JPH0322980A - Production of tryptophanase - Google Patents

Abstract

PURPOSE:To obtain the title enzyme economically in high yield by culturing Escherichia coli having tryptophanase promoter and tryptophanase structural gene using acetic acid or an acetate salt as a carbon source. CONSTITUTION:A microorganism in Escherichia coli having tryptophanase promoter and tryptophan structural gene which can be expressed by the promoter is cultured using only acetic acid or an acetate salt as a carbon source. The culture is conducted aerobically under agitation at 25 to 40 deg.C medium temperature at 5 to 9pH. The microorganism is preferably Escherichia coli ATCC 27325 or the like.

Description

DETAILED DESCRIPTION OF THE INVENTION (Field of Industrial Application) The present invention relates to a method for producing tryptophanase. Tryptophanase is a useful enzyme that is suitably used in the production of L-1 ribotophan, which has recently attracted attention as a feed additive.

(Conventional technology) Conventionally, Escherichia coli (Escherichia coli)
It is known that the production of tryptophanase within the cells of microorganisms belonging to h i a co l i ) is inhibited by the addition of glucose.

For this reason, Escherichia coli (Escherichia coli), which has a tryptophanase promoter and the tryptophanase structural gene expressed by the promoter,
co! When culturing microorganisms belonging to category i) to produce tributophanases within the microbial cells, glucose, which is inexpensive and widely used industrially, cannot be used as the main carbon source for the culture, and succinic acid and bilbinase cannot be used. Contains M such as acids
1r! Culture II using a polyhydric alcohol such as Ili or glycerol as a carbon source [Demoss, R. D. ；Jou
RNA of Bacteriology, Vol.
t05 33 (1971)]. However, when culturing is performed using these organic acids or polyhydric alcohols as carbon sources, the cost of raw materials becomes expensive, and it is difficult to produce and utilize tryptophanase industrially and economically. In addition, ethanol, which is used as an inexpensive carbon source, is hardly affected by microorganisms belonging to Escherichia and cannot be used for culturing these microorganisms. As described above, it has not been possible to culture microorganisms belonging to Escherichia using carbon sources commonly used industrially and to produce tryptophanase economically and in high yields within the cells of the microorganisms.

A method for producing tryptophanase, which comprises culturing a microorganism belonging to Escherichia coli to produce tryptophanase within the microorganism, using substantially only acetic acid or its salt as a carbon source. This is what we provide.

According to the present invention, tryptophanase can be produced economically and in high yield.

(Problems to be Solved by the Invention) As a result of intensive study on the type of carbon source that does not adversely affect the intracellular production of tryptophanase, the present inventors found that acetic acid or its salt was used as the carbon source for cultivation. The present inventors have discovered that microbial cells containing tributophanars at high concentrations can be obtained by doing so, and have completed the present invention.

(Structure and Effect of the Invention) That is, the present invention provides a tryptophanase promoter and a tryptophanase structural gene expressed by the promoter. Explain.

Escherichia coli used in the present invention
The microorganism belonging to Chia coli is not particularly limited as long as it has a tryptophanase promoter and a tryptophanase structural gene expressed by the promoter, but for example, Escherichia coli )A
TCC 27325. IFO 3301, YK
3002 [Feikoken Jyoyori No. 1733 (Feikoken Bichiyori
Transferred from No. 8844), same YK 3003 [I! l
Koken Jyoyori No. 1734 (transferred from FEIKEN JOYORI No. 8845), YK 3007 (FEIKEN BIYOYO No. 10543), etc. are preferably used. Escherichia coli mentioned above
ia coli) can be cultured in a medium to which substantially only acetic acid or its salt is added as a carbon source, and further contains nutrients such as a nitrogen source, inorganic salts, and growth-promoting substances.

Nitrogen sources to be included in the medium include, for example, ammonium salts such as ammonium sulfate, ammonium chloride, ammonium nitrate, and ammonium phosphate; nitrates such as potassium nitrate, sodium nitrate, and ammonium nitrate; organic nitrogen such as glutamic acid, glutamine, aspartic acid, and asparagine;
Ammonia etc. can be used alone or in combination, and examples of inorganic salts include potassium monohydrogen phosphate, potassium dihydrogen phosphate, magnesium sulfate, iron sulfate,
Manganese sulfate etc. can be used alone or in combination. As growth promoting substances for the microorganisms, vitamins such as thiamine and biotin; amino acids such as methionine and cysteine; or yeast extracts, polypeptones, meat extracts, corn staple liquor, casamino acids, etc. containing all or part of these are used. ．． Furthermore, it is preferable for the production of tryptophanase to add L- or DL-tributophane to the medium.

The amount of L- or DL-}lyptophan added to the medium is:
Although not particularly limited, o. oi~
2% by weight, more preferably 0.05-0.5% by weight.

The medium temperature is in the range of 10 to 45°C, preferably 25 to 40°C, and the pH of the medium is in the range of 3 to 10, preferably 5.
It is in the range of ~9. If the pH changes during culture, it is desirable to maintain it constant within the above range using ammonia, caustic soda, caustic potassium, etc. Culture time is usually 5 to 48 hours. Furthermore, it is desirable that the culture be carried out under aerobic conditions, with aeration and agitation so that dissolved oxygen during the culture does not become a rate-limiting factor.

The amount of acetic acid or its salt added to the culture solution is not particularly limited, but is usually 0.05 to 20% by weight, preferably 0.1 to 5% by weight. In addition, as the salt of acetic acid, sodium acetate, potassium acetate, rlFm ammonium, etc. are preferably used. Hereinafter, the present invention will be briefly explained with reference to Examples.

In the Examples below, tryptophanase activity was measured by the following method.

Centrifuge 1i (4000 m
RPM, 15 minutes, 4°C) and 100 mmoQ
Phosphate buffer (pH 8.0) 20m2! l! ! After turbidity, centrifuge again (4000 RPM, 15 minutes, 4°C) to collect bacterial cells. After suspending the collected bacteria in the above-mentioned buffer solution 2 tn jl, ultrasonic disruption treatment (Sonifier 200 manufactured by Branson) was performed, and the treated material was centrifuged (1 20
00 RPM, 40 minutes, 4°C) was used as the test enzyme solution.

For the reaction, add the above enzyme solution to 100 mmos! After diluting an appropriate amount with phosphate buffer (pH 8.0), the dilution}1
Add 0.1 m2 of reaction solution [L - tributophane lO mm
o2, pyridoxal phosphorus MO. 100 mmol 2 phosphate buffer containing 0.4 mmol (pH 8.04 mmol).
0) 1 to a test tube containing 0.9 m2, and a shaking reaction is performed in a constant temperature water bath at 37°C for 20 minutes. The reaction was stopped by adding 1 m2 of 10% trichloroacetic acid, and then centrifuged.
The amount of indole produced in the supernatant obtained at 4000 RPM, 15 minutes, 4° C. was determined by gas chromatography (Shimadzu, GC-38F). In addition, the amount of protein in the test enzyme solution can be measured using Low
The method of ry et al. (Journal of Biolo
logical Che+sistry, vol 193
, 11. 265, 195t). Tryptophanase activity was expressed as the amount of indole produced per hour per unit amount of protein. Implementation r141 Medium 100 mS! shown in Table 1. 500
Escherichia coli, a tryptophanase-producing bacterium, was dispensed into a 2-volume Erlenmeyer flask and sterilized at 120°C for 15 minutes.
i) Inoculate with ATCC 27325, 37
After shaking culture for 1 day at °C, 20 ml of the is
was similarly inoculated into 1000 m2 of a culture medium prepared by straining, and heated at 37°C at 6 rotations using an aerated stirring culture tank.
Culture was carried out for 8 hours at 0 O RPM, aeration volume of 1 V, and pH 7.2 (adjusted with 28% ammonia water).

Table 1 Sodium acetate 10gL,
- Tributophan 0.5gKH2P
O4 1.6g1'<28
PO4 5.5g (N }
h) 2 S 04
3. O g M g S O 4
・7H20 0.1gF e SO
a ・7 H20 8 0m gforcezaS)rll (D IFCO) 5.
Add 0g distilled water 1 0 0 0
m St (pH. 7.2) After completion of culture, t1! Perform centrifugation from 20 m2 of nutrient solution,
Tryptophanase activity was measured by the method described above.

As a comparative example, culture was performed using a medium in which sodium acetate was replaced with 10 g of glucose in the medium shown in Table 1.

The results are shown in Table 2. Table 2 Example 2 Escherichia coli (Escherichia coli) as a bacterial strain
The same operation as in Example 1 was performed except that A coli) YK 3007 (*Koken Bacteria No. 1 0543) was used. The results are shown in Table 3.

Table 3

Claims (1)

[Claims] (1) Escherichia coli possessing a tryptophanase promoter and a tryptophanase structural gene expressed by the promoter.
A method for producing tryptophanase, which comprises culturing a microorganism belonging to li) to produce tryptophanase in its cells, using substantially only acetic acid or its salt as a carbon source.