JPS60168394A - Production of l-lysine - Google Patents

Abstract

PURPOSE:To improve the production efficiency in the production of L-lysine by fermentation, by using obly the microorganisms essentially of the secretion phase. CONSTITUTION:An L-lysine-producing microbial strain [e.g. Corynebacterium glutamicum (ATCC-21513)] is cultured in a medium added with sugars (e.g. glucose, blackstrap molasses, etc.) and a nitrogen source (e.g. ammonium sulfate, urea, etc.) at about 6-9pH and about 15-37 deg.C under aeration. After the completion of fermentation, the microorganisms are separated from the medium, and the produced L-lysine is collected. In the above process, only the microorganisms essentially of the secretion phase (i.e. the cells proliferated separately and finished its proliferation phase, or the cells separated from the conventional L- lysine fermentation system after the completion of fermentation) is used as the microbial strain.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing L-lysine 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-lysine 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 terms of the proportion per single cell, most of the raw wood (sugar) taken into the bacterial body for assimilation is used for bacterial cell formation by bacterial cell proliferation. Ru.

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 phosphorus outside the bacterial body rather than for bacterial cell formation.

This will be explained in more detail with reference to FIG. Figure 1 is a graph showing the results of measuring the amount of glucose, amount of L-lysine, and amount of bacterial cells over time in a fermentation production system of L-phosphorus using glucose as log 9I using the conventional method. be. The region (5) 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 20 hours after the start of culture. .

On the other hand, the region in the middle of FIG. 1 is in the secretion phase, where the bacterial cell growth has almost finished and reached a saturated state, and most of the consumed glucose is used to produce and accumulate L-phosphorus.

In FIG. 1, this secretion phase corresponds to the region approximately 35 hours after the start of culture.

However, in conventional methods, once the growth phase and secretion phase are completed, the used bacterial cells are usually discarded before or after the separation and collection of lysine produced and accumulated in the fermentation system. The duration of the growth phase, the secretion phase, and the accumulated amount of L-lysine produced are uniquely determined by the metabolic ability of the bacterial cell itself. This was because it was impossible to force them to do so.

In other words, when producing L-lysine by fermentation methods, various studies and improvements have been made in microorganisms, additives, fermentation conditions, etc. in order to increase the amount of L-lysine produced and accumulated, but no improvements have been made. It was previously thought that it was almost impossible to improve L-lysine productivity 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 conducted extensive research with the aim of improving the productivity of L-lysine beyond the metabolic ability of bacterial cells, and as a result, the present invention was achieved.

In other words, the present invention provides a method for separating and collecting lysine produced and accumulated in a system by subjecting microorganisms capable of producing and accumulating L-lysine to aeration fermentation,
This is a method for producing lysine by a fermentation method, characterized in that only microorganisms that are substantially in the secretion phase are used as the microorganisms.

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

Such studies have been carried out on the fermentation method of (1)-glutamic acid (Japanese Patent Publication No. 38-25292), but not on the fermentation method of L-rylene, and this study has been achieved for the first time by the present invention. .

In the present invention, first, only microorganisms capable of producing and accumulating L-lysine, which are substantially in the secretion phase 3-, are aerated and stirred in the fermentation system.

In the present invention, microorganisms capable of producing and accumulating L-lysine include alkaline earth metals, Acinetobacter genus, Nigierisia spp., Brevibacterium spp., Bacillus spp., Corynebacterium spp., Flavobacterium spp. Examples include microorganisms belonging to the genus Thropacter, Cyclobacterium, Ncrochosoccus, or Pseudomonas. A typical example is Corynebacterium glutamicum (ATCC-21513, ATCC-130
32), Brevibacterium Q lactofermentum (ATCC-13869), Prehybacterium bravum (ATCC-13826), Corynebacterium
Acetamidephilum (ATCC-21476), Cyclobacterium ammoniaphyllum (ATCC-1)
5354), etc. These microorganisms may be obtained from natural soil, and may also include various amino acid-requiring mutant strains (Japanese Patent Publication No. 3.6-6499),
Other auxotrophic mutants (Japanese Patent Publication No. 48-28677), 5-2-aminoethyl-L-instine and other Norizin 4-nalog resistant mutants (Japanese Patent Publication No. 48-28078), or penicillin G or polymyxin resistant Stock (U.S. Patent No. 36
87810) etc. can also be used.

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).
Refers to the area in which the weight percentage is greater than or equal to % by weight.

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. More 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. , Denopnolysis solution, etc. are preferably used. As the nitrogen source, soluble inorganic salts containing nitrogen atoms 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 L-lysine 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 used for new fermentation +W, and the effects of the present invention can be exhibited by this reuse. The fermentation system may be a horizontal 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 system while being maintained in the secretion phase, and the amount of raw sugar is adjusted as needed. In other words, it has become possible to carry out the fermentation of one lysine in a continuous manner, which had previously only been done in a batch manner. Moreover, by applying the method of the present invention to a fermentation system, the fermentation productivity, which is expressed as the fermentation production rate, and the fermentation yield relative to sugar, which is expressed per unit raw material sugar, can be significantly improved.

EXAMPLES The present invention will be specifically explained below with reference to Examples.

Example 1 Using a 1#8E Nija Farmer as a fermenter, a medium solution having the following medium composition was sterilized by steaming at 120° C. for 20 minutes.

Medium composition Glucose (separately sterilized) 100 f (NH11+2S
O4501KH2PO, L2y Mg5O,,'yH,
00,5yFeSO4・7H2057H2O5q・4～
5I] 205 ~ Polypeptone S 159 Hyothine 1
Dilute 50 μf thiamino salt 250711 to 11 with ion exchange water.

-7 = Inoculum of monolysine-producing bacteria, Corynebacterium glutamicum (
ATCC-21513) was added to the culture medium at 30°C, pH
Aerated culture was performed in 6 and 9.

40 hours after the start of aerated culture, the culture solution is aseptically grown. Filtered. The filtration machine is a polymer hollow fiber membrane mainly made of polymethyl methacrylate-1 with an average pore diameter of 03μ and a porosity of 75%, an inner diameter of 370μ, a membrane thickness of 85μ, and an effective area of 0.
．． It was 5-. Through this filtration process, most of the soluble inorganic salts and organic substances, proteins, and sugars other than the bacterial cells were removed from the system. The remaining bacterial cells had an amount per unit volume of 83% of the amount at the time of saturation of growth, and were in the secretion phase.

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

Through this fermentation, 798 g of glucose was consumed in 22 hours, and L-lysine was 395 fim+ on a hydrochloride basis. L
-The yield of lysine based on sugar is 39.5 / 79.8 = 49
．． 5%, and the L-lysine production rate was 39.58-g L-lysine/l·22 hours, or 1.80 g L-lysine/e·hr. At 550 mμ, the fermentation liquid's rest stop value i (0, D, 550) is 3 at the early stage of fermentation.
02. At the end of fermentation, the temperature was 36.6. 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. 44 hours after the start of aerated culture, the glucose consumed in the fermentation system was 93. The amount of lysine produced by fermentation was 379 g as L-lysine hydrochloride.

The yield of lysine based on sugar obtained by this conventional fermentation method was 407%-37.9/93.0. Also, L-
The lysine production rate was 37.9 yL-lysine/e.times.44 hours, or 0.86 g L-lysine/l.

The amount of bacterial cells produced at the final stage was 320 as measured by optical density (0, D, 550) using transmitted light of 550 mμ. The results are summarized in Table 1.

Table 1 Example 2 In Example 1, the bacterial cells after completion of fermentation were harvested by the same one-pass process as in Example 1, and a sterile culture solution was added thereto again in the same manner as in Example J to carry out fermentation. Repeatedly. 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) Table 2 Example 3 Instead of adding fresh culture solution at the end of each fermentation in Example 21, the culture solution was added continuously at 0.05 l/hr and at the same time the product was added at 0.05 l/hr. 05 (fermentation was carried out by a continuous method by taking out at a rate of 1/hr. Table 3 shows the results of the improved fermentation method by continuous fermentation.

Table 3

[Brief explanation of drawings]

Figure 1 shows the amount of glucose, L, in the fermentation production system in the conventional method.
-i:,;7ri, The results of measuring the amount of bacterial cells over time are shown in graphs. (C), Proliferation Phase (B) 1 Secretion Phase Patent Applicant Higashi Shi Co., Ltd. Shed 1 Kayayama Donai (hrs)

Claims (1)

[Claims] In a method for producing and accumulating L-lysine in a system by aeration fermentation of microorganisms capable of producing and accumulating L-lysine, and separating and collecting L-lysine, only microorganisms that are substantially in the secretion phase are used as the microorganisms. A method for producing lysine by a fermentation method characterized by the following.