JPS5813155B2 - L- Cysteine - Google Patents

Description

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

The L-cysteine derivative obtained by the method of the present invention is useful as a pharmaceutical intermediate.

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

The present inventors found that a specific microorganism produces cysteine desulfhydrase, and also found that this enzyme produces L-cysteine derivatives from β-halogen-L-alanine and thiol in good yield ( Special request 1977
-51926).

As a result of further research, it was discovered that the reaction rate increases when the above reaction is carried out in the presence of pyridoxal phosphoric acid, leading to the present invention.

To explain the present invention in detail, β-halogeno-L-alanine used as a raw material in the present invention includes β-chloro-L-alanine, β-promo-L-alanine, β-iodo-L-alanine, etc. β-chloro-L-alanine is preferred.

Thiols used as another raw material in the present invention include methyl mercaptan, ethyl mercaptan, n-
Alkyl mercaptans such as propyl mercaptan and n-octyl mercaptan; aryl mercaptans such as phenyl mercaptan and naphthyl mercaptan; aralkyl mercaptans such as benzyl mercaptan and phenethyl mercaptan; and alkenyl mercaptans such as allyl mercaptan.

The cysteine desulfhydrase used in the present invention is
It is a known enzyme that catalyzes the reaction that decomposes L-cysteine into pyruvic acid, ammonia, and hydrogen sulfide.

(Biochem.Biophys.Res.Comm.
un. 59, 789 (1974)).

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

Specifically, Sarcina lutea (IAM 1099), Corynebacterium equi (IAM 1038), Arthrobacter symplex (IFO 3530), and Previbacterium ammoniagenes (IFO 12)
071), Pseudomonas fluorescens (IFO
3081), Proteus morganii (IFO
3848), Micrococcus roseus (IFO
3764), Citrobacter freundii (IF
O 12681), Esierisia coli (IFO
3301), Serratia marsetuscens (IFO 3)
054), Alcaligenes faecalis (IAM 1
015), Bacillus subtilis (IFO 3009
), Agrobacterium tumefaciens (IAM
1037), Enterobacter aerogenes (IFO 3320),
Enterobacter cloacae (IFO 12009
), Klebscilla pneumoniae (IFO 3512
), Salmonella typhimurium (IFO 125
29), 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 acids such as acetic acid and fumaric acid as carbon sources. 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, corn steep liquor, yeast extract, meat extract,
Examples include yeast powder, cottonseed flour, soybean flour, soybean hydrolyzate, peptone, and polypeptone.

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-
Cystine, S-methyl-L-cysteine, S-ethyl-
The presence of at least one type of amino acid selected from L-cysteine, L-serine, and O-methyl-L-serine can further increase the production amount 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 molecular weight of the obtained enzyme is 150,000 to 500,000.

To produce L-cysteine derivatives according to the method of the present invention, β
- React halogen-L-alanine and thiol.

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 about 0.1 to 20 g/l, preferably about 1 to 5 g/l, as a dry bacterial cell amount.

The reaction temperature is suitably 20 to 80°C, preferably 30 to 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 substrates β-halogeno-L-alanine and thiol are each about 1 to 40% by weight, preferably about 3 to 20% by weight.

Pyridoxal phosphate added according to the method of the present invention has the effect of activating cysteine desulfhydrase as a coenzyme.

Although cysteine desulfhydrase produced by microorganisms already contains a small amount of pyridoxal phosphate, the activity of this enzyme can be further increased by adding pyridoxal phosphate.

The concentration of pyridoxal phosphate in the reaction solution varies depending on the amount of enzyme used and the type of enzyme-producing bacteria, but is usually 0.001 to 1 mM, preferably 0.005 to 0.
．． It is 1mM.

In addition, instead of adding the enzyme and pyridoxal phosphate to the reaction solution separately, pyridoxal phosphate is added to the culture solution of a microorganism capable of producing cysteine desulfhydrase. Similar effects can be obtained by adding bacterial cells, dried bacterial cells, etc. to the reaction solution.

When added to the culture solution, cheaper pyridoxal, pyridoxine, or pyridoxamine, which is converted to pyridoxal phosphate in the system, can be used instead of pyridoxal phosphate.

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

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.

The L-cysteine derivative produced was qualitatively and quantitatively determined using an amino acid analyzer.

Reference example 1 L-cysteine/HCl 0.1%, yeast extract 0.5%
, meat extract 0.5%, polypeptone 0.5%, NaCl
100 ml of a medium with a pH of 7.5 having a composition of 0.2% was poured into a 500 ml shaking flask, and after sterilization, the first
Inoculate the various bacteria shown in the table from the agar slope for 30 minutes.
After culturing at ℃ for 16 hours, the bacterial cells were collected by centrifugation.

The obtained bacterial cells were washed with physiological saline, and phosphate buffer solution 1
After suspending the suspension to 0 ml, the suspension was 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, 1 ml of the above extract was mixed with 1 x 10-1M Tris-HCl buffer (pH 9).
) 2.0ml, 1 x 10-3M pyridoxal phosphate 0
．． 4 ml, 1 x 10-2 ML - Add to 4.0 ml of a solution containing 1.0 ml of cysteine, perform a cysteine decomposition reaction at 30°C for 20 minutes, and remove the generated pyruvic acid with Fried
-mann. T. E. and Haugan. G. E. method (J. Biol. Chem., 147, 415 (19
43)).

The enzyme activity is expressed as 1 u, which is the enzyme activity that decomposes 1 μmol of L-cysteine per minute.

Reference example 2 Sarcina lutea (
Sarcina lutea IAM 1099) was inoculated and cultured at 30°C for 15 hours.

The obtained bacterial cells were suspended in 0.1 M phosphate buffer (pH 7.0), treated with ultrasound, and centrifuged to obtain a bacterial cell extract.

This 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 0.3M phosphate buffer (pH 7.0).
It was eluted.

Ammonium sulfate was added to the thus obtained cysteine desulfhydrase-containing solution to 50% saturation, the enzyme was concentrated, and then dialyzed.

The dialyzed enzyme solution was passed through Sephadex G-150 and then G-2.
00 gel filtration and the active fraction was collected 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 preparation of cysteine desulfhydrase thus obtained behaved as a single protein in disk electrophoresis analysis, and exhibited an activity of about 25 u/mg.

Example L-cysteine 0.2%, yeast extract 0.5%, meat extract 0.5%, polypeptone 0.5%, glycerin 0.1
Enterobacter aerogenes (IFO3
320) was cultured aerobically at 30°C for 16 hours, and the culture solution was centrifuged to collect the bacteria.

Bacterial cells obtained from 10 ml of culture medium (enzyme activity approximately 6 units)
s), 2 mmole of β-chloro-L-alanine,
2 mmole of various thiols shown in the table, surfactant SDS
5 mg and 0.001 mmol of pyridoxal phosphate
5 x 10-1M ammonia buffer (pH 9.5) containing
), and the reaction was carried out at 30°C for 1 hour.

As a result, L-cysteine derivatives were produced in the amounts shown in Table 2.

For comparison, without adding pyridoxal phosphate,
The reaction was carried out under the conditions described above.

The results are also shown in Table 2.

Claims (1)

[Scope of Claims] 1 β-halogeno-L-alanine represented by the following general formula [■] (in the above formula, X represents a halogen atom) is synthesized by the following general formula [■] in the presence of cysteine desulfhydrase. ■] R-SH [■] (In the above formula, R represents an alkyl group, an aryl group, an aralkyl group, or an alkenyl group.) By reacting with a thiol represented by the following general formula [■] (In the above formula, R represents an alkyl group, an aryl group, an aralkyl group, or an alkenyl group. R has the same meaning as R in the above general formula [■]. Manufacturing method.