JPH0616704B2 - Bacterial catalase resistant to fluoride ions and Micrococcus sp. KWI-5 strain - Google Patents

Description

TECHNICAL FIELD The present invention relates to a novel fluoride ion-resistant catalase and a new strain belonging to the genus Micrococcus that produces it.

(Prior Art) Water treatment field of wastewater containing hydrogen peroxide such as semiconductor process,
Catalase is widely used in the field of food such as residual hydrogen peroxide due to bleaching of Kazunoko. Catalase is an enzyme that catalyzes the field of hydrogen peroxide and is known to be produced by many organisms. For example, horses (Deutsch, 1952), Saccharomyces cerevisiae (Seeh et al., 1973), Bacillus No. KU.
1 (Kurono et al., 1973), Escherichia coli (Claiborne et al., 1979), and the like.

(Problems to be Solved by the Invention) It is known that catalase is generally inhibited by fluoride ion, cyanide ion, sodium azide, β-mercaptoethanol and the like. In the application of catalase in the field of water treatment, it has been required to use catalase resistant to fluoride ions contained in wastewater. B as a catalase resistant to fluoride ions
Acillus No. KU1 has been known, but since the optimum pH of action is alkaline, a strain producing a catalase which is resistant to fluoride ion and has neutral activity has been required.

(Means for Solving the Problem) The present inventors extensively searched for catalase-producing microorganisms having fluoride ion resistance in nature, and isolated Micrococcus sp. The present invention was completed by finding that the KWI-5 strain produces catalase satisfying the above-mentioned requirements.

Next, an activity measuring method, physicochemical properties of the fluoride ion-resistant catalase of the present invention, and Micrococcus sp. KWI
-5 Describe the mycological properties of the bacterium.

(Activity measuring method) A phosphate buffer solution (pH 6.
8) 1 ml of enzyme solution was mixed with 1 ml of enzyme solution, reacted at 30 ° C. for 5 minutes, and then the reaction was stopped by adding 1 ml of titanium sulfate solution (20% sulfuric acid solution containing 0.8% sulfuric acid 2nd titanium sulfate solution). Then, color is developed, and the absorbance at 440 nm is measured. Substituting this measured value into the following formula, the activity (Kat-f
Value). (Vol. 2, "Enzyme Research Method" by Shiro Akahori,
Asakura Shoten, published in 1956, pages 324 to 325) Kat-f = log (A / B) × C / D [min] ^-1 [ml] ^-1 where A: absorbance of control (not containing catalase) B : Absorbance of sample (reacted with catalase) C: Dilution rate of enzyme solution D: Reaction time Physicochemical properties of the fluoride ion-resistant catalase of the present invention are as follows.

(Physical and chemical properties) (1) Action and substrate specificity It acts on hydrogen peroxide and decomposes into water and enzymes.

There is no peroxidase activity.

(2) Optimum pH and stable pH range The effect on pH was examined based on the above-mentioned activity measuring method.
FIG. 1 shows the relative activity at each pH when the maximum activity was 100. It can be seen that the optimum pH is pH 5.5 to 11.0.

Next, FIG. 2 shows the stability of pH. Enzyme for each p
After maintaining at 30 ° C. for 1 hour in H, the residual activity measured at 30 ° C. at pH 6.8 was measured by measuring the residual enzyme activity as 100%.
Indicated as.

From FIG. 2, the catalase of the present invention is stable at pH 5.5-10. The buffer solution used had a pH of 4.5 to 5.5.
(50 mM acetate buffer), pH 5.5-8.0 (50 m
M phosphate buffer), pH 7.5 to 9.0 (50 mM Tris buffer), pH 9.0 to 13.0 (50 mM glycine buffer).

(3) Range of suitable temperature of action According to the above-mentioned activity measuring method, enzyme reaction was carried out at each temperature for 5 minutes. FIG. 3 shows the activity ratio at each temperature when the activity at 30 ° C. is 100. It can be seen from FIG. 3 that the optimum action temperature of this enzyme is 30 to 35 ° C.
Further, the working temperature at which the activity is 80% or more of the optimum temperature is
It can be seen that the temperature is about 20 to 50 ° C.

(4) Thermal stability After heat treatment at pH 6.8 for 20 minutes at each temperature, the residual activity was examined. The untreated enzyme activity was set to 100 and its residual activity is shown in FIG. From Fig. 4, this enzyme is 10 ℃ ~ 50 ℃
It turns out that it is stable at.

(5) Deactivation condition As shown in Fig. 2, the catalase of the present invention is completely inactivated when treated at pH 4.0 for 1 hour at 30 ° C. As shown in FIG. 3, at pH 6.8, 70 ° C. 20
It is completely deactivated in minutes.

(6) Inhibition Catalase activity was measured in the presence of the drugs shown in Table 1,
The activity in the absence was defined as 100, and the residual activity was shown as a percentage. As can be seen from the table, this enzyme is extremely resistant to fluoride ions.

(7) Isoelectric point As a result of performing isoelectric focusing using a gradient polyacrylamide gel having a pH of 3.5 to 9.5, the isoelectric point was pH 4.4. As a standard substance, BIOR
AD IEF standard was used.

(8) Molecular weight A molecular weight of 210,000 was obtained by gel filtration using Super Rose 10 manufactured by Pharmacia. As a molecular weight marker, thyroglobulin (molecular weight 664,00
0), apoferritin (molecular weight 446,000), β-
Amylase (molecular weight 200,000), γ-globulin (molecular weight 150,000), bovine serum albumin (molecular weight 67,000), ovalbumin (molecular weight 45,000)
0) and cytochrome C (molecular weight 12,400) were used.

(9) Subunit structure As a result of SDS-polyacrylamide electrophoresis, one catalase molecule of the present invention was composed of four subunits having a molecular weight of 58,000. As the molecular weight markers, bovine serum albumin (molecular weight 67,000), ovalbumin (molecular weight 45,000), α-chymotrypsinogen (molecular weight 25,700), ribonuclease (molecular weight 1)
4,700), cytochrome C (molecular weight 12,400)
Was used.

(10) Specific activity The specific activity of the purified enzyme is 4,000 Kat-f / m.
g protein.

The enzyme can be purified by adding a lysing agent to the culture broth after the completion of culturing and extracting, and then using an isolation / purification means known in the art, for example, an ion exchanger or a gel filtration material. The method for producing this enzyme will be specifically described in Examples below.

Table 2 shows a comparison between the catalase obtained according to the present invention having the above-mentioned properties and the conventionally known catalase.

To produce the catalase of the present invention, Micrococcus sp. Belonging to the genus Micrococcus is used. The KWI-5 strain can be used.

The mycological properties of this strain are shown below. The test and classification method of the mycological properties were performed based on the following documents.

Takeharu Hasebe et al., “<Revised Edition> Identification of Microorganisms” (1984) published by the Society Publishing Center and Burgers Manual of Systematic Bacteriology (Be).
rkey'S Manual of System
ic Bacteriology (1984) (1) Morphology Cell shape and size Cocci having a size of 0.8 to 1.2 μm and no flagella.

Motility: None.

Spores: None.

Gram stain is positive.

The acid resistance is negative.

(2) Growth state (medium pH: 6.8) Meat broth agar plate culture Round, flat or ridged with smooth edges and gloss. It turns yellow as the culture time elapses.

Meat broth agar slope culture It grows in a wide band and the edges are smooth and shiny. It turns yellow as the culture time elapses. No pigment is required in the medium.

Broth liquid culture Good growth and light yellow color.

Litmus milk Alkaline.

(3) Physiological properties Reduction of nitrate: Positive.

Denitrification reaction: Negative MR test: Negative.

VP test: negative.

Formation of indole: negative.

Hydrogen sulfide formation: Negative.

Starch hydrolysis: negative.

Utilization of citric acid Koser's medium: used.

Christensen's medium: used.

Simon's Medium: Used.

Use of inorganic nitrogen source Nitrate: Negative.

Ammonium salt: Positive.

Dye formation: negative.

Urease: Weakly positive.

Oxidase: negative.

Catalase: positive.

Range of growth pH: Grow at 6-11. 6-8 is the best.

Temperature: Grow at 15-30 ° C. Best at 30 ° C.

Attitude toward oxygen: aerobic.

OF test (by Hugh Leifson method): O.

Presence or absence of generation of acid and gas from saccharides D-glucose, D-fructose, D-maltose,
Glycerol is used to generate an acid, but no gas is observed. D-arabinose, D-xylose, D
-Mannose, D-galactose, D-trehalose,
D-sucrose, D-lactose, D-sorbitol, D-mannitol, D-inositol and starch are not used.

(4) Other properties Sodium chloride tolerance: Grows in the presence of 10% sodium chloride.

Liquefaction of gelatin: Positive.

Tween degradation: Tween 40, 60 and 80 are degraded.

Tyrosine degradation: positive.

Casein degradation: Positive.

The strains of the present invention are similar to Micrococcus variance when compared with the known strains of the genus Micrococcus according to the classification method of the above-mentioned literature based on the above-mentioned mycological properties. However, there is a clear difference in the use of glycerol (without Micrococcus variance) and the tolerance of salt (Micrococcus variance is not resistant to 10% sodium chloride). In addition, the strain of the present invention has a physiological property very similar to that of Micrococcus roseus, but the color of the colony (Micrococcus roseus is red, whereas this strain is yellow), gelatin degradation (Micrococcus roseus is Not) and the use of citric acid (Micrococcus roseus does not grow in Simons medium) is clearly different.

As described above, since this strain is distinguished from known strains,
It was determined to be a new strain belonging to the genus Micrococcus, and Micrococcus sp. It was named KWI-5. This strain was deposited on February 6, 1990 at the Institute for Microbial Technology, Ministry of International Trade and Industry, Institute of Industrial Technology.

The microorganism accession number is Microbiology Research Institute No. 11258.

(Function) The fluoride ion-resistant catalase of the present invention is active even in the presence of 10 mM fluoride ion, and has a wide range of p
Acts on H. Therefore, hydrogen peroxide can be decomposed in waste water containing fluoride ions. In addition, the Micrococcus sP. The KWI-5 bacterium can be used to produce the fluoride ion-resistant catalase of the present invention.

The culture conditions for producing fluoride ion-resistant catalase are as follows. First, as the medium, a medium containing components necessary for the growth of microorganisms such as a carbon source, a nitrogen source, and an inorganic salt is used. As the carbon source, glucose, oleic acid, malic acid, glycerol and the like can be used, as the nitrogen source, fish meat extract, peptone, casamino acid, corn steep liquor, soybean powder and the like, as the inorganic salt, potassium dihydrogen phosphate, Magnesium sulfate, ferric chloride, etc. are used. It is desirable that the pH of the medium is around 6.8.

When this strain is cultivated aerobically in the above medium at 30 ° C., the production of catalase becomes maximum about 24 hours after the start of culturing. When the culture temperature is high, for example, the production amount is 30 at 37 ° C.
It becomes 60% of ℃, and the production amount decreases even if the temperature is low.

The culture solution thus obtained can be treated with a lytic enzyme, a nucleolytic enzyme, an organic solvent or the like to destroy the cells, and then the solid content can be separated by centrifugation or the like to obtain a crude enzyme solution. . The crude enzyme solution can be purified by a known means such as ultrafiltration, gel filtration, ion exchange, or electrophoresis to obtain a purified enzyme.

Example 1 Sterile liquid medium (pH 6.8) 1 consisting of 2% glucose, 1.6% polypeptone and 0.4% yeast extract 1
00 ml was placed in a Sakaguchi flask, and Micrococcus sp.
Inoculated with KWI-5, 30 ° C on rotary shaker
It was cultured in. This culture solution was treated with a lytic enzyme, a nucleolytic enzyme, and an organic solvent, and then centrifuged to remove solids to obtain a crude enzyme solution. The catalase activity was 400 Kat-f / mg.

Example 2 20 ml of the crude enzyme solution of Example 1 was subjected to gel filtration by preparative liquid chromatography (column: Tosoh KK, TSKg.
(elG3000SW) was fractionated. The obtained enzyme fraction was dialyzed in 0.05 mM phosphate buffer for 24 hours, and then subjected to ion exchange (column: Tosoh KK, TSKgel DEAE5PW) by fractionation liquid chromatography. The enzyme thus obtained showed a single band on polyacrylamide gel electrophoresis. The specific activity is 4,000 Kat
-f / mg protein.

(Effect) According to the present invention, Micrococcus sp. The KWI-5 strain is cultured, and fluoride ion-resistant catalase can be obtained from the culture solution. Since this catalase has excellent resistance to fluoride ions, it can be widely used for water treatment of wastewater containing hydrogen peroxide, such as in the semiconductor industry.

[Brief description of drawings]

FIG. 1 shows the optimum pH of the enzyme of the present invention, and FIG. 2 shows the stable pH range of the enzyme of the present invention. FIG. 3 shows the optimum temperature range for the action of the enzyme of the present invention, and FIG. 4 shows the thermostability of the enzyme of the present invention.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Office reference number FI technical display location C12R 1: 265)

Claims (2)

[Claims] 1. A bacterial catalase resistant to fluoride ions having the following physicochemical properties. Action and substrate specificity It acts on hydrogen peroxide and catalyzes a reaction that decomposes into water and oxygen. Optimal pH and stable pH range Optimal pH is 5.5 to 11.0, stable pH is 5.0 to 10
Is. Range of optimum temperature for action The optimum temperature is 30 to 35 ° C. Inhibition Sodium azide (1 mM) causes significant inhibition. It is not inhibited by potassium fluoride, hydrazine, β-mercaptoethanol, sodium ethylenediaminetetraacetate (1 mM each). Molecular weight According to SDS polyacrylamide gel electrophoresis, this enzyme consists of 4 subunits with a molecular weight of 58,000, and the molecular weight by gel filtration method is 210,000. Isoelectric point The isoelectric point by isoelectric focusing is pH 4.4. Specific activity Specific activity is 4,000 Kat-f / mg protein. 2. Micrococcus sp. Which belongs to the genus Micrococcus and has the ability to produce a fluoride ion-resistant catalase according to claim 1. KWI-5 strain.