DE2159179B2 - PROCESS FOR THE MANUFACTURING OF L-ISOLEUCINE - Google Patents

Description

25th

L-isoleucine is one of the essential amino acids and has been used as an active ingredient in medicinal preparations and as an additive to food or feed used. The known chemical synthesis of this amino acid has the disadvantage that four stereoisomers of isoleucine are formed during the reactions and that complicated workings necessary to separate the biologically active L-isoleucine from the mixture of isomers are. For this reason, it was recognized that a fermentative method in industrial production of L-isoleucine is most beneficial and practical.

There are already three methods of fermentative production of L-isoleucine known, these methods however, absolutely require the use of a precursor compound in fermentation. The known Methods can be divided into two groups. One method is in the advertised Japanese patent application 6 593/1963, the breeding of a strain of Pseudomonas or Serratia is applied in a nutrient medium containing D-threonine. The other The method is described in the published Japanese patent applications 18 511/1961 and 5 167/1962, wherein the cultivation of a strain of Bacillus or Pseudomonas in a DL-s-aminobutyric acid containing nutrient medium is applied. Furthermore, L-threonine cannot be used as a precursor compound in the method of the first group, d. H. at the well-known Japanese Patent application 6 593/1963.

It has now been found that the mutant of Serratia marcescens which is resistant to isoleucine hydroxamate and rt-aminobutyric acid: Serratia marcescens ATCC 21 740, or the mutant of Serratia marcescens which is resistant to isoleucine hydroxamate: Serratia marcescens ATCC 21 741, an excellent yield ail L -Isoleucine yields from a normal carbon source. Furthermore, gefnj ·. the fact that the productivity of these mutants can be increased considerably if the fermentation is carried out in the presence of 0.05 to 5% by weight / volume of L- ^ KO >> ¹ "· L-Homoserm or LAspraj

from

Serratia marcescens can be obtained by either shake cultivation or immersion under aerobic fermentation Conditions to be carried out. The Fermenäe-Tung is preferably carried out at 25 to 37 ° C and at a pH of 6 to 9. Potassium carbonate and ammonia can be used to adjust the pH of the medium. The fermentation medium contains a carbon source, a nitrogen source and other elements. Suitable Sources of carbon for fermentation include glucose, starch hydrolyzate, fumaric acid, citric acid and glycerin. Examples of suitable nitrogen sources are urea, ammonium salts of organic acids, e.g. 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 nitrogen source in the medium are within in the range from 2 to 15 w / v --°/o or 0.5 to 2 w / v --°/o. In addition, organic Nutrients, e.g. B. corn steep liquor, peptone, yeast extracts and / or inorganic elements, e.g. B. potassium phosphate, Magnesium sulfate, can be added to the medium.

When carrying out the fermentation according to the invention, the L-isoleucine yield can be the the above two mutants further by adding about 0.05 to 5 w / v-°/o L-threonine, L-homoserine or L-aspartic acid can be increased. The fermentation according to the invention can be carried out in be carried out for about 24 to 96 hours. L-isoleucine accumulates in the fermentation broth.

After the fermentation is complete, cells and other solid culture components are released from the fermentation broth through usual working methods, e.g. B. by heating followed by filtration or centrifugation, severed. Known working methods can be used in the production and / or purification of L-isoleucine from the filtrate or the supernatant solution. For example, the filtered Fermentation broth passed through a strong cation exchanger resin or hereby treated. Then the resin is washed with a dilute, alkaline solution such as aqueous ammonia eluted. The eluates containing L-isoleucine are combined and concentrated. To the concentrated solution an alkanol such as methanol or ethanol is added. The precipitated crystals are made from a aqueous alkanol such as aqueous methanol and aqueous ethanol recrystallized to give pure crystals of Get L-isoleucine.

The invention is illustrated by the following examples ^ ', explained in more detail.

example 1

The isoleucine hydroxamate-resistant mutant ATCC " No. 21741 of Serratia marcescens and the isoleucine Hydroxamate- and a-aminobutyric acid-resistant mu

a ATCC No. 21740 from Serratia marcescens were in each case 48 hours at 30 ° C in a JEtanedium (pH value = 7.0; components: glucose - 2 wt./VoL- ⁰ / o; dextrin = 7 wt./VoL- ° / o by weight of urea 0.5 ZVoL- · / "; Maisqueüwasser = 0.35 Gew./ s ⁰ vol /»; dibasic Kaüumphosphat = 0.1 w / v ⁰ Ze; magnesium sulfate = 0.5 Wt / vol, - "/"; potassium carbonate = 2 wt / vol - "/ o) or in a nutrient medium (pH = 7.0) that contains the above-mentioned components and 1.0 wt. / Vol -" / eL -Threonine, L-homoserine or L-aspartic acid, cultured. The amounts of L-isoleucine accumulated in the medium are shown in the following table.

Amount of accumulated in the medium
L-isoleucine (mg / ml)

Added amino acid Tribes ATCC ATCC No. 21740 No. 21741 2.5 No addition 0.1 7.0 L-threonine 4.0 5.0 L-homoser 2.0 3.5 L-aspartic acid 1.5

get leucine. {&) $ = + 40.1 ° (C = 1,6N hydrochloric acid).

Example 3

An aqueous nutrient wediura is produced, which contains the following components:

Glucose 5

Amraonium fumarate 2

Urea 0.5

Dibasic potassium phosphate 0.1

Magnesium sulfate 0.05

Potassium carbonate 2

Example 2

An aqueous nutrient medium was prepared which comprises the following components:

Weight / volume volume

L-threonine 2

Glucose 2

Dextrin 10

Urea 0.5

Dibasic potassium phosphate 0.1

Magnesium sulfate 0.05

Potassium carbonate 2

The aforementioned medium is adjusted to pH = 7.0. 15 ml of the medium are poured into a SOO ml shake bottle and its components sterilized by autoclaving. The contents of the loop of an inoculation needle to isoleucine hydroxamate-resistant Mutant ATCC No. 21,741 from Serratia marcescens is produced under aseptic conditions inoculated into the medium. The medium is then cultivated with shaking at 30 ° C. for 48 hours. That fermentation medium obtained in this way contains 7 mg / ml L-isoleucine.

1000 ml of the fermentation medium are 10 minutes heated to 100 ° C and then nitrided. The FiI kicked is introduced into a column (3 cm X 45 cm) of a strong cation exchange resin in the Η form. After washing with water, the column is eluted with 5% aqueous ammonia. The L-isoleucine containing fractions are collected and concentrated under reduced pressure to about 80 ml. 100 ml of aqueous methanol are added to the solution. The crystals will precipitate collected by filtration and then from aq Recrystallized methanol. 5 g L * Iso < The medium described above is adjusted to a pH of 7.0. Be 15 ml of the medium placed in a SQO ml sachet bottle, and its Contents are sterilized by autoclave treatment. The contents of the loop of an inoculation needle of isoleucine hydroxamate and α-aminobutyric acid resistant Mutant ATCC No. 21,740 from Serratia marcescens

»5 is aseptically inoculated into the medium. Then it will be the medium was cultivated in the same manner as described in Example 2. The fermentation medium thus obtained contains 2.5 mg / ml L-isoleucine.

Example 4

15 ml of the nutrient medium, which contains the same components, as described in Example 2, with the exception that contains 4 w / v --°/o DL-threonine Place of L-threonine are produced. After sterilization, the contents of the loop an inoculation needle on isoleucine hydroxamate-resistant mutant ATCC No. 21 741 from Serratia marcescens aseptically inoculated into the medium. The medium is then cultivated with shaking at 30 ° C. for 72 hours. The fermentation medium obtained in this way contains 13 mg / ml L-isoleucine.

Example 5

15 ml nutrient medium is prepared, which contains the same ingredients as described in Example 4, contains. After sterilization, the contents of the loop of an inoculation needle are isolated from the isoljucinhydroxamat- and α-aminobutyric acid-resistant mutant ATCC No. 21 740 from Serratia marcescens aseptically into the Inoculated medium. Then the medium is cultivated in the same manner as described in Example 2. The fermentation medium obtained in this way contains 25 mg / ml L-isoleucine.

"Example 6

An aqueous nutrient medium is produced which contains the following components:

Weight / volume volume

L-homoserine 1

Sorbitol. · .... 8

Spring water From powdered corn 0.35

Urea .... 0.5

⁶ S dibasic calcium phosphate 0.1

Magnesium sulfate 0.05

Calcium carbonate 2

The medium described above is adjusted to pH = 7.0 Example 8

set. Throw 15 ml of the medium into a 500-

ml shake flask, and the contents of which is produced throw 15 ml of a nutrient medium, wel-

sterilized by autoclaving. The contents contain the same components as in Example 2 in a loop of an isoleudnhydrox- 5 needle. After the sterilization of the amat- and a-aminobutyric acid-resistant mutant, throw the contents of a loop of an inoculation needle from the isokucin-ATCC No. 21 7jjv from Serratia marcescens throw asep-hydroxamate and * -aminobutyric acid-resistant table into the MpRlfi . Then the Me mutant ATCC No. 21 740 from Serratia marcescens dium is aseptically inoculated into the medium in the same way as described in Example 2. Then practice is cultivated. The Fennentiemngsme- io medium thus obtained in the same way as in Example 2 contains 4.5 mg / ml L-Isoleudn. wrote, cultivated the fermentation

. . The storage medium contains 15 mg / ml of L-isoleucine.

Example 7 Throw in 1000ml of fermentation medium

An aqueous nutrient medium is produced in the same way as described in Example 2, cups contains the following components: 15 acts. Throw in 12 g of L-isolates.

r _a ] a> = + 40.5 ° (C = 4.6 N hydrochloric acid).

Glucose 5 Example 9

Glycerin 1 An aqueous nutrient medium containing the following ingredients

_ "','"'""."." _ ,,. Contains 10 sentences, is made:

Spring water of powdered corn 0.35 wt

Urea 0.5 glucose 4

Dibasic potassium phosphate 0.1 dextrin 10

Magnesium sulfate 0.05 35 urea 1

Calcium carbonate 2 Secondary potassium phosphate 0 "l

Magnesium sulfate 0.05

The medium described above is adjusted to pH = 7.0 calcium carbonate 2

set. Throw 15 ml of the medium in a 500-30

ml shake flask is entered and its contents are This nutrient medium is adjusted to pH = 7.0,

sterilized by autoclaving. Throw the contents of 15 ml of the medium into a 500 ml shaker Loop of an inoculation needle is inserted into the isoleucine hydroxide bottle, and the contents are placed in the autoclave amate- and α-aminobutyric acid-resistant mutants sterilized. A spoonful of isoleucine hydroxamate-ATCC No. 21 740 from Serratia marcescens becomes asep-35 and α-aminobutyric acid-resistant mutant ATCC table inoculated into the medium. Then the Me No. 21 740 from Serratia marcescens is aseptically in Cultivated for 72 hours at 30 ° C with shaking. the medium introduced, which then took 48 hours The fermentation medium thus obtained contains shaking grown throws at 30 ° C. The thus obtained 2.0 mg / ml L-isoleucine. Fermentation medium contains 3.8 mg / ml L-isoleucine.

Claims (2)

Patent claims: I. Date of manufacture of msotacin sssszssz one compared to isoleucine hydroxamate and α-Aminobu ^ ric acid resident mutant of Serratia marcescens: Serratia marcescens ATCC No. 21740 or the opposite fcoleurinfayoro * - amat resistant mutant of Serratia marcesceas. Serratia marcescens ATCC No. 21,741 uses 2. The method according to claim!, Characterized in that that the cultivation in the presence of an additional 0.05 to 5 wt./Vol./o L-threonine, L-homoserine or L-aspartic acid is carried out ao