JPH0611232B2 - Method for producing L-sulfur-containing amino acid - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial field of application) The present invention provides a method for converting β-substituted-L-alanine into a metal sulfide, a metal hydrosulfide, a metal polysulfide, ammonium sulfide, water in the presence of tryptophan synthase. It relates to a method for producing L-cysteine and / or L-cystine by reacting with ammonium sulfide or ammonium polysulfide.

L-Cysteine and L-cystine are widely used for cosmetics, food additives, etc., as well as for medical use such as infusion components.

(Prior Art) Conventionally, as a typical production method of L-cysteine and L-cystine, (1) a method of extracting from natural products such as hair,
(2) Chemical synthesis method (for example, JP-A-57-200356),
(3) Method of enzymatically synthesizing from DL-2-aminothiazoline-4-carboxylic acid (Japanese Patent Publication No. 54-2272), (4) β
-A method in which a substituted alanine is reacted with a metal sulfide or a metal hydrosulfide in the presence of cysteine desulfhydrase (Japanese Patent Publication No. 57-21311) is known, but it is not always an advantageous industrial method. I don't think so.

(Problems to be Solved by the Invention) Under these circumstances, the present inventors have conducted research on a new method for producing inexpensive L-cysteine and / or L-cystine, and as a result, tryptophan synthase Reacting β-substituted-L-alanine with metal sulfide, metal hydrosulfide, metal polysulfide, ammonium sulfide, ammonium hydrosulfide or ammonium polysulfide in the presence of L-cysteine and / or L- It was found that cystine was produced, and the present invention was completed based on this finding.

(Means for Solving Problems) It is known that tryptophan synthase is widely present in microorganisms, higher plants, etc. (for example, Bacter
Iological Reviews, Vol. 39, No. 2, p. 87-120 (1975)), the source of the enzyme is not particularly limited in the present invention as well, but a source of microbial origin is usually used. As a strain producing tryptophan synthase, for example, Escherichia
Escherichia coli MT-10232 (FERM BP-19), Escherichia coli MT-10242 (FERM BP-20), Neurospora crassa ATCC-14692, Saccharomyces cerevis-iae ATCC 2678
There are 7 and so on. For the method of extracting tryptophan synthase from cultured Escherichia coli cells, see The Jour.
nal of Biological Chemistry, Vol.249, No.24, p.7756-7
763 (1974), regarding the extraction method from cultured cells of Neurospora crassa, Vol. 250, No. 8, p. 2941-2946 (197).
(5 years), for the extraction method from cultured cells of Saccharomyces cerevisiae, see European Journal of Biochemistry, V.
ol.102, p.159-165 (1979) and is known.

However, the tryptophan synthase used in the present invention does not necessarily have to be an extracted pure product. That is, a culture of a tryptophan synthase-producing bacterium, viable cells collected from the culture by a method such as centrifugation,
Treated cells obtained by grinding, autolyzing, or sonicating the dried cells or cells, and a crude product of an extract obtained from these cells and an enzyme obtained from the extract Even things can be used. Of course, these immobilized products may be used.

As the medium for culturing the tryptophan synthase-producing bacterium, either a synthetic medium or a natural medium can be used as long as it contains a carbon source, a nitrogen source, an inorganic substance and, if necessary, a small amount of a micronutrient. It may be useful to add trace amounts of tryptophan or indole to the medium. In addition, the amount of tryptophan synthase produced may be increased by adding a small amount of indole acrylic acid to the medium.

The culture is performed under aerobic conditions such as shaking culture or aeration-agitation culture. The culture temperature is in the range of 20 to 40 ° C, usually 25 to 37 ° C. The pH of the culture solution is 5-8.

It is well known that tryptophan synthase is a multifunctional enzyme that catalyzes various reactions in addition to the reaction of synthesizing L-tryptophan from indole-3-glycerophosphate and L-serine. [For example, Advances in Enzymology and
Related Areas of Molecular Biology, Vol.49, p.127-1
85 (1979)) However, the reaction of the present invention by tryptophan synthase was first discovered by the present inventors.

Examples of β-substituted-L-alanine that is a reaction substrate include β-halogeno-L-alanine such as β-chloro-L-alanine and β-bromo-L-alanine, and O-methyl-L.
-O-alkyl-L-serine such as serine and O-ethyl-L-serine, S-methyl-L-cysteine, S-alkyl-L-cysteine such as S-ethyl-L-cysteine, O-acetyl-L -Serine, O-benzyl-L-serine, S-benzyl-L-cystine L-serine O-
Sulfate, etc. can be used.

As the other substrate, sulfide, hydrosulfide, etc.,
For example, metal sulfides such as sodium sulfide, potassium sulfide, lithium sulfide, sodium hydrosulfide, potassium hydrosulfide,
Metal hydrosulfides such as lithium hydrosulfide, metal polysulfides such as sodium polysulfide, potassium polysulfide, ammonium sulfide, ammonium hydrosulfide, ammonium polysulfide and the like can be used. Further, these sulfides, hydrosulfides, etc. may be those produced from metal hydroxide or ammonium hydroxide and hydrogen sulfide in the reaction solution.

In the present invention, β-substituted-L-alanine is reacted with sulfide, hydrosulfide or polysulfide in the presence of tryptophan synthase, usually in an aqueous medium of pH 6-10.
A suitable reaction temperature is 20 to 60 ° C. The reaction time varies depending on the enzyme titer, the substrate concentration, and other conditions, but is usually 1 to 100 hours in the batch reaction. The reaction is allowed to stand or with gentle stirring. The concentration of β-substituted-L-alanine as a substrate and sulfide, hydrosulfide or polysulfide is not particularly limited, but is usually about 0.1 to 30% by weight. All of the substrate may be added to the reaction solution at the start of the reaction, or may be added in portions as the reaction progresses. In the reaction solution, sulfide, hydrosulfide or polysulfide is β-substituted-
It is desirable to be present in an equimolar amount or more relative to L-alanine. In the reaction, it is desirable to add a small amount of pyridoxal phosphate which is a coenzyme in addition to the substrate.

When the reaction is carried out in this way, L-cysteine is produced in the reaction solution, but since L-cysteine is easily oxidized and converted to L-cystine, L-cysteine is usually added to the reaction solution as the reaction progresses. -Coexistence of cysteine and L-cystine,
The amount of L-cystine gradually increases. However, it is also possible to change the concentration ratio of L-cysteine and L-cystine by controlling the reaction conditions.

A usual method can be used to collect L-cysteine or L-cystine from the reaction solution. For example, after completion of the reaction, the reaction solution is aerated to remove most of L-cysteine from L-
If oxidized to cystine, L-cystine is poorly soluble in water and can be easily isolated. Further, L-cysteine can be obtained by subjecting L-cystine thus obtained to electrolytic reduction.

L-Cysteine and L-cystine were quantified by liquid chromatography. The generated cysteine and cystine
The L-form was confirmed by liquid chromatography using a column for separating optical isomers.

(Examples) Hereinafter, the present invention will be specifically described with reference to Examples.

Example 1 1% meat extract, 0.5% peptone, 0.1% yeast extract, KH ₂ P
Escherichia coli MT-10 in liquid medium containing 0.2% O ₄ and pH 7.0
242 (FERM BP-20) was inoculated and cultured at 30 ° C. for 20 hours with shaking. After the completion of the culture, the cells were collected by centrifugation and collected by the method of O. Adachi et al. (The Journal of Biological Chemistry, Vol.
9, No. 24, p.7756-7763 (1974)), to obtain tryptophan synthase with a specific activity of 9.2 units / mg, and perform the following reaction using this enzyme. . The activity of tryptophan synthase is determined by the method of C. Yanofsky et al. (Methods in Enzymology, Vo1.5, P.801-807 (196
2)), 1 μmol / mi at pH 7.8, 37 ℃
The amount of enzyme that synthesizes n tryptophan from L-serine and indole was defined as 1 unit.

O-methyl-L-serine 50 mM, the sulfides shown in Table 1,
Hydrosulfide or polysulfide 100 mM, pyridoxal phosphoric acid 0.1
0.5 mg of tryptophan synthase was added to 10 ml of the reaction solution containing mM and adjusted to pH 8.5 with 1N-HCl, and gently shaken at 35 ° C for 10 hours.

Table 1 shows the concentrations of L-cysteine and L-cystine produced and the yield of O-methyl-L-serine, which is the sum of both concentrations.

Example 2 WHMatche using Neurospora crassa ATCC-14692
The method of tt et al. (The Journal of Biological Chemistry, Vo
l.250, No.8, P.2941-2946 (1975)), culture and enzyme purification were carried out to obtain tryptophan synthase with a specific activity of 1.3 units / mg, and this enzyme solution was used. The following reaction was performed.

O-methyl-L-serine 50 mM, sodium hydrosulfide 100 m
M, pyridoxal phosphate 0.1 mM, and tryptophan synthase 1.3 units at pH 8.5 containing 10 ml of reaction solution at 35 ° C.
Shake gently for hours. In the reaction solution, 12 mM L-cysteine and 4 mM L-cystine were produced.

Example 3 Saccharomyces cerevisiae ATCC-26787 was inoculated into a liquid medium containing 1% of peptone, 0.5% of yeast extract, 2% of glucose, 0.01% of indole acrylic acid and pH 6.0, and the mixture was incubated at 30 ° C. for 20 days.
Culture was performed with shaking for an hour.

After the completion of the culture, the cells were collected by centrifugation and the method of M. Dettwiler et al. (European Journal of Biochemistry, Vol. 102,
Enzyme purification according to P.159-165 (1979)) and specific activity of 1.
Tryptophan synthase having a titer of 2 units / mg was obtained, and the following reaction was performed using this enzyme solution.

O-methyl-L-serine 50 mM, sodium hydrosulfide 100 m
M, pyridoxal phosphate 0.1 mM, and tryptophan synthase 1.2 units at pH 8.5 containing 10 ml of reaction solution at 35 ° C.
Shake gently for hours. In the reaction solution, 11 mM L-cysteine and 3 mM L-cystine were produced.

Example 4 Meat extract 1%, peptone 0.5%, yeast extract 0.1%, KH ₂ P
Escherichia coli MT-10 in liquid medium containing 0.2% O ₄ and pH 7.0
242 (FERM BP-20) was inoculated and cultured at 30 ° C. for 20 hours with shaking. After the culture was completed, the cells were collected by centrifugation and used as an enzyme source for tryptophan synthase. The tryptophan synthase activity per 1 g of this wet microbial cell was 120 units.

O-methyl-L-serine 200 mM, sodium sulfide 300 mM,
Pyridoxal phosphate 0.1 mM, containing wet cells 5 g pH 8.5 (HC
100 ml of the reaction solution (adjusted by l) was shaken at 35 ° C. for 24 hours. After completion of the reaction, L-cystine in the reaction solution was reduced to L-cysteine with dithiothreitol, and the amount of L-cysteine produced was measured and found to be 98 mM.

Example 5 Using the tryptophan synthase obtained in Example 1, the following reaction was performed.

480m containing β-substituted-L-alanine 300mM, sodium hydrosulfide 600mM, pyridoxal phosphoric acid 0.1mM shown in Table 2.
To 100 ml of M trisaminomethane-HCl buffer (pH 8.5), 500 units of tryptophan synthase was added, and the mixture was gently shaken at 35 ° C. for 1 hour. After the reaction was completed, L-cystine in the reaction solution was reduced to L-cysteine with dithiothreitol, and then the amount of L-cysteine produced was measured. The results are shown in Table 2.

Example 6 Meat extract 1%, peptone 0.5%, yeast extract 0.1%, KH ₂ P
Escherichia coli MT-10 in liquid medium containing 0.2% O ₄ and pH 7.0
232 (FERM BP-19) was inoculated and shake-cultured at 30 ° C. for 20 hours. After the completion of the culture, the cells were collected by centrifugation and frozen and stored at -20 ° C to be used as an enzyme source for tryptophan synthase. The tryptophan synthase activity per 1 g of the wet cells was 89 units.

O-methyl-L-serine 200 mM, sodium sulfide 300 mM,
100 including pyridoxal phosphate 0.1 mM and wet cell 5 g
100 ml of mM Trisaminomethane-HCl buffer (pH 8.5), 35
It was shaken gently at 0 ° C for 10 hours. After the reaction was completed, L-cystine in the reaction solution was reduced to L-cysteine with dithiothreitol, and the amount of L-cysteine produced was measured and found to be 114 mM.

Example 7 Using the wet bacterial cells obtained in Example 6, the following reaction was performed.

O-methyl-L-serine 200 mM, sodium hydrosulfide 1000 m
M, pyridoxal phosphate 0.1mM, wet cell 5g pH8.
100 ml of the reaction solution of 0 was gently stirred at 35 ° C. for 10 hours.
2.1 g of L-serine 2 hours and 5 hours after the start of the reaction
Each was added to the reaction solution. During the reaction, the pH of the reaction solution was maintained at 8.0 by adding 6N phosphoric acid. After the reaction was completed, 4.3 g of L-cysteine and 1.1 were added to the reaction solution.
g of L-cystine had accumulated.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Internal reference number FI technical display area C12R 1: 645) (C12P 13/12 C12R 1: 865)

Claims (1)

[Claims] 1. A β-substituted-L-alanine represented by the general formula [I] in the presence of tryptophan synthase is converted to a metal sulfide, a metal hydrosulfide, a metal polysulfide, ammonium sulfide, ammonium hydrosulfide or A method for producing L-cysteine and / or L-cystine, which comprises reacting with ammonium polysulfide. General formula [I] (However, X shows a halogen atom, -OR group, or -SR group. (R shows an alkyl group, an acetyl group, a benzyl group, or a sulfonic acid group.))