JPS6123997B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention provides β-hydroxy-β-carboxy-
The present invention relates to a method for producing L-leucine by fermentation by culturing a microorganism capable of producing L-leucine from IPM in a medium containing isocaproic acid (hereinafter abbreviated as IPM).

The purpose of the present invention is to produce L-leucine, which is a type of amino acid that is important as an essential amino acid nutritionally and medicinally, and which has recently come to be used for seasonings, feeds, etc., by a fermentation method. The object of the present invention is to provide a manufacturing method that is economically advantageous.

Conventional methods for producing L-leucine using fermentation methods include using bacteria that require amino acids such as isoleucine and methionine, and bacteria that are resistant to 2-thiazolealanine, but the metabolic control is complicated. There were some difficulties in terms of productivity.

As a result of various studies on a more advantageous industrial production method of L-leucine using microorganisms, the present inventors found that a microorganism belonging to the genus Brevibacterium or Corynebacterium and having the ability to produce L-leucine from IPM. The present invention was completed based on the discovery that when cultured in a medium containing IPM, IPM is converted to L-leucine and a significant amount of L-leucine is produced and accumulated in the medium.

Although IPM is easily produced by a fermentation method, IPM recovered from fermentation broth or broth containing IPM can be appropriately added to the medium used in the method of the present invention. The timing of addition can be determined as appropriate depending on the ability of the microorganisms used, but it is usually best to add them after the microorganisms have grown considerably.

The production medium for leucine fermentation in the present invention contains conventional carbon sources, nitrogen sources, other inorganic and organic nutritional substances, and substances required by the bacteria used. As carbon sources, for example, not only monosaccharides and disaccharides such as glucose and sucrose, but also carbohydrates and hydrolysates such as starch, organic acids such as acetic acid, alcohols, and even hydrocarbons can be used depending on the bacteria. The nitrogen source may be a synthetic medium or a natural medium containing an ordinary inorganic or organic nitrogen source, nutrients or growth promoters for fermentation bacteria, inorganic salts, etc., as appropriate. Also, normal fermentation conditions may be used. In other words, aerated culture is best, the fermentation temperature is 24-37℃, and the number of fermentation days is usually 2-7.
It is day. The pH at the start of fermentation and during cultivation is preferably 5 to 9, and acidic or alkaline substances such as inorganic or organic acids, as well as urea, calcium carbonate, ammonia gas, etc., can be used to adjust the pH. IPM can be added by directly adding it in powder form and culturing it while controlling the pH, or by dissolving and neutralizing it and then adding it, either method may be used.

L-leucine can be collected from the fermentation broth by a combination of the ion exchange resin method and other known methods. Quantification of L-leucine was carried out by a bioassay method using Leuconostoc mesenteroides ATCC8042. The present invention will be specifically explained below with reference to Examples.

Example 1 Glucose 13g/dl, Ammonium sulfate 3g/dl,
KH ₂ PO ₄ 0.1g/dl, MgSO ₄・7H ₂ O0.04g/dl,
FeSO ₄・7H ₂ O1.0mg/dl, MnSO ₄・7H ₂ O1mg/dl
dl, biotin 100γ/dl, thiamine hydrochloride 350
γ/, soybean hydrochloric acid hydrolysis concentrate 0.5ml/dl, methionine 0.07g/dl and CaCl ₂ 2H ₂ O 0.4g/dl
(PH7.0 neutralizer KOH) to 300ml of medium
The mixture was dispensed into small jars and sterilized. Brevibacterium lactofamentum FERM-, which had been previously cultured at 30°C on a bouillon slant for 24 hours.
P2516 was inoculated and cultured at 31.5°C, aeration rate of 150 ml/min, and stirring at 1000 rpm. During culture, the pH was controlled at 7.0 with ammonia gas, and after 24 hours of culture, it was added to the IPM medium at a concentration of 1 g/dl and cultured for 60 hours. The accumulation of L-leucine in the medium after completion of the culture was 4.2 g/dl. On the other hand, it was 3.7 g/dl without IPM addition.

Example 2 Using the same medium as in Example 1, after 24 hours of culture
Added IPM3g/dl and glutamic acid 3g/dl 60
Cultured for hours. The amount of L-leucine accumulated in the medium after completion of the culture was 6.1 g/dl.

Example 3 Corynebacterium glutamicum FERM-
As a result of culturing P2518 in the same manner as in Example 1, 3.7 L-leucine was found in the medium after 72 hours of culture.
g/dl production and accumulation. On the other hand, 3.3g/dl without additives
It was an accumulation of

Example 4 Glucose 13g/dl, Ammonium sulfate 4g/dl,
KH ₂ PO ₄ 0.1g/dl, MgSO ₄・7H ₂ O0.04g/dl,
MnSO ₄ 7H ₂ O1mg/dl, FeSO ₄ 7H ₂ O1mg/dl, biotin 100γ/, thiamine hydrochloride 200γ/,
Soybean hydrochloric acid hydrolysis concentrate 1.1ml/dl, CaCl ₂ .
Medium 300 with a composition of _2H2O0.4g /dl (PH7.0)
ml was dispensed into 1-volume small jars and sterilized. Brevibacterium flavum cultured in advance
FERM-P420 (2-thiazole alanine resistance)
was inoculated and cultured for 60 hours using the same culture method as in Example 1. The amount of L-leucine accumulated in the culture solution is 2.0
g/dl. On the other hand, without additives, it was 1.6 g/dl.

Example 5 Glucose 10g/dl, KH ₂ PO ₄ 0.1g/dl,
MgSO ₄・7H ₂ O0.1g/dl, urea 0.6g/dl,
FeSO ₄・7H ₂ O1mg/dl, MnSO ₄・7H ₂ O1mg/dl,
Thiamine hydrochloride 100γ/, biotin 3γ/
dl, soybean hydrochloric acid hydrolyzate (TN) 36mg/ml, (PH
A medium having a composition of 7.0 (neutralizing agent KOH) was dispensed into small 300 ml jars and sterilized. Brevibacterium lactofamentum cultured in advance on this
Inoculated with ATCC13869, 28℃, aeration volume 150ml/
The cells were cultured under conditions of 1,000 rpm and stirring at 1,000 rpm. During culture, the pH was controlled to 7.0 with ammonia gas, and after 24 hours, 1 g/dl of IPM was added and cultured for 72 hours. The amount of L-leucine accumulated in the medium after completion of the culture was 0.34 g/dl. On the other hand, without IPM addition
It was 0.01g/dl.

Example 6 Glucose 13g/dl, Ammonium sulfate 2g/dl,
KH ₂ PO ₄ 0.15g/dl, K ₂ HPO ₄ 0.05g/dl,
MgSO ₄・7H ₂ O0.05g/dl, phenylalanine 150
γ/ml, isoleucine 100γ/ml, methionine 100
γ/ml, valine 100γ/ml, bithione 100γ/,
Thiamine hydrochloride 1000γ/, CaCl ₂・2H ₂ O0.4
300 ml of medium with a composition of g/dl (PH7.2)
The mixture was dispensed into small jars and sterilized. Corynebacterium glutamicum cultured in advance
Inoculate ATCC21301 (having relative requirement for isoleucine, methionine, phenylalanine and/or valine) at 28℃, aeration volume 150ml/
The cells were cultured under conditions of 1,000 rpm and stirring at 1,000 rpm. During culture, the pH was controlled to 7.0 with ammonia gas, and after 24 hours of culture, 2 g/dl of IPM was added and cultured for 72 hours. The amount of L-leucine accumulated in the culture solution was 2.14 g/dl. On the other hand, without IPM addition
It was 1.34g/dl.

Claims (1)

[Claims] 1. Microorganisms belonging to the genus Brevibacterium or Corynebacterium and having the ability to produce L-leucine from β-hydroxy-β-carboxy-isocaproic acid are grown in a medium containing β-hydroxy-β-carboxy-isocaproic acid. A method for producing L-leucine using a microorganism, which comprises culturing L-leucine using a microorganism and collecting L-leucine produced and accumulated in the culture solution.