WO2012105565A1 - Enzyme et son procédé de production - Google Patents

Enzyme et son procédé de production Download PDF

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WO2012105565A1
WO2012105565A1 PCT/JP2012/052159 JP2012052159W WO2012105565A1 WO 2012105565 A1 WO2012105565 A1 WO 2012105565A1 JP 2012052159 W JP2012052159 W JP 2012052159W WO 2012105565 A1 WO2012105565 A1 WO 2012105565A1
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enzyme
glycerol
seq
glycero
pla1
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PCT/JP2012/052159
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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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    • 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 phospholipid (glycerol phospholipid) to produce lysophospholipid (lyso form).
  • C Known as 3.1.1.32.
  • Phospholipase is a general term for enzymes that hydrolyze phospholipids.
  • Phospholipids have a fatty acid ester-bonded to one ⁇ (sn-1) and ⁇ (sn-2) hydroxyl groups of glycerol, and the other ⁇ -position hydroxyl group via a phosphate group is choline. , Ethanolamine, inositol, and the like.
  • 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 Documents 3 and 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 Document 2).
  • 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 biological world, and in particular, those derived from microorganisms and snake venoms 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 applications.
  • 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.
  • JP-A-6-256366 JP 2008-11745 A JP-A-10-287686 International Publication No. 2007/010892 JP-A-6-62850 Japanese Patent Laid-Open No. 2004-261022
  • 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. I found it.
  • the enzyme which concerns on this invention contains the polypeptide in 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 set forth in SEQ ID NO: 2.
  • C a polypeptide having at least 75% homology with the amino acid sequence set forth in SEQ ID NO: 2.
  • the enzyme according to the present invention has the following physicochemical properties (1) and (2).
  • a preferable form of the above enzyme is that when egg yolk lecithin (L) (manufactured by Nacalai Tesque) is used as a substrate and the hydrolysis activity is 100% at 37 ° C. for 5 minutes at pH 5.6. A hydrolysis activity of 50% or more is exhibited within the range of 1 to pH 10.0.
  • L egg yolk lecithin
  • a preferred form of the above enzyme is when egg yolk lecithin (L) (manufactured by Nacalai Tesque) is used as a substrate, and the hydrolysis activity under the condition of pH 9.0 and 50 ° C. for 5 minutes is defined as 100%.
  • L egg yolk lecithin
  • a preferred form of the above enzyme is when egg yolk lecithin (L) (manufactured by Nacalai Tesque) is used as a substrate and the hydrolysis activity under the condition of 5 minutes at 50 ° C. at pH 5.6 is 100%. 100% or more (upper limit is 140%) based on 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 100% or less (lower limit is 60%) with respect to 1,2-dimyristoyl-sn-glycerol-3-phosphate (DMPA), 150 against 1,2-diacyl-sn-glycero-3-phospho- (1-rac-glycerol) (1,2-Diacyl-sn-glycero-3-phospho- (1-rac-glycerol): PG) % Or more (upper limit is 190%) 150% or more with respect to L- ⁇ -phosphatidylserine (PS) (upper limit is 190%), 100% or more based on 1,2-dioleoyl-s
  • the isoelectric point calculated from the mature amino acid sequence excluding the secretory signal sequence is in the range of 5.7 to 5.8.
  • 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.
  • C a polynucleotide having at least 75% sequence identity to the base sequence set forth in SEQ ID NO: 1.
  • 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 step of obtaining a transformant producing the enzyme by introducing the polynucleotide or the vector described above into a host; and culturing the transformant A step of producing the enzyme.
  • the host is a microorganism belonging to the genus Streptomyces.
  • a novel phospholipase A1 having a high enzyme activity Furthermore, it is possible to provide a method for efficiently producing this enzyme by a microorganism.
  • 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.
  • 2 is an electrophoresis photograph showing the results of SDS-PAGE analysis of a purified fraction obtained from a culture solution of Streptomyces albidoflavus. 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%). .
  • 3 is a graph showing the change over time in the production concentration of lysophosphatidylcholine (a sum of both 1-acyl-sn-glycero-3-phosphocholine and 2-acyl-sn-glycero-3-phosphocholine).
  • 2 is a graph showing changes over time in the production concentrations of 1-acyl-sn-glycero-3-phosphocholine and 2-acyl-sn-glycero-3-phosphocholine.
  • 6 is a graph showing the change over time in the molar ratio of 1-acyl-sn-glycero-3-phosphocholine and 2-acyl-sn-glycero-3-phosphocholine.
  • It is a ribbon model which shows the three-dimensional structure of phospholipase A1 derived from Streptomyces albidoflavus. It is an enlarged view of the part (active center) enclosed with the broken line of FIG. 12A.
  • 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.
  • the microorganism refers to any of wild strains, mutant strains (for example, those induced by ultraviolet irradiation), recombinants induced by genetic engineering techniques such as cell fusion or genetic recombination methods, and the like.
  • a stock of 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 the phospholipid (glycerol phospholipid) is called phospholipase A1 (PLA1), and the ⁇ -position (sn-2 position) of the glycerol group
  • A2 The enzyme that hydrolyzes the fatty acid ester group is referred to as A2 (PLA2).
  • PLB 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, as described in the instructions attached to the kit. Measure as follows. The amount of enzyme that produces 1 ⁇ mol of free fatty acid per minute is defined as 1 unit.
  • the enzyme according to the present invention is selected from lysophospholipid, glycerol-3-phosphate, and glycerol-3-phosphate ester compound by preferentially hydrolyzing the sn-1 position acyl group of phospholipid.
  • An enzyme that produces more than a species comprising a polypeptide according to any one 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 set forth in SEQ ID NO: 2.
  • C a polypeptide having at least 75% homology with the amino acid sequence set forth in SEQ ID NO: 2.
  • the enzyme according to the present invention has the following physicochemical properties (1) and (2).
  • 1-Palmitoyl-2-oleoyl-sn-glycero-3-phospho-rac- (1-glycerol) : POPG has a relative activity of 100% or more (upper limit is 300%).
  • SB-PC soybean-derived L- ⁇ -phosphatidylcholine
  • LPC L- ⁇ -Lysophosphatidylcholine
  • 1- (1Z-octadecenyl) -2-arachidonoyl-sn-glycero-3-phosphoethanolamine (1- (1Z-Octadecenyl) -2-arachidonoyl-sn-glycero-3-phosphoethanolamine: PlsPE (C18, 20: 4)) has a relative activity of 50% or more (upper limit is 90%).
  • 4-nitrophenylbutyrate pNPB
  • 4-nitrophenyloctanoate pNPO
  • 4-nitrophenyldecanoate pNPD
  • 4-nitrophenyl Laurate 4-Nitorophenyl laurate: pNPL
  • 4-nitrophenyl myristate pNPM
  • 4-nitrophenyl palmitate pNPP
  • 4-nitrophenyl stearate 4-N Nitorophenyl stearate (pNPS) has a relative activity of 15% or less (lower limit is 0%).
  • some secretory hydrolases derived from the Streptomyces albus strain J1074 show 99% or more sequence identity with the amino acid sequence of the enzyme (PLA1) according to the present invention.
  • the above-described secreted hydrase is merely presumed to be a lipase, and the physicochemical properties of the enzyme according to the present invention have not been clarified, and even a person skilled in the art is in accordance with the present invention. It is impossible to estimate the enzyme. This is clear from the fact that the enzyme according to the present invention does not exhibit lipase activity and hardly exhibits esterase activity.
  • the above-mentioned enzymes include, as buffer solutions, acetic acid-sodium acetate buffer (pH 4.1 to 5.6), Bistris-HCl (BisTris-HCl) buffer (pH 5.6 to 7.2), Tris-hydrochloric acid. (Tris-HCl) buffer solution (pH 7.2 to 8.8) and glycine-sodium hydroxide buffer solution (pH 8.8 to 10.5) are used to react the above egg yolk lecithin (L) with the enzyme. When placed below, it may exhibit PLA1 activity within its pH range (pH 4.1-10.5). The optimum pH is around pH 5.6, but shows substantially 100% activity at pH 5-8.
  • the hydrolysis activity at pH 5.6 is 100% under the condition where egg yolk lecithin (L) and the above enzyme are reacted at 37 ° C. for 5 minutes as described above, the enzyme has a pH of 4. It is preferable to exhibit an activity of 50% or more within the range of 1 to pH 10.
  • the above enzyme can act at about 20 to 65 ° C., for example, under the reaction conditions with the above egg yolk lecithin (L).
  • 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 enzyme when the above phospholipid and the above enzyme solution are placed under reaction conditions using an acetic acid-sodium acetate buffer (pH 5.6) as a buffer, the above enzyme has 100 mM EDTA. Are not inhibited, and preferably exhibit almost the same activity as when 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 above enzyme preferably has the following substrate specificity.
  • DPPC 1,2-dipalmitoyl-sn-glycero-3-phosphocholine
  • DMPA 1,2-dimyristoyl-sn-glycerol-3-phosphate
  • 1,2-diacyl-sn-glycero-3-phospho- (1-rac-glycerol) (1,2-Diacyl-sn-glycero-3-phospho- (1-rac-glycerol): PG) Preferably having a relative activity of 400% or more (upper limit is 450%).
  • it preferably has a relative activity of 450% or more (upper limit is 480%) with respect to L- ⁇ -phosphatidylserine (PS).
  • PI L- ⁇ -phosphatidylinositol
  • SB-PC soybean phosphatidylcholine
  • the above enzyme preferably has the following substrate specificity.
  • DPPC 1,2-dipalmitoyl-sn-glycero-3-phosphocholine
  • DMPA 1,2-dimyristoyl-sn-glycerol-3-phosphate
  • 1,2-diacyl-sn-glycero-3-phospho- (1-rac-glycerol) (1,2-Diacyl-sn-glycero-3-phospho- (1-rac-glycerol): PG)
  • the relative activity is preferably 150% or more (upper limit is 190%).
  • DOPE 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine
  • PI L- ⁇ -phosphatidylinositol
  • SB-PC soybean phosphatidylcholine
  • the relative activity with respect to triglyceride is preferably about 0% (for example, 5% or less, 3% or less, 1% or less, or the detection limit or less).
  • 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 5.7 to 5.8 (eg, 5.72).
  • the isoelectric point of the enzyme can be calculated by GENETYX from the mature amino acid sequence excluding the secretory signal sequence.
  • 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 / or an enzyme having an added amino acid sequence.
  • site-directed mutagenesis Nucleic AcidARes., 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: A Practical Approach, IRL Press, 1991, pp. 200), etc., as appropriate, and / or By introducing additional mutations, the structure of the protein can be altered.
  • 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, 20 or less, preferably 16 or less, more preferably 5 or less, and even more preferably. 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 such as SWISS-PROT, PIR, DAD and other protein amino acid sequences, DNA databases such as DDBJ, EMBL, Gene-Bank, etc., programs such as BLAST and FASTA For example, it can be performed through 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
  • Streptomyces albus Streptomycesalbus
  • Streptomyces albidoflavus NA297 (Accession Number: NITE BP-1014) is liquid-cultured in an appropriate nutrient medium to secrete the above enzyme outside the cells.
  • Those treated with lyophilization, salting out, organic solvent, etc. can be produced as PLA1 enzyme preparations.
  • the microorganism that can be used in the production of the PLA1 enzyme preparation is not limited to Streptomyces albidoflavus NA297, and may be a microorganism that belongs to the genus Streptomyces and 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.
  • the 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.
  • a suitable medium for example, a medium containing a suitable carbon source, nitrogen source, and inorganic salts to secrete the enzyme.
  • the carbon source starch, starch hydrolysate, sugars such as glucose and sucrose, alcohols such as glycerol, organic acids (for example, acetic acid and citric acid) or salts thereof (for example, sodium salt), and the like.
  • the nitrogen source examples include organic nitrogen sources such as yeast extract, peptone, meat extract, corn steep liquor, and soybean powder, 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.
  • the culture 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 according to the present invention encodes the above PLA1.
  • This polynucleotide preferably includes the polynucleotide described in any of (a) to (c) below.
  • 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.
  • C a polynucleotide having at least 80% sequence identity to 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 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.
  • 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 PLA1 activity Also included are polynucleotides that encode proteins having: A person skilled in the art can introduce substitution, deletion, insertion, and / or addition mutation as appropriate into the polynucleotide having the base sequence described in SEQ ID NO: 1 using a site-directed 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 target gene in the polynucleotide is based on the nucleotide sequence information described in the present specification, preferably the above microorganism, preferably a microorganism belonging to the genus Streptomyces, more preferably Streptomyces albidoflavus (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.
  • the probe is designed by selecting one or more, and includes the conditions described in the manual (for example, cleaning conditions: 42 ° C., 0.5 ⁇ SSC) using, for example, ECL directic acid labeling and detection system (manufactured by GE Healthcare) In primary wash buffer), it refers to a hybridizing polynucleotide.
  • 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 polynucleotide encoding PLA1 can be expressed in the same or different host using genetic recombination technology.
  • the vector according to the present invention contains the above-described 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.
  • a procedure for producing a transformant and 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, promoters and terminators known to function in microorganisms used as hosts are used.
  • 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.
  • the genus Escherichia the genus Bacillus, the genus Pseudomonas, the genus Serratia, the genus Brevibacterium, the genus Corynebacterium, Bacteria for which host vector systems such as Streptococcus and Lactobacillus are developed; Actinomycetes for which host vector systems such as Rhodococcus and Streptomyces are developed; Saccharomyces (Saccharomyces) Saccharomyces), Kluyveromyces, Shizosa S.
  • genus Schizosaccharomyces genus Zygosaccharomyces, genus Yarrowia, genus Trichosporon, genus Rhodosporidium, genus Rhodospodium, genus C And yeasts that have been developed for host vector systems such as the genus Neurospora, the genus Aspergillus, the genus Cephalosporum, the genus Trichoderma, and the like.
  • Escherichia coli is preferred for ease of gene recombination, and actinomycetes are preferred for ease of gene expression.
  • 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.
  • the PLA1 of the present invention includes various types of PE. It can act on phospholipids, especially on PI (phosphatidylinositol), PG (phosphatidylglycerol), and PS (phosphatidylserine). It works well for PI, PG, PS, DOPE, L at pH 5.6, and works well for PI, PG, PS, DMPA at pH 9.0. By controlling the pH, the acting substrate can be controlled.
  • phosphatidylcholine for example, 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine: DPPC
  • DPPC 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine
  • phosphatidylcholine having a high purity for example, 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine: DPPC
  • phosphatidylethanolamine for example, 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine: DOPE
  • DMPA 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 and cell lysing agents including detergents such as egg yolk stains and blood stains.
  • lysophospholipids are extremely excellent in emulsifying properties, they become emulsifiers and 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 lecithin (L) is used as a substrate.
  • Example 1 Purification of Streptomyces albidoflavus-derived 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.
  • an electrophoretically purified enzyme was obtained from Streptomyces albidoflavus NA297 strain.
  • 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.
  • Substrate specificity PLA1 activity was measured according to the above "PLA1 activity standard measurement method" except that various phospholipids were used as a substrate instead of egg yolk lecithin (L).
  • Table 2 shows the relative values when the yolk lecithin (L) is used as a substrate and the hydrolysis activity at 100 ° C. for 5 minutes at 50 ° C. with a pH of 9.0 and a final concentration of 1.0% Triton X-100. It is the result of activity.
  • Table 3 shows the results when the yolk lecithin (L) was used as a substrate, the hydrolysis activity at 100 ° C. for 5 minutes at 50 ° C. with a pH of 5.6 and a final concentration of 1.0% Triton X-100. It is the result of relative activity.
  • PLA1 activity standard measurement method except that various phospholipids were used as a substrate instead of egg yolk lecithin (L), PLA1 was prepared in a reaction solution containing a final concentration of 0.005% Triton X-100. Activity was measured.
  • Table 4 shows the relative results when the yolk lecithin (L) 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%. It is the result of activity.
  • Tables 5 and 6 show the case where egg yolk lecithin (L) is used as a substrate and the hydrolysis activity at pH 7.2, final concentration 1% Triton X-100 at 50 ° C. for 5 minutes is defined as 100%. It is a result of relative activity with respect to various substrates.
  • the composition of the reaction solution when the substrate is shown in Table 5 and the soybean oil and olive oil shown in Table 6 is shown in Table 7, and the substrates are pNPB, pNPO, pNPD, pNPL, pNPM, pNPP, pNPS (hereinafter “ The composition of the reaction solution in the case of “pNPB” is shown in Table 8.
  • the enzyme solutions in Table 7 are obtained by diluting 1279 U / ml (106.6 U / mg) enzyme solution 2000 times with 20 mM Tris-HCl buffer (pH 9.0). The enzyme activity before dilution was measured at pH 7.2 and 50 ° C. using egg yolk lecithin (L) as a substrate according to the above “PLA1 activity standard measurement method” (see Table 7).
  • the enzyme solutions in Table 8 are obtained by diluting the above 1279 U / ml (106.6 U / mg) enzyme solution 4000 times with 20 mM Tris-HCl buffer solution (pH 9.0).
  • Example 1 As described above, the enzyme obtained in Example 1 exhibited substrate specificity as shown in Tables 2 to 6, FIG. 7 and FIG.
  • Lysophospholipid (lyso form) Lysophospholipid was produced by allowing the purified enzyme obtained in Example 1 to act on L- ⁇ -phosphatidylcholine (PC) (manufactured by Nacalai Tesque, purity 98%). Table 9 shows the composition of the reaction solution.
  • the enzyme solutions in Table 9 are obtained by diluting 1279 U / ml (106.6 U / mg) enzyme solution 1500 times with 20 mM Tris-HCl buffer solution (pH 9.0).
  • the enzyme activity of the enzyme solution before dilution was measured at pH 7.2 and 50 ° C. using egg yolk lecithin (L) as a substrate according to the above “PLA1 activity standard measurement method” (see Table 7). That is, the enzyme activity of the enzyme solution before dilution was measured by diluting 2000 times with egg yolk lecithin (L).
  • the above reaction was performed at pH 7.2 and 37 ° C. Then, 50 ⁇ l of the reaction solution is taken out at regular time intervals (0 minutes, 5 minutes, 10 minutes, 20 minutes, 30 minutes), and a solution of CHCl 3 : CH 3 OH (volume ratio 2: 1) is added to the reaction solution. Then, the reaction was stopped, the organic phase was extracted, the solvent was removed with a vacuum concentrator, and the lyso form was dissolved again with the solvent, and then analyzed by HPLC. The results are shown in FIGS. As is clear from FIGS. 9 to 11, a lysophospholipid with a yield of 16.5% was obtained in the reaction for 20 minutes.
  • This yield was calculated from the mass (w / v%) of lysophospholipid (lyso form) generated from the mass 0.5 w / v% of the substrate (phospholipid) charged in the reaction solution shown in Table 9. .
  • the breakdown is about 2-acyl-sn-glycero-3-phosphocholine (2-Acyl-sn-glycero-3-phosphocholine), 1-acyl-sn-glycero-3-phosphocholine (1-Acyl -sn-glycero-3-phosphocholine) was about 20%.
  • Table 10 shows the manufacturers and product numbers of the substrates described in this specification.
  • the above-mentioned phospholipase A1 is mainly Ser11, Ser216, His218 (the mature enzyme is from position 34 to position 269 of SEQ ID NO: 2, and therefore the amino acid residue at position 34 of SEQ ID NO: 2).
  • Ser11, Ser216, and His218 are obtained.
  • Ser at position 44, Ser at position 249, and His at position 251 are located in the active center, and phospholipid hydrolysis reaction occurs. It is thought that it catalyzes. This was supported by the results that the mutant enzymes in which Ser11, Ser216, and His218 were substituted with each alanine lost the activity of the enzyme produced as an enzyme.
  • 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 % 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-HCl buffer (pH 7.5) and 1 mg / ml lysozyme, and treated overnight at 37 ° C.
  • 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 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 (Tris-HCl) buffer (pH 8.0) and 1 mM EDTA.
  • Tris-HCl Tris-HCl
  • Example 5 Cloning of the core region of 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 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 a template DNA of 200 ng, 2 ⁇ MightyAmp Buffer 25 ⁇ l, each primer 300 nM, and MightyAmp DNA Polymerase 1.25 unit to a total volume of 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 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. Is shown.
  • the arrangement 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 consistent (see FIG. 6). 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).
  • the above deduced amino acid sequences were compared with sequences in four types of protein sequence databases (PTR, PRF, UNI-PROT and SWISS-PROT).
  • PTR protein sequence databases
  • PRF UNI-PROT
  • SWISS-PROT the mature enzyme excluding the secretory signal sequence at positions 1 to 33 of the amino acid sequence of SEQ ID NO: 14 (FIG. 6) among the above estimated amino acid sequences is Lipase_GDSL (DDBJ accession) estimated by genome analysis of Streptomyces albus J1074. No. D6BAL1) was 100% consistent.
  • 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, but the BglII recognition sequence was replaced by the following method because the BglII site was included in the structural gene of PLA1 derived from Streptomyces albidoflavus. .
  • 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 site containing the BglII site in this structural gene
  • Antisense primer C2 “primer C2” SEQ ID NO: 17
  • the antisense primer C2 “primer C2” was designed so that the BglII recognition sequence was eliminated and the designated amino acid was not changed. That is, the 696th g in SEQ ID NO: 1 was substituted with a (the designated amino acid remained glutamic acid). 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.
  • Template chromosomal DNA 200 ng, 10 ⁇ PCR Buffer 2.5 ⁇ l, primers 1200 nM each, dNTP mixture 0.3 mM, MgCl 2 1.2 mM, DMSO 4%, and KOD DNA Polymerase 1.25 units, distilled water 25 ⁇ l in total It added so that it might become.
  • 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.
  • a specific amplification product of about 600 bp was obtained by this PCR.
  • sense primer C3 “primer C3” (SEQ ID NO: 18) designed to replace the BglII recognition sequence in this structural gene, and an antisense primer in which a BglII site is added to the downstream sequence of the PLA1 structural gene C4 “primer C4” (SEQ ID NO: 19) was designed. 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.
  • Template chromosomal DNA 200 ng, 10 ⁇ PCR Buffer 2.5 ⁇ l, primers 1200 nM each, dNTP mixture 0.3 mM, MgCl 2 1.2 mM, DMSO 4%, and KOD DNA Polymerase 1.25 units, distilled water 25 ⁇ l in total It added so that it might become.
  • 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.
  • a specific amplification product of about 100 bp was obtained by this PCR.
  • a sense primer C1 “primer C1” (SEQ ID NO: 16) with an NheI site added to the upstream region sequence of the PLA1 structural gene, and an antisense primer C4 “with a BglII site added to the downstream region 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, distilled water The total amount was 25 ⁇ l.
  • 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 the 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.
  • tryptic soy media Becton Dickinson
  • the recovered precipitate is dissolved in 20 mM Tris-HCl (Tris-HCl) buffer (pH 8.0), dialyzed using 20 mM Tris-HCl (Tris-HCl) buffer (pH 8.0) as an external solution, and then enzyme solution.
  • Tris-HCl Tris-HCl
  • Tris-HCl Tris-HCl
  • the enzyme activity of this enzyme solution was measured at pH 5.6 and 50 ° C. using egg yolk lecithin (L) as a substrate according to 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 lysophospholipid, 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, DOPE, L at pH 5.6 and on PI, PG, PS, DMPA at pH 9.0.
  • lysophosphatidylinositol is an antifungal 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.
  • SEQ ID NO: 1 PLA1 gene
  • SEQ ID NO: 2 Full length of PLA1 (polypeptide)
  • 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 gene
  • SEQ ID NO: 10 primer SE1 Sequence number 11: Primer AN1 SEQ ID NO: 12: part of PLA1 (polypeptide) SEQ ID NO: 13: primer A2

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Abstract

L'invention concerne une nouvelle PLA1 et son procédé de production. L'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 représentée par SEQ ID NO : 2. (b) un polypeptide ayant une séquence d'acides aminés obtenue par la substitution, l'insertion, la délétion ou l'addition d'un ou plusieurs acides aminés dans la séquence d'acides aminés représentée par SEQ ID NO :2, (c) un polypeptide ayant au moins 75 % d'homologie, la séquence d'acides aminés étant représentée par SEQ ID NO :2. Ainsi, il est possible de fournir une nouvelle phospholipase A1 ayant une activité enzymatique élevée. La PLA1 comprend une spécificité de substrat pour les phospholipides.
PCT/JP2012/052159 2011-02-01 2012-01-31 Enzyme et son procédé de production WO2012105565A1 (fr)

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JPH10513343A (ja) * 1994-12-19 1998-12-22 コールド スプリング ハーバー ラボラトリー テロメラーゼの蛋白質成分
JP2000060576A (ja) * 1998-03-13 2000-02-29 F Hoffmann La Roche Ag 遺伝子操作されたｌ―ソルボ―スレダクタ―ゼ欠失突然変異体
JP2002291487A (ja) * 2000-12-15 2002-10-08 Mitsui Chemicals Inc コクラウリン−ｎ−メチルトランスフェラーゼ

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JPS58212783A (ja) * 1982-06-07 1983-12-10 Kyowa Hakko Kogyo Co Ltd 発酵法によるホスホリパ−ゼａの製造法

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JPH10513343A (ja) * 1994-12-19 1998-12-22 コールド スプリング ハーバー ラボラトリー テロメラーゼの蛋白質成分
JP2000060576A (ja) * 1998-03-13 2000-02-29 F Hoffmann La Roche Ag 遺伝子操作されたｌ―ソルボ―スレダクタ―ゼ欠失突然変異体
JP2002291487A (ja) * 2000-12-15 2002-10-08 Mitsui Chemicals Inc コクラウリン−ｎ−メチルトランスフェラーゼ

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