JPS608119B2 - Method for producing L-sulfur-containing amino acids - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention is directed to L-sulfur-containing amino acids, specifically L-cysteine and a process for producing L-cystine.

Conventionally, L-cysteine and L-cystine have been obtained by hydrolyzing L-cysteine in hair and natural products with a high content of L-cystine with key acid, and isolating and obtaining L-cystine from the resulting amino acid mixture. It is produced by reducing the L-cystine thus obtained.

In contrast, the present inventors have discovered a large number of microorganisms that have the ability to convert lower alkyl esters of 2-aminothiazoline-4-carboxylic acid (hereinafter referred to as ATC) into L-cysteine or L-cystine. As a result of further research based on this knowledge, we have completed a new method for producing L-cystine or L-cystine as described below.

The microorganisms used in the present invention are AT from wild strains existing in nature, strains stored in public microorganism preservation institutions, or microorganisms artificially mutated from these.
It is isolated and collected by examining the presence or absence of cystine or (and) cystine production ability from C lower alkyl esters, such as Achromobacter, Alcaligenes, Bacillus, Brevi/Decterium, Enterobacter, Erwinia, Etschierichia, They are microorganisms belonging to the genera Flavo/Acterium, Micrococcus, Mycoplana, Busoidomonas, Sarcina, or Serratia. More specific examples include the following strains: Sartina lutea AJ-1217 (Sarcj
nalutea) ATCC 272 Achromobacter
Delmarvaa AJ-1983 (AchremeMete
rdelmarvae) FERM-P3483 Alcaligenes denitrophicans AJ-2553 (Ncal
ATCC151
73 Bacmus brevis AJ-1282 (Bacmus
Brevis) ATCC8185 Brevibacterium
Brevibaeterium 1516
fla skin m) ATCC 13826 Enterobacter aerogenes AJ-2643 (En obacterae
rohoko neslAM IOI9) FERM-P 276
4 Erwinia Power Rotobora AJ-12753 (EMin
iacarotvoreCCM873) FERM-P
2766 Busoidomonas thiazolinophyllum AJ
-PseMomonasthiazolinop
hilum) FERM-P 2810Etsushi Erichia Cori AJ-2592
hiacoli lAM I132) FERM-P27
63 Miku0 Coccus sodonensis Yamaichi 1753 (M
iCr ratio oCC female SodonenSiS) ATCCI1
880 Mycoplana dimorpha AJ-2809 (M
ATCC4279
Serratia Marsetusenne AJ-12698 (Sena
tia marcescenelAM 1206) FE
RM-P2765 Flavobacterium acidophilicum AJ-2494 (rimm ac to F1avobac
idific town m) ATCC8366 Busoidomonas obalis AJ-2236 (PSaudome ship so
valiSlAM 1454) FERM Mountain P2762 ATC lower alkyl ester, which is a raw material, is supplied at low cost through a synthetic method and is generally in a racemic form.

However, according to the method of the present invention, both the D-form and the L-form are L-form.
Converted to monocystine or cysteine. As a medium for culturing the above-mentioned microorganisms, a normal nutrient medium may be used as appropriate. For example, as a carbon source, glucose, bran such as sucrose, xylose, and molasses, organic acids such as acetic acid, ethanol, glycerol, and methanol are used. Nitrogen sources such as ammonium sulfate and ammonium chloride; organic nutritional sources such as yeast extract, veptone, meat extract, corn, and stable liquor; inorganic ions such as magnesium, iron, manganese, potassium, sodium, and phosphoric acid; ions such as pyridoxine, pyridoxal phosphoric acid, etc. are used as vitamins.

Cultivation may be carried out by a conventional method, for example, the pH of the medium is 6 to 9.
After inoculation, the cells are cultured aerobically for 1 to 3 days at 20 to 40 qo. The culture obtained in this way may be physically, chemically or biochemically It can be made to act on the ATC lower alkyl ester in the state of destroyed microbial cells, extracts, crude products, purified products, purified protein preparations, or immobilized products such as microbial cells or purified products. The substrate concentration differs depending on whether it is a batch method or a continuous method, but in a batch method, it is generally 0 in the aqueous medium.
．． In a continuous system, it is preferable to lower the concentration slightly from 1 to 30%, preferably about 0.5 to 10%.

The reaction is usually carried out in aqueous soot at a temperature of 15 to 60 mm, preferably around 30 mm to 50 mm, with a range of 6 to 10, preferably 7.0 to 9. It is held near the castle.

The reaction time is not uniform as it varies depending on the means such as static, filtration, and flowing, as well as the form and potency of the enzyme preparation, but in a batch method it is usually about 10 minutes to 7 hours. As the reaction progresses, L-cystine and L-cystine coexist in the reaction solution, but normally L-cystine is easily oxidized by oxygen in the air and changes to L-cystine.
The amount of L-cystine increases over time. However, it is also possible to change the concentration ratio of L-cysteine and L-cystin by changing reaction conditions and the like. Generally, L-cystine can be obtained by a method in which most of the L-cystine is converted to L-cystine by aeration oxidation, and then crystallized using its key solubility.

In addition, in the case of L-cysteine, it can be collected as L-cysteine by processing it by a normal method using electroreduction. In addition, by adding hydroxylamine or semicarbazide to the reaction solution, L-cysteine and/or
Alternatively, the yield of L-cystine can be improved. L
- Cystine and L-cysteine were determined by liquid chromatography and microbial quantification.

The latter is the lactic acid bacterium Leuconostoc titroporum ATC0.
Although this method uses L-cystine, since this bacterium is given some growth by both L-cystine and L-cystine, the sum of both amino acids is quantified and is usually expressed as L-cystine. Note that this bacterium cannot grow on D-form cystine or cysteine. The fact that the enzymatic reaction product from DL-ATC lower alkyl ester is L-cystine is confirmed by the NMR spectrum, X-ray diffraction image, liquid chromatography, and bioassay of the cystine crystals obtained in the examples below. It was estimated from the quantitative value of and data such as specific rotation.

Note that the optical rotation purity was 96.3%. Example The microorganisms shown in Table 1 were added to an enzyme production plant (yeast extract 0.5
%, beptone 0.5%, glycerol 1%, meat extract 0
．． 5%, NaCIO. 5% and 0.2% of DL-2-aminothiazoline-4-carboxylic acid trihydrate.

The enzyme source was a broth obtained by inoculating 1 platinum loopful of the mixture at pH 7.0 (50 broth/500 flasks) and culturing it at 30 qo for 1 hour. One part enzyme solution was mixed with one part aqueous solution of 2% concentration of ATC-killester or ethylester, and a static reaction was carried out at 40°C. After 2 unique moments, Boukon
L-cysteine and L-cysteine produced by bioassay using ocstoccitrovor mATCC8081
- Cystine was quantified and expressed as L-cystine.

Table 1

Claims (1)

[Claims] 1 Sartina sp., Achromobacter sp., Alcaligenes sp., Baltis sp., Brevibacterium sp., Enterobacter sp. , L-cysteine or L-cysteine is produced from 2-amino-thiazoline-4-carboxylic acid lower alkyl ester by the action of microorganisms of the genera Erwinia, Essielibia, Micrococcus, Mycoplana, Serratia, Flavobacterium, and Pseudomonas. 1. A method for producing an L-sulfur-containing amino acid, characterized by producing the following.