WO2013145289A1

WO2013145289A1 - Lysophospholipid-containing cleaning agent

Info

Publication number: WO2013145289A1
Application number: PCT/JP2012/058661
Authority: WO
Inventors: 大助杉森
Original assignee: 国立大学法人福島大学
Priority date: 2012-03-30
Filing date: 2012-03-30
Publication date: 2013-10-03

Abstract

Provided is a lysophospholipid-containing cleaning agent that easily destroys cellular membranes such as of red blood cells in the blood and has an excellent cleaning effect on blood stains. This lysophospholipid-containing cleaning agent comprises a lysophospholipid obtained by allowing, as an enzyme, at least one of a phospholipase (A1), a phospholipase (A2) and a phospholipase (B) to act with a phospholipid, and hydrolyzing the phospholipid.

Description

Lysophospholipid-containing detergent

The present invention relates to a lysophospholipid-containing detergent containing lysophospholipid obtained by degrading phospholipid with phospholipase (phospholipid degrading enzyme).

Conventionally, as a cleaning agent for medical devices such as endoscopes, a cleaning composition comprising a nonionic surfactant, a protease, a sequestering agent, benzotriazole, and the like is known (for example, see Patent Document 1). A cleaning composition containing a protease, a nonionic surfactant, and an enzyme stabilizer is also known (see, for example, Patent Document 2).

JP-A-5-279700 JP 2001-31999 A

However, with conventional cleaning agents, for example, when cleaning an endoscope, it is difficult to sufficiently wash off dirt such as blood and other cells adhering to the tip portion having a lens or the like and the forceps opening. In particular, blood stains are likely to remain in a constricted portion such as the inside of a forceps opening even if it is cleaned by immersion, and cleaning by brushing is essential. Further, when the temperature becomes low such as in winter, the cleaning efficiency of the conventional cleaning agent decreases, so that blood stains are more likely to remain. In addition, there are similar cleaning problems in cleaning medical equipment and equipment to be reused. Further, there is a demand for a technique for removing coagulated blood (blood clot) generated in the body at the time of laparoscopic surgery as quickly as possible, but such a technique has not been developed at present.

The present invention has been made in view of the above points, and an object thereof is to provide a lysophospholipid-containing cleaning agent that easily destroys cell membranes such as red blood cells in blood and has an excellent blood stain cleaning effect. is there.

The lysophospholipid-containing detergent according to the present invention contains a lysophospholipid obtained by hydrolyzing the phospholipid by allowing at least one of phospholipase A1, phospholipase A2 and phospholipase B to act on the phospholipid as an enzyme. It is characterized by.

The lysophospholipid-containing detergent according to the present invention contains a lysophospholipid obtained by hydrolyzing the phospholipid by allowing at least one of the following phospholipase A1 and phospholipase B to act on the phospholipid as an enzyme. It is what.

Phospholipase A1: containing the following polypeptide (a1-1), (a1-2) or (a1-3), wherein the sn-1 position acyl group of the phospholipid is preferentially compared to the sn-2 position acyl group Disconnect.

(A1-1) A polypeptide having the amino acid sequence set forth in SEQ ID NO: 1.

(A1-2) 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: 1.

(A1-3) A polypeptide having at least 75% homology with the amino acid sequence set forth in SEQ ID NO: 1.

Phospholipase B: contains the following polypeptide (b1-1), (b1-2) or (b1-3), and cleaves both the sn-1 position acyl group and the sn-2 position acyl group of the phospholipid.

(B1-1) A polypeptide having the amino acid sequence set forth in SEQ ID NO: 2.

(B1-2) 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.

(B1-3) A polypeptide having at least 75% homology with the amino acid sequence set forth in SEQ ID NO: 2.

Phospholipase A1: containing the following polypeptide (a2-1), (a2-2) or (a2-3), wherein the sn-1 position acyl group of the phospholipid is preferentially compared to the sn-2 position acyl group Disconnect.

(A2-1) A polypeptide produced from a microorganism having the nucleotide sequence set forth in SEQ ID NO: 3 as a polynucleotide.

(A2-2) A polypeptide produced from a microorganism having a polynucleotide that hybridizes with a base sequence complementary to the base sequence set forth in SEQ ID NO: 3 under stringent conditions.

(A2-3) A polypeptide produced from a microorganism having a polynucleotide having at least 75% sequence identity with the nucleotide sequence set forth in SEQ ID NO: 3.

Phospholipase B: contains the following polypeptide (b2-1), (b2-2) or (b2-3), and cleaves both the sn-1 position acyl group and the sn-2 position acyl group of the phospholipid.

(B2-1) A polypeptide produced from a microorganism having the nucleotide sequence set forth in SEQ ID NO: 4 as a polynucleotide.

(B2-2) A polypeptide produced from a microorganism having a polynucleotide that hybridizes with a base sequence complementary to the base sequence set forth in SEQ ID NO: 4 under stringent conditions.

(B2-3) A polypeptide produced from a microorganism having a polynucleotide having at least 75% sequence identity with the nucleotide sequence set forth in SEQ ID NO: 4.

In the lysophospholipid-containing detergent, the enzyme is preferably derived from a microorganism belonging to the genus Streptomyces.

The lysophospholipid-containing detergent according to the present invention is particularly apt to destroy cell membranes such as red blood cells and white blood cells constituting blood. For example, blood and other cells contaminated with a medical device such as an endoscope Can be sufficiently washed out in a short time even at a low temperature, and has an excellent blood stain cleaning effect. Moreover, since the lysophospholipid-containing detergent according to the present invention has very low toxicity to humans, medical devices after washing with such a lysophospholipid-containing detergent can be reused without further treatment. Is something that can be done. For example, in endoscopic and laparoscopic surgery, cells such as red blood cells and white blood cells attached to the lens at the tip of these devices are quickly dissolved and removed using a lysophospholipid-containing detergent, or coagulation that occurs in the body. The dissolved blood (blood clot) can be quickly dissolved and removed using a lysophospholipid-containing detergent.

It is a graph which shows the relationship between a lysophosphatidylcholine (LPC) density | concentration and a hemolysis rate. It is a graph which shows the relationship between the heat processing time in 100 degreeC, and the ratio of the hemolysis rate after the heating with respect to before a heating. It is a graph which shows the relationship between the reaction time of an enzyme (PLA1) and a substrate (PC), and the LPC production | generation density | concentration which is a lysophospholipid of a decomposition product. Other substrates with reference to enzyme activity of PS under conditions of pH 9.0, 50 ° C., 5 minutes (100%) and enzyme activity of PI under conditions of pH 5.6, 50 ° C., 5 minutes (100%) It is a graph which shows the relative activity of. It is a graph which shows the relative activity of another substrate on the basis of the enzyme activity of POPA in the conditions of pH 7.2, 50 degreeC, and 5 minutes (100%). 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. 6A.

[Raw material for lysophospholipid-containing detergent]
The lysophospholipid-containing detergent according to the present invention causes phospholipase (particularly 1,2-diacylglycerophospholipid) to act on at least one of phospholipase A1 (PLA1), phospholipase A2 (PLA2) and phospholipase B (PLB) as an enzyme. And lysophospholipids obtained by hydrolyzing phospholipids. The lysophospholipid includes all or a part of phospholipid degradation products.

[enzyme]
Enzymes are not limited to purified enzymes, but also include crudely purified products and immobilized products. The enzyme can be purified, for example, by using a culture solution of microorganisms and using a method such as ammonium sulfate precipitation, ion exchange chromatography, hydrophobic chromatography, or the like. As a result, enzymes having various degrees of purification (including enzymes purified to almost a single level) can be obtained.

The microorganism may be any strain such as a wild strain, a mutant strain (for example, induced by ultraviolet irradiation), or a recombinant derived by genetic engineering techniques such as cell fusion or genetic recombination. May be. A genetically engineered microorganism such as a recombinant is used, for example, using a technique described in Molecular Cloning A Laboratory Manual, 2nd edition (Sambrook, J. et al., Cold Spring Harbor Press, 1989). Created easily. 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.

As the enzyme, an enzyme that exhibits hydrolytic activity against phospholipids is used. It is an enzyme called so-called phospholipase (PL). As the phospholipase, phospholipase A1 (PLA1), phospholipase A2 (PLA2), phospholipase B (PLB), or the like can be used. Examples of such enzymes include PLA2 derived from porcine pancreas, muphecobra derived from Streptomyces violaceoruber, commercially available from Sigma Aldrich, PLA1 derived from Thermomyces lanuginosus, PLA1 derived from Lecitase Ultra, etc. Is mentioned. Alternatively, PLA1 manufactured by Mitsubishi Chemical Foods Co., Ltd., lecitase manufactured by Novozymes, PLA2 Lipomod 699L manufactured by Genencor Kyowa Co., Ltd., PLA2 Nagase manufactured by Nagase ChemteX Corporation, PLA2 Maxa Pearl A2 manufactured by DSM Japan Co., Ltd., Asahi Kasei Industrial enzymes such as PLA2 manufactured by Pharma Co., Ltd. and PLB manufactured by Asahi Kasei Pharma Co., Ltd. may be used.

In producing a lysophospholipid-containing detergent, it is particularly preferable to use at least one of phospholipase A1 and phospholipase B described below as an enzyme. These enzymes can eliminate the addition of an inorganic salt such as a calcium salt, can be reacted at a low temperature to a high temperature (20 to 70 ° C.), and can improve the efficiency of the enzyme treatment. Further, since the reaction can be carried out at pH 3 to 11, the reaction can be easily controlled. Moreover, since the titer of the enzyme is extremely high, the reaction efficiency is remarkably high. Furthermore, since it acts on many types of phospholipids, the reaction yield is high.

[Phospholipase A1]
The phospholipase A1 includes the following polypeptide (a1-1), (a1-2) or (a1-3), and the sn-1 position acyl group of the phospholipid is preferential to the sn-2 position acyl group: It is an enzyme that cleaves.

(A1-1) A polypeptide having the amino acid sequence set forth in SEQ ID NO: 1.

Phospholipase A1 includes the following polypeptide (a2-1), (a2-2) or (a2-3), and the sn-1 position acyl group of the phospholipid has priority over the sn-2 position acyl group: It may be an enzyme that cleaves.

The polynucleotide having the base sequence described in SEQ ID NO: 3 can encode the polypeptide (amino acid sequence) described in SEQ ID NO: 1.

Phospholipase A1 is an enzyme that preferentially hydrolyzes the fatty acid ester bond at the α-position (sn-1 position) of the glycerol group in phospholipid (predominantly over the sn-2 position). Therefore, phospholipase A1 is a phospholipid that is at least one of glycerol-3-phosphodiester compounds such as 2-acylglycerol-3-phosphodiester compounds, glycerol-3-phosphate, glycerol-3-phosphocholine, preferably It is an enzyme that produces all three types.

PLA1 activity can be confirmed by the following method, but the confirmation method is not limited to this. For example, PLA1 activity can be confirmed by measuring the amount of free fatty acid produced as a result of the enzymatic reaction.

As a specific method, first, 1 g of egg yolk phosphatidylcholine (manufactured by Nacalai Tesque) was dissolved in 10 mL of 0.02% (w / v) Triton X-100 (manufactured by Nacalai Tesque), and 10% (W / v) Prepare egg yolk phosphatidylcholine. To 0.025 mL of 10% (w / v) egg yolk phosphatidylcholine, 0.060 mL of 0.2 M Tris-HCl buffer (pH 8.0), 0.5 M disodium EDTA (manufactured by Wako Pure Chemical Industries, Ltd.) Add 005 mL. And after preheating at 37 degreeC for 5 minute (s), 0.010 mL of samples which confirm enzyme activity are added, and it is made to react at 37 degreeC for 5 minutes. After the enzyme reaction, the reaction is stopped by heating at 100 ° C. for 5 minutes. After stopping the reaction, the amount of free fatty acid contained in 5 μL of the reaction solution is described in the instructions attached to the kit using, for example, “NEFA C Test Wako” (manufactured by Wako Pure Chemical Industries, Ltd.) which is a free fatty acid measurement kit. Measure as follows. The amount of enzyme that produces 1 μmol of free fatty acid per minute is defined as 1 unit (1 U).

For example, phospholipase A1 can be used as a buffer solution of 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). 8.8) and glycine-sodium hydroxide buffer solution (pH 8.8 to 10.5), when placed under the reaction conditions with the above egg yolk phosphatidylcholine, within the pH range (pH 4.1 to 10.5). ) May exhibit PLA1 activity. The optimum pH is around pH 5.6, but shows substantially 100% activity at pH 5-8.

Phospholipase A1 is, for example, 50% within the range of pH 4.1 to pH 10 when the hydrolysis activity at pH 5.6 is 100% under the conditions of reaction with egg yolk phosphatidylcholine at 37 ° C. for 5 minutes as described above. It is preferable to exhibit the above activity.

Phospholipase A1 can act at about 20 to 65 ° C., for example, against the above egg yolk phosphatidylcholine. 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.

For example, when treated with 120 mM acetic acid-sodium acetate buffer (pH 5.6) for 30 minutes, phospholipase A1 is stable with almost no decrease in activity from 4 ° C. to 40 ° C., and 80 ° C. even at 45 ° C. % Of activity (for example, 75%) or more remains.

Phospholipase A1 is not inhibited even in the presence of 100 mM EDTA when subjected to reaction conditions with the above-described phospholipid using, for example, acetic acid-sodium acetate buffer (pH 5.6) as a buffer. It is preferable to show almost the same activity as when no is 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.

When phospholipase A1 is reacted with a substrate at 50 ° C. for 5 minutes under the conditions of pH 7.2 as described above, assuming that the hydrolysis activity when egg yolk lecithin (L) is a substrate is 100%, 1- 50% or more for palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 500% for 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate (POPA) More than 150% for 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), more than 300% for L-α-phosphatidylinositol (PI), L-α-phosphatidyl- 300% or more of L-serine (PS), 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-ra 100% or more for c- (1-glycerol) (POPG), 100% or more for soybean-derived L-α-phosphatidylcholine (SB-PC), 30% or more for soybean lecithin (SBL), L More than 250% for α-lysophosphatidylcholine (LPC), 50 for 1- (1Z-octadecenyl) -2-arachidonoyl-sn-glycero-3-phosphoethanolamine (PlsPE (C18, 20: 4)) %, 1-hexadecyl-2- (9Z-octadecenoyl) -sn-glycero-3-phosphoethanolamine (PlsPE (C16,18: 1)), 200% or more, 1- (1Z-octadecenyl) -2 -0% to arachidonoyl-sn-glycero-3-phosphocholine (PlsPC (C18, 20: 4)), soybean oil And 10% or less of olive oil, 4-nitrophenyl butyrate (pNPB), 4-nitrophenyl octanoate (pNPO), 4-nitrophenyl decanoate (pNPD), 4-nitrophenyl laurate (pNPL) , 4-nitrophenyl myristate (pNPM), 4-nitrophenyl palmitate (pNPP) and 4-nitrophenyl stearate (pNPS) have a relative activity of 15% or less (see FIG. 5, provided that FIG. Shows the relative activity of other enzymes, with the enzyme activity of POPA as 100%.)

When phospholipase A1 is reacted with a substrate at 50 ° C. for 5 minutes under the condition of pH 9.0 as described above, assuming that the hydrolysis activity when egg yolk lecithin (L) is a substrate is 100%, 1 , 2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) of 50% or less (lower limit is 30%), 1,2-dimyristoyl- 350% or more (upper limit is 400%) based on sn-glycerol-3-phosphate (1,2-Dimyristoyl-sn-glycerol-3-phosphate: DMPA), 1,2-diacyl-sn-glycero-3-phospho -(1-rac-glycerol) (1,2-Diacyl-sn-glycero-3-phospho- (1-rac-glycerol): PG) is 400% or more (upper limit is 450%), L-α- 450% against phosphatidylserine (L-α-Phosphatidylserine: PS) Above (upper limit is 480%), more than 100% with respect to 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) (upper limit is 140 %), 350% or more (up to 380%) with respect to L-α-phosphatidylinositol (PI), soybean-derived L-α-phosphatidylcholine (SB-PC) 300% or more (upper limit is 360%) with respect to soybean lecithin (SBL) and 75.0% or more (upper limit is 90%) (see FIG. 4; however, in FIG. 4, in the case of pH 9.0, The relative activity of other enzymes is shown with the enzyme activity of PS as 100%.)

In addition, when phospholipase A1 is reacted with a substrate at 50 ° C. for 5 minutes under the condition of pH 5.6 as described above, the hydrolysis activity when egg yolk lecithin (L) is a substrate is defined as 100%. , 2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) of 100% or more (up to 140%), 1,2-dimyristoyl- 100% or less based on sn-glycerol-3-phosphate (DMPA) (lower limit is 60%), 1,2-diacyl-sn-glycero-3-phospho- (1-rac-glycerol) (1,2-Diacyl -sn-glycero-3-phospho- (1-rac-glycerol): PG) 150% or more (upper limit is 190%), L-α-Phosphatidyl-L-serine: PS ) Over 150% (upper limit is 190%) , 2-dioleoyl-sn-glycero-3-phosphoethanolamine (1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine: DOPE) 100% or more (upper limit is 120%), L-α-phosphatidylinositol (L-α-Phosphatidylinositol: PI) 250% or more (upper limit is 290%), soybean-derived L-α-phosphatidylcholine (L-α-Phosphatidylinositol: PI) (250% or more (upper limit) 290%), having an activity of 250% or more (upper limit is 280%) with respect to soybean lecithin (SBL), and having an activity with respect to triglyceride (olive oil) is almost 0% (for example, 5% or less, 3% or less, or 1%) (See FIG. 4. However, in FIG. 4, when the pH is 5.6, the enzyme activity of PI is defined as 100% and the relative activity of other enzymes is shown.)

For example, some secretory hydrolases derived from the Streptomyces albus strain J1074 have a sequence identity of 99% or more with the amino acid sequence of the above-mentioned phospholipase A1 (PLA1). However, the secretory hydrase is merely presumed to be a lipase, and the physicochemical properties of the phospholipase A1 have not been clarified, and even those skilled in the art can use the phospholipase A1. It is impossible to estimate. This is also clear from the fact that the above-mentioned phospholipase A1 shows no lipase activity and little esterase activity. Even if such homology is high, other factors such as the three-dimensional structure have a great influence on the physicochemical properties of the enzyme, so an enzyme having 99% or more sequence identity with the amino acid sequence of the phospholipase A1 described above. Even if it exists, it is thought that it does not have substrate specificity like said phospholipase A1. The steric structure of the phospholipase A1 will be described later as an example.

Phospholipase A1 may vary slightly depending on electrophoresis conditions and the like, but preferably exhibits a molecular weight in the range of 25,000 to 30,000 (for example, about 28,000 or about 27,000) in SDS-PAGE. Phospholipase A1 preferably has a molecular weight calculated from the amino acid composition in the range of 25,000 to 30,000. For example, 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.

Phospholipase A1 preferably exhibits an isoelectric point in the range of 6.0 to 6.1 (eg, 6.06). The isoelectric point of the enzyme can be calculated from the amino acid sequence by GENETYX.

One embodiment of phospholipase A1 consists of the amino acid sequence set forth in SEQ ID NO: 1. The phospholipase A1 preferably has an amino acid sequence from position 34 to position 269 of SEQ ID NO: 1 (hereinafter also referred to as “amino acid sequence described in SEQ ID NO: 1”).

As long as phospholipase A1 has PLA1 activity, one or more amino acids are substituted, deleted, inserted and / or substituted with respect to the amino acid sequence shown in SEQ ID NO: 1 or the amino acid sequence shown in SEQ ID NO: 1. It may be an enzyme having an added amino acid sequence. For example, site-directed mutagenesis (Nucleic Acid Ａ Res., 1982, 10, pp. 6487; Methods in Enzymol., 1983, 100, pp. 448; Molecular Cloning: A Laboratory Manual, Second Edition, ColdSpring Laboratories, Cold Spring Harbor, NY, 1989; PCR: APPROCICAL Approach, IRL Press, 1991, pp. 200), etc., by introducing substitutions, deletions, insertions and / or addition mutations as appropriate. The structure of (protein) can be modified. 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, and even more preferably 0 to 3. is there. In addition, 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 phospholipase A1.

A protein having an amino acid sequence having homology to the amino acid sequence shown in SEQ ID NO: 1 or the amino acid sequence shown in SEQ ID NO: 1 is also included in phospholipase A1 as long as it has PLA1 activity. The phospholipase A1 is preferably at least 75%, preferably at least 80%, more preferably at least 85%, even more preferably at least 90% of the amino acid sequence set forth in SEQ ID NO: 1 or the amino acid sequence set forth in SEQ ID NO: 1. %, Even more preferably at least 95%, even more preferably at least 99% homologous protein having an amino acid sequence.

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 Can be done through the Internet. The protein activity can be confirmed using the procedure described above.

The supply source of phospholipase A1 is not particularly limited, but phospholipase A1 can be obtained from living cells such as microorganisms. Examples of such microorganisms include microorganisms belonging to the genus Streptomyces. Streptomyces albidoflavus (Streptomyces albidoflavus) and Streptomyces albidoflavus (Streptomyces albidoflavus) NA297 strain (Accession number: NITE BP-1014) NA297 strain "). This Streptomyces albidoflavus NA297 strain has a polynucleotide represented by the nucleotide sequence of SEQ ID NO: 3 in its DNA.

For example, the Streptomyces albidoflavus NA297 strain (accession number: NITE BP-1014) described above secretes phospholipase A1 outside the cells by liquid culture in an appropriate nutrient medium. A product obtained by treating Kiyo with freeze-drying, salting out, an organic solvent, etc. can be produced as an enzyme preparation.

The microorganism that can be used in the production of the enzyme preparation is not limited to the Streptomyces albidoflavus NA297 strain, and may be a microorganism that belongs to the genus Streptomyces and can produce phospholipase A1. In addition, natural or artificial mutants of these species or gene fragments necessary for the expression of PLA1 activity may be artificially extracted and used for the production of phospholipase A1 even in other species incorporating them. it can. Moreover, even if it does not belong to Streptomyces genus, if it is microorganisms which can produce said phospholipase A1, it can also be used.

An example of an enzyme preparation using Streptomyces albidoflavus NA297 strain will be described.

Since this bacterium secretes phospholipase A1 out of the microbial cells by liquid culture in a nutrient medium, the culture supernatant is treated with lyophilization, salting out, an organic solvent, or the treated product is immobilized. Thus, an enzyme preparation can be produced. More specifically, first, this bacterium is cultured in a suitable medium, for example, a medium containing a suitable carbon source, nitrogen source, and inorganic salts to secrete phospholipase A1. Examples of the carbon source include starch and 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. It is done. Examples of the nitrogen source include organic nitrogen sources such as yeast extract, peptone, meat extract, corn steep liquor and soy flour, and inorganic nitrogen compounds such as ammonium sulfate, ammonium nitrate and urea. Examples of inorganic salts include sodium chloride, monopotassium phosphate, magnesium sulfate, manganese chloride, calcium chloride, and ferrous sulfate. The concentration of the carbon source is, for example, in the range of 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 phospholipase A1 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 for sufficiently producing phospholipase A1, for example, about 1 to 7 days. Culturing can be performed preferably under aerobic conditions, for example, with aeration stirring or shaking.

Polypeptides contained in phospholipase A1 are fractionated by protein solubility (precipitation with organic solvents, salting out by ammonium sulfate, etc.); cation exchange, anion exchange, gel filtration, hydrophobic chromatography; chelate, dye, antibody It can be purified by appropriately combining methods such as affinity chromatography using the above. 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 polypeptide constituting phospholipase A1 can be estimated as a monomer by HPLC analysis and gel filtration chromatography analysis.

[Phospholipase B]
Phospholipase B contains the following polypeptide (b1-1), (b1-2), or (b1-3), and an enzyme that cleaves both the sn-1 and sn-2 acyl groups of phospholipids It is.

(B1-1) A polypeptide having the amino acid sequence set forth in SEQ ID NO: 2.

Phospholipase B contains the following polypeptide (b2-1), (b2-2), or (b2-3), and cleaves both the sn-1 position acyl group and the sn-2 position acyl group of the phospholipid. It may be an enzyme.

The polynucleotide having the base sequence described in SEQ ID NO: 4 can encode the polypeptide (amino acid sequence) described in SEQ ID NO: 2.

Phospholipase B hydrolyzes both fatty acid ester groups at the α-position (sn-1 position) of the glycerol group and the fatty acid ester group at the β-position (sn-2 position) of the glycerol group. Enzyme activity to That is, phospholipase B is an enzyme having both PLA1 activity and PLA2 activity. Accordingly, phospholipase B is derived from phospholipids from glycerol-3- such as 1-acylglycerol-3-phosphodiester compound, 2-acylglycerol-3-phosphodiester compound, glycerol-3-phosphate, glycerol-3-phosphocholine and the like. It is an enzyme that produces at least one, preferably all three of the phosphodiester compounds.

The PLB activity can be confirmed by the following method, but the confirmation method is not limited to this. For example, PLB activity can be confirmed by measuring the amount of free fatty acid produced as a result of the enzyme reaction.

Specifically, first, 1 g of dimyristoylphosphatidic acid (manufactured by Funakoshi Co., Ltd.) was dissolved in 10 mL of 10% (w / v) Triton X-100 (manufactured by Nacalai Tesque Co., Ltd.), and 10% ( w / v) Prepare dimyristoyl phosphatidic acid. To 0.005 mL of 10% (w / v) dimyristoylphosphatidic acid, 0.025 mL of 0.2 M Tris-HCl buffer (pH 8.4) and 0.5 M disodium EDTA (manufactured by Wako Pure Chemical Industries, Ltd.) Add 0.002 mL and 0.063 mL of distilled water. And after preheating at 37 degreeC for 5 minute (s), 0.005 mL of samples which confirm enzyme activity are added, and it is made to react at 37 degreeC for 5 minutes. After the enzyme reaction, the reaction is stopped by heating at 100 ° C. for 5 minutes. After stopping the reaction, the amount of free fatty acid contained in 5 μL of the reaction solution is described in the instructions attached to the kit using, for example, “NEFA C Test Wako” (manufactured by Wako Pure Chemical Industries, Ltd.) which is a free fatty acid measurement kit. Measure as follows. The amount of enzyme that produces 1 μmol of free fatty acid per minute is defined as 1 unit (1 U).

Phospholipase B, for example, as a buffer solution, 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). 8.8) and glycine-sodium hydroxide buffer solution (pH 8.8 to 10.5), when subjected to the reaction conditions with the above phosphatidic acid, within the pH range (pH 4.1 to 10. 5) may show PLB activity. The optimum pH can be around pH 8 to 8.8.

Phospholipase B is, for example, in the range of pH 7.2 to pH 10.0 when the hydrolysis activity at pH 8.4 is 100% under the condition of reacting with dimyristoyl phosphatidic acid for 5 minutes at 37 ° C. as described above. Of these, it is preferable to exhibit an activity of 50% or more.

Phospholipase B can act at about 20 to 65 ° C. against dimyristoyl phosphatidic acid as described above, for example. The optimum temperature can be within this range. Preferably it is in the range of about 37-60 ° C, more preferably in the range of 45-55 ° C, and even more preferably about 50 ° C.

For example, when treated with 160 mM Tris-HCl buffer (pH 8.4) for 30 minutes, phospholipase B is stable from 4 ° C. to 45 ° C. with almost no decrease in activity, and preferably 50 ° C. However, an activity of about 80% (for example, 75%) or more remains.

Phospholipase B is not inhibited by 10 mM EDTA and added with no EDTA when subjected to the above phospholipid and reaction conditions using, for example, Tris-HCl buffer (pH 8.4) as a buffer. It is preferable to show almost the same activity. Further, in the presence of 10 mM Ca ²⁺ , it is preferable to show about 90% activity (for example, about 85 to 95% activity). On the other hand, 10 mM Mg ²⁺ , Mn ²⁺ , Co ²⁺ , Cu ²⁺ , Zn ²⁺ , Fe ³⁺ , and Fe ²⁺ can inhibit the activity.

When phospholipase B is reacted with a substrate at 50 ° C. for 5 minutes under the condition of pH 8.4, dimyristoyl phosphatidic acid (1,2-dimyristoyl-sn-glycero-3-phosphate) is the substrate. Assuming that the hydrolysis activity at a certain time is 100%, L-α-phosphatidylinositol (L-α-phosphatidylinositol (L-α-Phosphatidylinositol) is 95% or more with respect to L-α-phosphatidylcholine (SB-PC) derived from soybean. : PI) and 1,2-diacyl-sn-glycero-3-phospho- (1-rac-glycerol) (1,2-Diacyl-sn-glycero-3-phospho- (1-rac-glycerol): PG) It is preferable to have an activity of 20% or more against L-α-phosphatidylserine (L-α-Phosphatidyl-L-serine: PS). Under the same conditions, activity against sphingomyelin, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), tristearin and dipalmitoyl glyceride Is preferably 0% (for example, 5% or less, 3% or less, or 1% or less, or the detection limit or less). It is preferable to have such substrate specificity.

Phospholipase B may vary slightly depending on electrophoresis conditions, but preferably has a molecular weight in SDS-PAGE in the range of 38,000 to 40,000 (for example, about 39,000 or 38,900). Phospholipase B preferably has a molecular weight calculated from the amino acid composition in the range of 41,000 to 43,000. For example, a natural enzyme derived from Streptomyces sp. NA684 strain has a molecular weight of about 39,000, specifically 38,900 in SDS-PAGE. In the natural enzyme derived from this Streptomyces sp. NA684 strain, the molecular weight calculated from its amino acid composition is about 42,000.

Phospholipase B preferably exhibits an isoelectric point within the range of 6.2 to 6.6 (for example, 6.4). The isoelectric point of the enzyme can be calculated from the amino acid sequence by GENETYX.

One embodiment of phospholipase B consists of the amino acid sequence set forth in SEQ ID NO: 2. Phospholipase B preferably has an amino acid sequence from position 31 to position 412 of SEQ ID NO: 2 (hereinafter also referred to as “amino acid sequence in SEQ ID NO: 2”).

As long as phospholipase B has PLB activity, one or more amino acids are substituted, deleted, inserted and / or substituted with respect to the amino acid sequence shown in SEQ ID NO: 2 or the amino acid sequence shown in SEQ ID NO: 2. It may be an enzyme having an added amino acid sequence. For example, site-directed mutagenesis (Nucleic Acid Ａ Res., 1982, 10, pp. 6487; Methods in Enzymol., 1983, 100, pp. 448; Molecular Cloning: A Laboratory Manual, Second Edition, ColdSpring Laboratories, Cold Spring Harbor, NY, 1989; PCR: APPROCICAL Approach, IRL Press, 1991, pp. 200), etc., by introducing substitutions, deletions, insertions and / or addition mutations as appropriate. The structure of (protein) can be modified. 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, and even more preferably 0 to 3. is there. In addition, amino acid mutations include not only artificially mutated enzymes but also naturally mutated enzymes as long as they have PLB activity in phospholipase B.

A protein having an amino acid sequence having homology to the amino acid sequence shown in SEQ ID NO: 2 or the amino acid sequence shown in SEQ ID NO: 2 is also included in phospholipase B as long as it has PLB activity. The phospholipase B is preferably at least 75%, preferably at least 80%, more preferably at least 85%, even more preferably at least 90% of the amino acid sequence set forth in SEQ ID NO: 2 or the amino acid sequence set forth in SEQ ID NO: 2. %, Even more preferably at least 95%, even more preferably at least 99% homologous protein having an amino acid sequence.

The supply source of phospholipase B is not particularly limited, but phospholipase B can be obtained from living cells such as microorganisms. Examples of such microorganisms include microorganisms belonging to the genus Streptomyces. Preferably, Streptomyces sp., Streptomyces chattanoogensis and Streptomyces lydicus are used. Since these strains are closely related, it is considered that phospholipase B having the same kind of activity can be obtained.

For example, the above Streptomycess sp. NA684 strain (Accession No .: NITE BP-1015) secretes phospholipase B outside the cells by liquid culture in an appropriate nutrient medium. A supernatant obtained by lyophilization, salting out, organic solvent or the like can be produced as an enzyme preparation. The Streptomyces sp. NA684 strain has a polynucleotide represented by the nucleotide sequence of SEQ ID NO: 4 in DNA.

In addition, it has been confirmed through experiments that Streptomyces schattanoogensis NBRC12754 has the same activity as NA684 strain. Therefore, Streptomyces sp. NA684 strain and Streptomyces chattanogensis show similar activities.

The microorganism that can be used for the production of the enzyme preparation is not limited to the Streptomyces sp. NA684 strain, and may be a microorganism that belongs to the genus Streptomyces and can produce phospholipase B. In addition, natural or artificial mutants of these species or gene fragments necessary for the expression of PLB activity can be artificially extracted and used for the production of phospholipase B even in other species incorporating them. it can. Moreover, even if it does not belong to Streptomyces genus, if it is microorganisms which can produce said phospholipase B, it can also be used.

An example of an enzyme preparation using Streptomycessp. NA684 strain will be described.

Since this bacterium secretes phospholipase B outside the cell by liquid culture in a nutrient medium, the culture supernatant is treated with lyophilization, salting out, an organic solvent, or the treated product is immobilized. Thus, an enzyme preparation can be produced. More specifically, first, this bacterium is cultured in a suitable medium, for example, a medium containing a suitable carbon source, nitrogen source, and inorganic salts to secrete phospholipase B. Examples of the carbon source include starch and 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. It is done. Examples of the nitrogen source include organic nitrogen sources such as yeast extract, peptone, meat extract, corn steep liquor and soy flour, and inorganic nitrogen compounds such as ammonium sulfate, ammonium nitrate and urea. Examples of inorganic salts include sodium chloride, monopotassium phosphate, magnesium sulfate, manganese chloride, calcium chloride, and ferrous sulfate. The concentration of the carbon source is, for example, in the range of 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 phospholipase B 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 phospholipase B is sufficiently produced, and is preferably about 1 to 7 days, for example. Culturing can be performed preferably under aerobic conditions, for example, with aeration stirring or shaking.

Polypeptides contained in phospholipase B are fractionated by protein solubility (precipitation with organic solvents, salting out by ammonium sulfate, etc.); cation exchange, anion exchange, gel filtration, hydrophobic chromatography; chelate, dye, antibody It can be purified by appropriately combining methods such as affinity chromatography using the above. 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 polypeptide constituting phospholipase B can be estimated as a monomer by HPLC analysis and gel filtration chromatography analysis.

[Microorganisms]
Phospholipase A1, phospholipase A2 and phospholipase B can be produced by microorganisms. In particular, for phospholipase A1 and phospholipase B, a polypeptide can be artificially generated from the amino acid sequence information described in SEQ ID NO: 1 and SEQ ID NO: 2 and the base sequence information described in SEQ ID NO: 3 and SEQ ID NO: 4. Alternatively, an enzyme (polypeptide) obtained by artificial synthesis may be used for the production of a lysophospholipid-containing detergent. However, when an enzyme is produced by a microorganism, the above enzyme can be easily obtained.

As the microorganism, the above-mentioned bacteria belonging to the genus Streptomyces can be used, but a microorganism into which the above-described polynucleotide is introduced may be used. That is, the microorganism hybridizes under stringent conditions with a polynucleotide having the base sequence described in SEQ ID NO: 3 or SEQ ID NO: 4 and a base sequence complementary to the base sequence described in SEQ ID NO: 3 or SEQ ID NO: 4. A microorganism into which at least one polynucleotide selected from a polynucleotide having at least 75% sequence identity with the nucleotide sequence set forth in SEQ ID NO: 3 or SEQ ID NO: 4 has been introduced. The microorganism into which the above polynucleotide is introduced can be obtained by a vector or a transformant. That is, a transformant having the ability to produce phospholipase A1 or phospholipase B can be produced by introducing a polynucleotide or a vector into a host.

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: 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)”.

In order to express a polynucleotide encoding the above enzyme in a microorganism, 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. Regarding the vectors, promoters, and terminators that can be used in these various microorganisms, “Basic Course of Microbiology 8, Genetic Engineering, Kyoritsu Shuppan”, especially for actinomycetes, “PRACTICAL STREPTOMYCES GENETICS (Kieser et al., John Inns Foundation, 2000) It is possible to use this method. Moreover, it can be efficiently secreted and produced extracellularly by using a signal sequence as necessary. The signal sequence used at this time may be a phospholipase or another.

The host to be transformed is not particularly limited as long as it is an organism that can be transformed with a vector containing a polynucleotide encoding the enzyme and express the enzyme activity. For example, bacteria, actinomycetes, Bacillus subtilis, Escherichia coli, yeast, mold and the like can be mentioned. More specifically, for example, 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 as 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.

In addition to microorganisms, various host / vector systems have been developed in plants and animals. For example, insects such as moths (Nature 315, 592-594 (1985)), rapeseed, corn, potatoes, and other plants in large quantities. Systems for expressing heterologous proteins have been developed, and these may be used.

The obtained transformant can be used for enzyme production as described above. Specifically, the transformant is liquid-cultured in an appropriate nutrient medium, the expressed polypeptide is secreted outside the cell, and the culture supernatant is lyophilized, salted out, treated with an organic solvent, etc. Can be manufactured.

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

[Lysophospholipid-containing detergent]
The lysophospholipid-containing detergent according to the present invention hydrolyzes phospholipids into lysophospholipids by causing at least one of phospholipase A1 (PLA1), phospholipase A2 (PLA2) and phospholipase B (PLB) to act on the phospholipid as an enzyme. Can be obtained.

The phospholipid is not particularly limited, and examples thereof include egg yolk lecithin (L), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dimyristoyl-sn-glycerol. -3-phosphate (DMPA), 1,2-diacyl-sn-glycero-3-phospho- (1-rac-glycerol) (PG), L-α-phosphatidyl-L-serine (PS), 1,2- Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), L-α-phosphatidylinositol (PI), soy-derived L-α-phosphatidylcholine (SB-PC), soy lecithin (SBL), 1-palmitoyl-2 Oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1-palmitoyl-2-oleo Ru-sn-glycerol-3-phosphate (POPA), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho- rac- (1-glycerol) (POPG), 1- (1Z-octadecenyl) -2-arachidonoyl-sn-glycero-3-phosphoethanolamine (PlsPE (C18, 20: 4)), 1-hexadecyl-2- ( 9Z-octadecenoyl) -sn-glycero-3-phosphoethanolamine (PlsPE (C16, 18: 1)) and the like can be used.

As phospholipase A1 (PLA1), phospholipase A2 (PLA2), and phospholipase B (PLB), which are phospholipid-degrading enzymes, commercially available products can be used. Particularly, phospholipase A1 (PLA1) and phospholipase B (PLB) It is preferable to use those already described (see SEQ ID NOS: 1 to 4).

In the lysophospholipid-containing detergent, the concentration of lysophospholipid is preferably 0.2 mg / mL or more, more preferably 0.25 mg / mL or more, and even more preferably 0.3 mg / mL or more. .

The lysophospholipid-containing detergent can be produced as follows. First, an aqueous solution (enzyme reaction solution) containing phospholipid and a phospholipid-degrading enzyme (at least one of phospholipase A1, phospholipase A2 and phospholipase B) is prepared. Next, the enzyme reaction solution is adjusted to an optimum temperature (for example, 37 ° C.) of the phospholipid degrading enzyme, and the enzyme reaction is performed, for example, for 30 to 120 minutes. Then, if necessary, the enzyme reaction solution may be filtered through a filter paper or filter to obtain a lysophospholipid-containing detergent as a filtrate, or may be centrifuged to obtain a lysophospholipid-containing detergent as a supernatant. it can. Filtration is performed to remove unreacted substrates and impurities that cause turbidity, and is particularly effective when using a substrate with a large amount of impurities such as soybean lecithin (SBL). Filtration is unnecessary if turbidity is not a concern, especially when the substrate is egg yolk lecithin (L), and if there is an unreacted substrate, a sufficiently transparent lysophospholipid-containing detergent is obtained by filtration or centrifugation. (See Table 7).

The lysophospholipid-containing detergent obtained as described above is particularly prone to destroying cell membranes such as red blood cells and white blood cells constituting blood, such as blood adhering to medical devices such as endoscopes and other It is possible to sufficiently wash off dirt such as cells in a short time even at a low temperature, and is excellent in blood dirt washing effect. Moreover, since the lysophospholipid-containing detergent according to the present invention has very low toxicity to humans, medical devices after washing with such a lysophospholipid-containing detergent can be reused without further treatment. Is something that can be done. For example, in endoscopic and laparoscopic surgery, cells such as red blood cells and white blood cells attached to the lens at the tip of these devices are quickly dissolved and removed using a lysophospholipid-containing detergent, or coagulation that occurs in the body. The dissolved blood (blood clot) can be quickly dissolved and removed using a lysophospholipid-containing detergent.

[Purification of phospholipase A1]
(A) Microbial culture Streptomyces albidoflavus NA297 strain (accession number: NITE BP-1014) was used as the cell.

First, 500 mL of tryptic soy medium (Becton Dinkinson) was prepared, and 50 ml was dispensed into a 500 mL Erlenmeyer flask with a baffle, and further 1% soybean lecithin (SBL) and 0.1% tween (Tween). ) After adding 80, steam sterilization was performed at 121 ° C. for 15 minutes.

Then, an appropriate amount of the above-mentioned colonies of the cells grown on the plate medium was taken in advance and inoculated into a φ18 test tube (18 × 180 mm) containing 5 mL of tryptic soy medium (Becton Dinkinson) at 28 ° C. Shake culture until good growth was obtained. 0.5 ml of this culture solution was inoculated into 50 mL of the previously sterilized medium and cultured with shaking at 28 ° C. for 55 hours. The supernatant was recovered from this culture using a centrifuge.

(B) Ammonium sulfate fraction To the culture supernatant collected in (a) above, ammonium sulfate was added so as to be 80% (w / v) saturated, and the resulting precipitate was centrifuged (15,000 rpm, 30 minutes, 4 minutes). C). This precipitate was solubilized with 30 ml of 20 mM Tris-HCl buffer (pH 9.0) and dialyzed with 20 mM Tris-HCl buffer (pH 9.0) to obtain a crude enzyme solution.

(C) Toyopearl Phenyl-650M column chromatography To the crude enzyme solution obtained in (b) above, 1.5 M ammonium sulfate was added at a final concentration, and 20 mM Tris-HCl buffer (pH 8.0) containing 1.5 M ammonium sulfate was added. ) Was previously applied to a Toyopearl Phenyl-650M column (inner diameter 26 mm, height 38 mm, manufactured by Tosoh Corporation). After washing the column with the same buffer, the active fraction was eluted with a linear gradient of ammonium sulfate (from 1.5 M to 0 M).

(D) HiTrap SP HP column chromatography The active fractions obtained in (c) above were collected and concentrated and desalted using Viva spin (manufactured by Sartorius). To this, 20 mM MES-sodium hydroxide buffer (pH 6.0) was added. This was applied to a HiTrap SP (5 ml) column (manufactured by GE Healthcare Biosciences) pre-equilibrated with 20 mM MES-sodium hydroxide buffer (pH 6.0), and the column was washed with the same buffer. The active fraction was eluted with a linear gradient of sodium chloride (0M to 1M).

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

(F) SDS-PAGE
The active fraction eluted in (e) above was collected and analyzed by SDS-PAGE (15% (w / v) polyacrylamide gel). As a result, the elution fraction had a molecular weight of about 28,000 Da and a single band. Observed. As a result, an electrophoretically purified polypeptide was obtained from Streptomyces albidoflavus NA297 strain. This polypeptide was estimated to be monomeric by HPLC analysis and gel filtration chromatography analysis.

(G) Analysis of amino acid sequence Using the above polypeptide, the N-terminal amino acid sequence was analyzed by a protein sequencer. The internal amino acid sequence was analyzed by nanoLC-MS / MS. The analysis confirmed that the N-terminal amino acid sequence of the purified polypeptide obtained from Streptomyces albidoflavus NA297 strain is that shown in SEQ ID NO: 1. Therefore, it was confirmed that said polypeptide is phospholipase A1.

In addition, as a result of predicting the isoelectric point based on the amino acid sequence of the enzyme using Genetyx, the isoelectric point of the above polypeptide was 6.06.

[Purification of phospholipase B]
(A) Microbial culture Streptomycessp. NA684 strain (Accession number: NITE BP-1015) was used as the cells.

First, NB medium “1% peptone (Becton Dinkinson), 1% meat extract (Kyokuto Pharmaceutical Co., Ltd.), 0.5% sodium chloride (Wako Pure Chemical Industries, Ltd.), pH 7.2 Prepare 300 mL, dispense 100 mL each into a 500 mL Erlenmeyer flask, add 1% soybean lecithin (SBL) and 0.1% Tween 80, and steam sterilize at 121 ° C for 15 minutes It was.

Then, an appropriate amount of the above-mentioned colonies of the cells grown on the plate medium was taken in advance and inoculated into a φ18 test tube (18 × 180 mm) containing 5 mL of tryptic soy medium (Becton Dinkinson) at 28 ° C. Shake culture until good growth was obtained. 1 ml of this culture solution was inoculated into 100 mL of the previously sterilized medium and cultured with shaking at 28 ° C. for 108 hours. The supernatant was recovered from this culture using a centrifuge.

(B) Ammonium sulfate fraction To the culture supernatant collected in (a) above, ammonium sulfate was added so as to be 80% (w / v) saturated, and the resulting precipitate was centrifuged (10,000 rpm, 30 minutes, 4 minutes). C). This precipitate was solubilized and dialyzed against 20 mM Tris-HCl buffer (pH 9.0) to obtain a crude enzyme solution.

(C) DEAE-Toyopearl column chromatography The “DEAE-Toyopearl 650M column” (inner diameter 26 mm, high height) obtained by equilibrating the crude enzyme solution obtained in (b) above with 20 mM Tris-HCl buffer (pH 9.0) in advance. To 55 mm, manufactured by Tosoh Corporation). After washing the column with the same buffer, the active fraction was eluted with a linear gradient of sodium chloride (from 0 M to 1 M).

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

(E) RESOURCE PHE column chromatography The active fractions obtained in (d) above were collected and concentrated and desalted using Viva spin. To this, 20 mM Tris-HCl buffer (pH 8.0) containing 1 M ammonium sulfate was added. The column was applied to a “RESOURCE PHE” (1 ml) column (manufactured by GE Healthcare Biosciences) pre-equilibrated with 20 mM Tris-HCl buffer (pH 8.0) containing 1 M ammonium sulfate, and the column was washed with the same buffer. Thereafter, the active fraction was eluted with a linear gradient of ammonium sulfate (from 1 M to 0 M).

(F) Mono S column chromatography The active fractions obtained in (e) above were collected and concentrated and desalted using Viva spin. To this was added 20 mM female-sodium hydroxide buffer (pH 6.0). After applying to a “Mono S” (1 ml) column (manufactured by GE Healthcare Biosciences) pre-equilibrated with 20 mM female-sodium hydroxide buffer (pH 6.0), the column was washed with the same buffer, The active fraction was eluted with a linear gradient of sodium chloride (0M to 0.5M).

(G) SDS-PAGE
The active fraction eluted in (e) above was collected and analyzed by SDS-PAGE (12% (w / v) polyacrylamide gel). As a result, in the eluted fraction, a single band with a molecular weight of about 39,000 Da was obtained. Observed. This gave a polypeptide that was electrophoretically purified to a single one. This polypeptide was estimated to be monomeric by HPLC analysis and gel filtration chromatography analysis.

(H) Analysis of amino acid sequence Using the above polypeptide, the N-terminal amino acid sequence was analyzed by a protein sequencer. The internal amino acid sequence was analyzed by nanoLC-MS / MS. The analysis confirmed that the N-terminal amino acid sequence of the purified polypeptide obtained from Streptomyces sp. NA684 strain was as shown in SEQ ID NO: 2. Therefore, it was confirmed that said polypeptide B is phospholipase B.

In addition, as a result of predicting the isoelectric point based on the amino acid sequence of the enzyme using Genetyx, the isoelectric point of the above polypeptide was 6.4.

[Microbial DNA]
The base sequence encoding the polypeptide of SEQ ID NO: 1 is the base sequence of SEQ ID NO: 3, and therefore, the Streptomyces albidoflavus NA297 strain is presumed to have the polynucleotide of SEQ ID NO: 3 as DNA. . And when the DNA of the core region in this Streptomyces albidoflavus was amplified by PCR and analyzed, the polynucleotide of SEQ ID NO: 3 was confirmed. Therefore, it was confirmed that the Streptomyces albidoflavus NA297 strain has the polynucleotide of SEQ ID NO: 3 as part of the DNA.

The base sequence encoding the polypeptide of SEQ ID NO: 2 is the base sequence of SEQ ID NO: 4. Therefore, it is estimated that Streptomyces sp. NA684 strain has the polynucleotide of SEQ ID NO: 4 as DNA. And when the DNA of the core region in this Streptomyces sp was amplified by PCR and analyzed, the polynucleotide of SEQ ID NO: 4 was confirmed. Therefore, it was confirmed that Streptomyces sp. NA684 strain has the polynucleotide of SEQ ID NO: 4 as part of the DNA.

[Phospholipase activity]
About said phospholipase A1 and phospholipase B, the phospholipase activity and activity conditions were confirmed by the following method.

(A) Phospholipase A1
10 g of 0.02% (w / v) Triton X-100 (manufactured by Nacalai Tesque) dissolved in 1 g of egg yolk phosphatidylcholine (L-α-phosphatidylcholine (egg yolk)), 10 % (W / v) egg yolk phosphatidylcholine was prepared. To 0.025 mL of 10% (w / v) egg yolk phosphatidylcholine, 0.060 mL of 0.2 M Tris-HCl buffer (pH 8.0), 0.5 M EDTA disodium (manufactured by Wako Pure Chemical Industries, Ltd.) 005 mL was added. And after preheating at 37 degreeC for 5 minute (s), 0.010 mL of the sample containing phospholipase A1 was added, and it was made to react at 37 degreeC for 5 minutes. Here, the mass of phospholipase A1 with respect to 100 parts by mass of phospholipid is 4 parts by mass when the specific gravity of 0.01 ml is all enzyme, but the mass of phospholipase A1 varies depending on the purity of phospholipase A1 in the sample used. Therefore, attention was paid to the adjustment of the addition amount of the phospholipase A1-containing sample. After the enzyme reaction, the enzyme reaction was stopped by heating at 100 ° C. for 5 minutes. After stopping the reaction, the amount of free fatty acid contained in 5 μL of the reaction solution was determined using the “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. Measured as follows. The amount of enzyme that produces 1 μmol of free fatty acid per minute was defined as 1 unit.

This enzyme test confirmed that phospholipase A1 exhibits PLA1 activity.

As a pH condition, it was confirmed that phospholipase A1 exhibits strong PLA1 activity at least in the range of pH 4.0 to 10.5.

As the temperature condition, it was confirmed that phospholipase A1 exhibits strong PLA1 activity in the range of at least 20 to 60 ° C.

When the purified enzyme obtained as described above was subjected to X-ray crystallographic analysis, the three-dimensional structure (ribbon model) of phospholipase A1 derived from Streptomyces albidoflavus was as shown in FIG. 6A. It turned out to be. The resolution of the X-ray crystal structure analysis was 2.2 mm. From this three-dimensional structure, it was found that the above phospholipase A1 has seven α-helices, six β-sheets, and three SS bonds (disulfide bonds). And as for said phospholipase A1, as shown to FIG. 6B, it is thought that Ser11, Ser216, and His218 are mainly located in an active center, and catalyze the hydrolysis reaction of phospholipid. 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.

(B) Phospholipase B
1 g of dimyristoyl phosphatidic acid (manufactured by Funakoshi Co., Ltd.) was dissolved in 10 mL of 10% (w / v) Triton X-100 (manufactured by Nacalai Tesque Co., Ltd.), and 10% (w / v) dimyristoyl phosphatidic acid was dissolved. Prepared. To 0.005 mL of this 10% (w / v) dimyristoyl phosphatidic acid, 0.025 mL of 0.2 M Tris-HCl buffer (pH 8.4) and 0.5 M EDTA disodium (manufactured by Wako Pure Chemical Industries, Ltd.) 0.002 mL and 0.063 mL of distilled water were added. And after preheating at 37 degreeC for 5 minute (s), 0.005 mL of the sample containing phospholipase B was added, and it was made to react at 37 degreeC for 5 minutes. Here, the mass of phospholipase B with respect to 100 parts by mass of phospholipid is 2 parts by mass when the specific gravity is 0.001 with all the enzyme being 0.005 ml, but the mass of phospholipase B varies depending on the purity of phospholipase B in the sample used. Therefore, attention was paid to the adjustment of the addition amount of the phospholipase B-containing sample. After the enzyme reaction, the enzyme reaction was stopped by heating at 100 ° C. for 5 minutes. After stopping the reaction, the amount of free fatty acid contained in 5 μL of the reaction solution was determined using the “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. Measured as follows. The amount of enzyme that produces 1 μmol of free fatty acid per minute was defined as 1 unit.

This enzyme test confirmed that phospholipase B exhibits PLB activity.

As the pH condition, it was confirmed that phospholipase B exhibits strong PLB activity at least in the range of pH 6.0 to 10.5.

As the temperature condition, phospholipase B was confirmed to exhibit strong PLB activity in the range of at least 20 to 65 ° C.

[Effective concentration of LPC]
In producing a lysophospholipid-containing detergent, a commercially available lysophospholipid was used, and the concentration of lysophospholipid effective for obtaining a high detergency was examined in advance as follows.

Multiple samples of commercially available LPC (L-α-Lysophosphatidylcholine) were prepared from human whole blood (Kohjin Bio, with anticoagulant) diluted approximately 100-120 times with physiological saline. , See Table 12) 6 μL of an aqueous solution was added, immediately mixed, and allowed to stand at room temperature (about 20 ° C.) for 5 minutes. Then, centrifugation was performed, the supernatant was taken, and the hemolysis rate was examined by measuring the absorbance (A ₅₇₆ ) at a wavelength of 576 nm. The hemolysis rate was calculated from the following formula. This examined the effective concentration of LPC. Although hemolysis usually means a phenomenon in which red blood cells are destroyed, in this specification, it means a phenomenon in which cells other than red blood cells in blood are also destroyed.

Hemolysis rate = A / B
A = (A _{576 of} blood containing sample solution) − (A _{576 of} blood containing physiological saline)
B = (A _{576 of} blood containing distilled water) − (A _{576 of} blood containing physiological saline)
FIG. 1 is a graph showing the relationship between the LPC concentration and the hemolysis rate. From this graph, it was found that the effective concentration of LPC was 0.2 mg / mL or more, preferably 0.25 mg / mL or more, more preferably 0.3 mg / mL or more.

[LPC heat resistance test]
An aqueous LPC solution (0.25 mg / mL) was heat-treated at 100 ° C. for different times, and then the hemolysis rate was calculated in the same manner as described above to examine the heat resistance of LPC. As a result, as shown in FIG. 2, it was confirmed that even when an LPC aqueous solution (0.25 mg / mL) was heat-treated at 100 ° C. for 6 hours, the hemolysis ability was not changed and the heat resistance was extremely excellent.

[Differences depending on the type of lysophospholipid]
Human whole blood (Kohjin Bio, containing an anticoagulant) was placed in a ceramic (white porcelain) plate, and the blood was coagulated by heating at about 70-80 ° C. for about 15 minutes. Thereto, 40 μL of a commercially available lysophospholipid aqueous solution (0.1 mg / mL) (see Table 12) was dropped and allowed to stand for 10 minutes, and then washed with 400 μL of distilled water. Judged. As a result, as shown in Table 1, LPG, LPC, and LPA showed almost the same detergency. Speaking strongly, the detergency was strong in the order of LPG>LPC>LPA> LPI = LPS> LPE.

[Change in lysophospholipid production over time]
Using phosphatidylcholine (PC) (see Table 12) as a substrate and phospholipase A1 (PLA1) as an enzyme, an enzyme reaction was performed at 37 ° C. for 30 minutes at the reaction solution composition shown in Table 2, and LPC (1-acyl and The total change in 2-acyl lysophosphatidylcholine) formation was examined over time. The result suggested that an enzyme reaction of about 20 minutes was preferable as shown in FIG. As a result, it was found that in producing a lysophospholipid-containing detergent, it is necessary to trace the enzyme reaction and stop at a reaction time at which the yield of lysophospholipid is maximized.

[Lysophospholipid-containing detergent]
(Sample 1)
Sample 1 was prepared as follows. After soy lecithin (SBL) as a substrate and phospholipase A1 (PLA1) or phospholipase B (PLB) as an enzyme, an enzyme reaction was performed at 37 ° C. for 60 to 120 minutes at the reaction solution composition shown in Table 3. The enzyme reaction solution was filtered through various filter membranes (see Table 4 described later) to obtain a filtrate. This filtrate was designated as Sample 1.

(Sample 2)
Sample 2 was prepared as follows. Using soybean lecithin (SBL) as a substrate and phospholipase A1 (PLA1) or phospholipase B (PLB) as an enzyme, an enzyme reaction was carried out at 37 ° C. for 60 to 120 minutes at the reaction solution composition shown in Table 3. After extracting this enzyme reaction solution with chloroform / methanol (volume ratio 2/1), the chloroform layer containing lysophospholipid was recovered, the solvent was removed under reduced pressure, and distilled water was added to about 10-18 mg of the obtained lysophospholipid. Sample 2 was obtained at 0.3 g / mL.

(Sample 3)
Sample 3 was prepared as follows. Using an egg yolk lecithin (L) as a substrate and phospholipase A1 (PLA1) or phospholipase B (PLB) as an enzyme and carrying out an enzyme reaction at 37 ° C. for 60 minutes with the reaction solution composition shown in Table 3, Similarly, 0.663 g of the extract obtained by extraction with chloroform / methanol was purified by silica gel column chromatography. 13.3 g of silica gel (Wakogel C-200 Model No. 237-00075, manufactured by Wako Pure Chemical Industries, Ltd.) having a mass 20 times that of the extract was packed in a column (inner diameter: 23 mm) (gel height: 85 mm), and eluent (chloroform / methanol / Elution was performed with water at a volume ratio of 70/25/3), and 8 mL each was collected. Among these, the fraction containing lysophospholipid was collected, and the solvent was removed under reduced pressure. As a result, 18 mg of lysophospholipid could be obtained from 1.05 g of the substrate. Sample 3 was obtained by adding distilled water to the lysophospholipid to a concentration of 20 to 100 mg / mL.

The material of the filter membrane optimum for filtration was examined for sample 1, and the detergency (hemolytic power) of blood stains was examined in the same manner as described above for the filtrate (lysophospholipid-containing detergent) obtained by filtration. As shown in Table 4, the filtrate filtered using a PES (Polyethersulphone) membrane and a PVDF (PolyVinylidene DiFluoride) membrane having a pore diameter of 0.45 μm has little turbidity and strong detergency. From these results, it was found that these two types of membranes are preferably used for the filtration of the enzyme reaction solution. There are other filter membrane pore diameters used for filtration, such as 0.2 μm and 0.8 μm, which may be appropriately selected according to the purpose. In addition, almost the same detergency was obtained for the lysophospholipid-containing detergents prepared without adding Triton X-100 contained in the reaction solutions shown in Table 3.

The enzyme used to produce the lysophospholipid-containing detergent may be phospholipase A1 (PLA1), phospholipase A2 (PLA2), phospholipase B (PLB), or a mixture thereof. For example, soy lecithin (SBL) or egg yolk lecithin (L) is used as a substrate, and commercially available phospholipase A2 (PLA2) is used, and an enzyme reaction is performed at 37 ° C. for 60 minutes using the reaction solution composition shown in Table 5. The detergency of the filtrate obtained by filtering the reaction solution using a PVDF membrane was almost the same as the detergency of the filtrate obtained in the same manner using phospholipase A1 (PLA1) and phospholipase B (PLB).

[Detergency of samples 1 to 3]
As shown in Table 6, sample 1-1 obtained by filtration through a PVDF membrane with an enzyme reaction time of 60 minutes, sample 1-2 obtained by filtration through a PVDF membrane with an enzyme reaction time of 90 minutes, enzyme Samples 1-3 obtained by filtration through a PVDF membrane with a reaction time of 120 minutes all showed stronger detergency than commercially available LPC (0.1 mg / mL) (see Table 12).

In addition, the cleaning power of Sample 1, Sample 2, and Sample 3 was almost the same.

In addition, sample 2 was appropriately diluted with distilled water, and the effective concentration of lysophospholipid when blood stains were washed was examined. As a result, it was found that a concentration of at least 30 mg / mL or more was necessary.

In addition, the sample 3 was appropriately diluted with distilled water to examine the effective concentration of lysophospholipid when washing blood stains and the like. As a result, it was found that the cleaning power was the same as that of

Samples

1 and 3 for Samples 3-1 to 3-5.

From the above, it was confirmed that the method for producing Sample 1 was excellent as a method for producing a lysophospholipid-containing detergent at a low cost.

Also, as shown in Table 7, when a lysophospholipid-containing detergent is produced using soybean lecithin (SBL) as a raw material (substrate), filter filtration with a PVDF membrane is necessary. On the other hand, when producing a lysophospholipid-containing detergent using egg yolk lecithin (L) as a raw material (substrate), the supernatant liquid after centrifugation can be used as it is, without performing filter filtration in particular. May be. In addition, the lysophospholipid-containing detergent obtained from soybean lecithin (SBL) is yellow and slightly turbid, but the lysophospholipid-containing detergent obtained from egg yolk lecithin (L) is only slightly cloudy and turbid. There are few features. In addition, all the lysophospholipid-containing detergents had the same detergency.

[Cleaning effect confirmation test 1]
Using Sample 1-1 (obtained by filtration through a PVDF membrane with an enzyme reaction time of 60 minutes) and a commercially available endoscope / surgical instrument cleaner (Daisho Shoji Co., Ltd., Medipol EX-1) A test was conducted to confirm the cleaning effect of the blood contamination test piece (NITI-ON, cleaning indicator TOSI). Specifically, the sample 1-1 and a commercially available cleaning agent were dropped on the blood contamination site on the blood contamination test piece, and left at room temperature (about 20 ° C.) for 10 minutes. Thereafter, as a result of washing with water, blood stains were confirmed to remain in the commercially available cleaning agent, whereas blood stains and fibrin were not observed at all in Sample 1-1. Also, as shown in Table 8, Sample 1-2 (obtained by filtration through a PVDF membrane with an enzyme reaction time of 90 minutes), Sample 1-3 (filtered through a PVDF membrane with an enzyme reaction time of 120 minutes) Sample 1-4 (obtained by filtration through a PVDF membrane with an enzyme reaction time of 30 minutes), Sample 1-5 (obtained by filtration through a PVDF membrane with an enzyme reaction time of 40 minutes) As a result of performing the same test as described above, the detergency was almost the same. From this, it was found that the enzyme reaction time is sufficient for 30 minutes.

[Cleaning effect confirmation test 2]
A blood contamination test piece (NITI-ON, washing indicator TOSI) is placed in two sample bottles, and sample 1-1 (obtained by filtering through a PVDF membrane with an enzyme reaction time of 60 minutes) in one sample bottle. Put a commercially available cleaning agent for endoscopes and surgical instruments (Daisho Shoji Co., Ltd., Medipol EX-1) in the other sample bottle, and place both sample bottles at room temperature (24 ° C) and 40 ° C. A test for confirming the cleaning effect was performed by stirring with a stirrer (about 500 rpm). As a result, as shown in Tables 9 and 10, at 40 ° C., both the sample 1-1 and the commercially available cleaning agent could be completely removed to fibrin on the basal plane of blood contamination in 10 minutes. On the other hand, at room temperature (24 ° C.), the sample 1-1 was able to clean blood stains completely with a cleaning time of about 13 minutes and the commercially available cleaning agent with about 15 minutes.

From the above, the cleaning power of Sample 1-1 and the commercially available cleaning agent is equivalent at 40 ° C, but there is a difference in the cleaning power of both at room temperature (24 ° C), which is lower than that of the commercially available cleaning agent. Thus, it was revealed that Sample 1-1 had higher detergency. As a result, it was found that the lysophospholipid-containing cleaning agent is not easily affected by temperature and exhibits a sufficient cleaning effect in a wide temperature range.

[Cleaning effect confirmation test 3]
A test was conducted to confirm the cleaning effect of blood stains attached to the endoscope (manufactured by Olympus Corporation) and tweezers. Specifically, the tip of the endoscope (where the camera and forceps are located) and stainless steel tweezers are immersed in whole human blood (Kohjin Bio, no anticoagulant), and the blood is inserted into the endoscope tip and stainless steel tweezers. Attached to the tip. Then, the blood was solidified by leaving still at room temperature for 3 hours, and the blood stain at the time of operation was reproduced. Next, about 30 mL of sample 1-1 (obtained by filtering with a PVDF membrane with an enzyme reaction time of 60 minutes) is placed in the sample bottle, and the blood-contaminated endoscope tip and stainless tweezer tip The sample was immersed and lightly stirred in the sample bottle at room temperature (24 ° C.). As a result, blood stains at the endoscope tip and the stainless tweezer tip were completely removed within a few seconds.

[Cleaning effect confirmation test 4]
A test was conducted to confirm the cleaning effect of blood stains remaining in the tube (about 2 mm) into which the endoscopic forceps are inserted. Specifically, instead of the endoscopic forceps insertion tube, a transparent plastic tube (TYGON R-3603, ID, 3/32 ", OD, 5/32", Wall, 1/32 ", inner diameter 2.3 mm) The test for confirming the cleaning effect was carried out as follows: First, the inside of two transparent plastic tubes was filled with human whole blood (Kohjin Bio, no anticoagulant) for 10 minutes at room temperature (24 ° C.). After being allowed to stand, the blood was solidified by heating for 2 hours at 40 ° C. Next, sample 1-1 (obtained by filtration through a PVDF membrane with an enzyme reaction time of 60 minutes) was sucked up with a syringe. Filled one tube and allowed to stand for 2 minutes at room temperature (24 ° C.) Similarly, suck up a commercially available endoscope / surgical instrument cleaner (Daisho Co., Ltd., Medipol EX-1) with a syringe. Like the other Chu The tube was filled and allowed to stand at room temperature for 2 minutes, after which the liquid in both tubes was removed and washed with water, and as a result, as shown in Table 11, sample 1-1 was either room temperature (24 ° C.) or 40 ° C. While blood stains could be completely removed, commercially available cleaning agents had significant residual blood stains and could not completely remove blood stains. It was found that the cleaning ability was extremely excellent.

[Clot degradation test]
Take a blood clot of human whole blood (Kohjin Bio, with anticoagulant) on a ceramic (white porcelain) plate, add an appropriate amount of Sample 1-1 shown in Table 6 and leave it at room temperature, then clot The detergency of sample 1-1 was determined by visually confirming the degree of disintegration (decomposition). Here, clot disintegration (decomposition) means that the clot's firmness is reduced and the clot is loosened and becomes smaller (subdivided). As a result, the clot firmness decreased in about 10 minutes, and the clot was loosened and subdivided. Further, in order to decompose the clot in a short time, physical treatment with an ultrasonic oscillator (ultrasonic vibrator) (Tomy Seiko Co., Ltd., Handy Sonic UR-21P, 28 kHz · 10 W) was used in combination. As a result, the blood clot can be instantaneously decomposed and further easily removed by suction with a syringe (eg, Terumo Corporation, NN-2138R, 21G × 1 1/2 ″, 0.80 × 38 mm). From the above, for example, by simultaneously injecting a lysophospholipid-containing cleaning agent from the forceps of an endoscope or laparoscope, and simultaneously using physical treatment with an ultrasonic oscillator (ultrasonic vibrator), It is considered that the blood clot can be decomposed and removed more rapidly.

Table 12 shows the manufacturers and product numbers of the substrates and enzymes described in this specification.

[Streptomyces albidoflavus NA297 (Accession number: NITE BP-1014)]
Accession Number: NITE BP-1014
Name of depositary institution: National Institute for Product Evaluation Technology Patent Microorganism Depositary (NPMD)
Name of depositary institution: Japan 2-5-8 Kazusa Kamashitsu, Kisarazu City, Chiba Prefecture 292-0818, Japan
Date of deposit: January 26, 2011.
[Streptomyces sp. NA684 (Accession number: NITE BP-1015)]
Accession Number: NITE BP-1015
Name of depositary institution: National Institute for Product Evaluation Technology Patent Microorganism Depositary (NPMD)
Name of depositary institution: Japan 2-5-8 Kazusa Kamashitsu, Kisarazu City, Chiba Prefecture 292-0818, Japan
Date of deposit: January 26, 2011.

SEQ ID NO: 1: full length of PLA1 (polypeptide) SEQ ID NO: 2: PLA1 gene SEQ ID NO: 3: full length of PLB (polypeptide) SEQ ID NO: 4: PLB gene

Claims

A lysophospholipid-containing detergent characterized by containing lysophospholipid obtained by hydrolyzing the phospholipid by allowing at least one of phospholipase A1, phospholipase A2 and phospholipase B to act on the phospholipid as an enzyme.
A lysophospholipid-containing detergent, comprising lysophospholipid obtained by hydrolyzing the phospholipid by allowing at least one of the following phospholipase A1 and phospholipase B to act on the phospholipid as an enzyme.
Phospholipase A1: containing the following polypeptide (a1-1), (a1-2) or (a1-3), wherein the sn-1 position acyl group of the phospholipid is preferentially compared to the sn-2 position acyl group Disconnect.
(A1-1) A polypeptide having the amino acid sequence set forth in SEQ ID NO: 1.
(A1-2) 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: 1.
(A1-3) A polypeptide having at least 75% homology with the amino acid sequence set forth in SEQ ID NO: 1.
Phospholipase B: contains the following polypeptide (b1-1), (b1-2) or (b1-3), and cleaves both the sn-1 position acyl group and the sn-2 position acyl group of the phospholipid.
(B1-1) A polypeptide having the amino acid sequence set forth in SEQ ID NO: 2.
(B1-2) 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.
(B1-3) A polypeptide having at least 75% homology with the amino acid sequence set forth in SEQ ID NO: 2.
A lysophospholipid-containing detergent, comprising lysophospholipid obtained by hydrolyzing the phospholipid by allowing at least one of the following phospholipase A1 and phospholipase B to act on the phospholipid as an enzyme.
Phospholipase A1: containing the following polypeptide (a2-1), (a2-2) or (a2-3), wherein the sn-1 position acyl group of the phospholipid is preferentially compared to the sn-2 position acyl group Disconnect.
(A2-1) A polypeptide produced from a microorganism having the nucleotide sequence set forth in SEQ ID NO: 3 as a polynucleotide.
(A2-2) A polypeptide produced from a microorganism having a polynucleotide that hybridizes with a base sequence complementary to the base sequence shown in SEQ ID NO: 3 under stringent conditions.
(A2-3) A polypeptide produced from a microorganism having a polynucleotide having at least 75% sequence identity with the nucleotide sequence set forth in SEQ ID NO: 3.
Phospholipase B: Contains the following polypeptide (b2-1), (b2-2) or (b2-3), and cleaves both the sn-1 and sn-2 acyl groups of the phospholipid.
(B2-1) A polypeptide produced from a microorganism having the nucleotide sequence set forth in SEQ ID NO: 4 as a polynucleotide.
(B2-2) A polypeptide produced from a microorganism having a polynucleotide that hybridizes under stringent conditions with a base sequence complementary to the base sequence set forth in SEQ ID NO: 4.
(B2-3) A polypeptide produced from a microorganism having a polynucleotide having at least 75% sequence identity with the base sequence set forth in SEQ ID NO: 4.
The lysophospholipid-containing cleaning agent according to any one of claims 1 to 3, wherein the enzyme is derived from a microorganism belonging to the genus Streptomyces.