JPS62239990A - Novel lipase and production thereof - Google Patents

Abstract

PURPOSE:To obtain lipase to decompose triglyceride in serum completely, by cultivating a strain capable of producing lipase having properties wherein lipase is activated with a surface active agent and activity is not inhibited in <=5wt% concentration range of the surface active agent. CONSTITUTION:A strain such as Penicillium cyclopium ATCC34613 belonging to the genus Penicillium, capable of producing lipase having properties wherein lipase acts on triglyceride well, has >=5% ratio of glycerol forming activity to fatty acid forming activity, is activated with a surface active agent and not substantially inhibited in <=5wt% concentration range of the surface active agent is cultivated by an ordinary culture method for molds. Then, the above- mentioned lipase is collected from the culture mixture. The lipase decomposes triglyceride and can be preferably used especially for decomposing triglyceride in serum.

Description

DETAILED DESCRIPTION OF THE INVENTION [Object of the Invention] Industrial Application Field The present invention relates to a novel lipase and a method for producing the same. The lipase of the present invention is used for the decomposition of triglycerides, particularly preferably for the decomposition of triglycerides in serum.

Conventional Technology It is known to measure triglycerides by decomposing triglycerides present in biological body fluid samples such as serum using the enzyme lipase and quantifying the released fatty acids or glycerol, but there are no methods that have been put to practical use. Most of these methods quantify glycerol. The first lipases used for this purpose were lipases derived from animal pancreas. However, this enzyme was unable to completely degrade triglycerides. In order to completely enzymatically decompose triglycerides in serum, researchers investigated lipases originating from microorganisms, decomposition using combinations of lipases with different properties, and decomposition using combinations of lipases and chemical agents. For example, Rhizopus alizas (R
hizopus arrhizus) lipase (JP 59-15638, JP 52-25693), Pseudomonas lipase (JP 49-50990, JP 49-69186, JP 49-89596, JP 49-113) 695, Same 57-
58898), combined use of lipase and protease (Japanese Patent Publication No. 54-9518, Japanese Patent Publication No. 53-114493'),
A combination of lipase from Rhizopus alizas, carboxylesterase derived from pig liver, and alkyl sulfate of an alkali metal or alkaline earth metal
-64495), a combination of Candida lipase, pancreatic lipase and bile salts (Japanese Patent Application Laid-Open No. 52-11987), Chromobacterium (
Lipase of the genus Chromobacterium (JP-A-51-68297, JP-A-51-74692, JP-A-57-
58898), Rhizopus alizas lipase and Candida cylindracea (C. cylindracea)
) combination of lipases (JP-A-52-25694,
Tokuko Sho 56-29), Candida rugosa (Cor
ugosa) lipase and surfactant combination (
Special Publication No. 57-39158), Lipase of Rhizopus alizas and Pseudomonas fluorescens (P.
fluorescens) lipase combination (Japanese Patent Publication No. 57-28276), combination of lipase such as Rhizopus and cholesterol esterase (Japanese Patent Publication No. 56-46799), lipase and surfactant.

A combination of phenol derivatives or aniline derivatives (Japanese Patent Publication No. 5B-5677) is known.

It is also known that certain species of the genus Pseudomonas produce lipases in which the ratio of glycerol-producing activity to fatty acid-producing activity is 1% or more (Japanese Patent Laid-Open No. 187780).

It is also known that Penicillium species produce lipase. Iwai et al.
Waist ring (Penicillium cyclo)
-pium Westring) strain produces two types of lipases (Agr, BiolCh
em, + pp. 1063-1070, Volume 39, 198
0 years). They also have Penicillium Cyclopium M1
reported that the strain produces two types of lipase (J
, Biochem, + pages 205-211, Volume 87,
(1980).

Problems to be Solved by the Invention In the prior art related to the decomposition of triglycerides in serum as described above, lipases derived from microorganisms used alone are still unable to sufficiently decompose triglycerides or the rate of decomposition is slow. Ta. Another drawback of these lipases is that their activity is inhibited by the surfactant contained in the reagent composition for triglyceride measurement. Furthermore, when multiple lipases from different origins are used in combination, the production is complicated.

The present invention solves these problems and uses surfactants such as polyethylene glycol alkyl phenyl ether, which can sufficiently decompose triglycerides even when used alone, and which are used to emulsify fat in the measurement of triglycerides. The object of the present invention is to provide a novel lipase whose activity is not substantially inhibited by surfactants and a method for producing the same.

[Structure of the invention]

Means for Solving the Problems According to the present invention, a surfactant that acts on triglycerides, has a ratio of glycerol-generating activity to fatty acid-generating activity of 5% or more, is activated by a surfactant, and has a concentration of at least 5% or less. Provided are a novel lipase whose activity is not substantially inhibited within this range, and a method for producing the same.

The lipase of the present invention can be obtained by culturing a strain belonging to Penicillium genus. As a strain that belongs to the genus Penicillium and produces the lipase of the present invention, particularly preferred is Penicillium cyclopium ATCC 34613 (Penicillium cyclopium ATCC 34613).
illium cyclopium ATCC34
613).

It was confirmed that Penicillium cyclopium ATCC 34613 produces at least three types of lipases with different properties. The lipase of the present invention is one of them, and is a novel lipase that is clearly different from the two known types mentioned above. The lipase of the present invention exhibits the following physicochemical properties.

(1) Action: Acts on nitriglycerides, and the ratio of glycerol production activity to fatty acid production activity is 5% or more.

(2) Substrate specificity: Easily decomposes triglycerides of fatty acids having 4 to 18 carbon atoms.

(3) Optimal pH range: pH 5 to 7 (shown in Figure 1)
(4) Stable pi (range: pH 3 to 8, after treatment at 37°C for 30 minutes, residual activity was measured, and it was found to be stable in the pH range of approximately 4.5 to 6 (as shown in Figure 2). ).

(5) Range of action heating: 35-40℃ (shown in Figure 3)
(6) Temperature stability: At pH 7.0, residual activity was measured after treatment at each temperature of 0 to 50°C for 30 minutes, and it was found to be stable up to about 35°C (shown in Figure 4).

(7) Inhibition, activation and stabilization: activated by surfactants and whose concentration is at least 5
% or less, the activity is not substantially inhibited.

(8) Molecular weight: Approximately 110,000 (by gel filtration method using Sephadex G-100) (9) Isoelectric point: pH 3,84 (by isoelectric focusing method using Ampholine) (10) Crystal Shape: rhombus, plate The reactivity of the lipase of the present invention to triglycerides was determined using known lipases derived from the genus Pseudomonas (trade name: Riboprotein Lipase Type A, manufactured by Toyobo Co., Ltd.) and lipases derived from the genus Chromobacterium (Toyobo Co., Ltd.). Table 1 shows the results in comparison with those manufactured by Jozo Co., Ltd.).

(The following is a blank space) Table 1 The lipase of the present invention and the above-mentioned known lipase differ most in sensitivity to surfactants and production rate of fatty acids and glycerol. That is, the lipase of the present invention has the property that its activity is not substantially inhibited even in the presence of a surfactant at a high concentration of about 5%, and it rapidly produces glycerol. This property is useful when lipase is used to degrade triglycerides in serum.

In the present invention, the Penicillium genus strain is cultured by a general culture method for filamentous fungi. For example, as the nutrient medium, a synthetic medium or a natural medium containing a carbon source, a nitrogen source, inorganic substances, etc. that can be assimilated by Penicillium strains is used. Glucose, fructose, maltose, sucrose, molasses, starch, dextrin, organic acids, glycerin, and the like are used as carbon sources. As nitrogen sources, organic nitrogen sources such as malt extract, peptone, yeast extract, dried yeast, meat extract, corn steep liquor, casein, amino acids, and inorganic nitrogen sources such as nitrates and ammonium salts are used.

Inorganic substances include potassium, sodium, magnesium,
Inorganic salts such as calcium, zinc, iron, etc. are used as necessary. Antifoaming agents such as surfactants, silicones, and vegetable oils can also be added to suppress foaming during culture.

Cultivation is usually carried out under aerobic conditions with shaking or aeration. The culture conditions, such as culture temperature and pH of the medium, may be any conditions as long as they are within the range in which the bacteria can grow and lipase can be produced.

The lipase of the present invention can be collected from the culture obtained as described above by appropriately combining known protein purification methods by taking advantage of its physicochemical properties.

For example, after removing bacterial cells by filtering or centrifuging the culture, salting out using ammonium sulfate, sodium sulfate, etc., organic solvent precipitation using ethanol, methanol, acetone, etc., activated carbon, silica gel, alumina, hydroxyapatite, etc. , adsorption chromatography using cellulose, ion exchange chromatography using ion exchange resin, ion exchange cellulose, ion exchange Sephadex, etc., gel filtration and electrophoresis using Sephadex, biogel, etc., ultrafiltration, dialysis. Purification is carried out by appropriately combining or repeating known methods such as in any order.

In the present invention, lipase activity is expressed as 1 unit, which is the amount of 1 microequivalent of glycerol produced per minute when lipase is allowed to act on the substrate olive oil emulsion at 37°C.

Enzyme activity measurement method 1) Glycerol production activity Glycerol produced is measured by an enzymatic method using an olive oil emulsion as a substrate.

(1) Reagent a) Substrate Niolive oil (manufactured by Hanui Chemical Co., Ltd.) 10g, Triton X-10010g, purified water 30r11! Stir and emulsify for 30 minutes using a stirrer. Next, 50mM IJ acid buffer (pH 6.5) containing 10% bovine serum albumin (fraction ■) was added and mixed.

b) Glycerol measurement reagent: 100-MES (2-(
N-monopholino)ethane-sulfonic acid] buffer (pH 6
, 5) dissolve the following reagents.

Triton X-1000, 1g, N-ethyl-N-(2
-Hydroxy-3-sulfopropyl)-m-) Rubidine 64.6■, 4-aminoantipyrine 10.2■, E
DTA disodium 37.2■, adenosinate phosphate disodium 200■, magnesium chloride hexahydrate 40.7■, glycerol kinase 50 units, glycerophosphate oxidase 400 units, peroxidase 200 units.

(2) Manipulation Place 1.0- of the substrate in a test tube and prewarm it at 37°C. Add 0.1rI11 of the diluted enzyme solution to this to start the reaction.

After reacting at 37°C for 15 minutes, 2.0 ml of 0.2M trichloroacetic acid solution was added to stop the reaction. The reaction stop solution is filtered using Toyo filter paper (m131). Filtrate 0.02mf
was added to glycerol measurement reagent 3- and incubated at 37°C for 10
Heat for a minute and measure absorbance at 555 nm.

(3) Activity display The amount of enzyme that produces 1 micromole of glycerol per minute was defined as 1 unit.

2) Fatty acid production activity Using the olive oil emulsion as a substrate, the fatty acids produced are measured by titration with a sodium hydroxide solution.

(1) Procedure Place 1.0 mt' of the above substrate in a test tube and prewarm it at 37°C. Add 0.1 - of the diluted enzyme solution to this to start the reaction. After reacting at 37°C for 15 minutes, 2.5 mf' of an ethanol-acetone mixture (1:1) was added to stop the reaction.

Add a few drops of phenolphthalein as an indicator and add 1/20
Titrate with M sodium hydroxide.

(2) Activity display The amount of enzyme that produces 1 micromole of fatty acids per minute is 1
It was taken as a unit.

Example 1 Culture medium (pH 6.0) consisting of 2% rice bran and 1.5% corn stewley strength 20jl! Penicillium cyclopium in a jar containing ^TCC3461
3 was inoculated and cultured at 25°C for 24 hours to prepare a seed culture. The seed culture solution was inoculated into a fermenter containing 500ml of a medium with the same composition as above, and cultured at 25°C for 40 hours. The culture solution was filtered to remove bacterial cells, and the resulting filtrate was concentrated by ultrafiltration. 75% ammonium sulfate in concentrate
In addition to saturation, the generated precipitate was collected and dissolved in 10 mM phosphate buffer (pH 7,0) 201.

After desalting this solution by ultrafiltration, DEAE-cellulose 2k+r equilibrated in advance with the same buffer was added. After washing DEAE-cellulose with the same buffer solution 30/, the same buffer solution containing 0.25M sodium chloride was added, and the obtained eluate was desalted and concentrated by ultrafiltration. This solution was mixed in advance with 10mM phosphate buffer (pH 7).
The mixture was passed through a column packed with DEAE-Sepharose (manufactured by Pharmacia) equilibrated with . column to 0.1
After washing with the same buffer containing M sodium chloride, elution was performed by a linear concentration gradient method in which the concentration of sodium chloride was increased from 0.1 to 0.25M. Lipase activity was divided into three peaks (shown in Figure 5). Both parts of the second active peak were collected and subjected to ammonium sulfate weir fractionation, and the precipitate produced in the range of 55% saturation to 75% saturation was collected. The precipitate was dissolved in 10 mM phosphate buffer (p)l 7.0), desalted by ultrafiltration, and then freeze-dried to obtain a purified lipase preparation. This purified sample was subjected to column chromatography using hydroxyapatite, and then crystallized from ammonium sulfate solution. The crystal shape was rhombic and plate-like.

The purified sample obtained above has a specific activity of 9.2 U/■ protein, is purified approximately 130 times from the culture solution, and has a yield of approximately 1
It was 7%.

Reference Example 1 In Example 1, both the first and third active peaks obtained by DEAE-Sepharose column chromatography were further purified.

The sample obtained from the first activity peak has a strong effect on tributyrin and weak effect on fatty acid methyl esters, while the sample obtained from the third activity peak has a strong effect on tributyrin and weak effect on fatty acid monoglycerides. It works well, but
The effect on triglycerides was weak. These properties correspond to the lipases disclosed in the prior art cited above.

Reference Example 2 The reactivity of lipase to lipids was examined by allowing the lipase of the present invention to act on serum lipids and measuring the produced glycerol.

i.e. 0.5U/-glycerol kinase, 4U/-α
- Glycerophosphate oxidase, 2U/- peroxidase, 3.3mM adenosine triphosphate, 0.5m
M4-aminoantipyrine, 2.0mM TOO3 (
N-ethyl-N-(2-hydroxy-3-sulfopropyl)-m-)luidine), 2 mM magnesium hexahydrate, 0.5 U/- of the lipase of the present invention, and 0.05 to 5. 0.1 M P I PEI buffer (pl+ 6.5) containing polyethylene glycol p-isooctylphenyl ether (trade name Nitriton X-100, manufactured by Rohm & Haas) in a concentration range of 0%
1.0- and standard serum sample (trademark: Lipid Serum ■”
Eiken”, manufactured by Eiken Chemical Co., Ltd.) 0.01- and heated to 37°C.
Incubate for 10 minutes. 555 of reaction solution
The absorbance at nm was measured, and the triglyceride (triolein equivalent) concentration in the sample was calculated.

The concentration and measured values of Triton X-100 used are shown in Table 2. It can be seen that the activity of the enzyme of the present invention is not inhibited even by a surfactant at a high concentration of 5%.

Table 2 Reference Example 3 0.5 U/rn1 glycerol kinase, 4U/+n
eα-glycerophosphate oxidase, 2U/ml
Peroxidase, 3.3mM adenosine triphosphate, 0
．． 5mM 4-aminoantipyrine, 2.0mM TO
O3, 2mM magnesium chloride hexahydrate, and 0.1
0.1 containing Triton X-100 at a concentration of ~1.0%
M-PI PES buffer (pfl 6.5) 1.0
- and 25 U/- of the lipase of the present invention 0.02- were mixed and incubated for 5 minutes at 37°C. Then,
Serum sample 0.011n1 was added and 555nl was added over time.
The absorbance at m was measured.

The relationship between the concentration of Triton X-100 and the reactivity of lipase is shown in FIG. It can be seen that the enzyme of the present invention is activated by Triton X-100, and its effect hardly changes in the range of 0.1 to 1.0%.

Comparative Example 1 When the glycerol production activity and fatty acid production activity of various lipases were measured, this enzyme was found to be superior to commercially available lipases derived from the genus Pseudomonas (manufactured by Toyobo Co., Ltd., Ohno Pharmaceutical Co., Ltd.) and chromobacterium genus (manufactured by Toyo Jozo Co., Ltd.). , has a characteristic that glycerol production activity is significantly higher than fatty acid production activity.

(Margin below) Table 3 Comparative Example 2 In Reference Example 3, a commercially available lipase derived from Pseudomonas (trade name: LPL'') was used instead of the lipase of the present invention.
Amano''■, manufactured by Ohno Pharmaceutical Co., Ltd.), lipase derived from Chromobacterium (manufactured by Toyo Jozo Co., Ltd.), or lipase derived from Pseudomonas genus (trade name: riboprotein lipase type A, manufactured by Toyobo Co., Ltd.) with the indicated activity. 50
The same procedure was carried out except that U/me, 100 U/min and 0.2 U/min were used.

The relationship between the concentration of Triton X-100 and the reactivity of each lipase is shown in FIGS. 7A to 7C. Figure 7 A is LPL″A
Fig. 7B shows the case where lipase derived from Chromobacterium genus is used, and Fig. 7C shows the case where riboprotein lipase type A is used.

It can be seen that the activity of all of these known enzymes is inhibited as the concentration of Triton X-100 increases.

Reference Example 4 In Reference Example 3, Triton X-100 was replaced with 0.
1 to 0.5% nonylphenol ethoxylate surfactant (trade name Niadecatol NP-700, manufactured by Asahi Denka Co., Ltd.) or 0.1 to 0.5% secondary linear alcohol ethoxylate surfactant ( Trade name: Niadecatol So
, 135, manufactured by Asahi Denka Co., Ltd.) was used.

The relationship between the concentration of surfactant and the reactivity of the lipase of the present invention is shown in FIGS. 8 and 9. FIG. 8 and FIG. 9 show the case where a nonylphenol ethoxylate surfactant and a secondary linear alcohol ethoxylate surfactant are used, respectively.

It can be seen that the enzymes of the invention are not substantially inhibited by these surfactants.

〔Effect of the invention〕

According to the present invention, a novel lipase and a method for producing the same are provided. The lipase of the present invention almost completely decomposes triglycerides in serum, and its activity is not substantially inhibited even by high concentrations of surfactants, so it is extremely useful for measuring triglycerides.

[Brief explanation of drawings]

Figure 1 is a diagram showing the optimal pH range of the lipase of the present invention, and Figures 2.3.4 are diagrams showing the stable pH range, optimal action temperature range, and temperature stability, respectively. Figure 5 shows Penicillium Cyclopium ATCC34
613 is a diagram showing column chromatography patterns of three types of lipase produced by DEAE-Sepharose. FIG. 6 is a diagram showing the relationship between the concentration of Triton X-100 and the reactivity of the lipase of the present invention. Similarly, FIG. 7A shows lipase derived from the genus Pseudomonas (LPL" Aman
Fig. 7B shows the reactivity of lipase derived from the genus Chromobacterium, and Fig. 7C shows the reactivity of the lipase derived from the genus Pseudomonas (lipoprotein lipase type A). FIG. 9 is a diagram showing the relationship between the concentration of a nonylphenol ethoxylate surfactant and the reactivity of the lipase of the present invention. It is a diagram showing relationships.

Claims (1)

[Claims] 1. A substance that acts well on triglycerides, has a ratio of glycerol-generating activity to fatty acid-generating activity of 5% or more, is activated by a surfactant, and has a concentration of at least 5%.
A lipase whose activity is not substantially inhibited within the following range. 2. The lipase according to claim 1, wherein the surfactant is a nonionic surfactant. 3. The lipase according to claim 2, wherein the nonionic surfactant is a polyethylene glycol alkyl ether surfactant. 4 Belongs to the genus Penicillium, acts well on triglycerides, has a ratio of glycerol-generating activity to fatty acid-generating activity of 5% or more, is activated by surfactants, and is substantially inhibited when its concentration is at least 5% or less. 1. A method for producing lipase, which comprises culturing a strain that produces a lipase having properties that do not affect the production of lipase, and collecting the lipase from the resulting culture. 5. The method for producing lipase according to claim 4, wherein the strain belonging to the genus Penicillium is Penicillium cyclopium.