JPS58146286A - Preparation of selenium-containing amino acid - Google Patents

Abstract

PURPOSE:To prepare a selenosysteine derivative from methaneselenol or benzylselenol and L-serine inexpensively, by using tryptophan synthetase. CONSTITUTION:Methaneselenol or benzylselenol is reacted with L-serine in the presence of tryptophan synthetase at 20-60 deg.C, preferably by adding pyridoxal phosphate, a coenzyme.

Description

[Detailed description of the invention] The present invention relates to a method for producing selenium-containing amino acids.

More specifically, the object is a method for producing Se-methylselenocysteine or Se-benzylselenocysteine by reacting methaneselenol or pendylselenol and L-serine in an aqueous solution by the action of tryptophan synthetase. The aim is to produce inexpensive selenocysteine derivatives.

Selenocysteine derivatives cure various physiological activities and are expected to be used as reagents, medicines, or pharmaceutical intermediates, and the development of inexpensive production methods has been desired.

The present inventors investigated various methods for producing selenocysteine derivatives using enzymatic methods, and found that L-selJ
The present inventors have discovered that selenocysteine derivatives can be easily produced by reacting methane selenol and methaneselenol with the action of tryptophan φ ncetase, and have completed the present invention. All of the selenocysteine derivatives produced are L-type, and D-type is not produced.

As the tryptophan synthetase producing strain used in the present invention, for example, Echerichia coli MT-1o
2ss (FERM p-6145), Neurospora Clap ATCC 14692, etc. A method for extracting tryptophan synthetase from cultured specimens of Escherichia coli is described in The Journal of
Biological Chemistry, Vol.
, 252. A19, pp. 6594-6599 (1977), Vol.

250, Sake 8, pp. 2941-2946 (1975). However, the tryptophan synthetase used in the present invention does not necessarily have to be pure. That is, a culture of l-lyptophan synthetase producing strain, live bacterial cells collected from the culture by a method such as prior to centrifugation, dried bacterial cells or crushed bacterial cells,
Treated bacterial cells obtained by autolysis, sonication, etc.
Furthermore, extracts from these bacterial cells as well as crude enzymes obtained from the extracts can be used. Of course,
These identified enzymes or immobilized bacterial cells may be used.

As a medium for culturing tryptophan synthetase-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. However, it is generally necessary to add trace amounts of tryptophan, anthranilic acid, or indonyl to the medium. The carbon source and nitrogen source used in the culture medium may be of a type that can be used by the bacteria used. That is, various carbohydrates such as glucose, glycerol, fructose, sucrose, maltose, mannose, starch, starch hydrolyzate, and molasses can be used as carbon sources. Nitrogen sources include various inorganic and organic ammonium salts such as ammonia, ammonium chloride, ammonium nitrate, ammonium carbonate, ammonium acetate, ground meat extract, yeast extract, corn steep liquor, casein hydrolyzate, phynon meal or its digestion. Natural organic nitrogen sources such as soybean flour, defatted soybean meal, or its digested products 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 submerged culture with aeration. Culture temperature is 20-50℃, usually 60-50℃
It is in the range of 67°C. It is desirable to maintain the 'tI'i and pH of the culture medium near neutrality. Culture period (ri is usually 1 to 3 days.

In addition to the β-substitution reaction that synthesizes L-tryptophan from indole glycerophosphate and L-serine, l-lyptophan synthetase also performs α,β-de#ift.

It is a multifunctional pyritoxal enzyme that catalyzes reactions such as transamination.

The enzymatic chemical properties and catalytic mechanism of this enzyme were reported in detail by Miles et al., and Advances in Knzy
M-ology and Re1ated Areas
of Molecular Biology, Vol.
, 49, pp. 127-185 (1979). However, the reaction of the present invention using tryptophan synthetase is the first reaction discovered by the present inventors.

The serine that can be used in the present invention is L-type serine. D-
Serine cannot be used because it is not affected by driftfan synthetase.

There is no particular restriction on the amount of substrates such as L-serine and methaneselenol or benzylselenol in the reaction solution, but they can be used in a concentration range of 0.001 to 10% by weight in the solution.

During the reaction, in addition to the substrate, pyridoxal phosphate, which is a coenzyme, is added in a trace amount, for example, at a concentration of 01 to 1 in the liquid.
It is desirable to add in a range of 0 ppm.

The amount of tryptophan synthetase 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 ratio to substrate, enzyme activity, and other conditions. The reaction temperature in this reaction is usually in the range of 20 to 60°C.

The reaction is carried out either by a batch method or, in the case of immobilized enzymes, by a continuous method using a column, either standing still or under gentle stirring. The yield of the product increases if the enzyme and methaneselenol or pendylselenol are added sequentially during the reaction. The reaction time varies depending on the reaction conditions, but is usually 5 to 40 hours in the case of a batch method.

To isolate the reaction product selenocysteine derivative,
This can be easily carried out by a conventional method such as adsorption/desorption treatment using an ion exchange resin or activated carbon. The reaction product thus obtained was identified by chromatography, amino acid analysis, NMR, and other instrumental analyses, and it was confirmed that the reaction product of the present invention was definitely Se-methylselenocysteine or [Se-penfluor It has been confirmed that it is selenocysteine.

Example 1 Enerihia coli MT-10238 (FKRM P
-6145) was used to carry out the following reaction.

Contains L-Serif 30mM, Methaneseleno-#50mM Ma & Haben/Lucelenol 50mM, Pyridoxal Phosphate at 0.01mM concentration, I) Hs, o (KOH
) Tryptophan synthetase powder was added to the reaction solution adjusted to 10 m9/dt. 10ml of the reaction solution was shaken at 30°C for 24 hours. The molar yields of the produced selenocysteine derivatives are shown in Table 1.

Table-1 Example 2 Energia φ Cori MT-10238 (FEGRM
A loopful of P-6145) was inoculated into 5 oml of a medium having medium composition I, and cultured with shaking at 50°C for 20 hours.

Medium composition Meat extract Peptone for 10 Yeast extract for 05 Yeast extract for O1 KH2PO40.2% Initial pH 7.0 1 ton of culture solution was centrifuged to collect bacterial cells, which were mixed with tryptophan L-serine 30mM, methanesenol 50mM-j
H or hene/lucelenol 50mJ Contains 0.01mM concentration of pyridoxal phosphate and pHs. o (KOH)
Add 100 ie of culture solution to 100+yl # of reaction solution adjusted to
The centrifuged cells were added for 48 hours at 60° C. and the reaction was carried out with shaking for 48 hours. The molar yields of the selenocysteine derivatives produced are shown in Table 2.

Clothes - 2 patent applicant Mitsui Toatsu Chemical Co., Ltd. 461

Claims (1)

[Claims] Methaneselenol or ben/ruselenol and L-serine were reacted in an aqueous solution in the presence of tryptophan synthetase to produce Se-methylselenocystine.
A method for producing a selenium-containing amino acid, which comprises producing e-benzylselenocysteine.