JPH0158956B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing L-tryptophan, and more specifically, the present invention relates to a method for producing L-tryptophan, and more particularly, the present invention relates to a method for producing L-tryptophan, and more particularly, the present invention relates to a method for producing L-tryptophan, which contains a plasmid containing genetic information that regulates the production of tryptophan, and which is derived from a strain of Escherichia sp. lacking tryptophanase. The present invention relates to an improved method for producing L-tryptophan, which is characterized in that anthranilic acid and/or indole, as well as glycine and glucose are added when producing L-tryptophan by culturing the relevant microorganism in a nutrient medium. Conventionally, when producing L-tryptophan (hereinafter simply referred to as "tryptophan") using microorganisms, anthranilic acid and indole are added to a nutrient medium and cultured (e.g.,
12384), or culturing microorganisms in a medium containing L-serine (Japanese Patent Application Laid-Open No. 162771/1983),
etc.) or a method in which indole and L-serine are biochemically reacted in the presence of microbial cells (for example, Japanese Patent Publication No. 1836-1836, Japanese Unexamined Patent Publication No. 56-1982).
137894, etc.) are known. The present inventors have previously developed a plasmid containing genetic information that regulates the production of tryptophan,
In addition, we proposed a means for creating a microorganism belonging to the genus Escherichia that lacks tryptophanase, and a method for producing tryptophan using the microorganism (see JP-A-57-80398). According to this proposed method, by adding anthranilic acid to the culture medium and culturing, it has been possible to dramatically increase the amount of tryptophan accumulated in the culture. Therefore, when the concentration of indole becomes high, the growth of tryptophan-producing bacteria becomes poor, and furthermore, there are problems such as a long period of time required for culturing to increase the productivity of tryptophan. The accumulation of indole is due to the fact that the ability to supply the substrate L-serine is weak compared to the amount of tryptophan synthesizing enzymes. Therefore, it is thought that adding L-serine to the medium eliminates indole and increases the growth of tryptophan-producing bacteria and the productivity of tryptophan, but L-serine is expensive and is not practical.
Therefore, in order to solve this problem, we investigated the composition of the medium and found that adding glycine and glucose along with the addition of anthranilic acid to the medium increases the conversion rate of anthranilic acid to tryptophan in the fermentation, They discovered that the productivity of tryptophan was increased and completed the present invention. A feature of the present invention is that the yield of tryptophan can be increased by coexisting glycine and glucose instead of expensive L-serine. The microorganism that can be used in the present invention has a tryptophan biosynthesis system enhanced by containing a plasmid having genetic information that regulates tryptophan production, has a serine biosynthesis system, and has a tryptophanase production system. The genus does not matter as long as the deletion occurs, but bacteria belonging to the genus Escherichia, which has a well-defined tryptophan biosynthesis system, are suitable, and tryptophan in which the feedback inhibition of anthranilate synthase by tryptophan has been released. Escherichia coli that has a plasmid with the Juan operon and is deficient in tryptophan repressor and tryptophanase can be cited as suitable for the production of tryptophan, and more specifically, the present inventor Esierihia Collie W3110, which was first proposed by et al.
The trpAE1 trpR tnaA (pSC101-trp·I15) bacterium is deposited with the American Type Culture Collection on October 28, 1980 and has the accession number ATCC31743. In the method of the present invention, in order to produce tryptophan using the above-mentioned microorganism, the microorganism is cultured in a nutrient medium using a known method using a medium composition known per se. This can be carried out by adding indole and indole, and culturing with the addition of appropriate amounts of glycine and glucose, taking into account the amount of indole added or the amount of indole produced. Such cultivation can be carried out using a nutrient medium to which anthranilic acid or/and indole, glycine and glucose are added from the beginning of the culture, but a nutrient medium without these additions, such as a carbon source or nitrogen used in the cultivation of ordinary microorganisms, can be used. Culture the bacteria in a medium containing nutrients necessary for the growth of the bacteria, such as mineral salts, vitamins, etc., and wait for the growth of the bacteria.
It can also be carried out by a method of adding anthranilic acid or/and indole as well as glycine and glucose. When using this method, glucose used as the initial carbon source in the culture solution,
When the glucose content produced from starch or the like is low, it is essential to add glucose in addition to glycine. It is also possible to adopt a method in which, after collecting the proliferating bacterial cells, the culture is further continued in a medium to which anthranilic acid or/and indole, glycine, and glucose have been newly added and adjusted. in this case,
The ratio of anthranilic acid or indole or glycine or glucose to be added is not critical because the optimum amount changes based on the characteristics of the microorganism used, but it is usually about equimolar to 10 times the molar amount relative to anthranilic acid or indole. It is preferable to culture by adding glycine and about equimolar to 40 times the molar amount of glucose. In addition, the culture time is determined according to the above culture method.
Although it is different, when culturing is carried out by adding anthranilic acid etc. at the initial stage of culture, it takes about 24 hours to about 120 hours, and when culturing is carried out by adding indole etc. after the bacterial cells have multiplied, it is about 2 hours to about 2 hours after addition. It is best to culture for 24 hours. Further, the culture is preferably carried out under aerobic conditions such as shaking or aeration, and the culture temperature is usually 20° to 40°. PH is preferably 6 to 9. Thus, tryptophan accumulated in the culture solution can be easily isolated and purified by known methods, such as adsorption to activated carbon or ion exchange resin, and elution. The method of the present invention will be explained in detail below using examples. Example 1 Eacherichia coli W3110 trpAE1 trpR
tnaA (pSC101-trp·I15) was inoculated into L-broth (20 ml in a 100 ml flask) containing 10 g of tetracycline and cultured at 37°C for 14 hours. 1 ml of this culture solution contains 10 mg/L of tetracycline.
- inoculate broth (100 ml in a 500 ml flask) 37
The cells were incubated at ℃ for 6 hours. The entire amount of the seed culture solution obtained in this way was transferred to a jar fermenter containing 1 part of medium K (Marubishi Science Equipment Research Institute, MD-250 model, 2 volumes).
and cultured at 37°C for 18 hours. The pH of the medium during cultivation was adjusted to 7 with 14% ammonia water, and stirring was not performed.
500 rpm and ventilation was at 1 v.vm. After culturing, the cells were centrifuged and washed twice with 0.9% saline to obtain 30 g of wet cells. Transfer 0.6 g of the obtained moist bacterial cells to 20 ml of medium KW in a 500 ml flask.
suspended in. Indole added as a substrate, L-
The concentrations of serine, glycine, and glucose are each
0.5g/, 1g/, 2g/, 30g/. Table 1 shows the results of quantifying L-tryptophan produced after stirring at 33° C. for 4 hours by thin layer chromatography. [Table] Molar conversion rate
b) The mark ○ indicates that it was added to the medium KW. The L-broth, medium K, and medium KW described above are mediums having the following compositions. [Table] Example 2 0.6 g of the wet bacterial cells obtained in Example 1 were suspended in 20 ml of medium KW in a 500 ml flask. The concentrations of anthranilic acid, L-serine, glycine, and glucose added as substrates were 1 g, 1 g, and 2 g, respectively.
g, 30g/. Table 2 shows the results of quantifying L-tryptophan produced after stirring at 33° C. for 4 hours by thin layer chromatography. [Table] Example 3 E. coli W3110 trpAE1 trpR tnaA
(pSC101-trp·I15) was precultured in L-broth in the same manner as in Example 1. 2 ml of the obtained seed culture solution as a medium
Inoculate KF (20ml in a 500ml flask) and store at 33℃ for 72 hours.
Cultured for hours. A KF medium to which glycine or L-serine was added, and a control medium to which neither of the two were added were used. Further, a test was also conducted in which 2 g/g of glycine or L-serine was added after 20 hours of culture. Table 3 shows the results of quantifying L-tryptophan and indole produced after 72 hours by thin layer chromatography. [Table] [Table] The above medium KF is a medium having the following composition. Medium KF: KH ₂ PO ₄ 2g (NH ₄ ) ₂ SO ₄ 10g MgSO ₄・7H ₂ O 1g FeSO ₄・7H ₂ O 10mg MnCl ₂・4H ₂ O 10mg Casamino acid 8g Yeast extract 1g Anthranilic acid 4g Glucose 20g CaCO ₃ (Separate sterilization) 30g Tetracycline 10mg/PH7.0

Claims (1)

[Scope of Claims] 1. A method for producing L-tryptophan by culturing in a nutrient medium a microorganism belonging to the genus Escherichia containing a plasmid having genetic information for regulating the production of tryptophanase and lacking tryptophanase. , anthranilic acid and/or indole, and glycine and glucose. 2. The production method according to claim 1, characterized in that anthranilic acid and/or indole, glycine and glucose are added after the microorganism has grown. 3. The production method according to claim 2, wherein the microorganism is Escherichia coli containing a plasmid having a tryptophan operon in which feedback inhibition by tryptophan to anthranilate synthase has been released. 4 The microorganism is Escherichia coli W3110
(Esherichia coli) trpAE1 trpR tnaA
(pSC101-trp・I15)
Manufacturing method described in section.