AU628598B2 - Process for producing l-glutamic acid - Google Patents

Description

6285q8

AUSTRALIA

PATENTS ACT 1952 COMPLETE SPECIFICATION Form

(ORIGINAL)

FOR OFFICE USE Short Title: Int. Cl: Application Number: Lodged: t a t Complete Specification-Lodged: Accepted: Lapsed: Published: Priority: Related Art: TO BE COMPLETED BY APPLICANT Name of Applicant: Address of Applicant: Actual Inventor: Actual Inventor: AJINOMOTO CO., INC.

NO 5-8 KYOBASHI 1 CHOME

CHUO-KU

TOKYO

JAPAN

GRIFFITH HACK CO., 601 St. Kilda Road, Melbourne, Victoria 3004, Australia.

Address for Service: Complete Specification for the invention entitled: PROCESS FOR PRODUCING L-GLUTAMIC ACID.

The following statement is a full description of this invention including the best method of performing it known to me:- -2 PROCESS FOR PRODUCING L-GLUTAMIC ACID This invention relates to a process for producing L-glutamic acid and especially to a process which comprises growing a microorganism belonging to the genus Brevibacterium or the genus Corynebacterium, resistant to prumycin and derivatives thereof, and capable of producing L-glutamic acid; and recovering L-glutamic acid formed and accumulated in the culture liquor. The invention also relates to novel microorganisms which are useful in this process.

DETAILED DESCRIPTION OF THE INVENTION (Technical Field) L-glutamic acid is an important amino acid useful as a seasoning and for other purposes. This invention provides an improved fermentation process for producing this amino acid.

9 t (Prior Art) Already known are the fermentation methods for producing L-glutamic acid, which employ a wild strain of Brevibacterium or Corynebacterium, a strain resistant to glutamic acid analogues, a strain resistant to respiratory inhibitors, such as keto-malonic acid and fluoroacetic acid, or a strain resistant to esculetin.

(Problem to be Solved by the Invention) The object of this invention is to enhance the I fermentation yield of L-glutamic acid, thereby reducing its production cost.

(Means to Solve the Problem) Studies to solve the above-mentioned problem have led us to find that L-glutamic acid can be produced with a higher yield by the use of mutants resistant to prumycin and derivatives thereof and derived from the already known, L-glutamate-producing microorganisms Brevibacterium and Corynebacterium. Prumycin is an antibiotic produced by microorganisms belonging to the genus Bacillus.

3 Listed below are some examples of the strains of Brevibacterium and Corynebacterium resistant to prumycin and derivatives thereof used in this invention.

Brevibacterium lactofermentum AJ 12475(FERM P-10827, FERM BP-2922) Brevibacterium lactofermentum AJ 12476(FERM P-10828, FERM BP-2923) Brevibacterium flavum AJ 12477(FERM P-10829, FERM BP-2924) Corynebacterium glutamicum AJ 12478(FERM P-10830, FERM BP-2925) o Deposits of these strains under the Budapest Treaty were lodged in the Fermentation Research Institute of the Agency o o lo \c' S0 for Industrial Science and Technology onAi-ts MsSS, under the above-mentioned accession numbers.

SThese strains have been derived from Brevibacterium lactofermentum ATCC 13869, Brevibacterium flavum ATCC 14067 and Corynebacterium glutamicum ATCC 13032, respectively, by mutation. All of the starting strains were available to the public before 19th July 1989.

0eo0 0 The mutation may be effected by ordinary methods, such s o as irradiation with ultraviolet rays or X-rays, or by treatment with a mutagen (for example, treatment with 200 pg/ml nitrosoguanidine at 0°C for 20 minutes).

'Table 1 shows the experimental result on the prumycin resistance of these strains.

1 j X I lli ~plr irrrwur~-- la~QPI'IL-~Y 4- Table 1 0 er t000 Brevibacterium Brevibacterium Corynebacterium lactofermentum flavum glutamicum prumycin (mg/ml) ATCC AJ AJ ATCC AJ ATCC AJ 13869 12475 12476 14067 12477 13032 12478 0 100 100 100 100 100 100 100 2 51 115 110 40 100 53 105 3 23 110 110 0 98 2 99 4 8 105 104 0 88 0 6 100 90 0 70 0 1j *i '0 L-glutamic acid is produced by using these strains as ,i described below. Cultivation is carried out by using a liquid medium containing saccharic materials (such as sugar juice or waste molasses of sugar canes or beets, and starch hydrolyzates) and non-saccharic materials (such as acetic S°o acid, ethanol and paraffin). Nitrogen sources which may be o used include ammonium salts, ammonia water and urea (compounds used in ordinary L-glutamic acid fermentation), as °0 well as CSL and amino acids obtained by protein degradation.

In addition, inorganic salts (such as phosphates and magnesium salts) and trace nutrients (such as thiamin and biotin) may also be used as required. Furthermore, inhibitory substances against the action of biotin (such as polyoxysorbitan monopalmitate and penicillin) may also be added to the medium when required. Cultivation should preferably be carried out under aerobic conditions at a temperature of 240 to 370C while maintaining the pH within the range from 6 to 9 by the use of an inorganic or organic acid or base, urea, calcium carbonate or ammonia gas.

L-glutamic acid accumulated in the fermentation liquor can be recovered by an appropriate combination of ion-exchange resin treatment and other known techniques.

i;; 5 The growth of the strains listed in Table 1 was examined according to the procedure described below. Each strain was slant cultured in natural medium (containing 1 g/dl peptone, 1 g/dl yeast extract and 0.5 g/dl NaC1; pH for 24 hours, a suspension of the grown cells in sterile water was inoculated to a medium containing 0.5 g/dl glucose, 0.15 g/dl urea, 0.15 g/dl ammonium sulfate, 0.3 g/dl KH 2 P0 4 0.1 g/dl K 2 HP0 4 0.01 g/dl MgSO 4 .7H 2 0, 0.1 mg/dl CaC1 2 .2H 2 0, pg/dl thiamin hydrochloride, 3 pg/dl biotin, 0.44 mg/dl Na 2

B

4 07.10H 2 0, 4.85 mg/dl FeC1 2 .6H 2 0, 1.95 mg/dl CuSO 4 .5H 2 0, 0.185 mg/dl (NH 4 2 Mo70 24 .4H 2 0, 44 mg/dl ZnSO 4 .7H 2 0, 0.36 mg/dl MnCl 2 .4H 2 0, and prumycin in an amount shown in the a" table (pH and cultivation was continued for 24 hours.

0o o 0 0 S EXAMPLE 4o e 9 Example 1 A culture medium containing 10 g/dl glucose, 0.1 g/dl

KH

2

PO

4 0.1 g/dl MgSO 4 .7H 2 0, 20 pg/dl thiamin hydrochloride, 36 mg/dl concentrate of bean degradation solution, 2.0 g/dl ammonium sulfate, 1 mg/dl FeSO 4 .7 2 0, 1 mg/dl MnSO 4 .4H 2 0 and 3 ug/dl biotin (pH 7.0) was prepared, and 20 ml of the medium thus obtained was placed in each of 500-ml shake flasks and sterilized by heating at 115 0 C for 0 0 ten minutes. Each of the strains was inoculated into the sterilized medium and grown at 31.5 0 C by using a reciprocating shake culture machine.

The pH of the culture liquor was maintained within the S0', range from 6.5 to 8.0 by addition of 5 g/dl calcium carbonate; 2.5 g/dl ammonium sulfate was added after 24 hours, fermentation was terminated after 46 hours, and the yield of L-glutamic acid accumulated was measured.

The result obtained is summarized in Table 2.

L

6 Table 2 Total Yield of L-Glutamic Acid Brevibacterium lactofermentum ATCC 13869 48 AJ 12475 54 AJ 12476 51 Corynebacterium glutamicum ATCC 13032 45 AJ 12478 48 00 00 0 000 0 0090 09 0 00 0 0 90 0 00 0 00 5 Example 2 A culture medium containing 100 mg/ml beet molasses (as reducing sugar) and 1 mg/dl KH 2

PO

4 (pH 7.0) was prepared, and 30 ml of the medium thus obtained was placed in each of 500-mi shake flasks and sterilized by heating at 115°C for ten minutes. Each of the strains was inoculated into the sterilized medium and grown at 31.5°C by using a reciprocating shake culture machine, and the pH of the culture liquor was maintained within the range from 6.5 to by addition of 400 mg/dl aqueous solution of urea.

Polyoxysorbitan monopalmitate was added to a concentration of 4 mg/ml when the cells were grown to a predetermined level and fermentation was terminated 30 hours after inoculation, and the yield of L-glutamic acid accumulated was measured.

The result obtained is summarized in Table 3.

I I -7 Table 3 Total Yield of L-Glutamic Acid Brevibacterium lactofermentum ATCC 13869 57 AJ 12475 61 AJ 12476 62 Brevibacterium. flavum ATCC 14067 56 AJ 12477 60 Corynebacterium glutamicum ATCC 13032 53 AJ 12478 58 o 6 4. d* 00 0 -0 0 It will be clearly understood that the invention in its general aspects is not limited to the specific details ref erred to hereinabove.

Claims (7)

1. A microorganism which is capable of producing L- glutamic acid and which is resistant to prumycin and derivatives thereof, produced by the step of subjecting an initially L-glutamic acid-producing microorganism of the genus Brevibacterium or of the genus Corynebacterium to a mutagenic stimulus, and selecting mutants which are resistant to prumycin or a derivative thereof. s o a S 10

2. A microorganism according to Claim 1, wherein the S°"initially L-glutamic acid-producing microorganism is selected from the group consisting of Brevibacterium o. lactofermentum ATCC 13869, Brevibacterium flavum ATCC 14067, and Corynebacterium glutamicum ATCC 13032. 15

3. A microorganism according to Claim 1 or Claim 2, selected from the group consisting of Brevibacterium lactofermentum FERM BP-2922, S" Brevibacterium lactofermentum FERM BP-2922 Brevibacterium lactofermentum FERM BP-2923 0:00 •Brevibacterium flavum FERM BP-2924, and Corynebacterium glutamicum FERM BP-2925, as hereinbefore described.

4. A process for producing L-glutamic acid, comprising the steps of growing a microorganism of the genus Brevibacterium of the genus Corynebacterium which is resistant to prumycin and derivatives thereof, and which is capable of producing L-glutamic acid, and recovering L- glutamic acid from the culture liquor.

5. A process according to Claim 4, in which the microorganism is as defined in any one of Claims 1 to 3. i -r j- Eurerc~---I~~U~ 9

6. L-glutamic acid produced by the process of Claim 4 or Claim

7. A process substantially as hereinbefore described with reference to the examples. DATED THIS 30TH DAY OF JUNE 1992 AJINOMOTO CO., INC. 10 By Its Patent Attorneys: o GRIFFITH HACK CO SFellows Institute of Patent S 15 Attorneys of Australia a 0 o e e os o 0 0 0 LL