JPS62143690A - Production of sulfur-containing l-amino acid by enzymatic process - Google Patents

Abstract

PURPOSE:To produce L-cystine, etc., in high concentration at a low cost, by reacting L-serine with a metal sulfide, etc., in the presence of tryptophan synthase while keeping the L-serine concentration in the reaction liquid below a specific level. CONSTITUTION:L-serine is made to react with a metal sulfide, metal hydrosulfide, ammonium sulfide, ammonium hydroxide or ammonium polysulfide in the presence of cultured product of tryptophan synthase-producing microbial strain, collected living cell thereof or treated cell, etc. The L-serine concentration in the reaction liquid is maintained to <=0.5M and the reaction system is continuously or intermittently added with L-serine. The objective L-cysteine and/or L-cystine are produced by this process and the product is separated from the system by conventional method.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention involves reacting L-serine with a metal sulfide, metal bisulfide, ammonium sulfide, ammonium bisulfide or ammonium polysulfide in the presence of tryptophan synthase, and reacting L-serine with a metal sulfide, a metal bisulfide, ammonium sulfide, ammonium bisulfide or ammonium polysulfide to produce a monocysteine-cysteine and/or ammonium polysulfide. Or it relates to a method for producing L-cystine.

Cysteine and L-cysteine are widely used in pharmaceutical applications such as components of infusions, as well as in cosmetics and food additives.

(Prior Art) Conventionally, typical methods for producing L-cysteine and I,-cystine include (1) extraction from natural products such as hair, and (2) chemical synthesis (for example, JP-A-57-2003).
56), (Method of enzymatic synthesis from 31DL-2-aminothiazoline-4-carboxylic acid (Japanese Patent Publication No. 54-22)
72), (4) A method in which β-1f (tA alanine is reacted with a metal sulfide or metal hydrosulfide in the presence of cysteine desulfhydrase (Japanese Patent Publication No. 57-21311) is known; It does not necessarily seem to be an advantageous manufacturing method.

(Problems to be Solved by the Invention) Under these circumstances, the present inventors have conducted extensive research into a new method for producing cystine and/or L-cystine, which is inexpensive. A new method for producing cystine and/or L-cystine has been completed.

That is, the previously completed invention reacts L-serine with a metal sulfide, metal bisulfide, ammonium sulfide, ammonium bisulfide or ammonium polysulfide in the presence of tryptophan synthase, L-cysteine and /
Alternatively, it is a method for producing L-cystine.

The present inventors have discovered this 1. - cysteine and/or L
- As a result of further research to revise the cystine production method, we found that by continuously or intermittently adding L-serine while keeping the cystein concentration in the reaction solution below 0.5M, L-cysteine and The present inventors have discovered that L-cystine can be accumulated in a reaction solution at a high concentration, and have completed the present invention.

(Means for solving the problem) Tryptophan synthase is known to be widely present in microorganisms, higher plants, etc. (for example, Ila
cteriological Reviews, Vol.
, 39+No.

2, p. 87-120 (1975)), although the enzyme source is not particularly limited in the present invention, those derived from microorganisms are usually used.

Tryptophan sinter! As a strain that produces
For example, Escherichia coli (1!5 cber-i
chia coli) MT-10232 (FERM B
P-19), Escherichia coli MT-10242 (
FERM mouth P-20), Neurospora crassa (Ne
urospora crassa) 8TCC-1469
2. Saccharomyces “Saccharomyces”
yces cerev-isiae) ATCC-26
787 etc. Regarding the extraction method of tryptophan synthase from cultured cells of Escherichia coli, please refer to Th
e Jourr+al or BiologicalC
hemistry, Vol. 249. No. 24. p,
7756-7763 (1974), Vol. 1 for the extraction method from cultured cells of Neurospora crassa.
250. No...8. p, 2941-2946 (
1975), and the extraction method from cultured cells of Satucharomyces □cerevisiae is described in European Jou.
rnalof Biochemistry, Vol, 1
02. p, 159-165 (1979).

However, the tryptophan synthase used in the present invention does not necessarily have to be extracted and pure. That is, cultures of tryptophan synthase-producing bacteria, viable bacterial bodies collected from the culture by methods such as centrifugation, dried bacterial bodies, or bacteria obtained by grinding, autolysis, ultrasonication, etc. of the bacterial bodies. In addition, extracts from these microbial cells and crude enzymes obtained from the extracts can also be used. Of course, these immobilized products may also be used.

As a medium for culturing tryptophan synthase-producing bacteria, 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 micronutrients. It may be helpful to add trace amounts of tryptophan or indole to the culture medium. Furthermore, the production of tryptophan synthase may be increased by adding a small amount of indole acrylic acid to the medium.

Cultivation is performed under aerobic conditions such as shaking culture or aerated agitation culture. Culture temperature is 20-40℃, usually 25-3
It is in the range of 7°C. The pH of the culture solution is 5-8.

L −-1! which is a reaction substrate! The concentration of phosphorus in the reaction solution is kept at 0.5M or less. This point is a feature of the present invention, and I,-serine is added continuously or intermittently so that this condition is satisfied. The concentration of serine in the reaction solution is 0.
■ When 7-serine is added to a concentration of 5M or more, the progress of the reaction is significantly reduced, and L-cysteine and/or L-cysteine
-It is not suitable because the yield of cystine becomes low.

The other substrate, such as sulfide, is usually 0, 1 to 3
is added to the reaction solution at a concentration of M. It is desirable to add sulfide and the like in an amount equivalent to or more than the equivalent mole of L-serine. The sulfide etc. may be added in its entirety to the reaction solution at the start of the reaction, or may be added in portions as the reaction progresses.

The reaction is usually carried out at a temperature of 20 to 60°C and a pH of 6 to 10 while standing still or with gentle stirring.

When the reaction is carried out in this way, 14-cysteine is produced in the reaction solution, but 14-cysteine is oxidized and L-cysteine is produced.
Since L-cysteine is easily converted to cystine, L-cysteine and L-cystine usually coexist in the reaction solution as the reaction progresses, 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.

Conventional methods can be used to collect L-cysteine or L-cystine from the reaction solution.

For example, if most of L-cysteine is oxidized to L-cystine by aerating the reaction solution after the reaction is completed, L-cystine can be easily isolated because it is poorly soluble in water. Further, by electrolytically reducing 1,-cystine thus obtained, L-cysteine can be obtained.

To quantify L-cysteine, use methyl iodide to determine S-methyl-L
- After converting to cysteine, liquid chromatography was performed. L-cysteine was reduced to L-cysteine with dithiothreitol, and then quantified by the L-cysteine quantitative method.

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

Example 1 and Comparative Example 1 Escherichia coli MT-10242 (F[ERM B
P-20) was purified according to a conventional method from cultured cells, and the specific activity 2 was 9.2 units/mg () One unit of liptophan synthase was obtained by converting 1 μmol/min of tryptophan to L-serine at 37°C. Tryptophan synthase was obtained with a titer of (amount of enzyme synthesized from indole), and the following reaction was performed using this enzyme.

L-serine 400mM (4.2g/l, 00ml),
Sodium hydrosulfide 1000mM, pyridoxal phosphate 0
．． ], 400 units of tryptophan synthase was added to 100 ml of a reaction solution containing mM. The ptt of the reaction solution is lN
-Always adjusted to 8.5 with 11cI, reaction temperature 35℃
The reaction was carried out under mild stirring conditions. After 5 hours, L-serine in the reaction solution was analyzed by liquid chromatography and found to be 85 mM, so 4.2 g of L-serine was further added. The L-serine concentration in the reaction solution immediately after the addition of L-serine was 465 mM. The reaction was further continued for 7 hours, and after the completion of the reaction, the cystine in the reaction solution was reduced to L-cysteine, and the amount of L-cysteine produced was measured and found to be 7.5 g. (For monoserine yield 80
%) On the other hand, when 8.4 g of L-serine was added at once at the start of the reaction (the L-serine concentration at the start of the reaction was 800 mM) and the reaction was continued for 12 hours, the amount of L-cysteine produced was 4.8 g. (Yield of serine: 50%) Example 2 Meat extract 1%, Vep I・N 0.5, Yeast extract 0.1χ
, KHgPO* 0.2χ, pH 7, o Escherichia coli MT-10232 (F[ERM RP
-19) was inoculated and cultured with shaking at 30°C for 20 hours.

After completion of the culture, the cells were collected by centrifugation and stored frozen at -20°C, which was used as an enzyme source for tryptophan synthase.

100 ml of reaction solution containing 12+lomM thorium sulfide, 0.1 mM pyridoxal phosphate, and 5 g of wet bacterial cells
1 (pH 8.0), L-serine was added to the concentration shown in Table 1. The pH of the reaction solution is IN-HC.
It was constantly adjusted to 8.2 to 8.4 by I. The reaction is 35
The test was carried out at ℃ under mild stirring conditions.

At 11e and IO hours after the start of the reaction, the concentration of serine in the reaction solution was measured by liquid chromatography, and L-serine was added so that the L-serine concentration was the same as the concentration at the start of the reaction. . The reaction was carried out for 20 hours, and after the reaction was completed, cystine in the reaction solution was reduced to L-cysteine, and the amount of L-cysteine produced was measured. The results are shown in Table 1.

Table 1

Claims (1)

[Claims] When producing L-cysteine and/or L-cystine by reacting L-serine with metal sulfide, metal hydrosulfide, ammonium sulfide, ammonium hydrosulfide or ammonium polysulfide in the presence of tryptophan synthase, the reaction solution 1. A method for producing L-cysteine and/or L-cystine by an enzymatic method, characterized in that the concentration of L-serine in the L-cysteine is maintained at 0.5% or less.