KR860000153B1 - Preparing process of cholesterol oxidase from the microorganism(kctc 8176 p) - Google Patents

Abstract

Cholesterol oxidase, useful as a diagnostic reagent for cholesterol in the bood, was prepd. by fermn. of Streptomyces sp. K-02. Thus Streptomyces sp. K-02 was cultivated in a medium contg. glucose 5, (NH4)2SO4 0.2, KCl 0.01, FeSO4 0.001, KH2PO4 0.2, MgSO4 0.02, and a yeast extract 2% to give a cholesterol oxidase at 30≰C for 18 hrs. For enzyme assay, 0.05ml of the enzyme prepn. were added to 3ml of the assay mixt. contg. 100mM phosphate buffer (pH7.0), 2.5ml; 10mM ortho-dianisidine, 0.1ml; and a peroxidase soln., 0.1ml. Absosrbance of the reaction rate per min. was measured at 460nm. One unit of cholesterol oxidase was defined as the enzyme amount that forms 1umole of H2O2 at 37≰C. 2-5 units of cholesterol oxidase/ml were obtd.

Description

Method for preparing cholesterol oxidase

Figure 1: Streptomyces sp. NO. K-02 (Streptomyces sp. NRRL NO. K-02 15218): Yeast juice-wort agar medium at 270 times magnification of KCTC8176P).

Figure 2: Streptomyces sp NO. Oatmeal agar medium magnified 270 times of K-02 (Streptomyces sp. NRRL NO. K-02 15218: KCTC 8176P).

Figure 3: Streptomyces sp. NO. Starch agar medium at 270 times larger than K-02 (Streptomyces sp. NRRL NO. K-02 15218: KCTC 8176P).

Figure 4: Streptomyces sp. NO. Glycerine-asparagine agar medium at 270 times larger than K-02 (Streptomyces sp. NRRL NO. K-02 15218: KCTC 8176P).

Figure 5: Streptomyses sp. NO. K-02 (Streptomyces sp. NRRL NO. K-02 15218: KCTC 8176P) Spore morphology magnified 20,000 times.

The present invention relates to a novel method for preparing cholesterol oxidase and microbial strain Streptomyces sp. A new and advanced process for producing cholesterol oxidase by culturing K-02 (Streptomyces sp. NRRL NO. K-02 15218: KCTC 8176P) in conventional nutrient media containing carbohydrates, nitrogen sources and minerals.

Cholesterol oxidase acts as a catalytic enzyme to accelerate this reaction when cholesterol is broken down to produce cholesterol and hydrogen peroxide. Therefore, it is widely used in the enzymatic quantification method of cholesterol. Cholesterol oxidase has been applied to the measurement of cholesterol content in the blood in the pathology test, as a means for diagnosing diseases such as hypertension and heart disease is used as a faster and more accurate measurement method than the conventional chemical color reaction.

Cholesterol oxidase has been found in soil microorganisms with cholesterol degrading ability to produce a high purity forest enzyme from these microorganisms is currently known as genus Nocardia, Mycobacterium genus Streptomyces genus and Brevibacte Cholesterol oxidase is produced from cells or cultures by culturing strains of the genus Brevibacterium, etc., but the yields vary widely depending on the species and the enzyme activity is low. The application and dissemination of clinical enzymes has become a major obstacle. For example, according to US Patent 4,144,129 (1979.3.13), the microbial strain Proactinomyces erythroplois ATCC17895 is inoculated into a nutrient medium and aerated by adding a cholesterol suspension as an enzyme-inducing substance to dry the enzyme yield per 1 liter of medium. Seven grams of cells and about 280 units of enzyme activity were obtained. According to Japanese Laid-Open Patent Publication 54-76893 (Jun. 9, 1979), strain Streptomyces biosense, H82N-SV7 (Streptomyces violascense) was inoculated into a nutrient medium and incubated at 27 ° C for 96 hours to obtain an enzyme yield of 813 units per liter of medium. Was obtained.

As described above, a conventional method for producing cholesterol oxidase derived from microorganisms has been produced according to a conventional purification method for extracting and purifying enzymes from microorganism cells or from cells. The former strains are mainly Nocardia strains and the latter strains are known as Streptomyces and Brevibacterium strains. It was reported that a high yield of enzyme activity was obtained by culturing.

The present inventors have attempted to search for microorganisms that produce excellent cholesterol oxidase having a very high economic efficiency for the application of clinical enzymes for the purpose of industrial production of cholesterol oxidase. In other words, by separating the soil of each region in Korea, strains of the genus Streptomyces were isolated, and cultured in a conventional synthetic nutrient medium containing glucose as a carbon source. K-02 (Streptomyces sp. NRRL NO. 15218: KCTC 8176P) was isolated.

This strain was cultured in a synthetic medium using glucose as a carbon source, and the enzyme yield was 8 units per 1 milliliter of medium, and the activity was high, which was about 10 times higher than the conventional enzyme yield. It is an enzyme-inducing substance characterized by no need for cholesterol in the medium composition. The production of cholesterol oxidase by this strain is easy to secrete enzymes having cholesterol degrading ability in the medium, so that the enzymes can be recovered directly from the culture without destroying the bacteria and extracting the enzymes like the strains of the genus Nocardia. It is a representative advantage.

The purified enzyme produced according to the present invention can be used as a clinical enzyme for application in conventional cholesterol assay. In other words, the enzyme catalyzing the reaction of cholesterol is represented by the following reaction,

Cholesterol + Oxygen … … → cholesterone + hydrogen peroxide cholesterol oxidase

This enzyme is a redox enzyme that reacts in the presence of oxygen to produce hydrogen peroxide as a reaction product. Therefore, enzyme activity is easily measured by measuring the amount of hydrogen peroxide produced during the reaction. The microbial enzyme of the present invention has been identified as cholesterol oxidase because it has the specificity of degrading cholesterol only and not degrading other sterols.

Determination of the enzyme activity of cholesterol oxidase using a complex enzymatic assay using peroxidase and orhto-dianisidine, one enzyme per unit of enzyme that produces 1 micron of hydrogen peroxide per minute at 37 ° C It was.

Embodiments of the present invention are directed to Streptomyces sp. K-02 (NRRL NO. 15218: KCTC 8176P), which has the ability to produce cholesterol oxidase, to conventional synthetic media, such as glucose 5.0%, ammonium sulfate 0.2%, and calcium chloride 0.001. %, Ferrous sulfate 0.001%, ferric phosphate 0.2%, magnesium sulfate 0.02%, yeast extract 2.0% and neutral pH in liquid medium to accumulate enzymatic activity in the medium, and after completion of culture The present invention relates to a method for preparing cholesterol oxidase that recovers crude enzyme to easily recover purified enzyme having a high yield according to a conventional biochemical enzyme purification method.

The microbial properties of Streptomyces sp K-02 (NRRL NO. 15218: KCTC 8176P) isolated by the present inventors were as follows, which were isolated and identified according to the known international Streptonyces Project.

end. Morphological Properties (See Figure 5)

1.Bacillus mycelia: Bacillus mycelia, linear, without spiral.

2. Spore type and size: 0.6-0.8 (μ) × 1.0-1.5 (μ), surface is smooth

3. Mobility: None

4. Gram Dyeing: Positive

I. Culture properties

1) Water-soluble pigment is unchanged even when adding 0.05N NaOH, 0.05N, HCI

2) Observation after continuous incubation for 13 days at 27 ℃

All. Physiological properties

1) Growth temperature range: 25-37 ℃

2) Optimal growth temperature range: 30-35%

3) Growth PH Range: 4.0-9.5

4) Optimal growth pH range: 6.0-8.0

5) Oxygen Requirements: Aerobic

6) Death temperature: 60 ℃

7) litmus milk: unchanged

8) Catalase Production: Positive

9) Geladin Liquefaction: Positive

10) starch resolution: positive

11) Melanoid Pigment Production: Positive

12) Availability of Carbon Sources:

* Observed after 11 days incubation at 27 ℃.

As a result of examining the morphological characteristics and physiological characteristics of colonies according to the identification method of the isolated species of the present invention, it was determined that the strain belonged to Streptomyces soge.

Streptomyces K-02 (NRRL NO. 15218: KCTC 8176P) strains of the present invention were inoculated into a conventional nutrient medium containing glucose and yeast extract as a main nutrient source, as in the experiments of the examples, and under normal aeration culture conditions. When cultured, cholesterol oxidase having extremely high enzyme activity is induced in the medium. After completely removing the cells in the medium and purifying the crude enzyme according to the biochemical enzyme purification method can be easily obtained purified enzyme with a very high yield.

The embodiments described in detail the embodiments of the present invention are as follows, but the scope of the present invention is not limited thereto.

Example 1

Streptomyces sp K-02 (NRRL NO. 15218: KCTC 8176P) Platinum 50 mg of this medium (5% glucose, 0.2% ammonium sulfate 0.001% calcium chloride, 0.001% ferric sulfate, 0.2% carry monophosphate, 0.02% Magnesium sulfate, 2% enzyme extract) was incubated for 3 days at 30 ℃ shaking culture, 5 ml of this was used as seed stock inoculated in 100 ml of the same medium and cultured shaking at 30 ℃ 18 hours.

Enzyme activity was measured by mixing 0.05 ml of enzyme solution in 3 ml of reaction mixture substrate solution (0.1 mole phosphate buffer, PH 7.0, 2.5 ml, 0.01 mole Otso-Dianisidine 0.1, 0.1 ml peroxide enzyme solution) and then A conventional enzyme measuring method of measuring the reaction rate per minute of absorbance at 460 nm was used. One unit of the enzyme was 1 unit of the amount of the enzyme that produces 1 micromolar hydrogen peroxide at 37 ° C. The enzyme measurement result was 2-5 units per ml of the culture medium from which the cells were removed, producing a total of 200-500 units.

Example 2

5 ml of Streptomyces sp. K-02 (NRRL NO. K-02 15218: KCTC 8176P) was inoculated into 100 ml of Example 1 medium, followed by shaking culture at 30 ° C., and the production of enzyme according to incubation time. Is as follows.

The enzyme titer after 24 hours incubation time was 300 units per 100 ml of medium, and gradually increased as the culture time elapsed, reaching a maximum of 500 units per 100 ml after about 48 hours.

The strain culture medium was centrifuged to remove the cells, and then the culture medium was used as the coenzyme solution of cholesterol oxidase.

Example 3

100 ml of Streptomyces sp. K-02 (NRRL NO. K-02 15218: KCTC8176P) was inoculated into 400 ml of the medium of Example 1, and then in a 500 ml aeration fermenter at a temperature of 30 ° C., aeration rate of 1.5 VVM, and rotation speed of 300 rpm. As a result of incubation for about 48 hours, the enzyme production amount was 5 units per 1 ml of culture, which produced a total of 20.000 units of crude enzyme solution.

Example 4

Inoculated with 500 ml of the seedling solution of Example 3 in 15 l of the same nutrient medium and incubated in a 28-l aeration fermenter for about 48 hours at a culture temperature of 30 ° C., an aeration rate of 1.5 VVM, and a rotation speed of 300 rpm, the total enzyme production was 160,000 units of 8 ml of culture medium A unit of crude enzyme solution was produced. As a result of removing the enzyme from the crude enzyme solution completely removed from the cells according to the biochemical enzyme purification method, it was possible to produce 15-20 units of purified enzyme per gram of protein.

Claims (1)

Streptomyces sp. NO. K-02 (NRRL NO. K-02 15218: KCTC 8176P) A method for producing cholesterol oxidase, characterized by culturing the cholesterol oxidase by aeration in a known nutrient medium.

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