JPS61268175A - Cultivation of tryptophan synthetase-producing microbial strain - Google Patents

Abstract

PURPOSE:To culture a tryptophan synthetase-producing strain having high activity, in high efficiency and yield, by culturing a mutant of Escherichia coli in a specific medium. CONSTITUTION:A mutant of Escherichia coil capable of producing tryptophan synthetase is inoculated in a medium containing glucose as a carbon source at a concentration of <=10%, ammonium sulfate as a nitrogen source at a concentration of <=5% and organic acids, etc., and cultured aerobically at 4-9pH and 20-50 deg.C to obtain tryptophan synthetase. L-tryptophan can be produced by reacting indole with L-seselin in the presence of the above tryptophan synthetase as a catalyst.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to tryptophan synthetase (E.) belonging to the genus Escherichia.

C, 4, 2, 1, 20: L from indole and L-serine
-Enzyme that catalyzes tryptophan synthesis) 0 [Conventional technology] In recent years, L-)+3butophane has been used as a feed additive in addition to pharmaceuticals such as infusion components and tranquilizers and raw materials for pharmaceuticals. However, inexpensive production on an industrial scale is being considered. Conventionally, L-1 ribotophan has been produced mainly by chemical synthesis and fermentation methods. Chemical synthesis requires harsh conditions such as high temperature and high pressure, has a long reaction route, and the product is a racemic mixture of D and L forms, so it is difficult to obtain the required L form. In the 0-fermentation method, which required optical resolution to be carried out, the tryptophan produced by 0-con is a method of producing L-tributophane from sugars using the metabolism of microorganisms.
However, it is difficult to separate the desired product from a mixture of various substances and metabolites necessary for the growth of microorganisms, and since live microorganisms are used, it is not necessary to perform optical resolution. In addition, countermeasures against bacterial contamination are also necessary.

In contrast, in enzymatic synthesis, L-tryptophan is obtained from indole and L-serine in one step at room temperature and pressure using an enzyme involved in tryptophan metabolism as a catalyst, which requires less energy and cost during production. The method provides significant advantages.

The enzyme that catalyzes this reaction is tryptophan synthetase, which is produced by Escherichia.
The enzyme is produced by microorganisms belonging to the genus Ichia), but microorganisms that have been genetically improved from these microorganisms produce the enzyme more efficiently. The present enzyme can be used in a purified and isolated state, or in the form of cultured bacterial cells, cell-free extracts, immobilized products, etc. containing the enzyme.

In order to use this enzyme on an industrial scale, it is necessary to efficiently culture tryptophan synthetase-producing bacteria with high activity in an inexpensive medium with high yield. The properties of this enzyme are as described by E-Miles, Adr-
Enzymol, l 42+ 127 + (1979
)) etc. have been reported.

However, although natural or synthetic media have been used to culture this tryptophan synthetase-producing bacterium, there have been few reports on culture conditions, especially on culture medium composition, and it has not been determined empirically. In Japanese Patent Application Laid-open No. 58-20186, glucose is added continuously or intermittently to keep the glucose concentration in the medium below 1%. A method for culturing tryptophan synthetase-producing bacteria is disclosed.

[Problem that the invention seeks to solve]

In the method disclosed in JP-A-58-20186, it is necessary to quickly analyze the glucose concentration in order to keep the glucose concentration below 1 g.
There is no method to directly quantify glucose alone from medium components, and dissolved oxygen concentration CD, which has a correlation with glucose concentration.
Glucose concentration is regulated by changes in O value) and I)H. In this case, since the Do value will show that the glucose concentration has become zero, it should be set to 14 or less.0 For more detailed analysis, a glucose analysis device is attached to the culture device. Therefore, compared to the case where the sugar concentration is initially set to a predetermined amount, the equipment required for culturing on an industrial scale becomes more complicated. Furthermore, this publication does not discuss nitrogen sources. Therefore, the purpose of the present invention is to increase the concentration of glucose as a carbon source in the medium by 10% in the culture of an Escherichia coli mutant strain that produces tryptophan synthetase Below,
By setting the concentration of ammonium sulfate as a nitrogen source to 5% or less, there is no need to measure or adjust the glucose concentration during the culture, which simplifies the culture operation.
Moreover, it is an object of the present invention to provide a method for culturing tryptophan synthetase-producing bacteria that has high enzyme activity and good bacterial cell yield using an inexpensive medium.

[Means for solving problems]

In order to solve the above problems, the present invention aims at producing tryptophan synthetase, an enzyme used as a catalyst when producing L-tryptophan by reacting indole with L-serine.
hia-coli) mutant strain, the concentration of glucose as a carbon source in the medium is set to 10 parts or less, and the concentration of ammonium sulfate as a nitrogen source is set to 5 parts or less.

[Effect]

According to the present inventor's findings, as a result of examining the medium components in culturing an Escherichia coli mutant strain that produces tryptophan synthetase, it was found that the glucose concentration was reduced to 1.
0% or less, preferably 0.5 to 5%, and the concentration of ammonium sulfate as a nitrogen source to 5% or less, preferably 1.0 to 5%.
It has been found that by setting the concentration of f'L, L or less, a good yield of bacterial cells with high enzyme activity can be obtained, and the medium can be inexpensive. In addition, since it is only necessary to charge glucose and ammonium sulfate at once, and there is no need to measure the glucose concentration during culture, the culture equipment and operation are simple. [Specific Examples of the Invention] The present invention will be explained in more detail below. First, Referring to Figure 1, we will outline the steps from culturing tryptophan synthetase-producing bacteria to synthesis, isolation, and purification of L-1 liptophan. Source, N source.

Enzyme-producing bacteria are inoculated into a medium that has been adjusted (minerals, etc.), and sterile aerated culture is performed while controlling temperature and pH.

(2) Collect bacteria from the NFC culture solution obtained in step (1) by centrifugation (for example, 110 x 103 rp for 20 minutes), use this as an enzyme source for tryptophan synthetase, and in some cases immobilize it. (3) Synthesis reaction Next, using the enzyme obtained in (2) as a catalyst, indole and L
-React serine to synthesize L-)ributophane (4) Separate and purify the f'' L-tryptophan obtained in (3).

Next, the method for culturing the synthetase-producing bacteria according to the present invention will be described in detail.
This is an f'Lfc mutant strain derived from E.coli W3110, and when culturing the producing bacteria, the carbon sources used in the medium are sugars such as glucose, polyhydric alcohols such as glycerin, organic acids such as citric acid, etc. Substances that can be digested by microorganisms can be used, and the appropriate concentration varies depending on the added substance; in the case of glucose, the added concentration is 10% or less;
Preferably 0.5 to 5 is desirable. Here, the bacterial growth condition at glucose concentration 1 to 2 in the medium is used as a standard.
Up to 5%, an increase in activity and bacterial cell yield was observed as the concentration increased, but up to 10%, the concentration did not increase significantly compared to 5%.

Furthermore, if the concentration exceeds 10, the growth becomes very poor.Nitrogen sources include various organic and inorganic ammonium salts such as ammonia, ammonium chloride, ammonium sulfate, ammonium citrate, and ammonium phosphate, or meat extract, yeast extract, Natural organic nitrogen compounds such as casein hydrolyzate and corn f-7' liquor are used.
Many natural organic nitrogen compounds can be used not only as a nitrogen source but also as a carbon source at the same time. In this case as well, the appropriate concentration varies depending on the additive substance, and in the case of ammonium sulfate, it is 5% or less, preferably 1.0 to 5%.
A ratio of 0 is desirable.0 Growth is very poor in a medium with an ammonium sulfate concentration of more than 5.Furthermore, in the cultivation of tryptophan synthetase producing bacteria in the present invention, magnesium phosphate is added in order to culture more efficiently.

Potassium phosphate and inorganic substances such as metal ions such as iron, zinc, and manganese, as well as vitamins, amino acids, yeast extract containing these, casamino acids, polypeptone, etc. may be added. When culturing this enzyme-producing bacterium, it is necessary to add a sufficient amount to the medium so that it does not become a limiting substrate.

The culture temperature is 20-50°C, preferably 30-40°C. The pH of the medium is 4-9, preferably 7-8°C,
It is desirable to keep the concentration constant during culture using sodium hydroxide, potassium hydroxide, etc. The culture is preferably carried out aerobically, with aeration and stirring so that the dissolved oxygen concentration in the medium does not become a rate-limiting factor for the growth of the enzyme-producing bacteria during the culture period.

Furthermore, it is necessary to add L-tryptophan or indole to the culture of this enzyme-producing microorganism. The n-enzyme thus obtained serves as a catalyst for the synthesis of L-1 liptophan from indole and L-serine. In this case, it can be used in a purified and isolated state, or in the form of cultured bacterial cells containing enzymes, cell-free extracts, immobilized products, etc. Friend, if this enzyme is used as an enzyme in the production of L-1 liptophan by the enzymatic method described in Japanese Patent Application No. 59-264228 previously filed by the present applicant, L-1 liptophan can be synthesized efficiently.

〔Example〕

Next, the present invention will be further explained in detail in Examples, and its effects will be shown together with Comparative Examples.

(Example 1) Escherichia coli (Escherichia colt) tr which is a tryptophan synthetase producing bacterium
pR-W3110t" Seed culture for 20 minutes in the medium shown in Table 1.
medium 1 with the composition shown in Table 2.
In addition to 00d, main culture was performed at 30'C for 24 hours.

(pH 7,0) Table 2 (pH 8,0) After culturing, the bacterial cells were centrifuged (10xl O3 rpm 20 minutes), suspended in physiological saline, and the tryptophan synthetase activity was measured.The 0 bacterial cell concentration was 610 nm. It was determined from the absorbance at . Tryptophan synthetase activity was measured at 37°C using a reaction solution with the reaction composition shown in Table 3.
The reaction was carried out for 20 minutes and expressed as the amount of dry bacterial cells and the amount of decrease in indole per unit time. The results are shown in Table 4.

Table 3 (Example 2) Table 5 shows the results obtained by performing the same operation as in Example 1 with the ammonium sulfate concentration in Table 2 set to 1.0.

Table 5 (Example 3) The same operation as in Example 1 was performed except that the ammonium sulfate concentration in Table 2 was set to 5.0.

The results are shown in Table 6.

Table 6 Glucose concentration Dry bacterial mass Bacterial activity (indole reduction) (@ (mg) nmol Oki 9
m, 9dr7cellO, 554, 48-4 1.0 50.0
932.0 51.6
965.0 4'7-1
101 (Comparative Example) In Example 1, when the main culture was carried out in the same manner as shown in Table 1 (however, pH 8.0), the amount of bacterial cells was 73.4.
The enzyme activity in m& is 58 nmol/mm ・4-dry
It's easy to use cell.

〔Effect of the invention〕

As described above, according to the present invention, the culture operation is simple, and the tryptophan synthetase-producing bacteria exhibits enzyme activity 2 to 3 times higher than that of the conventional method using inexpensive medium components, and has good cell yield. can be cultured.

[Brief explanation of the drawing]

Figure 1 is a process diagram from culturing tryptophan synthetase-producing bacteria to synthesis, separation, and purification of L-toIJ butophane.0 Figure 1

Claims (1)

[Claims] (1) In the culture of an Escherichia coli mutant strain that produces tryptophan synthetase, an enzyme used as a catalyst when producing L-tryptophan by reacting indole and L-serine, carbon sources in the medium The glucose concentration as 10
% or less, and a method for culturing tryptophan synthetase-producing bacteria, characterized in that the concentration of ammonium sulfate as a nitrogen source is 5% or less.