JPS62265989A - Production of l-arginine by fermentation - Google Patents

Abstract

PURPOSE:To produce L-arginine, by culturing an L-arginine-producing microbial strain in a medium added with a carbon source substrate in a manner to maintain the carbon source concentration and lactic acid concentration in the medium within respective specific ranges. CONSTITUTION:A glutamic acid-producing Coryne-type bacterial strain is cultures in a nutrient medium used in the fermentative production of amino acids while maintaining a carbon source concentration to <=3% or a lactic acid concentration to <=2g/l by continuoulsy or intermittently adding a carbon source having a concentration of 20-40% to the medium when the concentration of the initial carbon source substrate is decreased to <=3% (w/v in terms of glucose). The liquid to be added to the medium contains about 3% ammonium sulfate in addition to the carbon source. The culture is carried out preferably under aerobic condition (agitation at 600-700rpm and aeration rate of 0.5-1.5l 1/min) preferably at 20-40 deg.C and preferably nearly neutral state for 1-5 days. L-arginine can be produced in high yield.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of the Invention The present invention relates to a method for producing L-arginine by fermentation.

L-arginine is an amino acid that has a variety of uses in a wide range of fields, including medicine, food, animal feed, and more.

Conventional technology Conventionally, as a method for producing L-arginine by fermentation using a strain belonging to the coryneform glutamic acid-producing strain, there is a method using a mutant strain with the ability to produce L-arginine derived from a wild-type strain. Are known.

Examples of L-arginine-producing mutant strains include arginine analog-resistant strains or strains in which a nucleic acid analog has been added to them [Agricultural Biological Chemistry (Agr, Biol, Chem, ), 36.1675
(1972>, Journal of General and Applied Microbiology (J, Gen.

Appl, Microbiol, H9,339 (19
73), Japanese Patent Publication No. 57-50479], etc. are known. However, there is little knowledge regarding the culture conditions, and it has been reported that when the agitation rate during culture in a shear fermenter is set low to lower the degree of oxygen sufficiency, the yield of L-arginine decreases and the accumulation of lactic acid increases. [Journal of Fermentation - John Chikunoroshii (J, Ferm
ent, Technol, ) 57.321 (1979
) There is no example in which E improves the production of L-arginine by feeding a carbon source.

Problems to be Solved by the Invention There is always a desire to develop a method for industrially producing L-arginine at low cost, which has various uses in a wide range of fields such as medicines, foods, and animal feeds.

Means for Solving the Problems The present inventor has conducted various studies on culture conditions for producing L-arginine using an L-arginine producing strain. As a result, the carbon source substrate was added so that the concentration of the carbon source substrate in the culture solution after consumption of the initially added carbon source substrate was maintained at 3% or less (W/V, glucose equivalent), and the lactic acid concentration was maintained at 2 g/β or less. The present inventors have discovered that the productivity of L-arginine can be significantly improved by culturing in this manner, and have completed the present invention.

The present invention will be explained in detail below.

In the present invention, when producing L-arginine by culturing L-arginine-producing bacteria in a medium, after consuming the initially added carbon S source substrate, a carbon source substrate is fed and cultured to produce L-arginine in the culture. Provided is a method for producing L-arginine by a fermentation method, which is characterized by accumulating L-arginine and collecting L-arginine from the culture.

In the method of the present invention, the carbon source substrate concentration in the medium is 3% or less (W/V, glucose equivalent), or the lactic acid concentration is 2% or less.
g/j! If the carbon source substrate is fed so as to maintain the following, L-arginine can be produced with higher yield.

To maintain the carbon source substrate concentration below 3% (1!l/V, glucose equivalent), from the time the initial carbon source substrate is consumed below 3%, a high concentration carbon source is continuously or intermittently added. Add the substrate solution.

The substrate concentration of the fed carbon source is usually 20-40% (W/V
) is used. The flow acceleration during continuous feeding may be determined as appropriate depending on the substrate concentration, culture volume, etc.

The fed-batch solution usually contains 3 ammonium sulfate in addition to the carbon source.
Contain about %. Other nutrients used in the fermentation production of amino acids can be added as appropriate and used as a fed-batch solution.

Any microorganism that can be used in the present invention can be used as long as it has the ability to produce L-arginine. For example, the following microorganisms belonging to coryneform glutamic acid-producing bacteria are suitable examples.

To produce L-arginine in the method of the present invention, a medium generally used for fermentative production of amino acids is used. That is, as shown in the Examples, either a synthetic medium or a natural medium can be used as long as the medium contains a suitable amount of a nitrogen source, inorganic substances, and other nutrients in addition to the main carbon source. As a carbon source,
Saccharides such as glucose, synicrose, starch, starch hydrolysates, and blackstrap molasses, various organic acids such as acetic acid and fumaric acid, and alcoholic acids such as ethanol and methanol can be used. Nitrogen sources include ammonia, ammonium salts, urea, ammonia gas, hefton, meat extract, yeast extract, corn steep liquor, casein hydrolyzate, fitschmeal or its digested product, defatted soybean meal or its digested product, Various natural products such as tub hydrolyzate can be used. Inorganic substances include potassium phosphate,
Magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, etc. can be used. The nutrients required by the microorganisms used for growth must be determined by adding appropriate amounts of nutrient sources to the culture medium to satisfy their requirements (although these substances must not be included in natural products that can be used as nitrogen sources). Sometimes added.

Culture was carried out under aerobic conditions (agitated at 600 to 700 rpm).
The culture is preferably carried out at an aeration rate of 0.5 to 1.51/m1n), and the culture temperature is generally preferably 20 to 40°C, but it may be carried out at a pond temperature as long as the temperature allows the bacteria to grow. It is desirable to maintain the pH in the culture solution near neutrality;
For adjustment, inorganic or organic acidic or alkaline substances, urea, ammonia gas, calcium carbon, etc. can be used. The number of days for culturing is usually 1 to 5 days. A conventional method such as an ion exchange resin method is used to recover L-arginine from the culture solution.

Examples of the present invention are shown below.

Example 1 Corynebacterium acetoacidophilum H-431
0 (FERM BP-1 (114)) was placed in a 21-volume flask and cultured at 30°C for 48 hours using 25 Qml of the following field medium.Meanwhile, 70 Qml of the following main fermentation medium was placed in a 21-volume jar flask and cultured for 48 hours. , the above seed culture solution 90
m1 was inoculated into this.

Adjust the pH to 6.6 under aerobic conditions (stirring number 700 r).
pm, ventilation amount IF! /min>, and culture was performed at 30°C.

At the time (15 hours later) when 70 g of blackstrap molasses/jl glucose equivalent) in the initial medium was consumed, the following fed-batch medium 700 g
Table 1 shows the results when culturing was carried out by adding the entire amount of ml at one time and when culturing was carried out by continuously adding small amounts.

Seed Medium Glucose 50g#Bebuton 10
g/fl yeast extract Log/j! N a C12.5g/j2 Biotin 50■/l
Urea 3 g/l Corn steep liquor 5 g/2p H7,2 Main fermentation medium (initial medium) Waste U Honey 70 g/l KH2Po
, 0.5g #ammonium sulfate
38g/(IMgSOa・7H200,5g/j2 F e So, ・7 H2O10mg/j! Thiamine hydrochloride 100■/l p 8 7.2 Fed-batch molasses 400g//!Ammonium sulfate 30g/(1 molasses concentration Both concentrations were in terms of glucose, and sterilization was performed at 120°C for 30 minutes.

Continuous addition of fed-batch medium maintained the carbon source substrate concentration between 0.5 and 3%, suppressed lactic acid accumulation, and significantly improved L-arginine yield.

Effects of the Invention According to the present invention, it is possible to obtain L-arginine with a high yield.

Claims (3)

[Claims] (1) When L-arginine-producing bacteria are cultured in a medium to produce L-arginine, after consuming the initially added carbon source substrate, the carbon source substrate is fed and cultured to produce and accumulate L-arginine in the culture. 1. A method for producing L-arginine by a fermentation method, characterized in that L-arginine is collected from the culture. (2) The scope of the patent is characterized in that the culture is carried out in such a way that the concentration of the carbon source substrate in the culture solution after consumption of the initially added carbon source substrate is maintained at 3% or less (W/V, glucose equivalent). The manufacturing method described in paragraph 1. (3) The production method according to claim 1 or 2, characterized in that the culture is carried out so that the lactic acid concentration in the medium is maintained at 2 g/l or less.