthermore, accurate quantification was difficult. Since no enzyme suitable for this purpose has been found so far, there has been no method for quantifying GPC or GPE in the prior art.

  An enzyme that acts on glycerol-3-phosphodiester to produce glycerol and a phosphoric acid ester compound such as phosphocholine or phosphoethanolamine is an enzyme similar to an enzyme having a catalytic action called phospholipase C. Phospholipase C (hereinafter also referred to as “PLC”) has an action of hydrolyzing a bond on the glycerol side of a phosphate group in a phospholipid or a hydrolyzate thereof (such as lysophospholipid). Among such enzymes, enzymes that act in a substrate-specific manner are particularly useful for industrial applications such as analysis. Various possibilities are expected by acting specifically on glycerophosphocholine.

  As enzymes having catalytic action similar to phospholipase C, glycerophosphocholine cholinephosphodiesterase (EC 3.1.4.38) and glycerophosphoinositol inositolphosphodiesterase (EC3.1.4.43) are known, but glycero-3-phospho other than GPC such as GPE The existence of an enzyme that acts on a diester is not known (from the enzyme database “BRENDA”). Non-patent document 2 is an enzyme derived from human, Non-patent document 3 is an enzyme derived from bovine, Non-patent document 4 is an enzyme derived from rat (English rat), and Non-patent document 5 is an enzyme derived from mouse. Is disclosed. Non-patent document 5 reports a rat rat (English rat) Rattus norvegicus-derived glycerophosphoinositol inositolphosphodiesterase. However, these enzymes selectively act on GPC or GPI, but all of them are derived from animal organs, and there are many problems such as low titer and production amount of the obtained enzymes and ethical problems. Is extremely difficult. Furthermore, there is a possibility that the metal ion requirement is limited (limited) in use.

  This invention is made | formed in view of said situation, and it aims at providing the novel enzyme which has a hydrolysis action with respect to glycerol-3-phosphodiester, and its manufacturing method.

  As a result of searching for a microorganism that produces the enzyme for the purpose of obtaining the novel enzyme as described above, the present inventors have found an enzyme having a novel property from microorganisms belonging to the genus Streptomyces. It was.

[enzyme]
The enzyme which concerns on this invention is an enzyme which has a hydrolysis action with respect to glycerol-3-phosphodiester, Comprising: The polypeptide in any one of the following (a) to (c) is included.
(A) a polypeptide having the amino acid sequence set forth in SEQ ID NO: 2;
(B) a polypeptide having an amino acid sequence in which one or more amino acids are substituted, inserted, deleted and / or added in the amino acid sequence of SEQ ID NO: 2 and exhibiting the hydrolysis activity; or ( c) A polypeptide having at least 90 % identity with the amino acid sequence set forth in SEQ ID NO: 2 and exhibiting the hydrolysis activity.

  A preferable form of the above enzyme is 20 ° C. to 60 ° C. when glycerol-3-phosphocholine is used as a substrate and the hydrolysis activity is 100% at pH 7.2 and 40 ° C. for 5 minutes. The hydrolysis activity of 40% or more is shown within the range of.

  A preferable form of the above enzyme is a range of pH 5 to pH 8 when glycerol-3-phosphocholine is used as a substrate and the hydrolysis activity is 100% at 37 ° C. for 5 minutes at pH 7.2. Among them, the hydrolysis activity is 10% or more.

  A preferred form of the above enzyme is that when glycerol-3-phosphocholine is used as a substrate, the hydrolysis activity under conditions of pH 7.2 and 37 ° C. for 5 minutes is 100%. Degradation activity is 90% or more for glycerol-3-phosphoethanolamine, 20% or less for glycerol-3-phosphate, and 1% or less for glycerol-3-phosphoglycerol and glycerol-3-phosphoinositol Approximately 0% for glycerol-3-phosphoserine, 40% or less for lysophosphatidylcholine, 3% or less for lysophosphatidylethanolamine, 1% or less for lysophosphatidic acid, lysophosphatidylinositol, lysophosphatidylserine And almost 0 for lysophosphatidylglycerol 5% or less for choline-type lysoplasmalogen, 1% or less for ethanolamine-type lysoplasmalogen, almost 0% for ethanolamine-type and choline-type plasmalogen, 0.5% for phosphatidylethanolamine % With a substrate specificity of 0.1% or less for phosphatidylserine and approximately 0% for phosphatidylinositol, phosphatidylcholine, phosphatidic acid and sphingomyelin.

  A preferred form of the above enzyme has a molecular weight in the range of 60,000 to 70,000 as measured by SDS-PAGE.

  A preferred form of the enzyme is derived from a microorganism belonging to the genus Streptomyces.

[Polynucleotide]
The polynucleotide according to the present invention encodes the enzyme according to any one of claims 1 to 6.

The preferable form of said polynucleotide contains the polynucleotide as described in the following (a) or (b).
(A) a polynucleotide comprising the base sequence set forth in SEQ ID NO: 1; or (b) a polynucleotide having at least 90 % sequence identity with the base sequence set forth in SEQ ID NO: 1.

  A preferable form of the above polynucleotide is derived from a microorganism belonging to the genus Streptomyces.

[Enzyme production method]
The method for producing an enzyme according to the present invention is a method for producing an enzyme having a hydrolyzing action on glycerol-3-phosphodiester, and includes a step of producing the enzyme from a microorganism having the above polynucleotide.

  ADVANTAGE OF THE INVENTION According to this invention, it becomes possible to provide the novel enzyme with a high substrate specificity which has a hydrolysis action with respect to glycerol-3-phosphodiester. In particular, it is possible to provide an enzyme that exhibits high substrate properties for glycero-3-phosphocholine and glycero-3-phosphoethanolamine. Furthermore, a method for efficiently producing the enzyme by a microorganism can be provided. Further, glycerol-3-phosphodiester can be analyzed efficiently. Moreover, the decomposition reaction of glycerol-3-phosphodiester can be utilized industrially.

It is an electrophoresis photograph which shows the result of the SDS-PAGE analysis about the refinement | purification fraction (GPC-CP) obtained from the Streptomyces sanglieri culture solution. It is a graph which shows the substrate specificity (relative comparison of a hydrolytic activity) of GPC-CP on the basis of enzyme activity at the time of using GPC as a substrate (100%). It is a graph which shows the relative activity of GPC-CP in various temperature on the basis of the enzyme activity in case reaction temperature is 40 degreeC (100%). It is a graph which shows the relative activity of GPC-CP in various pH on the basis of the enzyme activity in case pH is 7.2 (100%). It is a graph which shows the relative activity about GPC-CP after processing at various temperature on conditions whose pH is 7.2 and 30 minutes. It is a graph which shows the relative activity about GPC-CP after processing by various pH on conditions whose temperature is 4 degreeC and 3 hours. It is a graph which shows the relative activity about GPC-CP when a metal ion or EDTA is added. It is a graph which shows the relative activity about GPC-CP when a chemical substance is added.

[enzyme]
In the present specification, the enzyme is not limited to a purified enzyme, but includes a crude product, an immobilized product, and the like. The enzyme is purified by a method well known to those skilled in the art, such as ammonium sulfate precipitation, ion exchange chromatography, hydrophobic chromatography, etc., using a microorganism culture solution. Thereby, enzymes with various degrees of purification (including enzymes purified to approximately one) can be obtained.

  In the present specification, microorganisms are wild strains, mutant strains (for example, those induced by ultraviolet irradiation, etc.), recombinants induced by genetic engineering techniques such as cell fusion or genetic recombination methods, etc. Any strain may be used. Genetically engineered microorganisms such as recombinants are known to those skilled in the art, as described, for example, in Molecular Cloning A Laboratory Manual, 2nd edition (Sambrook, J. et al., Cold Spring Harbor Laboratory Press, 1989). Can be easily created using various techniques. The culture solution of microorganisms means both a culture solution containing microbial cells and a culture solution from which microbial cells have been removed by centrifugation or the like.

[GPC-CP]
Glycerophosphocholine cholinephosphodiesterase (EC 3.1.4.38) (hereinafter also referred to as “GPC-CP”) is an enzyme that exhibits enzyme activity similar to phospholipase C. According to the present invention, a novel GPC-CP is obtained. Phospholipase C acts on phospholipids and hydrolysates thereof (lysophospholipids, glycerophosphodiester) and has a function of hydrolyzing the phosphoester bond on the glycerol side at the phosphodiester site. The GPC-CP according to the present invention does not have high activity against phospholipids and lysophospholipids, and has specific hydrolysis activity against glycerophosphodiester compounds obtained by hydrolysis of them. When GPC-CP acts on glycerophosphodiester, it produces glycerol and phosphoric acid ester compounds such as phosphoric acid or phosphocholine and phosphoethanolamine. Furthermore, GPC-CP according to the present invention acts specifically on GPC among glycerol-3-phosphodiester.

  The chemical formula of GPC (glycerol-3-phosphocholine) is shown below. Glycerophosphocholine is an abbreviation for glycerol-3-phosphocholine. In addition, glycerol-3-phosphocholine includes glycero-3-phosphocholine, sn-glycero-3-phosphocholine, and sn-glycerol-3-phosphocholine as other names.

        GPC-CP is an enzyme that can catalyze the following reactions. That is, GPC is hydrolyzed by GPC-CP to produce glycerin and phosphocholine.

The enzyme in the present invention has a substrate-specific action. In the present invention, an enzyme (GPC-CP) that specifically acts on GPC (glycerol-3-phosphocholine) is obtained. In addition, GPC-CP in the present invention exhibits high activity against GPE (glycerol-3-phosphoethanolamine). And this GPC-CP is not so high in activity with respect to other glycerophospho compounds (glycerophosphodiester). By the way, the enzyme which acts on GPE can also be called glycerophosphoethanolamine ethanolamine phosphodiesterase (GPE-EP). Therefore, the enzyme in the present invention has both GPC-CP and GPE-EP properties. An enzyme that acts only on GPE is GPE-EP.

  Glycerol-3-phosphodiester (hereinafter also referred to as “GPX”) is a compound obtained by hydrolysis of phospholipid (glycerol phospholipid) or lysophospholipid. The enzyme that hydrolyzes the fatty acid ester bond at the α-position (sn-1 position) of the glycerol group in glycerol phospholipid is called phospholipase A1 (PLA1), and the fatty acid ester group at the β-position (sn-2 position) of the glycerol group. The enzyme that hydrolyzes is called phospholipase A2 (PLA2). An enzyme having both phospholipase A1 activity and phospholipase A2 activity is referred to as phospholipase B (PLB). That is, PLB has enzyme activity at both the α-position and the β-position. Therefore, GPC-CP can react with glycero-3-phosphodiester obtained by using phospholipase A1, A2, B, or by a chemical reaction or the like.

The enzyme in the present invention is an enzyme having a hydrolyzing action on glycerol-3-phosphodiester, and includes the polypeptide described in any of (a) to (c) below.
(A) a polypeptide having the amino acid sequence set forth in SEQ ID NO: 2;
(B) a polypeptide having an amino acid sequence in which one or more amino acids are substituted, inserted, deleted and / or added in the amino acid sequence of SEQ ID NO: 2 and exhibiting the hydrolysis activity; or ( c) A polypeptide having at least 80% homology with the amino acid sequence set forth in SEQ ID NO: 2 and exhibiting the hydrolysis activity.

  For example, the above-mentioned enzyme can act within a range of about 4 to 70 ° C. under the reaction conditions of GPC and the enzyme as described above. The optimum temperature can be within this range. Preferably it is in the range of about 20-65 ° C, more preferably in the range of 30-60 ° C, and even more preferably about 40 ° C.

  For example, when GPC is used as a substrate, the above enzyme has a hydrolysis activity of 100% at a pH of 7.2 and a temperature of 40 ° C. for 5 minutes. % Of hydrolysis activity is preferred. Further, it is more preferable that the hydrolysis activity is 70% or more within the range of 30 ° C to 60 ° C.

  For example, when the above glycero-3-phosphocholine and the enzyme are placed under reaction conditions at 37 ° C. using a HEPES buffer (pH 7.2) as a buffer, Can exhibit CP activity. As the buffer solution, Tris-HCl, Bis-Tris, acetic acid-Na acetate, Glycine-NaOH and the like can also be used.

  For example, when GPC is used as a substrate, the enzyme is 10% or more within the range of pH 5 to pH 8 when the hydrolysis activity at pH 7.2 is 5% at 37 ° C. for 5 minutes. It is preferable to show hydrolytic activity. Further, it is more preferable that the hydrolysis activity is 20% or more within the range of pH 6 to pH 8. The optimum pH can be around pH 7.2 (for example, within the range of pH 6.5 to 7.5).

  For example, the enzyme described above is obtained when glycerol-3-phosphocholine (GPC) is used as a substrate as described above, and the hydrolysis activity is 100% at 37 ° C. for 5 minutes at pH 7.2. Hydrolytic activity under these conditions is 90% or more for glycerol-3-phosphoethanolamine (GPE), 20% or less for glycerol-3-phosphate (GPA), glycerol-3-phosphoglycerol ( 1% or less for GPG) and glycerol-3-phosphoinositol (GPI), almost 0% for glycero-3-phosphoserine (GPS), 40% or less for lysophosphatidylcholine (LPC), lysophosphatidylethanolamine (LPE) 3% or less, lysophosphatidic acid (LPA) 1% or less, lysophosphati Almost 0% for luinositol (LPI), lysophosphatidylserine (LPS) and lysophosphatidylglycerol (LPG), 5% or less for choline-type lysoplasmalogens (Lyso-PlsPC), ethanolamine-type lysoplasmalogens ( 1% or less for Lyso-PlsPE), almost 0% for ethanolamine-type and choline-type plasmalogens (PlsPE, PlsPC), 0.5% or less for phosphatidylethanolamine (PE), phosphatidylserine (PS) ), And substrate specificity that is approximately 0% for phosphatidylinositol (PI), phosphatidylcholine (PC), phosphatidic acid (PA) and sphingomyelin (SM). Further, preferably, it has a substrate specificity of 0.5% or less with respect to phosphatidylcholine (PC (soy)) derived from soybean and almost 0% with respect to PAF. Here, the hydrolysis activity of approximately 0% may mean, for example, 1% or less, 0.1% or less, or a detection limit or less. An example of PC is DPPC, an example of PA is DMPA, an example of PS is POPS, an example of PG is POPG, and an example of PE is DPPE (see Table 3 below for abbreviations). ). The above enzyme preferably has such substrate specificity.

  The enzyme described above may vary slightly depending on electrophoresis conditions and the like, but preferably has a molecular weight in the range of 60,000 to 70,000 (Da) in SDS-PAGE. For example, a natural enzyme derived from the genus Streptomyces has a molecular weight of about 66 kDa in SDS-PAGE.

  One aspect of the above-described enzyme, that is, GPC-CP, consists of the amino acid sequence set forth in SEQ ID NO: 2.

  As long as the enzyme has GPC-CP activity, one or more amino acids are substituted, deleted, inserted into the amino acid sequence shown in SEQ ID NO: 2 or the amino acid sequence shown in SEQ ID NO: 2. And / or an enzyme having an added amino acid sequence. A person skilled in the art, for example, site-directed mutagenesis (Nucleic Acid Res., 1982, 10, pp. 6487; Methods in Enzymol., 1983, 100, pp. 448; Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1989; PCR: Atactical Approach, IRL Press, 1991, pp. 200), etc., as appropriate substitution, deletion, insertion, and / or addition mutation The structure of the protein can be modified by introducing. In the present specification, the number of amino acid residues that can be substituted, deleted, inserted and / or added is usually 50 or less, such as 30 or less, or 20 or less, preferably 16 or less, more preferably 5 or less, Preferably it is 0-3. In addition, amino acid mutations include not only artificially mutated enzymes but also naturally mutated enzymes as long as they have the same activity (GPC-CP).

  A polypeptide (protein) having an amino acid sequence having homology to the amino acid sequence described in SEQ ID NO: 2 is also included in the enzyme (GPC-CP) as long as it has GPC-CP activity. GPC-CP is preferably at least 80%, preferably at least 85%, more preferably at least 90%, more preferably at least 95%, even more preferably at least 98% with the amino acid sequence set forth in SEQ ID NO: 2. Even more preferably, it may be a protein having an amino acid sequence with at least 99% homology.

  Protein homology (homology) search, for example, a database such as Uniprot, SWISS-PROT, PIR, DAD, etc., or a database such as BLAST for DNA databases such as DDBJ, EMBL, or GenBank For example, it can be performed through the Internet. Confirmation of the activity of the protein can be performed using the procedure described above.

  Although the supply source of GPC-CP is not particularly limited, GPC-CP can be obtained from living cells such as microorganisms. Examples of such microorganisms include microorganisms belonging to the genus Streptomyces. Preferably, Streptomyces sanglieri (Streptomyces sanglieri) is mentioned. In addition, closely related strains such as Streptomyces atratus may also yield an enzyme having GPC-CP activity. In addition, Streptomyces sindenesis, Streptomyces badius, Streptomyces globisporus, Streptomyces pluricolorescens, Streptomyces rubiginosohelvolus, Streptomyces albovinaceus, Streptomyces anulatus, Streptomyces flavofuscus, Streptomyces microflavus, Streptomyces griseorubiginosus, Streptomyces puniceus, Streptomyces floridae, Streptomyces griseolus, Streptomyces halstedii, Streptomyces flavovirens, Streptomyces flavogriseus, Streptomyces pulveraceus, Streptomyces gelaticus, Streptomyces albus subsp. Albus, and the like may also yield an enzyme having GPC-CP activity. In addition, Streptomyces sp., Streptomyces chattanoogensis, Streptomyces lydicus, and the like may yield an enzyme having GPC-CP activity. There is. Since these strains are closely related, it is considered that GPC-CP having the same kind of activity can be easily obtained.

  For example, Streptomyces sanglieri strain A14 (accession number: NITE BP-1392) is secreted out of the cells by liquid culture in an appropriate nutrient medium, so the culture supernatant is frozen. What was processed with drying, salting out, an organic solvent, etc. can be manufactured as a GPC-CP enzyme formulation.

  Microorganisms that can be used for the production of GPC-CP enzyme preparations are not limited to Streptomyces sanglieri strain A14, but belong to the genus Streptomyces and can produce GPC-CP. Also good. In addition, natural or artificial mutant strains of these species, or gene fragments necessary for the expression of GPC-CP activity are artificially extracted, and GPC-CP can be produced even for other species incorporating them. Can be used. Moreover, even if it does not belong to Streptomyces genus, if it is microorganisms which can produce said GPC-CP, it can also be used.

  The production will be described by taking as an example a GPC-CP enzyme preparation using Streptomyces sanglieri A14 strain.

  Since this bacterium is secreted out of the bacterium by liquid culture in a nutrient medium, the culture supernatant is treated with lyophilization, salting out, an organic solvent, or the treated product is immobilized. Thus, an enzyme preparation can be produced. More specifically, first, this bacterium is cultured in a suitable medium, for example, a medium containing a suitable carbon source, nitrogen source, and inorganic salts to secrete the enzyme. Here, examples of the carbon source include starch and starch hydrolysate, sugars such as glucose and sucrose, alcohols such as glycerol, and organic acids (for example, acetic acid and citric acid) or salts thereof (for example, sodium salt). Can be mentioned. Examples of the nitrogen source include organic nitrogen sources such as yeast extract, peptone, meat extract, corn steep liquor, soybean flour, and inorganic nitrogen compounds such as ammonium sulfate, ammonium nitrate, and urea. Examples of inorganic salts include sodium chloride, monopotassium phosphate, magnesium sulfate, manganese chloride, calcium chloride, and ferrous sulfate. The concentration of the carbon source is, for example, in the range of 0.1 to 20% (w / v), preferably 0.4 to 10% (w / v). The concentration of the nitrogen source is, for example, in the range of 0.1 to 20% (w / v), preferably 0.4 to 10% (w / v). Specific examples of preferable medium compositions include, but are not limited to, 1% malt extract and 0.4% glucose. The culture temperature is preferably a temperature at which the above enzyme is stable and the cultured microorganism can sufficiently grow, and is preferably 20 to 40 ° C., for example. The culture time is preferably a time during which the enzyme is sufficiently produced, and is preferably about 1 to 7 days, for example. Culturing can be preferably performed under aerobic conditions, for example, with aeration stirring or shaking.

  GPC-CP uses protein fractionation (precipitation with organic solvents, salting out with ammonium sulfate, etc.); cation exchange, anion exchange, gel filtration, hydrophobic chromatography; chelate, dye, antibody, etc. Purification can be performed by appropriately combining known methods such as affinity chromatography. For example, after recovering the culture supernatant of the microorganism, it can be purified by ammonium sulfate precipitation, further anion exchange chromatography, hydrophobic chromatography, and / or cation exchange chromatography. Thereby, in polyacrylamide gel electrophoresis (SDS-PAGE), it can refine | purify to a substantially single band. That is, the enzyme (GPC-CP) can be estimated as a monomer by HPLC analysis and gel filtration chromatography analysis.

[Polynucleotide encoding GPC-CP]
The polynucleotide in the present invention encodes the above GPC-CP. This polynucleotide preferably includes the polynucleotide described in (a) or (b) below.
(A) a polynucleotide comprising the base sequence set forth in SEQ ID NO: 1; or (b) a polynucleotide having at least 80% sequence identity with the base sequence set forth in SEQ ID NO: 1.

  The above polynucleotide may be an artificial molecule including an artificial nucleotide derivative in addition to a natural polynucleotide such as DNA or RNA. The polynucleotide may be a DNA-RNA chimeric molecule. The polynucleotide encoding the GPC-CP has a base sequence from position 1 to position 1941 of SEQ ID NO: 1, for example. The base sequence described in SEQ ID NO: 1 can encode a protein containing the amino acid sequence described in SEQ ID NO: 2, and the protein containing this amino acid sequence constitutes a preferred form of GPC-CP.

  The polynucleotide encoding the GPC-CP includes an amino acid in which one or more amino acids are substituted, deleted, inserted, and / or added to the amino acid sequence set forth in SEQ ID NO: 2 as described above, and GPC -Polynucleotides encoding proteins with CP activity are also mentioned. A person skilled in the art may introduce substitution, deletion, insertion, and / or addition mutation as appropriate into the polynucleotide having the base sequence described in SEQ ID NO: 1 using the site-specific mutagenesis method (described above). Thus, it is possible to obtain a homologue of a polynucleotide.

  Based on the nucleotide sequence information described in the present specification, the polynucleotide is used to convert the target gene into the above microorganism, preferably a microorganism belonging to the genus Streptomyces, more preferably Streptomyces. It can be obtained from Streptomyces sanglieri. PCR or hybridization screening can be used for obtaining the gene. Alternatively, for obtaining genes, a technique for searching for ORFs and genes by in silico screening from information obtained by genome analysis using next-generation sequencing or the like can be used.

  Polynucleotides can also be obtained by chemically synthesizing the full length of a gene by DNA synthesis. Moreover, based on said base sequence information, the polynucleotide which codes said GPC-CP originating in organisms other than the above can also be acquired. For example, by designing a probe using the above base sequence or a part of the base sequence and performing hybridization under stringent conditions on DNA prepared from other organisms, GPCs derived from various organisms can be obtained. A polynucleotide encoding CP can be isolated.

  Furthermore, based on the above base sequence information, a primer for PCR should be designed from a region with high homology using sequence information registered in DNA databases such as DNA Database of Japan (DDBJ), EMBL, and GenBank. You can also. Isolation of the polynucleotide encoding GPC-CP from various organisms by performing PCR using such primers and all DNA (genomic DNA) or cDNA including chromosomal DNA or plasmid DNA as a template. You can also. Similarly, the polynucleotide encoding the GPC-CP can also be isolated from various organisms by performing PCR using DNA or RNA extracted from the environment as a template.

  The polynucleotide capable of hybridizing under stringent conditions is a sequence of at least 20, preferably at least 30, for example, 40, 60, or 100 consecutive sequences in the nucleotide sequence set forth in SEQ ID NO: 1. The probe is designed by selecting one or more, for example using ECL direct acid labeling and detection system (manufactured by GE Healthcare) (including washing conditions: 42 ° C., 0.5 × SSC). In primary wash buffer), it refers to a polynucleotide that hybridizes. More specifically, “stringent conditions” are, for example, usually 42 ° C., 2 × SSC, 0.1% SDS, preferably 50 ° C., 2 × SSC, 0.1% SDS. More preferably, the conditions are 65 ° C., 0.1 × SSC, and 0.1% SDS, but are not particularly limited to these conditions. Factors affecting the stringency of hybridization include a plurality of factors such as temperature and salt concentration, and those skilled in the art can realize optimum stringency by appropriately selecting these factors.

  Further, the polynucleotide encoding GPC-CP described above is at least 80%, preferably at least 85%, more preferably at least 90%, even more preferably at least 95%, even more as the amino acid sequence set forth in SEQ ID NO: 2. Preferably, it comprises a polynucleotide that encodes a protein having an amino acid sequence with at least 98%, even more preferably at least 99% homology, and having GPC-CP activity. The protein homology search is as described above.

  The polynucleotide encoding the GPC-CP is at least 80%, preferably at least 85%, more preferably at least 90%, more preferably at least 95%, and even more preferably the nucleotide sequence set forth in SEQ ID NO: 1. Also included are polynucleotides that encode a protein having a base sequence preferably having at least 98%, even more preferably at least 99% sequence identity, and having GPC-CP activity. The determination and search of the sequence identity of the base sequence is also as described above.

  The polynucleotide encoding the GPC-CP can be expressed in the same or different host using genetic recombination techniques.

[Enzyme production method]
In this invention, the method of manufacturing the enzyme which has a hydrolysis action with respect to glycerol-3-phosphodiester is provided. The method for producing an enzyme includes a step of producing the enzyme from a microorganism having the polynucleotide described above. With the method according to the invention, the GPC-CP described above is obtained. The microorganism having the above polynucleotide may be one that the microorganism itself naturally has, such as Streptomyces sanglieri, or one that has been mutated to have a polynucleotide, Alternatively, it may have a polynucleotide introduced therein.

  In introducing a polynucleotide, a vector, a transformant, or the like can be used. This vector contains the above-mentioned polynucleotide. In addition, by introducing a polynucleotide or a vector into a host, a transformant having the ability to hydrolyze glycerol-3-phosphodiester can be produced.

  The procedure for producing a transformant and the construction of a recombinant vector suitable for the host can be performed according to techniques commonly used in the fields of molecular biology, biotechnology, and genetic engineering (for example, Sambrook et al. , Molecular Cloning: Laboratory Manual 2nd edition, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1989). Particularly for actinomycetes, it can be performed with reference to “PRACTICAL STREPTOMYCES GENETICS (Kieser et al., John Inns Foundation, 2000)”.

  In order to express a polynucleotide encoding GPC-CP in a microorganism, this DNA is first introduced into a plasmid vector or a phage vector stably present in the microorganism, and the genetic information is transcribed and translated. Therefore, it is preferable to incorporate a promoter corresponding to a unit for controlling transcription / translation 5 'upstream of the DNA strand. Further, it is preferable to incorporate a terminator, which is a unit for controlling transcription / translation, downstream of the 3 ′ side of the DNA strand. More preferably, both the promoter and terminator are incorporated at each site. As the promoter and terminator, a promoter and terminator known to function in microorganisms used as hosts are used. Regarding vectors, promoters, terminators and the like that can be used in these various microorganisms, “Microbiology Basic Course 8 Genetic Engineering, Kyoritsu Shuppan”, especially regarding actinomycetes, “PRACTICAL STREPTOMYCES GENETICS (Kieser et al., John Inns Foundation, 2000) It is possible to use this method. Moreover, it can be efficiently secreted and produced extracellularly by using a signal sequence as necessary. The signal sequence used at this time may be the above GPC-CP or another.

  The host to be transformed is not particularly limited as long as it is an organism that can be transformed with a vector containing a polynucleotide encoding GPC-CP and express GPC-CP activity. For example, bacteria, actinomycetes, Bacillus subtilis, Escherichia coli, yeast, mold and the like can be mentioned. More specifically, for example, genus Escherichia, genus Bacillus, genus Pseudomonas, genus Serratia, genus Brevibacterium, genus Corynebacterium, genus Corynebacterium Bacteria for which host vector systems such as Streptococcus and Lactobacillus have been developed; Actinomycetes for which host vector systems such as Rhodococcus and Streptomyces have been developed; Saccharomyces (Saccharomyces) Saccharomyces), Kluyveromyces, Shizosa A genus of Schizosaccharomyces, Zygosaccharomyces, Yarrowia, Trichosporon, Rhodosporidium candy, and Pichia (Pi) Yeasts that have been developed; molds that have been developed for host / vector systems such as Neurospora genus, Aspergillus genus, Cephalosporum genus, Trichoderma genus, etc. . Escherichia coli is preferred for ease of gene recombination, and actinomycetes are preferred for ease of gene expression.

  In addition to microorganisms, various host / vector systems have been developed in plants and animals. For example, insects such as moths (Nature 315, 592-594 (1985)), rapeseed, corn, potatoes, etc. Systems for expressing heterologous proteins have been developed, and these may be used. Alternatively, an in vitro expression system such as a cell-free protein synthesis system derived from wheat germ may be used.

  The obtained transformant can be used for production of an enzyme preparation (GPC-CP) as described above. Specifically, the transformant is subjected to liquid culture in an appropriate nutrient medium, the expressed GPC-CP is secreted outside the cell, and the culture supernatant is treated with lyophilization, salting out, an organic solvent, etc. -CP enzyme preparations can be produced.

  Although the culture conditions can vary depending on the host cell, the culture can be performed under conditions commonly used by those skilled in the art. For example, when an actinomycete such as Streptomyces is used as a host, a tryptic soy medium containing an antibiotic such as thiostrepton (for example, Becton Dickinson) can be used. The enzyme produced by the transformant can be further purified as described above.

[Use of GPC-CP]
GPC-CP that can be produced as described above is an enzyme that specifically reacts with glycerol-3-phosphocholine and glycerophosphoethanolamine. Therefore, it can be used for analysis of glycerol-3-phosphocholine and glycerophosphoethanolamine, and can be quantified.

  Further, in combination with one or more enzymes selected from PLA1 (phospholipase A1), PLA2 (phospholipase A2), and PLB (phospholipase B), phosphoric acid, lysophospholipid, phosphoric acid, phosphocholine, phosphoethanolamine, etc. Can be generated. These can be used as fertilizers and physiologically active substances, and can also be used to actively produce them. Furthermore, it is also possible to produce monoglycerides and glycerin.

  Hereinafter, the present invention will be described in more detail with reference to examples. The present invention is not limited to these examples.

[Purification of enzyme derived from Streptomyces sanglieri]
Streptomyces sanglieri strain A14 was cultured, and the culture supernatant was purified using ammonium sulfate fractionation, anion exchange, hydrophobic interaction, and gel filtration column chromatography. Details are shown below.

(A) Culture ISP2 medium “1% malt extract (Becton Dickinson), 0.4% yeast extract (Nippon Pharmaceutical Co., Ltd.), 0.4% glucose (Wako Pure Chemical Industries, Ltd.) , PH 7.2 "was prepared in 500 mL, and 100 mL each was dispensed into a 500 mL Erlenmeyer flask, and further 0.1 to 1 mM Tween 80 and other biochemical surfactants were added, and then at 121 ° C for 15 minutes. Steam sterilization was performed. A suitable amount of a colony of Streptomyces sanglieri strain A14 previously grown on a plate medium was taken and inoculated into a φ18 test tube (18 × 180 mm) containing 5 mL of ISP2 medium, and good growth was obtained at 28 ° C. Shake culture until indicated. 1 ml of this culture solution was inoculated into 100 mL of the previously sterilized medium, and cultured with shaking at 28 ° C. for 48 hours. The supernatant (Supernatant) was recovered from this culture solution using a centrifuge.

(B) Ammonium sulfate fraction Ammonium sulfate was added to the culture supernatant collected in the above (a) so that the saturation amount was 30%, and the resulting precipitate was collected by centrifugation (10,000 rpm, 30 minutes, 4 ° C.). Removed. Ammonium sulfate was added to the obtained supernatant so as to reach 80% saturation, and the resulting precipitate was collected by centrifugation (10,000 rpm, 30 minutes, 4 ° C.). This precipitate was solubilized and dialyzed against 20 mM Tris-HCl buffer (pH 9.0) to obtain a crude enzyme solution.

(C) DEAE-Sepharose Column Chromatography The crude enzyme solution obtained in (b) above was applied to “DEAE-Sepharose” pre-equilibrated with 20 mM Tris-HCl buffer (pH 9.0). After washing the column with the same buffer, the active fraction was eluted with a linear gradient of sodium chloride (from 0 M to 1 M).

(D) HiTrap Butyl FF column chromatography The active fractions obtained in (c) above were collected and concentrated and desalted using Viva spin (manufactured by Sartorius). To this was added 20 mM Tris-HCl buffer and 1 M ammonium sulfate (pH 8.0). This was applied to a “HiTrap Butyl FF” (5 ml) column (manufactured by GE Healthcare Bioscience) previously equilibrated with the same buffer, washed with the same buffer, and then ammonium sulfate (from 1 M to 0 M). The active fraction was eluted with a linear gradient.

(E) HiTrap Q HP column chromatography The active fractions obtained in (d) above were collected and concentrated and desalted using Viva spin (manufactured by Sartorius). To this, 20 mM Tris-HCl buffer (pH 9.0) was added. This was applied to a “HiTrap Q” (5 ml) column (manufactured by GE Healthcare Bioscience) previously equilibrated with 20 mM Tris-HCl buffer (pH 9.0), and the column was washed with the same buffer. The active fraction was eluted with a linear gradient of sodium chloride (0M to 1M).

(F) Mono Q column chromatography The active fractions obtained in (e) above were collected and concentrated and desalted using Viva spin. To this, 20 mM Tris-HCl buffer (pH 9.0) was added. The column was applied to a “Mono Q” (1 ml) column (manufactured by GE Healthcare Bioscience) previously equilibrated with 20 mM Tris-HCl buffer (pH 9.0), washed with the same buffer, and then sodium chloride ( The active fraction was eluted with a linear gradient from 0M to 1M.

(G) Superdex200 10 / 300GL column chromatography The active fractions obtained in (f) above were collected and concentrated using Viva spin. To this, 20 mM Tris-HCl buffer and 0.15 M sodium chloride (pH 8.0) were added. The sample was applied to a “Superdex200 10 / 300GL” (24 ml) column (manufactured by GE Healthcare Bioscience) previously equilibrated with the same buffer and eluted with the same buffer.

(H) Mono P column chromatography The active fractions obtained in (g) above were collected and concentrated and desalted using Viva spin. To this, 25 mM bis-Tris buffer (pH 6.3) was added. This was applied to a “Mono P” (1 ml) column (manufactured by GE Healthcare Bioscience) previously equilibrated with 25 mM Bis-Tris buffer (pH 6.3), and the column was washed with the same buffer, and then Poly Buffer ( The active fraction was eluted with a pH linear gradient (pH 4.0) (manufactured by GE Healthcare Bioscience). At this time, the isoelectric point of the enzyme was expected to be around 4.8-5 from the elution position.

  As described above, a purified enzyme was obtained from the Streptomyces sanglieri A14 strain. The single purified enzyme was confirmed by the measurement of a single band in the molecular weight measurement. This enzyme was also estimated to be a monomer by HPLC analysis and gel filtration chromatography analysis.

  Table 1 shows the yield, yield and the like in GPC-CP purification.

In the above table, “30-80% ammonium sulfate fraction” is the value after (b) recovery by centrifugation in the ammonium sulfate fraction, and “dialysis” is the value after (b) dialysis in the ammonium sulfate fraction.

[Measurement of molecular weight of GPC-CP]
(Molecular weight)
About purified enzyme (GPC-CP) obtained by the above, molecular weight was analyzed by SDS-PAGE (12% (w / v) polyacrylamide gel).

  FIG. 1 is an electrophoresis photograph showing the results of analysis by SDS-PAGE of the eluted fraction. The lane on the left side of the figure is the molecular weight marker (M), and the lane on the right side of the figure shows the band of the elution fraction (GPC-CP). As shown in FIG. 1, a single band was observed in the eluted fraction, and the molecular weight of the purified enzyme (polypeptide) was about 66,000. The unit of molecular weight is Da (Dalton). Therefore, in the kDa notation, the molecular weight of this enzyme is about 66 kDa.

  Here, the molecular weight of the known GPC-CP enzyme is known to be 110 kDa from bovine brain, 230 kDa from rat brain, and 74 kDa from mouse brain by SDS-PAGE analysis. Therefore, it was confirmed that the enzyme is different from the known GPC-CP.

[Measurement of properties of enzyme (GPC-CP) derived from Streptomyces sanglieri]
(Enzyme activity (GPC-CP activity): substrate specificity)
In this example, the enzyme activity was basically measured as follows. This method is hereinafter referred to as “enzyme activity standard measurement method”. This method exemplifies a case where GPC (glycerophosphocholine) is used as a substrate.

  Purified enzyme (GPC-CP) was added to a solution of 0.8 mM substrate (GPC) and 50 mM HEPES buffer (pH 7.2, temperature 37 ° C.) to a concentration of 10% (v / v). A reaction solution was prepared. In the “enzyme activity standard measurement method”, the base mass is 0.8 mM. However, in the case of a substrate other than GPC (GPX or the like), Triton X-100 was added to the reaction solution in an amount of 0.5% (w / v) for solubilization. This reaction solution was stirred for 5 minutes (or 10 minutes) at a temperature of 37 ° C. to carry out an enzyme reaction. The time for the enzyme reaction was appropriately selected according to the degree of enzyme activity. Thereafter, as alkaline phosphatase treatment (AP treatment), 1 μL of AP and 94 μL of AP buffer solution (pH 9.6, temperature 56 ° C.) are added to 5 μL of the enzyme reaction solution to make a total of 100 μL. To stop the enzyme reaction. Next, as a color reaction, Biomol Green (BMG) is added to the liquid after stopping the enzyme reaction (AP reaction liquid) to prepare a color liquid, and the color reaction is performed by stirring at room temperature for 20 minutes. It was. For the BMG color solution, add either 225 μL of BMG to 25 μL of the AP reaction solution to make a total of 250 μL, or add 75 μL of BMG to 75 μL of the AP reaction solution to make a total of 225 μL. It selected suitably according to the degree. And the light absorbency in wavelength 620nm was measured with the absorptiometer about the colored liquid by BMG. In BMG, free phosphoric acid is colored and quantified.

  Table 2 (Tables 2A to 2E) shows the reaction solution composition in each of the above reactions.

Details of the substrate in the graph DPPC: 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine
PC (soy): soybean phosphatidylcholine (L-α-phosphatidylcholine) (manufactured by Sigma-Aldrich)
DPPE: 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine
PI: L-α-phosphatidylinositol (L-α-Phosphatidylinositol)
POPS: L-α-Phosphatidylserine
POPG: 1,2-diacyl-sn-glycero-3-phospho- (1-rac-glycerol) (1,2-Diacyl-sn-glycero-3-phospho- (1-rac-glycerol))
DMPA: 1,2-Dimyristoyl-sn-glycerol-3-phosphate (1,2-Dimyristoyl-sn-glycerol-3-phosphate)
PAF: β-Acetyl-γ-O-hexadecyl-L-α-phosphatidylcholine
SM: Sphingomyelinfrom chiken egg yolk
PlsPC: Choline-type lysoplasmalogen PlsPE: Ethanolamine-type lysoplasmalogen LPC: PC lyso form (see Table 3)
LPE: lyso form of PE (see Table 3)
LPI: PI lyso form (see Table 3)
LPS: PS lyso form (see Table 3)
LPG: lysozyme of PG (see Table 3)
LPA: lyso form of PA (see Table 3)
Lyso-PlsPC: Lyso form of the above PlsPC Lyso-PLsPE: Lyso form of the above PlsPE GPC: Glycero-3-phosphocholine GPA: Glycero-3-phosphate GPS: Glycero-3-phosphoserine GPG: Glycero-3-phosphoglycerol GPE: Glycero-3-phosphoethanolamine GPI: Glycero-3-phosphoinositol In addition, the thing with low relative activity (1% or less) performed enzyme reaction for 10 minutes, and set the time of enzyme reaction to 5 minutes otherwise.

  Table 3 shows the details of the results of the relative name in the enzyme reaction and the formal name and source of the substrate.

  Specifically, the hydrolysis activity of GPC-CP is 90% relative to glycerol-3-phosphoethanolamine (GPE), assuming that the activity when glycerol-3-phosphocholine (GPC) is used as a substrate is 100%. As described above, 20% or less of glycerol-3-phosphate (GPA), 1% or less of glycerol-3-phosphoglycerol (GPG) and glycerol-3-phosphoinositol (GPI), glycero-3-phosphoserine ( GPS) was almost 0%. Also, 40% or less for lysophosphatidylcholine (LPC), 3% or less for lysophosphatidylethanolamine (LPE), 1% or less for lysophosphatidic acid (LPA), lysophosphatidylinositol (LPI), lysophosphatidyl Nearly 0% for serine (LPS) and lysophosphatidylglycerol (LPG). In addition, 5% or less for choline-type lysoplasmalogens (Lyso-PlsPC), 1% or less for ethanolamine-type lysoplasmalogens (Liso-PlsPE), ethanolamine-type and choline-type plasmalogens (PlsPE, PlsPC) It was almost 0%. Moreover, 0.5% or less with respect to phosphatidylethanolamine (PE), 0.1% or less with respect to phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidylcholine (PC), phosphatidic acid (PA), and sphingomyelin It was almost 0% with respect to (SM). Moreover, it was 0.5% or less with respect to the phosphatidylcholine (PC (soy)) derived from soybean, and was almost 0% with respect to PAF. An example of PC is DPPC, an example of PA is DMPA, an example of PS is POPS, an example of PG is POPG, an example of PE is DPPE, and these are shown in a graph. .

  Here, in Table 3, “self-made” means that the substrate was produced by self-made. The self-made substrate is a so-called glycerophosphodiester (hereinafter also referred to as “GPX”). This glycerophosphodiester is obtained by allowing phospholipase A1 to act on lysophospholipid having a fatty acid in sn-1. Glycerophosphodiester has a structure represented by the following chemical formula.

The substituent X in the above glycerophosphodiester (GPX) has the structure shown in Table 4.

The above glycerophosphodiester (GPX) was prepared as follows. First, 20 mM Tris-HCl buffer (pH 7.2) was added to 8 mM lysophospholipid (LPX), phospholipase A1 was added at 1% (v / v), and the enzyme reaction was carried out overnight (over night). The substrate was reacted 100%. Thereafter, the enzyme was inactivated by heating to 100 ° C., and centrifuged (15 krpm, 5 min) to recover the supernatant. This was extracted with chloroform / methanol (2/1, volume ratio), and the aqueous layer was recovered. After removing moisture by drying, 20 mM Tris-HCl buffer (pH 7.2) in an amount equal to the reaction solution was added and dissolved.
As a result, a GPX-containing liquid was obtained. A 10% (volume ratio) GPX-containing solution was used for enzyme activity measurement. Table 5 shows the reaction composition of the enzyme reaction with PLA1.

(Enzymological properties)
The enzymatic properties of the purified enzyme (GPC-CP) were examined.

(1) Working temperature A purified enzyme (GPC-CP) is added to a solution of 8 mM substrate (GPC) and 50 mM Tris-HCl (pH 7.2, each temperature) so as to be 10% (v / v), A reaction solution having a total amount of 50 μL was prepared. This reaction solution was subjected to an enzyme reaction for 5 minutes under each temperature condition. Subsequent AP treatment and color reaction were carried out according to the “Enzyme Activity Standard Measurement Method” to measure enzyme activity.

  FIG. 3 is a graph showing enzyme activities at various reaction temperatures as relative activities based on the activity (100%) when the reaction temperature is 40 ° C. As shown in the graph of FIG. 3, the enzyme exhibited activity at 20 to 65 ° C., and the optimum temperature for the reaction was around 40 ° C. (eg, 35 to 60 ° C.).

(2) Working pH
10% of purified enzyme (GPC-CP) is added to a solution of 8 mM substrate (GPC) and 50 mM buffer (each pH) selected from Tris-HCl, acetic acid-Na acetate, Bis-Tris, or Glycine-NaOH. (V / v) was added, and a reaction solution having a total amount of 50 μL was prepared. This reaction solution was subjected to an enzyme reaction at a temperature of 37 ° C. for 5 minutes under each pH condition. Subsequent AP treatment and color reaction were carried out according to the “Enzyme Activity Standard Measurement Method” to measure enzyme activity.

Each pH condition is as follows.
Acetic acid-Na acetate buffer: pH 4.1, pH 4.4, pH 5, pH 5.6.
Bis-Tris buffer: pH 5.6, pH 6, pH 6.5, pH 7.2.
Tris-HCl buffer: pH 7.2, pH 8, pH 8.8, pH 9.
Glycine-NaOH buffer: pH 9, pH 9.5, pH 10.5.

  FIG. 4 is a graph showing enzyme activities at various reaction pHs as relative activities based on the enzyme activity when the reaction pH is 7.2 (100%). As can be seen from the graph of FIG. 4, this enzyme exhibited activity in a wide range from pH 4 to pH 9, and the optimum pH of the reaction was around 7 (for example, 6.5 to 7.2).

(3) Temperature stability Purified enzyme (GPC-CP) was added to Tris-HCl (pH 7.2, each temperature) 50 mM so that it might become 20% (v / v), and it kept at each temperature for 30 minutes. To a solution of 8 mM substrate (GPC) and 50 mM HEPES (pH 7.2), the enzyme (GPC-CP) after treatment at each temperature was added to 10% (v / v), and the total amount was 50 μL. A reaction solution was prepared. This reaction solution was subjected to an enzyme reaction for 5 minutes under the conditions of pH 7.2 and temperature of 37 ° C. Subsequent AP treatment and color reaction were carried out according to the “Enzyme Activity Standard Measurement Method” to measure enzyme activity.

  FIG. 5 is a graph showing the activity of the enzyme after treatment at pH 7.2 and at each temperature as a relative activity based on the activity (100%) when the enzyme reaction is performed at 37 ° C. without performing the temperature treatment. is there. As shown in the graph of FIG. 5, the activity of this enzyme was confirmed after treatment at a temperature from 4 ° C. to 60 ° C. In particular, after the treatment at 55 ° C. or lower, the activity of 40% or more before the treatment was maintained.

(4) pH stability Purified enzyme (GPC-CP) is added to 50 mM of a buffer solution (each pH) selected from Tris-HCl, HEPES, acetic acid-Na acetate, Bis-Tris, or Glycine-NaOH with 20% (v / V) and treated at 4 ° C. for 3 hours at each pH. Thereafter, the enzyme (GPC-CP) after treatment at each pH was added to a solution of 8 mM of substrate (GPC) and 50 mM of HEPES (pH 7.2) so as to be 10% (v / v), and the total amount A reaction solution of 50 μL was prepared. This reaction solution was subjected to an enzyme reaction for 5 minutes under the conditions of pH 7.2 and temperature of 37 ° C. Subsequent AP treatment and color reaction were carried out according to the “Enzyme Activity Standard Measurement Method” to measure enzyme activity.

  FIG. 6 is a graph showing the activity of the enzyme after treatment at various pHs as relative activity based on the activity (100%) when the enzyme reaction is carried out by pH treatment at pH 10.2. As shown in the graph of FIG. 6, the activity of this enzyme was confirmed even after pH treatment at least from pH 4 to 10.2.

(5) Effect of metal ions and EDTA To a solution of 8 mM substrate (GPC) and 50 mM HEPES (pH 7.2), purified enzyme (GPC-CP) was added to 10% (v / v), and Various metal ions or 2 mM of EDTA was added to prepare a reaction solution having a total amount of 50 μL. This reaction solution was subjected to an enzyme reaction for 5 minutes under the conditions of pH 7.2 and temperature of 37 ° C. Subsequent AP treatment and color reaction were carried out according to the “Enzyme Activity Standard Measurement Method” to measure enzyme activity.

FIG. 7 shows the results of relative activity when various additives were added when the condition (control) in which the additives (metal ions and EDTA) were not added was taken as 100%. In the presence of Ca ²⁺ , Mg ²⁺ , Na ⁺ , K ⁺ , and EDTA, the above enzyme showed almost the same activity as when no metal ion was added (control). In addition, in the presence of Mn ^2+, the activity tended to decrease slightly, and in Fe ²⁺ the tendency increased. Further, in the presence of Co ²⁺ , the activity was further reduced. Moreover, in the presence of Zn ²⁺ and Cu ²⁺ , the activity was greatly reduced and activity inhibition was observed.

  Here, it is known that known GPC-CP enzymes (derived from bovine brain, derived from rat brain, derived from mouse brain) are ion-requiring. On the other hand, the above enzyme has activity even in the absence of ions, and further maintains activity in the presence of EDTA. Therefore, the above enzyme is considered to be non-required for metal ions.

(6) Effect of chemical substance Purified enzyme (GPC-CP) was added to a solution of 8 mM substrate (GPC) and 50 mM HEPES buffer (pH 7.2) so as to be 10% (v / v). Various chemical substances were added at 2 mM to prepare a reaction solution having a total volume of 50 μL. This reaction solution was subjected to an enzyme reaction for 5 minutes under the conditions of pH 7.2 and temperature of 37 ° C. Subsequent AP treatment and color reaction were carried out according to the “Enzyme Activity Standard Measurement Method” to measure enzyme activity.

  FIG. 8 shows the results of relative activities to which various chemical substances were added when the condition (control) in which no chemical substance was added was 100%. In the presence of SDS (sodium dodecyl sulfate), 2-ME (2-mercaptoethanol), DTT (dithiothreitol), the above enzyme has almost the same activity as when no chemical substance is added (control). Indicated. In the presence of IAA (iodoacetamide), the activity tended to decrease slightly. Moreover, the tendency of the activity fall became large in the presence of PMSF (phenylmethylsulfonyl fluoride).

(7) Isoelectric point As a result of predicting the isoelectric point of the enzyme based on the amino acid sequence of the enzyme using Genetyx, the isoelectric point of the enzyme was 5.8.

[Analysis of N-terminal amino acid sequence of purified enzyme]
About said purified enzyme, electroblotting was performed after SDS-PAGE, and the target enzyme was transcribe | transferred to the PVDF membrane. This was analyzed for the N-terminal amino acid sequence by a protein sequencer. From the analysis, it was confirmed that the N-terminal amino acid sequence of this purified enzyme was as shown in SEQ ID NO: 3.

[Analysis of internal amino acid sequence of purified enzyme]
About said purified enzyme, after SDS-PAGE, the target enzyme was cut out and digested in gel using trypsin. And the internal amino acid sequence was analyzed with the mass spectrometer (nanoLC-MS / MS) about the obtained peptide sample. Thereby, it was confirmed that the internal amino acid sequences are those shown in SEQ ID NOs: 4, 5, and 6.

  Here, SEQ ID NO: 4 is the sequence shown from position 482 of SEQ ID NO: 2. SEQ ID NO: 5 is the sequence shown from position 576 of SEQ ID NO: 2. SEQ ID NO: 6 is the sequence shown from position 466 of SEQ ID NO: 2. In addition, since de novo amino acid sequence analysis by nanoLC-MS / MS cannot discriminate between Leu and Ile, there are places where these amino acids do not partially match.

[Isolation of chromosomal DNA of Streptomyces sanglieri strain A14]
Streptomyces sanglieri strain A14 was added to 50 μl of 2.5M magnesium chloride, 0.625 ml of 20% glycine (0.5% tryptone, 0.5% yeast extract, 0.3% malt extract, glucose 1 %, Sucrose 34%) was cultured at 28 ° C. for 3 days and collected.

  Then, the cells were suspended in 5 ml of a solution consisting of 75 mM NaCl, 25 mM EDTA, 20 mM Tris-hydrochloric acid (pH 7.5) and 1 mg / ml lysozyme, and treated at 37 ° C. overnight. To this, 750 μl of 10% (w / v) SDS and 5 mg of proteinase K were added and treated at 55 ° C. for 2 hours. To this solution, 7.5 ml of chloroform was added and stirred, and 5 ml of the aqueous phase was collected by centrifugation.

  To this aqueous phase, 3 ml of isopropanol was added and mixed to collect the DNA fraction, which was dissolved in 500 μl of a solution consisting of 10 mM Tris-HCl buffer (pH 8.0) and 1 mM EDTA. To this, RNase A was added to 20 μg / ml, treated at 37 ° C. for 1 hour, 500 μl of 13% PEG solution containing 0.8 M NaCl was added and stirred, and 500 μl of the aqueous phase was collected by centrifugation. did. 500 μl of a phenol / chloroform mixed solution was added thereto and stirred, and 500 μl of an aqueous phase was collected by centrifugation.

  To this aqueous phase, 50 μL of 3M sodium acetate (pH 5.2) and 1 ml of ethanol were added and mixed to recover DNA.

  This DNA was immersed in 70% (v / v) ethanol for 10 minutes, and then dissolved in 200 μl of a solution consisting of 10 mM Tris-HCl buffer (pH 8.0) and 1 mM EDTA.

[Cloning of core region of GPC-CP gene derived from Streptomyces sanglieri A14]
Primers were designed based on the N-terminal and internal amino acid sequences of GPC-CP and the codons used in the genus Streptomyces.

  In preparing the primer, the above three internal amino acid sequences (SEQ ID NOs: 4 to 6) were picked up. The one-letter code for these internal amino acid sequences (peptides) is shown below.

Peptide of SEQ ID NO: 4 LTFSAGDQAGAAF
Peptide of SEQ ID NO: 5 HLTVTNAY
Peptide of SEQ ID NO: 6 APYVDGAADLSTGK
For the peptide of SEQ ID NO: 4, a primer was designed based on the sequence of “GDQAGAAF”. For the peptide of SEQ ID NO: 5, a primer was designed based on the sequence of “TVTNAY”. For the peptide of SEQ ID NO: 6, a primer was designed based on the sequence of “PYVDGAAD”.

  The N-terminal amino acid sequence information was “Sense primer” and the internal amino acid sequence information was “Antisense primer”. Regarding degenerate codons, codons with high codon usage in the genus Streptomyces were selected for primer design. Also, mixed base primers were used for those with the same codon usage frequency. In addition, Leu and Ile, which are difficult to be separated by mass spectrometry, were excluded. Thus, degenerate oligonucleotide primer S1 (SEQ ID NO: 7) for PCR was designed as a sense primer. Moreover, as three types of antisense primers (Antisense primers), “primer A1-1” (SEQ ID NO: 8) designed from SEQ ID NO: 4 and “primer A1-2” (SEQ ID NO: 9) designed from SEQ ID NO: 5 And “primer A1-3” (SEQ ID NO: 10) designed from SEQ ID NO: 6. Here, s in the sequence represents c or g.

  Next, PCR was performed using the above primers. The composition of the reaction solution for PCR is as follows.

  100 ng of template chromosomal DNA obtained in the above [Separation of chromosomal DNA of Streptomyces sangulieri strain A14], 2 × KOD FX Buffer 25 μL, each primer 300 nM, and KOD FX DNA Polymerase 1 unit so that the total amount of distilled water is 50 μl. Added to.

  PCR reaction conditions are as follows.

Step 1: 94 ° C., 2 minutes;
Step 2: 98 ° C., 10 seconds;
Step 3: 68 ° C., 90 seconds;
Repeat steps 2 to 3 for 30 cycles;
Step 4: 68 ° C., 2 minutes.

  A specific amplification product of about 1500 bp was obtained by PCR using the primers of SEQ ID NOs: 7 to 10.

  The PCR reaction solution was subjected to agarose gel electrophoresis, and the target 1500 bp band portion was excised and bound to pGEM-T Easy Vector using pGEM-T Easy Vector System I (Promega) to transform Escherichia coli. The transformed strain was cultured in LB medium (tryptone 1%, yeast extract 0.5%, sodium chloride 0.4%, pH 7.2) containing ampicillin 50 μg / ml, and Miniprep DNA Purification Kit (manufactured by TaKaRa) was used. Plasmid for DNA sequencing was extracted and purified.

  Subsequently, the base sequence of the inserted fragment was determined by an automatic sequencer using T7 primer and SP6 primer derived from a vector (pGEM-T Easy Vector). This base sequence is shown in SEQ ID NO: 11.

[Cloning of Streptomyces sanglieri A14 strain-derived GPC-CP gene downstream of core region]
In order to clarify the sequence of the peripheral region of the gene sequence determined in the above [Cloning of core region of GPC-CP gene derived from Streptomyces sanglieri A14 strain], a DNA fragment including the downstream side was amplified by genome walking. . The chromosomal DNA obtained above was completely digested with PstI, and ligation high Ver. Cassette, Pst 1 (manufactured by Takara Bio Inc., product code: 3871) was ligated to the digested fragment using 2 (manufactured by Toyobo). Using this as a template, sense primers “Sense-Gw1” (SEQ ID NO: 12) and “Sense2-Gw2” (SEQ ID NO: 13) prepared based on the partial gene sequence of GPC-CP (SEQ ID NO: 11), antisense primer Using “Antisense1” (Pst 1-Cassette primer1) (SEQ ID NO: 14) and “Antisense2” (Pst 1-Cassette primer2) (SEQ ID NO: 15), downstream of GPC-CP The containing DNA fragment was amplified.

  The composition of the reaction solution for PCR is as follows.

  Distilled water was added to 30 ng of template DNA, 25 μl of 2 × KOD FX Neo Buffer, 300 nM of each primer, and 1 unit of KOD FX Neo DNA Polymerase to a total volume of 50 μl.

  PCR reaction conditions are as follows.

Step 1: 94 ° C., 2 minutes;
Step 2: 98 ° C., 10 seconds;
Step 3: 68 ° C., 2 minutes;
Repeat steps 2 to 3 for 25 cycles;
Step 4: 68 ° C., 2 minutes.

  By this PCR, a specific amplification product of about 750 bp was obtained, which was cloned with pGEM-T Easy Vector (manufactured by Promega), and the base sequence was determined.

  The base sequence obtained by analysis is shown in SEQ ID NO: 16, and the amino acid sequence is shown in SEQ ID NO: 17. This base sequence and amino acid sequence are sequences downstream of the core region.

[Cloning of the GPC-CP gene upstream of the GPC-CP gene derived from Streptomyces sanglieri A14 strain]
The chromosomal DNA obtained in the above [Separation of chromosomal DNA of Streptomyces sangulieri strain A14] was completely digested with SacII, and Ligation high Ver. 2 (manufactured by Toyobo Co., Ltd.). Using this as a template, a sense primer “primer InvN3S” (SEQ ID NO: 18) prepared based on a partial gene sequence of GPC-CP (SEQ ID NO: 11) and an antisense primer AN1 “primer InvN3A” (SEQ ID NO: 19) Inverse PCR was performed.

  The composition of the reaction solution for PCR is as follows.

  Distilled water was added to 30 ng of template DNA, 25 μl of 2 × KOD FX Neo Buffer, 300 nM of each primer, and 1 unit of KOD FX Neo DNA Polymerase to a total volume of 50 μl.

  PCR reaction conditions are as follows.

Step 1: 94 ° C., 2 minutes;
Step 2: 98 ° C., 30 seconds;
Step 3: 68 ° C., 2 minutes;
Repeat steps 2 to 3 for 25 cycles;
Step 4: 68 ° C., 7 minutes.

  By this PCR, a specific amplification product of about 600 bp was obtained, which was cloned with pGEM-T Easy Vector (manufactured by Promega), and the base sequence was determined.

  The base sequence obtained by analysis is shown in SEQ ID NO: 20, and the amino acid sequence is shown in SEQ ID NO: 21. This base sequence and amino acid sequence are sequences upstream of the core region.

[Determination of nucleotide sequence of GPC-CP gene derived from Streptomyces sanglieri A14]
Based on the base sequence determined above together with the base sequence determined in [Cloning of core region of GPC-CP gene derived from Streptomyces sanglieri A14 strain], derived from Streptomyces sanglieri A14 strain The base sequence of the region containing the GPC-CP gene was determined (SEQ ID NO: 22). The signal region (positions 1 to 13 of the sequence) was excluded from this sequence (SEQ ID NO: 22), and the base sequence (1941 base) from which the mature partial enzyme portion was extracted was determined (upper sequence number 23). Further, the amino acid sequence of the structural gene part was deduced from the base sequence (lower part of SEQ ID NO: 23, SEQ ID NO: 24).

  The result of sequence analysis revealed that the structural gene encoding GPC-CP consists of 1,941 bp nucleotides and encodes an amino acid of 646 residues. The amino acid sequence of SEQ ID NO: 21 is a secretory signal sequence.

  The N-terminal and internal amino acid sequences of the purified enzyme derived from Streptomyces sanglieri strain A14 determined above were present in the deduced amino acid sequence and were almost completely identical.

  That is, the amino acid sequence of SEQ ID NO: 24 is the sequence shown in the amino acid sequence of SEQ ID NO: 2. The amino acid sequence of SEQ ID NO: 4 is the sequence shown from position 482 of the amino acid sequence of SEQ ID NO: 2. The amino acid sequence of SEQ ID NO: 5 is the sequence shown from position 576 of the amino acid sequence of SEQ ID NO: 2. The amino acid sequence of SEQ ID NO: 6 is the sequence shown from position 466 of the amino acid sequence of SEQ ID NO: 2.

  Further, the purified enzyme derived from Streptomyces sanglieri A14 strain (ie GPC-CP) was calculated based on the amino acid composition of this deduced amino acid sequence, and was estimated to have a molecular weight of 70,400. It was.

[Sequence of GCP-CP and its expression gene]
As described above, the amino acid sequence of GCP-CP and its expressed gene were determined. When the sequence was performed twice, slightly different results were obtained. SEQ ID NO: 25 shows the base sequence of the GPC-CP expression gene. The corresponding codon is shown in SEQ ID NO: 26. SEQ ID NO: 27 shows the amino acid sequence of GPC-CP.

  The nucleotide sequence of SEQ ID NO: 25 differs from the nucleotide sequence of SEQ ID NO: 1 at position 1915 (a and g). Further, the amino acid sequence of SEQ ID NO: 27 is different from the amino acid sequence of SEQ ID NO: 2 at position 639 (Ser and Gly). The same position also differs in the corresponding codon of SEQ ID NO: 26. This analysis is due to the fact that “a” at position 166 in SEQ ID NO: 16 was replaced with “g”.

  Therefore, in the enzyme of the present invention, the GPC-CP expression gene may be the nucleotide sequence of SEQ ID NO: 25, and GPC-CP may be the amino acid sequence of SEQ ID NO: 27. Of course, since this base sequence and amino acid sequence are obtained by replacing only one base and amino acid, the sequence identity of the base sequence represented by SEQ ID NO: 1 and the amino acid sequence represented by SEQ ID NO: 2 and Homology is included in the range of 80% or more, and included in the range substituted with 3 or less.

  According to the present invention, a novel GPC-CP and a method for producing the same are provided. Since this GPC-CP specifically reacts with glycerol-3-phosphocholine, its quantification is possible. It can also be used for quantification of glycerol-3-phosphoethanolamine. In quantification using GPC-CP, colorimetric quantification is possible, and quantification can be easily performed. Glycerol-3-phosphate compounds such as glycerol-3-phosphocholine are useful as compounding substances for pharmaceuticals, foods and cosmetics. Among these, glycerol-3-phosphocholine (GPC) is expected to prevent dementia. Therefore, GPC-CP can be used as an enzyme for in-vitro diagnostic drugs and an enzyme for quantification of glycerol-3-phosphocholine. For example, substances such as GPC present in body fluids such as serum can be easily and accurately quantified.

  NITE BP-1392

SEQ ID NO: 1: GPC-CP expression gene SEQ ID NO: 2: GPC-CP (polypeptide)
SEQ ID NO: 3: N-terminal sequence of GPC-CP SEQ ID NO: 4: Internal sequence of GPC-CP SEQ ID NO: 5: Internal sequence of GPC-CP SEQ ID NO: 6: Internal sequence of GPC-CP SEQ ID NO: 7: Primer S1
Sequence number 8: Primer A1-1
Sequence number 9: Primer A1-2
Sequence number 10: Primer A1-3
Sequence number 11: GPC-CP expression gene (analysis result)
Sequence number 12: Primer Gw1
Sequence number 13: Primer Gw2
SEQ ID NO: 14: cassette primer 1
SEQ ID NO: 15: Cassette primer 2
SEQ ID NO: 16: GPC-CP expression gene (analysis result)
SEQ ID NO: 17: Terminal sequence of GPC-CP SEQ ID NO: 18: Inverse PCR primer Sense (InvN3S)
SEQ ID NO: 19: Inverse PCR primer Antisense (InvN3A)
SEQ ID NO: 20: Inverse PCR analysis result (base sequence)
SEQ ID NO: 21: Inverse PCR analysis result (amino acid sequence)
SEQ ID NO: 22: GPC-CP expression gene (analysis result)
SEQ ID NO: 23: GPC-CP expression gene (mature enzyme expression site)
SEQ ID NO: 24: GPC-CP (polypeptide analysis result, mature enzyme part)
SEQ ID NO: 25: GPC-CP expression gene SEQ ID NO: 26: GPC-CP expression gene (codon)
SEQ ID NO: 27: GPC-CP (polypeptide)

Claims (10)

An enzyme having a hydrolysis action on glycerol-3-phosphodiester, comprising the polypeptide according to any one of the following (a) to (c):
(A) a polypeptide having the amino acid sequence set forth in SEQ ID NO: 2;
(B) a polypeptide having an amino acid sequence in which one or more amino acids are substituted, inserted, deleted and / or added in the amino acid sequence of SEQ ID NO: 2 and exhibiting the hydrolysis activity; or ( c) A polypeptide having at least 90 % identity with the amino acid sequence set forth in SEQ ID NO: 2 and exhibiting the hydrolysis activity.   When glycerol-3-phosphocholine is used as a substrate, the hydrolysis activity under the condition of pH 7.2 and 40 ° C. for 5 minutes is defined as 100%, and it is 40% or more within the range of 20 ° C. to 60 ° C. The enzyme according to claim 1, which exhibits hydrolytic activity.   When glycerol-3-phosphocholine is used as a substrate, hydrolysis is 10% or more within the range of pH 5 to pH 8 when the hydrolysis activity under the condition of pH 7.2 and 37 ° C. for 5 minutes is 100%. The enzyme of Claim 1 or 2 which shows activity.   When glycerol-3-phosphocholine is used as a substrate and the hydrolysis activity under the condition of pH 7.2 and 37 ° C. for 5 minutes is defined as 100%, the hydrolysis activity under the conditions is glycerol-3- 90% or more for phosphoethanolamine, 20% or less for glycerol-3-phosphate, 1% or less for glycerol-3-phosphoglycerol and glycerol-3-phosphoinositol, for glycerol-3-phosphoserine Approximately 0%, lysophosphatidylcholine 40% or less, lysophosphatidylethanolamine 3% or less, lysophosphatidic acid 1% or less, lysophosphatidylinositol, lysophosphatidylserine and lysophosphatidylglycerol 0%, choline-type lysoplasma low 5% or less for ethanol, 1% or less for ethanolamine-type lysoplasmalogen, almost 0% for ethanolamine-type or choline-type plasmalogen, 0.5% or less for phosphatidylethanolamine, phosphatidylserine The enzyme according to any one of claims 1 to 3, which has a substrate specificity of 0.1% or less based on phosphatidylinositol, phosphatidylcholine, phosphatidic acid and sphingomyelin.   The enzyme according to any one of claims 1 to 4, having a molecular weight within a range of 60,000 to 70,000 as measured by SDS-PAGE.   The enzyme according to any one of claims 1 to 5, which is derived from a microorganism belonging to the genus Streptomyces.   A polynucleotide encoding the enzyme according to claim 1. The polynucleotide according to claim 7, comprising the polynucleotide according to the following (a) or (b):
(A) a polynucleotide comprising the base sequence set forth in SEQ ID NO: 1; or (b) a polynucleotide having at least 90 % sequence identity with the base sequence set forth in SEQ ID NO: 1.   The polynucleotide according to claim 7 or 8, which is derived from a microorganism belonging to the genus Streptomyces. A method for producing an enzyme having a hydrolysis action on glycerol-3-phosphodiester,
A method for producing an enzyme, comprising a step of producing the enzyme from a microorganism having the polynucleotide according to claim 7.