DE2159179A1 - Process for the fermentative production of L-isoleucine - Google Patents

Description

DR. MÜLLER-BORE DIPL.-PHYS. DR. MANITZ DIPL.-CHEM. DR. DEUFEL

DIPL.-ING. FINSTERWALD DIPL.-ING GRÄMKOW 2159179 PATENTANWÄLTE

25. KOV. 1971

Dek / Sh - T 1158

lanabe Seiyaku Co., Ltd.,.

21, TJoshomachi 3-chome, Higashi-ku, Osaka, Japan

Process for fermentative production of L-isoleucine

Priority: Japan v. November 30, 1970 No. 105605/70

The invention relates to a method for fermentative production of L-isoleucinο

L-isoleuein is and was one of the essential amino acids Used as an active ingredient in medical preparations and as an additive to food or fritters. the known chemical synthesis of this amino acid has the disadvantage that four stereoisomers of isoleucine during the Reactions are formed, and that complicated procedures for the separation of the biologically active L-isoleucine from the mixture of isomers are necessary. For this reason, it has been recognized that a fermentative method in the industrial Production of L-isoleucine is most beneficial and practical.

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Ea "three methods of fermentative production ^: of L-isoleucine are already known, but these methods absolutely require the use of a precursor compound" during fermentation. The known methods can be divided into two groups. One method is described in the known Japanese patent application 6593/1963, wherein the cultivation of a strain of Pseudomonas or flerratia genus in a nutrient medium containing D-threonine is used. The other method is described in the published Japanese patent applications 18511/1961 and 5167/1962, the cultivation of a strain of Bacillus or Pseudomonas φ genus in a nutrient medium containing Dir-Oo -aminobutyric acid being used. Furthermore, L-threonine cannot be used as a precursor compound in the method of the first-mentioned group, i.e. well-known Japanese patent application 6593/1963 - can be used. ^

Due to extensive investigations it has now been found that a mutant of Serratia marcescens, which opposite Isoleucine hydroxamate or both versus isoieueinhydroxamate as well as 4 * ~ Amiajj> l (utt er acid resistant, an excellent yield of ^ -Isoleucine from a normal source of carbon results. |> In addition, it was found that the fertility the mutant β in L-isoleucine can be remarkably increased which can be done by permeating in the presence of Ii-iDhreonin, L-homoserine or L-aspartic acid is carried out

The method according to the invention for the division of Ir-isoleucine is characterized in that a mutant γόη Serratia marcescens which is resistant to isoleucine hydroxamate or which is resistant to isoleucine hydroxamate and α-aminobutyric acid is cultivated in a nutrient medium under aerobic conditions and the accumulated L-isoleucine from the fermentation broth gt- * j is won. - \

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The isoleuein hydroxamate-resistant hutante of the invention can be obtained by UT irradiation of a wild strain of ßerratia marcescens or by treating this wild strain with a courage. treated with nitro-N-nitrosoguanidine and then ^{cultivated on agar plates for 3 ms 5 days at 50 0} O, which has the following composition: K ₂ HPO ₄ = 0.7 ßew / ¥ ol-Sfc | IHgH) ^ «0.3 & ew / vol-% t MgSO ^ * 7H ₂ O = 0.01 wt / tol-tff (HH ₄ I ^ O _4. « 0.1 wt / ltol- # s glucose = 0, The isoleucine hydroxamate-resistant mutant of Serratia marcescens can be isolated in the form of large colonies Colonies are isolated when the isoleucine hydroxamate-resistant llutant 2 to 3 position is cultivated at .30 ° 0 on agar plates which contain the same ingredients mentioned above with the exception that 10 mg / ml OC -aminobutyric acid is used instead of isoleucine hydroxamate. A viable culture of an isoleucine hydroxamate resistant lutant from Serratia marcescens has been deposited with the American Type Culture Collection - hereinafter referred to as ATOO - under the number 2174-1, and a viable culture of an isoleucine hydroxamate and (tf-aminobutyric acid resistant llutant from ßerratia marc escens has also been deposited with the same depository under ATOO No. 2174-0.

The periaentation of the mat an te of ßerratia marcescens can be carried out either by shaking cultivation or immersion fermentation under aerobic conditions. The fermentation is preferred at 25 ° to 37 ° G and at a pH value performed from 6 to 9. Calcium carbonate and ammonia can be used to adjust the pH of the medium. The ferment! The production medium contains a carbon source, a Nitrogen source and other elements. Suitable carbon

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Sources for fermentation include glucose, starch hydrolyzate, iHimaric acid, citric acid and glycerin. Example ^ .. ■ sources of suitable nitrogen are urea, ammonium salts - '; -.: ■ of organic acids, z. B. ammonium acetate, ammonium oxalate and ammonium salts of inorganic acids, e.g. B. ammonium sulfate, ammonium nitrate. Preferred amounts of the carbon source and the nitrogen source in the medium are within the range of 2 to ^: 5 w / v and 0.5 to 2 w / v, respectively. Vol-% 0 In addition, organic nutrients, e.g. corn steep liquor, peptone, yeast extracts, and / or inorganic elements, e.g. B. potassium phosphate, magnesium sulfate, can be added to the medium.

In carrying out the fermentation according to the invention , the L-isoleucine fertility of the above mutants further by adding about 0.05 to 5 wt / vol% L-threonine, L-homoserine or L-aspartic acid can be increased. The inventive Fermentation can take about 24 to 96 hours be performed. In the fermentation broth there is L-isoleucine accumulated.

After the fermentation is complete, cells and others become solid Culture components from the fermentation broth by usual Working methods, e.g. B. by heating followed by filtration or centrifugation. Known ways of working can be used in the recovery and / or purification of L-isoleucine from the filtrate or the supernatant solution »For example the filtered fermentation broth is passed through a resin of a strong cation exchanger or hereby treated. The resin is then eluted with a dilute, alkaline solution such as aqueous ammonia. The L-isoleucine containing eluates are combined and concentrated. An alkenol such as methanol is added to the concentrated solution or ethanol added. The precipitated crystals are made from an aqueous alkanol such as aqueous methanol and aqueous Recrystallized ethanol to give pure crystals of L-Isolen.cin to obtain,

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* ⁴ - "BAD ORIGINAL

The invention is further illustrated by the following examples explained.

Test execution ;

Serratia marcescens OUT (Faculty of Engineering, Osaka University) 8259, <the isoleucine hydroxamate-resistant mutant ATOO Kr. 21741 from Serratia marcescens and the isoleucine hydroxamate and tf-aminobutyric acid-resistant mutant ATCO No. 21740 from Serratia marcescens were each 48 hours " at 30 ⁰ O .- .;> il: .. ei .: Hähraedium (pH value = 7, Oj components: glucose = 2 w / yol%} dextrin = 7 w / yol% i urea 0, 5 wt / vol-%} corn steep water = 0.35 wt / vol-%} dibasic potassium phosphate I = 0.1 wt / vol-%; magnesium sulfate = 0.5 wt-vol-% $ calcium carbonate = 2 wt-vol-% ) or in a nutrient medium (pH = 7.0) containing the aforementioned components and 1.0% by weight of L-threonine, Ir-homoserine or L-aspartic acid of L-isoleucine are listed in the following table ₀

Tabel

Amount of L-isoleucine accumulated in the medium

(mg / ml)

Strains of added amino acid

no addition 0 0.1 L-threonine O 4.0 L-homoserine 0 2.0 L-aspartic acid 0 1.5

OUT 8259 ATGO No. 21741 ATOO No. 21740

7.0 5.0 3.5

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example Λ

An aqueous nutrient medium was prepared as follows

Components includes:

II-youronin 2 Wt / vol% glucose 2 Il Dextrin 10 Il urea 0.5 Il dibasic potassium phosphate 0.1 Il Magnesium sulfate 0.05 Il Potassium carbonate 2 Il

The aforementioned medium is adjusted to pH = 7 »0. 15 ml of the medium are placed in a 500 ml Schüitel bottle and its components are sterilized by autoclaving. The contents of the loop of an inoculation needle on isoleucine hydroxamate-resistant H & ante ATOÖ No. 21741 from Serratia marcescens is inoculated into the medium under aseptic conditions. The medium is then ^{cultivated for 48 hours at 30 °} C. with shaking. The fermentation medium obtained in this way contains 7 mg / ml L-isoleucine.

1000 ml of the fermentation medium are heated to 100 ° 0 for 10 minutes and then filtered. The filtrate is introduced into a column (3 cm 45 cm) of a strong cation exchange resin in the Η form (manufactured by Höhn & Haas Go. Under the trademark Amberlite IR-120). After washing with water, the column is eluted with 5 Seigers of aqueous ammonia. The reactions containing L-isoleucine are collected and concentrated under reduced pressure to about 80 ml. 100 ml of aqueous methanol are added to the solution. The crystals which precipitate are collected by filtration and then recrystallized from aqueous methanol. 5 S of L-isoleucine are obtained, [obJj ° = + 40.1 ° (0 = 1.6U-0hlorwas s er stoff acidest

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Example 2 ;

An aqueous nutrient medium is prepared, which follows Components contains:

glucose 5 Wt / vol-ff Ammonium fumarate 2 Il urea 0.5 Il dibasic potassium phosphate 0.1 Il Magnesium sulfate 0.05 Il Calcium carbonate 2 Il

The medium described above is brought to a pH of 7.0
set. 15 ml of the medium are placed in a 500 ml shaking ((bottle and the contents are sterilized / Aufcoclave treatment. The contents of the loop of an inoculation needle of isoleucine hydroxamate and egg-aminobutyric acid-resistant mutant ATOO No. 21740 from ßerratia marcescens is aseptically in the
Inoculated medium. Then the medium is cultivated in the same manner as described in Example 1. The fermentation medium thus obtained contains 2.5 mg / ml of L-isoleucine.

Example 3

15 ml of the nutrient medium, which contains the same ingredients as described in Example 1, with the exception that
4% by weight / volume of DL-ohracnine instead of L-threonine is produced. After sterilization, the contents of the loop of an inoculation needle are aseptically inoculated into the medium on isoleucine hydroxamate-recovered mutant ATOO No. 21741 from Serratia marcescens the medium at 30 ° 0 for 72 hours
cultivated with shaking. The fermentation medium obtained in this way contains 13 mg / ml L-isoleucine.

Example 4

15 ml culture medium is prepared, which contains the same constituents

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parts as described in Example 3 contains. Do that Sterilizing the contents of the loop of an inoculation needle Resistant to isoleucine hydroxamate and ^ -amino butyric acid Mutant ATCG Mr. 21740 of Serratia marcescens aseptically in inoculated the medium. Then the medium is in the same Manner as described in Example 1, cultivated * The thus obtained The fermentation medium contains 25 mg / ml L-isoleucine.

Example ^

An aqueous nutrient medium is prepared which follows Components contains:

L-homoserine 1 wt / vol%

Sorbitol 8 "

Powdered Corn Spring Water 0.25 "

Urea 0.5 "-

dibasic potassium phosphate 0.1 "

Magnesium sulfate 0, Q5 "

Potassium carbonate 2 "

The medium described above is adjusted to pH = 7-0 15 ml of the medium are placed in a 500 ml shake flask, and their contents sterilized by autoclaving. The contents of a loop of an isoleucine hydroxamate vaccination needle and j ^ -aminobutyric acid resistant mutant ATGO Fr. 21740 from Serratia marcescens is aseptically poured into the Medium put in »Then the medium is cultivated in the same manner as described in Example 1» The thus obtained Fermentation medium contains 4.5 mg / ml L-isoleucine.

Example 6

An aqueous nutrient medium is prepared, which follows Components contains:

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glucose 5 Wt / vol% Glycerin 1 (I. Spring water from powdered corn 0.35 Il urea 0.5 Il dibasic potassium phosphate 0.1 Il Magnesium sulfate 0.05 π Kai ziuHLC ax'b onat 2 Il

The medium described above is adjusted to pH = 7.10. 15 μl of the medium are placed in a 500 ml shake flask, and the contents are sterilized by autoclaving mu ä aunt Atog Ur. 2174-0 of Serratia marcescens is aseptically inoculated into the medium. the medium is then cultured for 72 hours at 30 with shaking OO. Since JFermentierungsinedium thus obtained contains 2.0 mg / ml L-isoleucine.

Example 7

15 ml of a culture medium are prepared, which the same Ingredients as described in Example 1 contains. After sterilization, the contents of a loop become an inoculation needle the isleucine hydroxamate and oi-aminobutyric acid resistant Mutant A'HCG Mr. 2174-0 of Serratia marcescens aseptically in the medium is inoculated "> Then the me <ium is inoculated in the same way, as described in Example 1, cultivated. The standardized medium obtained in this way contains 15 mg / ml of L-isoleucine.

100 ml of the fermentation medium are treated in the same way as described in Example 1. 12 g of L-irjoleucine are obtained. M ^ ⁰ = '+ 40.5 ° (0 = 4-, 6H-chlorinated water-

Pu bofitanr / pi'iicho:

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Claims (1)

Claims 1ο method for the production of L-isoleucine, thereby characterized in that one opposite isoleucine hydroxamate resistant or a mutant resistant to isoleucine hydroxamate and itf-aminobutyric acid of Serratia marcescens in a nutrient medium under aerobic conditions Cultivated conditions and the accumulated L-isol.eucin is recovered from the fermentation broth. Method according to claim 1, characterized in that the mutant ATCQ Hr. 21740 of Serratia marcescens is used. Jo method according to claim 1, characterized in that g e k e η η - draws that the mutant ATCO ITr. 21741 by Serratia marcescens is used. Method according to claim 1, characterized in that the cultivation in the presence from 0.05 to 5% by weight / volume of L-threonine, II-homoserine or L-aspartic acid is carried out. 5. The method according to claim 1, characterized in that the cultivation at about 25 ° "to about 35 ⁰ O is carried out. 6ο The method according to claim 1, characterized in that the cultivation at a pH from 6 to 9 is performed. - 10 - 209826/091 2 7. The method according to claim 1, characterized in that the cultivation in the presence of .0.05 to 5 wt / vol% Ii-threonine, L-homoserine or! -Aspartic acid at about 25 ° to about 35 ⁰ O and is carried out at a pH of 6 to 9 8. The method according to claim 7 »thereby g e k e η η - ζ eich.net that the courage ante ATOG No. 2174-0 of Serratia marcescens is used 9. The method according to claim 7 »characterized in that the mutant ATOG No. 21741 of Serratia marcescens is used «. 209826/0912