JPH09251A - Production of cholesterol esterase - Google Patents

Abstract

PURPOSE: To mass-produce a new cholesterol esterase, useful as a clinical testing agent for determining cholesterol and having high specific activities at a low cost by culturing Pseudomonas stutzeri. CONSTITUTION: A microorganism belonging to Pseudonmonas stutzeri such as Pseudomonas stutzeri PL-836 strain (FERM P-14968) is cultured. A cholesterol esterase collected from the resultant culture solution has activities against an ester of a fatty acid with cholesterol having a wide range of number of carbon atoms, high specific activities, 7-8 optimum pH, 35-40 deg.C optimum temperature and 6.7 isoelectric point and is hardly inhibited by metallic ions.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a novel method for producing cholesterol esterase.

[0002]

2. Description of the Related Art Cholesterol is widely distributed in various organs and organs of the human body, and it is important clinically to grasp the amount of cholesterol present in blood. That is, in the case of hypercholesterolemia, it causes arteriosclerosis, myocardial infarction and other diseases, and in the case of hypocholesterolemia, it is associated with cachexia and thyroid function enhancement.

In recent years, an enzymatic method using cholesterol oxidase, which is easy to operate and has high sensitivity, has been widely adopted for quantifying cholesterol. Most of blood cholesterol exists as an ester with fatty acid, but cholesterol oxidase acts only on free cholesterol. Therefore, in the above enzymatic method, cholesterol ester is hydrolyzed to release cholesterol. There must be. Cholesterol esterase is used for this hydrolysis.

As a cholesterol esterase of microbial origin, Pseudomonas sp.
No. 8, JP-A-56-42586, JP-A-52-7483, etc.), Streptomyces (JP-A-53-109992), Nocardia (JP-A-57-43686) ), Basidiomycetes such as Kawaratake (Japanese Patent Application Laid-Open No. 55-114288) and Suehirotake (Japanese Patent Application Laid-Open No. 53-9391). In addition, Japanese Patent Laid-Open No. 62-36200 discloses many cholesterol esterases derived from microorganisms.

The fatty acid components of blood cholesterol ester are mainly linoleic acid, oleic acid and palmitic acid, and arachidonic acid, palmitoleic acid,
It also contains stearic acid and myristic acid. Therefore, a cholesterol esterase that acts on a fatty acid ester having a relatively wide number of carbon atoms is desired. It is also necessary to have the ability to quantitatively degrade all cholesterol esters present at approximately the same rate and accuracy. However, cholesterol esterases that are active against such a wide range of fatty acid esters are limited. It is also important that cholesterol esterase has a high enzyme activity (specific activity) per unit protein. Further, in industrial use, it is required that the enzyme production amount per unit culture of the microorganism is large. However, the above-mentioned known cholesterol esterases have problems in either their properties or production.

[0006]

SUMMARY OF THE INVENTION Therefore, an object of the present invention is to provide a method for efficiently producing a cholesterol esterase which exhibits activity with respect to fatty acid esters having a wide range of carbon atoms and has a high specific activity. is there.

[0007]

Means for Solving the Problems The present inventors newly searched for a cholesterol esterase-producing bacterium in order to inexpensively obtain cholesterol esterase satisfying the above-mentioned conditions on an industrial scale. As a result, they have found that Pseudomonas stutzeri isolated from soil has an activity of producing a wide range of fatty acid esters and the ability to produce cholesterol esterase having a high specific activity, and completed the present invention.

The present invention provides a method for producing cholesterol esterase, which comprises culturing Pseudomonas stutzeri and collecting cholesterol esterase from the culture.

The microorganism used in the production method of the present invention belongs to Pseudomonas stutzeri, and any strain can be used as long as it has broad substrate specificity and is capable of producing cholesterol esterase with high specific activity. Pseudomonas stutzeri PL-836 strain can be mentioned as a specific example, and its variant or mutant strain can also be used. This Pseudomonas stutzeri PL-836 strain was newly collected from soil, and its mycological properties are shown below. (a) Morphological properties 1. Cell shape and size: bacillus, 0.5 × 1 to 3 μm 2. 2. Cell polymorphism: not often observed, but rarely long ones are included. Motility: Yes 4. Flagella: Extreme flagella 5. Spores: do not form

(B) Growth state in each medium 1. Meat broth agar plate medium: The colony is raised in a convex shape, the surface is smooth, and the peripheral edge is wavy. No diffusible dye is observed. 2. Broth agar slope medium: Fungal moss grows in a diffused manner. When observed under light, it is a translucent brown color, but no production of water-soluble dye is observed. 3. Broth liquid medium: The entire medium becomes cloudy. The fungus grows in a white ring in the upper layer, and a large amount of fungi are produced in the lower layer. 4. Meat broth gelatin puncture medium: The puncture part is filamentous, the surface growth is good, but no liquefaction is observed. 5. Litmus milk: Produces alkali, but neither coagulation nor decomposition is observed.

(C) Physiological properties 1. Gram stainability: negative 2. Anti-acidity: None 3. Urease: Positive 4. Oxidase: Positive 5. Catalase: Positive 6. OF test: oxidation 7. Attitude toward oxygen: Aerobic 8. Growth range pH: 6.5 to 9.0 Temperature: 15 to 40 ° C 9. Formation of indole: negative 10. Methyl red test: negative 11. VP test: negative 12. Use of citric acid: negative 13. Generation of hydrogen sulfide: slightly positive 14. Nitrate reduction: Positive 15. Denitrification reaction: Positive 16. Use of inorganic nitrogen source Nitrate: Positive Ammonium salt: Positive 17. Gelatin liquefaction: Negative 18. Hydrolysis of starch: Positive 19. Hydrolysis of milk casein: negative 20. GC content of intracellular DNA: 63-65% 21. Quinone molecular species: Q ₉ 22. The DNA of this bacterium is Pseudomonas stutzeri IAM 12668 ^T
Shows a homology of 84 to 92% with the DNA of. twenty one. Assimilation negative: L-arabinose, saccharose, D-lactose, D-sorbitol, inositol, raffinose Positive: galactose, maltose, glucose, trehalose, mannitol, D-xylose, glycerin,
D-mannose, dextrin, fructose

[0012] This Pseudomonas Stutzeri P
The L-836 strain was deposited at the Institute of Biotechnology, Institute of Biotechnology, Institute of Industrial Science on June 1, 1995, and the deposit number was FERM P-14968.

In order to culture Pseudomonas stutzeri according to the present invention, an ordinary nutrient medium can be used, and either a natural medium or a synthetic medium can be used as long as it appropriately contains a carbon source, a nitrogen source, inorganic salts and the like. Can be used.

As the carbon source, glucose, glycerol, sucrose, molasses, sugars such as starch, alcohols, organic acids, fats and oils such as olive oil and soybean oil, and combinations thereof can be used. As the nitrogen source, organic nitrogen such as corn steep liquor, soybean flour, peptone, meat extract and yeast extract, and inorganic nitrogen such as ammonium sulfate, ammonium nitrate and urea can be used. As the inorganic salts, common salt, potassium chloride, magnesium sulfate, potassium dihydrogenphosphate, potassium dihydrogenphosphate, ferrous sulfate, and the like can be used. Emulgen, Triton, Tween, to further promote growth and enhance enzyme productivity.
A suitable surfactant such as span may be added.

For the culture, aerobic culture such as aeration and stirring in a liquid medium containing such components is carried out at 15 to 35 ° C. and pH 5 to 9 for 1 to 1.
Do it for 3 days. To collect cholesterol esterase from the culture broth, the culture broth is separated from cells by filtration or centrifugation, and ammonium sulfate salting out from the filtrate or the supernatant, solvent precipitation using alcohol, acetone, or the like, by an ultrafiltration membrane. An enzyme preparation is obtained by a known method such as separation and concentration. To extract the cholesterol esterase accumulated in the cells, use a cell-free enzyme solution by a known method such as cell grinding, autolysis, sonication, etc., and then purify it in the same manner as the enzyme outside the cells. Can be used for In order to obtain a highly purified enzyme preparation, it may be purified by an enzyme purification method which has been conventionally used such as ion exchange chromatography, adsorption chromatography, gel filtration chromatography and the like.

The activity of cholesterol esterase according to the present invention was measured by the following measuring method. The physicochemical properties of the cholesterol esterase of the present invention are shown below.

(1) Measurement of Cholesterol Esterase Activity Cholesterol esterase produced according to the production method of the present invention using Pseudomonas stutzeri strain PL-836 and, as a comparative example, a commercially available cholesterol esterase, Boehringer Mannheim 691941 [Pseudomonas fluorescens] (From Pseudomonas fluorescens)], Sigma C 1403 (from Pseudomonas sp.), And
The activity of Boehringer Mannheim 161772 (derived from Candida cylindracea) was measured by the following measuring methods and, respectively. As a substrate, Table 1
The cholesterol ester shown in 1 above (all manufactured by Sigma) was used.

[0018]

[Table 1]

Measurement method Cholesterol ester solution [concentration 20 mM, solvent dioxane: Thesit (polyoxyethylene dodecyl ether having an average degree of polymerization of 9; manufactured by Boehringer Mannheim) =
2: 3 mixed solvent] 50 μl and 0.1 M phosphate buffer (pH
7.0) 400 μl was mixed. Cholesterol ester concentration after mixing is 2 mM and Thesit concentration is 6%.
After incubating this mixture at 37 ℃ for 5 minutes, 50 μl of cholesterol esterase solution (0.03-0.3u / ml)
Was added and incubated at 37 ° C. for 5-10 minutes. Then, the enzyme reaction was stopped by heating at 100 ° C. for 4 minutes.
Cholesterol color-developing solution for free cholesterol determination [free cholesterol E-Test Wako; manufactured by Wako Pure Chemical Industries; 50 mM MES buffer (pH 6.1), 4-aminoantipyrine 0.19 mM, 3,5-dimethoxy- N-Ethyl-N- (2-hydroxy-3-sulfopropyl) -aniline sodium (D
AOS) 0.98 mM, cholesterol oxidase (Streptomy
ces) 0.29u / ml and peroxidase (horseradish) 4.7
u / ml] 2.0 ml was added and incubated at 37 ° C. for 10 minutes, and then the absorbance at 600 nm was measured. The cholesterol esterase activity (u / ml) per 1 ml of the cholesterol esterase solution was determined by the following formula: activity (u / ml) = A × 0.321 × 20 / t × (dilution rate) A: absorbance t: reaction time (min) . still,
The amount of enzyme required to release 1 μmol of cholesterol from cholesterol ester in 1 minute is 1 unit (1un
it).

Measurement method 40 μl of a solution of cholesterol ester [concentration 20 mM, solvent is a mixed solvent of dioxane: Thesit = 2: 3] and cholesterol coloring solution for quantitative determination of free cholesterol [free cholesterol C-Test Wako; manufactured by Wako Pure Chemical Industries, Ltd. ; 0.1M
Phosphate buffer (pH 7.0), phenol 10.6 mM, 4-aminoantipyrine 0.74 mM, cholesterol oxidase (Pse
udomonas) 0.034 u / ml and peroxidase (horseradish) 0.68 u / ml] 2.0 ml were mixed. The cholesterol ester concentration after mixing is 400 μM, and Thesit concentration is
It is 1.2%. After incubating this mixed solution at 37 ° C for 5 minutes, 50 µl of cholesterol esterase solution (0.05-0.5u / ml) was added while maintaining the temperature at 37 ° C to start the reaction, and the absorbance at 2-9 minutes after the reaction was started ( 505 nm)
Was measured continuously. The measurement results were plotted with the reaction time on the horizontal axis and the absorbance on the vertical axis, and the increase amount ΔA in the linear portion of the absorbance per minute was determined. The following formula: Activity (u / ml) = ΔA × 20/3 × (dilution rate) ΔA: The cholesterol esterase activity (u / ml) per 1 ml of the cholesterol esterase solution was determined by the increase amount of the absorbance per minute.

Calculation method of specific activity From the activity determined by the above assay method or by the following formula, the enzyme activity per unit protein of cholesterol esterase in the assay method or [specific activity (u / mg)]
I asked.

The protein concentration in the above formula is the concentration of the protein contained in 1 ml of the cholesterol esterase solution, and the protein concentration reagent (BCA Protein Assay Reagent) is used.
agent: Pierce 23225X).

The purity of cholesterol esterase in the above formula was measured by reverse phase HPLC. Table 2 shows the results. The measurement conditions are as follows. Column: Resource RPC (1.0 ml), Pharmacia Solvent: Acetonitrile 32-56% Linear concentration gradient 0.05% trifluoroacetic acid in 1% acetonitrile /
min Flow rate: 0.7 ml / min Detection: UV220nm

Further, the results of measuring the molecular weight of each cholesterol esterase by SDS-PAGE are shown in Table 2. still,
Pre-cast gel Resofine G (8 × 8c
m, 10-20%) ACI was used.

[0025]

[Table 2]

Table 3 shows the results of determining the specific activity of cholesterol esterase in the assay method using the above formula for calculating the specific activity.

[0027]

[Table 3]

Table 4 shows the results of determining the specific activity of cholesterol esterase in the assay method using the above formula for calculating the specific activity.

[0029]

[Table 4]

(2) Action Hydrolyze cholesterol ester to release fatty acid and cholesterol.

(3) Substrate specificity From the results of (1) above, it is understood that the cholesterol esterase of the present invention has a higher specific activity for most substrates than other cholesterol esterases. In particular, it can be seen that the activity of the fatty acid component is high for cholesterol ester having 12 or more carbon atoms.

(4) Optimum pH The activity of this enzyme at each pH was measured according to the measuring method of (1) above using cholesterol oleate as a substrate. The activity ratio (relative activity) at each pH was calculated when the activity at pH 8 was 100%. pH
The activity curve is shown in FIG. The optimum pH is 7-8.

(5) Stable pH After the enzyme was treated at each pH for 24 hours at 8 ° C., the activity was measured according to the method described in (1) above using cholesterol oleate as a substrate. The activity ratio (residual activity) at each pH when the activity of the enzyme before treatment was taken as 100% was determined. The pH stability curve is shown in FIG. The stable pH is 6-9.5.

(6) Optimum temperature The activity of this enzyme at each temperature was measured according to the method of the above-mentioned (1) using cholesterol oleate as a substrate. The activity ratio (relative activity) at each temperature was calculated when the activity at 35 ° C was 100%. The temperature activity curve is shown in FIG. The optimum temperature is 35-40 ° C.

(7) Stable temperature This enzyme was added to a phosphate buffer (pH 7.0, 0.1M) at 30 ° C at each temperature.
After treatment for minutes, the activity was measured according to the method of the above (1) using cholesterol oleate as a substrate. The activity ratio (remaining activity) at each temperature was calculated when the activity of the enzyme before treatment was 100%. The temperature stability curve is shown in FIG. This enzyme is stable up to 35 ° C.

(8) Isoelectric point As a result of measurement with a liquid isoelectric focusing apparatus (Rotophor, manufactured by Bio-Rad Laboratories Co., Ltd.), 6.
It was 7.

(9) Effects of various substances In order to investigate the effects of various substances on the present enzyme, Table 5
The additives shown in (1) above were added at the concentrations shown in Table 5, respectively.
Was added to the enzyme reaction solution of the above measurement method, and the activity was measured by the same method. Table 5 shows the activity ratios (relative activities) when various additives were added, with the activity when no additives were added as 100%.

[0038]

[Table 5]

From the above results, it can be seen that the cholesterol esterase according to the present invention is hardly inhibited even in the presence of metal ions.

(10) Amount of required enzyme In 3 ml of a cholesterol color-developing solution (free cholesterol C-Test Wako) for quantifying free cholesterol used in the measuring method of the above (1), cholesterol derived from the microorganisms shown in Table 6 below. Esterase solution (each 0.16u /
Solutions of ml, 0.04u / ml and 0.016u / ml were used. ) 50 μl
And standard serum (Lipid Salem I, Eiken Chemical Co., Ltd.) 50μ
l was added and the absorbance at 505 nm was measured over time while reacting at 37 ° C. Table 6 shows the time until the coloration reaches a certain level (time until the cholesterol ester is completely decomposed). FIG. 5 is a plot of the measurement results, where the horizontal axis is the reaction time and the vertical axis is the absorbance.

[0041]

[Table 6]

From the above results, the cholesterol esterase obtained by the present invention has a constant coloration in a short time even when the amount of enzyme used is small (for example, 0.016 u / ml) as compared with other cholesterol esterases. You can see that That is, it was confirmed that the cholesterol esterase obtained by the present invention can decompose cholesterol ester in serum in a short time even in a small amount.

[0043]

【Example】

[Example 1] In a Sakaguchi flask having a capacity of 500 ml, glycerol 1% and corn steep liquor 2 were used as a preculture medium.
%, Citric acid 0.5%, and urea 0.5%, and 100 ml of a liquid medium (pH 6.5) was added thereto, and after steam sterilization, 1 platinum loop of Pseudomonas stutzeri PL-836 strain was inoculated.
Then, preculture was obtained by shaking culture at 25 ° C. for 24 hours. In a 2 liter capacity jar, Kinako 3%, Glycerol 3%, Corn steep liquor 2%, Urea
1.5%, sodium chloride 0.5%, dipotassium phosphate 0.2
% And Emulgen 430 0.5% in liquid medium, 1
After autoclaving at 21 ° C for 15 minutes, the above preculture liquid was inoculated, and aeration culture was carried out at 26 ° C, 700 rpm, 0.7 vvm for 36 hours. After culturing, centrifuge to remove solids containing bacterial cells,
A culture supernatant was obtained. The cholesterol esterase activity in the obtained culture supernatant was 320 u / ml.

[Example 2] To 1 liter of the culture supernatant obtained in Example 1, ammonium sulfate was added and dissolved so as to be 35% saturated, and the mixture was allowed to stand at 5 ° C for 6 hours. The resulting precipitate was collected by centrifugation and dissolved in 25 mM Tris-HCl buffer (pH 7.5) containing 0.5% tesit (polyoxyethylene dodecyl ether). Next, the obtained solution is
DEAE-Toyopearl equilibrated with the same buffer was loaded and adsorbed. The active fraction eluted by the concentration gradient of sodium chloride was further loaded onto hydroxyapatite equilibrated with 10 mM sodium phosphate buffer (pH 7.0) for adsorption. By elution with a concentration gradient of sodium phosphate buffer, a purified enzyme with a purity of 95% or more was obtained. Purification ratio is 30
The activity recovery rate of the produced enzyme from the culture supernatant obtained in Example 1 was 20%, and the specific activity was 600 u / mg.

[0045]

Industrial Applicability According to the method of the present invention, a large amount of cholesterol esterase having a high specific activity, which is active against fatty acid and cholesterol esters having a wide range of carbon atoms and has a high specific activity, can be produced inexpensively. be able to. Further, the cholesterol esterase according to the present invention is not easily inhibited by metal ions and can decompose cholesterol ester even in a small amount, and therefore it is useful as a clinical test drug used for quantifying cholesterol in blood.

[Brief description of drawings]

FIG. 1 pH of cholesterol esterase according to the present invention
It is a figure which shows an activity curve.

FIG. 2 pH of cholesterol esterase according to the present invention
It is a figure which shows a stability curve.

FIG. 3 is a diagram showing a temperature activity curve of cholesterol esterase according to the present invention.

FIG. 4 shows a temperature stability curve of cholesterol esterase according to the present invention.

FIG. 5 is a view showing a time-dependent change in absorbance with decomposition of cholesterol ester in serum when various cholesterol esterases were added.

Claims (1)

[Claims] 1. Pseudomonas stutzeri (Pse
A method for producing cholesterol esterase, which comprises culturing udomonas stutzeri) and collecting cholesterol esterase from the culture.