JPH0731472A - Phospholipase a1 and its utilization - Google Patents

Abstract

PURPOSE:To obtain an acidic phospholipase A1, having the function to hydrolyze a phospholipid and produce a lysophospholipid, specific physical properties and a high ratio of conversion into a lyso derivative, excellent in temperature stability and useful for producing, etc., the lysophospholipid of good quality. CONSTITUTION:This acidic phospholipase A1 has the function to hydrolyze a phospholipid and produce a lysophospholipid, produced by a mold of the genus Aspergillus, further action optimum pH at 4.0-5.0, action optimum temperature of 50-60 deg.C, molecular weight of 32000 and isoelectric point (pI) of 3.0. Furthermore, a lysophospholipid is obtained by treating a phospholipid with this acidic phospholipase A1.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a phospholipase A1 which selectively hydrolyzes an acyl group at the 1-position of a phospholipid to produce a 2-acyl type lysophospholipid, particularly a filamentous fungus of the genus Aspergillus. To phospholipase A1 and the use of such enzymes.

[0002]

2. Description of the Related Art Phospholipids are widely used in the fields of food, feed, cosmetics, pharmaceuticals, etc. because they have surface activity, antioxidant activity and physiological activity.

On the other hand, since the surfactant activity of phospholipids is generally inferior to that of other synthetic surfactants, attempts have been made to improve the emulsifying properties using enzymes.

Lysophospholipid is a partially decomposed phospholipid obtained by hydrolyzing a part of the fatty acid bonded to the glycerin residue of the phospholipid. This lysophospholipid has a stronger 0 / W emulsifying property than the original phospholipid and forms a more stable emulsion with respect to changes in pH and temperature.
It has been reported to have excellent properties such that the emulsifiability does not decrease even when metal ions such as calcium and magnesium coexist in high concentrations. Therefore, lysophospholipids can be used more widely than phospholipids.

Further, the lyso-formation of phospholipids in the phospholipid-containing substance may lead to improvement of the physical properties of foods by the phenomenon alone or by a synergistic effect with other coexisting substances.

Phospholipase is known as an enzyme that decomposes phospholipids, and is further classified according to its action site. Of these, phospholipase A1 or phospholipase A2 is known as a substance that decomposes phospholipids into lysophospholipids, and any of them can be used for the purpose of lysopherizing phospholipids.

Phospholipase A already produced by microorganisms
(JP-A-58-212783) and lipases (JP-A-63-4)
Japanese Patent No. 2691) discloses a method for decomposing phospholipids,
Also, the presence of lipase containing phospholipid-degrading activity has been known for a long time in Takaji Astase (registered trademark) derived from Aspergillus oryzae, which is a koji mold called "enzyme treasure trove" (Biochem. Z., 26
1,275 (1933)), the method using these enzymes has a lower enzyme activity than pancreatin, or when an enzyme with poor substrate specificity is used, the yield of lysophospholipid is deteriorated, There are drawbacks such as deterioration of the quality of lysophospholipid due to the coexistence of organisms.

[0008] Therefore, in order to overcome the above problems and drawbacks, it has been desired to develop an enzyme which has excellent activity and selectivity and can be stably supplied at a low cost.

At present, lysophospholipids obtained by decomposing phospholipids using enzymes are commercially available. This is phospholipase A2 contained in pancreatin-derived enzyme agent porcine pancreas.
Is used. However, pancreatin
It has been pointed out that there are odors peculiar to organs, that the source of production is porcine pancreas, and that there is a limit to the reduction of production costs. Furthermore, the following problems have been pointed out.

The first point is that, in general, phospholipase A1,
This is the point where pH adjustment is required during A2 action.
When phospholipases A1 and A2 act, the free fatty acid lowers the pH of the reaction system, but the optimum pH of porcine pancreatic phospholipase A2 is neutral to weakly alkaline.
Therefore, it is necessary to adjust the pH during the enzyme action, and a step for removing the substance added for pH adjustment after the enzyme reaction is required.

To solve this problem, solvent fractionation (US Pat. No. 3,652,397 etc.), enzymatic reaction in a nonionic non-polar organic solvent (JP-A-3-98590) or free fatty acid and metal soap A method of adding a compound which makes the compound into the reaction solution (Japanese Patent Laid-Open No. 62-14790, Japanese Patent Publication No. 4-81).
No. 431) is proposed. However, these methods are complicated in operation, and the safety of the product may be a problem in some cases.

The second point is that after the enzymatic reaction, porcine pancreatic phospholipase A2, which has extremely high stability to heat and organic solvents, remains in the obtained product. If phospholipids that have not been hydrolyzed are present in the final product, phospholipase A2 may newly release fatty acids from the phospholipids, which may significantly impair their commercial value.
Therefore, a method of deactivating the residual enzyme by heat treatment is conceivable. However, if the enzyme reaction product is heat-treated at 95 ° C. for about 30 minutes, porcine pancreatic phospholipase A2 can be obtained.
Does not inactivate sufficiently, and when it is inactivated by heating at about 120 ° C., problems such as deterioration of phospholipids or free fatty acids occur.

Therefore, in order to obtain a lysophospholipid-containing substance having substantially no residual activity of phospholipase A2,
Various methods have been tried. For example, a method combining solvent fractionation using various solvents and column treatment using silica gel or the like in the production process, a method of drying a phospholipid treated with phospholipase A2, and then fractionating using a polar solvent ( JP-A-62-262998, Japanese Patent Publication
2-11234) or an enzyme treatment and then treating the phospholipase A2 in the reaction product with a protease, and then inactivating the protease by heating (JP-A-63-233750).
Gazette). However, these operations have various problems such as time and cost and lower yield.

[0014]

DISCLOSURE OF THE INVENTION For many years, the present inventors have conducted extensive studies on phospholipase-producing microorganisms and phospholipases, and a method for producing lysophospholipids from phospholipids using phospholipases. By isolating and purifying an enzyme produced by a filamentous fungus of the genus Aspergillus ), a highly purified acid phospholipase A1 having excellent activity and selectivity was found, and by treating a phospholipid with the acid phospholipase A1, The present invention has been completed by finding a method for producing high quality lysophospholipids which can deactivate the residual activity by mild heat treatment without requiring pH adjustment during action, have a high lysolysis rate, and have good quality. .

[0015] Accordingly, the present invention is Aspergillus (Asperg
illus ) filamentous fungi produced by phospholipase A1.

The invention further enables the use of acid phospholipase A1, in particular acid phospholipase A1 obtained from filamentous fungi of the genus Aspergillus , in the production of lysophospholipids from phospholipids.

[0017]

[Constitution of the invention]

[0018]

The phospholipase A1 of the present invention has a molecular weight of 32,000 and an isoelectric point of pI.
It is an enzyme protein having a pH of 3.0 and has the following properties.

1. Action and Substrate Specificity This enzyme hydrolyzes only the acyl group at the 1-position of phospholipids, and its action is phospholipase A1 (enzyme number EC
3.1.1.32).

2. Optimum pH of action The optimum pH of action of this enzyme is the usual activity measurement system (reacted at 37 ° C for 10 minutes in the presence of Triton X-100 (registered trademark)).
Then, it is 4 to 5. Further, in the activity measurement system not containing Triton X-100 (reaction at 37 ° C. for 10 minutes),
It is 4.5 to 5.5.

3. pH stability The stable pH range of the present enzyme changes depending on the concentration of the enzyme itself, the type and concentration of coexisting substances, and the like.

4. Optimal temperature of action The present enzyme has an action at 30 ° C to 65 ° C, the optimal temperature is 50 ° C to 60 ° C, and the maximum activity of the enzyme is shown at around 55 ° C.

5. Inhibition, activation and stabilization This enzyme is not specifically inhibited by metal polyvalent ions such as mercury, lead and iron. In addition, no change in activity due to ethylenediamine tetraacetic acid (EDTA) was observed.

6. Purification method To a koji culture extract of a filamentous fungus of the genus Aspergillus , cold acetone was added with stirring to a final concentration of 60 to 80 (v / v)%, and the mixture was allowed to stand for a while.
Centrifuge to collect the precipitate. Also, if ethanol is used instead of acetone, a precipitate is obtained in the same manner.
This precipitate is dissolved in 50 mM acetate buffer (pH 5.5), salted out with ammonium sulfate, and centrifuged to separate the precipitate. The obtained precipitate was dissolved in a 50 mM acetate buffer solution (pH 5.5) containing 1 M ammonium sulfate, and the insoluble matter was filtered off, followed by column chromatography [column:
Butyl Toyopearl pak 650S
S), manufactured by Tosoh Corporation, effluent solvent: ammonium sulfate-containing 5
0 mM acetate buffer (pH 5.5)]. The obtained semi-purified enzyme was added to 20 mM acetate buffer (pH
After dialysis with 5.5), column chromatography [column: Q-Sepharose (Pharmacia), eluent: salt-containing 20 mM acetate buffer (pH)
5.5)] to obtain phospholipase A1.
Further, this was salted out with ammonium sulfate and subjected to column chromatography [column: Superose 12 (Superose 1
2), manufactured by Pharmacia Ltd.] to obtain the enzyme protein phospholipase A1.

7. Crystal structure Since the enzyme has not been obtained in crystalline form, its crystal structure cannot be determined.

[0026] As-producing bacteria which produce phospholipase A1 of the present invention, for example, Aspergillus niger (Aspe
rgillus nige r: For example, ATCC-9642, IFO-4407, etc.),
Aspergillus oryzae (Aspergillus oryzae: for example, SANK-11870, IFO-30102, etc.), Aspergillus Usamii (Aspergillus usamii,: for example, IAM-2414,
IFO-6082 etc.), Aspergillu
s awamori : For example, IAM-2112, IFO-4033, etc., Aspergillus sojae : For example, IAM-
2666), Aspergillus
phoenicis : For example, IAM-2215), Aspergillus wentii (for example, IAM-2133), and other filamentous fungi belonging to the genus Aspergillus, preferably Aspergillus niger or Aspergillus oryzae.

The above-mentioned filamentous fungi are all known fungi (for example, Japanese Patent Publication No. 46-32792, J. Gen. Appl. Microbiol., 1).
7,281 (1971), Biochem.Z., 261 275 (1933), etc.),
American Type Culture Collection (Amer
ican Type Culture Collection: ATCC, Institute for Fermentation Osaka: IF
O), Institute of Applied Microbiology (IA), Institute of Applied Microbiology, University of Tokyo
It is stored under each number in a storage organization such as M) and can be freely distributed.

Aspergillus oryzae ( A. oryzae )
SANK 11870 has been deposited with the Institute of Microbial Science and Technology (now renamed as the Institute of Biotechnology, Institute of Biotechnology), Agency of Industrial Science and Technology, as Micro Engineering Research Institute No. 3887 (FERM BP-3887).

In addition to natural original strains, these strains include artificial mutants thereof.

The phospholipase A1 of the present invention is, for example,
It is manufactured by the following method.

That is, the culture of the strain was carried out by adding water to the bran.
5 to 2 times the weight (preferably the same amount) is added, and the solid medium obtained by steaming is inoculated with the inoculum and the koji culture method is performed. As the medium raw material, in addition to bran, cereals (for example, rice, wheat, etc.), various minor cereals (for example, corn, soybean, sesame, etc.), cottonseed meal, etc. can be used alone or in combination.

The culture temperature is 10 ° C to 40 ° C (preferably 15 ° C to 35 ° C, particularly preferably 18 ° C to 32 ° C).
C.), and the culturing time is usually 3 to 20 days (preferably 4 to 8 days), varying depending on the medium composition, culturing temperature and the like.

After completion of the culture, 1 to 20 water or an appropriate buffer solution (eg, acetate buffer solution, phosphate buffer solution) is added to the koji.
An enzyme solution can be obtained by adding twice the weight, stirring well, and squeezing and filtering. Furthermore, a conventional method for collecting and purifying the enzyme, for example, a salting-out method, an organic solvent precipitation method, an adsorption method using an ion exchanger or the like as an adsorbent, an ultrafiltration method, a vacuum drying method, etc., alone or in appropriate combination. By using
The enzyme of the present invention is collected from the above enzyme solution and isolated and purified.

The phospholipase A1 of the present invention is an enzyme which has excellent activity and selectivity and which can be constantly and inexpensively supplied from microorganisms and is an extremely useful phospholipase.

Furthermore, the present invention uses phospholipase A1 having the same enzymatic properties as those of phospholipase A1 described above in the production of lysophospholipids, thereby eliminating the need for pH adjustment during enzyme action and deactivating residual activity by mild heat treatment. It is possible to produce lysophospholipids having high lysolysis rate and high quality.

The molecular weight and isoelectric point of phospholipase A1 which can be used in the method for producing lysophospholipid of the present invention vary depending on the producing strain, and the molecular weight is about 30,000-40,
000 is preferably about 32,000 to 37,000,
Isoelectric point (pI) is p by the isoelectric focusing method.
I2.8-4.5, preferably pI3.0-4.3.

Further, the present enzyme has the following characteristics.

1. Action and Substrate Specificity This enzyme hydrolyzes only the acyl group at the 1-position of phospholipids, and its action is phospholipase A1 (enzyme number EC
3.1.1.32).

2. Active region The enzyme has a pH of 2.5 to 6, preferably pH 3 to 5.
It has 5 active areas.

3. Stability upper limit temperature The enzyme is 45 ° C to 90 ° C, preferably 50 ° C to 80 ° C.
It has a stable upper limit temperature of ° C.

4. Optimum pH of action The optimum pH of action of the present enzyme is in the usual activity measuring system (reacting in the presence of Triton X-100 at 37 ° C. for 10 minutes).
The pH is 3.2 to 5.0.

5. pH stability The stable pH range of the present enzyme changes depending on the concentration of the enzyme itself, the type and concentration of coexisting substances, and the like.

6. Optimal temperature of action The enzyme acts at 30 ° C to 65 ° C, and the optimal temperature of action is 50 ° C to 60 ° C.

7. Temperature stability The enzyme is stable at about 30 ° C to 55 ° C when treated for 30 minutes at pH 4.0.

Phospholipase A1 which can be used in the present invention
The reaction for decomposing phospholipids is carried out by contacting the enzyme with a substrate in an aqueous medium or in a wet state, and in some cases, a nonionic nonpolar organic solvent (for example, ether such as diethyl ether or dioxane). It is also possible to carry out the enzyme reaction in the coexistence of compounds, hydrocarbons such as hexane, benzene and toluene, and esters such as ethyl acetate and butyl acetate.

The substrate used is phospholipid itself or a phospholipid-containing substance, regardless of its source, content or property. They are, for example, soybean, wheat, barley, corn, rapeseed, sunflower, peanut, plant phospholipids such as cottonseed, egg yolk, animal brain (cow, sheep, pig, chicken, etc.) animal phospholipids, It may be chlorella cells, microbial cell phospholipids such as filamentous fungal cells, etc., and is preferably soybean, wheat or egg yolk phospholipid.

The aqueous or wetting medium can be, for example, ion-exchanged water, distilled water, well water, tap water, etc., and if necessary,
Acid (eg acetic acid etc.), alkali (eg sodium hydroxide etc.) or buffer (eg acetate buffer) is added to give a pH of 2.5 to 6.5 (preferably a pH of 3.5 to 5.). It can also be adjusted to 5). When soft water such as ion-exchanged water or distilled water is used, it may be preferable to add calcium chloride or the like.

The amount of phospholipase A1 added varies depending on the reaction temperature, the reaction time, the pH during the reaction, the properties and quality of the substrate, the substances contaminated, the degree of the required effect, etc., but preferably 1,000 units 0.05% to 10% by weight (preferably 0.2% to 2% by weight) based on the phospholipid (for example, soybean phospholipid) using / g of enzyme.
Is.

The reaction temperature of this enzyme is 10 ° C to 70 ° C.
(Preferably 20 ° C. to 65 ° C., particularly preferably 30 ° C.
To 60 ° C.), the time required for the reaction depends on the reaction temperature,
Although it varies depending on the pH and the like, it is usually 10 minutes to 10 days (preferably 1 hour to 2 days).

It is usually not preferable that the phospholipase A1 activity remains in the obtained lysophospholipid,
After performing the enzymatic reaction, the residual phospholipase A1 activity in the enzymatic reaction product can be easily inactivated by a simple operation, that is, a mild heat treatment, if necessary. This treatment is performed at 45 ° C to 90 ° C (preferably 50 ° C).
To 80 ° C.) for 5 minutes to 5 hours (preferably 10 minutes to 2 hours).

In addition to the heat treatment, a conventional method for inactivating the enzyme, such as pH treatment or pressure treatment, may be used alone or in combination, and the residual phospholipase A1 may be used.
The activity can be deactivated.

By the reaction of the present enzyme, the reaction product can be used as it is, but if necessary, concentration or purification by a concentrator such as thin layer concentration, further spray drying or freeze drying, etc. It is concentrated by applying the usual method such as
It can also be a refined or dried product.

Phospholipase A1 that can be used in the present invention
Is preferably partially purified before use. For example, when the present enzyme is directly added to wheat flour, it is appropriate that the protease or the like is not contaminated.

Further, when it is added directly to the dough for the purpose of improving the physical properties of flour products such as bread and noodles, phospholipase D and an emulsifying agent (eg, monoglyceride, calcium stearyl lactylate, etc.) may be added, if necessary. It is possible to add. In addition, the effect of using the present enzyme appears in the physical properties of the dough and the physical properties of the product, and the addition of the present enzyme causes the dough to have appropriate elasticity and extensibility,
Further, the adhesiveness is also suppressed, and the operation in each work process becomes easy.

[0055]

EXAMPLES Among the present invention, phospholipase A1,
Particularly, the phospholipase A1 produced by a filamentous fungus of the genus Aspergillus will be described more specifically, but the present invention is not limited thereto.

[0056] [Example 1] from Aspergillus niger (Aspergillus niger)
Manufacturing Aspergillus niger crude phospholipase A1 (A. Niger) ATCC 9642 strain in a medium 12g consisting of an equal mixture of bran and water, were cultured preincubated for 6 days at 30 ° C., resulting whole cell 600 g of a mixture of bran and water was placed in a metal dish [42 × 24 × 7 (depth) cm] and inoculated into the main culture medium that had been boiled at 120 ° C. for 30 minutes, then at 30 ° C. for 15 hours, then at 19 ° C. for 5 hours.
Cultured for a day. Add 3 liters of water to the koji thus prepared,
Mix well, leave at 37 ° C for 2 hours, filter,
An enzyme extract was obtained. 1N acetic acid was added to this enzyme extract,
Adjust the pH to 4.0, add double volume of cooled acetone,
I left it in a cold place overnight. Then, the supernatant was discarded, the precipitate was thoroughly washed with acetone, and vacuum dried to obtain 19.2 g of an enzyme sample containing phospholipase A1. Table 5 shows the main enzyme activity per 1 g of this enzyme. Each enzyme activity was measured by the method described below.

[0057] Hosuhoripa from [Example 2] Aspergillus niger (A. Niger)
Over crude phospholipase A1 10 g about water 10 of Example 1 Ze A1
The mixture was dissolved in 0 ml, 1N acetic acid was added to adjust the pH to 4.0, water was added to make 200 ml, 200 ml of cold acetone was added and mixed, and the mixture was left for 1 hour. Then, the mixed solution was centrifuged to obtain a first precipitate. 600 ml in the supernatant
Cold acetone was added and mixed, and the mixture was left for 1 hour. Then, the mixed solution was centrifuged to obtain a second precipitate. This second
The precipitate is added to 500 ml of 50 mM acetate buffer (pH 5.5).
And ammonium salt was added thereto for salting out. The precipitate obtained by centrifugation was dissolved in 50 mM acetate buffer (pH 5.5) containing 1M ammonium sulfate, the insoluble matter was filtered off, and then Butyl Toyopearl Pack 650S (Butyl
Purified by column chromatography using Toyopearl pak 650S, manufactured by Tosoh Corporation, Q-Sepharose (Q-Sepharose, manufactured by Pharmacia) and Superose 12 (Superose 12, manufactured by Pharmacia), and phospholipase A.
An enzyme sample containing 131.5 units was obtained.

Table 5 shows the main enzyme activities per 1 g of this enzyme. Each enzyme activity was measured by the method described below.

It was confirmed that the enzyme sample prepared in Example 2 above had almost no lipase activity (about 0.1% or less of phospholipase A1 activity).

Next, the present inventor has used L-α in which 0.0236 units of the enzyme sample prepared in Example 2 above was labeled with (C) ¹⁴ C (NEN, NEC 764) of the palmitoyl group at the (I) position. -Dipalmitoylphosphatidylcholine (1mC
i / mmole) 0.1 μmole, and (II) L-α-dipalmitoylphosphatidylcholine (2 mC) in which the palmitoyl groups at the 1- and 2-positions are labeled with ¹⁴ C (NEN, NEC 682).
i / mmole) 0.1 μmole and reacted for 10 minutes.
The final reaction solution at that time was 90 μl. As a result, 1
When the 2-position of palmitoyl groups ¹⁴ C from the labeled phosphatidylcholine only ¹⁴ C-labeled palmitic acid is liberated, the enzyme sample was confirmed to be acting only on the 1-acyl group.

The characteristics of the phospholipase A1 prepared in Example 2 are shown below. Phospholipase A1 molecular weight: 32,000 (by gel filtration method using Superose 12). Isoelectric point: pI 3.0 (by isoelectric focusing). Optimum pH of action: In the presence of Triton X-100, FIG.
As shown in FIG. 2, the pH is 4 to 5, and in the absence of Triton X-100, the pH is 4.5 to 5.5 as shown in FIG. The phospholipase A1 activity was measured by a method described below after preparing an enzyme solution using a 0.2 M acetate buffer, and the activity at pH 4 was set to 100%.

PH stability: The pH stability is shown in FIG. The phospholipase A1 activity was 33 mM acetate buffer (pH 3.2 to 6.0), 33 mM trisaminomethane-maleate buffer (pH 5.5 to 8.5), and 33 mM glycine / salt-sodium hydroxide ( pH8 ~
12.5) with phospholipase A1 about 10 units /
An enzyme solution (ml) was prepared, left at 37 ° C. for 60 minutes, and then the activity was measured by the method described below. The activity when it was most stable was defined as 100%.

Optimum temperature of action: The optimum temperature of action is 50 ° C. to 60 ° C. as shown in FIG. The activity at 37 ° C was 100%. Temperature stability: The temperature stability at pH 4 is as shown in FIG. In addition, phospholipase A1 activity
After heat treatment at 30 to 70 ° C. for 30 minutes, the activity was measured by the method described below, and the activity when no heat treatment was performed was defined as 100%.

The phospholipase A1 of the present invention will be described below.
The use of is further described, but the invention is in no way limited thereby.

[Example 3] Production of lysophospholipid Aspergillus niger ( A. nige) prepared in Example 2
r ) -derived phospholipase A1 p for phospholipid degradation
The following tests were conducted to examine the effect of H. The following experiment numbers 1-A and 2-A 50 ml and calcium chloride 0.2
g was placed in a 100 ml beaker and sufficiently dispersed with an ultrasonic homogenizer. After preheating at 37 ° C., 900 units of phospholipase A1 prepared in Example 2 was added to each of Experiment Nos. 1-A and 2-A, and the enzymatic reaction was allowed to proceed at 37 ° C. while stirring with a stir bar. Let During the progress of the enzymatic reaction, a beaker was covered with a Cylon film (manufactured by Fuji Film Co., Ltd.) to prevent evaporation of water.

Experiment No. 1-A: 10% (w / w) of 5 g of SLP-white (manufactured by Trurecitin Industry Co., Ltd.) added to 45 ml of water.
w) Suspension Experiment No. 2-A: 20 mM acetate buffer (pH 3.5) 4
10% (w / w) suspension in which 5 g of SLP-white was added to 5 ml. At the start of the reaction, 1 hour, 2 hours, and 3 hours later, the reaction solution was collected, and the free fatty acid in the sample was assayed for free fatty acid quantification reagent. It was quantified using Determiner NEFA (manufactured by Kyowa Medix). The apparent phospholipid lysolysis rate was calculated by the formula A / Bx100 A: the number of moles of lysophospholipid (the number of moles of fatty acid released by the enzymatic reaction), assuming that the average molecular weight of the phospholipid in SLP-white is 765. B: Calculated using the number of moles of substrate phospholipid. The results are shown in Table 1.

[0067]

[Table 1]

The phospholipase A1 derived from Aspergillus niger ( A. niger ) showed an excellent phospholipid lysation rate even when a pH adjusting agent was not used.

[Example 4] Production of lysophospholipid Aspergillus oryzae prepared in Production Example 2 described later.
The following tests were conducted to examine the effect of pH on phospholipid degradation of phospholipase A1 derived from ( A. oryzae ).

The following experiment numbers 3-A, 4-A, 1-B, 2-B and 3-B (50 ml) and calcium chloride (0.2 g) were added to 100 m.
It was placed in a 1-liter beaker and sufficiently dispersed with an ultrasonic homogenizer. After pre-warming at 37 ° C, experiment number 3-A and
4-A includes phospholipase A18 prepared in Production Example 2.
65 units, 865 units of porcine pancreas-derived phospholipase A2 (manufactured by Sigma) were added to Experiment Nos. 1-B, 2-B and 3-B, respectively, and the enzyme reaction was carried out at 37 ° C while stirring with a stir bar. Advanced. During the progress of the enzymatic reaction, a beaker was covered with a Cylon film (manufactured by Fuji Film Co., Ltd.) to prevent evaporation of water. However, the activity of porcine pancreas-derived phospholipase A2 was measured at pH 8.0.

Experiment No. 3-A: SLP-white (manufactured by Trurecitin Industry Co., Ltd.) 5 g was added to 45 ml of water to obtain 10% (w /
w) Suspension Experiment No. 4-A: 20 mM acetate buffer (pH 3.5) 4
10% (w / w) suspension obtained by adding 5 g of SLP-white to 5 ml Experiment number 1-B: 10% (w / w) suspension obtained by adding 5 g of SLP-white to 45 ml of water Experiment number 2-B : 20 mM Tris-HCl buffer (pH 9.
0) 5% of SLP-white was added to 45 ml of 10% (w /
w) Suspension Experiment No. 3-B: SLP-white (5 g) was added to water (45 ml), and sodium hydroxide was appropriately added to maintain the pH during the enzyme reaction at 7.5 to 8.5. / w) suspension.

At the start of the reaction, 1 hour, 2 hours and 3 hours later, the reaction solution was sampled, and the free fatty acid in the sample was quantified using the free fatty acid quantification reagent Determiner NEFA.

The apparent phospholipid lysation rate was determined according to Example 3. The results are shown in Table 2.

[0074]

[Table 2]

[0075] Aspergillus oryzae (A. Oryzae) derived phospholipase A1, even if you do not use a pH adjusting agent, as compared with porcine pancreatic phospholipase A2, showed excellent phospholipid lyso conversion ratio.

FIG. 6 shows the changes with time in the apparent phospholipid lysolysis rate and pH of Experiment No. 3-A.

[Example 5] Inactivation of residual enzyme by heat treatment Phosphorus obtained from Aspergillus niger ( A. niger )
Holipase A1 10 ml of the suspension described in Experiment No. 5-A below and 0.04 g of calcium chloride were placed in a beaker and sufficiently dispersed with an ultrasonic homogenizer. After preheating at 37 ° C,
Phospholipase A1 (151 units) of Example 2 was added, and the enzyme reaction was allowed to proceed at 37 ° C. for 5 hours while stirring with a stirring bar.

Experiment No. 5-A: 5 mM acetate buffer (pH
4.5) 10% by adding 1 g of SLP-white to 9 ml
(W / w) suspension.

The apparent phospholipid lysation rate was 90% or more 5 hours after the start of the reaction.

After the enzymatic reaction, about 2 g of the reaction solution was dispensed into several test tubes, covered with a Cylon film (manufactured by Fuji Film Co., Ltd.) so as to prevent water from evaporating, and heated at 50 to 80 ° C. for 3 minutes.
It was left for 0 minutes.

Phospholipase A1 remaining in each reaction solution
The activity was measured by the method described below and expressed as a percentage of the phospholipase A1 activity in the reaction solution which was not heat-treated. The results are shown in Table 3.

[0082]

[Table 3]

When phospholipase A1 is used,
The enzyme activity could be completely deactivated at 80 ° C.

[Example 6] Deactivation of residual enzyme by heat treatment Phosphorus obtained from Aspergillus oryzae ( A. oryzae )
Folipase A1 50 ml of the suspension described in Experiment Nos. 6-A, 7-A, 4-B and 5-B below and 0.2 g of calcium chloride were placed in a 100 ml beaker and sufficiently dispersed with an ultrasonic homogenizer. . After preheating at 37 ° C., 2160 units of phospholipase A1 of Production Example 2 or porcine pancreatic phospholipase A
2160 units (manufactured by Sigma) was added, and the enzyme reaction was allowed to proceed at 37 ° C. for 5 hours while stirring with a stirring bar.

Experiment No. 6-A: 5 mM acetate buffer (pH
4.5) SLP-white 5g was added to 45ml 10%
(W / w) suspension Experiment No. 7-A: 5 mM acetate buffer (pH 4.5) 25
50% (w / w) suspension obtained by adding 25 g of SLP-white to ml Experiment No. 4-B: 5 mM Tris-HCl buffer (pH 8.
0) 5% of SLP-white was added to 45 ml of 10% (w /
w) Suspension Experiment No. 5-B: 5 ml Tris-HCl buffer (pH 8.
0) 25% SLP-white 25 g was added to 25 ml 50%
(W / w) suspension.

The apparent phospholipid lysation rate 5 hours after the start of the reaction was about 65% in the case of Experiment No. 5-B, ie, a system in which 50% SLP-white was treated with porcine pancreatic phospholipase A2.
And in other systems, namely 6-A, 7-A and 4-B, 90
% Or more.

After 5 hours of enzyme reaction, about 7 g of each reaction solution
Dispense each into several Somogyi test tubes, cover with Ceylon film to prevent water evaporation, and
It was left for 30 minutes at 0 to 80 ° C.

Phospholipase A remaining in each reaction solution
The 1 and A2 activities were measured by the method described below and expressed as percentages of the phospholipase A1 and A2 activities in the reaction solution which were not heat-treated. The results are shown in Table 4. However,
The activity of porcine pancreatic phospholipase A2 was measured at pH 8.

[0089]

[Table 4]

When phospholipase A1 is used,
Compared with the case of using porcine pancreatic phospholipase A2,
The enzyme activity could be completely inactivated at low temperature.

[0091] [Production Example 1] from Aspergillus oryzae (Aspergillus oryzae)
Instead of producing Aspergillus niger crude phospholipase A1 (A. Niger), except for using the Aspergillus oryzae (A. Oryzae) SANK 11870 strain, by performing the same operation as in Example 1, enzymes including phospholipase A1 11.1 g of sample was obtained.

Table 5 shows the main enzyme activities per 1 g of this enzyme. Each enzyme activity was measured by the method described below.

Production Example 2 Phospholipa from Aspergillus oryzae ( A. oryzae )
Except for using the crude phospholipase A1 10 g of Production Example 1 over Ze A1 performs the same acetone fractionation procedure as in Example 2 to obtain a second precipitate. The second precipitate was dissolved in 500 ml of 50 mM acetate buffer (pH 5.5), and 300 g of ammonium sulfate was added for salting out. 1 precipitate obtained by centrifugation
50 ml acetate buffer containing M ammonium sulfate (pH
5.5), insoluble material is filtered off, and then column chromatography [column: Butyl Toyopearl Pack 650S
(Butyl Toyopearl pak 650S, manufactured by Tosoh Corporation, solvent spill:
50 mM acetate buffer containing ammonium sulfate (pH 5.
5) Ammonium sulfate concentration, 1 to 0 M; gradient elution] was carried out. The phospholipase A1 active fraction was eluted at an ammonium sulfate concentration of 0.6 M or less. This fraction was desalted and concentrated to obtain 40 mg of enzyme sample. The molecular weights of the two types of phospholipase A1 contained in the present enzyme sample were measured by SDS-polyacrylamide gel electrophoresis, and the values were 37,000 and 35,000, respectively, which were determined by Ampholine electrophoresis. The isoelectric points are pI3.9 or pI4.3, respectively.
Met.

Table 5 shows the main enzyme activity per 1 g of this enzyme. Each enzyme activity was measured by the method described below.

It was confirmed that the phospholipase A1 prepared in the above Production Example 2 had almost no lipase activity (about 0.1% or less of the phospholipase A1 activity).

Furthermore, 0.0236 unit of this enzyme was added to (I) 2
L-α-dipalmitoylphosphatidylcholine (1 in which the palmitoyl group at the position is labeled with ¹⁴ C (NEN, NEC 764)
mCi / mmole) 0.1 μmole, and (II) 1- and 2-
L-α-dipalmitoylphosphatidylcholine (2 with palmitoyl group at position ¹⁴ C (NEN, NEC 682) labeled)
mCi / mmole) 0.1 μmole and reacted for 10 minutes. The final reaction solution at that time was 90 μl. as a result,
It was confirmed that ¹⁴ C-labeled palmitic acid was released only from phosphatidylcholine labeled with ¹⁴ C at the 1- and 2-position palmitoyl groups, and that this enzyme acts only on the 1-acyl group.

The characteristics of the phospholipase A1 enzyme prepared in Production Example 2 are shown below.

Optimum pH of action: The optimum pH of action in a usual activity measuring system is pH 3.2 to 5, as shown in FIG. pH stability: The pH stability is pH 3 to 8.5 as shown in FIG. The phospholipase A1 activity was 0.
2M acetate buffer (pH 3.2-6.5) and 0.01
A 1 (w / v)% enzyme solution was prepared using M Tris-hydrochloric acid buffer solution (pH 6.5 to 9.0), allowed to stand at 37 ° C. for 60 minutes, and then measured by the method described below to obtain pH 5. In Example 5, the activity when left at 37 ° C. for 60 minutes was defined as 100%.

Optimum temperature of action: The optimum temperature of action is 50 ° C. to 60 ° C. as shown in FIG. The activity at 37 ° C was 100%. Temperature stability: The temperature stability at pH 4 is 30 ° C. to 55 ° C. as shown in FIG. The phospholipase A1 activity was measured by the method described below after heat treating the enzyme solution at 30 to 70 ° C. for 30 minutes, and the activity without heat treatment was defined as 100%.

The main enzyme activity of the enzyme samples prepared in Examples and Production Examples was measured by the following method.

Phospholipase A 1 activity test 2.0 (w / v)% SLP-white (manufactured by Trurecitin Industry Co., Ltd.) and 4 (v / v)% Triton X-100 aqueous solution 0.5 ml, 0.1M. 0.05 ml of calcium chloride
0.25 ml of 0.2 M acetate buffer (pH 4.0) was added. Next, 0.1 ml of the enzyme solution was added, and the mixture was stirred to make it uniform, and allowed to stand at 37 ° C. to carry out an enzyme reaction for 10 minutes. Then, 0.1 ml of 1N hydrochloric acid was added to stop the enzymatic reaction. 0.02 ml of the reaction solution was sampled, and free fatty acid was quantified using a free fatty acid quantification reagent Determiner NEFA (manufactured by Kyowa Medix). The enzyme activity that produces 1 μmol of fatty acid per minute of the enzyme reaction was defined as 1 unit.

Lipase activity test 2.0 (w / v)% olive oil (manufactured by Wako Pure Chemical Industries, Ltd.) emulsion [to 0.2 g of olive oil, 10 ml of 0.5 m 15 (w / w)% arabic gum aqueous solution was added, and an ultrasonic homogenizer was used. Dispersed for 5 minutes] 0.5 ml 0.1 M calcium chloride 0.05 ml 0.2 M7 acetate buffer (p
H6.0) 0.25 ml was added. Then, the enzyme solution 0.
1 ml was added, and the mixture was stirred to make it uniform, and allowed to stand at 37 ° C. for 10 minutes for enzymatic reaction. Then, 1N hydrochloric acid 0.
The enzymatic reaction was stopped by adding 1 ml. Reaction solution 0.0
2 ml was taken and the free fatty acid was quantified using the free fatty acid quantification reagent Determiner NEFA. The enzyme activity that produces 1 μmol of fatty acid per minute of the enzyme reaction was defined as 1 unit.

Amylase (saccharification type) activity test tube [20 g of water (2 g of soluble starch) (appropriate amount) was added to the substrate and dissolved by heating, and after cooling, 0.2 M acetate buffer (pH 4.5) was added. 20 ml was added, and water was further added to adjust the volume to 50 ml], 5 ml was added, and after preheating in a 37 ° C. constant temperature water bath, 0.25 ml of the enzyme solution was added. After the enzymatic reaction for 30 minutes, the reaction was stopped by adding 0.25 ml of 0.5N sodium hydroxide. Using this as an enzyme reaction solution, Somogyi-Nelson reducing sugar assay (J. Biol. Chem., 160, 61-68 (1945), J. Biol. Che
m., 153,375-380 (1944)) was used to quantify the reducing sugar produced by the enzymatic reaction. The enzymatic activity for producing a reducing sugar equivalent to 1 μmol of glucose per minute of the enzymatic reaction was defined as 1 unit.

Protease activity test Hagiwara method (Enzyme Research Method 2 Asakura Shoten, 237-246 (1956))
It was measured by. The substrate pH was 3.0 (acidic protease) or 7.0 (neutral / alkaline protease). The unit of enzyme activity is Northro
p, J. Gen. Physiol., 16, 41 (1932)) and Ans (Ans.
on, J.Gen.Physiol., 22,79 (1938)), 1 μg of 27 tyrosine equivalent per minute under standard conditions.
The enzyme activity for producing a non-protein substance showing absorption at 5 nm was defined as 1 unit.

[0105]

[Table 5]

*: Fatty acid release activity from phospholipids **: Below the lower limit of measurement (10 units)

[Brief description of drawings]

FIG. 1 is a pH activity curve of phospholipase A1 obtained in Example 2 (◯) and Production Example 2 (●) (in the presence of Triton X-100).

FIG. 2 shows a pH activity curve of phospholipase A1 of Example 2 (◯) (in the absence of Triton X-100).

FIG. 3 shows the pH stability of phospholipase A1 obtained in Example 2 (◯) and Production Example 2 (●).

FIG. 4 is a temperature activity curve of phospholipase A1 of Example 2 (◯) and Production Example 2 (●).

FIG. 5: Temperature stability of phospholipase A1 of Example 2 (◯) and Production Example 2 (●)

FIG. 6 shows changes with time in apparent phospholipid lysolysis rate (●) and pH (◯) by phospholipase A1 of Production Example 2.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification number Office reference number FI technical display location (C12N 9/16 C12R 1: 685)

Claims (10)

[Claims] 1. It has an action of hydrolyzing a phospholipid to produce a lysophospholipid, and has an optimum action pH of 4.0 to 5.
0, the optimum action temperature is 50 ° C to 60 ° C,
Acid phospholipase A1 having a molecular weight of 32,000 and an isoelectric point of pI 3.0. 2. A method for producing lysophospholipid, which comprises treating phospholipid with acid phospholipase A1. 3. The acidic phospholipase A1 has a pH of 2.5.
The method for producing a lysophospholipid according to claim 2, which is a phospholipase A1 having an active region of 1 to 6. 4. The method for producing a lysophospholipid according to claim 2, wherein the acid phospholipase A1 is phospholipase A1 having a stable upper limit temperature of 45 ° C. to 90 ° C. 5. Acid phospholipase A1 has a molecular weight of 3
10,000 to 40,000, and the isoelectric point pI 2.8
A phospholipase A1 having a pI of 4.5.
3. The method for producing a lysophospholipid according to 3 or 4. 6. Acid phospholipase A1 has a molecular weight of 3
2,000 to 37,000 with an isoelectric point pI3.0
To phospholipase A1 having a pI of 4.3.
3. The method for producing a lysophospholipid according to 3 or 4. 7. The acidic phospholipase A1 has a pH of 3.2.
The method for producing a lysophospholipid according to claim 2, 3, 4, 5 or 6, which is phospholipase A1 having an optimum pH of action of 5 to 5, an optimum temperature of 50 to 60 ° C and a temperature stability of 55 ° C or lower. 8. The method for producing a lysophospholipid according to claim 1, 2, 3, 4, 5, 6, or 7, wherein the acid phospholipase A1 is phospholipase A1 produced by a filamentous fungus of the genus Aspergillus . 9. The acid phospholipase A1 is phospholipase A1 produced by Aspergillus niger, which is 1, 2, 3, 4, 5, 6 or 7.
Method for producing lysophospholipids. 10. The phospholipase A1 produced by Aspergillus oryzae , wherein the acid phospholipase A1 is a phospholipase A1 produced by Aspergillus oryzae.
Method for producing lysophospholipids.