KR100464674B1 - A method for producing cholesterol oxidase from Streptomyces sp. - Google Patents

Abstract

The present invention relates to a method for producing cholesterol oxidase from a strain derived from the genus Streptomyces, to search for actinomycetes that produce cholesterol oxidase, and among them, Streptomyces polychromogins IFO 13072 is selected. Investigate the effects of carbon and nitrogen sources on cholesterol oxidase, and then culture the specimens in the above-described production optimum medium to purify cholesterol oxidase, and then use SDS-polyacrylamide gel electrophoresis of the purified enzyme. After measuring the degree of purification and molecular weight, the temperature stability and pH stability of the purified enzyme were investigated, and the reaction temperature and reaction optimum pH on the activity of the enzyme were investigated, and then the reaction rate according to the substrate concentration of the enzyme was investigated. Investigate the effect of metal ions on the activity of Can be obtained, the yield is excellent production conditions of the cholesterol oxidase by measuring the enzyme activity by the addition of various inhibitors to examine the characteristics of the active site of the enzyme group is an excellent agricultural and food effect industrially.

Description

A method for producing cholesterol oxidase from Streptomyces sp.

The present invention relates to a method for producing cholesterol oxidase from a strain derived from Streptomyces spp. More specifically, the present invention relates to a method for producing cholesterol oxidase which can enhance the yield of oxidase by providing optimum conditions for producing cholesterol oxidase from a strain derived from Streptomyces sp.

Cholesterol oxidase (3β-hydroxysteroid oxidase) is an enzyme that performs oxidation and isomerization of steroids having a trans A: B ring junction. 5-cholesten-3β-ol) is catalyzed by oxidation of 4-cholesten-3-one to 4-cholesten-3-one.

The study of Cholesterol oxidase is a study of steroid-degrading microorganisms in Turfitt.ArthrobacterStarting from Turfitt, G. E .; The microbiological degradation of steroids (1944), Richmond, W .; Properation and properties of bacterial cholesterol oxidase from 1973Norcardiasp. and its application to the enzymatic assay of total cholesterol in serum; 1973) and Preg (Flegg, H. M .; An investigation of the determination of serum cholesterol by enzymatic method; 1973). Microorganisms producing cholesterol oxidase include Streptomyces (StreptomycesFukuda, H. et al (1973), PseudomonasPseudomonas) Genus (Allain, C. C. et al; 1974 and Lee, S. Y. et al., 1989), Brevibacterium (BrevibacteriumGenus (Uwajima, T. et al .; 1973), Norcadia (Norcardia) (Richmond, W .; 1973 and Weyman, A. E .; 1974), Asrobacter (ArthrobacterTurfitt, G. E. (1944) and CorynebacteriumCorynebacteriumProduction in a variety of microorganisms, including Tolalay, P. et al. (1953).

In particular, cholesterol oxidase produced by Streptomyces genus was isolated and purified by Kamei (T. et al .; Purification of 3β-hydroxysteroid oxidase of Streptomyces violascens origin by affinity chromatograpy on cholesterol; 1978). Recently, active research, including cloning of cholesterol oxidase biosynthesis genes, has been performed by Muroka (Y. et al .; 1986). In Korea, Lee et al. (1992) report on the isolation and purification of cholesterol oxidase from actinomycetes isolated from soil and Lee, SY et al. (1989), Pseudomonas sp. In addition to the separation and purification of enzymes in the plant, there are reports of enzyme production from Rhodococcus sp. Isolated from Pak, SH [22]. Although studies on the production, purification and characterization of cholesterol oxidase produced by Streptomyces sp. 4 (Kim, HS et al .; Production and Characterization of Cholesterol oxidase from Streptomyces sp. 4; 1999 and Kim, HS et al .; Purification and Characterization of Cholesterol oxidase by Streptomyces sp. No. 4; 1999).

In particular, actinomycetes streptomyces (Streptomyces) Cholesterol oxidase Pseudomonas fluorescens (Pseudomonas fluorescens)My noccia erythropolis (Nocardia erythropolisR & D for mass production is being conducted as a clinical enzyme preparation because it shows economic cost and long enzyme stability (Lolekha, P. H. et al .; 1996) compared to enzymes produced by

Cholesterol oxidase has become an indicator for measuring blood cholesterol levels in the clinical field, which may contribute to the prevention of atherosclerosis, arterial stenosis and hypertension. In addition, the structural gene cho M in Streptomyces sp. A19249 (Corbin, DR et al .; 1994) codes for cholesterol oxidase with insecticidal activity, which is called Escherichia coli. coli ) is also expressed as an enzyme with the same pesticidal activity, attracting attention as a new pesticide in the agricultural field. In the food sector, a broad industrial application (Murooka Yoshikatsu; 1996) is expected, along with its availability as cholesterol-lowering foods for cheese, ham, sausage and various dairy products. In this recent research report, mass production technology is being researched for the use of new functional aspects of cholesterol oxidase, and in Korea, it is difficult to use because it relies on imports at high cost as a clinical diagnostic reagent.

In view of the above, the present inventors search for actinomycetes producing cholesterol oxidase, and among them, streptomyces polychromogins IFO 13072, which is excellent in the production of enzymes, is selected and oxidized cholesterol against the selected strains. Investigation of the effects of carbon and nitrogen sources on the enzyme and then cultivating the test bacteria in the production optimum medium determined above, purifying cholesterol oxidase and purifying the purified enzyme using SDS-polyacrylamide gel electrophoresis and After the molecular weight was measured, the temperature stability and pH stability of the purified enzyme were investigated, and the reaction temperature and reaction optimum pH on the activity of the enzyme were investigated, and then the reaction rate according to the substrate concentration of the enzyme was investigated. The effect of metal ions on the active site In order to investigate the present invention, various enzymes were added to measure the enzyme activity.

Accordingly, an object of the present invention is to provide a method for producing cholesterol oxidase from a strain derived from Streptomyces sp.

The object of the present invention is to search for actinomycetes producing cholesterol oxidase and to examine the effects of the carbon source and nitrogen source on cholesterol oxidase in the selected strains, and then cultivating the test bacteria in the production optimum medium determined above. After purifying cholesterol oxidase, the purity and molecular weight of the purified enzyme were measured by SDS-polyacrylamide gel electrophoresis, and then the temperature and pH stability of the purified enzyme were investigated and After examining the optimum temperature and the optimum pH of the reaction, the reaction rate according to the substrate concentration of the enzyme was investigated, the effect of metal ions on the activity of the enzyme was investigated, and various inhibitors were added to investigate the characteristics of the active site of the enzyme. By measuring enzymatic activity.

Hereinafter, the configuration of the present invention.

1 is a graph showing the results of affinity column chromatography for purification of the present invention cholesterol oxidase.

Figure 2 shows the results of the SDS-polyacrylamide gel electrophoresis of purified cholesterol oxidase.

Figure 3 is a graph showing the result of determining the molecular weight of purified cholesterol oxidase using SDS-PAGE.

Figure 4 is a graph showing the effect of temperature on the stability of the cholesterol oxidase of the present invention.

5 is a graph showing the pH stability of the present invention cholesterol oxidase.

Figure 6 is a graph showing the effect of temperature on the cholesterol oxidase activity of the present invention.

Figure 7 is a graph showing the effect of pH on cholesterol oxidase activity of the present invention.

Figure 8 is a graph showing the results of determining the Km value of cholesterol oxidase by the Lineweaver-Burk plot (Lineweaver-Burk plot).

The present invention comprises the steps of searching for actinomycetes producing cholesterol oxidase; Investigating the effect of carbon source on cholesterol oxidase on the selected coliforms in the above step; Investigating the effect of nitrogen source on cholesterol oxidase on the selected coliform bacteria in the step; Purifying cholesterol oxidase after culturing the test bacteria in the production optimum medium determined in the step; Measuring the purity and molecular weight of the purified enzyme using SDS-polyacrylamide gel electrophoresis; Examining the temperature stability of the purified enzyme; Examining the pH stability of the enzyme; Investigating a reaction optimum temperature on the activity of the enzyme; Investigating the optimum pH of the reaction on the activity of the enzyme; Examining the reaction rate according to the substrate concentration of the enzyme; Examining the effect of metal ions on the activity of the enzyme; In order to investigate the properties of the active site of the enzyme consists of measuring the enzyme activity by adding various inhibitors.

Streptomyces polychromogins IFO 13072 of the present invention can be cultured under conventional general culture conditions, but preferably can be optimally cultured in a medium containing 1% dextrin as a carbon source and 0.5% kazamino acid as a nitrogen source. Do.

Hereinafter to describe in detail by Examples and Experimental Examples for the present invention The following Examples are only for illustrating the present invention and do not limit the scope of the invention.

Example 1 Screening of Cholesterol Oxidase-producing Actinomycetes

To detect actinomycetes producing cholesterol oxidase, the actinomycetes possessed by the Department of Microbiology, Keimyung University College of Natural Science, were incubated for 3 to 5 days in a plate medium using cholesterol oxidase production basal medium, and then 0.5% cholesterol was added. Cholesterol oxidase-producing actinomycetes were selected using a plate assay method according to the Allain method (Fukuda, HM; 1973), which produced a red color by placing a paper disc soaked in the reaction buffer (pH 7.0). Among them, Streptomyces polychromogenes IFO 13072 (excellent enzyme production ability) was used as the test specimen of the present invention.

Example 2 Effect of Carbon Source on Cholesterol Oxidase

Cultivation of selected specimens was carried out from slant cultured for 7-10 days, 1% soluble starch, 2% peptone, 0.1% KH ₂ PO _4, 0.1% NaNO ₃ , MgSO ₄ · 7H ₂ O 0.05% (pH 7.3) in the medium was inoculated with platinum from the slant (slant) and stored at -70 ℃ until incubation for 36 to 48 hours at 28 ℃, 150 rpm.

In order to examine the conditions for enzyme production, the growth base medium prepared above was dextrin, fructose, glucose, glucose, glycerol, lactose, maltose and sodium as carbon sources. Inoculate 3% of precultured bacteria in 25 mL of each culture medium containing 1% of acetate, soluble starch, sucrose, and xylose. Enzyme activity was measured by incubating at 150 rpm for 2 to 5 days.

To measure the activity of cholesterol oxidase, Allene et al. (Allain, CC et al .; 1974) were used in consideration of the effect of pigments produced in the culture medium. 1 mL of 0.1 M phosphate buffer (pH 7.0) containing 2 μM 4-aminoantipyrine, 35 μM phenol, 2,375 units / L peroxidase (Sigma Co.) 0.25 mL of each enzyme solution was added to the solution, preheated at 37 ° C for 3 minutes, and then 0.05 mL of substrate solution containing 13 μmol / mL of cholesterol (Sigma Co.) was added to 10% Triton X-100 (Merck Co.). And reacted at 37 ° C. for 30 minutes. The quinoneimine dye produced in the reaction solution was measured at an absorbance of 500 nm, and the absorption of the dye present in the enzyme solution was excluded. Activity of the cholesterol oxidase is from 37 ℃ was the amount of enzyme that generates 1 μmol of H ₂ O ₂ with 1 unit, the amount of generated H ₂ O ₂ were calculated using the curve amount of H ₂ O _2.

Furthermore, the optimum concentration of enzyme production was determined by adding carbon sources having excellent enzyme productivity at concentrations of 0.5, 1, 2, and 3%.

As a result, as shown in Table 1, dextrin, maltose, sodium acetate, and sucrose showed relatively high enzyme productivity, lactose and soluble starch. Soluble starch was also judged to have good enzyme productivity. Fructose, glucose, glycerol and xylose did not show enzyme productivity. The results are as follows (Lee, IA et al .; Studies on the Separation and Enzyme Production of Soil Microorganisms Producing Cholesterol oxidase; 1992) For the Actinomycetes HSL-613 strain reported, galactose, glucose ), Xylose, and glycerol have different characteristics from the test specimens due to the difference between the results of good enzyme production. Soluble starch, glucose, dextrins were also found in the Streptomyces sp. No. 4 strain of Kim, HS et al .; Production and Characterization of Cholesterol oxidase from Streptomyces sp. The enzyme production was good at, but the present strain did not produce the enzyme in glucose.

Effect of Carbon Sources on the Production of Cholesterol Oxidase Carbon source Cholesterol Oxidase Activity (munit / mL) dextrin 38 Fructose - Glucose - Glycerol - Lactose 22 Maltose 29 Sodium acetate 28 Soluble starch 21 Sucrose 28 Xylose -

Table 1 shows the difference in enzyme production according to the type of carbon source, the enzyme productivity according to the concentration of dextrin, maltose, sodium acetate, sucrose, the carbon source excellent in the enzyme productivity was examined. The results showed the highest enzyme productivity when 1% dextrin was added as shown in Table 2.

Effect of Carbon Source Concentration on the Production of Cholesterol Oxidase Carbon source concentration (%) Cholesterol Oxidase Activity (munit / mL) dextrin 0.5 19 One 38 2 37 3 33 Maltose 0.5 20 One 29 2 36 3 34 Sodium acetate 0.5 - One 28 2 29 3 30 Sucrose 0.5 21 One 28 2 23 3 19

Example 3 Effect of Nitrogen Sources on Cholesterol Oxidase

In the case of the nitrogen source, the organic nitrogen source was used as the growth base medium used to investigate the effect of the carbon source in Example 2, and the organic nitrogen source was corn steep liquor, beef extract, malt extract, soybean. 2% of soybean meal, casamino acid, yeeast extract, peptone, tryptone, urea, sodium nitrate and nitrate as inorganic nitrogen sources Ammonium (ammonium nitrate), ammonium sulfate (ammonium sulfate) and ammonium chloride (ammonium chloride) were each added at a concentration of 0.5% to measure the enzyme activity in the same manner as the carbon source. In addition, the optimum concentration was determined by adding the nitrogen source having the highest enzyme productivity for each concentration of 0.5, 1, 2, 3, 4, 5%.

Among the effects of carbon source on cholesterol oxidase production, as shown in Table 2, the effect of nitrogen source in the presence of 1% dextrin as carbon source was investigated based on the result of high enzyme productivity when adding dextrin. As shown in Table 3, the production of cholesterol oxidase was the best when adding kazamino acid and beef extract (beef extract) as organic nitrogen sources, and yeast extract, tryptone, and peptone were also good, but urea, corn steep liquor, malt The malt extract did not produce enzymes. In the case of inorganic nitrogen source, sodium nitrate was good, but productivity was relatively low compared to organic nitrogen source. The results indicate that the isolates of Lee et al. (Lee, IA et al .; 1992) show that the yeast extract is excellent in enzyme production and that the strain strain Streptomyces No. 4 of Kim et al. (Kim, HS et al .; 1999) is isolated. The results showed the difference between the best enzyme production in corn steep liquor.

Effect of Nitrogen Sources on the Production of Cholesterol Oxidase Nitrogen source Cholesterol Oxidase Activity (munits / mL) Organic nitrogen source Beef extract 40 Kazamino 40 Corn Soak - Malt Extract - peptone 33 Soy flour 19 Trypton 34 Element - Yeast extract 36 Inorganic nitrogen source Ammonium chloride 18 Ammonium Nitrate 23 Ammonium sulfate 31 Sodium nitrate 35

Enzyme productivity was compared by adding organic nitrogen source and inorganic nitrogen source with high enzyme productivity to each production medium to which dextrin 1% having high enzyme production capacity was added as a carbon source. As shown in Table 4, the production of enzyme was the best when both nitrogen sources were added 0.5%, and the organic nitrogen source was better than the inorganic nitrogen source.

Effect of Nitrogen Source Concentration on the Production of Cholesterol Oxidase Nitrogen source Nitrogen source concentration (%) Cholesterol Oxidase Activity (munits / mL) Kazamino 0.5 40 One 35 2 29 3 33 4 31 5 27 Sodium nitrate 0.5 35 One 33 2 24 3 - 4 - 5 -

Example 4 Purification of Cholesterol Oxidase

For the purification of cholesterol oxidase, 100 mL (500 mL) of optimal medium containing 1% of dextrin, 0.5% of kazamino acid, 0.1% of KH ₂ PO ₄ , 0.5% of NaNO ₃ , 0.05% of MgSO ₄ · 7H ₂ O (pH 7.3) Erlenmeyer flasks) were inoculated to 3% precultured bacteria and incubated at 28 ° C. and 150 rpm for 2 days. The enzyme was purified by culturing the culture solution with a filter paper and removing the cells from the culture filtrate as a crude enzyme solution. Purification of the enzyme was carried out by adding 30% saturated (NH ₄ ) ₂ SO ₄ to 1 L of the crude enzyme solution and leaving it at 4 ° C. for 3 hours, followed by centrifugation at 12,000 rpm (4 ° C.) for 15 minutes to remove the precipitate, and the supernatant. Was added to 80% saturated (NH ₄ ) ₂ SO _{4 It} was left at 4 ℃ 24 hours. A precipitate obtained by centrifuging 30-80% saturated (NH ₄ ) ₂ SO ₄ fraction under the same conditions was dissolved in a small amount of 10 mM phosphate buffer (pH 7.0) and dialyzed (2.582 units / mg) (NH ₄ ) ₂ Used as SO ₄ precipitate fraction. Cholesterol affinity column chromatography precipitated by heating 10 g of cholesterol (Sigma Co.) in 100 mL of distilled water according to the method of Kamei et al. (1978). After filling into a column (φ2.4 × 30 cm) and equilibrating with 10 mM phosphate buffer (pH 7.0), a dialysis (NH ₄ ) ₂ SO ₄ precipitate fraction was injected. Elution of the enzyme protein was sufficiently washed with the used buffer, eluted with 10 mM phosphate buffer (pH 7.0) containing 0.1% Triton X-100, and aliquots of 3 mL per tube.

As a result of measuring the enzyme activity of each of the eluted fractions (3 mL / tube), as shown in FIG. 1, the activity of cholesterol oxidase was excellent in fraction numbers 11 to 17. Therefore, the active fractions were collected and concentrated by Amicon ultrafiltration kit (24.4 units / mg). As shown in Table 5, purified cholesterol oxidase had a degree of purification of 24.4 times and a recovery of 23.2%.

Protein quantification during purification was performed using a protein assay kit (Bio rad Co.) according to the Bradford method (Bradford, MM; 1976), and the standard protein is bovine serum albumin (bovine serum). albumin) (Bio rad Co.) was used.

Summary of Cholesterol Oxidase Purification step Total protein (mg) Total activity (units) Specific activity (units / mg) Purification (Fold) Yield (%) Culture broth 167.8 142.2 0.85 1.0 100.0 (NH ₄ ) ₂ SO ₄ fraction (30-80%) 20.5 53.0 2.58 3.1 37.3 Cholesterol Column 1.4 33.0 24.4 28.8 23.2

Experimental Example 1 Measurement of Purity and Molecular Weight of Cholesterol Oxidase of the Present Invention

12.5% SDS-polyacrylamide using a mini-gel kit (Hoefer Co.) according to the method of Laemmli, UK; 1970, to determine the purity and molecular weight of the purified enzyme. Gel (SDS-polyacrylamide gel) electrophoresis was performed. As shown in Figure 2 (lane B; lane B), the purified enzyme of the present application showed a single band (purity) was purified as a monomer (monomer) protein. In FIG. 2, lane A corresponds to a standard protein and lane C represents 30-80% ammonium sulfate {(NH ₄ ) ₂ SO ₄ } precipitate fraction. Protein detection was stained with Coomassie brilliant blue R-250, phosphorylase b (MW 94,000), albumin (albumin) (marker protein) MW 67,000), ovalbumin (MW 43,000), carbonic anhydrase (MW 30,000), trypsin inhibitor (MW 20,100) and α-lactoalbumin (α 14,400) ) Was used. As a result of measuring the molecular weight of the enzyme, it was estimated to be about 52,000 Da as shown in FIG. 3.

These results indicate that the molecular weight of cholesterol oxidase produced by Streptomyces violascens of Kamei et al. (1978) derived from actinomycetes is 61,000 Da, Lee et al. al .; 1992), the molecular weight of the enzyme produced by Streptomyces sp. HSL613 of 59,500 Da, Kim, HS et al. (1999), Streptomyces sp. No. 4) is eoteuna indicate the report to the difference of molecular weight 60,000 Da of the enzyme production, such as Inoue (Inouye, Y. et al .; 1982 ) reported by other actinomycetes Streptomyces lay suberic tisil Solarium cholesterol rikum (Streptoverticillium cholesterolicum) Results were somewhat smaller than the derived molecular weight of 56,000 Da. In addition, the molecular weight of enzymes produced by bacteria other than actinomycetes is 57,000 from Shirokane et al. (1977), 56,000 from Lee et al. (Ye et al; 1989), Fukuyama et al. Similar to 55,000 in M. et al. (1979) and 52,000 in Park, SH et al. (1998), and somewhat less than 31,000 Da in Uwajima, T. et al. (1973). It was assumed to be a large molecular weight cholesterol oxidase.

Experimental Example 2 investigation of temperature stability of the present invention cholesterol oxidase

In order to investigate the temperature stability of the cholesterol oxidase of the present invention, the enzyme solution obtained by adding the enzyme to 100 mM phosphate buffer solution (pH 7.0) was 10 minutes, 20 minutes, 30 minutes, and 40 minutes at each temperature from 25 ° C to 60 ° C. After heat treatment for 50 minutes, the solution was quenched and the residual enzyme activity was measured at 37 ° C.

The temperature stability of the cholesterol oxidase of the present invention was almost stable at 25 ° C. up to 30 minutes as shown in FIG. 4, but thereafter, it was rapidly deactivated to 50% or lower, and the enzyme activity was rapidly decreased even after heat treatment for 10 minutes at other temperatures. . The results were as follows: Lee, HS et al .; Purification and Chracterization of Cholesterol Oxidase Produced by Soil Microorgarnism HSL613; 1992 reported that 15 minutes of cholesterol oxidase activity of Streptomyces sp. Rhodococcus 3T6-5MJ reported by Park et al. (Park, SH et al .; Purification and characterization of cholesterol oxidase produced by Rhodococcus sp. 3T6-5MJ isolated from Changran-jeot; 1998) ( Rhodococcus sp. 3T6-5MJ) derived enzymes are stable up to 30-45 ° C and reported by Kim et al. (Product and Characterization of Cholesterol oxidase from Streptomyces sp. No. 4; 1999) The enzyme from Streptomyces sp. No. 4 was stable up to 30-40 ° C., and was found to be low in heat stability.

Experimental Example 3: pH stability investigation of the present invention cholesterol oxidase

In order to investigate the pH stability of the cholesterol oxidase of the present invention, a mixture of various buffers and enzyme solutions having different pH from pH 4 to pH 10 was treated at 4 ° C. for 10 minutes, 20 minutes, 30 minutes, 40 minutes, and 50 minutes. After that, the remaining enzyme activity was measured. The buffer used was 50 mM citric acid buffer (pH 4.0-6.0), 50 mM phosphate buffer (pH 6.0-8.0) and 50 mM glycine-NaOH (pH 8.0-10.0). ) Were used respectively. The pH stability of the enzyme was found to be 70% or more stable in the range of pH 6-7 as shown in FIG. The results showed that the enzymes derived from Streptomyces sp. HSL613 of Streptomyces sp. HSL613, reported by Lee, HS et al. (1992), were stable up to pH 6-11 and Inouye, Y. et. al .; 1982) showed that the enzyme produced by Streptoverticillium cholesterolicum was stable up to pH 4-12.5, and Kim, HS et al. (1999), Streptomyces genus number 4 The enzyme produced by Streptomyces sp. No. 4 is stable to pH 6.0-9.0.

Experimental Example 4 investigation of the optimum temperature of the cholesterol oxidase of the present invention

In order to investigate the optimal reaction temperature on the activity of cholesterol oxidase of the present invention, 13 mM cholesterol was used as a substrate in 100 mM phosphate buffer (pH 7.0) and the activity was measured after reacting at each temperature from 25 ° C. to 60 ° C. . As shown in FIG. 6, the optimum reaction temperature was 37 ° C., which was Liu et al. (Liu, W. et al .; 1985), Lee et al., Lee et al. (1992), Watanabe et al. (Watanabe, K. et al .; 1989), Park et al. (Park, SH et al .; 1998) differed from the report that the optimum reaction temperature for enzymes was around 50 ° C. Lee et al. (Lee, SY et al .; 1989), Kim et al. Similar results reported by (Kim, HS et al .; 1999).

Experimental Example 5 investigation of the optimal pH of the present invention cholesterol oxidase

In order to investigate the effect of pH on the activity of cholesterol oxidase of the present invention, the enzyme reaction solution was reacted with a substrate at 37 ° C. using each buffer solution up to pH 5-10, and then the activity was measured. The buffer used was 50 mM citric acid buffer (pH 4.0-6.0), 50 mM phosphate buffer (pH 6.0-8.0) and 50 mM glycine-NaOH (pH 8.0-10.0). ) Were used respectively.

As a result, as shown in Figure 7, the enzyme activity was the best at pH 7.0, the results are different from the optimum pH of the reaction of the enzyme separated by Fukuyama (M. et al .; 1979) is 5, most The report shows that the optimum pH of cholesterol oxidase ranges from 6.0 to 7.5 (Kim, HS et al., 1999; Lee, HS et al., 1992; Lolekha, PH et al., 1996; Park, SH et al). , 1998; Stadtman.TC et al., 1954; Weyman, AE, 1974).

Experimental Example 6: Investigation of the change in reaction rate according to the substrate concentration of cholesterol oxidase of the present invention

In order to investigate the reaction rate according to the substrate concentration of cholesterol oxidase of the present invention, the concentration of cholesterol as a substrate was added to the reaction solution to 1, 2.5, 5, 10, 13 mM to carry out the enzymatic reaction, and a Linnewer-Buck plot ( Lineweaver-Burk plot) was used to analyze the Km value of 25 mM. In FIG. 8 the velocity {velocity (V)} is expressed as specific activity (units / mg).

Experimental Example 7: Investigation of the effect of metal ions on cholesterol oxidase of the present invention

In order to examine the effect of metal ions on the present enzyme activity, enzyme activity was measured in the presence of 1 mM of each metal ion. As shown in Table 6, the enzyme was slightly promoted in the presence of Mn ions and inactivated by 40-80% in the presence of Cu, Fe, and Hg ions, especially by 1 mM HgCl ₂ . More than 84% of enzyme activity was inhibited. These results are somewhat different from those reported by Lee et al. (1992) and Inouye et al. (1982), but the strong inhibition of cholesterol oxidase by Hg ions is almost all. Consistent with the report.

Effect of Metal Ions on Cholesterol Oxidase Metal ions (1 mM) Relative activity (%) None 100.0 CaCl ₂ 97.15 CdCl ₂ 96.21 CuSO ₄ 60.52 FeCl ₃ 39.27 HgCl ₂ 16.61 MnCl ₂ 106.85 PbCl ₂ 93.83 ZnSO ₄ 98.58

Experimental Example 7: Investigation of the Effect of Inhibitors on Cholesterol Oxidase of the Present Invention

In order to investigate the characteristics of the active site of cholesterol oxidase of the present invention, the activity of the enzyme according to the addition of various inhibitors was examined. The effect of 1 mM of each inhibitor on the enzyme activity was 91% by mercaptoethanol, almost 100% in the presence of dithiothreitol, and ρ-chloromercurybenzoic acid, as shown in Table 7. (ρ-Chloromercuribenzoic acid) and isonicotinic acid were inhibited by 30% and 78%, respectively. Other inhibitors had little effect on enzyme activity. The results suggest that disulfide bonds exist in the active site of the enzyme, and that chelating agents do not show inhibition by Shirokane et al. (1977). Similar to the report, but reports of cholesterol oxidase are somewhat inhibited by EDTA (Lee, HS et al .; 1992) or iodine sensitively Inouye, Y. et al .; 1982 In comparison with the report of), the enzymatic properties were estimated to be enzymes of different properties.

Effect of Inhibitors on Cholesterol Oxidase Inhibitor (1 mM) Relative activity (%) None 100.00 ρ-Chloromercuribenzoic acid 69.41 N-ethylmaleimide 100.27 Isonicotinic acid 22.37 EDTA 103.20 2,2'-bipyridine 103.70 PMSF 104.38 Iodoacetic acid 101.76 Trichloroacetate 100.80 ο-phenanthroline 100.00 8-Hydroxyquinoline 92.69 Mercaptoethanol 8.65 Sodium azide 90.18 Dithiothreitol 0.04

As described above, the method for producing cholesterol oxidase from the strain derived from Streptomyces sp. Of the present invention as described above using the Streptomyces polychromogens IFO 13072 strain as a co-bacteria, a carbon source for cholesterol oxidase and Investigation of the effect of the nitrogen source has the effect of providing the optimum production medium conditions, so it is a very useful invention for agriculture and food industry.

Claims (1)

Streptomyces polychromogenes IFO 13072 is inoculated in a medium containing essentially 1% dextrin and 0.5% kazamino acid and incubated at 28 ° C. and 150 rpm for 2 to 5 days. How to produce cholesterol oxidase.