JP2005328781A - New phospholipase c - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain a new phospholipase C (PLC) derived from a new microorganism. <P>SOLUTION: The new phospholipase C is derived from Pseudomonas sp. KS3.2 strain (FERM P-19732). The phospholipase C has high specificity to phosphatidylcholine, and low activity to other phospholipids, and comprises a subunit having about 17-19 kDa molecular weight. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a novel phospholipase C. More particularly, it relates to a novel phospholipase C derived from a novel microorganism.

  Phospholipase C [EC 3.1.4.3] (hereinafter referred to as PLC) acts on phospholipids, hydrolyzes its phosphate ester bond, and 1,2-diacylglyceride (hereinafter referred to as 1,2-DG) and phosphoryl. It is a hydrolase that liberates bases. PLC has been reported to be useful in the food industry such as bread production and the oil and fat industry such as ceramide production (Patent Documents 1 and 2). In recent years, signal transduction systems have been widely studied in the field of cell biology, and the demand for PLC as a research reagent is also increasing. Thus, development of PLC as an industrial enzyme and research reagent is strongly desired.

So far, PLC derived from animal liver and spleen, Clostridium perfriongens, Bacillus cereus, Pseudomonas fluorescens, Pseudomonas schuylkilliensis PLC derived from microorganisms such as) are known (Non-Patent Documents 1 to 5). However, PLCs derived from animal internal organs have not been industrialized due to the complicated supply of organs as raw materials, complicated enzyme purification, and religious reasons. For microorganism-derived PLCs, research related to pathogenicity has been conducted, but research for industrial use has hardly been conducted (Non-patent Document 1). As for industrial use, PLC derived from Aspergillus niger has been reported (Patent Document 3), but since its optimum pH is as low as 2 to 5, it is actually difficult to use.
JP-A-8-266213 JP-A-6-61247 JP 2000-166543 A Titball RW, "Microbiol. Rev.", 1993, 57, pp.347-366 Crevel IUS et al., “Eur. J. Biochem.”, 1994, 224, pp.845-852 Ivanov A. et al., “Microbiologica”, 1996, 19, pp. 113-121 Osamu Doi et al., “Biochim. Biophys. Acta”, 1971, 248, pp.234-244 Motoo Arai et al., “Journal of Japanese Society for Agricultural Chemistry”, 1974, 48, pp.409-415

  An object of the present invention is to provide a novel phospholipase C (PLC).

  As a result of investigating whether or not various oil-degrading bacteria have PLC activity, the present inventors have found that Pseudomonas sp. KS3.2 strain has a PLC having hydrolytic activity specifically for phosphatidylcholine. It was discovered that it can be produced. This bacterium is deposited as FERM P-19732 at the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology.

The present invention provides a novel phospholipase C having the following characteristics (1) to (8):
(1) Action: acts on phospholipids and hydrolyzes its phosphate ester bond to release 1,2-diacylglycerides and phosphoryl bases;
(2) Substrate specificity: Acts specifically on phosphatidylcholine but has little effect on phosphatidic acid, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, and sphingomyelin, and on p-nitrophenylphosphorylcholine Has virtually no effect;
(3) Optimal pH: 7.2;
(4) Stable pH: 8.2 to 8.8;
(5) Optimal temperature: about 50 ° C .;
(6) Stable temperature: 20 ° C. or lower;
(7) Inhibition: inhibited by metal ions excluding calcium, phenylmethanesulfonyl fluoride, dithiothreitol, 2-mercaptoethanol, SDS, and EDTA, but not iodoacetamide; and (8) Molecular weight: about 17 kDa ~ 19 kDa (SDS-PAGE).

  In a preferred embodiment, the phospholipase C is derived from Pseudomonas sp. KS3.2 strain (FERM P-19732).

  The present invention also provides Pseudomonas sp. KS3.2 strain (FERM P-19732) having the ability to produce phospholipase C.

  According to the present invention, there is provided a PLC that specifically acts on phosphatidylcholine (lecithin) that is most abundant as a phospholipid, and a Pseudomonas sp. KS3.2 strain that can produce the PLC. .

(Strain producing phospholipase C)
The Pseudomonas sp. KS3.2 strain of the present invention is a strain obtained by screening 256 strains of oil-degrading bacteria obtained from the soil by the present inventors for PLC activity, and independently. Deposited as FERM P-19732 on March 11, 2004 at the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology.

The Pseudomonas sp. KS3.2 strain has the following mycological properties:
Cell shape and size: Neisseria gonorrhoeae, 0.7-0.8 × 1.5-2.0 μm
Gram staining:-
Sporulation:-
Mobility: +
Colony morphology: round, smooth all edges, low convex, shiny, yellow (medium: Nutrient Agar; 24 hours culture)
Culture temperature: 28 ° C: +, 37 ° C:-, 42 ° C:-, 45 ° C:-
Catalase: +
Oxidase:-
Urease:-
Acid / gas production (glucose):-/-
O / F test:-/-
Growth under anaerobic conditions:-
Viability on MacConkey agar medium: +
Fluorescent dye production on King's B agar medium: +
Other physiological properties: shown in Table 1 below.

  Chemical taxonomic properties: In the homology search of the full-length base sequence of 16S rRNA gene (1528 bp: SEQ ID NO: 1), 99.3% against Pseudomonas monteilii and Pseudomonas putida KT2440 With 99.2% homology.

(Phospholipase C of the present invention)
The phospholipase C (PLC) of the present invention is produced from a microorganism belonging to Pseudomonas sp., And preferably produced from Pseudomonas sp. KS3.2 strain. Further, as described later, these recombinant enzymes can also be used. In this case, the produced PLC may be accumulated in the cells, but is preferably secreted outside the cells.

(Manufacturing of Pseudomonas sp.-derived PLC)
The PLC of the present invention can be produced by the following commonly used culture means.

  The microorganism belonging to Pseudomonas sp. Is cultured in an appropriate medium containing lecithin for 1 to 5 days, preferably 1 to 3 days under aerobic conditions at 20 to 35 ° C., preferably around 28 ° C. . An example of a suitable medium containing lecithin is a lecithin-containing broth medium.

  Bacteria are removed from the obtained culture solution by filtration or centrifugation to obtain a filtrate or supernatant. The filtrate or supernatant thus obtained is subjected to enzyme purification methods commonly used by those skilled in the art, such as salting out with ammonium sulfate, solvent precipitation with ethanol, acetone, etc., ultrafiltration, ion exchange chromatography, etc. The PLC can be purified.

  For example, Pseudomonas sp. Strain KS3.2 is cultured in a lecithin-containing broth medium under aerobic conditions, and the cells are separated and removed from the obtained culture solution to obtain a culture supernatant. . The culture supernatant is then fractionated with ammonium sulfate, anion exchange chromatography (eg, DEAE-Sepharose Fast Flow column (Amersham Biosciences Corp.)), and then hydrophobic interaction chromatography (eg, Phenyl-Sepharose High performance). By purifying with a column (manufactured by Amersham Biosciences Corp.), a purified enzyme having a molecular weight of about 17 to 19 kDa can be obtained by SDS-polyacrylamide gel electrophoresis (hereinafter referred to as SDS-PAGE).

(Measurement of PLC activity)
Using phospholipid (typically phosphatidylcholine) as a substrate, the measurement is performed by quantifying the phosphate released by the enzymatic reaction. Specifically, 10 μL of enzyme solution, 0.2% (w / v) L-α-phosphatidylcholine (hereinafter referred to as PC), 0.285% (v / v) Triton X-100, and 46 mM Tris-HCl buffer (pH 8. Incubate 300 μL of the enzyme reaction solution containing 0) at 37 ° C. for 10 minutes, and heat the reaction solution at 100 ° C. for 10 minutes to stop the reaction. Using the boiled enzyme, the substrate and the buffer solution are similarly incubated, and the reaction is stopped by heating to make a blank. After completion of the reaction, 10 μL of this reaction solution was added to 190 μL of alkaline phosphatase solution (0.2 units alkaline phosphatase, 1 mM MgCl ₂ , 0.1 mM ZnCl ₂ , 50 mM glycine-NaOH buffer (pH 9.6)) at 37 ° C. for 20 minutes. Perform dephosphorylation. PLC activity is determined by quantifying the released phosphoric acid. 1U of PLC is the amount of enzyme that liberates 1 μmol of phosphoryl base per minute.

  When phosphatidic acid is used as a substrate, the phosphoric acid liberated is quantified immediately after the enzymatic reaction as described above, without being treated with alkaline phosphatase. In addition, when p-nitrophenylphosphorylcholine (hereinafter referred to as pNPPC), which is an artificial phospholipid, is used as a substrate, 50 μL of the enzyme solution is 1.45 mL of enzyme reaction solution (3.6 mM pNPPC, 50 mM Tris-HCl buffer (pH 7.2)). And incubated at 50 ° C. for 10 minutes before quantifying free p-nitrophenol. Again, 1U of PLC is the amount of enzyme that liberates 1 μmol of p-nitrophenol per minute.

(Physicochemical properties of PLC derived from Pseudomonas sp. KS3.2)
The physicochemical properties of the PLC derived from Pseudomonas sp. KS3.2 strain of the present invention are as follows (1) to (8).

(1) Action It acts on phospholipids and hydrolyzes its phosphate bond to release 1,2-diacylglycerides and phosphoryl bases.

(2) Substrate specificity Acts specifically on phosphatidylcholine but has little effect on phosphatidic acid, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, and sphingomyelin, and on p-nitrophenylphosphorylcholine Substantially does not work (see Example 6).

(3) Optimal pH: 7.2
When the relative activity of this enzyme was reacted at 37 ° C. for 10 minutes in various buffers having a final concentration of 46 mM at pH 4 to 11, the optimum pH was 7.2 as shown in FIG.

(4) Stable pH: 8.2 to 8.8
About this enzyme, after incubating at 5 ° C. for 3 hours in various buffers having a final concentration of 45 mM at pH 4 to 12, the residual activity at pH 7.2 and 50 ° C. was measured. As shown in FIG. Is 8.2 to 8.8.

(5) Optimal temperature: about 50 ° C
This enzyme was reacted at a temperature of 20 to 70 ° C. for 10 minutes in a final concentration of 46 mM Tris-HCl buffer (pH 7.2) containing 0.01% (v / v) Triton X-100 and 0.2 mM ammonium sulfate. When relative activity is measured, the optimum temperature is about 50 ° C. as shown in FIG.

(6) Stable temperature: 20 ° C. or less The enzyme was incubated at a temperature of 4 to 70 ° C. for 30 minutes in a Tris-HCl buffer solution (pH 7.2) having a final concentration of 46 mM, and then at pH 7.2 and 50 ° C. When the residual activity is measured, the stable temperature is 20 ° C. or less as shown in FIG.

(7) Inhibition This enzyme is inhibited by metal ions other than calcium, phenylmethanesulfonyl fluoride, dithiothreitol, 2-mercaptoethanol, SDS, and EDTA, but not iodoacetamide (see Example 7). .

  (8) Molecular weight: about 17 kDa to 19 kDa (SDS-PAGE) (see Example 3).

(Usage of PLC)
The PLC of the present invention has high substrate specificity for phosphatidylcholine (lecithin). Therefore, it can be used for removing phospholipids.

  As the usage form of the PLC of the present invention, (i) a PLC-producing cell is immobilized by a normal method, (ii) the PLC-producing cell is mechanically, enzymatically, surfactant or organic solvent, etc. Treated, or those immobilized, (iii) pulverized PLC-producing cells to remove crushing residues, or immobilized from it, (iv) enzyme purified product, or immobilize it (V) a culture medium from which a PLC-producing bacterium has been removed or a culture supernatant from which a PLC-producing microbial cell has been removed, or a product obtained by concentrating, drying and / or immobilizing it. The immobilization is performed by an ordinary immobilization method generally used by those skilled in the art, such as polyacrylamide, calcium alginate, carrageenan, ion exchange carrier, chitosan beads and the like.

(Example 1: Screening of PLC-producing microorganisms)
1% lecithin emulsified normal bouillon agar [1% (w / v) soybean lecithin, 0.5% (w / v) bile powder, 1% (w / v) extract beef, 1% (w / v ] When growing in bactopeptone, 0.5% (w / v) NaCl, 1.5% (w / v) agar, pH 7.2], the cloudy white halo around the colony due to the PLC released outside the cells Form. Utilizing this, primary screening was performed on 256 strains of oil-degrading bacteria obtained from soil. Each strain was cultured at 28 ° C. using the above 1% lecithin emulsified ordinary bouillon agar medium, and 29 strains that formed a cloudy halo were selected.

A secondary screen was then performed. The selected 29 strains were treated with 1% lecithin-containing normal broth medium [1% (w / v) extract beef, 1% (w / v) bactopeptone, 1% (w / v) soy lecithin, 0.5% (w / v v) NaCl, 0.025% (w / v) antifoam SI, pH 7.2] The cells were cultured at 28 ° C. for 24 hours with shaking at 166 rpm in 100 mL. 20 μL of the culture supernatant obtained by centrifugation (18760 × g, 5 minutes) was added to lecithin emulsified agar plate [0.3% (w / v) soybean lecithin, 9 mM CaCl ₂ · 2H ₂ O, 2.73 mM sodium taurocholate, 47 mM. In a well provided in Tris-HCl buffer (pH 8.0), 1.5% (w / v) agar], the plate was incubated at 37 ° C. for 24 hours, and 19 strains that formed a cloudy halo around the well were selected.

Furthermore, the tertiary screening was performed for 19 strains as follows. The selected 19 strains were cultured in 100 mL of a normal bouillon medium containing 1% lecithin for 24 hours at 28 ° C. with shaking at 166 rpm, and then a culture supernatant was obtained by centrifugation (18760 × g, 5 minutes). . 30 μL of culture supernatant of each strain, 300 μL of enzyme reaction solution containing 0.2% (w / v) PC, 9 mM CaCl ₂ · 2H ₂ O, 2.34 mM sodium deoxycholate, 41 mM Tris-HCl buffer (pH 8.0) And incubated at 37 ° C. for 30 minutes. The reaction was heated at 100 ° C. for 5 minutes to stop the reaction, and then an equal volume of chloroform / methanol (2: 1, v / v) was added and shaken vigorously for 1 minute. After centrifugation at 17860 × g for 5 minutes at 4 ° C., the chloroform layer was subjected to thin layer chromatography (TLC) using petroleum ether / diethyl ether / acetic acid (7: 3: 0.025, v / v) as a developing solvent. went. The product 1,2-diacylglyceride (hereinafter referred to as 1,2-DG) was detected by iodine vapor. On the other hand, TLC using n-butanol / ethanol / acetic acid / water (8: 2: 1: 3, v / v) as a developing solvent was performed on the dried concentrate of the reaction solution, and another product, phosphorylcholine, was obtained. Detection was performed. Thus, as a result of examining the presence or absence of the production | generation of 1, 2-DG and phosphorylcholine, it turned out that 1 strain | stump | stock called KS3.2 is producing PLC out of a microbial cell.

(Example 2: Properties of Pseudomonas sp. KS3.2 strain)
The physiological property test of KS3.2 strain was commissioned to MC Japan Co., Ltd. As a result, the KS3.2 strain had the physiological properties as described above. Furthermore, the full-length base sequence (1528 bp: SEQ ID NO: 1) of the 16S rRNA gene was analyzed by consigning to MC Japan Ltd. As a result of homology search for all DNA databases of Japan DNA Data Bank using BLAST as a search algorithm, 99.3% for Pseudomonas monteilii and Pseudomonas putida KT2440 99.2% homology was observed. From the above, it became clear that the KS3.2 strain is a novel strain belonging to Pseudomonas. Therefore, the PLC-producing bacterium according to the present invention is named Pseudomonas sp. KS3.2 strain, and is registered with the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology on March 11, 2004. Deposited as -19732.

(Example 3: Purification of PLC)
(A) Culture Pseudomonas sp. KS3.2 was inoculated with 1 platinum ear in a normal bouillon medium containing 1% lecithin (100 mL × 5) in a 500 mL baffle flask and cultured at 28 ° C. for 48 hours while shaking at 166 rpm. did. The culture solution was centrifuged (15200 × g, 15 minutes) to obtain 440 mL of culture supernatant.

(B) Ammonium sulfate fraction After adding 0.5 M Tris-HCl buffer (pH 8.0) to the culture supernatant to a final concentration of 10 mM, add ammonium sulfate under ice-cooling until 35% saturation is reached. , Dissolved. After stirring at 4 ° C. for 3 hours, the supernatant was collected by centrifugation (18780 × g, 20 minutes). Ammonium sulfate was added to the obtained supernatant until reaching 80% saturation, and the mixture was stirred overnight at 4 ° C. A precipitate was obtained by centrifugation (18780 × g, 20 minutes), suspended in 10 mM Tris-HCl buffer (pH 8.0), and dialyzed with the same buffer at 4 ° C. for 16 hours.

(C) Anion exchange chromatography After dialysis, a final concentration of 0.1% (w / v) Triton X-100 was added to solubilize the aggregate. Subsequently, the mixture was centrifuged (20000 × g, 15 minutes) and then filtered through a 0.45 μm filter to remove insoluble matters. The filtrate was applied to a DEAE-Sepharose Fast Flow column (25 × 80 mm, Amersham Biosciences Corp.) equilibrated with 10 mM Tris-HCl buffer (pH 8.0), washed with the same buffer (flow rate: 3.0 mL / min, Elution was performed with a linear gradient of 0 to 1 M NaCl (flow rate: 3.0 mL / min, 10 times the column bed volume).

For the eluted fraction, protein concentration and PLC activity were measured as follows. The protein concentration was measured using BCA protein assay kit (Pierce Biotechnology) using bovine serum albumin as a standard protein. For PLC activity, first, 300 μL of enzyme reaction solution containing 10 μL of eluate, 0.2% (w / v) PC, 0.285% (v / v) Triton X-100, and 46 mM Tris-HCl buffer (pH 8.0) was used. The mixture was incubated at 37 ° C. for 10 minutes, and the reaction solution was heated at 100 ° C. for 10 minutes to stop the reaction. Next, 10 μL of this reaction solution was added to 190 μL of alkaline phosphatase solution [0.2 units alkaline phosphatase, 1 mM MgCl ₂ , 0.1 mM ZnCl ₂ , 50 mM glycine-NaOH buffer (pH 9.6)] and dephosphorized at 37 ° C. for 20 minutes. An oxidation reaction was performed, and the released phosphoric acid was quantified using a Biomol Green reagent (BIOMOL Research Laboratories) to determine PLC activity. The blank was treated in the same manner using the boiled eluate. 1U of PLC is the amount of enzyme that liberates 1 μmol of phosphoryl base per minute. The elution profile of PLC is shown in FIG.

(D) Hydrophobic interaction chromatography After collecting the PLC active fractions by the above anion exchange chromatography and adding each to a final concentration of 0.5 M ammonium sulfate and 0.1% (w / v) Triton X-100 On a Phenyl-Sepharose High performance column (15 x 120 mm, Amersham Biosciences Corp.) equilibrated with 10 mM Tris-HCl buffer (pH 8.0) containing 0.5 M ammonium sulfate and 0.1% (w / v) Triton X-100 Provided. After washing with the same buffer (flow rate: 3.0 mL / min, 3 times the column bed volume), elution was performed with a linear gradient of 0.5 to 0 M ammonium sulfate (flow rate: 2.2 mL / min, 10 times the column bed volume). For the eluted fraction, protein concentration and PLC activity were measured in the same manner as described above. The PLC active fraction was a fraction from which a highly hydrophobic protein was eluted. The elution profile of PLC is shown in FIG.

(E) SDS-polyacrylamide gel electrophoresis (SDS-PAGE)
About the PLC active fraction by the above-mentioned hydrophobic interaction chromatography, SDS- is used according to Laemmli's method (Nature, 1970, Vol. 227, pp. 680-685) using 15% (w / v) polyacrylamide. PAGE was performed. As a molecular weight marker, SDS-PAGE Molecular Weight Standard, Broad Range (BIO-RAD) was used, and the gel was stained using a silver staining kit (BIO-RAD). The PLC activity was measured at pH 8.0 and 37 ° C. using PC as a substrate. The results of purification of PLC are shown in Table 2, and a photograph of SDS-PAGE is shown in FIG. Lane 1 is a molecular weight marker and Lane 2 is purified PLC (50 ng protein).

  Using 48-hour culture supernatant as a starting material, the specific activity could be purified up to 107 times, and the recovery rate was 36%. As a result of analyzing the purified sample by SDS-PAGE, as shown in FIG. 7, bands were detected at positions of about 18.6 kDa and about 17.5 kDa. Of the two bands, about 18.6 kDa was the major band. However, these bands were not separated at all using gel filtration chromatography. That is, it was not eluted in these molecular weight fractions, but was eluted and aggregated at a very large molecular weight position (almost in the vicinity of the pass-through fraction). From this, it is estimated that the PLC of the present invention consists of subunits of at least about 18.6 kDa or about 17.5 kDa. This molecular weight is completely different from the known PLC molecular weight (about 28.5-78 kDa). The PLC of the present invention was not stained with Coomassie Brilliant Blue, which is commonly used for protein staining of SDS-PAGE gels.

  The active fractions were then collected and stored at −20 ° C. and used in the following examples.

(Example 4: Effect of pH on PLC activity)
The purified enzyme obtained in Example 3 was reacted for 10 minutes at 37 ° C. in various buffers having a final concentration of 46 mM at pH 4 to 11 using PC as a substrate, and the relative activity was measured. As shown in FIG. 1, the optimum pH was 7.2. Incubate for 3 hours at 5 ° C in various buffer solutions with a final concentration of 45 mM at pH 4-12, and then react for 10 minutes at 50 ° C in a Tris-HCl buffer solution (pH 7.2) with a final concentration of 46 mM. Residual activity was measured to examine pH stability. As shown in FIG. 2, it was stable at pH 8.2 to 8.8, but deactivated in other pH ranges. According to Non-Patent Document 5, it is reported that the Pseudomonas sylkiriensis-derived PLC does not deactivate at pH 7-10. Therefore, the PLC of the present invention seems to have low pH stability.

(Example 5: Effect of temperature on PLC activity)
The purified enzyme obtained in Example 3 was reacted for 10 minutes in a Tris-HCl buffer solution (pH 7.2) with a final concentration of 46 mM at a temperature of 20 to 70 ° C. using PC as a substrate, and the relative activity was measured. . The optimum temperature was about 50 ° C. as shown in FIG. Next, after incubation for 30 minutes in a final concentration of 46 mM Tris-HCl buffer (pH 7.2) containing 0.01% (v / v) Triton X-100 and 0.2 mM ammonium sulfate at a temperature of 4 to 70 ° C., the final concentration The reaction was carried out in 46 mM Tris-HCl buffer (pH 7.2) at 50 ° C. for 10 minutes, and the residual activity was measured to examine the temperature stability. As shown in FIG. 4, it turned out that it deactivates at 20 degreeC or more. It has been reported that Pseudomonas sylquiriensis-derived PLC is not inactivated at all by treatment at 60 ° C. for 30 minutes, whereas in Non-Patent Document 4, it is derived from Pseudomonas fluorescens A-3-1. PLC has been reported to be deactivated to about 70% relative activity at 30 ° C. by treatment at pH 6.8 for 5 minutes. From this, it is considered that the PLC of the present invention has relatively low temperature stability.

(Example 6: Substrate specificity of PLC)
The substrate specificity of the PLC of the present invention was examined. Enzyme solution 300 μL containing 10 μL enzyme solution, 0.2% (w / v) substrate, 0.285% (v / v) Triton X-100, and 46 mM Tris-HCl buffer (pH 7.2) was added at 50 ° C. The reaction was stopped for 10 minutes by heating at 100 ° C. for 10 minutes. After completion of the reaction, 10 μL of this reaction solution was added to 190 μL of alkaline phosphatase solution [0.2 units alkaline phosphatase, 1 mM MgCl ₂ , 0.1 mM ZnCl ₂ , 50 mM glycine-NaOH buffer (pH 9.6)] at 37 ° C. for 20 minutes. The dephosphorylation reaction was performed, and the PLC activity was determined by quantification using Biomol Green reagent (BIOMOL Research Laboratories). The amount of the enzyme that releases 1 μmol of phosphoryl base per minute was defined as 1 U for the activity of PLC. When phosphatidic acid was used as a substrate, the phosphoric acid liberated was quantified immediately after the enzymatic reaction as described above without being treated with alkaline phosphatase. When p-nitrophenylphosphorylcholine (pNPPC) is used as a substrate, 50 μL of the enzyme solution is added to 1.45 mL of enzyme reaction solution [3.6 mM pNPPC, 50 mM Tris-HCl buffer (pH 7.2)], and the mixture is heated to 50 ° C. After incubation for 10 minutes, the absorbance of free p-nitrophenol at 400 nm was measured, and the amount of free p-nitrophenol was determined using the molar extinction coefficient (14700 M ⁻¹ cm ⁻¹ ). The results are shown in Table 3.

  The PLC of the present invention showed the highest activity against phosphatidylcholine (PC). Activity against other phospholipids was low and showed little activity against the artificial substrate pNPPC. Known PLCs are known to be active against both PC and phosphatidylethanolamine. Therefore, it was further clarified that the PLC of the present invention is a novel PLC having high specificity for PC as compared with the known PLC.

(Example 7: Influence of various compounds on PLC)
Using PC as a substrate, various compounds described in Table 4 below were added, and the activity of PLC was measured at pH 7.2 and 50 ° C. in the same procedure as in Example 6 above. Relative activity was determined by setting the activity when no additive was added as 100%. The results are shown in Table 4.

The PLC of the present invention is considered to have an SH group because it is strongly inhibited by Cu ²⁺ , Fe ²⁺ , and Hg ²⁺ . Furthermore, it was inhibited by dithiothreitol and 2-mercaptoethanol, which are reductive cleavage agents of protein disulfide bonds, but was not inhibited by iodoacetamide, a modified protective agent for SH groups. Therefore, it is considered that the PLC of the present invention is not a thiol enzyme having an SH group at the active center but a disulfide group is involved in maintaining the steric structure and activity. Moreover, it was not activated by Mg ²⁺ and Ca ²⁺ and was different from the known PLC. Since it is strongly inhibited by phenylmethanesulfonyl fluoride (PMSF), it is considered that active serine is involved in the enzyme activity. In addition, the PLC of the present invention is strongly inhibited by SDS, which is the same as that derived from Pseudomonas sylkiriensis. Furthermore, since it is also strongly inhibited by EDTA, metal ions may be required for enzyme activity. As described above, the PLC of the present invention exhibited characteristics different from those of known PLCs with respect to the influence of various compounds.

  The PLC of the present invention has high substrate specificity for phosphatidylcholine (lecithin), which has the highest abundance ratio among phospholipids, and thus is useful in the food industry, the fat and oil industry, and the like. Specifically, for example, in the food industry, when PLC is added during bread production, plump bread can be produced, and in the production of edible oils and ceramides, removal of phospholipids to prevent quality degradation Useful for. It can also be used to synthesize various phosphate esters using phospholipids such as phosphatidylcholine as substrates. Alternatively, it can be used as a detergent for removing stains due to adhesion of chicken eggs rich in lecithin and sebum containing phospholipids, and removal of wood pitch during pulp production. Furthermore, it can be used as a reagent for research such as research on signal transduction systems.

2 is a graph showing the relative activity of the PLC of the present invention at various pHs. It is a graph which shows the residual activity of PLC of this invention when it incubates at 5 degreeC at various pH for 3 hours. 2 is a graph showing the relative activity of the PLC of the invention at various temperatures at pH 7.2. It is a graph which shows the residual activity of PLC of this invention when it incubates for 30 minutes at various temperature with pH7.2. It is a graph which shows the elution profile of DEAE-Sepharose Fast Flow column chromatography in the purification process of PLC derived from Pseudomonas sp. KS3.2 strain | stump | stock of this invention. It is a graph which shows the elution profile of Phenyl Sepharose column chromatography in the purification process of PLC derived from Pseudomonas sp. KS3.2 strain | stump | stock of this invention. It is a photograph of SDS-polyacrylamide gel electrophoresis of the purified PLC of the present invention.

Claims (3)

Novel phospholipase C having the following characteristics (1) to (8):
(1) Action: acts on phospholipids and hydrolyzes its phosphate ester bond to release 1,2-diacylglycerides and phosphoryl bases;
(2) Substrate specificity: Acts specifically on phosphatidylcholine but has little effect on phosphatidic acid, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, and sphingomyelin, and on p-nitrophenylphosphorylcholine Has virtually no effect;
(3) Optimal pH: 7.2;
(4) Stable pH: 8.2 to 8.8;
(5) Optimal temperature: about 50 ° C .;
(6) Stable temperature: 20 ° C. or lower;
(7) Inhibition: inhibited by metal ions excluding calcium, phenylmethanesulfonyl fluoride, dithiothreitol, 2-mercaptoethanol, SDS, and EDTA, but not iodoacetamide; and (8) Molecular weight: about 17 kDa ~ 19 kDa (SDS-PAGE).   The phospholipase C of Claim 1 which is derived from Pseudomonas sp. KS3.2 strain (FERM P-19732).   Pseudomonas sp. KS3.2 strain (FERM P-19732) having the ability to produce phospholipase C.

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