WO2012104987A1 - Enzyme et son procédé de production - Google Patents

Enzyme et son procédé de production Download PDF

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WO2012104987A1
WO2012104987A1 PCT/JP2011/052003 JP2011052003W WO2012104987A1 WO 2012104987 A1 WO2012104987 A1 WO 2012104987A1 JP 2011052003 W JP2011052003 W JP 2011052003W WO 2012104987 A1 WO2012104987 A1 WO 2012104987A1
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enzyme
seq
pla1
activity
amino acid
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PCT/JP2011/052003
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English (en)
Japanese (ja)
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大助 杉森
功大 加納
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国立大学法人福島大学
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Priority to PCT/JP2011/052003 priority Critical patent/WO2012104987A1/fr
Priority to PCT/JP2012/052159 priority patent/WO2012105565A1/fr
Publication of WO2012104987A1 publication Critical patent/WO2012104987A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/18—Carboxylic ester hydrolases (3.1.1)
    • C12N9/20—Triglyceride splitting, e.g. by means of lipase

Definitions

  • the present invention relates to a novel phospholipase A1 and a method for producing the same.
  • Phospholipase A1 is an enzyme that acts on glycerol phospholipid to produce lysophospholipid.
  • C Known as 3.1.1.32.
  • Phospholipase is a general term for enzymes that hydrolyze phospholipids.
  • Phospholipid glycerol phospholipid
  • PDA1 The enzyme that hydrolyzes the fatty acid ester bond at the ⁇ -position of the glycerol group in glycerol phospholipid is called phospholipase A1 (PLA1), and the enzyme that hydrolyzes the fatty acid ester group at the ⁇ -position of the glycerol group is called phospholipase A2 (PLA2).
  • PLB phospholipase B
  • lysophospholipid a phospholipid from which only one of ⁇ -position or ⁇ -position fatty acyl group in phospholipid is removed is called lysophospholipid, and acts on lysophospholipid to hydrolyze the remaining fatty acid ester bond. Is also included in the PLB because the decomposition product is the same as in the case of the PLB. The action on lysophospholipid is called lysophospholipase activity.
  • PLC phospholipase C
  • PLD phospholipase D
  • Non-patent Document High density lipoprotein cholesterol, generally referred to as good cholesterol
  • lysophosphatidylethanolamine has been reported to have a neuronal differentiation inducing action and an action to protect neuronal cells from apoptosis (apoptosis inhibitory action) as a neuronal nutrient (Non-patent Document 3, 4).
  • lysophosphatidylethanolamine was found to have a translocational activity suppression effect, a nerve activation effect, a cytoprotective effect, and a central nerve calming effect (Non-patent Document 4). From these reports, lysophosphatidylethanolamine is expected to be used for the prevention and treatment of Alzheimer's disease that occurs after ischemic brain injury and neurological disorders such as neuronal cell death.
  • lysophosphatidylinositol has an antifungal action (Patent Document 1).
  • lysophosphatidic acid is known to have a vasoconstrictive action (Non-patent Document 5), a strong cell proliferation action and a DNA synthesis promoting action (Non-Patent Document 6).
  • 1-lysophosphatidic acid has been reported to be effective in hair formation (hair growth, hair growth) (Non-patent Document 7).
  • lysophosphatidylserine has been reported to be involved in mast cell activation (allergy and atopic dermatitis) (Non-patent Document 5).
  • lysophosphatidylglycerol has been reported to have an excellent noodle modification action as a noodle modifier (Patent Documents 2 and 7).
  • lysophosphatidylglycerol has a strong surface activity and is known to have a strong foaming action. For example, strengthening O / W emulsification with strong emulsifying power, improving thermal stability of O / W emulsions, forming emulsions with excellent storage stability, excellent binding properties with proteins and starches, antibacterial effects, excellent Moisturizing properties and antioxidative effects are known.
  • Soy lysolecithin has (1) strong O / W emulsifying properties, (2) high emulsion stability in the presence of acids and in the presence of salts, and (3) excellent ability to bind to proteins and starch. It has been described that the demand has been increasing in recent years because it has such features as (4) excellent mold release action and the like (Patent Document 3).
  • Phosphatidylethanolamine is a component of phospholipids contained in soybean lecithin and egg yolk lecithin, and is known to have an emulsion stabilizing effect.
  • a lot of unsaturated fatty acids are bound to phosphatidylethanolamine, particularly in the ⁇ (sn-2) position of egg yolk lecithin, and when this unsaturated fatty acid is liberated, it becomes a source of bitterness. Therefore, in food processing, it can be said that it is desirable that hydrolysis of phosphatidylethanolamine does not occur.
  • no enzyme suitable for this purpose has been found so far.
  • no enzyme capable of controlling substrate specificity by pH has been found so far.
  • PLA1 is widely distributed in the living world, and in particular, the properties of microorganisms and snake venom-derived substances have been studied in detail, and there are sales as biochemical reagents and sales as industrial enzymes.
  • industrial PLA1 there is PLA1 (manufactured by Mitsubishi Chemical Foods) derived from Aspergillus oryzae.
  • Aspergillus aspergillus oryzae-derived PLA1 has a problem of being restricted in use because its working pH is in the acidic range.
  • metal ions such as Ca 2+ ions are required for the catalytic action, there is a problem depending on the application.
  • the kind of phospholipid which can act is not specified (patent document 5).
  • glycerol-3-phosphocholine is expected to prevent dementia (Patent Document 4).
  • Patent Document 4 no enzyme or technology for efficiently producing a glycerol-3-phosphate compound such as glycerol-3-phosphocholine has been developed.
  • the present invention has been made in view of the above circumstances, and an object thereof is to provide a novel PLA 1 and a manufacturing method thereof.
  • PLA1 having novel properties from microorganisms belonging to the genus Streptomyces. .
  • the enzyme according to the present invention preferentially hydrolyzes the acyl group at the sn-1 position of phospholipid, and is selected from lysophospholipid, glycerol-3-phosphate, and glycerol-3-phosphate ester compound.
  • polypeptides which is an enzyme that produces one or more of the following polypeptides (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 75% homology with the amino acid sequence shown in SEQ ID NO: 2 and exhibiting the hydrolysis activity.
  • a preferred form of the above enzyme is that when egg yolk phosphatidylcholine (manufactured by Nacalai) is used as a substrate and pH is 5.6 and the hydrolysis activity under the condition of 5 minutes at 37 ° C. is 100%, pH 4.1 It exhibits a hydrolysis activity of 50% or more within a pH of 10.0.
  • a preferred form of the above enzyme is that when egg yolk phosphatidylcholine (manufactured by Nacalai, purity of about 70%) is used as a substrate and the hydrolysis activity is 100% at pH 9.0 and 50 ° C. for 5 minutes.
  • egg yolk phosphatidylcholine manufactured by Nacalai, purity of about 70%
  • 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) (lower limit is 30%), 350 for 1,2-Dimyristoyl-sn-glycerol-3-phosphate (PA) % Or more (upper limit is 400%), 1,2-Diacyl-sn-glycero-3-phospho- (1-rac-glycerol) (PG) is more than 400% (upper limit is 450%), L- ⁇ - 450% or more for Phosphatidylserine (PS) (upper limit is 480%), 100% or more for 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (PE) (upper limit is 140%), L- ⁇ - Phosphatidylinositol (PI) 350% or more (upper limit is 380%), soybean phosphatidylcholine (SIGMA, L- ⁇ -Phosphatidylc Holine) has a substrate specificity of 300% or more (SIGMA
  • a preferable form of the above enzyme is that when yolk phosphatidylcholine (manufactured by Nacalai) is used as a substrate, and the hydrolysis activity under the condition of 5 minutes at 50 ° C. at pH 5.6 is 100%, it is 100% or more (upper limit is 140%), 100% or less for PA (lower limit is 60%), 150% or more for PG (upper limit is 190%), 150% or more for PS (upper limit is 190%) ), 100% or more for PE (upper limit is 120%), 250% or more for PI (upper limit is 290%), 250% or more for soybean phosphatidylcholine (manufactured by SIGMA) (upper limit is 290%), soybean Has an activity of 250% or more (upper limit is 280%) for lecithin (Wako Pure Chemical Industries), and has substrate specificity that is almost zero for triglycerides (Wako Pure Chemical, Olive) Oil). .
  • the isoelectric point calculated from the amino acid sequence is in the range of 6.0 to 6.1.
  • a preferred form of the above enzyme has a molecular weight in the range of 25,000 to 30,000 as measured by SDS-PAGE, and a molecular weight in the range of 25,000 to 30,000 as analyzed from the amino acid composition. It is.
  • the preferred form of the enzyme is derived from a microorganism belonging to the genus Streptomyces.
  • the polynucleotide according to the present invention is a polynucleotide encoding the above enzyme.
  • the preferable form of said polynucleotide contains the polynucleotide in any one of the following (a) to (c).
  • A a polynucleotide comprising the base sequence set forth in SEQ ID NO: 1;
  • B a polynucleotide that hybridizes with a base sequence complementary to the base sequence described in SEQ ID NO: 1 under stringent conditions; or
  • the preferred form of the above polynucleotide is derived from a microorganism belonging to the genus Streptomyces.
  • the vector according to the present invention is a vector containing the above-described polynucleotide.
  • the transformant according to the present invention is one or more selected from phospholipids, lysophospholipids, glycerol-3-phosphate, and glycerol-3-phosphate compounds into which the above-described polynucleotide or the above-described vector is introduced. It is a transformant possessing the ability to produce an enzyme that produces.
  • a preferred form of the transformant is a microorganism in which the host of the transformant belongs to the genus Streptomyces.
  • the method for producing an enzyme according to the present invention acts on a phospholipid and hydrolyzes the phospholipid to select one or more selected from lysophospholipid, glycerol-3-phosphate, and glycerol-3-phosphate ester compound
  • a method for producing an enzyme that produces the enzyme wherein the polynucleotide or the vector described above is introduced into a host to obtain a transformant that produces the enzyme; And a step of producing an enzyme.
  • the host is a microorganism belonging to the genus Streptomyces.
  • a novel phospholipase A1 having a high enzyme activity Furthermore, a method for efficiently producing the enzyme by a microorganism can be provided. Thereby, a novel phospholipid processing agent having high PLA1 activity and having substrate specificity for phospholipid can be obtained without the addition of a metal salt.
  • glycerol-3-phosphate compounds such as lysophospholipid, glycerol-3-phosphate, and glycerol-3-phosphocholine can be efficiently produced.
  • highly purified phosphatidylethanolamine, glycerol-3-phosphate, or glycerol-3-phosphocholine can be efficiently produced.
  • FIG. 2 is an electrophoresis photograph showing the results of SDS-PAGE analysis of a purified fraction obtained from a Streptomyces albidoflavus culture solution. It is a graph which shows the relative activity of phospholipase A1 about the purified enzyme derived from Streptomyces albidoflavus in various pH on the basis of the enzyme activity when pH is 5.6 (100%) . It is a graph which shows the relative activity of phospholipase A1 about the purified enzyme derived from Streptomyces albidoflavus in various temperature on the basis of the enzyme activity in case reaction temperature is 50 degreeC (100%). .
  • 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.
  • 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, for example, as described in Molecular Cloning A Laboratory Manual, 2nd edition (Sambrook, J. et al., Cold Spring Harbor 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.
  • PLB phospholipase A1
  • PLB1 The enzyme that hydrolyzes the fatty acid ester bond at the ⁇ -position (sn-1 position) of the glycerol group in glycerol phospholipid is called phospholipase A1 (PLA1), and the fatty acid ester group at the ⁇ -position (sn-2 position) of the glycerol group
  • A2 The enzyme that hydrolyzes.
  • A2 PLB2
  • 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 ⁇ -position and the ⁇ -position.
  • PLA1 generates at least one, preferably all three of glycerol-3-phosphate compounds such as 2-lysophospholipid, glycerol-3-phosphate, and glycerol-3-phosphocholine from phospholipids. Is an enzyme.
  • the PLA1 activity can be confirmed, for example, as follows, but the confirmation method is not limited to this. Specifically, for example, PLA1 activity can be confirmed by measuring the amount of free fatty acid produced as a result of the enzyme reaction.
  • the amount of free fatty acid contained in 5 ⁇ L of the reaction solution can be determined using, for example, “NEFA C Test Wako” (manufactured by Wako Pure Chemical Industries, Ltd.), which is a free fatty acid measurement kit, in the instructions attached to the kit. Measure as described.
  • the amount of enzyme that produces 1 ⁇ mol of free fatty acid per minute is defined as 1 unit.
  • the enzyme in the present invention is one kind selected from lysophospholipid, glycerol-3-phosphate, and glycerol-3-phosphate ester compound by preferentially hydrolyzing the acyl group at the sn-1 position of the phospholipid.
  • 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 75% homology with the amino acid sequence shown in SEQ ID NO: 2 and exhibiting the hydrolysis activity.
  • the above-mentioned enzymes include, as buffer solutions, acetic acid-sodium acetate buffer (pH 4.1-5.6), bistris-hydrochloric acid buffer (pH 5.6-7.2), Tris-hydrochloric acid buffer (pH 7. 2 to 8.8) and glycine-sodium hydroxide buffer (pH 8.8 to 10.5), the egg yolk phosphatidylcholine and the enzyme are put under reaction conditions within the pH range ( It can exhibit PLA1 activity at pH 4.1-10.5). The optimum pH is around pH 5.6, but shows substantially 100% activity at pH 5-8.
  • the above enzyme is, for example, in the range of pH 4.1 to pH 10 when the hydrolysis activity at pH 5.6 is 100% under the condition that egg yolk phosphatidylcholine and the enzyme are reacted at 37 ° C. for 5 minutes as described above. It is preferable that the activity of 50% or more is shown.
  • the above enzyme can act at about 20 to 65 ° C. under the reaction conditions of the above egg yolk phosphatidylcholine and the enzyme, for example.
  • the optimum temperature can be within this range. Preferably it is in the range of about 30-55 ° C, more preferably in the range of 40-55 ° C, and even more preferably about 50 ° C.
  • the enzyme when the enzyme is treated with 120 mM acetic acid-sodium acetate buffer (pH 5.6) for 30 minutes, it can be stable with almost no decrease in activity from 4 ° C. to 40 ° C. and 45 ° C. However, an activity of about 80% (for example, 75%) or more remains.
  • the above phospholipid and enzyme solution are placed under reaction conditions using an acetic acid-sodium acetate buffer (pH 5.6) as a buffer, the above enzyme is inhibited even in the presence of 100 mM EDTA. It is preferable that the activity is almost the same as that in the case where EDTA is not added. In the presence of 10 mM Ca 2+ and Zn 2+ , it is preferable to exhibit about 80% activity (for example, about 80 to 95% activity). On the other hand, 10 mM Fe 3+ and Fe 2+ can inhibit the activity.
  • the hydrolysis activity with respect to the case where egg yolk phosphatidylcholine is a substrate is 100%. 50% or less for DPPC (lower limit is 30%), 350% or more for PA (upper limit is 400%), 400% or more for PG (upper limit is 450%), 450% or more for PS ( The upper limit is 480%), 100% or more for PE (upper limit is 140%), 350% or more for PI (upper limit is 380%), 300% or more for soybean phosphatidylcholine (manufactured by SIGMA) (upper limit is 360) %) And 75.0% or more (upper limit is 90%) relative to soybean lecithin (manufactured by Wako Pure Chemical Industries, Ltd.).
  • the above enzyme is 100% or more (upper limit is 140%) with respect to DPPC, assuming that egg yolk phosphatidylcholine is used as a substrate and the hydrolysis activity is 100% at 50 ° C. and pH 5.6 for 5 minutes. %), 100% or less for PA (lower limit is 60%), 150% or more for PG (upper limit is 190%), 150% or more for PS (upper limit is 190%), 100 for PE % Or more (upper limit is 120%), 250% or more (upper limit is 290%) for PI, 250% or more (upper limit is 290%) for soybean phosphatidylcholine (manufactured by SIGMA), soy lecithin (manufactured by Wako Pure Chemical Industries)
  • the activity against triglyceride (olive oil) is almost 0 (for example, 5% or less, 3% or less, or 1% or less, or less than the detection limit). ) It is preferred that. It is preferable to have such substrate specificity.
  • the above enzyme may vary slightly depending on electrophoresis conditions, etc., but the molecular weight in SDS-PAGE is within the range of 25,000 to 30,000 (for example, about 28,000 or about 27,000). Is preferred.
  • the enzyme preferably has a molecular weight calculated from the amino acid composition in the range of 25,000 to 30,000.
  • a natural enzyme derived from Streptomyces albidoflavus strain NA297 has a molecular weight of about 28,000, specifically 28,000 in SDS-PAGE.
  • the natural enzyme derived from this Streptomyces albidoflavus NA297 strain has a molecular weight of 27,199 calculated from its amino acid composition.
  • the above enzyme preferably exhibits an isoelectric point in the range of 6.0 to 6.1 (for example, 6.06).
  • the isoelectric point of the enzyme can be calculated from the amino acid sequence by GENETYX.
  • phospholipase A1 consists of the amino acid sequence shown in SEQ ID NO: 2.
  • the phospholipase A1 preferably has an amino acid sequence from the 34th position to the 269th position of SEQ ID NO: 2 (also referred to herein as “the amino acid sequence described in SEQ ID NO: 2”).
  • one or more amino acids in the amino acid sequence shown in SEQ ID NO: 2 or the amino acid sequence shown in SEQ ID NO: 2 are substituted, deleted, inserted, and It may also be 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 Laboratories, Cold Spring Harbor, NY 1989; PCR: A Spring Practical Approach, IRL Press, 1991, pp. 200), etc.
  • the structure of the protein can be altered by introducing additional mutations.
  • 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.
  • amino acid mutations include not only artificially mutated enzymes but also naturally mutated enzymes as long as they have PLA1 activity, and are included in the enzyme (PLA1).
  • a protein having an amino acid sequence shown in SEQ ID NO: 2 or an amino acid sequence having homology to the amino acid sequence shown in SEQ ID NO: 2 is also included in the enzyme (PLA1) as long as it has PLA1 activity.
  • PLA1 is preferably at least 75%, preferably at least 80%, more preferably at least 85%, even more preferably at least 75% of the amino acid sequence set forth in SEQ ID NO: 2 or the amino acid sequence set forth in SEQ ID NO: 2. It may be a protein having an amino acid sequence with 90%, even more preferably at least 95%, even more preferably at least 99% homology.
  • Protein homology (homology) search for example, databases related to amino acid sequences of proteins such as SWISS-PROT, PIR, DAD, or DNA databases such as DDBJ, EMBL, Gene-Bank, etc. BLAST, FASTA, etc. This program can be used, for example, via the Internet. Confirmation of the activity of the protein can be performed using the procedure described above.
  • the supply source of PLA1 is not particularly limited, but PLA1 can be obtained from living cells such as microorganisms.
  • microorganisms include microorganisms belonging to the genus Streptomyces. Streptomyces albidoflavus (Streptomyces albidoflavus) and Streptomyces albidoflavus (Streptomyces albidoflavus) NA297 (Accession number: NITE BP-1014, hereinafter referred to as “NAITE BP-1014 strain” Stock ").
  • Streptomyces albidoflavus NA297 (accession number: NITE BP-1014) is liquid-cultured in an appropriate nutrient medium to secrete the enzyme outside the cells, so that the culture supernatant is frozen.
  • a product treated with drying, salting out, an organic solvent or the like can be produced as a PLA1 enzyme preparation.
  • Microorganisms that can be used for the production of PLA1 enzyme preparations are not limited to Streptomyces albidoflavus NA297, and may be microorganisms that belong to the genus Streptomyces and that can produce PLA1.
  • natural or artificial mutants of these species or gene fragments necessary for the expression of PLA1 activity can be artificially extracted and used for the production of PLA1 even in other species incorporating them. .
  • even if it does not belong to Streptomyces genus if it is a microorganism which can produce said PLA1, it can also be used.
  • PLA1 enzyme preparation using Streptomyces albidoflavus NA297 will be described as an example.
  • 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.
  • a suitable medium for example, a medium containing a suitable carbon source, nitrogen source, and inorganic salts to secrete the enzyme.
  • 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).
  • the nitrogen source examples 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.
  • 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 1 to 20% (w / v), preferably 1 to 10% (w / v).
  • the concentration of the nitrogen source is, for example, in the range of 1 to 20% (w / v), preferably 1 to 10% (w / v).
  • 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 37 ° C., for example.
  • the culture time is preferably a time during which the enzyme is sufficiently produced, for example, about 1 to 7 days. Culturing can be preferably performed under aerobic conditions, for example, with aeration stirring or shaking.
  • PLA1 is fractionated by protein solubility (precipitation with organic solvents, salting out with ammonium sulfate, etc.); cation exchange, anion exchange, gel filtration, hydrophobic chromatography; affinity chromatography using chelates, dyes, antibodies, etc. It can be purified by appropriately combining known methods such as graphy. 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, it can be purified to almost a single band in polyacrylamide gel electrophoresis (SDS-PAGE). That is, the enzyme (PLA1) can be estimated as a monomer by HPLC analysis and gel filtration chromatography analysis.
  • SDS-PAGE polyacrylamide gel electrophoresis
  • the polynucleotide in the present invention encodes the above PLA1.
  • This polynucleotide preferably includes the polynucleotide described in any of (a) to (c) below.
  • 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 above-mentioned polynucleotide encoding PLA1 has, for example, the base sequence from position 1 to position 816 of SEQ ID NO: 1 (also referred to herein as “base sequence described in SEQ ID NO: 1”).
  • the base sequence described in SEQ ID NO: 1 encodes a protein including the amino acid sequence described in SEQ ID NO: 2, and the protein including this amino acid sequence constitutes a preferred form of PLA1.
  • the polynucleotide encoding PLA1 includes an amino acid in which one or more amino acids are substituted, deleted, inserted, and / or added to the amino acid sequence shown in SEQ ID NO: 2 as described above, and has PLA1 activity. Also included are polynucleotides that encode the proteins they have. 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.
  • polynucleotide encoding PLA1 is also a protein capable of hybridizing under stringent conditions with a polynucleotide having a base sequence complementary to the polynucleotide having the base sequence shown in SEQ ID NO: 1 and having PLA1 activity Also included are polynucleotides encoding.
  • 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 albidoflavus NA297. PCR or hybridization screening can be used for obtaining the gene.
  • 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 PLA1 derived from 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 conditions stringent to DNA prepared from other organisms, PLA1 derived from various organisms can be obtained. The encoding polynucleotide can be isolated.
  • PCR primers are designed from regions with high homology using sequence information registered in DNA databases such as DNA Databank of Japan (DDBJ), EMBL, and Gene-Bank. You can also By using such a primer and performing PCR using chromosomal DNA or cDNA as a template, the polynucleotide encoding PLA1 can also be isolated from various organisms. Similarly, the polynucleotide encoding PLA1 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.
  • Select one or more probes and design the probe for example, using the conditions described in the manual (for example, cleaning conditions: 42 ° C., 0.5 ⁇ SSC) using ECL directic acid labeling and detection system (manufactured by GE Healthcare). It includes a polynucleotide that hybridizes in a primary wash buffer.
  • the “stringent conditions” are usually conditions of 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.
  • the above-mentioned polynucleotide encoding PLA1 is at least 75%, preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably the amino acid sequence set forth in SEQ ID NO: 2.
  • the protein homology search is as described above.
  • the polynucleotide encoding PLA1 is at least 75%, preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably the nucleotide sequence set forth in SEQ ID NO: 1. Also included are polynucleotides that have a base sequence with at least 95%, even more preferably at least 99% sequence identity, and encode a protein having PLA1 activity. The determination and search of the sequence identity of the base sequence is also as described above.
  • the above-mentioned polynucleotide encoding PLA1 can be expressed in the same or different host using genetic recombination technology.
  • the vector in the present invention contains the above-mentioned polynucleotide.
  • any of glycerol-3-phosphate ester compounds such as lysophospholipid, glycerol-3-phosphate, and glycerol-3-phosphocholine acts on phospholipids by introducing a polynucleotide or a vector into a host.
  • a transformant possessing the ability to produce an enzyme that produces one or more can be made.
  • 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: A Laboratory Manual 2nd edition, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1989). In particular, for actinomycetes, it can be performed with reference to “PRACTICAL STREPTOMYCES GENETICS (Kieser et al., John Inns Foundation, 2000)”.
  • this DNA is first introduced into a plasmid vector or a phage vector that is 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.
  • promoters and terminators that can be used in these various microorganisms, “Microbiology Basic Course 8 Genetic Engineering, Kyoritsu Shuppan”, especially for actinomycetes, “PRACTICAL STREPTOMYCES GENETICS (Kieser et al., John s Foundation, 2000) ) "And the like, and the method can be used. 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 PLA1 or the other.
  • the host to be transformed is not particularly limited as long as it is a living organism that can be transformed with a vector containing a polynucleotide encoding PLA1 and express PLA1 activity.
  • Escherichia coli, Bacillus subtilis, bacteria, actinomycetes, yeast, mold and the like can be mentioned.
  • insects such as moths (Nature 315, 592-594 (1985)), rapeseed, corn, potatoes and other plants.
  • Systems for expressing large amounts of heterologous proteins have been developed and may be used.
  • the obtained transformant can be used for production of an enzyme preparation (PLA1) as described above. Specifically, the transformant is subjected to liquid culture in an appropriate nutrient medium, the expressed PLA1 is secreted outside the cell, and the culture supernatant is treated with lyophilization, salting out, an organic solvent, etc. Can be manufactured.
  • PHA1 enzyme preparation
  • the culture conditions may vary, but the culture can be performed under conditions commonly used by those skilled in the art.
  • an actinomycete such as Streptomyces
  • a tryptic soy medium containing thiostrepton for example, manufactured by Becton Dickinson
  • the enzyme produced by the transformant can be further purified as described above.
  • PLA1 has a low catalytic activity in the absence of metal ions such as calcium ions. Therefore, in food processing, it is necessary to add a metal salt to a food material and perform enzyme treatment. In contrast, PLA1 of the present invention does not require the addition of a metal salt for the enzyme reaction. Therefore, the food material can be processed as it is, and the range of use can be expanded, and it can be expected to prevent the deterioration of food quality due to the metal salt and improve the safety. Furthermore, when the enzyme treatment is performed using a reactor, it is not necessary to add a metal salt, so that scales do not adhere to the reactor, maintenance is easy, and running costs are kept low.
  • PLA1 of the present invention has various phospholipids including PE.
  • PE phosphatidylethanolamine
  • PLA1 of the present invention has various phospholipids including PE.
  • PI phosphatidylinositol
  • PG phosphatidylglycerol
  • PS phosphatidylserine
  • PI, PG, PS, PE and PC pH 5.6
  • PI, PG, PS and PA pH 9.0.
  • the acting substrate can be controlled.
  • phosphatidylcholine for example, 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine
  • high purity phosphatidylcholine for example, 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine
  • phosphatidylethanolamine for example, 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine
  • phosphatidic acid for example, It is possible to keep the enzyme action against 1,2-Dimyristoyl-sn-glycerol-3-phosphate
  • PLA1 of the present invention exhibits substantially 100% activity in the pH range of 5-8. Furthermore, since it exhibits an activity of 50% or more with respect to the maximum activity in the range of pH 4.1 to pH 10, the usable pH range is extremely wide compared to the conventional PLA1, and it can be used under weakly acidic to alkaline conditions. Therefore, reaction control is easy and a wide range of applications can be expected.
  • PLA1 of the present invention exhibits an activity of 50% or more in a range exceeding the range of 40 to 55 ° C. Furthermore, the conventional enzyme (PLA1 manufactured by Mitsubishi Chemical Foods) has an activity of 50% or less at 30 ° C. or lower, whereas PLA1 of the present invention exhibits an activity of 50% or higher at 20 ° C. Low temperature processing is possible. Therefore, the usable temperature range is wide as compared with the conventional PLA1.
  • the conventional PLA1 (PLA1 manufactured by Mitsubishi Chemical Foods) has a stable temperature of 55 ° C., whereas the stable temperature of the PLA1 of the present invention is 40 to 45 ° C. Therefore, it is possible to deactivate the enzyme at a lower temperature.
  • PLA1 can decompose the cell membrane. PLA1 then lyses the cells. Therefore, it can be used for cell degrading agents or cell lysing agents including detergents such as egg yolk stains and blood stains.
  • lysophospholipids are extremely excellent in emulsifying properties, they become emulsifiers or surfactants that can be used in various fields.
  • lysophospholipids have been discovered as various physiological functions in recent years, and thus can be used as pharmaceuticals and supplements. Therefore, various highly functional lysophospholipids can be produced using PLA1 of the present invention.
  • PLA1 of the present invention By adding PLA1 of the present invention to flour such as bread dough or noodle dough, the phospholipid originally contained in the flour is converted to lysolecithin. By forming lysolecithin in the dough, it binds to proteins and starch to help form a gluten network, improve the physical properties of bread and noodles, and prevent aging.
  • Cyclic phosphatidic acid having excellent properties and effects as a cosmetic material can be produced from lysophospholipid. Therefore, cyclic phosphatidic acid can be efficiently produced by using PLA1 of the present invention.
  • Glycerol-3-phosphocholine is expected to prevent dementia.
  • Other glycerol-3-phosphate compounds can be expected to have high functions. Therefore, the PLA1 of the present invention can produce a highly functional glycerol-3-phosphate ester.
  • Glycerol-3-phosphate is a glycolytic metabolic intermediate. Therefore, PLA1 of the present invention can produce glycerol-3-phosphate having the possibility of enhancing metabolism.
  • PLA1 activity standard measurement method This method illustrates the case where egg yolk phosphatidylcholine is used as a substrate.
  • Example 1 Purification of Streptomyces albidoflavus enzyme
  • A Cultivation 500 mL of tryptic soy medium (Becton Dinkinson) is prepared, and 50 ml each is dispensed into a 500 mL baffle Erlenmeyer flask, and further 1% soybean lecithin and 0.1% Tween (Tween) After adding 80, steam sterilization was performed at 121 ° C. for 15 minutes.
  • FIG. 1 is an electrophoresis photograph showing the results of analysis by SDS-PAGE of the eluted fraction.
  • Lane 1 (left side of the figure) is a molecular weight marker, and lane 2 (right side of the figure) shows a band of the eluted fraction. As shown in FIG. 1, a single band was observed in the elution fraction.
  • This enzyme was estimated to be a monomer by HPLC analysis and gel filtration chromatography analysis.
  • Example 2 Measurement of properties of enzyme derived from Streptomyces albidoflavus] (PLA1 activity)
  • the purified enzyme obtained in Example 1 and 1-palmitoyl-2-oleoylphosphatidinecholine as a substrate were subjected to an enzymatic reaction under the reaction conditions of “PLA1 activity standard measurement method”.
  • reaction solution was extracted with chloroform / methanol (2/1, v / v).
  • the extract was analyzed by gas chromatography and the fatty acid produced by hydrolysis was quantitatively analyzed to confirm that it had PLA1 activity.
  • FIG. 2 is a graph showing enzyme activities at various reaction pHs as relative activities based on the enzyme activity when the reaction pH is 5.6 (100%). As can be seen from the graph of FIG. 2, this enzyme showed an activity with a relative activity of 50% or more with respect to the maximum activity (100%) in a wide range from pH 4.1 to pH 10.0.
  • 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 50 ° C. As shown in the graph of FIG. 3, this enzyme exhibited activity at 20 to 60 ° C., and the optimum temperature for the reaction was around 50 ° C. (eg, 45 to 55 ° C.).
  • FIG. 4 is a graph showing the residual activity of the enzyme after treatment at various temperatures. As shown in the graph of FIG. 4, after the treatment at a temperature of 4 ° C. to 40 ° C., 90% or more of the enzyme remained before the treatment. After the treatment at 45 ° C., about 80% (about 75 to 80%) of the activity before the treatment remained.
  • FIG. 5 is a graph showing the residual activity of the enzyme after treatment at various pHs. As shown in the graph of FIG. 5, this enzyme remained active from pH 3.99 to 10.5.
  • Table 1 shows the results of relative activities with various additives added, assuming that the condition in which no chemical substance is added is 100%.
  • the enzyme obtained in Example 1 is not inhibited by 50 mM EDTA and exhibits the same activity as when not added, and is hardly affected by 100 mM EDTA. Moreover, it was not inhibited by 2 mM dithiothreitol. Further, 10 mM Ca 2+, in the Zn 2+ presence, showed about 80% active. On the other hand, it was confirmed that the activity could be inhibited by 10 mM Fe 3+ , Fe 2+ , 2 mM 2-mercaptoethanol, iodoacetamide, PMSF (phenylmethylsulfonyl fluoride), and SDS.
  • Table 2 shows the results of relative activity when egg yolk phosphatidylcholine is used as a substrate and the hydrolysis activity at 100 ° C. for 5 minutes at 50 ° C. with pH 9.0 and final concentration of 1.0% Triton X-100. It is.
  • Table 3 shows the relative activity when egg yolk phosphatidylcholine is used as a substrate and the hydrolysis activity at pH 5.6, final concentration 1.0% Triton X-100 at 50 ° C. for 5 minutes is defined as 100%. It is a result.
  • PLA1 activity was measured in a reaction solution containing a final concentration of 0.005% Triton X-100 according to the above "PLA1 activity standard measurement method" except that various phospholipids were used as a substrate instead of egg yolk phosphatidylcholine. did.
  • Table 4 shows the results of relative activity when egg yolk phosphatidylcholine is used as a substrate, and the hydrolysis activity at pH 8.0, final concentration 0.23% Triton X-100 at 37 ° C. for 5 minutes is defined as 100%. is there.
  • Example 1 the enzyme obtained in Example 1 exhibited substrate specificity as shown in Tables 2 to 4.
  • Example 3 Analysis of N-terminal amino acid sequence of enzyme derived from Streptomyces albidoflavus
  • the N-terminal amino acid sequence was analyzed with a protein sequencer.
  • the internal amino acid sequence was analyzed by nanoLC-MS / MS. 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.
  • the internal amino acid sequences were confirmed to be those shown in SEQ ID NOs: 4 and 5.
  • SEQ ID NO: 3 is a sequence shown from position 34 of SEQ ID NO: 2 (positions 1 to 33 are secretory signal sequences that have been removed by the mature (purified) enzyme).
  • SEQ ID NO: 4 is the sequence shown from position 145 of SEQ ID NO: 2.
  • SEQ ID NO: 5 is the sequence shown from position 165 of SEQ ID NO: 2.
  • Streptomyces albidoflavus NA297 was added to YEME medium (0.3% yeast extract, 0.5% peptone, 0.3% malt extract, 1% glucose, 34% sucrose, 5 mM MgCl 2 , 0. 5 ml of 5% glycine) was cultured at 28 ° C. for 4 days and collected.
  • 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.
  • 750 ⁇ l of 10% (w / v) SDS and 5 mg of proteinase K were added and treated at 55 ° C. for 2 hours.
  • 7.5 ml of chloroform was added and stirred, and 5 ml of the aqueous phase was collected by centrifugation.
  • the DNA fraction was collected by adding 3 ml of isopropanol to this aqueous phase, and dissolved in 500 ⁇ l of a solution consisting of 10 mM Tris-HCl buffer (pH 8.0) and 1 mM EDTA.
  • RNase A was added to 20 ⁇ g / ml, treated at 37 ° C. for 1 hour, 500 ⁇ l of 13% PEG solution containing 0.8 M NaCl was added and stirred, and 500 ⁇ l of the aqueous phase was collected by centrifugation. did. 500 ⁇ l of a phenol / chloroform mixed solution was added thereto and stirred, and 500 ⁇ l of an aqueous phase was collected by centrifugation.
  • This DNA was immersed in 70% (v / v) ethanol for 10 minutes, and then dissolved in 200 ⁇ l of a solution composed of 10 mM Tris-HCl buffer (pH 8.0) and 1 mM EDTA.
  • Example 5 Cloning of the core region of the PLA1 gene derived from Streptomyces albidoflavus NA297] Based on the N-terminal and internal amino acid sequences of PLA1 and the codons used in the genus Streptomyces, degenerate oligonucleotide primer for PCR S1 sense primer “primer S1” (SEQ ID NO: 6), and three types of A1 antisense primers “Primer A1-1” (SEQ ID NO: 7) and “primer A1-2” (SEQ ID NO: 8) were designed.
  • s in the sequence represents c or g
  • w represents a or t
  • k represents g or t.
  • the PCR reaction solution composition is as follows.
  • Distilled water was added to 50 ⁇ l of template chromosomal DNA 50 ng, 2 ⁇ MightyAmp Buffer 25 ⁇ L obtained in Example 4, 300 nM of each primer, and MightyAmp DNA Polymerase 1.25 unit.
  • PCR reaction conditions are as follows.
  • Step 1 98 ° C., 2 minutes; Step 2: 98 ° C., 10 seconds; Step 3: 65 ° C., 15 seconds; Step 4: 68 ° C., 1 minute; Repeat steps 2 to 4 for 30 cycles; Step 4: 68 ° C., 1 minute.
  • a specific amplification product of about 300 bp was obtained by PCR using SEQ ID NOs: 6-8.
  • the PCR reaction solution was subjected to agarose gel electrophoresis, the target 300 bp band portion was cut out, and bound to pMD20-T Vector using pMD20-T Vector (manufactured by TAKARA) to transform Escherichia coli.
  • the transformed strain is cultured in LB medium (tryptone 1%, yeast extract 0.5%, sodium chloride 0.4%, pH 7.2) containing ampicillin 50 ⁇ g / ml, and using Miniprep DNA Purification Kit (TaKaRa). Plasmid for DNA sequencing was extracted and purified.
  • the base sequence of the inserted fragment was determined by an automatic sequencer using M13 primerM4 derived from a vector (pMD20-T Vector). This base sequence is shown in SEQ ID NO: 9.
  • Example 6 Cloning around the core region of the PLA1 gene derived from Streptomyces albidoflavus NA297
  • a DNA fragment including the upstream side and the downstream side was amplified by inverse PCR.
  • the chromosomal DNA obtained in Example 4 was completely digested with SacII, and Ligation high Ver. 2 (manufactured by Toyobo Co., Ltd.). Using this as a template, Inverse PCR using sense primer SE1 “primer SE1” (SEQ ID NO: 10) and antisense primer AN1 “primer AN1” (SEQ ID NO: 11) prepared based on the partial gene sequence of phospholipase A1 Went.
  • the PCR reaction solution composition is as follows.
  • Distilled water was added to 200 ng of template DNA, 25 ⁇ l of 2 ⁇ MightyAmp Buffer, 300 nM of each primer, and 1.25 units of MightyAmp DNA Polymerase so that the total amount was 50 ⁇ l.
  • PCR reaction conditions are as follows.
  • Step 1 98 ° C., 2 minutes
  • Step 2 98 ° C., 10 seconds
  • Step 3 68 ° C, 4 minutes
  • Step 4 68 ° C., 7 minutes.
  • the base sequence on the 5 'region side of the region containing the PLA1 gene derived from Streptomyces albidoflavus was determined. Further, the amino acid sequence of the structural gene portion was deduced from the base sequence (SEQ ID NO: 12).
  • Example 7 Cloning around the core region of PLA1 gene derived from Streptomyces albidoflavus NA297] Since the digestion site of SacII used for inverse PCR was contained in the PLA1 gene, the 3 ′ region side (C-terminal side) was determined only halfway.
  • the amino acid sequence that showed 100% homology by BLAST (Lipase # GDSL (DDBJ estimated by genome analysis of Streptomyces albus J1074)
  • the nucleotide sequence is specified from the accession number D6BAL1)
  • the primer A2 (SEQ ID NO: 13) on the 3 ′ region side (C-terminal side) is designed, and the DNA fragment is amplified by PCR with the S1 primer (SEQ ID NO: 6). did.
  • the PCR reaction solution composition is as follows.
  • Distilled water was added to 50 ⁇ l of template chromosomal DNA 50 ng, 2 ⁇ MightyAmp Buffer 25 ⁇ L obtained in Example 4, 300 nM of each primer, and MightyAmp DNA Polymerase 1.25 unit.
  • PCR reaction conditions are as follows.
  • Step 1 98 ° C., 2 minutes
  • Step 2 98 ° C., 10 seconds
  • Step 3 68 ° C, 1 minute
  • Step 4 68 ° C., 5 minutes.
  • This PCR yielded a specific amplification product of about 700 bp, which was cloned with the pMD20-T vector (TaKaRa) and the base sequence was determined.
  • the base sequence of the region containing the PLA1 gene derived from Streptomyces albidoflavus NA297 was determined (FIG. 6A and FIG. 6). 6B (hereinafter collectively referred to as FIG. 6)). Further, the amino acid sequence of the structural gene portion was deduced from the base sequence (FIG. 6, SEQ ID NOs: 14 and 15).
  • FIG. 6 shows the base sequence of the region containing the determined PLA1 gene and the deduced amino acid sequence of the structural gene deduced from this base sequence.
  • the base sequence is shown in the upper row, and the deduced amino acid sequence (SEQ ID NO: 15 in the lower row). ).
  • the sequence in FIG. 6 is DDBJ ACCESSION No. It is registered privately as AB605634.
  • PLA1 is composed of 807 bp nucleotides and encodes 269 amino acids.
  • 236 residues are positions 34 to 269 of the amino acid sequence of SEQ ID NO: 14 (FIG. 6).
  • Positions 1-33 of the amino acid sequence of SEQ ID NO: 14 (FIG. 6) are a secretory signal sequence.
  • N-terminal and internal amino acid sequences of the purified enzyme derived from Streptomyces albidoflavus NA297 determined in Example 3 were present in the above deduced amino acid sequence and were completely in agreement (in FIG. 6). Is underlined).
  • amino acid sequence of SEQ ID NO: 3 is a sequence shown from position 34 of the amino acid sequence of SEQ ID NO: 14 (FIG. 6).
  • the amino acid sequence of SEQ ID NO: 4 is the sequence shown from position 145 of the amino acid sequence of SEQ ID NO: 14 (FIG. 6).
  • the amino acid sequence of SEQ ID NO: 5 is the sequence shown from position 165 of the amino acid sequence of SEQ ID NO: 14 (FIG. 6).
  • sequence similarity search programs BLAST and FASTA By using the sequence similarity search programs BLAST and FASTA, the above-mentioned deduced amino acid sequences were compared with sequences in four kinds of protein sequence databases (PTR, PRF, UNI-PROT and SWISS-PROT). As a result, the deduced amino acid sequence was 100% identical with Lipase # GDSL (DDBJ accession number D6BAL1) estimated by Streptomyces albus J1074 genome analysis. However, the second and third residues at the N-terminal of the signal sequence were different.
  • the purified enzyme derived from Streptomyces albidoflavus (ie, PLA1) was calculated based on the amino acid composition of this deduced amino acid sequence, and was estimated to have a molecular weight of 27,199.
  • Example 8 Preparation of recombinant plasmid containing PLA1 gene derived from Streptomyces albidoflavus
  • PLA1 derived from Streptomyces albidoflavus
  • the restriction enzyme sites that can be used for recombination were NheI and BglII.
  • the BglII site was included in the structural gene of PLA1 derived from Streptomyces albidoflavus, this BglII recognition sequence was replaced by the following method. did.
  • it includes a sense primer C1 “primer C1” (SEQ ID NO: 16) in which an NheI site is added to the upstream region of the structural gene of PLA1 derived from Streptomyces albidoflavus, and a BglII site in this structural gene.
  • Antisense primer C2 “primer C2” was designed from the site.
  • the antisense primer C2 “primer C2” was designed so that the BglII recognition sequence was eliminated and the designated amino acid was not changed. Subsequently, PCR was performed using these primers with the chromosomal DNA obtained in Example 4 as a template.
  • the PCR reaction solution composition is as follows.
  • PCR reaction conditions are as follows.
  • Step 1 98 ° C., 2 minutes; Step 2: 98 ° C., 15 seconds; Step 3: 72 ° C., 2 seconds; Step 4: 74 ° C., 25 seconds; Repeat steps 2 to 4 for 30 cycles; Step 5: 74 ° C., 10 seconds.
  • the PCR reaction solution composition is as follows.
  • PCR reaction conditions are as follows.
  • Step 1 98 ° C., 2 minutes; Step 2: 98 ° C., 15 seconds; Step 3: 72 ° C., 2 seconds; Step 4: 74 ° C., 25 seconds; Repeat steps 2 to 4 for 30 cycles; Step 5: 74 ° C., 10 seconds.
  • PCR a specific amplification product of about 100 bp was obtained.
  • a sense primer C1 “primer C1” (SEQ ID NO: 16) in which an NheI site is added to the upstream sequence of the PLA1 structural gene, and an antisense primer C4 in which a BglII site is added to the downstream sequence of the PLA1 structural gene
  • PCR was performed using the amplification products of about 600 bp and about 100 bp obtained by the above PCR as templates.
  • the PCR reaction solution composition is as follows.
  • Template 600 bp DNA 300 ng, Template 100 bp DNA 340 ng, 10 ⁇ PCR Buffer 2.5 ⁇ l, Primer 1200 nM, dNTP mixture 0.3 mM, MgCl 2 1.2 mM, DMSO 4%, and KOD DNA Polymerase 1.25 units, total amount of distilled water It added so that it might become 25 microliters.
  • PCR reaction conditions are as follows.
  • Step 1 98 ° C., 2 minutes
  • Step 2 98 ° C., 15 seconds
  • Step 3 72 ° C., 2 seconds
  • Step 4 74 ° C., 25 seconds
  • Step 5 74 ° C., 10 seconds.
  • a specific amplification product of about 700 bp was obtained by this PCR.
  • the amplified fragment was digested with NheI and BglII and inserted into the NheI-BglII site of the actinomycete plasmid as an expression vector to obtain a recombinant plasmid.
  • the signal sequence and terminator are those of the phospholipase enzyme gene, but those derived from other genes may be used in appropriate combination.
  • Example 9 Production of recombinant actinomycetes expressing PLA1 gene derived from Streptomyces albidoflavus NA297
  • Example 10 Measurement of enzyme activity of recombinant actinomycetes expressing PLA1 gene derived from Streptomyces albidoflavus NA297
  • the recombinant actinomycetes obtained in Example 9 were cultured in four 100 mL tryptic soy media (Becton Dickinson) containing 12 ⁇ g / mL thiostrepton. The supernatant was recovered from the obtained culture solution (340 mL) by centrifugation (15000 rpm, 5 minutes, 4 ° C.), and the precipitate was recovered by ammonium sulfate fractionation. The collected precipitate was dissolved in 20 mM Tris-HCl buffer (pH 8.0), and dialyzed using 20 mM Tris-HCl buffer (pH 8.0) as an external solution to obtain an enzyme solution.
  • the enzyme activity of this enzyme solution was measured at pH 5.6 and 50 ° C. using egg yolk phosphatidylcholine as a substrate according to the enzyme activity measurement method described in the above “PLA1 activity standard measurement method”. As a result, a culture supernatant showing strong activity exceeding 100 U / ml (lower limit is 60 U / ml) was obtained. Moreover, the enzyme activity (80 mL) after ammonium sulfate fractionation showed a stronger activity (244 U / ml or more, the lower limit was 200 U / ml). In addition, PLA1 activity was not detected in actinomycetes transformed using the plasmid as a vector as it was.
  • a novel PLA 1 and a manufacturing method thereof are provided.
  • Glycerol-3-phosphate compounds such as lysophospholipids, glycerol-3-phosphate, and glycerol-3-phosphocholine produced by causing PLA1 to act on phospholipids are useful as base materials for foods and cosmetics. is there.
  • the enzyme according to the present invention works particularly well on PI, PG, PS, PE, PC at pH 5.6 and on PI, PG, PS, PA at pH 9.0.
  • Lysophosphatidylinositol is an anti-fungal agent
  • lysophosphoglycerol LPG
  • LPG lysophosphoglycerol
  • LPE lysophosphatidylethanolamine
  • LPA lysophosphatidic acid
  • LPS lysophosphatidylserine
  • glycerol-3-phosphocholine which can be produced using the enzyme according to the present invention, is expected to prevent dementia and the like.
  • Glycerol-3-phosphoinositol can be expected to be useful because it has an effect of increasing good cholesterol.
  • NITE BP-1014 National Institute for Product Evaluation Technology Patent Microorganism Depositary
  • Sequence number 1 PLA1 (polypeptide) SEQ ID NO: 2: PLA1 expression gene SEQ ID NO: 3: N-terminal sequence of PLA1 SEQ ID NO: 4: Internal sequence of PLA1 SEQ ID NO: 5: Internal sequence of PLA1 SEQ ID NO: 6: Primer S1 SEQ ID NO: 7: Primer A1-1 SEQ ID NO: 8: Primer A1-2 SEQ ID NO: 9: Internal sequence of PLA1 expression gene SEQ ID NO: 10: Primer SE1 Sequence number 11: Primer AN1 Sequence number 12: PLA1 expression gene Sequence number 13: Primer A2 SEQ ID NO: 14: PLA1 expression gene SEQ ID NO: 15: PLA1 (polypeptide) Sequence number 16: Primer C1 Sequence number 17: Primer C2 SEQ ID NO: 18: Primer C3 SEQ ID NO: 19: Primer C4

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Abstract

L'invention concerne une nouvelle PLA1 et son procédé de production. Cette enzyme est obtenue par l'hydrolyse en priorité du groupe acyle à la position sn-1 d'un phospholipide pour générer un ou plusieurs des composés suivants : un lysophospholipide, le glycérol-3-phosphate et un composé ester glycérol-3-phosphorique. L'enzyme contient l'un quelconque des polypeptides décrits dans (a) à (c) ci-dessous. (a) un polypeptide ayant une séquence d'acides aminés décrite par SEQ ID NO:2 ; (b) un polypeptide ayant une séquence d'acides aminés obtenue par la substitution, l'insertion, la délétion et/ou l'addition d'un ou plusieurs acides aminés dans la séquence d'acides aminés représentée par SEQ ID NO:2, et qui présente une activité d'hydrolyse de l'ADN ; (c) un polypeptide ayant au moins 75% d'homologie avec la séquence d'acides aminés représentée par SEQ ID NO:2 et qui présente une activité d'hydrolyse de l'ADN. Ainsi, il est possible de fournir une nouvelle phospholipase B ayant une activité enzymatique élevée. La PLB a une spécificité de substrat pour les phospholipides.
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