AU616168B2 - Novel microorganisms and a method for production of glutamic acid using the same - Google Patents

Description

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AUSTRALIA

PATENTS ACT 1952 COMPLETE SPECIFICATION 616168 Form

(ORIGINAL)

FOR OFFICE USE Short Title: Int. Cl: Application Number: Lodged: Complete Specification-Lodged: Accepted: Lapsed: Published: Priority: Related Art: ttIt 4 49 TO BE COMPLETED BY APPLICANT

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Name of Applicant: Address of Applicant: AJINOMOTO CO., INC.

NO 5-8 KYOBASHI 1 CHOME

CHUO-KU

TOKYO

JAPAN'

CLEMENT HACK CO., 601 St. Kilda Road, Melbourne, Victoria 3004, Australia.

Actual Inventor: Address for Service: Complete Specification for the invention entitled: NOVEL MICROORGANISMS AND A METHOD FOR PRODUCTION OF GLUTAMIC ACID USING THE

SAME

The following statement is a full description of this invention including the best method of performing it known to me:- GRIFFITH HACK Co PATENT AND TRADE MARK ATTORNEYS MELBOURNEP S D TH /9j 10-356-0 TITLE OF THE INVENTION NOVEL MICROORGANISMS AND A METHOD FOR PRODUCTION OF GLUTAMIC ACID USING THE SAME BACKGROUND OF THE INVENTION Field of the Invention The present invention relates to a novel I Imicroorganism capable of accumulating a marked amount of glutamic acid by fermentation and a method for producing the desired product by culturing the 0 microorganism.

0 Background of the Prior Art (8 In a method which comprises accumulating a marked amount of glutamic acid and collecting the same, (8namely, production of glutamic acid fermentation in an industrial scale, microorganisms capable of 1800 $set accumulating a marked amount of glutamic acid in 0 medium, so called glutamic acid-producing bacteria, are cultured in a fermentation tank using an appropriate medium under suitably controlled culture conditions such as pH, temperature, amount of dissolved oxygen, etc. Many reports have been made on classification of glutamic acid-producing bacteria hitherto known and, for example, the following publications are given.

Ai i 0 99 i 0 0 I 69 is s S or 9 5 1. Kinoshita et al., Amino Acids, 2, 42 (1960) (hereafter referred to as Publication 2. Okumura et al., J. Agr. Chem. Soc. Japan, 36, 141 (1962) (hereafter referred to as Publication 3. Takayama et al., J. Agr. Chem. Soc. Japan, 39, 328 (1965) (hereafter referred to as Publication 4. Takayama et al., J. Agr. Chem. Soc. Japan, 39, 335 (1965) (hereafter referred to as Publication Takayama et al., J. Agr. Chem. Soc. Japan, 39, 342 (1965) (hereafter referred to as Publication 6. Komagata et al., J. Gen. Appl. Microbiol., 243 (1969) (hereafter referred to as Publication 7. Yamada et al., J. Gen. Appl. Microbiol., 16, 103 (1970) (hereafter referred to as Publication 8. Yamada et al., J. Gen. Appl. Microbiol., 16, 215 (1970) (hereafter referred to as Publication 9. Yamada et al., J. Gen. Appl. Microbiol., 18, 399 (1972) (hereafter referred to as Publication i i: f il i g i

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ri ii I Ih -3- Yamada et al., J. Gen. Appl. Microbiol., 18, 417 (1972) (hereafter referred to as Publication In these reports, the glutamic acid-producing bacteria are not necessarily defined strictly to be bacteria capable of accumulating a marked amount of glutamic acid in medium. However, as a measure for the production amount, the glutamic acid-producing bacteria are reasonably interpreted to refer to industrially 0 4 utilizable microorganisms capable of accumulating at 9 o g least 30 g/l of glutamic acid in a medium in a yield of a at least 30% based on glucose, as described in So.* Publication These known glutamic acid-producing bacteria are 4* all aerobic, gram-positive and non-sporeforming rods and classified in a bacterial group named coryneform bacteria. In addition, there are known facts regarding morphological properties and physiological and o biological properties including that they are capable of accumulating a marked amount of glutamic acid in a medium, are biotin auxotrophic, contain mesodiaminopimelic acid in cell walls and have GC content of about 55% in DNA are similar to each other, etc. From these facts, it is widely admitted that known microorganisms called glutamic acid-producing I bacteria are taxonomically akin to each other. In C *i S-4spite of the fact that they are considered to be microorganisms akin to each other, known glutamic acidproducing bacteria are identified to be in different genera such as the genus Brevibacterium, the genus Corynebacterium, the genus Microbacterium, etc. A major cause for this phenomenon of giving various classifications is believed to be earlier practices in identification which was made, inter alia, on a S different classification system and a classification ft standard of the newest version of Bergey's Mannual of S Determinative Bacteriology at that time, which is the most authoritative identification book for bacteria in the world, a difference in an identifier's weight on classification standard, etc. Further Kinoshita et al.

(Publication made taxonomical research on approximately 20 glutamic acid-producing bacteria and using common properties possessed by these bacteria as rt4, the classification standard, proposed to create the genus of glutamic acid-producing bacteria. However, this proposal has not been adopted widely to date.

In production of glutamic acid by fermentation in an industrial scale, the aforesaid glutamic acidproducing bacteria are cultured in a medium containing components such as glucose, sucrose, acetic acid, etc.

under aerobic conditions, using ammonia, urea, ammonium sulfate, etc. as nitrogen sources to accumulate a I t 440w 4 a a a 0D 0 a a a a O a *o a a0, marked amount of glutamic acid in a medium. The amount of glutamic acid to be accumulated varies depending upon composition of medium, pH for incubation, culture temperature, amount of dissolved oxygen, means for secreting the glutamic acid produced in cells into medium, etc. However, by setting forth these factors in optimum ranges, glutamic acid can be accumulated in a yield of 30% or more based on glucose in a concentration of accumulated glutamic acid of 30 g/l or more.

Glutamic acid has been industrially produced by the fermentation method described above not only in Japan but also in many other countries. In the industrial production, one of the most important factors is high production rates. The present invention is to provide a highly economical technique for producing glutamic acid by fermentation in an industrial scale.

SUMMARY OF THE INVENTION In the industrial production of glutamic acid by fermentation, there are some technical standards to measure improvement from an economical standpoint. For example, these factors are an increase in yield based on glucose, an increase in concentration of glutamic acid accumulated, shortening of incubation time, etc.

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-i i -6- An additional important factor is elevation of the incubation temperature. Incubation is carried out at an optimum temperature for fermentation of glutamic acid; in the case of using conventional glutamic acidproducing bacteria, this temperature is generally at 31 to 32 0 C. When incubation is initiated, fermentation heat generates so that if the system is allowed to stand as it is, a temperature of the culture solution i4 C will increase so that production of glutamic acid will be markedly reduced. In order to maintain the temperature of the culture solution in an optimum range, it is necessary to set a heat exchanger in a 4, fermenter and recycle chilled water to the exchanger.

S ,In order to obtain chilled water, a freezer must be C* 4 used but because of a vast amoun't of the fermentation 4 heat generated, the electric energy consumed by the freezer is also large. Accordingly, if it is possible to elevate the incubation temperature in fermentation of glutamic acid higher than the conventional temperature, the burden of cooling can be reduced thereby to improve the industrial production from an i economical viewpoint.

As a result of various investigations to solve the problem described above, the present inventors have found a novel microorganism capable of producing glutamic acid equal to conventional glutamic acid- -7producing bacteria (yield of 30% or more based on glucose, amount of accumulated glutamic acid of 30 g/1 or more) and capable of accumulating a marked amount of glutamic acid in a high temperature region for example, 43 0 C at which conventional glutamic acidproducing bacteria do not grow and fermentation of glutamic acid is impossible. These microorganisms are grown at 45°C in which conventional glutamic acid- A producing bacteria cannot grow and the inventors, have found conditions for accumulating a marked amount of S glutamic acid in a medium by glutamic acid fermentation, using the microorganism and have thus conmp to the present invention.

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DETAILED DESCRIPTION OF THE INVENTION' With respect to the growth temperature of conventional glutamic acid-producing bacteria, the growth temperature is a property common to glutamic acid-producing bacteria in any report, as far as it is mentioned in the reports referred to above. Namely, it is reported in Publication that glutamic acidproducing bacteria grow well at 28 to 37 0 C. In Publication the bacteria grow well at 30 to 37 0

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but many bacteria hardly grow at 42 0 C. It is also reported in Publication that the optimum growth temperature is 25 to 37 0 C but bacteria hardly grow at Si j1 i ir iI

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8 42°C. It is reported in Publication that no bacteria grow at 420C. Even conceded that methods and standard for judging growth may be different in the respective publications, it is assumed that the highest growth temperature of conventional glutamic acid-producing bacteria would be about 42C. The present inventors have determined the highest growth temperature of each of strains shown in Table 1 which are almost all bacterial strain accessible from known bacteria deemed to be glutamic acid-producing bacteria, by two methods, a shake liquid thermostat method using nutrient broth as a medium, and by plate culture with a high accuracy gaseous thermostat using nutrient agar as a medium. As a result, no growth of the strains tested was noted at 42 0 C at all by S* 15 any of liquid culture and plate culture. It has thus been judged that the highest growth temperature of conventional S glutamic acid-producing bacteria was below 420C.

According to one aspect of the present invention there is provided a biologically pure culture of Corynebacterium thermoaminogenes being of the strain selected from the group consisting of FERM BP-1539, FERM BP-1540, FERM BP-1541 and FERM BP-1542, as hereinbefore described and mutants or derivatives thereof having the characteristics of the natural strain, having a maximum 25 temperature for growth of no lower than 43 C and thermoresistance at 55°C for 10 minutes or longer and capable of accumulating at least 30 g/dl of glutamic acid in a medium in a yield of at least 30% based on glucose.

According to a second aspect of the invention, there is provided a method for production of glutamic acid which comprises performing glutamic acid fermentation at 4 from 43 C to 55 C in a culture solution, using a Corynebacterium thermoaminogenes being of the strain selected from the group consisting of FERM BP-1539, FERM BP-1540, FERM BP-1541 and FERM BP-1542, and collecting iK glutamic acid from said culture solution.

I I V L'U NT01 -9- [Table 11 Known Glutamic Acid-Producing Bacteria Measured on the Highest Growth Temperature and Temperature Resistance 41Brevibacterium armoniagenes ATCC 13745 Brevibacterium divaricatum, NRRL B2312 Brevibacterium flavum ATCC 13826 Brevibacterium flavurn ATCC 14067 Brevibacterium glutamigenes ATCC 13747 Brevibacterium immariophilum. ATCC 14068 IBrevibact .ri..m lactofermentum ATCC 13869 Brevibacterium roseum ATCC 13825 Brevibacterium saccharolyticum ATCC 14066 Brevibacterium taipei ATCC 13744 Brevibacterium thiogenitalis ATCC 19240 Corynebacteriun acetoacidophilum ATCC 13870 2::Corynebacterium callunae NRRL B2244 Coyeatru ltmcmAC 33 Corynebacterium glutamicum ATCC 13032 Corynebacterium herculis ATCC 13868 Corynebacterium lilium NRRL B2243 Corynebacterium melassecola ATCC 17965 Corynebacterium sp. -ATCC 14747 Microbacterium a~tioriaphilum ATCC 15354 j* S 5 On the assumption that in order to perform glutamic acid fermentation in a temperature range higher than conventional incubation temperatures, microorganisms having a growth temperature maximum higher than that of conventional glutamic acidproducing bacteria would be required, the present inventors isolated microorganisms that grow at 43°C from various samples in the natural world as sources for isolation and made a survey of strains capable of i accumulating a marked amount of glutamic acid in medium :and acquired 14 strains from different sources for isolation of the microorganisms. As a result of assay and identification of bacteriological properties of t S these strains, it has been judged that the isolated strains are akin to each other and classified in the 4 a same species. Bacteriological properties of four representative strains (Strain Nos. AJ 12308, AJ 12309, AJ 12310 and AJ 12340) are described below.

1* 9. 4 4 9 4 9 94 o 9, 9 4. 44a a 44 4 499 4 AJ 12308 AJ 12309 AJ 12310 AJ 12340 Morphological characteristics: Shape and size of cell Pleomorphism Motility Spore formation Gram staining rod of (0.7-1.0) x round at both ends of cells; V-shaped arrangement based on snapping division is observed.

No pleomorphism is noted but depending upon period of culture, long rod cells, cystite cells and rudimentary branching cells are rarely noted.

none none positive same as in the left column same as in the left column same as in the left column same as in the left column same as in the left column same as in the left column same as in the left column same as in the left column same as in the left column same as in the left column same as in the left column said as in the left column same as in the left column same as in the left column same as in the left column r .1,

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c a a 4 f a a 4 r *cr 4 4 4 4 4 4 41)44 I'LI 4 4 *a a -a a a a 1 a 4 a a 4 a aaQ a baa a AJ 12308 negative Acid fast staining Cultural characteristics: AJ 12309 same as in the left column AJ 12310 same as in the left column AJ 12340 same as in the left column Nutrient-agar plate culture Nutrient-agar slant culture Nutrient broth abundant or moderate growth; colonies are round, smooth, entire,convex, glistening, opaque or translucent, dull yellow and butyrous.

abundant or moderate growth; colonies are filiform, glistening and dull yellow.

moderate growth; almost uniformly turbid but sone cells precipitate.

same as in the left column same as in the left column same as in the left column same as in the left column same as in the left column same as in the left column abundant or moderate growth; colonies are round, smooth lustrous, opaque or translucent, dull yellow and flakelike.

same as in the left column moderate growth; cells tend to gather and also precipitate.

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AJ 12308 Nutrient-gelatin stab culture Litmus milk moderate growth; non liquefaction made very weakly alkaline; neither liquefaction nor coagulation is noted.

Physiological and biological characteristics: Reduction of nitrates reduced AJ 12309 same as in the left column same as in the left column same as in the left column same as in the left column negative negative same as in the left column AJ 12310 same as in the left column same as in the left column same as in the left column same as in the left column negative or weakly positive positive same as in the left column AJ 12340 same as in the left column same as in the left column

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Denitrification MR test negative negative or weakly positive same as in the left column same as in the left column positive negative same as.

in the left column VP test positive negative Indole formation

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S 0 a a S C S a *050 q S a a a a a a a a AJ 12308 Formation of hydrogen sulfide Hydrolysis of starch Utilization of citrates Utilization of inorganic nitrogen positive negative It does riot grow in Koser's medium but grows in Christensen's medium to render the medium alkaline.

It does not utilize nitrates, but utilize ammonium salt.

No extracellular formation of pigment.

negative or weakly positive negative o ao as 06a aD a AJ 12309 same as in the left column same as in the left column same as in the left column same as in the left column AJ 12310 same as in the left column same as in the left column same as in the left column same as in the left column same as in the left column negative or weakly positive same as in the left column AJ 12340 same as in the left column same as in the left column same as in the left column p Formation of pigment (11) Urease test (12) Oxidase same as in the left column negative same as in the left column same as in the left oIumn same as in the left column positive same as in the left column

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AJ 12308 (13) Catalase (14) Growth range Behavior to (16) O-F test (glucose) positive It grows well at pH 7-9.5; grows well at 25-45°C; Slight growth is noted at 460C.

aerobic or facultative anaerobic It grows fermentatively to produce acid.

AJ 12309 same as in the left column same as in the left column same as in the left column same as in the left column negative negative positive positive positive negative positive positive negative negative negative negative negative negative negative AJ 12310 same as in the left column same as in the left column same as in the left column same as in the left column negative negative positive positive positive negative positive negative negative positive negative negative negauive negative negative AJ 123140 same as in the left column It grows well at pH 7-9.5; grows well at 25-441C; Slight growth is noted at 450C.

same as in the left column 4 (17) Acid (1) (2) (3) (4) (6) (7) (8) (9) (10) 11) (12) (13) (14) formation from sugars: L-Arabinose D-Xylol D-Glucose D-Mannose D-Fructose D-galactose Maltose Sucrose Lactose Trehalose D-Sorbitol D-Mannitol Inositol Glycerine Starch negative negative positive positive positive negative positive positive negative negative negative negative negative negative negative same as in the left column negative negative positive positive positive negative negative positive negative negative negative positive positive negative negative Ii W J~Wij±. 4 4 p 44 0 *4 00 0 4 P 4 0 0 40* 44 0 ~0 000 0 *40 4 AJ 12308 Other characteristics: Temperature resistance It survives in skim milk at 60*-10 minutes by capillary method; It is dead at 65°C for 10 minutes.

AJ 12309 It survives in skim milk at 55°C for 10 minutes by capillary method; it is dead at 600C for min.

AJ 12310 It survives in skim milk at 60'C for 10 minutes by capillary method it is dead at 65°C for 10 min.

same as in the left column same as in the left column 59.0% GC same as in the left column AJ 12340 It survives in skim milk at 55°C-10 minutes by capillary method; it is dead at 600C for 10 minutes.

Resistance to sodium chloride Auxotrophy It grows in 5% salt-containing medium.

It requires biotin for growth.

same as in the left column same as in the left column same as in the left column same as in the left column 56.8% GC same as in the left column

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o i- 0 t r Base composition of DNA (Tm method) Dibasic amino acid contained in cell wall Source for isolation 60.2% GC mesodiaminopimelic acid fruit 59.5% GC same as in the left column vegetable soil fruit P!k _P,.Z

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ijj liii.i Fil' ii i i pm il I L' I As shown above, these bacterial strains (hereafter referred to as the bacteria of the present invention) are all gram-positive, non-sporeforming rods which grow aerobically and therefore, belong to coryneform bacteria group. In addition, the bacteria of the present invention have characteristics that mode of cell division is of snapping type, dibasic amino acid contained in cell wall is mesodiaminopimelic acid, they are osmosis-resistant bacteria capable of growing in salt-containing medium, they require biotin for growth, they produce a marked amount of glutamic acid from sugars in high yield and accumulate in medium as shown St«, in examples later described, etc.; and these properties are identical with those of conventionally known S glutamic acid producing bacteria.' Further in the other i :morphological properties, physiological and biological properties, the bacteria are common in many properties to known glutamic acid-producing bacteria.

From the foregoing, it is considered that the bacteria of the present invention would reasonably belong to the same genus as that of known glutamic acid-producing bacteria on a genus level. As has been described above, opinion may be divided on in what genus the known glutamic acid-producing bacteria are to be classified but according to the newest Bergey's Manual, 8th edition, in relation to coryneform It' 1 1 bacteria group edited with reference to Publications through etc., known glutamic acid-producing bacteria are recited by dividing into the genus Corynebacterium and the genus Brevibacterium. Taking into account that the genus Brevibacterium per se is treated as Genus incertae sidis from a taxonomical viewpoint and the genus Corynebacterium as a regular genus, however, it is considered to be most reasonable at this point of time that the bacteria of the present invention belong to the genus Corynebacterium.

r Next, taxonomical examination on a species level t f is given on the bacteria of the present invention. The following three points in bacteriological properties are different between the bacteria of the present 4 invention and known glutairic acidproducing bacteria in I common. A first characteristic point is that the highest temperature showing clearly observable growth A4 A is 43 0 C or higher. As has been described above, the highest growth temperature of known glutamic acidt#4 producing bacteria is about 42 0 C or lower but no 4 bacteria capable of growing at 43 0 C or higher exist.

A second characteristic point is resistance to temperature. In resistance to temperature, accurate results can be obtained only with difficulty in the case of performing test in a mass scale using a test tube, etc. because time for thermal conduction and the -19suspension in a glass capillary is considered to be Slikbest (Publication The bacteria of the present invention can all survive in skim milk after treatment at 55 0 C for 10 minutes by the capillary tube assay method. To the contrary, most of known glutamic acidproducing bacteria are dead after treatment at 55 0 C for minutes according to this method; but there is a report that some bacteria show slight survival (Publication With respect to this report, the present inventors .l duplicated the experiment. Test on thermostability was performed with all of the known glutamic acid-producing bacteria shown in Table 1 under the same conditions as in the bacteria of the present invention; as a result, the known glutamic acid-producing bacteria were all dead by treatment at 55 0 C for 10 minutes. On the other hand, in the bacteria of the present invention, all of t I9 t'ft the isolated 14 strains survived by the treatment at 0 C for 10 minutes. Further 11 out of these strains also survived even by treatment at 60 0 C for 10 minutes.

A third characteristic point is that the bacteria of the present invention can accumulate a marked amount of glutamic acid even at 43°C. Conditions for the experiment and amounts of glutamic acid accumulated are

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glutamic acid equal to conventional glutamic acidi ;j as shown in the examples. Accumulation of glutamic acid by the known glutamic acid producing bacteria shown in Table 1 under the same conditions was assayed but all of the bacterial strains did not grow and glutamic acid accumulated was substantially zero.

The properties of the bacteria of the present invention described above are not observed with known glutamic acidproducing bacteria; in particular, it is impossible to raise or improve the properties of the highest growth temperature and thermostability by operation for variation of microorganisms and these S; properties are considered to be stable. Accordingly, i, m it can be interpreted to have a basis sufficient to consider that the bacteria of the present invention are different from any of the known glutamic acid-producing' bacteria. In addition, slight differences in S r morphological properties such as growth condition, t color hue of colonies, etc. and differences in physiological and biochemical properties such as acid formation from sucrose, maltose, trehalose, D-mannitol, Sinositol, etc., MR test, VP test, nitrate reduction and urease test, etc. are noted between the isolated 14 strains of the present invention. These differences are on a strain level but considered to be inadequate for classifying them into different species. Thus, the bacteria of the present invention were all identified I to belong to the same species.

Survey was made by comparing the bacteria of the q present invention with bacteria belonging to coryneform bacteria group other than glutamic acid-producing bacteria; however, no bacteria in the corresponding species was found. From the foregoing, the bacteria of the present invention were all identified to be novel species belonging to the genus Corynebacterium and named Corynebacterium thermoaminogenes nov. sp.

(fit tff Representative strains belonging to this species are AJ 12308, AJ 12309, AJ 12310 and AJ 12340, which have been flflflna fl flf A A n< i, rAt r lA 4 4 4 t l' i£ a I If I 64 It 4 I 440 4 6 4 4 94 *t de i-yj .Lted das zza j c y i rCju DC -sD U, XZr~ -tirui J %D (FERM BP-1541), FERM P-9246 (FERM BP-1542) and FERM P- 9277 (FERM BP-1539) respectively.

These strains identified above by FERM P-9244, 9245, 9246 and 9277 were originally deposited on March 10, 1987 (FERM P-9244-9246) and March 13, 1987 (FERM P- 9277) at the Fermentation Research Institute, Agency of Industrial and Technology, Ministry of International Trade and Industry (FHI), 1-3, Higashi 1-chome, Tsukuba-shi, Ibaragi-ken 305, Japan, and were accorded the FERM P-9244, 9245, 9246 snd 9277 indicated above.

These strains deposits were then converted into deposits under the Budapest Treaty on October 27, 1987. And strains of FERM P--9244, P-9245, P-9246 and

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t Ti r j~b ""CI- I -22.- P-9277 were accorded the corresponding FERM BP-1540, 1541, 1542 and 1539, respectively.

[Example 1] In a small fermenter having a volume of 1 liter was charged 300 ml of culture liquid having a medium composition shown in Table 2 and, Corynebacterium thermoaminogenes AJ 12308 was cultured at a temperature of 43 0 C, while appropriately supplementing ammonia gas to keep pH of the culture liquid at 7.5 to 8.0. Five hours after the initiation of the culture, penicilline was added in a concentration of 3 U/ml when t nephelometry reached 0.6 and culture was further t I continued. The culture solution obtained after incubation for 16 hours was analyzed by high performance liquid chromatography. As a result, glutamic acid was accumulated in a concentration of 39.1 g/l. An amount of glutamic acid accumulated was 0.1 g/l or lower in a run simultaneously performed using Brevibacterium flavum ATCC 13826 under the same conditions.

i 1 -i t 2- 2 S' -23- [Table 2] Composition of Medium Used in Glutamic Acid Fermentation Test. Glucose 100 g Soybean hydrolysate (as total nitrogen 0.36 g 4 1 g MgS04.7H 2 0 1 g Fe++ Mn++ 2 mg each Vitamin B 1 .HC1 100 Biotin 100 y Ammonium sulfate 5 g Water 1,000 ml t 4 pH 7.8 S [Example 2] Glutamic acid fermentation was carried out using Corynebacterium thermoaminogenes AJ 12309 in a manner similar to Example 1. As a result, 40.0 g/l of glutamic acid was accumulated in the culture solution obtained after incubation for 19 hours.

[Example 3] Glutamic acid fermentation was carried out using Corynebacterium thermoaminogenes AJ 12310 in a manner similar to Example 1. As a result, 35.2 g/l of I k I i -2 glutamic acid was accumulated in the culture solution obtained after incubation for 17 hours.

similar to Example 1. As a result, 38.1 g/l of glutamic acid was accumulated in the culture solution obtained after incubation for 18 hours.

[Example In a small fermenter having a volume of 1 liter was charged 300 ml of culture liquid having a medium composition shown in Table 2 from which glucose and ammonium sulfate were removed and to which 20 g of ammonium acetate was supplemented and, Corynebacterium thermoaminogenes AJ 12308 was cultured at a temperature S of 40 0 C, while appropriately supplementing acetic acid or ammonia gas to keep pH of the culture liquid at to 8.0. Eight hours after the initiation of the culture, penicilline was added in a concentration of 3 U/ml when nephelometry reached 0.6 and culture was further continued. The culture solution obtained after incubation for 24 hours was analyzed by high performance liquid chromatography. As a result, glutamic acid was accumulated in a concentration of'32 g/l, which corresponded to yield of 31.5% based on acetic acid.

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Obviously, numerous modifications and variations of the present invention are possible in light of the above teachings. It is therefore to be understood that within the scope of the appended claims, the invention may be practiced otherwise than as specifically described herein.

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Claims (2)

1. A biologically pure culture of Corynebacterium thermoaminogenes being of the strain selected from the group consisting of FERM BP-1539, FERM BP-1540, FERM BP- 1541 and FERM BP-1542, as hereinbefore described and mutants or derivatives thereof having the characteristics of the natural strain, having a maximum temperature for growth of no lower than 43 0 C and thermoresistance at 55 C for 10 minutes or longer and capable of accumulating at least 30 g/dl of glutamic acid in a medium in a yield of at least 30% based on glucose.

2. A method for production of glutamic acid which comprises performing glutamic acid fermentation at from 40"C to 55*C in a culture solution, using a Corvnebacterium thermoaminoqenes being of the strain selected from the group consisting of FERM BP-1539, FERM BP-1540, FERM BP- S1541 and FERM BP-1542, as hereinbefore described, and collecting glutamic acid from said culture solution. t K DATED this 21st day of May 1991 AJINOMOTO CO., INC. By Their Patent Attorneys: GRIFFITH HACK CO Fellows Institute of Patent Attorneys of Australia a( I~ a I II A U 4 tuj i1