JPS5928485A - Preparation of l-cysteine - Google Patents

Abstract

PURPOSE:To prepare efficiently L-cysteine by the action of a microorganism, by using 2-aminothiazoline-4-carboxylic acid as a raw material. CONSTITUTION:A microorganism, e.g. Achromobacter delmarvae (FERM-P No. 3483), belonging to the genus Achromobacter, Alcaligenes or Bacillus, etc. and having the ability to hydrolyze 2-aminothiazoline-4-carboxylic acid to produce L-cysteine or L-cystine is reacted with an aqueous solution of the 2-aminothiazoline-4-carboxylic acid in 0.1-30% substrate concentration containing 2-40% acetone to produce 2,2-dimethylthiazolidine-4-carboxylic acid, which is then hydrolyzed under acidic conditions to produce the aimed L-cysteine.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for efficiently producing L-cysteine using the action of microorganisms.

Conventionally, L-cysteine (hereinafter abbreviated as cysteine) and L-cystine (hereinafter abbreviated as cystine) have been used in hair, etc., which have a high content of cysteine and cysteine.

Cystine was produced by hydrolyzing natural substances with mineral acids, separating cystine from the resulting amino acid mixture, and reducing cystine thus obtained.

In contrast, the present applicant has already developed a method for producing cystine or cystine using 2-amino-thiazoline-4-carboxylic acid (hereinafter referred to as ATO) as a raw material and utilizing the action of microorganisms (Unexamined Japanese Patent Publication No. Showa 51-70881.51-
54983 and Japanese Patent Publication No. 54-2272).

Although this method is suitable for industrial production of L-cysteine, cysteine cannot be directly obtained because the ice-melting reaction of ATO to cysteine is strongly inhibited by cysteine produced in the reaction solution. Therefore, in this method, the reaction of hydrolyzing ATO to produce cysteine and the oxidation reaction of cysteine to cystine are carried out simultaneously to produce cystine, which has no inhibitory effect, and the produced cystine is reduced to obtain cystine. I have no choice but to adopt it. Therefore,
Since this production method requires long and complicated steps and is not necessarily satisfactory in terms of yield, further improvements in these points have been desired.

Therefore, the present inventors conducted intensive research to develop a method to efficiently generate cysteine from ATO, and found that ATO
When cysteine or a microorganism capable of producing cystine is allowed to act on ATC in the presence of acetaton, 2.
2-dimethylthiazoline-4-carboxylic acid (hereinafter referred to as DT
They discovered that DTO (abbreviated as O) was produced in good yield, and that by hydrolyzing this DTO under acidic conditions, cystine could be produced in a short time and in high yield. The present invention was completed based on this discovery. That is, a microorganism according to the present invention having the ability to hydrolyze 2-amino-thiazoline-4-carboxylic acid to produce cysteine or cystine is allowed to act on ATO in the presence of acetone to produce DTO.
The present invention relates to a method for producing cysteine, which is characterized in that TO is hydrolyzed under acidic conditions to produce cysteine.

The microorganism used in the present invention is a microorganism that has the ability to hydrolyze ATO to produce cysteine or L-cystine, such as JP-A-51-54983 and JP-A-51-70.
Achromobacter, Alcaligenes, Bacillus, Brevibacterium, Enterobacter, Erwinia, Etsuchierichia, 72 Bobacterium, Micrococcus, which are described in No. 881 and Japanese Patent Publication No. 54-2272.
It is a microorganism that belongs to the genera Mycograna, Pseudomonas, Sarcina, or Serratia and has the ability to hydrolyze ATO to produce cysteine or cystine. Specific examples include the following strains: Sarcina・Lutia ATCC272 (5a
rcina lutθa) Achromobacter delmalvaa Fl! i
RM-P3483 (Achromobacter de
lmarvae) Alcaligenes tenitrificasis ATOO15173 (Alcaligen
es denitrificans ) Bacillus plebis ATOO8185 (Bacillus
brevis) Flavobacterium 7lavum
ATC! 013826 (Brevibac
terium flavum) Enterobacter aerokenes F'ERM-P 2764 (E
(enterolncter aerogenes) Erpinia carotovo-y FERM-P2766
(Whnia carotovora) Pseudomonas thiazolinophilum IRM-P2810
(Pseudomonae thiazolinoph
ilum) Pseudomonas desmolytica
FBRM-P2816 (Pseudomonas
desmolytica) Escherichia lily
FIRM-P 2763 (Eeche
richia coli ) Micrococcus sodonensis ATOo 11B! 'lO (Micr
ococcus 5odonensis ) Mycoplana dimol 7ar ATOO4249 (My
coplana dimorpha) Serratia marcescens FERM-P 2765 (5e
rratia marcescene) Flavobacterium acidificum ATOO8366 (
F avobacterium acidificum
) Pseudomonas obalis FERM
-P 2762 (Peudornonas oval
is), etc.

The medium for culturing microorganisms should contain a carbon source, a nitrogen source, inorganic ions, and if necessary organic micronutrients as appropriate.For example, as a carbon source, glucose, sucrose, xylose, honey, etc. , organic acids such as acetic acid,
Alcohols such as ethanol, glycerol, and methanol; nitrogen sources such as ammonium sulfate and ammonium chloride; organic nutritional sources such as yeast extract, peptone,
Ions such as magnesium, iron, manganese, potassium, sodium, and phosphoric acid are appropriately used as inorganic ions in meat extracts, corn staple liquor, and the like.

The microorganisms can be cultured according to the appropriate method, and the pH of the medium is adjusted to 6 to 9, and the microorganisms are cultured aerobically at 20 to 40C.

When culturing, it is desirable to add a small amount of ATO.
TC! Microbial cells with high water-splitting activity can be obtained. In the method of the present invention, the microorganism thus obtained is used as an enzyme source. As the enzyme source, the culture solution obtained in this way, isolated bacterial cells isolated from the culture solution, washed viable bacterial cells,
Freeze-dried product, acetone-dried bacterial cells, physically, chemically or biochemically destroyed bacterial cells, extracts, crudely purified products, purified products, purified protein preparations, or immobilized bacterial cells or purified products etc. are used as enzyme sources.

ATO, which is a raw material for cysteine, can be used in either the D-unit, L-unit, or racemic form, which is supplied by a synthetic method.

The negative a degree differs depending on whether it is a batch type or a continuous type, but in a batch type it is generally 0.1 to 30% in an aqueous medium, preferably about 05 to 10%, and in a continuous type it is slightly lower than this. It is preferable to

The reaction is carried out in an aqueous solution containing 2 to 40% acetone, and the yield can be improved by adding hydroxylamine or semicarbatide to the reaction solution.

Reaction temperature is 15-60C1, preferably 30-50tl:
', pH is preferably in the range of 6 to 10, preferably 7.0 to 9.5.

When A'TOK microorganisms are used under these conditions, A
TO is hydrolyzed to cysteine and condensed with acetone in the reaction solution to produce DTC. DTO is rapidly and regularly hydrolyzed to cysteine under acidic conditions.

Unlike cysteine, DTC generated from ATO is
TC! Since it does not inhibit the ice-melting reaction to cysteine at all, the reaction yield of DTO, that is, the yield of cysteine, is high, reaching over 90%, and the reaction time from ATO to DTO is short. can be manufactured. This is shortened to about % compared to the known production of cystine from ATO, which requires 2 to 50 hours.

Furthermore, in the conventional method, there was a risk that the produced cysteine would be decomposed by harmful enzymes, resulting in the generation of hydrogen sulfide, but in the method of the present invention, there is no risk of generation of hydrogen sulfide.
This point can also be said to be advantageous for industrial production.

Cysteine is collected from the ice-melting solution after DTO is hydrolyzed under acidic conditions by removing acetone and then using a known method for separating cysteine, such as a method using a cationic resin or a crystallization method.

Cysteine emplacement is carried out by high performance liquid chromatography using a cation exchange resin or by microbial quantification using lactic acid bacteria (Leuconostoc titroborum ATOOso s 1).

Examples will be described below.

Example 1 A culture medium having the composition shown in Table 81!1 was prepared, 50ml was dispensed into a 500mJ shaking 7 flask, and 12011:1 was prepared.
Heated for 0 minutes.

Table 1 Medium composition (pH 7,0) Glycerol 1.0 Chi yeast extract
0.5〃 Peptone 0.5〃 Meat extract 0.5 Mail 0.
5 Pseudomonas desmolytica AJ 38 in this medium
68 FERM-P2816 was inoculated and cultured with shaking at 30C for 24 hours. The culture solution was centrifuged to collect bacterial cells, which were added to a substrate solution having the composition shown below, maintained at 30C, and stirred occasionally for 16 hours to carry out a reaction.

DL-ATO2, Ot/Oval KW, FO41, ott Hydroxylamine hydrochloride 0.15I After the reaction is completed, hydrochloric acid is added to the reaction solution to adjust the pH to 3.0 to generate cysteine. was placed in place. The results are shown in Table 3.

Cystine in reaction solution Cystine 0
0.1 1.02
1.1
-5 12
-10 1.2
-15 1.3
-20 1
．． 1 -30
0.8 -40
0.6 -50
0.3 Example 2 The wet bacterial cells 5.0 obtained by the method of Example 1 were freeze-dried, or the wet bacterial cells 5.0 were dissolved in a 0.1 M, pH 7.0 phosphate buffer solution 50; Suspended in J and subjected to ultrasonic treatment (
Tomy Seiko ML Kou R-200P type, 20KO.

5 minutes), and furthermore, wet bacterial cells 5.02 with deionized water 20m? KWA was made cloudy, 3.8 tons of acrylamide and 2257 methylenebisacrylamide were added thereto, oxygen was removed by flowing nitrogen gas, and 17.5 tons of ammonium persulfate was added.
y and N', N'-dimethylamine propionitrile 4
An immobilized product was prepared by adding 0 sf, and a substrate solution having the composition shown in Table 2 (the amount of acetone was 5.0%) was prepared.
The enzymatic reaction was carried out by adding 100 liters of water and keeping it at 30C for 16 hours. After the reaction is completed, insoluble materials are removed from the reaction solution,
The pH was adjusted to 3.0 to generate cysteine, and the cysteine was then stabilized.

The results are shown in Table 4.

Freeze-dried specimen 1.3 Ultrasonication 1.4 Immobilization 12 Example 3 The microorganisms shown in Table 5 were cultured in the medium shown in Table 1 according to the method of Actual Leakage Example 1. Centrifuge each culture solution to remove wet bacterial cells.5. oyt- was added to the substrate solution shown in Table 2 (acetone: 5.0%), and heated to 30C for 2 hours.
The reaction was carried out by holding for 4 hours.

After the reaction was completed, the pH of the reaction solution was adjusted to 3.0 to generate cysteine, and the target of cysteine formation was determined. The results are shown in Table 5.

S, 1uteaATOO2720,15A, del
marvae FIRM-P 3483
0.13A, denitrificans ATC!
Of 5173 0.68B, brevis
ATC! 08185 0.12Br
evi, flavum ATOO138260,10
Rht, aerogenes FJ! [M-P2764
At 0.35, carotovora F
KRM-P2766 0.10E, col
i FKRM-P2763 0.12
M, sodonengie ATOO118800,
81Myco, dimorpha ATOO4279
0,48S, marcesaens FERM-P27
65 0.18F, aoidificum
ATOO83660, 16Pseu, ovalis
FIRM-P2762 0.72Pseu
, thiazolinophl 1um F Fri
RM-P 2B10 0.88 Procedure Correction Yen (Method) % Formula % 2, Title of Invention 1--Production of Cysdine; Kaho 3, ? +Relationship with Ministerial Affairs 1'1 Special nT Column Address Tokyo Chuo 1 Nukyo (8-chome 5-8 Yumi (1 for shipping November 30, 1980)!i, ?i
li positive 1+'! Adding invention n None 6, n city stop rows clear hand Il 1 yen

Claims (2)

[Claims] (1) A microorganism that has the ability to hydrolyze 2-amine-thiazoline-4-carboxylic acid to produce L-cystine or L-cystine was used to react on 2-amino-thiazoline-4-carboxylic acid in the presence of acetone. to produce 2,2-dimethylthiazolidine-4-carboxylic acid, and the 2,2-dimethylthiazolidine-4-carboxylic acid
-Dimethylthiazolidine-4-carboxylic acid is hydrolyzed under acidic conditions to produce L-cysteine! lf
A method for producing L-cysteine. (2) Achromobacter range, alkaline earth metals, Bacillus genus, Brevipuffbrilum genus, Enterobacter genus, Nirubini gamma genus, Etschierichia genus, 7Labobactericum genus, Micrococcus genus, Mycoplana cormorant, Pseudomonas spp., Sarcina genus, or Claim M (claim 0) characterized by using a microorganism belonging to the genus Serratia and having the ability to hydrolyze 2-amino-thiazoline-4-carboxylic acid to produce L-cystine or L-cystine. Method for producing L-cysteine.