JPS6050435B2 - Type C cytochrome of highly thermophilic bacteria - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention provides highly thermophilic bacteria
This relates to the C-type cytochrome of HB-8.

Cytochrome is a heme protein whose unique function is to transfer reduction equivalents through redox of heme iron, and it mainly works in energy exchange fields such as respiration and photosynthesis, and also in the production of other substances necessary for living organisms. It is a substance in living organisms that is widely involved in life phenomena associated with the production of electrons and electron transfer. As for the pharmacological action of cytochrome C, it has been reported that it is effective in treating heart disease and gas intoxication, and it is also commercially available as enzyme preparations such as cell metabolism activators and fibrous respiration activators.

In comparative biochemical research, the present inventors discovered highly thermophilic bacteria (
Therm1J Sthermophilus) HB-8
We focused on the C-type cytochrome, attempted to isolate it, and arrived at the present invention.

That is, the gist of the present invention is that highly thermophilic bacteria (Tlle
rmusthermophilus) HB-8, and the cytochrome dan-5
54 has the following physical and chemical properties.

(a) Absorption maximum of oxidized form: 410 nm (b) Absorption maximum of reduced form: 554, 522 and 4l7n
7-rl, (the α absorption band is asymmetric and a clear shoulder can be seen at 549n771).

Ar/Aα■8.8)←→ Millimolar molecular extinction coefficient by pyridine hemochrome method: 19.0 Isoelectric point: 4.9 (e) Molecular weight by gel filtration method: Approximately 30,000N stable p
H1-10.5 (g) Stable temperature: about 80°C To describe the method for obtaining cytochrome C-554 of the present invention, first, highly thermophilic bacteria
us) HB-8 (ATCC 27634) was cultured at around 75°C for about 8 hours using a medium consisting of 5 fl polypeptone, 4 Da yeast extract, 1 Da glucose, 2 g sodium chloride, and 1000 ml water, and then isolated. After purifying and crushing the resulting bacterial cells with alumina, soluble components were extracted with Tris-HCl buffer containing EDTA) magnesium chloride, potassium chloride, and β-mercaptoethanol, and the extract was purified with methacrylic acid (Rohm & Haas). After treatment with Amberlite CG-50 (type ■) (trademark), a weakly acidic cation exchange resin, and fractionating the resulting Oki liquid with ammonium sulfate, it was treated with a molecular sieve of dextran gel Molecular Sieve Sephadex G manufactured by Pharmacia.
lOO (trademark), manufactured by Jujo Paper Co., Ltd., anion exchange resin DE
AE-Cellulose (trademark) and Fatman anion exchange resin DE-32 (trademark) are used for chromatography.

As shown in the examples below, cytochrome (-55
4 comes out without being adsorbed to the DE-32 column. Then, it is purified to uniformity by isoelectric focusing. Example (1) Cultivation of highly thermophilic bacteria HB-8 (ATCC 27634) containing 1 g of polypeptone 5y1 yeast 4y1 glucose.
and cultured using a medium containing 2y of sodium chloride.

That is, first, the above bacteria were inoculated into two 20 ml test tubes containing about 3 ml of the above medium, and the tubes were left in an incubator (75 tube C) for two days and nights. Next, they were planted in eight 5e Sakalokolben plants containing the above-mentioned medium 1e-, and cultured with shaking at 75°C overnight. Furthermore, this was planted in four 20' jar fermenters, aerated culture was carried out at 75°C for about 8 hours, and bacteria were collected at the late stage of logarithmic growth (Cretz number about 400) (using a Sharpless type continuous centrifuge). I washed it lightly with about 2 hours of physiological saline. The bacterial cells were stored frozen at -20°C. The bacterial cell yield was approximately 700 y in wet weight per 80 f of the culture medium. (2) Grinding of bacterial cells 500q (wet weight) of bacterial cells stored frozen at -20°C
The cells were left at room temperature for about 3 minutes, and before the cells had melted, they were cut into dice with a knife (from this point onwards, the cells were kept at 4°C).

This was placed in a large mortar and ground with aluminum oxide for approximately 500 m, and the bacterial cells were mechanically ground using a pestle. After about 3 millimeters, the above oxidation a. An appropriate amount of aluminum (about 125y) was added, and the grinding was continued for about 1 hour. Once the bacterial cells were absorbed and the mixture became thick, we moved on to the next extraction procedure. (3) Extraction of soluble components 1mM EDTA1107TLM magnesium chloride, 13
50mM Tris-HCl buffer (PH7.8) containing 5mM potassium chloride and 7mM β-mercaptoethanol was added to
．． The mixture was added to a 3F mortar and continued stirring using a single pestle for about 30 minutes.

The thick suspension was centrifuged at 10500Xy for 4 cycles and the supernatant was further centrifuged at 22000Xg for u hours.

A supernatant of approximately 1.2' was obtained. 1) Purification The above supernatant was dialyzed overnight against 10 mM potassium phosphate buffer (PH7.O) containing 17TLMEDTA17TrLMβ-mercaptoethanol.

Add cation exchange resin Amperlite CG-50 (type ■) to the dialysate and wash it with alkali and acid, approx. 30077
! After draining the water and adding the mixture, the mixture was stirred appropriately and filtered with suction using a Buchner. Add powdered ammonium sulfate to the solution 100ml
It was added at a rate of 16.4 y per portion while stirring with a stirrer. After standing overnight, centrifugation was performed for three times at 10,500Xf1 to obtain a supernatant. Add powdered ammonium sulfate to this supernatant again.
It was added in the same manner as before at a rate of 11.7y/ml and centrifuged to obtain a precipitate. This precipitate was lightly dialyzed against 10TrLM Tris-HCl buffer (PH7.8), the dialysate was centrifuged, and the supernatant was transferred to a Sephadex G-100 column (φ6.0 x 120c7n) equilibrated with the above buffer.
) gel? I passed away. The amount of processing at one time is approximately 125m
It was t. Collect the reddish fractions at the end of the chromatography, concentrate with ammonium sulfate, and add 10T
rL. Dialysis was performed with M Tris-HCl buffer (PH7.8), and after centrifugation, the supernatant was transferred to a DEAE-cellulose column (φ3.6 x 3
5 cm) and a linear concentration gradient of potassium chloride (0 to 0.5M1
Perform chromatography of each 2') and perform potassium chloride 0
．． Fractions around 0.02 to 0.06 M were collected, concentrated with ammonium sulfate, and concentrated at 10 m. When dialysis was performed with M potassium phosphate buffer (PH7.O) and chromatography was performed on a DE-32 column (φ2.5 x 40Cr11) equilibrated with the above buffer, a fraction containing cytochrome C-554 was detected. The amount came out without being absorbed.

This was chromatographed again in the same manner using a DE-32 column (φ1.6 x 30 cm), and cytochrome C-
When fractions of 554 were collected and subjected to isoelectric focusing at pH 4-6, homogeneous cytochrome q-55 was observed at pH 4.9f4'.
An S product was obtained. 0.3mg from the wet weight of bacterial cells 500y
of cytochrome (-55g amount.1) Cytochrome C
- Measurement of the properties of 554 (a) Absorption band of oxidized form Potassium ferricyanide was added to the sample solution to a concentration of approximately 10 μM, and 107n,M tris-hydrochloric acid buffer (PH7
．． 8) and the spectrum was measured.

It had an absorption maximum at 410r1m.

(b) Absorption band of reduced form A trace amount of hydrosulfite powder was added to the sample in (a) above, and the spectrum was measured.

554, 522 and 417nrrL. An absorption maximum is seen at , and the α absorption band is asymmetric at 549r177! ．． The shoulders were clearly visible.

Also, A4l7nTrL (=Ar) and A554n7Tl
．． The ratio (Ar/Aα) of (=Aα) was 8.8. (
c) Millimolar molecular extinction coefficient J. BlOl. Chem. When the millimolar molecular extinction coefficient was measured by the pyridine hemochrome method according to the method described in Kaze 605 (1942), a value of 19.0 was obtained.

(d) Isoelectric point ActaChem. Scand. ,? , 820 (196
When the isoelectric point was measured at pH 4 to 6 according to the method described in 6), it was 4.9.

(e) Molecular weight BlOchem. J. The molecular weight was determined to be about 30,000 by gel filtration method according to the method described in , 2, 222 (1964).

(f) Stable pH: 1-10.5

Claims (1)

[Claims] 1. Highly thermophilic bacteria (Thermusthermoph
illus) Cytochrome ¥C¥-554 obtained from HB-8 and having the following physical and chemical properties (a) to (f). (a) Absorption maximum of oxidized form: 410nm (b) Absorption maximum of reduced form: 554, 522 and 417n
m (c) Millimolar molecular extinction coefficient by pyridine hemochrome method: 19.0 (d) Isoelectric point: 4.9 (e) Molecular weight by gel filtration method: about 30,000 (f) Stable pH: 1 to 10.5