JPH0611231B2 - Method for producing L-sulfur-containing amino acid by enzymatic method - Google Patents

Description

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

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

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

(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, have found that tryptophan synthase By reacting L-serine with metal sulfide, metal hydrosulfide, metal polysulfide, ammonium sulfide, ammonium hydrosulfide or ammonium polysulfide in the presence thereof, L-cysteine and / or L-cystine is produced. I found that

As a result of further research into improvement of this method, the present inventors have found that L-cysteine and / or L-cysteine can be reacted by keeping the pH of the reaction solution constant by supplying hydrogen sulfide to the reaction solution. It has also been found that the yield of cystine is significantly increased and the amount of metal sulfide, metal hydrosulfide, metal polysulfide, ammonium sulfide, ammonium hydrosulfide or ammonium polysulfide added to the reaction solution can be reduced.

The present invention has been completed based on these findings.

(Means for Solving Problems) It is known that tryptophan synthase is widely present in microorganisms, higher plants, etc. (for example, Bacter
Iological Review, Vol. 39, No. 2, p. 87-120 (1975), the source of the enzyme is not particularly limited in the present invention as well, but normally, a source derived from a microorganism is preferably used.

Examples of strains producing tryptophan synthase include Escheri-chia coli MT-10.
232 (FERM BP-19), Escherichia coli MT-10242 (FERM B
P-20), Neurospora crassa AT
CC-14692, Saccharomyces
crevis-iae) ATCC-26787 etc.

For the method of extracting tryptophan synthase from cultured Escherichia coli cells, see The Journal of Biologi.
cal Chemistry, Vol.249, No.24, p.7756-7763 (1974),
Regarding the extraction method from cultured cells of Neurospora crassa, Vol.250, No.84, p.2941-2946 (1975), the method of extraction from cultured cells of Saccharomyces cerevisiae is described in European Journal of. Biochemistry, Vol.102, p.159
-165 (1979).

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 carbonic acid 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.

Tryptophan synthase is known to be a multifunctional enzyme that catalyzes various reactions in addition to the synthesis of L-tryptophan from indole-3-glycerophosphate and L-serine (for example, Advances in Enzymology and Relat
ed Areas of Molecular Biology, Vol.49, p.127-185 (197
9)].

The reaction for producing cysteine by tryptophan synthase was first discovered by the present inventors.

Examples of sulfides, hydrosulfides and the like which are substrates include metal sulfides such as sodium sulfide, potassium sulfide and lithium sulfide, metal hydrosulfides such as sodium hydrosulfide, potassium hydrosulfide and lithium hydrosulfide, sodium polysulfide, Metal polysulfides such as potassium polysulfide, ammonium sulfide, ammonium hydrosulfide, ammonium polysulfide and the like can be used.

In the present invention, L-in the presence of tryptophan synthase, usually in an aqueous medium of pH 6-10, preferably 7-9.
Reacts serine with sulfides, hydrosulfides or polysulfides.

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 L-serine 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.

It is desirable that sulfides, hydrosulfides or polysulfides are present in the reaction solution in an equimolar amount or more relative to L-serine.

In the reaction, it is desirable to add a small amount of pyridoxal phosphate which is a coenzyme in addition to the substrate.

As the reaction progresses, metal hydroxide (for example, sodium hydroxide when sodium hydrosulfide is used as hydrosulfide) or ammonium hydroxide is generated in the reaction solution, and the pH of the reaction solution increases. In the present invention, hydrogen sulfide is supplied to the reaction solution to keep the pH of the reaction solution constant.

Hydrogen sulfide supplied to the reaction solution reacts with metal hydroxide or ammonium hydroxide in the reaction solution to form a sulfide or hydrosulfide that can be used as a reaction substrate. It is also possible to reduce the amount of sulfide and hydrosulfide added.

The pH of the reaction solution can be kept constant by adding a mineral acid such as hydrochloric acid or sulfuric acid or an organic acid such as acetic acid, but the yield of L-cysteine and / or L-cystine does not depend on hydrogen sulfide. This is not suitable because it becomes lower than that when it is used, and this is not done in the present invention.

Since L-cysteine has a —SH group, it functions to lower the pH of the reaction solution as it is produced, and therefore it may be effective to add an alkali to the reaction solution as the reaction progresses.

Further, when using a culture medium or cells of a microorganism having tryptophan synthase-producing ability as an enzyme source, by adding one or more alcohols, esters, ketones or surface-active compounds to the reaction mixture, The yield may increase.

When the reaction is carried out in this manner, L-cysteine is produced in the reaction solution, but L-cysteine is easily oxidized and converted to L-cystine. -Cysteine and L-cystine coexist, and 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, if most of L-cysteine is oxidized to L-cystine by aeration in the reaction solution, L-cystine is hardly soluble in water and can be easily isolated. Also obtained in this way
If L-cystine is electroreduced, L-cysteine can be obtained.

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 and Comparative Examples.

Example 1 and Comparative Example 1 1% meat extract, 0.5% peptone, 0.1% yeast extract, KH ₂ P
Escherichia coli MT- in a liquid medium containing 0.2% O ₄ and pH 7.0.
10242 (FERM BP-20) was inoculated and shake-cultured at 30 ° C. for 20 hours.

After completion of the culture, the cells were collected by centrifugation and the method of O. Adachi et al. (The Journal of Biological Chemistry, Vol. 249, p.
Purified according to 756-7763 (1974), the specific activity is 9.2.
Obtain tryptophan synthase with a titer of unit / mg,
The following reaction was performed using this enzyme.

The activity of tryptophan synthase was measured by the method of C. Yanofsky et al. (Methods in Enzymology, Vo15, P.801-807 (1962)), and 1 μmol / min of tryptophan was added to L-serine and indole at pH 7.8 and 37 ° C. The amount of enzyme synthesized from was 1 unit.

250 units of tryptophan synthase was added to 100 ml of a reaction solution having a pH of 8.5 and containing 400 mM of L-serine, 1000 mM of sodium sulfide, and 0.1 mM of pyridoxal phosphate, and gently stirred at 35 ° C for 10 hours.

During the reaction, the pH of the reaction solution was maintained at 8.5 by supplying hydrogen sulfide to the reaction solution.

After the reaction was completed, 3.25 g of L-cysteine and 0.6
1 g of L-cystine had accumulated.

On the other hand, instead of hydrogen sulfide, 6N hydrochloric acid
When the pH was maintained at 8.5, in the reaction solution after the reaction was completed,
2.23 g of L-cysteine and 0.55 g of L-cystine accumulated.

Example 2 and Comparative Example 2 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
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 toptophan synthase.

The tryptophan synthase activity per 1 g of the wet cells was 89 units.

L-serine 200mM, sodium hydrosulfide 300mM, pyridoxal phosphoric acid 0.1mM, pH 8.3 reaction solution containing 2g wet cells 100ml
The mixture was gently stirred at 35 ° C for 10 hours.

After the reaction was completed, 1.85 g of L-cysteine and 0.2
2 g of L-cystine had accumulated.

On the other hand, instead of hydrogen sulfide, 1N hydrochloric acid
Those who maintain the pH at 8.3 have 0.9% in the reaction solution after completion of the reaction.
Accumulation of g L-cysteine and 0.28 g of L-cystine was accumulated.

Example 3 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 culturing, the cells were collected by centrifugation to collect 1 g of wet cells.
The tryptophan synthase activity per unit was 120 units.

100 ml of a reaction solution having a pH of 8.5 and containing 200 mM (2.1 g) of L-serine, 500 mM of sodium hydrosulfide, 0.1 mM of hyridoxal phosphate, 5 g of wet cells and 0.3 g of butyl acetate was stirred at 35 ° C. After the start of the reaction, a 2M L-serine solution (pH was adjusted to 9.5 with NaOH) every 2 hours.
5 ml) was added to the reaction solution. During the reaction, the pH of the reaction solution was maintained at 8.5 by supplying hydrogen sulfide to the reaction solution.

The reaction was continued for 15 hours, during which 35 ml of L-serine solution (7.35 g as L-serine) was added to the reaction solution.

After the reaction was completed, 7.8 g of L-cysteine and 1.4 g were added to the reaction solution.
L-cystine was accumulated.

Example 4 and Comparative Example 3 The following reaction was carried out using the cells obtained in Example 2.

L-serine 200 mM, sulfide, hydrosulfide or polysulfide 400 mM shown in the table below, pyridoxal phosphate 0.1 mM, wet bacterial cells
100 ml of a reaction solution containing 2 g and having a pH of 8.5 was gently stirred at 30 ° C. for 10 hours.

During the reaction, the pH of the reaction solution was maintained at 8.5 by supplying hydrogen sulfide to the reaction solution.

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 by liquid chromatography. The results are shown in the table on the next page.

On the other hand, instead of hydrogen sulfide, 1N hydrochloric acid
The results when the pH was maintained at 8.5 are also shown in the table.

Claims (1)

[Claims] 1. L-serine and / or L-cysteine by reacting L-serine with a metal sulfide, a metal hydrosulfide, a metal polysulfide, ammonium sulfide, ammonium hydrosulfide or ammonium polysulfide in the presence of tryptophan synthase.
Alternatively, in the method for producing L-cystine, the pH of the reaction solution
-L-cysteine and / or L-cysteine, which are reacted by supplying hydrogen sulfide to the reaction solution while keeping it constant
Alternatively, a method for producing L-cystine.