JPS61242589A - Production of l-sulfur-containing amino acid - Google Patents

Abstract

PURPOSE:To obtain L-cysteine and L-cystine useful as a transfusion component, cosmetic, food additive, etc., by reacting L-serine with a specific sulfide such as metal sulfide in the presence of tryptophan synthase. CONSTITUTION:The objective compound can be produced by reacting L-serine with a metal sulfide (e.g. sodium sulfide), metal hydrosulfide (e.g. sodium hydrosulfide), ammonium sulfide, ammonium hydrosulfide or ammonium polysulfide in the presence of tryptophan synthase. The tryptophan synthase can be produced by culturing a microbial strain such as Escherichia coli MT-10242 (FERM BP-20).

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Application Field) The present invention relates to a method for producing L-cysteine and/or L-cystine using tryptophan synthase.

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

(Prior art) Conventionally, known methods for producing L-cysteine include 1) extraction from natural products, 2) organic synthesis, 3) fermentation, and 4) enzymatic methods. As for the manufacturing method,
A method of extracting from natural products or a method of oxidizing L-cysteine is known. Among these methods, the method of extracting from natural products has an unstable supply of raw materials and is contaminated with other unnecessary amino acids. Organic synthesis methods involve complicated steps and require L-splitting. Furthermore, fermentation methods have low accumulation levels.

Due to these drawbacks, it is difficult to say that it is an industrially advantageous manufacturing method. As a method for synthesizing L-cysteine using an enzyme,
) Synthesis from L-serine and hydrogen sulfide using cysteine synthase, 2) Synthesis from L-serine and hydrogen sulfide or β-chloroalanine and hydrogen sulfide using cysteine sulfhydrase, 3) Cystine
Known methods include synthesis from β-substituted alanine and metal sulfide using desulfhydrase, and 4) synthesis from 2-amino-thiazoline-4-carboxylic acid, but these are industrial methods. It doesn't necessarily seem like an advantageous method.

(Problems to be Solved by the Invention) The present inventors have discovered that cysteine and/or L
-: As a result of intensive research into a new method for producing insulin, we learned of a new reaction catalyzed by tryptophan synthase, and based on this knowledge, we completed the present invention.

(Means for Solving the Problems) As the strain producing tryptophan synthase used in the present invention, for example, Escherichia coli MT-1
0242 (FERM BP-20), Neurospora cranna ATCC14,692, etc. For the extraction method of tryptophan synthase from cultured cells of Escherichia coli, please refer to The Journal of Bi.
Logical Chemistry, Vol.
252, A1.9.

pp. 6594-6599 (1977), Neurospora
Regarding the extraction method from cultured bacterial cells of Clap, see Vol.
, 250, A8, pp. 2941-2946 (197
5) and is known. However, the tryptophan synthase used in the present invention does not necessarily need to be extracted and pure. Namely.

Cultures of tryptophan synthase-producing strains, live bacterial cells collected from cultures by methods such as centrifugation, dried bacterial cells, or processed bacterial cells obtained by grinding, autolysis, or sonication of bacterial cells; Furthermore, extracts from the bacterial cells and crude enzymes obtained from the extracts can also be used. Of course, these immobilized enzymes or immobilized bacterial cells 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, inorganic substances, and, if necessary, small amounts of micronutrients. 1 In the case of a synthetic medium, it may be effective to add trace amounts of tryptophan, anthranilic acid or indole to the medium. As the carbon source and nitrogen source used in the culture medium, any type of carbon source and nitrogen source that can be used by the bacteria used may be used. That is, carbon sources include glucose, glycerol, fructose,
Various carbohydrates can be used, such as sucrose, maltose, mannose, starch, starch hydrolyzate, and molasses. Nitrogen sources include various inorganic and organic ammonium salts such as ammonia, ammonium chloride, ammonium sulfate, ammonium carbonate, ammonium acetate, or meat extract, yeast extract, corn steep lJ carr, casein hydrolyzate, and finocycin. Natural organic nitrogen sources such as meal or its digestate, defatted soybean meal or its digestate can be used.

Many natural organic nitrogen sources can be both nitrogen and carbon sources. Further, as inorganic substances, potassium hydrogen phosphate, potassium dihydrogen phosphate, potassium chloride, magnesium sulfate, sodium chloride, ferrous sulfate, etc. can be used as necessary.

Cultivation is performed under aerobic conditions such as shaking culture or aerated agitation culture. Culture temperature is 20-50℃, usually 30-37℃
°C range. It is desirable to maintain the pH of the medium during culture near neutrality. The culture period is usually 1 to 3 days.

Tryptophan synthase is a multifunctional pyridoxal enzyme that catalyzes reactions such as α, β-dissociation, and transamination in addition to the β-substitution reaction that synthesizes L-) liptophan from indole glycerophosphate and L-serine. The enzymatic chemical properties and catalytic mechanism of this enzyme were investigated in detail by Miles et al., and published in books (e.g., Advances in Enzyme).
Zymology and Re1atedAreas
of Molecular Biology, V
ol, 49, pp. 127-185 (1979)). However, the reaction of the present invention using tryptophan synthase is the first reaction discovered by the present inventors.

Serine that can be used in the present invention is L-type serine, and D-
Serine cannot be used because it is not affected by tryptophan synthase.

The substrate concentration of L-serine and various sulfides in the reaction solution is not particularly limited, but the concentration in the solution is usually from 0.1 to
It can be used within the range of 30 weight units. Further, during the reaction, it is desirable to add a trace amount of pyridoxal phosphate, which is a coenzyme, in addition to the substrate, for example, at a concentration in the solution in the range of 0.1 to 50 pP. Further, the amount of tryptophano synthase in the reaction solution varies depending on the enzyme separation and purification or processing method as described above, but is not particularly limited and can be changed as appropriate depending on the concentration of the substrate, the activity of the enzyme, and other conditions. Therefore, the reaction temperature in this reaction is usually in the range of 20 to 60°C, and 1 reaction p [-1 is usually 6
~10.

The reaction is carried out by a batch method or, in the case of immobilized enzymes, by a continuous method using a column, either by standing still or by gentle stirring. The reaction time varies depending on the reaction conditions, but in the case of a batch method it is usually about 5 to 72 hours.

L-7steine is produced in the reaction solution, but since L-cysteine is easily oxidized by oxygen in the air and converted to L-cystine, as one reaction progresses, the reaction solution usually contains
L-cysteine and L-cystine coexist, and the amount of L-cystine increases with the passage of one hour. However, by controlling the reaction conditions, I,-cysteine and L-
It is also possible to vary the concentration ratio of cystine.

■J-cysteine or L-cysteine can be easily collected from the reaction solution by a conventional method.

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 since it is poorly soluble in water. Moreover, L-cysteine can be obtained by electrolytically reducing the monocystine thus obtained.

Quantification of L-cysteine was performed by liquid chromatography after converting it to S-methyl-L-cysteine with methyl iodide. In addition, by this method, it was confirmed that it was in the L form.

Quantification of L-cysteine was performed by reducing L-cysteine with dithiothreitol and then quantifying L-cysteine.

(Example) Examples of the present invention are shown below.

Example 1 Escherichia coli MT-10242 (FERMBP
-20) from the cultured bacterial cells according to a conventional method,
We obtained pure tryptophan synthase with a specific activity of 9.2 units/m9 (one unit of tryptophan synthase is an enzyme synthesized from tryptophan serine and indole at 18 m01/min at 37°C), and The following reaction was carried out using

L-serine 50mM, various sulfides separately 1.00mM, pyridoxal phosphate Q, lmM
Contains a concentration of r)r-I in a reaction solution adjusted to 8,5 (HCI).

Tryptophan synthase powder was added at 5 m9/old. Reaction solution 10- was shaken at 35°C for 10 hours. Table 1 shows the concentrations of cystein and L-cysteine produced and the sum of both yields relative to serine.

Table-1 Example 2 Meat extract 1 part, peptone 0.5%, yeast extract 0.1 part, potassium dihydrogen phosphate 0.2 part t' pt-t 7,0
Escherichia coli MT-1024-2 (FER, M
BP-20) was inoculated and cultured with shaking at 30°C for 20 hours. After culturing, the cells were collected by centrifugation and used as an enzyme source for tryptophan synthase.

L-Serif 200mM, Sodium Sulfide 300mM
m'M.

Contains a 0.1 mM concentration of pyridoxal phosphate, p[(
Wet bacterial cells were added to the reaction solution adjusted to 8.5 (HCI) at a concentration of 50 l/l. 100ml of reaction solution at 35°
The mixture was shaken at C for 24 hours. After the reaction was completed, L-cysteine in one reaction solution was reduced to L-cysteine, and the amount of mustard-cysteine produced was measured to be 98 mM, and the yield relative to serine was 49 moles.

Patent Applicant: Mitsui Toatsu Chemical Co., Ltd. Procedural Amendment Written on June 1985, Director General of the Patent Office, 1. Indication of the Case, Patent Application No. 84545, 1985. 2. Name of the Invention: Production of monosulfur-containing amino acids. Method 6, Relationship with the person making the amendment Patent applicant address 3-2-5 Kasumigaseki, Chiyoda-ku, Tokyo Name (312) Mitsui Toatsu Chemical Co., Ltd. Representative Kasama
Yuichiro Telephone: 593-7416 4. Detailed explanation of the invention column 5 of the specification subject to amendment 5 Contents of the amendment (1) "Cysteine sulfhydrase" from line 11 to line 12 of page 2 of the specification [ Serine sulfhydrase,” he corrected.

(2) "L-type" on page 6, line 11 of the specification is corrected to 1L-type 1.

(3) Change the “L body” on page 8, line 10 of the specification to [L body-1
I am corrected.

(4) "1 serine" on page 9, line 2 of the specification is corrected to "L-serine".

Claims (1)

[Claims] L-serine in the presence of tryptophan synthase,
A method for producing L-cysteine and/or L-cystine, which comprises reacting with a metal sulfide, metal hydrosulfide, ammonium sulfide, ammonium hydrosulfide or ammonium polysulfide.