DE1088447B - Process for the biotechnological production of 1-glutamic acid - Google Patents

Description

GERMAN

The invention relates to a method for the biotechnological production of 1-glutamic acid.

It is known that microorganisms in a suitable medium absorb various types of amino acids can generate. However, the amounts produced here are extremely small, and it was never reported that any particular type of amino acid was found in large amounts in the medium could be accumulated through fermentation. -

The reason for the extraordinary difficulties in obtaining any amino acids in A fermentation medium is that amino acids are the components of proteins and that all once in Amino acids generated in a medium are easily re-synthesized into proteins, polypeptides or the like or converted into other substances or decomposed into other substances by various biochemical reactions can be. In other words: an amino acid can hardly be in a monomeric state in can be accumulated in a culture medium. The word "monomeric state" is used to denote this of the monomolecular state of both the free acid and a salt are used.

This is the reason why a fermentation process, i.e. the direct use of biochemical activities a living culture of microorganisms, never to the biosynthesis and accumulation of 1-glutamic acid has been proposed. The known biosynthesis takes place in an enzymatic system, i. H., a special type of enzyme is derived from microorganisms or from certain animal or animal species plant tissues are extracted, and the enzyme is treated with suitable substrates, e.g. B. α-ketoglutaric acid and Ammonium compounds, implemented under very limited reaction conditions.

For example, processes used for the production of α-ketoglutaric acid and 1-glutamic acid have become known, one of which produces the desired product through fermentation from glucose and the other of which itself an enzymatic reduction and amination of α-ketoglutaric acid. The known procedures however, proceed in two stages, and it is not possible to use 1-glutamic acid by simply combining them to be generated directly in a single step.

The invention discloses a process for the biotechnological production of 1-glutamic acid, the first time the large-scale production of glutamic acid by fermentation in a particularly advantageous way and also made possible in an economically sensible way.

In a further embodiment of the invention, the nutrient substrate is used as the carbon source for the Micrococcus species at least one · carbohydrate, namely glucose; Fructose, sucrose, maltose, Xylose, or hydrolyzed starch, is used.

Process for the biotechnological production of! -Glutamic acid

Applicant:

Kyowa Hakko Kogyo Kabushiki Kaisha, Tokyo

Representative: Dipl.-Ing. H. Leinweber, patent attorney,
Mündien 2, Rosental 7

Claimed priority:
Japan May 17, 1956

Shukuo Kinoshita, Shigezo Udaka and Sadao Akita,

Tokyo,
have been named as inventors

Finally, according to a further feature of the invention, any mutant of Micrococcus glutamicus No. 534 is used as the organism. '"'
It has been found that a microorganism which fulfills two biochemically specific requirements, namely on the one hand an ability to form α-ketoglutaric acid from sugary substances and on the other hand a strong 1-glutamic acid dehydrogenase activity, in particular a strong activity against the reverse reactions of the above reversible enzymatic reaction, ie, having reductive amination activity of α-ketoglutaric acid, is suitable for direct production of substantial amounts of 1-glutamic acid by fermentation processes.

The characteristic feature of the present invention resides in generation and accumulation of 1-glutamic acid in the culture medium by the biochemical activities during growth and the propagation of the species. The essential difference of the present invention from the one mentioned enzymatic production in which 1-glutamic acid is extracted by the conversion of a Enzyme and is synthesized with specific substrates is therefore obvious.

The inventors found a new species belonging to the genus Micrococcus, called Micrococcus No. 534 or "Mikrococcusglutamicus" is called. The general characteristics of the species are described below: Most of the attempts

009 590/69

10

were employed according to the "Manual of Methods for Pure Culture Study of Bacteria" (Society of American Bacteriologists, 9th Edition).

A. Morphological observations I. Microscopic observations: (agar medium, 37 ° C, 18 hours)

1st form: micrococci ,. individually, in pairs or

irregular masses, spherical or slightly ellipsoidal. Size of the majority: 0.6 to 1.2 μ.

2. Mobility: None.

3. Spores: None.

4. Grief Reaction: Positive.

II. Agar smear:

(Agar medium, 37 ° C, 18 hours)

1. Growth: Moderate.

2. Form of growth: offshoot form.

3. Gloss: dull.

4. Color development: milky white to weak

yellowish white.

5. Odor: None.

6. Consistency: buttery to slightly sticky

viscous.

7. Color of medium: unchanged.

III. Agar colonies:

(Agar medium, 37 ° C, 20 hours)

1. Shape: circular.

2. Surface: Smooth.

3rd margin: Completely.

■ 4th elevation: Slightly rising. 5. Appearance: Opaque.

IV. Agar sting:

(Agar medium, 37 ° C, 18 hours)

1. Growth: Preferably on the surface.

11. Citric acid consumption: Negative.

12. Starch Liquefaction: None.

13. Milk: No change or weak

alkaline.

14. Catalase: Very weak.

15. Urease: Strong.

16. 1-Glutamic acid dehydrogenase activity: Very

strong.

17. Acid formation: ct-ketoglutaric acid, milk

acids are formed. Other organic acids in traces.

18. Sugar fermentation:

35 arabinose
Xylose ...
Glucose ...
Fructose.
Galactose.
Mannose.
Lactose ..
Dextrin ..
Glycerin ..
Sucrose
Maltose ...
Trehalbse.
Raffinose.
Melecitosis

strength

Mannitol ...

Sorbitol

Salicin ...

Inulin

Glycogen.

Acid | Gas development)

V. Nutrient solution:

(Nutrient solution, 37 ° C, 18 hours)

1. Surface growth: Annular.

2. Turbidity: Low.

3. Odor: None.

4. Separation: flaky, slight.

B. Physiological properties

- 1st growth temperature: no growth above 47 ° C; low growth around 42 ^° C .; good growth between 27 and 37 ° C; Optimal around 30 ° C.

2. Oxygen: Fully aerobic.

3. Nitrate reduction: starch.
.4. Indole formation: none.

5. Hydrogen sulphide: Not formed.

6. Indicator reduction: methylene blue, 2,6-di-

chlorophenol indophenol,. Janus green, 2,3,5-triphenyl tetrazolium chloride are reduced. 6g

7. Gelatin Liquefaction: Negative or Very

weak.

8. Casein degradation: weak.

9. Voges-Proskauer reaction: negative.

10. Methyl red reaction: weakly acidic.

In view of the properties and observations listed above, the inventors realized that the present breed belonged to a new species of the genus Mikrococcus because it was not associated with any of the in Bergey's Manual listed could be identified.

Fermentation process

The composition of the medium can be any known and suitable for the growth of the micrococcus. All fermentable sugars that can be seen in the table above can be used as a carbon source. There are no particular restrictions on the nitrogen sources and other nutrients as there are on the carbon source. In order to maintain a vigorous carbohydrate metabolism in the culture, various types of organic or inorganic nitrogenous substances and various minerals that are needed by the organism should be added to the culture medium. The culture is inoculated under aerobic conditions at 27 to 30 ° C and. increased. In the course of fermentation, organic acids tend to form and make the medium acidic. It has been found that the formation of 1-glutamic acid by the _pH value of the medium is greatly affected during the course of fermentation. The formation of 1-glutamic acid is favorably influenced, when the _pH value is maintained from 6.0 to 9.0; the most favorable P _n values appear to be between about 7.0 and 8.5. According to the present invention, the P ₁₁ value is determined by adding one of the compounds

which contain a basic, nitrogen-containing radical, brought into the range from 6.5 to 8.5 and held there. Thus, through the catalytic effect of the strong reductive amination of 1-glutamic acid dehydrogenase, which is contained in the breed, once 5-ct-ketoglutaric acid is converted with the ammonium ions resulting from the added "basic nitrogen-containing residue, and 1-glutamic acid is formed. Because of the strong reductive Aminierungsaktivität the 1-glutamic acid dehydrogenase of the present race and by maintaining the p _H -value in the favorable range, the various unfavorable side reactions are kept to a minimum. the 1-Glutaminsäurebildung therefore exceed side reactions and leads to accumulation of 1-glutamic acid in a large amount.

Various methods can be used to neutralize the culture medium. In any case, it should a required amount of ammonium ions can be added to the medium. Simultaneous use of ammonium salts and alkalis is also within the scope of this invention. It is at least It is essential for the present invention that the culture conditions are not very far outside the Range for the growth of the present breed. As any skilled person will easily see, Mutants of the present breed can also be used within the scope of the present invention will.

The accumulated in the medium 1-glutamic acid - or its salt - is according to a known, suitable Process isolated, e.g. B. by ion exchange, such a process per se is not an important one Is part of the present invention.

The invention is illustrated by the following examples.

example 1

Shake culture fermentation through a synthetic medium

40

Micrococcus No. 534 was shaken in glucose broth by shaking culture at 28 ° C. for 24 hours and used as an inoculation base. 30 ecm of fermentation medium were distributed in a 250 ccm Koiben and inoculated from the above stock culture and shaken with a rotating shaker at 220 revolutions per minute. The composition of the fermentation medium was as follows: glucose 50 g, urea 8 g, K ₂ HPO ₄ 0.5 g, MgSΟ ₄ · 7H ₂ O 0.1 g and FeCl ₃ -OH ₂ O 5 mg on 11.

The analytical results are fine Breeding
management
duration Table 1 Rest
glucose in table 1 yield
to ⁵⁵
needed
glucose given. PH-
value based Days g / 100 ecm Vo Culture-
tempe-
rature 1 2.8 1-GIut-
amine
acid 60
30.0 2 8.6 mg / 22.0 ⁰ C 2 7.3 2.3 100 ecm 22.6 33 { 3 8.2 1.2 660 21.3 f 4th 6.8 0.2 700 23.5 65 28 \ 8.7 610 1 810 1130

Example 2

Shake culture fermentation
through composite medium

Stock culture (inoculum) and procedures were the same as in Example 1. The fermentation medium was composed as follows: glucose 100 g, meat extract g, NZ amine 5 g, urea 15 g, K ₂ HPO ₄ 1 g and MgS O ₄ -7 H _? O 0.25 g at 11th temperature at 28 ° C.

The analytical results are given in Table 2.

p _H value Tabel 2 yield
in "/ ο, on Breeding Rest 1-glutamine consumed
sugar management
duration- 8.4 sugar acid based Days 8.2 g / 100 ecm mg / 100 ecm 13.6 1 7.3 7.2 370 28.4 2 6.2 5.5 1280 30.1 3 3.5 1960 35.2 4th 2.9 2480

The above examples are intended to be illustrative only. There can be various other sugary ones Substances and various other organic and inorganic nitrogen sources, e.g. B. ammonia, Ammonium salts such as ammonium chloride, ammonium sulfate, ammonium carbonate, ammonium acetate and the like, Peptone, digested soybean meal, digested fish meal, casein hydrolyzate, and corn steep liquor Place the nutrients in the fermentation medium of Example 2 can be used.

As can be seen from the above descriptions, in a synthetic medium as well as in in an organic medium, an amount of up to 20 to 30% of the sugar consumed in 1-glutamic acid being transformed.

Claims (3)

Patent claims: 1. A process for the biotechnological production of 1-glutamic acid, characterized in that the new, in the description morphologically defined species of micrococcus »Mikrococcus glutamicus« in a common nutrient substrate, the fermentable carbon sources, nitrogen sources and inorganic Contains substances grown under aerobic conditions and the pg value of the nutrient substrate in the range from 6 to 9 by adding neutralizing agents is held. 2. The method according to claim 1, characterized in that that as a carbon source of the nutrient substrate at least one carbohydrate, namely Glucose, fructose, sucrose, maltose, xylose or hydrolyzed starch is used. 3. The method according to claim 1, characterized in that the organism is any mutant Micrococcus No. 534 is used. Considered publications:
Journal Am. Chem. Soc, Vol. 74, 5142 (1952);
German patent specification No. 931 582. 009 590/69 8.60