JPS582679B2 - L- Cysteine - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention mainly relates to improvements in a method for producing L-cysteine using cysdine desulfhydrase produced by microorganisms.

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.

Furthermore, sulfoxides obtained by oxidizing S-methyl-L-cysteine and S-aryl-L-cysteine are known to suppress increases in cholesterol in the blood and liver.

Conventionally, methods for producing L-cysteine include (1) extracting cystine from the hydrolyzate of natural products such as hair and reducing it; (2) condensation of penzyl chloride methyl sulfide and diethyl phthalimide malonate; Methods of 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.

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

Examples of synthesis methods using enzymes include: (1) A method of synthesizing cysteine from serine and hydrogen sulfide using cysteine synthetase of Neurospora crassa (OL8 2,225.797
;J. Biol. Chem. , 242.12
(1967) ) (2) Synthesis method from serine and hydrogen sulfide in the presence of serine sulfhydrase (Bioc
hemi sche Zeitschrift, 336
, 258-273 (1962)) (3) Production of O-acetylserine sulfhydrylase from 〇1-acetyl-L-serine obtained by reacting L-serine and acetyl-CoA in the presence of serine transacetylase and hydrogen fluoride. Method for synthesizing L-cysteine in the presence of L-cysteine (J. Biol.
Chem- + Hatori, 4955 (1966)) (4) Method for synthesizing cysteine from methionine and serine using enzymes in animal epidermal tissue (Special Publication No. 47
-1 6 0 1 9; Chemical Abst
lacts, 57 + 5] 50 i) and (5) synthesis from β-chloroalanine and hydrogen sulfide in the presence of serine sulfhydrase. This method is not necessarily suitable as an industrial method for producing cysteine.

On the other hand, according to the method of the present invention, L-cysteine can be synthesized in high yield and industrially advantageously.

The present inventors found that certain microorganisms produce cysteine desulfhydrase, while the enzyme is capable of producing β-monosubstituted mono-L-alanine and hydrogen sulfide, metal sulfide, metal hydrosulfide, ammonium sulfide, water Patent Application C. 1972-., which discovered that L-cysteine could be produced in good yield from ammonium sulfide or ammonium polysulfide. 51496, patent application No. 49-51926, patent application No. 51-48440).

After that, as a result of further research, it was discovered that the yield of L-cysteine increases when carbonyl compounds such as aldehydes, ketones, and α-keto acids are added when carrying out the -L reaction, and the present invention was achieved. It is.

To explain the present invention in detail, the β-monosubstituted-L-alanine used as a raw material in the present invention includes, for example, β-chloro-L-7lanine, β-bromo-L-alanine, β-iodo-L-alanine, etc. No β-halogen No L-alanine, 0
-Methyl-L-serine, 0-ethyl-L-serine, 0-
O-alkyl-L- such as (n-hexyl)-L-serine
Serin,? -0-aryl-L-serine such as phenyl-L-serine, O-aralkyl-serine such as 0-benzyl-L-serine, -serine, S-methyl-L-cysteine, S-ethyl-L-cysteine, S-(n-hexyl) One L-
S-alkyl groups such as cysteine, S-aryl groups such as L-cysteine, S-phenyl-L-cysteine, S-aryl groups such as cysteine, S-benzyl-cysteine, S-aralkyl groups such as cysteine, S-aryl-cysteine, S-aryl-L-cysteine, and L-cysteine.
Examples include -serine, 0-aryl-L-serine, and particularly preferred are β-chloro-L-alanine, S-alkyl-L-cysteine, and L-serine.

The sulfide, hydrosulfide, or ammonium polysulfide used as another raw material in the present invention is preferably one that is soluble in water, such as sodium sulfide, potassium sulfide, lithium sulfide, magnesium sulfide, and calcium sulfide. , sulfides such as strontium sulfide, barium sulfide, ammonium sulfide, hydrogen sulfide; sodium bisulfide, potassium bisulfide, lithium bisulfide, magnesium bisulfide, calcium bisulfide, strontium bisulfide, barium bisulfide, ammonium bisulfide, etc. Hydrosulfides; mention may be made of ammonium polysulfides, including ammonium disulfide, ammonium trisulfide, ammonium tetrasulfide, ammonium pentasulfide, ammonium disulfide or mixtures thereof.

The cysteine desulfhydrase used in the present invention is
It is a known enzyme that catalyzes the reaction that decomposes L-cysteine into pyruvate, ammonia, and hydrogen sulfide (Bi
ochem. Biophys. &s. Commun. 5
9,789 (1974)).

This enzyme is easily produced by microorganisms, and examples of microorganisms that produce this enzyme include, for example, Plevibacterium, Sarcina, Corynebacterium, Arsostobacter, Pseudomonas, Proteus, Micrococcus,
Ethierisia, Serratia, Alcaligenes, Bacillus, Agrobacterium, Enterobacter (
Examples include microorganisms belonging to the genus Aerobacter (genus Aerobacter), genus Citrobacter, genus Klebscilla, and genus Salmonella, but the invention is not limited to these, and any microorganism that produces the present enzyme may be used.

Specifically, Sarcina lutea (IAM 1099), Corynebacterium equi (IAM 1038), and Earthmouth Bacter symplexus (IF0 353).
0″). Previbacterium ammoniagenes (I
FO12071), Pseudomonas fluorescens (IFO 3081), Proteus morganii (I
F0 3848), Micrococcus roseus (
IFO 3764), Citrobacter freundii (IFO 12681), Escherichia coli (I
FO 3301), Serratia marsetuscens (IF
O 3054), Alcaligenes faecalis (IA
M 1015), Bacillus subtilis (IFO300
9), Agrobacterium tumefaciens (IA
M 1037), Enterobacter senichirokenes (
Aerobaccu aerogenes) (IF03320),
Enterobacter cloacae (IFO12009),
Klebscilla pneumoniae (IFO 3512)
, Salmonella teifimurium (IFO 1252
9) etc.

The method for producing cysteine desulfhydrase used in the present invention using these microorganisms will be summarized as follows.

Nutrient sources necessary for culturing microorganisms include carbohydrates such as glucose, sucrose, fructose, mannose, mannitol, xylose, glycerol, sorbitol, molasses, and starch hydrolysates, and organic carbon sources such as acetic acid and fumaric acid. Acid and n-paraffin etc. are used.

Nitrogen sources include ammonia and ammonium salts of organic and inorganic acids such as ammonium chloride, ammonium sulfate, ammonium carbonate, and ammonium acetate, nitrates such as sodium nitrate, potassium nitrate, and ammonium nitrate, cornstarch liquor, yeast extract, meat extract,
Examples include yeast powder, cottonseed flour, soybean flour, soybean hydrolyzate, peptone, and polypebutone.

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

The culture temperature is preferably 20 to 80°C, particularly 25 to 50°C.

Cultivation is carried out aerobically for 10-72 hours.

Further, it is desirable to maintain the pH during culturing at 7 to 11.

About 0.1 to 1% by weight of L-cysteine, L-
The presence of at least one amino acid selected from cystine, S-methyl-L-cysteine, S-ethyl-L-cysteine, L-serine, and O-methyl-L-serine can further increase the production of the enzyme.

The cysteine desulfhydrase thus obtained exists mainly within the cells of microorganisms, and known methods such as ultrasonication, ammonium sulfate fractionation, and ion exchange chromatography can be applied to its isolation and purification.

The obtained enzyme has a molecular weight of 150,000 to 500,000, and pyridoxal phosphate is required as a coenzyme for its activity to be expressed.

In order to produce L-cysteine according to the method of the present invention, in the presence of cysteine desulfhydrase and carbonyl compound of microbial origin etc. obtained in this way, the pH is usually 6 to 6.
12, preferably 7 to 11 in an aqueous medium β-monosubstituted -L
- Reacting alanine with sulfide, hydrosulfide or ammonium polysulfide.

The enzyme used is not limited to purified and crystallized ones, but any enzyme that has enzyme activity, such as microbial culture solution, live bacterial cells, dried bacterial cells, crushed bacterial cells, and bacterial cell extracts, may be used. The amount is usually 01 to 20 g/t as dry bacterial mass.
Preferably it is about 1 to 5&/t.

The appropriate reaction temperature is 20-80°C, preferably 30-50°C.

The reaction time varies depending on enzyme activity, substrate concentration and type, and reaction temperature, but is usually 1 to 100 hours.
-48 hours.

The concentrations of the substrate β-monosubstituted L-alanine and sulfide, hydrosulfide or ammonium polysulfide are each about 1 to 40% by weight, preferably about 3 to 20% by weight.

Carbonyl compounds added according to the method of the present invention include α-1-keto acids having 3 to 20 carbon atoms such as pyruvic acid, α-ketobutyric acid, and α-ketoglutaric acid; carbon atoms such as formaldehyde, acetaldehyde, propionaldehyde, isobutyraldehyde, and benzaldehyde. 1 to 20 aldehydes; acetone, methyl ethyl ketone, diethyl ketone,
Examples include ketones having 3 to 20 carbon atoms, such as methyl isobutyl ketone, cyclohexanone, penduphenone, acetophenone, and benzyl, with acetone, pyruvic acid, α-ke1-butyric acid, and the like being particularly preferred.

The concentration of the carbonyl compound in the reaction solution is usually 0.0.
1-20M, preferably 0.05-IOM.

Usually 0.001 to 1mM, preferably 0.
．． The presence of 0.005 to 0.1 mM pyridoxal phosphate can further enhance the activity of cysteine desulfhydrase and further increase the reaction rate of this reaction.

After the reaction is completed, L-cysteine is separated according to a conventional method, for example, by treatment with an ion exchange resin.

The method for obtaining L-cysteine by reacting β-1-L-alanine with sulfide, hydrosulfide, or ammonium polysulfide in the presence of cysteine desulfhydrase has the following advantages: (1) the raw material is inexpensive; (2) It has the advantage that the reaction conditions are mild and that only the 3-ru type can be obtained, but if this reaction is carried out in the presence of a carbonyl compound by the method of the present invention, a higher yield of L-cysteine can be obtained. can be obtained.

Next, reference examples and examples will be shown to explain the method of the present invention in more detail, but the present invention is not limited to the following examples unless it exceeds the gist thereof.

Note that the produced L-cysteine was qualitatively and quantitatively determined using an amino acid analyzer.

Reference example I L-cysteine/HCI 0.1%, yeast extract 0.
5%, meat extract 0.5%, polypep 1hen 0.5%, .
NaClO. pH consisting of 2% composition 7. 100rrL of the medium from No. 5 was injected into a 500RL shaking flask, and after sterilization, the various bacteria shown in Table 1 were inoculated from the agar slant, cultured at 30°C for 16 hours, and the bacterial bodies were collected by centrifugation. Bacteria were collected.

The obtained bacterial cells were washed with physiological saline, and phosphate buffered i1
After suspending in 1077Il, the cells were disrupted by sonication and centrifuged to obtain a cell extract exhibiting cysteine desulfhydrase activity.

The results are shown in Table 1. To measure the activity, add 1 rrtl of the above extract to 1 x 10'Ml-Lys-HCl buffer (
1) H9) 2. omt:, IXIO-3M pyridoxal phosphate 0. 4 m. 4. Solution containing IXIO-2ML-cysteine lornl. Ord plus 30
The cysteine decomposition reaction was carried out at ℃ for 20 minutes, and the generated pyruvic acid was purified by Friedmann, T. E. and Haug
an, G. E. method (J. BioiChem.1
47,415 (1943).

Enzyme activity is expressed as 1 u of enzyme t-<t'+ which decomposes 1 μmol of L-cysdeine per minute.

Reference Example 2 Sarcina lutea (
Sarcina lutea IAM1099) was inoculated and cultured at 30°C for 15 hours.

The obtained bacterial cells were reduced to 0. 1 M IJ acid buffer (pH 7
．． After suspending in 0), a bacterial cell extract was obtained by ultrasonication and centrifugation.

The bacterial cell extract was subjected to ammonium sulfate fractionation, and the cysteine desulfhydrase activity fraction (0 to 0.4 saturation) was dialyzed and then passed through a DEAE-Sephadex column to adsorb the enzyme.

After washing the column with 0.1M phosphate buffer (pH 7.0), the enzyme was washed with 0.1M phosphate buffer (pH 7.0). 3M IJ acid buffer iffl
(pH 7.0).

Ammonium sulfate was added to the cysteine desulfhydrase-containing eluate thus obtained to 50% saturation to concentrate the enzyme, which was then dialyzed.

The dialyzed enzyme solution was passed through Sephadex G-150 and then G-2.
The gel was filtered at 0.00 and the active fraction was separated using a fraction collector.

The cysteine desulfhydrase-containing eluate thus obtained was again subjected to ammonium sulfate fractionation (0 to 30% saturation).
A purified enzyme preparation was obtained.

The purified enzyme standard of cysteine desulfhydrase thus obtained behaved as a single protein in disc electrophoresis analysis and exhibited an activity of about 2 5 u/m9.

Example I L-cysteine 0.2%, glucose 1.0%, yeast powder 1.0%, soybean hydrolyzate 4.0%, KH2P040
．． 2%, Mg SO4 ・7 H2 0 0. 1%,
FeSO, -7H200.001%, (NH4)280
4 pH consisting of 0.1% composition 7. Enterobacter cloacae (IF0 12009) was cultured aerobically at 30° C. for 18 hours in the medium of No. 2, and the culture solution was centrifuged to collect the bacteria.

Bacterial cells obtained from medium LOrul (enzyme activity 20 uni
ts), β-chloroL-alanine 5 mmol
e. Sodium sulfide} IJum 5 mmole, surfactant S
DS5■, pyridoxal phosphate 0.0 0 1 m
5X10-'M ammonia buffer (p
H9.5) ■ In addition to 0rrLl, the reaction was carried out at 30° C. for 3 hours in the presence of various carbonyl compounds at the concentrations shown in Table 2.

As a result, L-cysteine was produced in the reaction solution in the amount shown in Table 2.

For comparison, the reaction was carried out without or with the addition of a carbonyl compound.

The results are also shown in Table 2.

Example 2 Enterobacter cloacae (IFO 12009) was cultured under the same conditions as in Example 1, and the culture solution was centrifuged to collect the bacteria.

Bacterial cells obtained from 10ml of culture medium (enzyme activity 20un)
its), 5 mmole of β-chloroL-alanine,
5 mmole of various sulfides or hydrosulfides shown in Table 3,
5×10'M ammonia buffer (pH 9.5) containing surfactant SDS57Q and 0.001 mmole of pyridoxal phosphate]. In addition to Oml, the reaction was carried out at 30° C. for 6 hours in the presence of various carbonyl compounds at the concentrations shown in Table 3.

As a result, L-cysteine was produced in the reaction solution in the amount shown in Table 3.

For comparison, the reaction was carried out with or without the addition of a carbonyl compound.

The results are also shown in Table 3.

Claims (1)

[Claims] 1 The following general formula (1) (In the above formula, X is a halogen atom, -OR group, or -S
Indicates an R group. (However, in the above 10R group and -SR group, R represents a hydrogen atom, an alkyl group, an aryl group, an aralkyl group, or an allyl group.) In the presence of lase, hydrogen sulfide, metal sulfides,
1. A method for producing L-cysteine, which comprises performing the reaction in the presence of a carbonyl compound when producing L-cysteine by reacting with metal hydrosulfide, ammonium sulfide, ammonium hydrosulfide, or ammonium polysulfide.