Microorganisms for production of glutamic acid

Abstract

Novel bacteria identified as effective in glutamic acid production through fermentation chemistry conducted at temperatures above 42.degree. C.

Description

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a novel microorganism capable of accumulating a marked amount of glutamic acid by fermentation and a method for producing the desired product by culturing the microorganism.

2. Background of the Prior Art

In a method which comprises accumulating a marked amount of glutamic acid and collecting the same, namely, production of glutamic acid fermentation in an industrial scale, microorganisms capable of accumulating a marked amount of glutamic acid in 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.

1. Kinoshita et al., Amino Acids, 2, 42 (1960) (hereafter referred to as Publication (1)) 2. Okumura et al., J. Agr. Chem. Soc. Japan, 36, 141 (1962) (hereafter referred to as Publication (2)) 3. Takayama et al., J. Agr. Chem. Soc. Japan, 39, 328 (1965) (hereafter referred to as Publication (3)) 4. Takayama et al., J. Agr. Chem. Soc. Japan, 39, 335 (1965) (hereafter referred to as Publication (4)) 5. Takayama et al., J. Agr. Chem. Soc. Japan, 39, 342 (1965) (hereafter referred to as Publication (5)) 6. Komagata et al., J. Gen. Appl. Microbiol., 15, 243 (1969) (hereafter referred to as Publication (6)) 7. Yamada et al., J. Gen. Appl. Microbiol., 16, 103 (1970) (hereafter referred to as Publication (7)) 8. Yamada et al., J. Gen. Appl. Microbiol., 16, 215 (1970) (hereafter referred to as Publication (8)) 9. Yamada et al., J. Gen. Appl. Microbiol., 18, 399 (1972) (hereafter referred to as Publication (9)) 10. Yamada et al., J. Gen. Appl. Microbiol., 18, 417 (1972) (hereafter referred to as Publication (10))

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 utilizable microorganisms capable of accumulating at least 30 g/l of glutamic acid in a medium in a yield of at least 30% based on glucose, as described in Publication (1).

These known glutamic acid-producing bacteria are 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 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 bacteria are taxonomically akin to each other. In spite of the fact that they are considered to be microorganisms akin to each other, known glutamic acid-producing 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 different classification system and a classification standard of the newest version of Bergey's Mannual of 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 (1)) made taxonomical research on approximately 20 glutamic acid-producing bacteria and using common properties possessed by these bacteria as 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 acid-producing 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 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. 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 acid-producing bacteria, this temperature is generally at 31.degree. to 32.degree. 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 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 fermenter and recycle chilled water to the exchanger. In order to obtain chilled water, a freezer must be used but because of a vast amount of the fermentation 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 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-producing bacteria (yield of 30% or more based on glucose, amount of accumulated glutamic acid of 30 g/l or more) and capable of accumulating a marked amount of glutamic acid in a high temperature region--for example, 43.degree. C. at which conventional glutamic acid-producing bacteria do not grow and fermentation of glutamic acid is impossible. These microorganisms are grown at 45.degree. C. in which conventional glutamic acid-producing bacteria cannot grow and the inventors, have found conditions for accumulating a marked amount of glutamic acid in a medium by glutamic acid fermentation, using the microorganism and have thus come to the present invention.

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 (1) that glutamic acid-producing bacteria grow well at 28.degree. to 37.degree. C. In Publication (2), the bacteria grow well at 30.degree. to 37.degree. C. but many bacteria hardly grow at 42.degree. C. It is also reported in Publication (4) that the optimum growth temperature is 25.degree. to 37.degree. C. but bacteria hardly grow at 42.degree. C. It is reported in Publication (9) that no bacteria grow at 42.degree. C. 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 42.degree. C. The present inventors have determined the highest growth temperature of each of 20 strains shown in Table 1 which are almost all bacterial strains accessible from known bacteria deemed to be glutamic acid-producing bacteria, by two methods, i.e., 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.degree. C. at all by any of liquid culture and plate culture. It has thus been judged that the highest growth temperature of conventional glutamic acid-producing bacteria was below 42.degree. C.

[Table 1] Known Glutamic Acid-Producing Bacteria Measured on the Highest Growth Temperature and Temperature Resistance

______________________________________Brevibacterium ammoniagenes ATCC 13745Brevibacterium divaricatum NRRL B2312Brevibacterium flavum ATCC 13826Brevibacterium flavum ATCC 14067Brevibacterium glutamigenes ATCC 13747Brevibacterium immariophilum ATCC 14068Brevibacterium lactofermentum ATCC 13869Brevibacterium roseum ATCC 13825Brevibacterium saccharolyticum ATCC 14066Brevibacterium taipei ATCC 13744Brevibacterium thiogenitalis ATCC 19240Corynebacterium acetoacidophilum ATCC 13870Corynebacterium callunae NRRL B2244Corynebacterium glutamicum ATCC 13032Corynebacterium glutamicum ATCC 13761Corynebacterium herculis ATCC 13868Corynebacterium lilium NRRL B2243Corynebacterium melassecola ATCC 17965Corynebacterium sp. ATCC 14747Microbacterium ammoniaphilum ATCC 15354______________________________________

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 acid-producing bacteria would be required, the present inventors isolated microorganisms that grow at 43.degree. C. from various samples in the natural world as sources for isolation and made a survey of strains capable of 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 these strains, it has been judged that the isolated strains are akin to each other and classified in the same species. Bacteriological properties of four representative strains (Strain Nos. AJ 12308, AJ 12309, AJ 12310 and AJ 12340) are described below.

__________________________________________________________________________ AJ 12308 AJ 12309 AJ 12310 AJ 12340__________________________________________________________________________Morphological characteristics:(1) Shape and size of rod of (0.7-1.0) .times. same as same as same as cell (1.0-4.0)) in the in the in the round at both ends left left left of cells; V-shaped column column column arrangement based on snapping division is observed.(2) Pleomorphism No pleomorphism is same as same as same as noted but depend- in the in the in the ing upon period of left left left culture, long rod column column column cells, cystite cells and rudimentary branching cells are rarely noted.(3) Motility none same as same as same as in the in the in the left left left column column column(4) Spore formation none same as same as same as in the in the in the left left left column column column(5) Gram staining positive same as same as same as in the in the in the left left left column column column(6) Acid fast negative same as same as same as staining in the in the in the left left left column column columnCultural characteristics:(1) Nutrient-agar abundant or same as same as abundant or plate culture moderate growth; in the in the moderate colonies are left left growth; round, smooth, column column colonies are entire, convex, round, smooth glistening, opaque lustrous, or translucent, opaque or dull yellow and translucent, butyrous. dull yellow and flake- like.(2) Nutrient-agar abundant or same as same as same as slant culture moderate growth; in the in the in the colonies are left left left filiform, glistening column column column and dull yellow.(3) Nutrient broth moderate growth; same as same as moderate almost uniformly in the in the growth; turbid but some left left cells tend to cells precipitate. column column gather and also precipitate.(4) Nutrient-gelatin moderate growth; same as same as same as stab culture non in the in the in the liquefaction left left left column column column(5) Litmus milk made very weakly same as same as same as alkaline; in the in the in the neither lique- left left left faction nor column column column coagulation is noted.Physiological and biological characteristics:(1) Reduction of reduced same as same as same as nitrates in the in the in the left left left column column column(2) Denitrification negative same as same as same as in the in the in the left left left column column column(3) MR test negative or negative negative positive weakly positive or weakly positive(4) VP test positive negative positive negative(5) Indole formation negative same as same as same as in the in the in the left left left column column column(6) Formation of positive same as same as same as hydrogen sulfide in the in the in the left left left column column column(7) Hydrolysis of negative same as same as same as starch in the in the in the left left left column column column(8) Utilization of It does not grow same as same as same as citrates in Koser's medium in the in the in the but grows in left left left Christensen's column column column medium to render the medium alkaline.(9) Utilization of It does not same as same as same as inorganic nitrogen utilize nitrates, in the in the in the but utilize left left left ammonium salt. column column column(10) Formation of No extracellular same as same as same as pigment formation of in the in the in the pigment. left left left column column column(11) Urease test negative or negative negative positive weakly positive or weakly positive(12) Oxidase negative same as same as same as in the in the in the left left left column column column(13) Catalase positive same as same as same as in the in the in the left left left column column column(14) Growth range It grows well at same as same as It grows well pH 7-9.5; grows in the in the at pH 7-9.5; well at 25-45.degree. C.; left left grows well Slight growth is column column at 25-44.degree. C.; noted at 46.degree. C. Slight growth is noted at 45.degree. C.(15) Behavior to aerobic or same as same as same as facultative in the in the in the anaerobic left left left column column column(16) O-F test It grows same as same as same as (glucose) fermentatively in the in the in the to produce acid. left left left column column column(17) Acid formation from sugars: (1) L-Arabinose negative negative negative negative (2) D-Xylol negative negative negative negative (3) D-Glucose positive positive positive positive (4) D-Mannose positive positive positive positive (5) D-Fructose positive positive positive positive (6) D-galactose negative negative negative negative (7) Maltose positive positive positive negative (8) Sucrose positive positive negative positive (9) Lactose negative negative negative negative (10) Trehalose negative negative positive negative (11) D-Sorbitol negative negative negative negative (12) D-Mannitol negative negative negative positive (13) Inositol negative negative negative positive (14) Glycerine negative negative negative negative (15) Starch negative negative negative negativeOther characteristics:(1) Temperature It survives It survives It survives It survives resistance in skim milk in skim milk in skim milk in skim milk10 at 55.degree. C. 60.degree. at 60.degree. C. at 55.degree. C.-10 minutes by for 10 for 10 minutes by capillary method; minutes minutes capillary It is dead at 65.degree. C. by capillary by capillary method; it is for 10 minutes. method; method dead at 60.degree. C. it is it is for 10 minutes. dead at dead at 60.degree. C. for 65.degree. C. for 10 min. 10 min.(2) Resistance to It grows in same as same as same as sodium chloride 5% salt-containing in the in the in the medium. left left left column column column(3) Auxotrophy It requires same as same as same as biotin for growth. in the in the in the left left left column column column(4) Base composition 60.2% GC 59.5% GC 59.0% GC 56.8% GC of DNA (Tm method)(5) Dibasic amino mesodiamino- same as same as same as acid contained in pimelic acid in the in the in the cell wall left left left column column column(6) Source for fruit vegetable soil fruit isolation__________________________________________________________________________

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 5% 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 in examples later described, etc.; and these properties are identical with those of conventionally known glutamic acid producing bacteria. Further in the other 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 bacteria group edited with reference to Publications (1) through (10), 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.

Next, taxonomical examination on a species level is given on the bacteria of the present invention. The following three points in bacteriological properties are different between the bacteria of the present invention and known glutamic acid-producing bacteria in common. A first characteristic point is that the highest temperature showing clearly observable growth is 43.degree. C. or higher. As has been described above, the highest growth temperature of known glutamic acid-producing bacteria is about 42.degree. C. or lower but no bacteria capable of growing at 43.degree. 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 like greatly vary. Therefore, a test performed by suspending bacteria in skim milk and sealing the suspension in a glass capillary is considered to be best (Publication (3)). The bacteria of the present invention can all survive in skim milk after treatment at 55.degree. C. for 10 minutes by the capillary tube assay method. To the contrary, most of known glutamic acid-producing bacteria are dead after treatment at 55.degree. C. for 10 minutes according to this method; but there is a report that some bacteria show slight survival (Publication (3)).

With respect to this report, the present inventors 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.degree. C. for 10 minutes. On the other hand, in the bacteria of the present invention, all of the isolated 14 strains survived by the treatment at 55.degree. C, for 10 minutes. Further 11 out of these strains also survived even by treatment at 60.degree. 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.degree. C. Conditions for the experiment and amounts of glutamic acid accumulated are 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 acid-producing 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 properties are considered to be stable. Accordingly, 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 morphological properties such as growth condition, color hue of colonies, etc. and differences in physiological and biochemical properties such as acid formation from sucrose, maltose, trehalose, D-mannitol, inositol, 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 to belong to the same species.

Survey was made by comparing the bacteria of the 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. Representative strains belonging to this species are AJ 12308, AJ 12309, AJ 12310 and AJ 12340, which have been deposited as FERM P-9244, FERM BP-1540, FERM P-9245 (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 Mar. 10, 1987 (FERM P-9244-9246) and Mar. 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, Yatabemachi, Tsukuba-gun, Ibaragi-ken 305, Japan, and were accorded the FERM P-9244, 9245, 9246 and 9277 indicated above.

These strains deposits were then converted into deposits under the Budapest Treaty on Oct. 27, 1987. And strains of FERM P-9244, P-9245, P-9246 and 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.degree. 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 nephelometry reached 0.6 and culture was further 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.

TABLE 2______________________________________Composition of Medium Used in Glutamic AcidFermentation Test______________________________________Glucose 100 gSoybean hydrolysate (as total nitrogen 0.36 gKH.sub.2 PO.sub.4 1 gMgSO.sub.4.7H.sub.2 O 1 gFe.sup.++ Mn.sup.++ 2m g eachVitamin B.sub.1.HCl 100 .gamma.Biotin 100 .gamma.Ammonium sulfate 5 gWater 1,000 ml______________________________________ pH 7.8

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 glutamic acid was accumulated in the culture solution obtained after incubation for 17 hours.

EXAMPLE 4

Glutamic acid fermentation was carried out using Corynebacterium thermoaminogenes AJ 12340 in a manner 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 5

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 of 40.degree. C., while appropriately supplementing acetic acid or ammonia gas to keep pH of the culture liquid at 7.5 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.

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.

Claims

1. A biologically pure culture of a Corynebacterium thermoaminogenes selected from the group consisting of FERM BP-1539, FERM BP-1540, FERM BP-1541, and FERM BP-1542.