JPH1156355A - Production of cholesterol esterase - Google Patents

Abstract

PROBLEM TO BE SOLVED: To produce the subject enzyme having the optimum pH or a slightly acidic side excellent in thermal stability and useful for determination or the like of an ester-type cholesterol by culturing cholesterol esterase-producing microorganisms belonging to the genus Xanthomonas. SOLUTION: A strain [e.g. Xanthomonas sp. No. 81-13 (FERM P-16369)] belonging to the genus Xanthomonas and having cholesterol esterase-producing activities is cultured in a medium, and the objective cholesterol esterase is collected in the method for producing the cholesterol esterase. The cholesterol esterase derived from the Xanthomonas sp. No. 81-13 (1) strongly acts on cholesterol oleate, cholesterol linolate, and cholesterol palmitate in order, and further slightly acts on cholesterol acetate, (2) has the optimum pH at nearly pH 6. 5, and a stable pH region within 5.5-8. 5, (3) has the acting optimum temperature within a rage of 45-50 deg.C, and (4) has about 55,000 molecular weight (SDS-PAGE).

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD [0001] The present invention relates to cholesterol
A method for producing cholesterol esterase that hydrolyzes esters to produce cholesterol and fatty acids, more specifically, a cholesterol esterase-producing strain belonging to the genus Xanthomonas is cultured in a medium, and cholesterol esterase is collected from the culture. And a method for producing cholesterol esterase.

[0002]

2. Description of the Related Art The 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 quantitative values in arteries. 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), from the genus Kawatake (see Japanese Patent Publication No. 56-53355), and from the genus Streptomyces (see Japanese Patent Publication No. 56-53355). No. 60-15311, Nocardia cholestericum (Nocardia choles)
tericum) obtained from NRRL 5767 (see Japanese Patent Publication No. 60-58955). However, Xanthomonas (Xanthomonas)
There are no reports on cholesterol esterases derived from microorganisms belonging to the genus onas).

[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 extensively searching for microorganisms capable of producing cholesterol esterase from nature, Xanthomonas isolated from soil. That the strain belonging to the genus produces cholesterol esterase, and the optimal 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 is a method for producing cholesterol esterase, which comprises culturing a strain belonging to the genus Xanthomonas and capable of producing cholesterol esterase in a medium, and collecting cholesterol esterase from the culture. Hereinafter, the present invention will be described in detail.

[0006]

BEST MODE FOR CARRYING OUT THE INVENTION First, as a bacterium used in the present invention, any bacterium belonging to the genus Xanthomonas and capable of producing cholesterol esterase may be used. Good. And as a specific example, Xanthomonas sp. 81-13 (Xantho
monas sp. No. 81-13), and variants or mutants of the strain can also be used. This Xanthomonas sp. 81-13 (hereinafter referred to as "the present bacterium") is a bacterium obtained by the present inventors from the soil of a field on the outskirts of Kumamoto city and purely isolated, and the bacteriological properties thereof are as follows. .

<Xanthomonas sp. No. 81
Mycological properties of -13> (a) Morphological properties Microscopic observation (30 minutes on gravy agar medium (pH 7.0)
(Culturing at 20 ° C. for 20 hours) (1) Size and shape of cells: 0.3 to 0.6 × 1.5 to
1.7 μm bacilli. (2) Presence or absence of cell polymorphism: None. (3) Motility and flagellation: Motility.
It has one or several polar flagella. (4) Spore presence: None. (5) Gram staining: negative.

(B) Growth state (1) Gravy agar plate culture: A round colony having a diameter of about 0.8 to 1.0 mm is formed by culturing at 30 ° C. for 24 hours, and its surface is smooth, glossy, and convex. It has a slightly transparent lemon color. (2) Broth liquid culture: grows well and becomes turbid by shaking culture at 30 ° C. for 24 hours. In addition, the culture solution becomes viscous in the stationary culture for 48 hours or more. (3) Meat gelatin stab culture: Liquefaction at 30 ° C for 4 days. (4) Litmus milk: liquefaction is observed at 30 ° C. for 2 days, but no acid or alkali is produced.

(C) Physiological properties (1) Reduction of nitrate: positive. (2) Denitrification reaction: negative. (3) MR test: positive. (4) VP test: negative. (5) Indole formation: negative. (6) Production of hydrogen sulfide: negative. (7) Starch hydrolysis: positive. (8) Use of citric acid: positive for Coser's medium, positive for Christensen's medium. (9) Use of inorganic nitrogen source: Both nitrate and ammonium salts are positive. (10) Dye formation: negative. (11) Urease: negative. (12) Oxidase: positive. (13) Catalase: positive. (14) Range of growth: pH; 4.5-9.5, temperature;
~ 48 [deg.] C. (15) Attitude to oxygen: aerobic. (16) OF test: oxidative. (17) Nutritional requirements: None. (18) ONPG test (β-galactosidase production test): positive. (19) Extracellular DNase: positive. (20) Casein degradation: positive. (21) Degradation of esculin: positive. (22) Tyrosine degradation: positive. (23) Amino acid decarboxylase: negative when L-lysine is used as a substrate. Negative when using L-ornithine as substrate. (24) Arginine dihydrolase: negative. (25) Amino acid deaminase: When phenylalanine is used as a substrate, phenylpyruvic acid is generated. Negative formation of indolepyruvic acid when tryptophan was used as a substrate. (26) Lipase: positive. (27) Lecithinase: positive. (28) Quinone type: Q-8. (29) Intracellular main fatty acids: 13-methyltetradecanoic acid, 15-methylhexadecanoic acid, 12-methyltetradecanoic acid, tetradecanoic acid, hexadecanoic acid and the like. (30) Presence or absence of generation of acid and gas from sugar: L-arabinose, D-xylose, D-glucose, D-mannose, D-fructose, D-galactose, maltose, sucrose, lactose, trehalose, and starch Generate. No gas generation was observed from any of them.

[0010] The above properties are described in the Verges Manual.
Of Detaminative Bacteriology (Berg
eye's Manual of Determinat
live Bacteriology) 9th edition (1994)
Year), this strain is an aerobic gram-negative bacillus, having one to several polar flagella, having no fermentability,
The production of acids from various sugars and the quinone type of Q-8 indicate that Pseudomonas (Ps
eumonas) was expected to be a closely related bacterium.

Then, when the fatty acid composition of the cells was examined,
The composition was found to be composed mainly of branched fatty acids of 13-methyltetradecanoic acid and 15-methylhexadecanoic acid. Pseudomonas type bacteria exhibiting a branched fatty acid composition include Stenotrophomonas (Ste
a characteristic found in bacteria of the genera notrophomonas and Xanthomonas (Journal of G. Journal of General and Applied Microbiology).
general and Applied Microb
iology) 24, 199-213, 1978.
Year, and the International Journal of Systematic Bacteriology (International
nal Journal of Systematic
Bacteriology), vol. 46, 298-3.
04, 1996].

First, DNA-DNA hybridization was performed for comparison with Stenotrophomonas.
The genus Stenotrophomonas currently includes Stenotrophomonas maltophilia and S. africa.
Although two species na) have been reported, the latter was just announced as a new species, and the homology was examined only with Stenotrophomonas maltophilia because no type strain was available. As a result, the present bacterium showed only about 20% homology with Stenotrophomonas maltophilia, and thus was clearly different from Stenotrophomonas maltophilia, indicating that the bacterium belongs to the genus Stenotrophomonas. Was not considered. Next, comparison with Xanthomonas sp. 16S ribosomal DNA (rDNA)
Was performed with the nucleotide sequence of As a result, a homology search of the 16S rDNA nucleotide sequence revealed that the bacterium was closely related to a bacterium belonging to the genus Xanthomonas.
The homology was highest with X. campestris. Therefore, as described in the literature (International Journal of Systematic Bacteriology, Vol. 47, pp. 328-335, 1997), 13 representative strains of Xanthomonas bacteria and 2 strains of Stenotrophomonas bacteria are described. Neighbor-joining method using
Phylogenetic analysis was performed. As a result, this bacterium formed a phylogenetic tree different from Xanthomonas bacteria with a high bootstrap value (a value indicating the reliability of a phylogenetic tree created based on the neighbor-joining method). Next, three related strains of the genus Pseudomonas and Burkholderia.
ia) When a phylogenetic tree was similarly prepared including one strain of the genus Bacteria, it was found that the bacterium had a high bootstrap value and was located between Stenotrophomonas and Xanthomonas bacteria. The aforementioned Stenotrophomonas maltophilia was previously included in the genus Xanthomonas, but it is thought that this bacterium is more closely related to the genus Xanthomonas. However, this bacterium was determined to be a new species of the genus Xanthomonas because it was located outside the population of the genus Xanthomonas in all phylogenetic trees. Based on the above, the strain was transformed into Xanthomonas sp.
o. 81-13 (Xanthomonas sp. N)
o. 81-13). And this strain was sent to FERM P-16 by the Institute of Biotechnology and Industrial Technology, National Institute of Advanced Industrial Science and Technology.
369 has been deposited.

Next, a strain belonging to the genus Xanthomonas and capable of producing cholesterol esterase is cultured in a medium, and cholesterol esterase is collected from the culture to produce cholesterol esterase (hereinafter referred to as the present enzyme). First, as a culture method,
An ordinary solid culture method may be used, but a 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. As the carbon source, any carbon compound that can be assimilated may be used. For example, 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, salt, potassium chloride, magnesium sulfate, manganese chloride, ferrous sulfate, potassium phosphate,
Various salts such as dipotassium phosphate, sodium carbonate and sodium acetate, vitamins, antifoaming agents and the like are used. These nutrient sources can be used alone or in combination. To produce the enzyme using the medium thus prepared,
Aerobically cultured by aeration agitation, submerged culture, shaking culture, or the like. At that time, the initial pH of the medium is adjusted to about 5 to 9, Culture for 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.

<Xanthomonas sp. No. 81
Physicochemical properties of -13-derived cholesterol esterase> (1) Action: hydrolyzes cholesterol esters to produce fatty acids and cholesterol.

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

[0017]

[Table 1] Table 1

As can be seen from Table 1, this enzyme is composed of cholesterol / linoleic acid, cholesterol / oleic acid,
It acts on cholesterol / palmitic acid, cholesterol / octanoic acid and cholesterol / stearic acid, and most strongly on cholesterol / oleic acid. It also has a slight effect on cholesterol and acetic acid.

(3) Optimum pH and stable pH range: optimum 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 4.5-5.5), 50 mM
Sodium hydroxide buffer (pH 5.5-7.0), 50
mM Hepes-sodium hydroxide buffer (pH 7.0 to 7.0)
8.0) and 50 mM chess-sodium hydroxide buffer (pH 8.0-10.0), pH 4.5-10.
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 5.5 to 8.0.
5 In addition, in the mark shown in FIG.
Sodium acetate-acetate buffer (pH 4.5-5.5)
And ■ are 50 mM female-sodium hydroxide buffer (pH
5.5 to 7.0), ● represents a 50 mM Hepes-sodium hydroxide buffer (pH 7.0 to 8.0),
mM chess-sodium hydroxide buffer (pH 8.0-1
0.0) are shown.

(4) Range of suitable temperature for action: The same substrate and enzyme solution as used in the titer determination method described later are reacted at various temperatures (25 ° C. to 70 ° C.) to measure the activity of the enzyme. went. The results are as shown in FIG. 3, and the range of suitable temperature for the action of the present enzyme is 45 to 50 ° C.

(5) Deactivation conditions depending on pH, temperature, etc .:
This enzyme has a pH of 5.5 to 5.5 at 37 ° C. for 60 minutes.
The enzyme was stable at 8.5, and the thermostability of the present enzyme when treated with 50 mM Hepes-sodium hydroxide buffer (pH 7.0) at each temperature for 30 minutes was examined. The results are as shown in FIG. 4. The enzyme is stable up to 55 ° C. and is inactivated at 65 ° C. by about 40%.

(6) Inhibition, activation and stabilization: In the same substrate / enzyme mixture in the titer measurement method described later,
Table 2 shows the results obtained by adding various metal salts (concentrations of 1 mM each) and measuring the enzyme activity, and examining the influence of each metal salt.

[0023]

[Table 2] Table 2

[0024] As can be seen from Table 2, this enzyme is HgC
strongly inhibited by l _2, also in the ZnSO ₄ and CuSO ₄ 15
Although it is inhibited by about 25%, no metal salt that particularly contributes to activation and stabilization is found.

(7) Molecular weight: The molecular weight of the present enzyme determined by SDS-polyacrylamide gel electrophoresis is about 55,0
00.

(8) Method of measuring titer: cholesterol
Using linoleic acid as a substrate, cholesterol esterase is acted on, cholesterol generated is further oxidized by cholesterol oxidase, and the generated hydrogen peroxide is further quantitatively oxidatively condensed with 4-aminoantipyrine and phenol to form a quinone dye. Let it. The dye was measured at 500 nm to determine the enzyme titer. The composition and measurement procedure of the enzyme reaction solution are as follows. One unit of cholesterol esterase in the present invention is defined as the amount of enzyme required to produce 1 micromol of free cholesterol per minute under conditions of pH 7.0 and 37 ° C. using cholesterol / linoleic acid as a substrate.

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

<Measurement procedure> The above reaction mixture (2.75 ml) is 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 using a spectrophotometer controlled at 37 ° C. with water as a control.
The amount of change in absorbance per minute is determined.

(9) Purification method: 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.
For comparison, the genus Pseudomonas (1)
Pseudomonas fluorescens KY3955 described in JP-A-50-157588, (2) JP-B-58
No. 30033, Pseudomonas knob SP No. 109, (3) Agricultural and biological chemistry
ral and Biological Chemis
try) Volume 40, 1957-1964, 1976
Pseudomonas fluorescens ATCC listed
21156, and (4) those derived from Pseudomonas sp. Described in the Toyobo catalog. Also (5)
As the genus Fusarium, Journal of Biochemistry
ry) Vol. 81, pp. 1209-1215, 1977,
Fusarium oxysporum IG described in
The product derived from H-2 is referred to as (6) Schizophyllam sp. Schizophylic described in JP-B-55-34674.
from Ilum commune IFO4928, (7) As the genus Kawaratake, JP-B-56-5335
No. 5 publication Coriolus versicolor
r derived from K-1213 and (8) Streptomyces as described in JP-B-60-15311.
reptomyces lavendulae H-6
The properties of cholesterol esterase described in each document and those of the present enzyme were compared for those derived from 46-SY2.

[0031]

[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 heat stability of pH 7.0 when treated for 30 minutes at 55 minutes.
It is stable up to ° C and differs from that from other microorganisms.

[0033]

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
20 l of a medium (pH 7.5) composed of 0.005% (W / V) heptahydrate and tap water was placed in a 30 l jar,
Sterilized at 120 ° C. for 15 minutes. Xanthomonas sp. No. 81-13 (Xanthommon
as. sp. No. 81-13) Preservation slant (3
), A bacterial cell suspension prepared using physiological saline was inoculated, and this was stirred and cultured at 30 ° C. for about 24 hours with equal aeration. After the culture, 20 l of the culture solution was centrifuged to remove bacterial cells, and the supernatant was concentrated using an ultrafiltration membrane to give 2 mM.
It was dialyzed against 10 mM phosphate buffer (pH 7.5) containing ethylenediaminetetraacetic acid (hereinafter referred to as buffer A). Purification of the present enzyme was performed on 2 liters of a half of the concentrated dialysate (about 4 liters) by the following operation.

Step 1: (Phenyl-Sepharose C
L-4B / chromatography) Ammonium sulfate powder was added to the dialysate (2 l) so as to be 5% saturated and mixed. Liquid A
The column was washed with. Next, 0M to 1.0M urea and 3M
The enzyme was eluted by a linear gradient method of buffer A containing 5 mM sodium cholate, and the active fraction was ultra-concentrated and dialyzed against buffer A. Step 2: (DEAE-Sephacel chromatography) The dialysate (240 ml) was adsorbed on a DEAE-Sephacel column (6 × 20 cm), and the column was washed with buffer A. 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 (12 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 six times to obtain a purified enzyme preparation having a total protein amount of 9 mg and a total activity of 770 U.

[0035]

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.

────────────────────────────────────────────────── ─── front page continued (51) Int.Cl. ⁶ identifications FI C12R 1:64)

Claims (2)

[Claims] 1. A cholesterol / esterase-producing strain belonging to the genus Xanthomonas is cultured in a medium,
A method for producing cholesterol esterase, comprising collecting cholesterol esterase from the culture. 2. A strain having the ability to produce cholesterol esterase is Xanthomonas sp. 81
The method for producing cholesterol esterase according to claim 1, which is -13.