CS195554B1 - Method of producing l-lysine by fermentation - Google Patents

Description

The invention relates to a process for the production of L-lysine by fermentation of a liquid culture medium comprising sources of assimilable carbon and nitrogen and a mineral nutrient salt by means of L-lysine-producing microorganisms, in particular Corynebacterium sp. or Brevibacterium sp., under submerged conditions. The most commonly used assimilable carbon source is sucrose, the preferred complex nitrogen source is corn steep liquor, peanut flour hydrolyzates and the like.

Since sucrose is a relatively expensive and scarce raw material, efforts are being made to completely or at least partially substitute it for the production of L-lysine by industrial-scale fermentation. The fermentative preparation of L-lysine using acetic acid, in combination with ammonium acetate and glucose in batches for dosing during fermentation is described in French Pat. No. 2,033,119 and an analogous procedure in German Pat. Further, the fermentation process of L-lysine biosynthesis with Brevibacterium lactofermentum strains is either based on an acetic acid-ammonium acetate mixture or an ethanol-araonic salt mixture as disclosed in German Pat. No. 2,321,461. British Pat. No. 1,300,654 describes the preparation of L-lysine based on acetic acid and ammonium ions with strains of Corynebacterium acetophilum.

The application of two synthetic sources of assimilatable carbon or their combination with other synthetic sources of carbon which can also serve as assimilatable nitrogen sources and with inorganic nitrogen sources in L-lysine biosynthesis has not been described yet.

An important technical and economic task in the process of producing L-lysine by fermentation has been to replace sucrose as a carbon source with sources by others, either wholly or at least partially, while maintaining economically viable yields of L-lysine, extending the fermentation time.

This object is achieved by a process for the production of L-lysine by fermentation of a liquid culture medium containing sources of assimilable carbon and nitrogen and a mineral nutrient salt by means of L-lysine-producing microorganisms, in particular production strains of Corynebacterium sp. or Brevibacterium sp., under submerged conditions, according to the invention, characterized in that the fermentation is started in a culture medium containing, as the only defined source of assimilable carbon, an alkali metal salt of acetic acid, preferably sodium acetate, in an amount of 35 to 45 g / liter of soil, whereupon the pH of the soil is maintained at 6.5-7.5, preferably 6.9-7.2, by adding an aqueous solution of a mixture of assimilable carbon and nitrogen sources in the further course of the fermentation after reaching pH 8.0. containing acetic acid and / or lactic acid and at least one other assimilable carbon source which can simultaneously serve as an assimilable nitrogen source, such as ε-aminocaproic acid, ethanol, sucrose, ammonium acetate, ammonium nitrate, ammonium chloride or urea.

95554

In order to adjust the pH in the further course of the fermentation, an aqueous solution containing, in a volume of 40 ml, for example:

acetic acid conc. 10 ml ammonium acetate 2.5 g Lactic acid conc. 7.5 ml .beta.-aminocaproic acid 10 G water distilled to 40 ml

and / 10 g acetic acid

7.5 g lactic acid

2.5 g ammonium acetate a

10.0 g of ε-aminocaproic acid, or

b / 7.5 G acid dairy or acetic 8 G ethanol and 1,25 G urea, or c / 7.5 G loins of linen dairy or acetic 8 G ethanol, 1,25 G urea and 10 G sucrose or

d / 7.5 8 2.5 g of acetic acid, g of ethanol and g of ammonium chloride, or E/ 7.5 g of acetic acid 8 g e thano1u a 2.5 g of ammonium acetate, or F/ 7.5 g acetic acid, 8 g ethanol and 2.5 g of ammonium nitrate. How is evident ·? of the following examples

in which several possible combinations of synthetic carbon and nitrogen sources are shown, acceptable yields of L-lysine can be achieved. In addition to the nutrient composition, the amount of yield also depends on the production strain used and its adaptability to altered fermentation conditions. As further evident from the examples, complete replacement of sucrose in the broth is possible.

each time the pH reaches about 8.0; enough mixture is added to adjust the pH of the culture to 6.9-7.2. L-lysine production is 20.7-24.8 grams / liter medium / 72 hr fermentation.

Example 1 a d 2

The procedure is as in Example 1 except that the following mixture of nutrient sources is added to the fermentation broth from 6 hours of cultivation in the production phase:

ethanol 10 ml urea 1.25 g lactic acid (or acetic acid) 7.5 ml water distilled to 40 ml

It is dosed analogously to Example 1. The production of L-lysine is 17.7 to 20.7 g / liter of medium / 72 hours of fermentation.

Example 3

The procedure is the same as in Example 1, except that from the 6th hour of the culture in the production phase, it is added to the fermenter.

of soil nutrients: ethanol 10 ml urea 1, 25 G Lactic acid (or acetic acid) 7.5 ml sucrose 10 G water distilled to 40 ml

Example 1

Corynebacterium glutamicum is inoculated with 50 ml of inoculation medium, placed in a 500 ml flask, of the following composition:

sodium acetate sucrose corn liquor 60Z water pH after sterilization g

g 30 g to 1000 ml 6.9 to 7.2

Cultivation is carried out on a rotary shaker (220 rpm) at 29-30 ° C for 22-24 hours, then the pH of the culture is about 7.5. This culture is at 10%. Vol. inoculate 20 ml of fermentation medium, placed in 500 ml boiling flasks of the following composition:

sodium acetate 40 g corn steep liquor 60X 10 g peanut flour hydrolyzate 150 ml ammonium sulphate 10 g potassium phosphate acid 1 g magnesium sulphate crystals. 0.1 g calcium carbonate ground 30 g water to 1000 ml pH after sterilization 7.2

Cultivate on a rotary shaker at 29 to 30 ° C for 72 hours. From 6 hours on culture, the following mixture of nutrient sources is added to the flasks:

It is dosed analogously to Example 1. The production of L-lysine is 26.4 to 32.5 g / liter of medium / 72 hours of fermentation.

Example 4

The procedure is as in Example 1 except that from 6 hours of cultivation in the production phase the following mixture of nutrient sources is added to the fermentation broth:

ethanol 10 ml ammonium chloride 2.5 g acetic acid 7.5 ml water distilled to 40 ml

The dosage is analogous to Example 1. The production of L-lysine is 16.2 to 17.7 g / liter of medium / 48 hours of fermentation.

Example 5

The procedure is the same as in Example 1 except that the following mixture of nutrient sources is added to the fermentation broth from 6 o'clock.

ethanol 10 ml ammonium acetate 2.5 g acetic acid 7.5 ml water distilled to 40 ml

The dosage was similar to Example 1. The production of L-lysine was 19.2 to 22.0 g / liter of medium / 72 hours of fermentation.

Example 6

The procedure is the same as in Example 1 except that from the 6th hour of the culture in the production phase it is added to the fermentation broth.

this mix of resources nutrients: e thano1 10 ml ammonium nitrate 2.5 G acetic acid 7.5 ml water distilled to 40 ml

It is dosed analogously to Example 1. The production of L-lysine is 13.6 to 18.6 g / liter of medium / 48 hours of fermentation.

Claims (7)

CLAIMS 1. A method for producing L-lysine by fermentation of a liquid culture medium containing sources of assimilated carbon and nitrogen and mineral nutrient salts using L-lysine-producing microorganisms, in particular Corynebacterium sp. or Brevibacterium sp. , under submerged conditions, characterized in that the fermentation is initiated in a culture medium containing, as the only defined source of assimilable carbon, an alkali metal salt of acetic acid, preferably sodium acetate, in an amount of 35 to 45 g / liter of soil, during the fermentation, after reaching a pH of 8.0, it maintains the pH of the soil at 6.5 to 7.5, preferably 6.9 to 7.2, by dispensing an aqueous solution of a mixture of assimilable carbon and nitrogen sources containing acetic and / or lactic acid; and at least one other assimilable carbon source, which may simultaneously serve as a source of assimilable nitrogen, such as 6-aminocaproic acid, ethanol, sucrose, ammonium acetate, ammonium nitrate, ammonium chloride or urea, 2.5 g ammonium acetate and 10 g t-aminocaproic acid. OF THE INVENTION 2. A process according to claim 1, wherein the mixture of assimilable carbon and nitrogen sources is an aqueous solution containing in a volume of 40 ml 10 g acetic acid, 7.5 g lactic acid, 3. A process according to claim 1, wherein an aqueous solution containing 7.5 g of lactic or acetic acid, 8 g of ethanol and 1.25 g of urea is used as a mixture of assimilable carbon and nitrogen sources. 4. A process according to claim 1, wherein the mixture of assimilable carbon and nitrogen sources is an aqueous solution containing, in a volume of 40 ml, 7.5 g of lactic or acetic acid, 8 g of ethanol, 1.25 g of urea and 10 g. sucrose. 5. A process as claimed in claim 1, wherein an aqueous solution containing 7.5 g of acetic acid, 8 g of ethanol and 2.5 g of ammonium chloride is used as a mixture of assimilable carbon and nitrogen sources. 6. The process of claim 1, wherein the mixture of assimilable carbon and nitrogen sources is an aqueous solution containing in a volume of 40 ml 7.5 g acetic acid, 6 g ethanol and 2.5 g ammonium acetate. 7. A process according to claim 1, wherein the mixture of the sources of assimilable carbon and nitrogen is an aqueous vehicle containing in a volume of 40 ml 7.5 g acetic acid, 8 g ethanol and 2.5 g ammonium nitrate.