JPH10127278A - Production of cholesterol esterase - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject esterase capable of hydrolyzing an ester type cholesterol and having physicochemical properties useful for the diagnosis of arteriosclerosis and myocardial infarct and excellent in practical properties by culturing a specific strain belonging to the genus Burkholderia. SOLUTION: This cholesterol esterase is obtained by culturing strains of Burkholderia-sp. No.120-11 (FERM-P-15924), etc., and has the following properties: (1) hydrolyses a cholesterol ester to form a fatty acid and cholesterol; (2) affects cholesterol linoleate most strongly, affects cholesterol oleate, cholesterol palmitate and cholesterol octanoate, and only scarcely affects cholesterol acetate, etc.; (3) an optimum pH is in the vicinity of 6.5, suitable temperature range for action thereof is 43-47 deg.C, stable up to approximately 60 deg.C and excellent in thermal stability.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD [0001] The present invention relates to cholesterol
A method for producing cholesterol esterase, which hydrolyzes esters to produce cholesterol and fatty acids, and more specifically belongs to the genus Burkholderia,
A strain having an esterase-producing ability is cultured in a medium, and cholesterol esterase is collected from the culture.
The present invention relates to a method for producing cholesterol esterase.

[0002]

2. Description of the Related Art Quantification of total cholesterol (total amount of free cholesterol and ester cholesterol) and ester cholesterol in blood is very important in clinical diagnosis, and it is important to know the amount of cholesterol in the artery. It is useful for diagnosis of sclerosis, myocardial infarction, etc., and is regarded as important in health. Conventionally, as a method for quantifying free cholesterol, there is known a method for directly quantifying enzymatically by reacting cholesterol oxidase. However, since this enzyme does not act on ester cholesterol, it is necessary to quantify ester cholesterol. In this case, the ester bond is first hydrolyzed to form free cholesterol, and then cholesterol oxidase is acted on the free cholesterol to measure the hydrogen peroxide produced to quantify the ester cholesterol. The method is generally performed. Examples of the method of hydrolyzing the ester-type cholesterol include a method of chemically reacting with alcoholic potassium hydroxide to saponify, a method of enzymatically reacting cholesterol esterase, and the like. The latter method is preferably used because the decomposition operation is simple and direct and rapid quantification can be performed in an automatic analysis method or the like.

[0003] Cholesterol esterase is an enzyme that acts on ester-type cholesterol to produce free cholesterol and fatty acids, particularly those derived from animal pancreas and liver, and those derived from microorganisms. . Pseudomonas (Pseudomonas) include cholesterol esterases derived from microorganisms.
s) obtained from microorganisms belonging to the genus
7588), Fusarium (Fusariu)
m) obtained from a microorganism belonging to the genus (Journal of Biochemistry (Journal of Biochemistry)
Chemistry) Vol. 81, 1209-1215
P. 1977), Schizophyllam (Schizo).
phyllum) (see Japanese Patent Publication No. 55-34674), basidiomycetes obtained from the genus Kawatake (see Japanese Patent Publication No. 56-53355), Streptomyces (Streptomyces).
Genus (see Japanese Patent Publication No. 60-1531) and Nocardia cholestericum (Noca).
rdia cholesterolicum) NRRL
No. 5767 (see Japanese Patent Publication No. 60-58955) and the like are known. However, there is no report on cholesterol esterase derived from a microorganism belonging to the genus Burkholderia.

[0004]

SUMMARY OF THE INVENTION An object of the present invention is to provide a method for easily obtaining a cholesterol esterase having excellent physicochemical properties, which is excellent in practical use and is used for the determination of ester cholesterol. It was done as.

[0005]

Means for Solving the Problems The present inventors have conducted intensive studies in order to achieve the above-mentioned object, and as a result of searching for microorganisms capable of producing cholesterol esterase from the natural world, as a result, it has been found that the microorganisms isolated from soil That the strain belonging to the genus Horderia produces cholesterol esterase, and the optimum pH of the enzyme produced by the strain
Was found to be slightly acidic and excellent in thermal stability and suitable for practical use. Based on this finding, the present invention was completed. That is, the present invention provides a method for producing cholesterol esterase, which comprises culturing a strain belonging to the genus Burkholderia and having a cholesterol esterase-producing ability in a medium, and collecting cholesterol esterase from the culture. . Hereinafter, the present invention will be described in detail.

[0006]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS First, as a bacterium used in the present invention, Burkholderia
Any bacteria belonging to the genus and capable of producing cholesterol / esterase may be used, and their variants or mutants may be used. And, as a specific example, Burgholderia SP No. 128-11 (Bu
rkholderia sp. No. 128-11)
And a variant or mutant of the strain can also be used. This Burgholderia sp. 128
Numeral -11 is a bacterium obtained from the soil of a field on the outskirts of Nara city and purely isolated by the present inventors, and the bacteriological properties thereof are as follows.

<Burkholderia sp. No. 1
Bacteriological properties of 28-11> (a) Morphological properties Microscopic observation (30 minutes on broth agar medium (pH 7.0))
(C for 6 to 48 hours) (1) Cell shape and size: 0.7 to 0.8 x 1.4 to
1.6 μm bacilli. (2) Cell polymorphism: single or double linkage. (3) Mobility: Mobility. It has several polar flagella. (4) Spore presence: no spore. (5) Gram staining: negative. (6) Acid resistance: negative. (B) Growth state (1) Gravy agar plate culture: A circular colony having a diameter of about 1.5 to 2.0 mm is formed by culturing at 30 ° C. for 48 hours, the surface of which is smooth, glossy, and a convex circular protuberance. And the color tone is pale cream with no production of soluble pigment. (2) Gravy agar slant culture: grows in filament form at 30 ° C. for 24 hours. The surface is shiny and pale cream with good growth. (3) Liquid broth culture: Still culture at 30 ° C. for 48 hours causes slight turbidity and precipitates. (4) Meat gelatin stab culture: grown at 30 ° C. for 24 hours and grown only on the surface of the medium, not along the puncture hole. It also liquefies gelatin as it grows. (5) Litmus milk: It becomes slightly alkaline after standing at 30 ° C. for 3 days and liquefies but does not coagulate. (C) Physiological properties (1) Reduction of nitrate: negative. (2) Denitrification reaction: negative. (3) MR test: negative. (4) VP test: negative. (5) Indole formation: negative. (6) Production of hydrogen sulfide: negative. (7) Starch hydrolysis: negative. (8) Use of citric acid: Use. (9) Use of inorganic nitrogen source: nitrate is not used, but ammonium salt is used. (10) Dye formation: negative. (11) Urease: positive. (12) Oxidase: positive. (13) Catalase: positive. (14) Growth range: pH; 4.5-8.5, temperature;
42 ° C. (15) Attitude to oxygen: aerobic. (16) OF test: oxidative. (17) Generation of acid and gas from saccharides: L-arabinose, D-xylose, D-glucose,
D-mannose, D-fructose, D-galactose, lactose, trehalose, D-sorbitol, D
-It produces acids from mannitol, inositol, glycerol, maltose and sucrose and not from starch. No gas generation was observed from any of them.

[0008] The above properties are described in the Verges Manual.
Of Detaminative Bacteriology (Ber
gay's Manual of Determina
five Bacteriology) 9th edition (199)
4 years), it is a bacillus in form, is an aerobic bacterium, has several polar flagella,
Pseudomonas (Pseudo) due to being gram negative, motile, and catalase positive
omonas), but was considered to belong to the genus Oya.
Izu and Komagata report that differences in the composition of 3-hydroxy fatty acids correspond to the five ribosomal ribonucleic acid-deoxyribonucleic acid (rRNA-DNA) homology groups of the genus Pseudomonas (Journal of General and General).・ Applied microbiology (Journal of General)
and Applied Microbiology)
29, 17-40, 1983).

[0009] Then, when the intracellular fatty acid composition of this strain was examined, among the 3-hydroxy fatty acids, 3-hydroxymyristic acid (3OH-C14: 0) and 3-hydroxypalmitic acid (3OH-C16: 0) were determined. The presence was recognized. From the composition of the 3-hydroxy fatty acid, it was revealed that this strain is a strain belonging to the rRNA-DNA homology group II of the genus Pseudomonas. This rRNA-DNA homology group II is 1993
Independently from the genus Pseudomonas as the genus Burkholderia in 2004 (Microbiology and Immunology (Microbio
volume and Immunology) Vol. 36,
1251-1275, 1992), and thus the strain was determined to belong to the genus Burkholderia. This was supported by the main quinone system being Q-8. Furthermore, among intracellular fatty acids,
Lactobacillic acid (C19CPA) is replaced by stearic acid (C1
8: 0), the presence of Burkholderia cepacia is more than twice as large.
cepacia). As a result of electron microscopic observation, the strain was confirmed to have cilia (pili or fimbriae) around the cells in addition to several polar flagella. Such cilia are known as Burkholderia cepacia and Pseudomonas flagii.
onas fragi) (Journal of General Microbiology)
(biology) Vol. 58, pp. 227-237, 19
1969), whereas the growth temperature range of Pseudomonas flagii is 10 ° C to 30 ° C, whereas the growth temperature range of this strain is 5 ° C to 42 ° C, which is clearly different from Pseudomonas flagii. Was.

From the above results, it is recognized that this strain is closely related to Burkholderia cepacia, but the above literature and the International Journal of Systematic Bacteriology (Internationa
l Journal of Systematic Ba
cterology) Vol. 31, pp. 479-481,
Compared with the properties described in 1981, the properties such as pigment-producing ability, ornithine decarboxylation ability, urease-producing ability, and esculin-degrading property are clearly different, indicating that this strain is a new species belonging to the genus Burkholderia. As a result, Burgholderia SP No. 128-11
(Burkholderia sp. No. 128-1
1). This strain has been deposited with the National Institute of Bioscience and Biotechnology at the National Institute of Advanced Industrial Science and Technology as FERM P-15924.

Next, belonging to the above-mentioned genus Burkholderia,
A cholesterol / esterase producing strain is cultured in a medium, and a cholesterol / esterase (hereinafter referred to as “enzyme”) is produced using a strain that collects cholesterol / esterase from the culture. The solid culture method may be used, but the liquid culture method is preferred. As the medium, either a synthetic medium or a natural medium can be used as long as it contains a carbon source, a nitrogen source, an inorganic substance, and other nutrients appropriately.
The carbon source may be any assimilable carbon compound, such as maltose, glucose, starch hydrolyzate, glycerin, fructose, molasses, soybean oil,
Cholesteryl stearate and the like are used. As the nitrogen source, any available nitrogen compound may be used. For example, yeast extract, peptone, meat extract, corn steep liquor, soybean flour, amino acids, ammonium sulfate, ammonium nitrate and the like are used. In addition, various salts such as salt, potassium chloride, magnesium sulfate, manganese chloride, ferrous sulfate, potassium phosphate monobasic, potassium phosphate dibasic, sodium carbonate, sodium acetate, etc., vitamins, antifoaming agents, etc. are used. Is done. These nutrient sources can be used alone or in combination. In order to produce the present enzyme using the medium prepared in this way, aerobically culturing is performed by aeration stirring, submerged culture or shaking culture, and the initial pH of the medium is adjusted to about 5 to 8 at that time. Incubate at a temperature of about 20 to 37 ° C, preferably about 25 to 35 ° C for 10 to 50 hours, preferably 15 to 25 hours. By doing so, the enzyme is produced and accumulated in the culture.

Next, in order to collect the present enzyme from the culture, ordinary enzyme collecting means can be used. Since this enzyme is mainly an enzyme existing outside the cells, the cells are removed from the culture by, for example, filtration, centrifugation, etc., and then the organic solvent is precipitated from the cell-removed solution with acetone or alcohol. The enzyme is obtained by a commonly used method such as a salting out method using ammonium sulfate or other salts. In order to obtain a highly purified enzyme preparation, an adsorption elution method and an electrophoresis method utilizing hydrophobicity and ion exchange can be used. Next, an example of the physicochemical properties of the present enzyme obtained by the method of the present invention is as follows.

<Physicochemical properties of the present enzyme> (1) Action: hydrolyzes cholesterol esters to produce fatty acids and cholesterol.

(2) Substrate specificity: The activity of the present enzyme for various cholesterol esters was measured by the activity measuring method described later, and the relative activity (%) when the activity for cholesterol and linoleic acid was defined as 100. Table 1 shows an example of the obtained result.

[0015]

[Table 1]

As can be seen from Table 1, cholesterol
It acts on linoleic acid, cholesterol / oleic acid, cholesterol / palmitic acid and cholesterol / octanoic acid, and has the strongest effect on cholesterol / linoleic acid. It also slightly acts on cholesterol / stearic acid and cholesterol / acetic acid.

(3) Optimal pH and stable pH range: optimal p
H was determined by using a 200 mM phosphate buffer, adjusting the pH to each with diluted sodium hydroxide and hydrochloric acid, and measuring the activity of the enzyme at each pH. The result is shown in FIG.
The optimum pH of the present enzyme is around 6.5. The stable pH range is 50 mM sodium acetate-acetic acid buffer (pH 3.0-5.0), 50 mM female-sodium hydroxide buffer (pH 5.0-6.5),
0 mM Hepes-sodium hydroxide buffer (pH 6.5-
8.0) and 50 mM chess-sodium hydroxide buffer (pH 8.0-10.0), pH 3.0-10.
After treatment at 37 ° C. for 60 minutes at 0, the residual activity of the enzyme was measured and determined. The results are as shown in FIG. 2, and the stable pH range of the present enzyme is 4.5 to 7.0.
5 In addition, in the mark shown in FIG.
Sodium acetate-acetate buffer (pH 3.0-5.0)
And ■ are 50 mM female-sodium hydroxide buffer (pH
5.0 to 6.5), ● indicates a 50 mM Hepes-sodium hydroxide buffer (pH 6.5 to 8.0), and ○ indicates 5
0 mM chess-sodium hydroxide buffer (pH 8.0-
10.0) are shown.

(4) Suitable temperature range of action: The same substrate and enzyme solution as used in the titer determination method described later are treated at various temperatures (25 ° C. to 70 ° C.) to measure the activity of the enzyme. Was. The results are as shown in FIG. 3, and the range of suitable temperature for the action of this enzyme is 43 to 47 ° C.

(5) Deactivation conditions depending on pH, temperature, etc .:
This enzyme has a pH of 4.5 to 4.5 at 37 ° C. for 60 minutes.
The enzyme was stable at 7.5, and the thermostability of the present enzyme when treated at 50 ° C. for 30 minutes at each temperature using a 50 mM female-sodium hydroxide buffer solution (pH 6.0) was examined. The results are as shown in FIG. 4. The enzyme is stable up to about 60 ° C. and is inactivated at about 75 ° C. by about 30%.

(6) Inhibition, activation and stabilization: Various metal salts shown in Table 2 (each at a concentration of 1 mM) were added to the same substrate / enzyme mixture as used in the titer measurement method described later. Table 2 shows the results obtained by measuring the enzyme activity and examining the effect of each metal salt.

[0021]

[Table 2]

As can be seen from Table 2, this enzyme is HgC
Although strongly inhibited by l ₂ and inhibited by CuSO ₄ , no metal salt is found that contributes specifically to activation and stabilization.

(7) Method for measuring titer: cholesterol
Using this enzyme with linoleic acid as a substrate, the resulting cholesterol is further oxidized by cholesterol oxidase, and the resulting hydrogen peroxide is further quantitatively oxidatively condensed with 4-aminoantipyrine and phenol to form a quinoneimine dye. . 500 of this dye
Measured in nm and the enzyme titer was determined. The composition of the enzyme reaction solution and the reaction conditions are as follows. In addition, one unit of cholesterol esterase in the present invention is obtained by using cholesterol / linoleic acid as a substrate at pH 7.0, 3
The amount of enzyme required to produce 1 micromol of free cholesterol per minute at 7 ° C. was used.

<Composition of reaction solution> Cholesterol / linoleic acid (0.6 mM, 1% Triton X-100 solution); 1.0 ml 86.6 mM 4-aminoantipyrine aqueous solution;
05 ml 638 mM phenol aqueous solution; 0.1 ml peroxidase (150 units / ml, 0.1 M phosphate buffer, pH 7.0); 0.1 ml cholesterol oxidase (100 units / ml,
0.1 M phosphate buffer (200 mM, pH 7.0); 1.5 ml Enzyme solution; 0.1 ml phosphate buffer (200 mM, pH 7.0);
l Reaction liquid volume: 2.95 ml

<Measurement procedure> 2.75 ml of the above reaction mixture was placed in a test tube, preliminarily heated at 37 ° C. for about 5 minutes, added thereto, and further heated for 2 minutes. Next, the above was added, and after gentle mixing, the change in absorbance at 500 nm was recorded for 3 to 4 minutes with a spectrophotometer controlled at 37 ° C. using water as a control, and the change in absorbance per minute from the initial linear portion. Find the quantity.

(9) Purification method: The isolation and purification of the present enzyme can be carried out according to a conventional method, for example, ammonium sulfate precipitation, organic solvent precipitation, adsorption treatment with an ion exchanger, etc., ion exchange chromatography. , Hydrophobic chromatography, gel filtration chromatography, adsorption chromatography, affinity chromatography, electrophoresis and the like are used alone or in appropriate combination.

Although the main physicochemical properties of the cholesterol esterase obtained by the present invention are as described above, it is mainly determined that this enzyme is different from the conventionally known cholesterol esterase. The pH and thermal stability are shown in Table 3 and compared.
In addition, as a target of comparison, as a genus Pseudomonas,
(1) Pseudomonas fluorescens KY3955 described in JP-A-50-157588; (2) Pseudomonas knob SP No. described in JP-B-58-30033. 109, (3) Agricultural
And Biological Chemistry (Agricu
italal and Biological Che
misty) 40, 1957-1964, 19
Pseudomonas fluorescens AT described in 1976
CC21156 and (4) those derived from Pseudomonas sp. Described in the Toyobo catalog. (5) As a genus of Fusarium, Journal of Biochem (Journal of Biochem)
Istry) Vol. 81, pp. 1209-1215, 197
7 years, Fusarium oxysporum described in
The product derived from IGH-2 was used as (6) Schizophyllam in Schizo described in JP-B-55-34674.
The phyllumcommune IFO4928-derived product was designated as (7) Basidiomycete genus Kawaratake,
Coriolus ver. Described in JP-A-6-53355
and those derived from sicolor K-1213, and (8)
As the genus Streptomyces, Streptomyces lavendu described in JP-B-60-15311.
The properties of cholesterol esterase described in each document and those of the present enzyme were compared with those of the present enzyme, including those derived from lae H-646-SY2.

[0028]

[Table 3]

As shown in Table 3, the cholesterol esterase according to the method of the present invention has a slightly acidic optimum pH and a thermal stability of 60 when treated for 30 minutes at pH 6.0.
It is stable up to ° C and differs from that from other microorganisms.

[0030]

Next, the present invention will be described in more detail by way of examples, but the present invention is not limited to these examples. Example 1 Soybean oil 0.5% (W / V), Yeast extract 0.5% (W
/ V), monopotassium phosphate 0. 1% (W / V), dipotassium phosphate 0.1% (W / V), magnesium sulfate heptahydrate 0.01% (W / V), ferrous sulfate heptahydrate, 0 20 l of a medium (pH 7.5) consisting of 0.005% (W / V) and tap water was placed in a 30 l jar,
Sterilized at 0 ° C. for 15 minutes. In this medium, Burkholderia sp. 128-11 (Burkholde
ria. sp. No. A cell suspension prepared using physiological saline was inoculated from the storage slant (3 pieces) of 128-11), and this was cultured with stirring at 30 ° C. for about 24 hours with equal aeration. After completion of the culture, 20 l of the culture solution was removed by centrifugation, and the supernatant was concentrated using an ultrafiltration membrane to obtain 2 m
10 mM phosphate buffer (pH 7.5) containing M magnesium chloride and 0.5 mM ethylenediaminetetraacetic acid
(Hereinafter referred to as buffer A). Purification of the present enzyme was performed on 2.5 l of a half of 5 l of the concentrated dialysate by the following operation.

Step 1: (Phenyl-Sepharose C
L-4B / Chromatography) Ammonium sulfate powder was added to the dialysate (2.5 L) so as to be 5% saturated and mixed, and then phenyl-Sepharose CL-4B was added.
The present enzyme was adsorbed on a column (8 × 30 cm), and the column was washed with buffer A. Next, the enzyme was eluted by the linear concentration gradient method of buffer A containing 0 M to 1.0 M urea and 35 mM sodium cholate, and the active fraction was ultra-concentrated and dialyzed against buffer A. Step 2: (DEAE-Sephacel Chromatography) The dialysate (250 ml) was adsorbed on a DEAE-Sephacel column (6 × 21 cm), and the column was washed with buffer A, and then 0M to 2M. 0.0M urea and 70mM
The buffer A containing sodium cholate was eluted by a linear gradient method, and the active fraction was ultra-concentrated and dialyzed against the buffer A. Step 3: (Sephadex G-200 / chromatography) A portion (2 ml) of the concentrated solution (14 ml) was added to a column (2.5 × 95 cm) of Sephadex G-200, and 0.1 M potassium chloride was added. Gel filtration was performed using the contained buffer solution A, and 80 ml of the eluted active fraction was collected. The purification operation of the above Step 3 was repeated a total of seven times to obtain a purified enzyme preparation having a total protein amount of 10 mg and a total activity of 510 U.

[0032]

According to the present invention, cholesterol esterase having excellent heat stability can be easily produced. The enzyme is highly practical because of its excellent thermal stability, and has good storage properties when the enzyme is used as a liquid reagent. Can be

[Brief description of the drawings]

FIG. 1 is a graph showing the optimum pH of the present enzyme.

FIG. 2 is a graph showing a stable pH range of the present enzyme.

FIG. 3 is a graph showing a temperature range suitable for the action of the present enzyme.

FIG. 4 is a graph showing the thermostability of the present enzyme.

Claims (2)

[Claims] 1. A method for producing cholesterol esterase, comprising culturing a strain belonging to the genus Burkholderia and capable of producing cholesterol esterase in a medium, and collecting cholesterol esterase from the culture. 2. A strain having the ability to produce cholesterol esterase is Burkholderia sp. 1
The method for producing cholesterol esterase according to claim 1, which is 28-11.