JPS60180598A - Production of l-threonine - Google Patents

Abstract

PURPOSE:To produce L-threonine in high productivity, by culturing a microbial strain capable of producing and accumulating L-threonine and essentially in the secretion phase, in a nutrient medium under aeration, and producing and accumulating L-threonine. CONSTITUTION:A microbial strain capable of producing and accumulating L- threonine (Brevibacterium fermentum, FERM-P No.4182) is cultured in a nutrient medium under aeration. The microbial cell finished its proliferation phase and entered in the secretion phase is held in the fermentation system, and the reaction product is separated from the system while keeping the cell in the secretion phase. The nutrient medium containing saccharides is introduced into the fermentation system to effect the aerobic fermentation, and L-threonine is produced and accumulated in the culture liquid. The objective L-threonine is separated from the culture liquid by filtration, centrifugal separation, adsorption, etc.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing L-threonine by fermentation.

In general, in a fermentation system, there is a growth phase in which microorganisms (bacterial cells) assimilate fermentation raw materials (e.g. sugar) and multiply by division, and a second phase in which the proliferated bacterial cells further assimilate fermentation raw materials and undergo metabolic effects within the cells. It can be roughly divided into a secretion phase in which L-threonine is excreted from the bacterial body through the secretion phase.

Among these, the growth phase is located in the early stage of fermentation, and in this phase, most of the raw sugar taken into the bacterial cells for assimilation, based on the ratio per single cell, is used for bacterial cell formation by bacterial cell proliferation.

On the other hand, the secretion phase is located in the later stages of fermentation, where:
The raw sugar taken into the bacterial body is used for secretion of threonine outside the bacterial body rather than for bacterial cell formation.

This will be explained in more detail with reference to FIG. FIG. 1 is a graph showing the results of measuring the amounts of glucose IIk, L-threonine, and bacterial cells over time in a conventional fermentation production system for monothreonine using glucose as a raw material. The region (c) in FIG. 1 is the growth phase, in which consumed glucose is mainly used for bacterial cell formation, and in FIG. 1 corresponds to the region up to about 30 hours after the start of culture.

On the other hand, the region (c) in FIG. 1 is the secretion phase, where the growth of the bacterial cells has almost finished and reached a saturated state, and most of the consumed glucose is used to produce and accumulate L-threonine. In FIG. 1, this secretion phase corresponds to the region approximately 52 hours after the start of culture.

However, in conventional methods, once the growth phase and the secretion phase have been completed, the used bacterial cells are usually discarded before or after the separation and collection of threonine produced and accumulated in the fermentation system.

It depends on the time of proliferation phase, secretion phase and L
- The amount of threonine produced and accumulated is uniquely determined by the metabolic ability of the bacterial cell itself, and it was impossible to increase the amount of threonine produced and accumulated during the secretion phase even if the fermentation time was increased.

In other words, when producing L-threonine by conventional fermentation methods, various studies and improvements have been made on microorganisms, additives, fermentation conditions, etc. in order to increase the amount of L-threonine produced and accumulated. It was previously thought that it was almost impossible to improve the productivity of L-threonine beyond the metabolic ability of bacterial cells. Therefore, each time a new fermentation was carried out, a new bacterial cell was used, and the growth phase and secretion phase were repeated.

However, the present inventor has conducted extensive research aimed at improving the productivity of L-threonine beyond the metabolic ability of bacterial cells, and as a result has arrived at the present invention.

That is, the present invention provides a method for separating and collecting L-threonine produced and accumulated in a system by subjecting a microorganism capable of producing and accumulating L-threonine to aeration fermentation, wherein the microorganism is substantially in the secretion phase. This is a method for producing ni-threonine by a fermentation method, which is characterized by using only chlorine.

The main aim of the present invention is to avoid fermentation in the growth phase and to sustain fermentation in the secretion phase.

Although such studies have been carried out on the fermentation method of L-glutamic acid (Japanese Patent Publication No. 3'8-25292), they have not been carried out on the fermentation method of L-threonine, and this has been achieved for the first time by the present invention.

In the present invention, first, only microorganisms capable of producing and accumulating L-threonine that are substantially in the secretion phase are aerated and stirred in the fermentation system.

In the present invention, examples of microorganisms capable of producing L-threonine (gm-tt) include the genus Nizielisia, the genus Brevibacterium, the genus Aerobacter, the genus Serratia, and the genus Corynebacterium Iti. When attempting to produce L-threonine, wild strains hardly produce L-threonine outside their cells, so a method is used in which the wild strains are artificially mutated to give them the ability to produce L-threonine. Conventionally known artificial mutant strains having monothreonino-producing ability include the genus Brevibacterium (Japanese Patent Publication No. 45-26708) and the genus Enerina (Japanese Patent Publication No. 45-26708), which are resistant to α-anoβ-bitroxyvaleric acid. Microorganisms of the genus Corynebacterium (Japanese Patent Publication No. 47-34956) are mentioned, and representative examples include Corynebacterium spp.
Glutamate (ATCC-21660), Brevibaczolium-lactofermentum (FERM-p 41)
82), Brevibacterium brabum (FERM-
p 211), Corynebacterium acetoacidophyllum (ATCC-21270), and Clobacterium ammoniaphyllum (FERM-2331).

In the present invention, only those microorganisms that are substantially in the secretion phase are used.

In the present invention, the secretion phase means that the amount of bacterial cells per unit volume of microorganisms is 80% of the maximum amount of bacterial cells in the fermentation system (i.e., the amount of bacterial cells when bacterial cell growth reaches a saturated state).
% by weight or more.

In the present invention, the microorganisms that are substantially in the secretion phase used in the fermentation system may be grown separately in advance and used as they are after the growth phase has been completed, or they may be used as they are in the fermentation system after the normal threonine fermentation is completed. The isolated bacterial cells may be used again.

In the fermentation system of the present invention, it is necessary to add raw material sugar and a nitrogen source in the same way as in normal fermentation.

In the present invention, any raw sugar that can be used in normal fermentation can be used, and in particular, in addition to natural products or synthetic chemicals such as glucose, ethanol, and acetic acid, blackstrap molasses and cellulose decomposition liquid can be used. , starch decomposition liquid, etc. are preferably used. As the nitrogen source, soluble inorganic salts containing nitrogen molecules such as ammonium sulfate and urea are preferably used.

In the present invention, a conventional method can be used for aeration and stirring.

Fermentation is carried out while maintaining the pH at 6-9 and the temperature at 15-37°C.

After completion of the fermentation, the microorganisms in the fermentation system and the product 1-threonine are separated. As the separation method, conventional methods such as filtration method, centrifugation method, adsorption separation method, etc. are used.

The microorganisms obtained by separation can be reused for new fermentation, and the effects of the present invention can be exerted by this reuse. The fermentation system may be a -potency type or a multi-stage, multi-tank type.

According to the present invention, the producing bacteria that have completed the growth phase and entered the secretion phase are retained in the fermentation system, and the reaction product is taken out from the fermentation light source while being maintained in the secretion phase, and the reaction product is extracted as necessary. It is possible to adopt a method in which a certain amount of raw material sugar, etc. is input into the fermentation system, and in 1), it is also possible to perform the fermentation of L-threonine, which has conventionally only been carried out in a batch manner, in a continuous manner. It has become. Moreover, by employing the method of the present invention in a fermentation system, the production rate (i) expressed by the fermentation production rate and the fermentation yield for sugar (expressed per unit raw material sugar) can be significantly improved.

The present invention will be explained in detail below by way of examples.

Example 1 A 1β-volume mini-diaphragm was produced. A medium solution with the following medium composition was heated at 120°C for 20 minutes.
Sterilized by steaming for a minute.

Medium composition Glucose (separately sterilized) 100f (NH4) 2504
, 45fKH2PO4' 1.2 y MgSO4
・7H200,5fFe so4117 H2O5”f
Mn CRz ” '1~5H205q Polypebutono S 151 Biotin 150μf Thiamine Hydrochloride 2
50μ! to 14 with ion-exchanged water.

Separately, Brevibacterium lactofamentum (FERM-p4182), an inoculum of monothreonine-producing bacteria cultured in a broth medium (3% broth), was added to the medium solution Cζ.
Aerated culture was performed at 0°C and pH 6.9.

The culture solution obtained 72 hours after the start of aerated culture was filtered using an aseptic method. The filter has an average pore diameter of 0.3μ and a porosity of 75.
% polymethyl methacrylate as the main material, inner diameter 370μ, membrane thickness 85μ, effective neck area 0.5
Through this filtration process, most of the soluble inorganic salts and organic matter, white matter, and sugars other than the bacterial cells were removed from the system. The amount of bacterial cells was 92% of the amount at saturation and was in the secretion phase.

Simultaneously with the filtration process, the same amount of the above-mentioned sterile culture solution was added to carry out the fermentation of threonine in the secretion phase.

Through this fermentation, 95.0g of glucose was consumed in 35 hours, and L-threanine was 1111-18.5ttl1M.
did. The yield of L-threonine based on sugar is 8.5/95, 0
= 8.9%, and the L-threonine production rate was 8.9%. '
5fL-Sunonin/e・35 hours or 0.24fL-
It became threonine/l/hour.

Bacterial cell optical density of fermentation liquid at 550 mμ (0, D-5
50) was 29.5 at the beginning of fermentation and 32.1 at the end of fermentation. The results are summarized in Table 1.

Comparative Example 1 Aeration culture (fermentation) was carried out under the same conditions as in Example 1 using the same culture medium and strain. 72 hours after the start of aerated culture, the glucose consumed in the fermentation system was 96.0 g/
l and one threonino produced by fermentation is 5.1
It was 1/n. The yield of monothreonine based on sugar obtained by this conventional fermentation method is 5.1 / 96.0 =
It was 5.3%. In addition, the L-threonine conversion rate is 5
．． 1f L-threonino/1-72 hours or 0.07f
The L-thread was 1V/hour.

The amount of bacterial cells produced at this final stage is 30.5 as the optical density of bacterial cells (0, 'D, 560) measured using transmitted light of 550 mμ.
Met. The results are summarized in Table 1.

Table 1 Example 2 In Example 1, the bacterial cells after completion of fermentation were counted by the same filtration process as in Example 1, and sterile culture solution was added thereto again in the same manner as in Example 1, and fermentation was repeated 4 times. Ta. The results are shown in Table 2.

It was confirmed that the improved fermentation method of the present invention has extremely good production efficiency even when repeated fermentations are performed, and provides reproducible results.

(Blank below on this page) Table 2 Example 3 Instead of adding fresh culture solution at the end of each fermentation in Example 2, the product was continuously added at 0.25 (1/hr) and at the same time Fermentation was carried out using a continuous method with withdrawal at a rate of .25β/hr.

Table 3 shows the results of the improved fermentation method using continuous fermentation. −Table
3

[Brief explanation of the drawings] Figure 1 shows the amount of glucose, L, in the fermentation production system in the conventional method.
- This is a graph showing the results of measuring the amount of threonino and the amount of bacterial cells over time. (c): Proliferation phase 0: Secretion phase Patent applicant Toshi Co., Ltd. Sozu 1 acid 9 + Tomo (hrs)

Claims (1)

[Claims] In a method for separating and collecting L-threonine produced and accumulated in a system by subjecting microorganisms capable of producing and accumulating L-threonine to aerated fermentation, only microorganisms that are substantially in the secretion phase are used as the microorganisms. A method for producing shiichithreonino using a fermentation method characterized by: