2,971,890 United States Patent ()?ice Patented Feb. 14, 1961 1 2 which may be easily obtained by chemical synthesis or 2,971,890 by fermentation. FERMENTATIVE PROCESS FOR PRODUCHVG Further objects of this invention will become apparent L-GLUTAMIC ACID FROM FUMARIC ACID from the following description of processes embodying Tetsuo Ogawa, Tokyo, Toshinao Tsunoda, Zushi-shi, the invention and speci?c examples thereof. and Ryohei Aoki, Kazumoto Kinoshita, and Shinji Processes for producing L-glutamic acid in accordance Okumura, Tokyo, and Yasuhiro Kondo, Kawaguchi-shi, with this invention may be carried out with either a syn all in Japan, assignors to Ajinomotor Co., Inc., Tokyo, thetic medium which contains mainly fuman'c acid as the Japan, a corporation of Japan carbon source besides a nitrogen source, such as, am No Drawing. Filed Mar. 18, 1958, Ser. No. 722,147 10 monium nitrate or urea, as well as some mineral salts Claims priority, application Japan Apr. 16, 1957 essential to microorganism growth, such as, potassium phosphate, magnesium sulfate, ferrous sulfate, and the 3 Claims. (Cl. 195--30) like, and has been neutralized, or a complex medium which contains for the most part fumaric' acid and to The present invention relates to a process for produc 15 which has been added a small quantity of sugar (such as ing L-glutamic acid. More particularly, this invention re glucose, fructose, lactose, maltose, sucrose, pentose or lates to a process for the production of L-glutamic acid by molasses), organic ‘acids, organic or inorganic nitrogenous fermentation from fumaric acid which may be prepared nutrients (such as soybean cake, ?sh meal, casein, pep by either chemical synthesis or fermentation. tone, meat-extract, yeast extract, malt extract, koji-extract In the heretofore known processes for producing L-glu 20 or amino acid), inorganic salts and vitamins. Such a tamic acid by the utilization of microorganisms, there medium is inoculated with a strongly L-glutamic acid-pro have been used, as principal materials, carbohydrates such ducing bacterium selected from among above-listed micro as starch, glucose and other sugars or u-ketoglutaric acid. organisms and subjected to aerobic submerged culture or Having observed that all these processes are not satisfac static culture at its growth temperature. The inoculated tory from industrial and economical points of view, the 25 microorganisms grow in the medium, fumaric acid is as inventors conducted their exhaustive studies of the fer similated and L-glutamic acid is gradually accumulated mentation of fuman'c acid and succeeded in directly pro in the medium, its amount being increased by degrees as ducing L-glutamic acid with an extremely high yield. the fermentation period proceeds. Contrary to expecta When a compound the TCA-cycle is chosen as the sub tion, there is only slight formation of amino acids other strate, the amount of produced-L~glutamie acid is con 30 than L-glutamic acid and only a small quantity of aspartic sidered to depend upon the balance among conjugated acid is produced in those cases where certain kinds of the enzymes. Experiments with reactions of enzymes in ani microorganisms are used. Separation of L-glutamic acid mal tissues, e. g. liver, on the compounds in the TCA-cycle, from the fermentation broth and puri?cation thereof may which were chosen as the substrate, showed that the be easily performed by a conventional process well known amount of glutamic acid converted from fumaric acid is 35 in the art. For example, fermentation broth from which extremely small in comparison with that converted from bacterial cells have been separated is concentrated to citric acid, succinic acid or a-ketoglutaric acid. This is cause L-glutamic acid to crystallize out at its isoelectric because a remarkable quantity of aspartic acid is produced point. Ion-exchange resins may also be utilized for sep by the action of aspartase, so that the formation of glu aration of the L-glutamic acid. tamic acid is greatly suppressed, in the case where fumaric 40 The following examples are given to afford a clearer acid is chosen as the substrate. Also with certain bac understanding of the invention but are not to be con teria, e.g. Escherichia coli, similar results were observed. strued as limiting the same: The inventors have found that many kinds of microor ganisms produce L-glutamic acid if they are cultured Example 1 under proper conditions in media containing fumaric acid A culture medium, 100 cc. in volume, containing 1.0 g. as the main carbon source together with inorganic nitro 45 of fumaric acid, 0.1 g. of glucose, 0.1 g. of KH2PO4, 0.02 gen source compounds. Having in mind the favorable g. of MgSO4'7H2O and 0.1 g. of (NH4)2SO4 and neu prospect of industrial production of L-glutamic acid by tralized by sodium hydroxide to pH 7.0, was inoculated fermentation from fumaric acid as the main carbon with Bacillus pumilus and subjected to shaking culture at source, the inventors searched for the most suitable micro 30° C. After 38 hours, 0.22 g. of L-glutamic acid was organisms and successfully found many superior strains. 50 obtained in the fermentation broth. This yield shows al— The microorganisms newly isolated by the inventors most quantitative formation of L-glutamic acid from 21 which change fumaric acid into L-glutamic acid, in other mg. of nitrogen contained in said culture medium. words, the microorganisms which accumulate L-glutamic acid in culture media containing mainly fumaric acid as Example 2 i the carbon source, were found to be of wide distribution 55 Three culture media, each 100 cc. in volume and con in the natural world. They may be found almost every taining 4 g. of fumaric acid, 2 g. of glucose, 0.1 g. of where so far as usual microorganisms can live at all, for KH2PO4 and ‘0.04 g. of MgSO4-7H2O, with 3 cc. of am example, in soil, sewage and the like. They are mainly monia water (20 grams per deciliter) added thereto and aerobic bacteria or facultative anaerobic bacteria such neutralized with KOH aqueous solution was inoculated as Pseudomonas, Xanthomonas, Protam-inobacter, Mi 60 with the following strains respectively and subjected to crococcus, Corynebacterium, Serratia, Alcaligenes, Achro shaking culture at 30° C. After 72 hours, the following mobacter, Flavobacterium, Escherichia, Aerobacter, Sar amounts of glutamic acid was obtained in each culture. cina, Proteus, Bacillus, Bacterium and the like. It is a principal object of this invention to produce L-glutamic acid L-glutamic acid directly from a compound without pass 65 Microorganism: (grams per deciliter) ing through intermediate stages as is usual in chemical Bacillus pumilus variety 0: ______2.80 synthesis. Bacillus pumilus variety 5 ______..._ 3.60 It is a further object of the present invention to produce Bacillus pumilus variety 7 ______.._ 3.00 L-glutamic acid with ease and an extremely high yield. Example 3 70 It is a further object of the instant invention to produce 11 culture media each, 100 cc. in volume and contain L-glutamic acid from fumaric acid, as the carbon source, ing 4 g. of fumaric acid, 1 g. of glucose, 0.1 g. of KH2PO4 2,971,890 ' 3 4 and 0.04 g. of MgSO4.7H2O, with 3 cc. of ammonia water genes, Serratia marcescens, Bacillus natto, Bacillus mega therium, Bacillus cereus, Escherichia coli, M icrococcus (20 grams per deciliter) added thereto and neutralized pyogenes, Xanlhomonas pruni and Pseudomonus aeru with KOH aqueous solution was inoculated with the fol ginosa, subjecting the inoculated medium to a culture at lowing microorganism respectively and subjected to shak its growth temperature to accumulate L-glutamic acid, ing culture at 30° C. After 72 hours, the following and separating the L-glutamic acid from the fermentation amounts of glutamic acid was obtained in each culture. broth. L-glutamic acid 2. A process for producing L-glutamic acid as in claim Microorganism: (grams per deciliter) 1; wherein said nitrogen source is selected from the group Pseudomonas aeruginosa ______0.20 10 consisting of ammonium salts, ammonia and urea. Xanthomonas pruni ______0.20 3. A process for producing L-glutamic acid as in claim Micrococcus pyogenes ______. 0.30 1; wherein said culture medium is approximately neu Escherichia coli ______\____ 0.60 tralized with ammonia and alkali hydroxide. Aerobacter aerogenes ______0.50 References Cited in the ?le of this patent Serratia marcescens ______0.50 15 Bacillus subtilis No. 1 ______2.00 V UNITED STATES PATENTS Bacillus subtilis No. 2 ______1.10 Bacillus cereus ______0.20 2,749,279 Smythe ______June 5, 1956 Bacillus megatherium ______0.30 OTHER REFERENCES Bacillus natto ______._ 0.40 20 “Journal of Biological Chemistry,” vol. 187, pp. What we claim is: 1. A fermentive process for producing L-glutamic acid 821-830 (1950). comprising the steps of approximately neutralizing a cul “The Enzymes,” by Summer et -al., vol. II, part 2, Academic Press Inc., New York (1952), pp. 1117-1118. ture medium containing fumaric acid as the main carbon Proc. of the Int. Symposium on Enzyme Chemistry, source, a nitrogen source and mineral salts essential to living microorganisms, inoculating said medium with a T okylo and Kyoto, 1957, pages 464-468, Maruzen, Tokyo, living microorganism selected from the group consisting 1958. of Bacillus pumilus, Bacillus subtilis; Aerobacter aero Chemical Abstracts, vol. 51, pages 7643-7644, 1957.

fig‘... UNITED STATES PATENT OFFICE CERTIFICATION OF CORRECTION

Patent No‘, 2,971,890 ' February 14, 1961 Tetsuo Ogawa et a1; It is hereby certified'that error appears in the above numbered pat ent requiring correction and that the said Letters Patent should read as corrected below .

In the grant, lines 4 and I4, and in the heading to the printed specification, line7, name of assignee, for "Ajinomotor C0,, , Inca", each occurrence, read ' -- Ajinomoto C0,, , Inc, ——=.

Signed and sealed this 13th day of June 1961.,

( SEA L) Attest: Y Y ERNEST W. SWIDER DAVID L. LADD Attesting Officer Commissioner of Patents