DE2016496C - Process for the biotechnological production of L histidine - Google Patents

Description

The invention relates to a process for biotechnical production of i.-histidine by • cultivating microorganisms in carbon and Nutrient media containing nitrogen sources and mineral salts.

i.-Histidine, one of the protein-building amino acids, is used as a medicament, additive, for living beings used in animal feed or as an additive to food.

It has already been reported that i.-histidine can be removed by growing microorganisms such as the against Reagent resistant mutants of Escherichia coli and Salmonella typhiniurium (Science, 129, p.968 [1959], and Genetics, 50, p. 611 [1964]). However, that is accumulated around culture media Amount of i.-histidine is low, so that this process can be used for the technical production of i.-histidine are not suitable.

The invention is based on the object of a method for the production of L-histidine with considerably

ίο indicate increased yield.

According to the invention, this object is achieved by using Corynebacterium glutamicum ATCC 21339 breeds together with Torulopsis ATCC 20200. The chemism of the formation of i.-histidine by Cultivation of the above-mentioned microorganisms has not yet been elucidated. Based on the expiration of the Biosynthesis of u-histidine, however, is believed to be that L-histidinol, which is grown by Corynebacterium glutamicum ATCC 21339 is obtained,

by i.-histidinol dehydrase from Torulopsis ATCC 20200 is converted into i.-histidine. The i.-histidinol dehydrase has already been found in various microorganisms, the present invention has recognized, however, that microorganisms believed to contribute to i.-histidinoI dehydrase are capable, are not always capable of growing i.-histidinol-producing conditions under the conditions Strains i.-hislidinol in i.-histidine too with the exception of Torulopsis ATCC 20200, as shown in Table 1.

Table 1

Microorganism collection
of L-histidinol collection
of L-histidine Brevibacterium ammoniagenes
Serratia marcescens
Aspergillus niger
Streptomyces griseus
Streptomyces ambofaciens
Streptomyces kelicolor
Corynebacterium glutamicum ATCC21339 ..
Bacillus subtilis
Saccharomyces cerevisiae
Achromobacter
Torulopsis ATCC 20200 -I- + 4
+ + +
4 + 4
4- + 4 +
-r I I I I I! I I I +
+

1. Composition of the medium: glucose 12g / dl, ammonium sulfate 1.5 g / dl, ammonium chloride 0.5 g / dl, urea 0.3 g / dl, KH ₂ PO ₁ 0.15 g / dl, K ₂ HPO ₁ 0.5 g / dl, MgSO ₁ · _{7H 2} O 0.05 g / dl, FcSO ₁ · _{7H 2} O 0.002 g / dl, MnSO, -4H ₂ O 0.002 g / dl. Biotin 30 (ig / l, CaCO ₁₁ 2 g / dl, peptone 1 g / dl and meat extract 0.5 g / dl. The pH was adjusted to 7.2 with ammonia.

2. Stem and Heimpfungszeil: With Corynebacterium glutamicum ATCC No. 21399 was vaccinated at the beginning of the cultivation and after 4 days of cultivation vaccinated again with the various microorganisms listed in Table 1. the Cultures were continued for another 3 days.

3. / üehlungsbcdbedingungen: piston shaking, temperature in breeding 28 C.

As a source of cabbage, glucose, fructose, galactose can be used. Sucrose, mallose, mycosc, cellobiose, Arabinose, alcohols, acetic acid, molasses and / or starch hydrolyzate and the like, preferably at one concentration from 5 to 20 g / l can be used in the nutrient medium.

At least one of the organic or inorganic compounds, such as ammonia, Urea, ammonium sulfate, ammon chloride, ammon acetate, ammon citrate and the like can be used.

Inorganic mineral salts that can be used are hydrochloric acid, sulfuric acid, nitric acid and phosphoric acid salts des Na, K, Mn, Mg, Ca, Co, Ni, Zn, Cu and the like can be used. However, it is - as the i.-histidinol producer The microorganism has a characteristic requirement for the nutrients - that is necessary to add necessary organic nutrients to the medium. Since the yield of i.-histidinol and consequently i.-histidine will also depend on the amount of added nutrients, so it is necessary to add appropriate amounts of nutrients. So

is preferably L-histidine or i.-histidine-containing Material in amounts from 50 to 3000 ^ g / ml, based on on the histidine. It is also possible to add a small amount of L-histidinol or add an organic nutrient that effectively supports L-histidine formation; however has such an addition has no or only a partial effect on the growth of microorganisms. Such organic nutrients are generally amino acids, Vitamins and nucleic acid derived substances other than the above L-histidine. It is also possible to use natural organic materials, such as corn water, peptones, meat extracts, Containing yeast extract, a hydrolyzate of soybean meal or microbial cells and the like as nutrients Materials to use. These various organic nutrients can be used individually or together with used by others.

The cultivation is carried out under aerobic conditions at a preferred pH of about 5 to 9 in the course of the cultivation (in particular 7 to 9 after inoculation with Torulopsis ATCC 20200). The temperature during cultivation is preferably in the range from about 23 to 40 ^° C .; cultivation is generally carried out for 3 to 8 days. According to the invention, by cultivating the L-hisiidinol-producing microorganism Cjrynebacterium glutamicum ATCC 21339 and the yeast Torulcpsis A ^ CC 20200, which is imstp.iide to convert i.-histidinol into l-histidine, a considerable amount of i.-histidine. ^! in are accumulated, with both microorganisms being mixed together from the start of cultivation. However, it is preferable to start breeding with Corynebacterium glutamicum ATCC 21339 first and then continue after adding Torulopsis ATCC 20200.

The following example explains the invention

example

A medium containing glucose (4 g / dl), KH, PO ₄ (0.15 g / dl), K ₂ HPO ₄ (0.05 g / dl), MgSO ₄ -7 H ₂ O (0.05 g / dl), peptone (I g / dl). Meat extract (1g / dl), yeast extract (0.5 g / dl) and NaCl (0.3 g / dl) was adjusted to pH 7.2 with ammonia water and then inoculated with Corynebacterium glutamicum ATCC No. 21339. This cultivation was continued with shaking for 24 hours. The same medium was separately inoculated with Torulopsis ATCC No. 20200 and cultured with shaking for 24 hours. Both media were used as seed cultures.

A fermentation medium was then prepared by adding L-histidine, meat extract, corn water egg, a hydrolyzate from soybeans or from microbial cells from glutamic acid fermentation a medium made from molasses (12 g / dl) - as

ίο Glucose -, (NH ₄ ) ₂ SO ₄ (2 g / dl), KH ₂ PO ₄ (0.15 g / dl), K ₂ HPO ₄ (0.05 g / dl) MgSO ₄ 7H ₂ O ( 0.05 g / dl) in the U: th extent shown at a pH of 7.2 adjusted with ammonia water. Was added 10 ml of the fermentation medium in a 250 ml Erlenmeyer flask and sterilized, the medium contained therein was mixed with 0.5 ml of Saatkuhur of Corynebacterium glutamicum ATCC no. 21339, and incubated for 4 days cultured with shaking at 28 ^"J C. Thereafter, inoculated with 1 ml of the seed culture of Torulopsis ATCC No. 20200 together with added glucose (2 g / dl) and urea (0.3 g / dl) The cultivation was continued with shaking for an additional 3 days; Table 2 below gives the results on.

Table 2

Added material and amount A. B. L-histidine · HCl 350 [Lg / ml 6.4 8.4 Meat extract 0.2 g / dl 9.4 0.8 Meat extract 0.2 g / dl + L-histidine · HCl 100 µg / ml 11.3 2.3 Corn water 0.5 g / dl 4.8 0.2 Cornwaters 0.5 g / dl + L-histidine · HCl 150 µg / ml 12.4 2.4 Soybean hydrolyzate 0.5 g / dl 5.2 2.1 Soybean hydrolyzate 0.5 g / dl + L-histidine · HCl 200 µg / ml 6.5 6.9 Glutamic acid hydrolyzate producing cells 0.5 g / dl 5.4 1.6 Glutamic acid hydrolyzate producing cells 0.5 g / dl + L-histidine · HCl 200 [ig / ml 5.0 9.8

A - L-histidine accumulation in mg / ml (calculated as i.-histidine

din · HCl).
B r = L-histidine accumulation in mg / ml (calculated as L-hislidinol · HCl).

Claims (2)

Patent claims: 1. Process for the biotechnological production of L-histidine by growing microorganisms in Nutrient media containing carbon and nitrogen sources and mineral salts, characterized in that that Corynebacterium glutamicum ATCC 21339 together with Torulopsis ATCC 20200 breeds. 2. The method according to claim 1, characterized in that that the breeding started first with Corynebacterium glutamicum ATCC 21339 and then after the addition of Torulopsis ATCC 20200 will be continued.