JPS58179488A - Preparation of peroxidase - Google Patents

Abstract

PURPOSE:To prepare a peroxidase useful as an agent for clinical diagnosis and enzyme immunoassay, by culturing a microbial strain belonging to Bacillus genus and capable of producing peroxidase, and separating the accumulated enzyme. CONSTITUTION:A microbial strain belonging to Bacillus genus and capable of producing peroxidase, e.g. Bacillus cereus, Bacillus megaterium, etc. is cultured to produced and accumulate the peroxidase in the cultured product, and the peroxidase is separated therefrom. The cultivation can be carried out in a synthetic or natural medium containing nutrients and additives. The carbon source is, e.g. glucose, soluble starch, citric acid, etc. and the nitrogen source is, e.g. peptone, casein, meat extract, sodium nitrate, ammonium chloride, etc.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention provides a method for producing Veroin cytase using microorganisms,
For more details, check out the new Bell Saiju Jitter originating from Bacillus! ,
%, 4-aminoacytipyrine (hereinafter abbreviated as r4-AAJ)-phenolic, 3-methyl-2-besylytiarylinosyde 5-lysi (hereinafter abbreviated as "MBTII")-dimethylanilisi (hereinafter abbreviated as "DMAJ") ) system, 4-AA-DM
A series, and 4-AA-diethylanilisi (hereinafter referred to as rDEA)
This invention relates to a novel method for producing Beryl cytase that develops color using a hydrogen donor system (abbreviated as J).

Beryl oxidase is an enzyme that oxidizes various compounds in the presence of hydrogen peroxide, and has recently been used as a clinical diagnostic reagent in various ways to quantify talc, total cholesterol, free cholesterol, phospholipids, and uric acid. It is also used as a labeling enzyme for enzyme immunoassay. In the past, the sources of Beruoh+cytase used in these reagents were only plants such as radish and horseradish, and some Benoh+cytase derived from microorganisms was also known. is cytochrome C-berio+ sidase and NADH-beroo+ sidase produced by bacteria and filamentous fungi, and is not a non-specific beroo+ cytase found in plant origin, but is unique to the plants mentioned above in terms of its specificity. It cannot be used as a clinical diagnostic reagent that can replace the original Beluo+gitase. Also in recent years O
-O-dianisidine has been produced from Escherichia coli and microorganisms belonging to the genus Myrocesium, but O-dianisidine has carcinogenic effects.O-dianisidine tends to be avoided for industrial hygiene reasons, and its clinical use tends to be avoided. is not suitable for use as a diagnostic reagent.

In view of the above-mentioned current situation, the present inventors have developed Beruo bovine sitase, which has properties that can be used as a clinical diagnostic reagent and an enzyme immunoassay method, because it multiplies rapidly and can be produced in large quantities compared to plants. We are conducting intensive research from the viewpoint that it would be industrially beneficial if it could be found in microorganisms that have the potential to dramatically improve the ability of 4-AA.As a result, 4-AA, which is widely used as a clinical diagnostic agent - As microorganisms that produce Veroin cytase that develops color using color-forming agents such as phenol, 4-AA-DMA, and 4-AA-DEA, the present inventors have identified microorganisms that produce Veroin cytase, such as Alternaria, Alternaria, Cochliobolus, etc. He discovered microorganisms belonging to the genus Perichuria and Curbularia, and established a method for producing Verocytase by bioutilization of the fungus, and applied for a patent.
No. 7565).

The present inventors continued to isolate bacteria from the natural world over a wide range of areas, cultured the isolated bacteria, and investigated whether or not 4-AA-phenol-based substances occur in the culture in the presence of hydrogen peroxide. As a result, Bacillus spp. It was confirmed that the desired Bacterium cytase-producing bacteria existed, and the present invention was completed based on this new knowledge.

That is, the present invention provides a method for culturing a microorganism belonging to the genus Bacillus and having the ability to produce Pel-10-citase in a nutrient medium, producing and accumulating Pel-10-citase in the culture, and collecting the product. Relating to a method for producing cytase.

In the present invention, examples of microorganisms belonging to the genus Bacillus having the ability to produce Bacillus cytase include, for example, Bacillus 5LLbttLiz,
Bacillus cereus (B, ctrtu, ?), Bacillus megaterium (B, m-yatarium)
Various microorganisms belonging to the genus Bacillus can be used. Among these, one of the most suitable is:
The present inventors isolated Bacillus cereus in the soil and found that it was
) The strain named J. The mycological properties of the strain were investigated in accordance with the ``Medical Bacterial Identification Manual'' 2nd Edition (Kinda Publishing) and the ``Second Differential Table of Bacillus'' K, and the results are shown in Table 1 below. be.

Table 1 From Table 1 above, this bacterial strain is identified as Bacillus cereus. The strain was entrusted to the National Institute of Microbiology, Agency of Industrial Science and Technology as FERMSPA-6503.

Other microorganisms suitably used in the present invention include, for example, Bacillus cereus IFO3132, Bacillus cereus IP03836, Bacillus cereus IF013690, and Bacillus megaterium IFO39.
70 and Bacillus subtilis IP03936.

The present invention is carried out as follows using the above-mentioned various strains or mutant strains thereof. That is, first, the above-mentioned microorganism is cultured in a nutrient medium. Cultivation can be carried out in synthetic or natural media containing conventional nutrients and additives. Glucose, maltose, saccharose, galactose,
Any commonly used substances such as fructose, xylose, mannose, raffinose, soluble starch, liquefied starch, molasses, glycerol, sorbitol, quesic acid, cohelic acid, etc. can be used. Nitrogen sources include natural nitrogen sources such as Bezutoshi, Yeast Kojusu, defatted soybeans, Gazeishi, Meat Kojusu, Casamino acids, and Kosi Cheap Liquor, as well as inorganic sources such as sodium nitrate, ammonium nitrate, ammonium sulfate, and ammonium chloride. Nitrogen can be used. In addition, inorganic salts such as phosphates, magnesium sulfate, iron sulfate, steel sulfate, zinc sulfate, iron chloride, baltic acid chloride, and pita chloride can also be used as trace nutrient sources, if necessary. These medium components are used at concentrations that do not inhibit the growth of each microorganism to be cultured, and although this varies slightly depending on each microorganism, in general shaking culture or aerated agitation culture, the carbon source is generally 0. 1-10% by weight, preferably 0.5-10% by weight
7% by weight, nitrogen source 0.01-8% by weight, preferably 0
．． The concentration is preferably 1 to 5% by weight. Also, the pH of the medium
is 4 to 11, preferably 5 to 10, and the culture iI temperature is 2.
The temperature is preferably 0 to 45°C, preferably 25 to 38°C,
Cultivation is usually carried out for about 2 to 10 days.

By the above-mentioned culture, the desired Verocytase is produced and accumulated in the culture. The term "culture" as used herein means the nine microbial cells produced and the culture supernatant or culture p solution. The method for collecting Bell Saijucyse from these cultures may be in accordance with a conventional method, for example, by removing the bacterial cells and insoluble matter from the culture solution after the completion of culture by centrifugation and filtration. Get the liquid. The cytase contained in the bacterial cells can also be obtained as a crude enzyme solution by destroying the bacterial cells by grinding or ultrasonication and extracting the enzyme. Furthermore, it is also possible to obtain a crude enzyme solution by subjecting a culture solution containing microbial cells to ultrasonic treatment for a while to destroy the microbial cells and then removing insoluble matter. Purification of crude enzyme solution may be carried out by following conventional methods, such as fractional precipitation method of aci7nium sulfate, dialysis, fractionation method using adsorbent, organic solvent fractionation method, isoelectric focusing method, etc. Column chromatography using an ion exchanger, affinity chromatography, and the like can be used alone or in combination. Purified perio+cytase is thus obtained.

In the present invention, the activity of Bell's Cytase was measured using Ima-A,4-phenol, which is used in clinical diagnostic reagents, as a hydrogen donor. That is, o, +ysvH1
l buffer solution (PH7,0) IdKo, 08% 4-A A solution Q, 5 wl, 0.4% phenol solution 0.5 ml, 0.15 ml hydrogen peroxide solution 111! After preheating to 30° C., add 1 d of enzyme solution and react for 15 minutes, and immediately measure the absorbance at a wavelength of 5109. Separately, as a control, water was added in place of the hydrogen peroxide solution and the absorbance was measured by the same procedure.Next, the enzymatic chemical properties of the Bell Saiju Cydase obtained in the present invention will be shown.

(1) Specificity of action: This enzyme acts very specifically on hydrogen peroxide, and catalyzes the oxidation of various compounds that function as hydrogen donors in the presence of hydrogen peroxide. Its mechanism of action is as shown in the following equation.

Vero + Cytase H2O2 + AH22H20 + A [However, in the formula, J2 represents a hydrogen donor, and A represents an oxidized 9-hydrogen donor.] (2) Optimum pH; pH range of 4.5 to 10, wide range buffering liquid (0.04J/ricic acid-0.04M acetic acid-
A mixed solution of 0.04M boric acid and 0.2M caustic sorter) was used at the same mixing ratio as in the activity measurement. The results are shown in FIG.

(3) Optimum action temperature: Enzyme activity was measured at 30 to 70 CK and the results are shown in FIG.

(4) PH stability: Enzyme solution 1ILt was added to Zuridocyoshi buffer solution 411LIK with a pH of 4.0 to 10 and left at 30°C for 17 hours. When measuring enzyme activity, KO, 4M lysic acid buffer 5Il/
was added to adjust the pH to 7.0, and the residual enzyme titer was measured. The results are shown in FIG.

(6) Temperature stability: 5 m of 0.02 M lysic acid buffer (PH8,0)
The enzyme solution prepared by adding ml of the enzyme solution was kept at a temperature of 20 to 80°C for 30 minutes, and immediately after the treatment, it was cooled with ice water for 10 minutes, and then the residual enzyme activity was measured. The results are shown in FIG.

(6) Measurement method of titer; As mentioned above.

(7) Comparison of the degree of color development by various color formers Among the color formers used for clinical diagnostic reagents, the following four types of chemicals were investigated. That is, the conditions other than the color former were unified, and 4-AA-phenol was measured under the same conditions as the titer measurement described above. 4-AA-DMA system [Hygiene inspection 25. (5), 312), 4-AA-D
MA mixture C0,036% 4AA and 0.05% DMA)
Measure the absorbance of 560 genus with 2d formulation. 8-Hyde 0 + Norin (8fQ) -F-F Nishiji,,' (PA
”) system [Ckg abuse, Ple αrm, BuLL, .

27.568, 1979), 0.72% 8
-Measure the absorbance of 600% rich with Hyde 0 Beef Norishi solution Q, 5m, 0.56th
lol.

18.943.1972), MBTH-DMA
Mixture C0.04% METH and 0.061DMA) Im
The absorbance of 590F&llL was measured with the following formulation. The measurement results are shown in Table 2 below.

Table 2 Next, examples of the present invention will be shown.

Example ! Soluble starch41. 50% liquid culture medium CpH 7.0) containing 2% Bedstone and 0.31 cornstay suspension.
01I7! Mr. Yosakaro put 5011j in Kolbeshi, 120
After sterilizing at ℃ for 30 minutes, Bacillus cereus UP was cultured with shaking for 19 hours in a liquid culture solution with the same composition as above, and Q, 5 * 1 was inoculated using the 9 liquid as medium seed, and then incubated at 30℃. So 4
Culture with shaking for days. After completion of culture + 1 at 00 OrPM
A crude enzyme solution (p#8.8) was obtained by centrifugation for 5 minutes. This crude enzyme solution had a Bell's schizase activity of 0.54 units.

Example 2 Glyose! Excellent, soluble starch l-, beptoshi4chi, ]-
Sisti liquor〇, 1 vomit, 0. Acid buffer CpH
7-0), aci7nium nitrate 0.0411 and boric acid,
Chlorinated steel, calcium chloride and mangaka chloride 0.02M each
A liquid culture solution containing (7)H7,0)IOt was charged into a 301 volume jar, and heated at 120℃ for 40 hours.
After sterilization for 1 minute, culture solution 5017 obtained by culturing Bacillus cereus UP in the same composition medium for 19 hours with shaking was inoculated thereto, and cultured with aeration at 30°C with aeration rate of 0.25 sq. did. After 24 hours of culture, a portion of the culture solution was collected and centrifuged at +ooorpm for 15 minutes to separate the bacterial cells, which were then washed with physiological saline to obtain 5 tons of wet bacterial cells. Add 0.5t of sea sand and a small amount of 0.0Iffi phosphate buffer to this, grind it in a mortar, add the same buffer, and reduce the total volume to 100%.
After binding, the mixture was centrifuged at SOOO rpm for 15 minutes to obtain a crude enzyme solution as a supernatant.

This crude enzyme solution had a citase activity of 0.1 unit.

- In addition, the culture was further continued in the above manner, and the culture solution obtained after 72 hours of culture was similarly centrifuged or filtered by suction in the presence of calcium phosphate gel and a household aid to remove bacterial cells. A crude enzyme solution (12 units/d) was obtained.

Addition of 20,665% by weight of sulfuric acid to the obtained crude enzyme solution resulted in a salting out product, which was collected by suction filtration after addition of a p-superior. )50
0+j and dialyzed against the same buffer using 70 Fan Chews to remove acitium.

The obtained dialysate solution was equilibrated in advance with 0.02 J/) Lis-HCl buffer (pH 7.5) and placed in a six-hour timer dock.
B" (manufactured by Amikosi Far East Ltd.) to adsorb Vero + Cytase, wash with the same buffer, and add 30% thiethylene cyclolicol solution (pH 7).
, adjusted to 5), the eluate was dialyzed against pure water using a t0 fan tube, and then concentrated using a tO diosi membrane, and the concentrated solution was lyophilized to obtain a powder of Beruo bovine citase. Ta. The culture v/mo activity yield of this product was 48 cm, and the specific activity (unit: 10D28°) was 19.06.

Example 3 Liquid culture solution with a composition containing 41 parts of soluble starch, 3 parts of defatted soybean flour, and 3 parts of Kosi Stay Liquor (PH7.0)
Put 50wLl into 500m11 Mr. Sakaguchi Colben,
After sterilizing at 120°C for 30 minutes, Bacillus cereus IFO3132 (BαCilluC11lusc Fr
ankLand b FrankLand I F 0
3132) was inoculated and cultured at 30°C for 4 days, followed by centrifugation at + o o o o o rpm for 15 minutes to obtain a crude enzyme solution. The Vero+cytase activity of this crude enzyme solution was 0.094 units.

Examples 4 and 5 The same operations as in Example 3 above were repeated using the following microorganisms.

Example: Bacillus cereus IPOf3690 (B,
cgrt&&z FrankLancL&F
rankland zuhzp.

f ttortpcgnz Lα;bαC le IFO13
690) Real MNfl15 Bacillus cereus IFO
3836) (B, etrtuz Frathklan
ct & FratshLatsd zwbzp.

rxycoidmz Sm1th at al IFO
3836) The Vero+cytase activity of the obtained crude enzyme solution was 0.07 units in Example 4 and 0.105 units in Example 5.

Example 6 Bacillus methylium IFO3970CB.

ngtqattriwr'ILdt Bary I F
03970) to obtain a crude enzyme solution in the same manner as in Example 3. The Vero+cytase activity of the obtained crude enzyme solution was 0.015 units.

Example 7 Bacillus subtilis ry03936 (B.

zuhtilLiz (Ehrgnbgrg) Co
A crude enzyme solution was obtained in the same manner as in Example 3 using IFO3936).

The activity of this product was 0.021 units.

[Brief explanation of the drawing]

FIG. 1 is a diagram showing the optimum pH of the present invention Bell-saijucytase;
FIG. 2 is a diagram showing the optimum temperature of the present invention Bell's Cytase, and FIGS. 3 and 4 show the pH of the present invention's Bell's Cytase.
FIG. 3 is a diagram showing stability and thermal stability. (Above) Figure 1 Figure 12 Action 51JtG'' Figure 4 Figure 4 Kudamari Prostitute C゛Procedural Amendment (Toge) 1981 Type 6 i4 Day J') Director-General of the Agency 1. Display of 1 1981 Permanent Application No. 6'$692 Part 10-
Manufacturing method for Pesio Nakashi'-ze 3. Relationship with the case of the person making the amendment Patent applicant Ueda Kasanko Lebo Nishokusha (and 14 others) 4. Agent in Heiwa Building, 2-10 Hirano-cho, Higashi-ku, Osaka Electric fortune telling 06-. 2
03-0941 (Zt) 8, Contents of the Amendment As shown in the attached sheet, Spring (1) The statement in the specification is corrected as shown in the errata below. (that's all)

Claims (1)

[Claims] ■ A method for producing pero+sitase, which comprises culturing a microorganism belonging to the genus Bacillus and having the ability to produce pero+sitase in a nutrient medium, producing and accumulating pero+sitase in the culture, and collecting the same.