JPS63240779A - Novel microorganism and production of glutamic acid therewith - Google Patents

Abstract

NEW MATERIAL:Corynebacterium thermoaminogenes which has the optimal growing temperature higher than 43 deg.C, resists to 55 deg.C for more than 10min and produces a considerable amount of glutamic acid. EXAMPLE:Corynebacterium thermoaminogenes AJ 12308 (FERM-9244). USE:The production of glutamic acid by fermentation. The microorganism improves the economy of industrial production of glutamic acid, since it can accumulate a large amount of glutamic acid in a high-temperature range. PREPARATION:Microorganisms which can grow at 43 deg.C is separated from a variety of naturally occurring samples.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a novel microorganism that accumulates a significant amount of glutamic acid by fermentation, and a method for producing a desired product by culturing the microorganism.

[Conventional technology]

In industrial production of glutamic acid fermentation, which uses microorganisms to accumulate and collect the entire amount of glutamic acid, microorganisms that have the ability to accumulate a significant amount of glutamic acid in a culture medium, so-called glutamic acid producing bacteria, are used in fermentation tanks. The culture is carried out using a suitable medium and controlling the culture conditions such as -1 temperature and the amount of dissolved oxygen under appropriate conditions.

There are many reports regarding the classification of conventionally known glutamic acid producing bacteria, including the following.

1. Kinoshita et al. Am1noAcids 242 (1960)
, (Reference ■) 2, Oku et al. Agricultural Journal 36 141 (19
62), (Reference ■) 3, Takayama et al. Agricultural Journal 39 328
(1965), (Reference ■) 4, Takayama et al. Agricultural Journal 39
335 (1965), (Reference ■) 5, Takayama et al. Agricultural Journal 39 342 (1965), (Reference ■) 6, Komagata et al. J, Gen, Appl, Microblol,
, 15243 (1969).

(Reference ■ below) 7. Yamada et al. J, Gen, Appl, Microb
iol, 16103 (1970).

(References below) 8. Yamada et al. J, Gen, Appl, Werabi
ol, , 16215 (1970).

(Reference ■) 9. Yamada et al. J, Gsn, Appl, Mierob
lol, 18399 (1972).

(Reference ■ below) 10. Yamada et al. J, Gan, Appl, Micro
biol, 18417 (1972).

(Reference ■) In these reports, glutamic acid-producing bacteria are bacteria that accumulate a significant amount of glutamic acid in the culture medium, but a strict definition has not been made. However, as a quantitative guideline, it refers to industrially usable microorganisms that accumulate glutamic acid in the culture medium with a yield of 301/1 or more and a yield of 30% or more relative to glucose, as stated in 1. It can be interpreted.

All of these known glutamate-producing bacteria are aerobic, Durham-positive, non-spore-forming rods and are included in a group of bacteria called coryneform bacteria, but they also have the ability to accumulate significant amounts of glutamate in the culture medium. It is biotin-requiring. Contains mesodiaminopimelic acid in the cell wall. It is known that the Get and t of DNA are around 55%, and that their morphological characteristics and physiological and biochemical properties are similar to each other, and for these reasons, they are called glutamate-producing bacteria. It is widely accepted that known microorganisms are taxonomically related to each other.

Although these microorganisms are thought to be closely related to each other, the only known glutamate-producing bacteria are the genus Brevibacterium and the genus Corynebacterium.
Or Microbacterium F4
iumi) and have been identified in different genera. This is due to differences in the era of identification, and in particular, differences in the classification system and classification standards used in the latest edition of the Byrdes Manual Determined Native Pacteriology, the world's most authoritative bacterial identifier.

Differences in the weighting of classification criteria among identifiers are thought to be the cause of this phenomenon.

In addition, Kinoshita et al. (Reference ■) conducted a taxonomic study on about 20 strains of glutamate-producing bacteria, and advocated the establishment of a genus of glutamate-producing bacteria based on the common characteristics of these bacteria. Until now, this argument has not been widely adopted.

On the other hand, in the industrial production of glutamic acid fermentation, the above-mentioned glutamic acid-producing bacteria are grown in a medium containing ingredients such as sucrose and acetic acid, and ammonia is used as a nitrogen source.

Cultivate under aerobic conditions using urea or ammonium sulfate to accumulate a significant amount of glutamic acid in the medium. The amount of glutamic acid accumulated depends on factors such as medium composition, culture temperature, amount of dissolved oxygen, and means of excreting glutamic acid produced within the bacterial cells into the medium, but it is important to optimally set these factors. It is possible to easily accumulate glutamic acid at a glucose yield of 30% or more and an accumulation concentration of glutamic acid in the culture solution of 309/1 or more.

[Problem that the invention seeks to solve]

Glutamic acid is industrially produced in Japan and many other countries around the world by the above-mentioned fermentation method.

One of the most important factors in implementing industrial production is economy. The present invention provides a technology that improves the economic efficiency of industrial production of glutamic acid by fermentation.

[Means for solving problems]

There are several technical factors that increase the economics of industrial production of glutamic acid fermentation. For example, improvements in sugar yield, accumulation of glutamic acid concentration, shortening of culture time, etc. Another important factor is the increase in culture temperature. The culture temperature can be set to the optimum temperature for glutamic acid fermentation, but when all conventional glutamic acid producing bacteria are used, this temperature is usually 31 to 32°. When the culture is started, fermentation heat is generated, so if the culture is left as is, the temperature of the culture solution will rise and the production of glutamic acid will decrease significantly. In order to optimally maintain the temperature of the culture solution, it is necessary to install a heat exchanger in the fermenter and circulate cold water through it. To obtain cold water, a refrigerator must be used, but since the fermentation heat generated is high, the electrical energy consumed by the refrigerator is also large. Therefore, if the culture temperature for glutamic acid fermentation can be raised higher than before, the cooling burden will be reduced, thereby increasing the economic efficiency of this industrial production.

The present inventors conducted various studies to solve the above-mentioned problems, and as a result, we found a microorganism that can grow at 45 degrees, which conventional glutamic acid-producing bacteria cannot grow.

Glutamic acid production ability equivalent to conventional glutamic acid producing bacteria (sugar yield 36% or more, glutamic acid accumulation 301/1)
above), and in a high temperature range where conventional glutamate-producing bacteria cannot grow and glutamate fermentation cannot be carried out (for example, 4
The present invention was completed by discovering a new microorganism that has the ability to accumulate a significant amount of glutamic acid at 3°C), performing glutamic acid fermentation using the microorganism, and finding conditions for accumulating a significant amount of glutamic acid in the medium. reached.

The details of the present invention will be described below.

Regarding the growth temperature of conventional glutamate-producing bacteria, as far as the above-mentioned reports mention, all of the reports cited 1 as a common property of glutamate-producing bacteria.
According to literature ■, it grows well at 28°-37°. Literature■
There are many plants that grow well at 30°-37° and do not grow very well at 42°. In literature ■, the optimal growth temperature is 25°-37°, and at 42°, there is slight growth, and in literature ■, 4.
It has been reported that nothing grows on 2e'. Although there are differences in implementation methods and growth criteria in each report, it can be inferred that the maximum growth temperature of conventional glutamic acid-producing bacteria is approximately 42° at most. Therefore, the inventors used the 7'j20 strain shown in Table 1, which covers most of the strains currently available as glutamic acid-producing bacteria, and used Nutriendo broth as a medium in a shaking liquid constant temperature bath. The maximum growth temperature was determined using two methods: one using nutriendo agar as a medium, and the other using plate culture in a high-precision gas constant temperature bath. As a result, all of the tested bacterial strains were observed to grow at 42° in both liquid culture and plate culture. In other words, the maximum growth temperature of conventional glutabanic acid producing bacteria is 42c'
It was determined as follows.

Brevibacterium ammonium
es ATCC13745Brevibac
terium divaricatum N
RRL B2312Brevibacterium
flavum ATCC13826
Brevibacterium flavum
ATCC14067Brevibacte
rium glutamigenes ATC
C13747Brevibacterium imma
riophllum ATCC14068Bre
vibacterium lactoferment
um ATCC13869Brevi'bact
erium roseum ATC
C13825Brevibacterium sacc
halolyticum ATCC14066Br
evibacterium taipei
ATCC13744 Brevibacterium
m thiogenjtaljs ATCC192
40Corynebacterium acetoa
cidophilum ATCC13870Cor
ynebacterium callunae
NRRL B2244Corynebacte
rium glutamicum ATCC1
3032Corynebacterium gluta
micum ATCC13761Coryne
bacterium herculis A
TCC13868Corynebacterium l
ilium NRRL B2243C
orynebacterium melassecol
a ATCC17965Corynebact
erium sp, ATCC14747 Microba
cterlum ammoniaphilum
ATCC 15354 The present inventors hypothesized that in order to carry out glutamic acid fermentation at temperatures higher than conventional culture temperatures, it would be necessary for the microorganism to have a maximum growth temperature higher than that of conventional glutamic acid producing bacteria. Originally, various types of samples in the natural world were used as biological isolation sources.
We isolated microorganisms that grow at ℃ and searched for strains capable of accumulating significant amounts of glutamic acid in the culture medium, and obtained 914 strains from different sources of endangered organisms. The mycological properties of these strains were tested and identified, and the results were positive. As a result, it was determined that the isolated strains were similar to each other and included in the same species. Below are four representative strains (strain numbers: AJ12308, A
J12309.

The mycological properties of AJ12310, AJ12340) are fully described.

As shown here, all of these strains (hereinafter referred to as the present invention bacteria) belong to the Corynephono L/mu bacterial group because they are Durham-positive, non-spore bacilli that grow aerobically.

Furthermore, the mode of cell division is a snapping type.
The dibasic amino acid contained in the cell wall is mesodiaminopimelic acid. It is an osmotic resistant bacterium that can grow in a medium containing 5% salt. It requires biotin for growth. Although it has standby properties such as producing a remarkable amount of glutamic acid in high yield and accumulating it in the medium,
These properties are the same as those of the conventionally known glutamic acid producing moe. In addition, many of its other morphological and physiological and biochemical properties are common to known glutamate-producing bacteria. Based on the above, it is considered appropriate that the strain of the present invention belongs to the same genus as the known glutamine-producing bacteria. Although opinions are divided, the latest 8th edition of the source manual for the Coryneform bacteria group, which was compiled with reference to the literature ■～■, states that the known glutamate-producing bacteria are of the genus Corynebacterium. It is described separately from Plevipactericum B. However, in this manual, the genus Brevibacterium itself is treated as a taxonomically indeterminate genus (Genu@1ncertae 5idis), and the genus Corynebacterium is treated as a regular genus. Considering all of the above, it is not possible to use the bacterium of the present invention. At present, it is considered appropriate to assign it to the genus Corynebacterium.

Next, a taxonomic discussion of the present invention bacteria will be made at my level.

Commonly different mycological properties between the present bacterium and known glutamic acid producing bacteria are the following three properties. The first is that the maximum temperature at which clear growth occurs is 43° or higher. As mentioned above, the maximum growth temperature of known glutamic acid-producing bacteria is around 42° or below 42°, both from the literature and from the test results conducted by the inventors, and there are no bacteria that grow at temperatures above 43°. do not. The second is temperature resistance. When testing temperature resistance in a large capacity test tube, it is difficult to obtain accurate results due to variations in heat conduction time, etc.
The best method is to test by suspending the bacteria in skim milk and sealing it in a glass capillary. Survive after 10 minutes of treatment. On the other hand, if the known glutamic acid bacteria are used in their natural state, 5
There is a report (Reference ■) that most bacterial strains are killed by treatment for 5° to 10 minutes, but some strains leave a trace of survival.

The inventors conducted additional tests on this point as well.

A temperature resistance test was conducted on all of the known glutamic acid producing bacteria shown in Table 1 under the same conditions as the present invention, and all of the known glutamic acid producing bacteria were killed by treatment at 55° for 10 minutes. In contrast, the bacteria of the present invention are 55°-10
All 14 isolates survived after 10 minutes of treatment, and 11 of these also survived after 10 minutes of treatment at 60°.

The third characteristic property is that the bacterium of the present invention accumulates a significant amount of glutamic acid even at 43°. The conditions of this test and the amount of glutamic acid accumulated are as shown in the Examples.

The glutamic acid accumulation properties of the known daltamic acid producing strains shown in Cloth-1 were examined under the same conditions, but none of the strains grew, and virtually no glutamic acid was accumulated. The above-mentioned properties of the present bacterium are not found in known glutamic acid-producing bacteria, and in particular, the properties of maximum growth temperature and temperature resistance are considered to be stable properties that cannot be increased by microbial mutation manipulation. Therefore, it can be interpreted that there is sufficient evidence that the bacterium of the present invention is a different species from any known glumebanoic acid producing bacterium. In addition, the present invention bacterial isolate 1
Slight differences in morphological properties such as growth status and colony color among the four strains 1. Acid production from sucrose, malt, tolenose, mannitol, inosit, etc.

Differences in physiological and biochemical properties were observed in MR, VP, nitrate reduction, urease, etc., but all were recognized as belonging to the same species.

In addition, a comparison was made by comparing the Δ of the present invention with three species belonging to the Coryneform bacteria group other than glutamic acid producing bacteria, but no corresponding @ species was found. Based on the above considerations, we recognized that all three strains of the present invention are new species belonging to the genus Corynebacterium, and that Corynebacterium thermoaminodanes (Co
rynabacterium thermoaminog
enes nov, ip). Representative strains belonging to Book 11 are AJ12308 and AJ12309.
.

AJ12310 and AJ12340, these tension bodies are FERM 9244 + 9245 respectively.
．． 9246 p 9277.

Example-1 Corynebacterium ψ Thermoaminodanes AJ1230
8, fill a small fermentation groove with a capacity of 11 with 300 cups of culture solution having the culture medium composition shown in Table 2, and add ammonia gas to keep it OK. 9. Incubate at 43° while adding appropriate amount of water. 5 hours after the start of culture, when the turbidity reaches 0.6, add penicillin e 3.
It was added to the concentration of U7fnl and the culture was continued. After 16 hours of culture, the entire culture solution was analyzed by high-performance liquid chromatography, and the result was 39. Glutabanic acid was accumulated at a production level of IEl/l. At this time, when Glevipacterium flavum ATCC 13826 was used in parallel under the same conditions, the amount of glutamic acid accumulated was 0.11/l or less.

<Table 2> Composition of medium used for glutamic acid fermentation test Glucose 100I! soybean decomposition g
, (as total nitrogen) 0.36NKH2PO41
g MgSO4・7H20lF F−, Mnl+ 2 each ■ Vitamin・B1. Hctl 00 r Biotin 1001 Ammonium Sulfate 5II Water
1. OOOmj Example 2 Corynebacterium thermoaminogenes AJ1230
As a result of carrying out glutamic acid fermentation in the same manner as in Example 1 using 40. O1/l
Glutamic acid accumulated during the course of the study.

Example 3 Corynebacterium thermoaminodenes AJ1231
As a result of carrying out glutamic acid fermentation in the same manner as in Example 1 using
Glutamic acid accumulated at 'a degree.

Example 4 Corynebacterium thermoaminogenes AJ1234
As a result of performing glutamic acid fermentation in the same manner as in Example 1 using 0.38.17j in the culture solution after 18 hours of culture.
Glutamic acid accumulated at # degree of /l.

Example-5 Corynebacterium thermoaminodanes AJ1230
8, 300 d of culture solution having the culture medium composition shown in <Table 2> K except glucose and ammonium sulfate and addition of 20 g of ammonium acetate was poured into a small fermenter with a capacity of 11, and the - of the culture solution was 7. Culture was carried out at 40° while adding acetic acid or ammonia gas as appropriate to maintain the temperature between .5 and 8.0.

Eight hours after the start of the culture, when the turbidity reached 0.6, pear nigirin was added to the solution at a concentration of 3 U/rnl, and the culture was continued. After 24 hours of incubation, the culture solution was analyzed by high-performance liquid chromatography and found to have accumulated at a concentration of 32 y/l, which was 31.5% based on acetic acid.

Claims (2)

[Claims] (1) Maximum growth temperature is 43℃ or higher, temperature resistance is 55℃
- A novel microorganism with the ability to accumulate significant amounts of glutamate for more than 10 minutes;
oanimogenes) (2) A method for producing glutamic acid, which comprises performing glutamic acid fermentation using Corynebacterium thermoaminogenes and collecting glutamic acid from the culture solution.