KR910004362B1 - Novel superoxide dismutase - Google Patents

Abstract

A superoxide dismutase is produced by serratia sp. Y-4, donation No.KFCC 8276P. The dismutase has a mol. wt. of 54000. The strain is cultured on a seed medium at 28 deg.C with shaking for 18-24 hrs, seeded to fermentation medium by 5% at an air flow of 0.3-1.0 VVm and a stirring rate of 350-450 rpm. and cultured at 27-31 deg.C for 8 hrs. The strain is cultured further for 18-22 hrs. but with the stirring rate dropped to 200-300 rpm. paraquat is added with a ratio of 0.5 mM as a inducing substance within the initial 6 hrs. The dismutase is separated and purifyed from the culture cells. The dismutase is useful as antiinflammatory agent.

Description

Serratia sp. Novel Superoxide Dismutase Produced by Y-4 and Its Production Method

1 is a graph showing the optimal pH of a superoxide dismutase according to the present invention.

2 is a graph showing the pH stability of the superoxide dismutase according to the present invention.

3 is a graph showing the thermal stability of the superoxide dismutase according to the present invention.

4 is an infrared and visible light absorption spectrum of the superoxide dismutase according to the present invention.

5 is an infrared absorption spectrum of the superoxide dismutase according to the present invention.

The present invention is Serratia sp. It relates to a novel superoxide dismutase (Y-4) produced and a method for producing the same.

Ceratia sp. Used as a production strain of superoxide dismutase in the present invention. Y-4 is a novel strain invented by the present inventors, and has been entrusted to the Korea Advanced Institute of Science and Technology (Accession No .: KCTC 8267 P), which is already a domestic patent application as a protease producing strain (Application No. 87-6746). )

Superoxide dismutase is an enzyme that catalyzes the conversion of superoxide radicals (O ₂ ^_ ) to hydrogen peroxide and oxygen.

, 171, 1971).Superoxide radicals result from the reduction of oxygen, which is essential for cell survival, which directly or indirectly causes fatal injuries to the microstructure of living cells (Annu, Rev, Phys. Chem.,

, 171, 1971).

, 397, 1982), 미생물이나 세포의 화학적 상해도 바로 슈퍼옥사이드 라디칼과 관련되어 있다.This radical is also produced in many biological reactions (Am. J. Pathol.,

, 397, 1982), chemical injury of microorganisms and cells is also directly related to superoxide radicals.

, 55, 1980), 피라진 유도체인 파라쿠아트(Paraquat)가 생체내에서 비교적 안정한 자유 라디칼로 쉽게 환원됨으로서 치명적으로 작용하는 경우도 있다(J. Bacteriol.,

, 505, 1977).For example, when superoxide radicals are produced by streptozotocin, insulin secreting cells can cause plasma membrane injuries and cause diabetes (Diabetologia,

, 55, 1980). Paraquat, a pyrazine derivative, may act deadly by being easily reduced to a relatively stable free radical in vivo (J. Bacteriol.,

, 505, 1977).

, 5395, 1978), 류마티스성 관절염(Biochem. Pharmacol.,

, 535, 1987)지질의 과산화 및 간질환(Biochem. J.,

, 343, 1976) 동맥경화등의 심장질환(Proc. Soc. Exp. Biol. ＆ Med.,

, 30, 1978)과 항산화작용으로 인한 노화의 방지 및 조직내 생리학적 기능을 돕기도 한다.Superoxide dismutase inhibits the production of free radicals such as superoxide radicals, thereby preventing skin diseases (Proc. Natl. Acad. Sci., USA,

, 5395, 1978), rheumatoid arthritis (Biochem. Pharmacol.,

, 535, 1987) Lipid Peroxidation and Liver Diseases (Biochem. J.,

, 343, 1976) Heart diseases such as atherosclerosis (Proc. Soc. Exp. Biol. & Med.,

, 30, 1978) and antioxidants to help prevent aging and physiological functions in tissues.

As such, superoxide dismutase is essential for the survival of all organs that metabolize oxygen, and is expected to be widely used for medical treatment or prevention.

, 2865, 1983)등을 생산균주로 사용하거나 에세리키아 콜라이 비(Escherichia Coli B, J. Biol. Chem.,

, 4782, 1972)를 사용하는 방법등이 알려져 있다.In the past, various techniques for producing superoxide dismutase by microbiological methods have been introduced, for example, Serra tia marcescence ATCC 21074 (US Pat. No. 4,563,349) or Bacillus subtilis IFO. 3022, Agri, Biol. Chem.,

, 2865, 1983), etc. or as Escherichia coli B, J. Biol. Chem.

, 4782, 1972).

In addition, as a component of a medium that has been used for culturing these strains, a suitable carbon source, nitrogen source, various inorganic salts necessary for the growth of bacteria (sodium chloride, potassium chloride, magnesium sulfate, zinc carbonate, etc.), imino acid source and vitamins, Depending on the characteristics of the nucleic acid or a nucleic acid-containing material has been appropriately mixed, a medium has been used, and oil (soybean oil, corn oil) or synthetic antifoaming agents (Tweens, anti-foam, etc.) have been used to remove bubbles generated during the culture.

However, these conventional methods require more than 48 hours to ferment the strain, and the productivity of the enzyme is poor, and there is much room for improvement in industrial use thereof.

Thus, the present inventors have been studying how to produce a superoxide dismutase in a more economical manner, while the Serratia sp. Y-4 produces a new type of superoxide dismutase that is different from the conventional one, and it is possible to greatly reduce the incubation time when the composition of the bacterial culture medium is changed. Attention was drawn to the present invention.

An object of the present invention is Serratia sp. It is to provide a new type of superoxide dismutase characterized by the production of Y-4.

Another object of the present invention is to provide a more economical method for producing superoxide dismutase by using a strain, while significantly reducing the incubation time of the strain, and further improving the productivity of the enzyme.

Hereinafter, the present invention will be described in detail.

In the present invention, Seratia sp. After incubating the Y-4 at 28 ° C. for 18 to 24 hours with shaking, the fermentation medium was inoculated at a rate of 5% under aeration of 0.3 VVM to 1.0 VVM and a stirring rate of 350 rpm to 450 rpm, and then at 27 ° C. to 31 ° C. After 8 hours of incubation, the stirring speed was lowered to 200 rpm to 300 rpm, followed by further 18 to 22 hours of incubation, and 0.5 mM paraquat was added as an inducer between the initial 6 hours.

At this time, the composition of the seed medium is, for example, sodium chloride 0.1%, potassium chloride 0.05%, manganese chloride 0.02%, magnesium sulfate 0.02%, zinc carbonate 0.002%, skim soybean 1.0%, casein 1.0%, ammonium hydrogen phosphate 0.6% The composition of the fermentation medium is, for example, sodium chloride 0.1%, potassium chloride 0.05%, manganese chloride 0.02% magnesium sulfate, 0.02%, zinc carbonate 0.002%, defatted soybean meal 1.0%, casein 1.0%, ammonium hydrogen phosphate 0.6% Lactose 0.5% Paraquat 0.5 mM is used without pH adjustment.

Characterization of the fermentation broth composition component according to the present invention, instead of using calcium chloride, calcium carbonate, soybean oil, actocol, and the like, manganese salts and sugars and the addition of paraquat.

In the present invention, the addition of manganese salt into the fermentation broth is based on the fact that manganese is bound to the active site of the highly purified superoxide dismutase, and the appropriate saccharide raw material increases the growth of the strain. Attention is made to add one or more saccharides such as glucose, lactose, or galactose, and paraquat is newly added as an inducer because paraquat is easily reduced to superoxide radical in vivo.

According to the present invention, Serratia sp. The change in enzyme productivity according to the passage of fermentation time during the incubation of Y-4 is shown in Table 1 below.

TABLE 1

Seratia sp. Used as superoxide dismutase producing strain in the present invention. Y-4 is significantly higher in productivity and enzyme titer than the production strains used in the prior art, compared to Serratia Marssense ATCC 21074 and Bacillus subtilis IFO 3022 as shown in Table 2 below.

TABLE 2

Note) 1. Enzyme productivity is UNIT per 1ml of fermentation broth.

2. Enzyme titer is UNIT per 1mg of protein in cells.

3. In KCTC 8267 P, (1) uses a conventional protease production medium and (2) uses fermentation medium according to the present invention.

4. Fermentation medium of each strain.

a. Serratia sp. Y-4

(1) 0.1% sodium chloride, 0.05% potassium chloride, 0.02% magnesium sulfate, 0.02% calcium chloride, 0.5% soybean oil, 0.5% sodium hydrogen phosphate, 1.0% skim milk powder, pH 6.5.

(2) Shown in Example 1.

b. Serratia Marssense ATCC 21074

Casein 1.5%, solvent extracted soybean meal extract 1.0%, ammonium hydrogen phosphate 0.6%, sodium chloride 0.1%, potassium chloride 0.05%, calcium chloride 0.02%, magnesium sulfate 0.02%, calcium carbonate 1.0%, zinc carbonate 0.002%, soybean oil 0.6%, ill Tocol 0.1%, adjusted to pH 7.0.

c. Bacillis subtilis IFO 3022 peptone 1.5%, potassium monophosphate 0.1%, dibasic potassium phosphate 0.1%, sodium chloride 0.5%, magnesium sulfate 0.01%, yeast extract 0.01%, meat extract 0.01%, pH 7.2.

At this time, the enzyme titer can be measured according to the most commonly used method of McCord and Pridovic (J. Biol. Chem., 244, 6049, 1969), which is described in detail 0.216M-potassium phosphate 25 ml of buffer (pH 7.80), 1 ml of 10.7 mM-ethylenediaminetetraacetic acid, 1 ml of 1.1 mM-Cytochrome C, and 50 ml of 0.108 mM-Xanthin Sodium were added thereto. Make 100 ml and use this mixture as the reaction solution.

2.8 ml of this reaction solution was added to a cell (light, 1 Cm of light transmission distance) at a reaction temperature of 25 ° C, and 0.1 ml of xanthin oxidase solution of an appropriate concentration capable of measuring enzyme activity was reduced in cytochrome C. After measuring the absorbance at a wavelength of 550 nm for 1 minute, add 0.1 ml of superoxide dismutase solution and measure the increase in absorbance for 1 minute.

In this way, the reduction rate of the cytochrome C, that is, 50% of the increase in absorbance, was determined to be 1 UNIT.

Protein quantitation is determined by Bovine Serum Albumin as standard, by Coomassie Brilliant Blue G-250, Anal. Biochem., 79, 544, 1977.

On the other hand, Serratia sp. After fermentation of Y-4, the cells are recovered from the fermentation broth using centrifugation or other known separation methods, and the recovered cells are suspended in water or a buffer solution and closed using ultrasonic wave or grinding.

Then, the pulverized cells are removed by centrifugation or the like, and the precipitates formed by ammonium sulfate or acetone treatment are recovered by centrifugation. Then, they are dissolved in water or a buffer solution, and then the low-molecular substance is dialyzed and removed through the dialysis membrane. )

For reference, Serratia sp. When the protease was produced from Y-4, the protease was separated and purified from the culture medium from which the protease was exo-enzyme, but the superoxide dismutase was endo-enzyme. Dismutase is to be purified separately.

Finally, the crude enzyme solution is separated and purified by ion exchange resin chromatography and gel filtration or a similar method generally used for enzyme purification, so that the superoxide dismutase which is the object of the present invention can be separated and purified to high purity.

The enzymatic and physicochemical properties of the superoxide dismutase purified according to the present invention are as follows.

1. The action-superoxide radical (O ₂ ⁻ ) is converted into a hydrogen peroxide molecule and an oxygen molecule.

2. Acts on substrate specificity-superoxide radicals.

3. Optimal pH Range—The optimal pH for this enzyme is 7-9, which shows the optimal pH.

4. pH Stability- This enzyme is stable at pH 5-11, and Figure 2 shows the pH stability of this enzyme.

5. Range of functional temperature-This enzyme works between 10 ° C and 40 ° C.

6. Thermal Stability- The enzyme was treated with pH 8.0 buffer to measure the remaining activity over time. As a result, it was stable at 37 ° C for 60 minutes, at 50 ° C for 35 minutes, and deactivated at 60 ° C for 10 minutes. 42% deactivation when treated for 60 minutes and completely deactivated when left for 60 minutes at 70 ° C and 30 minutes at 80 ° C. 3 shows the thermal stability of this enzyme.

7. Elemental Analysis-This enzyme consists of 40.38 ± 0.65% carbon, 5.50 ± 0.03% hydrogen, and 11.81 ± 0.65% nitrogen.

8. Ultraviolet and Visible Absorption Spectrum—The enzyme exhibits the highest absorbance near 280 nm in the ultraviolet absorption spectrum and the highest absorbance near 475 nm in the visible absorption spectrum. Figure 4 shows the absorption spectra of the enzyme in the ultraviolet and visible light.

9. Infrared Absorption Spectrum-The infrared absorption spectrum of this enzyme is determined by the KBr method. 5 shows the infrared absorption spectrum of this enzyme.

10. Isoelectric Point-The isoelectric point of this enzyme is around pH 4.7.

11. Molecular weight- The specific molecular weight is 54,000 Daltons by gel filtration using Sephacryl S-200 Superfine.

12. Analysis of Containing Metal Ions-As determined by atomic absorption method, this enzyme contains 1.0 g of manganese. Therefore, it is certain that this enzyme is manganese superoxide dismutase.

As can be seen from the above, the superoxide dismutase produced according to the present invention was shown to have a different physical and chemical properties compared to the superoxide dismutase known in the prior art. The molecular weight of Superoxide Dismutase produced by Escherichia coli was 39,500, Superoxide Dismutase produced by Serratia Marssense ATCC 21074 had a molecular weight of 48,000 and Superoxide Dismux produced by Bacillus subtilis IFO 3022. The molecular weight of the other agent was 41,000, and the molecular weight of the superoxide dismutase produced by Streptococcus mutans was 40,250, but the molecular weight of the superoxide dismutase according to the present invention was 54,000. It was found to be significantly higher than the molecular weight.

For this reason, the superoxide dismutase according to the present invention was identified as a novel enzyme.

Hereinafter, examples of the present invention will be described.

Example 1

Used strain-Serratia sp.Y-4 KCTC 8267 P

Species medium-sodium chloride 0.1%, potassium chloride 0.05%, manganese chloride 0.02%, magnesium sulfate 0.02%, zinc carbonate 0.02%, defatted soybean meal 1.0%, casein 1.0%, ammonium hydrogen phosphate 0.6%, without pH adjustment.

Fermentation medium-sodium chloride 0.1%, potassium chloride 0.05%, manganese chloride 0.02%, magnesium sulfate 0.02%, zinc carbonate 0.02%, skim soybean meal 1.0%, casein 1.0%, ammonium hydrogen phosphate 0.6, lactose 0.5%, paraquaart 0.5 mM Use without pH adjustment.

Cultivation method-First, 400 ml of seed medium was dispensed into a 2 L shake flask to prepare a seed culture, and autoclaved at 121 ° C. for 15 minutes, inoculated with Seratia sp.Y-4 and 20 hours at 28 ° C. at 200 rpm. Shake culture was performed. Subsequently, 10 liters of fermentation medium was added to a small 14.5 L fermenter, and autoclaved at 121 ° C. for 15 minutes. The seed culture medium was inoculated at a rate of 5% of the fermentation medium and incubated at 28 ° C. at 400 rpm and 0.5 VVM for 8 hours. , rpm was reduced to 300 and further incubated for 19 hours. Enzyme production amount of superoxide dismutase in the culture complete solution was 1320 UNIT / ml.

Example 2

The composition of the strains used and the seed medium were the same as in Example 1, but during the fermentation medium composition, paraquat was sterilized separately and added to finally 0.5 mM between 4 hours and 6 hours after the start of the culture. Other conditions were the same as in Example 1.

Example 3

The fermentation broth obtained in Example 1 was continuously centrifuged at 25,000 rpm (15,000 x g) to obtain 212 g of the cells, and the cells were suspended in 500 ml of 0.05m potassium phosphate buffer (pH 7.8) containing 0.1 mM ethylenediaminetetraacetic acid and suspended at 4 ° C. Cells were crushed for 80 minutes by an ultrasonic crusher (all the following operations were performed at 4 ° C).

The crushed cells were centrifuged at 20,000 rpm (hereinafter, all centrifugation was performed at 20,000 rpm) to remove the crushed cells and only the supernatant (coenzyme solution) was recovered.

Subsequently, the supernatant was treated with ammonium sulfate up to 45%, and the precipitate thus formed was removed by centrifugation, and only the supernatant was recovered.

The recovered supernatant was treated with ammonium sulfate from 45% to 80%, and the resulting precipitate was recovered by centrifugation. The precipitate was dissolved in 150 ml of water and treated with ammonium sulfate from 0 to 70%, and the precipitate was recovered by centrifugation. The recovered precipitate was dissolved in 80 ml of 0.002 M potassium acetate buffer (pH 5.5) and dialyzed for two days with the same buffer through a cellulose membrane, but the precipitate formed during dialysis was removed by centrifugation.

The dialysate was then sucked through a DEAE-Sephacel Column (Pharmacia Fine Chemicals AB, 2.6 × 25 cm) equilibrated with 0.02 M Tris hydrochloric acid buffer (pH 8.5) and twice the column volume with the same buffer. The non-absorbed impurities were first washed out.

After washing, the concentration gradient from 0 to 0.1M was measured with sodium chloride, and the absorbance of the eluate was measured at a wavelength of 280 nm, and only the fractions having enzyme activity were collected separately.

The eluted enzyme active solution was dialyzed with distilled water through a cellulose membrane for two days, and then frozen and dried. The lyophilized enzyme was dissolved in 5 ml of 0.05 M Tris hydrochloric acid buffer (pH 8.0) and equilibrated with the same buffer. Gel filtration was performed through (Sphacryl S-200 Superfine, Phrmacia Chemicals AB, 1.6 × 85 Cm).

The obtained eluate was measured for absorbance at a wavelength of 280 nm, the fractions with enzyme activity were collected and again dialyzed through a distilled cellulose membrane for two days, and the purified enzyme solution was lyophilized to finally obtain 441.2 mg of shureoxide dismutase (7222UNT). / mg).

Example 4

205 g of cells were obtained from the fermentation broth obtained in Example 2 in the same manner as in Example 1.

The purification method was the same as in Example 3.

Finally, 412 mg (8984 UNIT / mg) of purified superoxide dismutase was obtained.

Example 5

Strains and seed media used were the same as in Example 1.

Enzyme production was measured for each concentration by changing the concentration of manganese salt in the fermentation medium as shown in Table 3 below. At this time, the composition of the other fermentation broth was the same as in Example 1 except that 400 ml of the fermentation broth was dispensed in a 2 L shake flask, autoclaved at 121 ° C. for 15 minutes, and then inoculated with a spawning broth at a rate of 5% and 200 rpm, Incubated for 30 hours at 28 ℃.

Enzyme production by concentration of manganese salt is shown in Table 3.

TABLE 3

Example 6

In order to measure the amount of enzyme production according to the concentration of the inducer paraquat, the concentration of paraquat was changed in the same manner as in Example 5 while changing as shown in Table 4 below.

Enzyme production by the paraquat concentration is shown in Table 4.

TABLE 4

Example 7

In order to measure the effect of lactose on the growth and enzyme production of the strain was carried out in the same manner as in Example 5 while changing the concentration of lactose as shown in Table 5.

Strain growth and enzyme production of lactose is shown in Table 5.

TABLE 5

Claims (4)

Serratia sp. A novel superoxide dismutase characterized by the production of Y-4 (Serratia sp. Y-4, Accession No .: KCTC 8267 P). The method of claim 1 wherein the superoxide dismutase has a molecular weight of 54,000. In fermentation broth, Serratia sp. A method for producing superoxide dismutase, wherein Y-4 is fermented and cultured for 26 to 30 hours, and superoxide dismutase is separated and purified from the cells. The method according to claim 3, wherein the fermentation medium contains manganese salt, a saccharide raw material, and paraquat.

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