JPS61170386A - Production of xanthine dehydrogenase by fermentation - Google Patents

Abstract

PURPOSE:To produce the titled substance capable of oxidizing hypoxanthine or xanthine to uric acid, by fermentation process, by culturing a particular microorganism belonging to Nocardioides genus or Nocardia genus in a nutrient medium. CONSTITUTION:A microbial strain belonging to Nocardioides genus (preferably Nocardioides albus ATCC 27980) or Nocardia genus (preferably Nocardia uniformis subsp. tsuyamanensis ATCC 21806) is cultured in a nutrient medium preferably at 25-30 deg.C and 6.0-8.0pH for 1-3 days under shaking to obtain the objective substance.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of the Invention The present invention relates to a method for producing xanthine dehydrogenase by fermentation.

Conventional technology Xanthine dehydrogenase is an enzyme (E) that catalyzes the oxidation of hypoxanthine or xanthine to uric acid.
When oxidizing each substance with C1, 2, 1, 37), NA
Reduces D and produces NADH. This NADH, N
Absorption by ADH, diaphorase and INT (2-p-
Hypoxanthine or xanthine in a sample can be quantified by measuring nitrophenyl-3-p-indophenyl-5-phenyltetrazolium chloride) as a formazan dye. It can also be applied to the measurement of guanase activity, which is an indicator of liver disease in the field of clinical chemistry. Typical xanthine dehydrogenases that use NAD as an electron acceptor are enzymes derived from bird and rat liver, but those derived from microorganisms include those of the genus Streptomyces (^grc, Rial, Chew, 41
．． 1161.1977), Micrococcus (J.
Biol, Chell, 242, 4108.19
67), Neuoxpora (J, Biol, Chew,
253.2604°1978), Pseudomonas (Biochim, Biophys.

^cta, 410.12.1975).

Problems to be Solved by the Invention It has recently been discovered that xanthine dehydrogenase is produced by microorganisms belonging to the genus Nocardioides or Nocardia.

Means for Solving the Problems The present invention involves culturing a microorganism belonging to the genus Nocardioides or the genus Nocardia and having the ability to produce xanthine dehydrogenase in a nutrient medium, producing and accumulating xanthine dehydrogenase in the culture, and The present invention relates to a method for producing xanthine dehydrogase, which comprises collecting xanthine dehydrogase.

The present invention will be explained in detail below.

The microorganism used in the present invention may be any strain that belongs to the genus Nocardioides or Nocardia and has the ability to produce xanthine dehydrogenase. An example of a suitable strain is Nocardioides albus^TCC2.
? 980, Nocardia uniformis subspicis tuyamanensis (14 (ICardiaun
iformis 5ubsp, tsu amane
nsis ) ATCC21806, Nocardia coeliaca IF
An example is M30.

As the medium used in the present invention, either a synthetic medium or a natural medium can be used as long as it contains an appropriate amount of carbon sources, nitrogen sources, inorganic substances, and other nutrients. As carbon sources, carbohydrates such as glucose, sicucrose, and blackstrap molasses, and nucleic acids such as xanthine, hypoxanthine, adenine, and inosine are used.

Ammonium chloride, ammonium sulfate,
Various inorganic and organic ammonium compounds such as ammonium phosphate, ammonium carbonate, and ammonium acetate, nucleic acid-related substances such as xanthine, hypoxanthine, adenine, and inosine, nitrogen compounds such as urea, peptone, enzyme extract, casein hydrolyzate, and meat. Nitrogen-containing natural products such as extracts, corn stave liquor, etc. can be used. As the inorganic substance, salts of metals such as sodium, potassium, manganese, magnesium, and calcium and inorganic acids such as phosphoric acid, sulfuric acid, nitric acid, and hydrochloric acid can be used.

The culture temperature is 20 to 35°C, preferably 25 to 30°C. The pH at the start of culture is usually 6.0 to 8.5,
It is preferably in the range of 6.5 to 8.0.

When cultured with shaking for 1 to 3 days under such conditions, xanthine dehydrogenase is produced and accumulated in the bacterial cells as a cell in the culture.

After completion of the culture, the enzyme can be collected from the culture using a conventional enzyme collection method. Usually, bacterial cells are obtained from the culture by centrifugation or the like, the bacterial cells are disrupted by an appropriate means, and a supernatant is obtained from the crushed liquid by centrifugation or the like. The supernatant liquid can be purified by methods commonly used for enzyme purification, such as salting out, dialysis, ion exchange chromatography using DEAE-cellulose, QAE-Sephadex, etc., gel filtration using Sephadex G-200, Sepharose 6BI, and A highly purified preparation of the present enzyme can be obtained by a combination of adsorption and elution methods using hydroxy abatide and the like.

The activity of xanthine dehydrogenase in the present invention is measured by the following method.

[Measurement method]

0.2M phosphate buffer (1) H7,5) 1. Oa+1
．． 10mM NAD” 0.2+11.5mM xanthine aqueous solution Q, la+1 with diaphorase 3.3u% IN
Add 0.4211 g of T and 0.2 ml of xanthine dehydrogenase solution to bring the total volume to 3. Qsl, the reaction was carried out at 37° C. for 5 minutes, and the enzyme activity was determined by measuring the absorbance of formazan at 490 nm.

One enzyme unit was defined as the amount of enzyme that oxidized 1μ1IIO1 of xanthine in 1 minute at 37°C.

The properties of xanthine dehydrogenase obtained by the present invention are shown below.

(1) Optimal pH and stable pH range The optimal pH of this enzyme is 8 to 8.5, and it is stable up to pH 5 to 11 when treated at 37°C for 30 minutes (Figure 1.2).

0) Optimal temperature and thermostability The optimal temperature of this enzyme is around 40°C, and it shows 90% residual activity even when treated with 0.1M phosphate buffer (pH 7.5) at 50°C for 30 minutes ( Figure 3.4).

(3) Substrate specificity The activity of this enzyme was measured using 0.11 of a 5mM substrate solution in the measurement method described above, and the activity of each substrate was expressed as relative activity, with the value when xanthine being used as a substrate being set as 100. The results are shown in Table 1.

(Table 2 shows the results of measuring the activity of this enzyme after adding the inhibitor at the concentration shown in Table 2.

Table 1: Xanthine 100 Hypoxanthine 67.6a)
Shin
6.8 Pudding
2.4 Xanthosine 2.1 Adenine
0.7 adenosine 0 guanine
0 Guanosine 0 Uric acid 0 8-Azaxanthin 0 8-Azaguanine 0 Table 2 Compound Added concentration (iM) Relative activity (%) A
gNOs 0.1 0 (3,5)0
CuS04' 5H*0 0.1 0 (
1, 2> ”ZnC1z 3' 92
．． 3p-chlormarcyl-0,10(0)”puenylsulnic acid NaN5 3
96.0EDT^ ・2Na
(5) Molecular weight The molecular weight of the enzyme was 540,000 as measured by gel permeation method using Sephadex G-200.

(6) Isoelectric Point The isoelectric point of this enzyme was found to be 4.1 by focusing electrophoresis using carrier amphorae F at pH 3.5 to 10.

(η Klm Vmax When the ω value and Vmax of this enzyme for xanthine were measured, they were 24 μM and 0.042 u/ml, respectively.

Examples of the present invention are shown below.

Example 1 Three strains shown in Table 3 were mixed with xanthine 0.5g/d1, yeast extract 0.5g/a, and peptone 0.5g#! .

28 in a medium (pH 7.0) containing 0.5 g/a of meat extract.
After culturing at ℃ for 2 days, the culture is centrifuged to obtain bacterial cells. The bacterial cells were dissolved in 0.02 M phosphoric acid all solution (p
After suspending in H7,5), the cells were crushed with glass beads and centrifuged to obtain a cell-free extract. The enzyme titer of the cell-free extract is shown in Table 3 in units per ml of culture fluid.

Table 3 Nocardioides 0.006 Co. IPM 30 Example 2 Nocardioides albus ATCC27980 was mixed with 0.5 g/a of xanthine, 0.5 g/J of yeast extract, 0.5 g/d1 of peptone, and 0.0 g of meat extract. 5g/di! (pH 7,0) 0.31, and cultured with shaking at 28°C for 3 days.The culture was inoculated into the above medium 151, and cultured with aeration and stirring at 28°C for 2 days (number of stirring: 35 O
r, p, a, ). Bacterial cells were obtained from the obtained culture by centrifugation (8, θ00r, p, m, xlS minutes),
After suspending in 0.02 M phosphate buffer (pH 7.5>) containing 0.1 Kasumi EDTA and 0.5 mM mercaptoethanol, it was crushed with a Dynomill and centrifuged (8.0 GOr,
p, ta, x 20 min) to add crude enzyme solution (0
, 8u/ml). Ammonium sulfate was added to the crude enzyme solution to saturation of 80% (W/V), and after leaving it at 4°C overnight,
Add filter aid and collect the precipitate fraction. After sufficiently dialyzing the precipitate with the above buffer solution, the dialyzed solution was centrifuged (10,00 Or,
p, m, x 20 min) and obtain the supernatant. Ammonium sulfate is added to the supernatant, and the fraction that precipitates at 50 to 75% saturation is sufficiently dialyzed with the above buffer solution and then passed through a column of HPA-75 (Mitsubishi Kasei). After thorough washing with the above buffer, 0 to I
Elute with concentration gradient method using M NaCl.

Combine the obtained active categories and add 75% ammonium sulfate.
Add to saturation and precipitate the enzyme. Next, the resulting precipitate was centrifuged (12,00 Or, p, III,
x 20 minutes), dissolved in 5 ml of the above buffer solution, and equilibrated Cef7 Defuku X G-200 (Pharm.
aciaFine Chen+1cals Co.) column. Collect the active fraction that flowed out, and add it to ^n+1con
Concentrate with a YM-10 membrane and pass through a hydroxyapatite column. The active fraction of the enzyme that passed through was collected and lyophilized to obtain powdered purified enzyme preparation 17■.

Example 3 Nocardia uniformis subspice ATCC 21806 was cultured in the same manner as in Example 2, and 90 g of Nocardia uniformis subspice ATCC 21806 was obtained from the 151 medium.

The cells were crushed and purified in the same manner as in Example 2 to obtain 91 g of a powdered purified enzyme preparation.

Effects of the Invention According to the present invention, xanthine dehydrogenase is produced by a microorganism belonging to the genus Nocardioides or the genus Nocardia.

[Brief explanation of drawings]

Figures 1 to 4 respectively show the optimum pH, stable pH range, optimum temperature, and heat resistance of xanthine dehydrogenase according to the present invention. In Figures 1 to 4, o-. indicates the case of phosphate buffer, and XX indicates the case of bicine-sodium hydroxide buffer. iJ÷! Jll Day pH No. 21!1 Place 3 Garden 4 Day Temperature (°0 Procedural amendment March 68, 1985 1, Indication of the case 1985 Patent Application No. 11319 2, Name of the invention Xanthine dehydrogenase by fermentation method Manufacturing method 3, relationship with the case of the person making the amendment Patent applicant postal code 100 Address 6-1 Otemachi-chome, Chiyoda-ku, Tokyo Name
(102) Claims column and Detailed Description of the Invention column 5 of the Kyowa Hakko Kogyo Co., Ltd. specification, Contents of amendment (1) The claims are amended as shown in the attached sheet. Claims: A microorganism belonging to the genus Nocardioides or Nocardia and having the ability to produce xanthine dehydrogenase is cultured in a nutrient medium, and xanthine dehydrogenase is produced and accumulated in the culture, which is then collected. A method for producing xanthine dehydrogenase.

Claims (1)

[Claims] A xanthine dehydrogenase characterized by culturing a microorganism belonging to the genus Nocardioides or the genus Nocardia and having the ability to produce quinsatin dehydrogenase in a nutrient medium, producing and accumulating xanthine dehydrogenase in the culture, and collecting the same. manufacturing method.