JPS5838154B2 - Method for producing L-cysteines - Google Patents

Description

[Detailed description of the invention] The present invention relates to a method for producing L-cysteines.

More specifically, the present invention involves reacting β-halogenoalayune with hydrogen sulfide, thiol, etc. in the presence of microorganisms to produce L-
The present invention relates to a method for producing cysteine or a derivative thereof.

L-cysteine is a semi-essential amino acid and is used as an infusion antidote,
It is useful as a food additive, etc., and is also useful as an intermediate in the production of pharmaceuticals.

Also, S-methyl-L-cysteine and S-allyl L
- Sulfoxide obtained by oxidizing cysteine is known to suppress the rise in cholesterol in the blood and liver.

Conventionally, methods for producing L-cysteine include (1) extracting cystine from a hydrolyzate of natural products such as hair and reducing it; (2) a method of extracting cystine from a hydrolyzate of natural products such as hair; (2) a method of extracting cystine from a hydrolyzate of natural products such as hair; A method of condensation followed by hydrolysis and reduction, (3) a method of hydrolyzing a thiazoline derivative obtained by reacting N-thiobenzoylserine and thionyl chloride, and (4) a method of synthesis using an enzyme are known. ing.

Among these methods, the method of obtaining L-cysteine by organic synthesis is not advantageous in terms of cost, as the process is complicated and requires D1L splitting.

As a synthesis method using an enzyme, (1) a method of synthesizing cysteine from serine and hydrogen sulfide using cysteine synthetase of Neurospora crassa (OL8 2, 2 2 5, 79
7;J. Bio1. Chem. 242.12 (1967
)) (2) A method of combining serine and hydrogen sulfide in the presence of serine sulfhydrase (Biochemi-sch
e Zeit'schrift, 336, 258~
273 (1962)) (3) 〇1 acetyl-L-serine obtained by reacting L-serine and acetyl-CoA in the presence of serine transacetylase and hydrogen sulfide to L- in the presence of O-acetylserine sulfhydrylase. Method for synthesizing cysteine (J. Biol. Chem241, 4955 (19
66)) (4) Method for synthesizing cysteine from methionine and serine using enzymes in animal epidermal tissue
-1 6 0 1 9 : Chemical Abst
lacts, 57, 5 150 i) and (5) synthesis from β-chloroalanine and hydrogen sulfide in the presence of serine sulfhydrase of animal origin. This method is not necessarily suitable as an industrial production method for cysteine.

The present inventors have achieved the present invention by discovering that L-cysteines are produced in good yield when β-halogenoalanine is reacted with hydrogen sulfide, thiol, etc. in the presence of specific microorganisms.

The purpose of the present invention is to industrially advantageously produce L-cysteine and its derivatives in high yield, and this purpose is to produce L-cysteine and its derivatives with high yield and advantageous industrially. β-halogenoalanine represented by Table 9) and hydrogen sulfide or a substance that generates hydrogen sulfide in the reaction system or thiol are combined with Hansenula, Cryptococcus, Debaryomyces, Candida, Rhodotorula, Trichosporon, and Satucharomyces. Genus, Neurospora; Geotrichum, Aspergillus, Stemphylum, Aureobasidium, Penicillium, Waldomyces, Absidia, Monascus, Achromobacter, Flavobacterium, Staphylocotcus or It is achieved by reacting in the presence of one or more types of microorganisms belonging to the genus Bacterium.

To explain the present invention in detail, β-halogenoalanine (I) used as a raw material in the present invention includes β-chloro L-alanine, β-bromo L-alanine, β-iodo L-alanine, etc. However, β-chloro L-alanine is preferred.

Among the other raw materials for the method of the present invention, substances that contain hydrogen sulfide in the reaction system include sulfides such as ammonium sulfide, sodium sulfide, potassium sulfide, magnesium sulfide, calcium sulfide, barium sulfide; ammonium hydrogen sulfide; , sodium bisulfide, potassium bisulfide, magnesium bisulfide, calcium bisulfide, barium bisulfide, and other hydrosulfides: polysulfides including ammonium disulfide, ammonium trisulfide, ammonium tetrasulfide, ammonium disulfide, or mixtures thereof. Ammonium; Metal polysulfides such as sodium polysulfide, potassium polysulfide, calcium polysulfide, etc. can be mentioned.

Examples of thiols include alkyl mercaptans such as methyl mercaptan, ethyl mercaptan, propyl mercaptan, and octyl mercaptan; aryl mercaptans such as phenyl mercaptan and naphthyl mercaptan;
Mention may be made of aralkyl mercaptans such as benzyl mercaptan and phenethyl mercaptan; and alkenyl mercaptans such as allyl mercaptan.

When β-halogenoalanine and hydrogen sulfide or a substance that generates hydrogen sulfide in the reaction system are reacted in the presence of the above microorganisms, L-cysteine is produced, and when β-halogenoalanine and thiol are reacted, L-cysteine is produced. In this case, an S-substituted mono-L-cysteine corresponding to the thiol used is produced.

The microorganisms used in the present invention include Hansenula, Cryptococcus, Debaryomyces, Candida, Rhodotorula, Trichosporon, Satuuromyces, Neurospora, Geotrichum, Alpergillus,
The microorganism is selected from the genus Stemphylum, Aureobasidium, Penicillium, Waldomyces, Absidia, Monascus, Achromobacter, Flavobacterium, Staphylococcus, or Bacterium.

Specifically, Cryptococcus albidus (IFO
0378), Debaryomyces hanzenii (IFO
0564), Rodo Torla Marina (IFO 143)
2), Rhodotorula rubra (IFO 1433), Candida humicola (IFO 1527), Candida tropicalis (IFO 1499), Candida giliamondii (IFO 0566), Candida intermedia (IFO 0761)
, Trichosporon ctaneus (IFO 0116)
, Satucharomyces cerevisiae (IFO 0334)
, Hansenula anomala (IFO 0118), Neurospora sitophylla (IFO 6069), Neurospora cratusa (IFO 6067), Geotrichum candidium (IFO 4597), Alpergillus wendii (IFO 8879), Alpergium usamii (IFO 8876), Alpergillus iizucae (IFO 8869), Alpergillus fumicatas (IFO 8866), Stemphyllium irissis (IFO 8824), Aureobasidium pullulans (IFO 6405), Penicillium purprogenum (IFO 4684), Penicillium crustozum (IFO 6014), Penicillium coriofilm (IFO 5791), Waldmyces symplexus (IFO 8909),
Absidia coerulea (IF0 5302), Monascus purpureuum (IF0 4513), Achromobacter parvulus (IF0 13181),
Achromobaccu liquefaciens (IFO1260)
5), Akuchimobacter liquidum (Engineering FO 3084)
), Flavobacterium arboretuscens (IFO
3750), Staphylococcus aureus (IF
O 3060), Bacterium cadaveris (IF0
3731), etc.

Cultivation methods for these microorganisms are not particularly limited, and commonly used known culture methods such as solid culture methods and liquid culture methods can be applied, but industrially, deep aeration liquid in fermenters is used. Culture methods are advantageous.

The pH of the medium is preferably 4-10, and the culture temperature is 20-5.
0°C, especially 20-37°C is preferred.

Cultivation is carried out aerobically for 10-120 hours.

Nutrient sources necessary for culture include glucose, sucrose, fructose, mannose, mannitol, xylose, glycerol, sorbitol, and carbon sources.
Carbohydrates such as molasses and starch hydrolyzate, organic acids such as acetic acid and fumaric acid, and n-paraffin are used.

Nitrogen sources include ammonia and ammonium chloride,
Ammonium salts of inorganic and organic acids such as ammonium carbonate, nitrates such as sodium nitrate, potassium nitrate, ammonium nitrate, cornstarch liquor, yeast extract, meat extract, yeast powder, cottonseed flour, soybean flour, soybean hydrolysis. Examples include peptone, polypeptone, and the like.

Further, as the inorganic salt, potassium phosphate, sodium phosphate, magnesium sulfate, ferrous sulfate, etc. are used.

To produce L-cysteines according to the method of the present invention,
β-halogenoalanine and the above-mentioned sulfides, thiols, etc. are reacted in the presence of the above-mentioned microorganisms in an aqueous medium having a pH of usually 6 to 12, preferably 7 to 11.

Microorganisms are used in the form of live cells, dried cells, ground microbial cells, cell extracts, etc., but a microbial culture solution may also be used.

The amount of microorganisms used is usually 0.1 to 20 as the amount of dry bacterial cells.
g/l1 is preferably about 1 to 5 g/12.

The reaction temperature is suitably 20 to 80°C, preferably 30 to 50°C.

The reaction time varies depending on the amount of microorganisms used, substrate concentration and type thereof, and reaction temperature, but is selected from the range of 1 to 100 hours, usually 2 to 48 hours.

The concentrations of the substrate β-halogenoalanine, sulfide, thiol, etc. are each 0.1 to 40% by weight, preferably 0.1 to 40% by weight.
It is about 3 to 10% by weight.

After the reaction is completed, L-cysteines are separated according to conventional methods, such as treatment with an ion exchange resin and oxidation to poorly soluble L-cysteine under aeration.

According to the method of the present invention, L-
Cysteines can be synthesized.

Next, the method of the present invention will be specifically explained using Examples, but the present invention is not limited to the following Examples unless the gist thereof is exceeded.

In the following examples, the qualitative and quantitative determination of the produced L-cysteines was carried out using an amino acid analyzer and M. K. Ga
itonde, Biochem. J. ，
1 0 4 +627 (1967) using acidic ninhydrin.

Example 1 Sucrose 10%, Asparagine 0.25%, IIG{
2P040.1%, MgS04 ・7H20 0.3%
pH consisting of the composition 5. 1001 nl of the medium from No. 5 was poured into a shaking flask, and after sterilization, various microorganisms shown in Table 1 were inoculated from an agar slant, cultured aerobically at 30°C for 48 hours, and the bacterial bodies were centrifuged. Bacteria were collected.

The obtained bacterial cells were mixed with 0.04Ml-IJ Soo H (l buffer (
pns. o) In addition to 10- cells, the cells were disrupted using a vibrogen cell mill to obtain a cell extract.

Add 1 ml of this extract to 500μ of β-chloroL-alanine.
Mo1, Na2S・9H20500μmol1, 0.04M Tris-H containing 1 μmol of pyrodoxal linic acid (l buffer (pHs.o)) Added to 30
The reaction was carried out at ℃ for 2 hours.

As a result, L-cysteine was produced in the amounts shown in Table 1.

Example 2 Sucrose 3%, NaNOa 0.2%, K2HPO4
0.1%, MgS04.7H200.05%, KC70
．． 05%, F e S 040. 50011 100ml of pH 5.5 medium consisting of 0 0 1%
After sterilization, various microorganisms shown in Table 2 were inoculated from the agar slant and cultured aerobically at 30°C for 48 hours. Live reaction of cysteine was performed.

The results are shown in Table 2. Example 3 Polypeptone 1.0%, meat extract 1.0%, yeast extract 1.0%, NaClO. pH 7.2 consisting of 5% composition
100 TL of the culture medium was poured into a 500 ml shaking flask, and after sterilization, various microorganisms shown in Table 3 were inoculated from the agar slant, and cultured aerobically at 30°C for 24 hours. Then, an L-cysteine production reaction was carried out.

Claims (1)

[Claims] 1 β-halogenoalanine represented by the following general formula (I) (in the above general formula (I), X represents a halogen atom) and hydrogen sulfide or hydrogen sulfide produced in a reaction system or thiol, Hansenula spp., Crypcochus spp., Debaryomyces spp., Candida spp., Rhodotorula spp., Trichosporon spp., Satucharomyces spp., Neurospora spp., Geotrichum spp., Aspergillus spp., Stemphyllum spp., Aureobasidium spp. L-, characterized in that the reaction is carried out in the presence of one or more microorganisms belonging to the genus Penicillium, Waldomices, Absidia, Monascus, Akumobacter, Flavobacterium, Staphylococcus, or Bacterium. Method for producing cysteines.