JPH0614868B2 - Method for producing L-sulfur-containing amino acid - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION Industrial Field of the Invention The present invention comprises reacting β-substituted-L-alanine with hydrogen sulfide in an aqueous medium in the presence of tryptophan synthase,
It relates to a method for producing L-cysteine and / or L-cystine. L-cysteine and L-cystine are
It is widely used for cosmetics, food additives, etc., as well as for medical uses 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 By reacting β-substituted-L-alanine with hydrogen sulfide in the presence of an aqueous medium in the presence of L-cysteine and / or
Alternatively, it was found that L-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 cerevisiae ATCC-26787, etc. There is. For the extraction method of tryptophan synthase from cultured Escherichia coli cells, see The Journal.
of Biological Chemistry, Vol.249, NO.24, p.7756-7763
(1974), about the extraction method from cultured bacterial cells of Neurospora crassa, Vol.250, No.8.p.2941-2946 (1975).
,) For the extraction of Saccharomyces cerevisiae from cultured cells, 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.

Culture is performed under aerobic conditions such as shaking culture or aeration-agitation culture. Culture temperature is in the range of 20-40 ° C, usually 25-37 ° 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.1
27-185 (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, 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-cysteine L-serine O-sulfate, L-serine and the like can be used.

In the present invention, β-substituted-L-alanine and hydrogen sulfide are reacted in the presence of tryptophan synthase, usually in an aqueous medium of pH 6-10. A reaction temperature of 20-60 ° C is suitable. 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 concentrations of the substrate β-substituted-L-alanine and hydrogen sulfide are not particularly limited. 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, 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-cystine can be obtained by subjecting L-cystine thus obtained to electrolytic reduction.

L-Cysteine and L-cystine were quantified by liquid chromatography. It was confirmed by liquid chromatography using a column for separating optical isomers that the produced cysteine and cystine were in the L-form.

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

Example 1 Meat extract 1%, peptone 0.5%, yeast extract 0.1%, KH
₂ PO ₄ 0.2%, pH 7.0 in liquid medium Escherichia coli
MT-10242 (FERM BP-20) was inoculated and cultured with shaking at 30 ° C. for 20 hours. After culturing, centrifuge to collect the cells and
achi's method (The Journal of Biological Chemistry,
Vol.249, NO.24, p.7756-7763 (1974)) to obtain tryptophan synthase with a specific activity of 9.2 units / mg, and use this oxygen to perform the following reaction. went. The activity of tryptophan synthase is C.Yanofs
ky et al. (Methods in Enzymology, Vol. 5, p. 801-807 (1
962)), and the amount of enzyme that synthesizes 1 μmol / min tryptophan from L-serine and indole at pH 7.8 and 37 ° C. was defined as 1 unit.

L-serine 50 mM, hydrogen sulfide 100 mM, pyridoxal phosphoric acid
1M trisaminomethane-HCl buffer containing 0.1 mM (pH 9.
0) Add 0.5 mg of tryptophan synthase to 10 ml, and add 35
Shake gently for 2 hours at ° C. 3.1 mM in the reaction solution
L-cysteine and 0.4 mM L-cystine were produced.

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 performed to obtain tryptophan synthase with a specific activity of 1.3 units / mg, and this enzyme solution was used. The following reaction was performed.

L-serine 50 mM, hydrogen sulfide 100 mM, pyridoxal phosphoric acid
10 ml of 1M trisaminomethane-HCl buffer (pH 9.0) containing 0.1 mM and 1.3 units of tryptophan synthase at 35 ° C.
It was shaken gently for 2 hours. 1.2 mM L-cysteine and 0.3 mM L-cystine were produced in the reaction solution.

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 indoleacrylic acid and pH 6.0, and then inoculated at 30.
Culture was performed with shaking for 0 hours.

After completion of the culture, the cells were collected by centrifugation and the method of M. Dettwiler et al. (European Journal of Biochemist
ry, Vol.102, p.159-165 (1979)) to obtain tryptophan synthase with a specific activity of 1.2 units / mg, and perform the following reaction using this enzyme solution. It was

L-serine 50 mM, hydrogen sulfide 100 mM, pyridoxal phosphoric acid
10 ml of 1M trisaminomethane-HCl buffer (pH 9.0) containing 0.1 mM and 1.2 units of tryptophan synthase at 35 ° C.
It was shaken gently for 2 hours. In the reaction solution, 1.0 mM L-cysteine and 0.2 mM L-cystine were produced.

Example 4 Meat extract 1%, peptone 0.5%, yeast extract 0.1%, KH
₂ PO ₄ 0.2%, pH 7.0 in liquid medium Escherichia coli
MT-10242 (FERM BP-20) was inoculated and cultured with shaking at 30 ° C. for 20 hours. 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.

1M trisaminomethane-H containing L-serine 100 mM, hydrogen sulfide 400 mM, pyridoxal phosphate 0.1 mM, and wet bacterial cells 5 g
100 ml of Cl buffer (pH 9.0) was shaken at 35 ° C. for 5 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 15 mM.

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

500 units of tryptophan synthase was added to 100 ml of 980 mM trisaminomethane-HCl buffer (pH 8.5) containing 100 mM of β-substituted-L-alanine shown in Table 1, 300 mM of hydrogen sulfide, and 1 mM of pyridoxal phosphate, and the mixture was added at 500C at 35 ° C. Shake gently for 1 hour. After completion of the reaction, dithiothreitol reduces L-cystine in the reaction solution to L-cysteine,
The amount of L-cysteine produced was measured. The results are shown in Table 1.

Table 1 β-Substituted-L-alanine L-Cysteine (mM) β-chloro-L-alanine 6.2 O-methyl-L-serine 10.4 O-acetyl-L-serine 8.5 O-benzyl-L-serine 2.3 S- Methyl-L-cysteine 2.5 S-ethyl-L-cysteine 1.9 S-benzyl-L-cysteine 0.7 L-serine O-sulfate 3.4 L-serine 11.3

Claims (2)

[Claims] 1. L-cysteine and / or L characterized by reacting β-substituted-L-alanine represented by the general formula [I] with hydrogen sulfide in an aqueous medium in the presence of tryptophan synthase. -Method for producing cystine. (However, X represents a halogen atom, -OR group, or -SR group. (R represents a hydrogen atom, an alkyl group, an acetyl group, a benzyl group, or a sulfonic acid group.)) 2. The method according to claim 1, wherein the general formula [I] is as shown below. (However, X is a halogen atom, -OR ₁ group, or -SR ₂
Indicates a group. (R ₁ represents a hydrogen atom, an alkyl group, an acetyl group, a benzyl group or a sulfonic acid group, and R ₂ represents an alkyl group, an acetyl group, a benzyl group or a sulfonic acid group.))