KR850001231B1 - Process for preparing l-lysine utilizing microorganism - Google Patents

Abstract

L-lysine was produced by fermentation using a microorganism having lysine-analogue resistance, threonine-resistance and homoserine, leucine and valine triple auxotrophic nutrients. Corynebacterium glutamicam ATCC 13286 was mutagenized with NTG and selected on a minimal agar medium containing AEC 0.3%. The selected analogue- resistant mutant was mutagenized again at 40≰C for 30 min. The mutagenized strain was cultured at 45≰C for 5 days to select a thermo-resistant mutant having high lysine-producing ability. The corynebacterium mutant having various properties was fermented to produce L-lysine of 50g/L at 40≰C.

Description

Method for producing L-lysine by microorganism

The present invention is a method for producing L-lysine by fermentation using a microorganism having lysine analog resistance and heat resistance and at the same time specifically requiring homoserine leucine valine for growth.

L-lysine is a kind of essential amino acid that must be supplied to humans and animals whose grains are staple foods, and is used as a raw material for medicines, foods, feeds, etc., and the global demand is increasing day by day.

To date, a method for producing L-lysine by fermentation is well known. Examples of the method for preparing lysine using nutrient-constituting mutants include Korean Patent Publication No. 73-126, Japanese Patent No. 48-28677, and US Patent 3524797. British Patent No. 1258380, Japanese Patent No. 53-1833, Sub 54-34835 and the like are known.

However, most of the well-known methods are fermented at about 30 ℃, which is a moderate temperature fermentation. In tropical regions such as Thailand, the Philippines, Taiwan, Indonesia, and India, where the atmospheric temperature is high, normal fermentation temperature is virtually impossible, and thus enormous scale fermentation. In addition, in Korea, fermentation in summer requires a considerable amount of cooling facilities, making it difficult to reduce costs in the production of L-lysine, and moreover, thermal shock when instant fermentation temperature rises during fermentation. Abnormal fermentation phenomenon and fermentation conversion were caused by the appearance of return mutant strain by (Heat Shock).

With this in mind, the present inventors have found that the strains that specifically require homoserine and leucine valine as strains having lysine analog resistance and temperature resistance during the heat resistant strain breeding experiment in order to develop a method for producing lysine by high temperature fermentation method. Not only was the lysine production rate high in fine cultures, but lysine was accumulated at that concentration and the present invention was completed.

The strain used in the present invention is the mutant strain Koranbacterium sp. TR-3579- (KFCC 10065) induced by mutation treatment with the parent strain of Koranbacterium glutamikim ATCC 13286 having lysine production ability. This mutant strain was bred in the following manner. A microbial solution treated with Corinbacterium glutimimim ATCC-13286 in 500 μg / ml concentration of N-methyl-N'-nitro-N-nitroso guanidine for 30 minutes at 37 ° C was placed on the agar plate of a minimal medium having the following composition: After plating, the cells were incubated at 37 ° C. for 4-5 days to obtain lysine analog and temperature resistant strains.

N-methyl-N'-nitro of 500μg / ml concentration in two steps to select the mutant strains from which the L-lysine production was recognized from the collected primary mutant strains to obtain nutritive constitutive mutants with excellent heat resistance and yield of L-lysine Treat for 30 minutes at 40.5 ° C. in -N-nitrosoguanidine. The second strain, TR-3579 (KFCC 10065), was obtained from colonies (Colony), which appeared by smearing the treated bacteria solution on a minimal medium agar plate containing nutrient-containing substances and incubating at 40.5 ° C for 5 days. Lysine production ability according to the culture temperature is shown in Example 1).

The secondary mutant strain TR-3579 (KFCC 10065) specifically required homoserine, leucine, and valine for growth, and their addition in moderation showed remarkable lysine production ability. The growth results of nutrients of mutant strain TR-3579 (KFCC 10065) are as follows.

The growth rate was measured by dispensing 5 ml of the basic medium with the following composition into a 50 ml triangular flask to sterilize and then inoculate one platinum tooth of TR-3579 (KFCC 10065) strain grown on an agar nutrient medium. The absorbance is measured at 570 μm with a 26-fold dilution of the cultured solution.

As described above, the mutant strain Corinbacterium TR-3579 (KFCC 10065) of the present invention is lysine analog resistant and heat resistant and is not affected by the metabolite during fermentation, and has excellent lysine formation at high temperature fermentation. It is suitable as a strain for lysine production in tropical regions with high atmospheric temperature because there is no return mutant caused by hoat shok, and it is possible to manage temperature effectively with small-sized cooling facilities even under summer temperature in Korea. Not only is it very convenient, it is also expected to make a significant contribution to the price reduction of lysine production.

As a result of identifying strain characteristics of the mutant strain Corinbacterium species TR-3579 (KFCC 10065) according to the Burgess manual is as follows.

1. Morphological Properties 1) Form: Bacillus 3) Gram Dyeing: Positive

2) Size (μ): 0.6-0.8 × 1.0-2.0 4) Mobility: None

2. Physiological properties 1) Growth temperature: 13 ℃ -48 ℃ Glucose: Yes

2) Optimal growth temperature: 37 ℃ -45 ℃ Sugar: Yes

3) Growth pH: 4.5-9.0 Fructose: Yes

4) Optimal growth: 7.3-7.5 Malt sugar: Yes

5) Death temperature: 63 ℃ × 5 minutes Galactose: Yes

6) Oxygen Requirements: Aerobic Mannose: Yes

7) Methylred: Positive Lactose: Yes

8) Pigment Production Capacity: Positive Raffinose: None

9) Gas Production Capacity: None Arabinos: None

10) urease: benign xylose: none

11) Catalase: Cationic Starch: None

12) Taamino acid production: yes ylin: no

13) Penicillin Resistance: Yes Inositol: No

14) Acid-producing ability of aspartic acid from carbon source: Yes

Succinic Acid: None

Citric Acid: None

Lactic Acid: None

3. Culture Properties 1) Juicy agar plate culture: good growth, round, slightly elevated

2) Juicy agar slope culture: Good growth

3) Juicy agar puncture: surface growth

4) Gelatin culture: no liquefaction

4. Comparison of parent strain Corinbacterium tolutamikim ATCC 13286 with variant strain Corinbacterium fish TR 3579 (KFCC 10065)

On the other hand, in the production of L-lysine by the present mutant strain Corinbacterium TR-3579 (KFCC 10065), sugars such as glucose, molasses and starch hydrolyzate are used as carbon sources, and ammonium sulfate, ammonium acetate ammonium phosphate, urea as nitrogen sources. , Ammonia and corn steep liquor, soybean meal hydrolyzate and the like can be used.

In performing the main culture, fermentation conditions are as follows.

Aeration rate is 0.8-1.6VVM, stirrer speed is 450RPM, fermentation temperature is incubated for 45-50 hours in the 38 ℃ -45 ℃ range. During incubation, pH is 7.0-7.3 and automatically adjusted with ammonia gas. L-lysine collected in the culture was crystallized with L-lysine hydrochloride using a conventional ion exchange resin method, and lysine analysis was performed by a bioassay method using Esch-erichia-col: Mutant LA-404. It was.

The present invention will be described in detail with reference to the following Examples.

Example 1

A medium having the following composition is prepared.

Glucose 13% Ammonium Sulfate 4%

Monophosphate 0.1% Magnesium Sulfate 0.04%

Fe Ion 2ppm Mn Ion 2ppm

Biotin 50μg / ℓ Chiamine Hydrochloride 300ug / ℓ

Corn Dipping 0.6% Soybean Paste 0.4%

Calcium Carbonate 5% pH 7.3

Dispense the medium into 500 ml shake flasks, each 20 ml and sterilize at 115 ° for 15 minutes and inoculate 100 ml of TR-3579 strain grown on agar nutrient medium and incubate for 96 hours at 40 rpm at 120 rpm. The culture broth contains 47.2 g / L of L-lysine. Meanwhile, L-lysine accumulation degree according to the culture temperature of the parent strain and the present invention mutant strain is shown in the following table.

Example 2

Prepare a medium having the following composition.

Molasses (as reducing equivalent) 6% Ammonium sulfate 1.2%

Monophosphate 0.05% Magnesium Sulfate 0.04

Fe · Mn ion 2ppm chimine hydrochloride 130μg / ℓ

Corn Dipping 0.8% Soybean Digestion 0.3%

β-alanine 13μg / L antifoam 0.02%

pH 7.1

12 l of the medium having the above composition was injected into a 30 l fermentation tube, sterilized at 115 ° C. for 15 minutes, and then inoculated with 4% of TR-3579 (KFCC 10065) seed culture medium grown in nutrient medium before inoculation at 38 ° C.-45 ° C. High temperature fermentation is performed for 50 hours while automatically adjusting to pH 7.2 ± 1 using ammonia gas in the temperature range.

Aeration rate is 1 vvm and the rotation speed of the stirrer is maintained at 450 rpm.

After 6 hours of fermentation, an additional amount of additional veggies having the following composition is periodically supplied to adjust the nutritional balance in the feed solution.

* Additional Vegetation

Molasses (in reducing equivalents) 40% Ammonium sulfate 2%

Corn Dipping Solution 1.4% Soybean Digestion Solution 0.5%

Monophosphate 0.05% Magnesium Sulfate 0.04%

Antifoam 0.4% pH 5.5

The fermentation broth produced and accumulated 51.8 g / l of L-lysine.

Example 3

The high temperature fermentation is carried out in the same manner as in Example 2) using a veggie added to the vegetation composition of Example 2 at a concentration of 0.005% lysine and 0.02% homoserine at 0.02% each.

59.9 g / l was produced and accumulated in the fermentation broth.

Claims (2)

Microbial Corinbacterium SPZ TR-3579 (KFCC 10065), belonging to the genus Corinthium, having lysine analog resistance and temperature resistance, and specifically requiring homoserine, leucine, and valine for growth, Method for producing L-lysine by the fermentation method for producing lysine. The method for preparing L-lysine by high temperature fermentation according to claim 1, wherein the culture temperature is adjusted to 37 ° C-45 ° C.