JP2011024454A - Method for producing s-position-substituted cysteine derivative and/or s-position-substituted homocysteine derivative by fermentation method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an economically advantageous method for producing a useful S-position-substituted cysteine derivative äS-allyl-L-cysteine, S-propyl-L-cysteine, S-(3-hydroxyhexyl)-L-cysteine or S-(3-hydroxy-1,1-dimethylpropyl)-L-cysteine} and S-position-substituted homocysteine derivative äS-allyl-L-homocysteine, S-propyl-L-homocysteine, S-(3-hydroxyhexyl)-L-homocysteine or S-(3-hydroxy-1,1-dimethylpropyl)-L-homocysteine}. <P>SOLUTION: The fermentation method includes accumulating the useful S-position-substituted cysteine derivative and/or S-position-substituted homocysteine derivative corresponding to a remarkable amount of an added organic compound having a mercapto group in a fermentation product and a fermentation liquid by carrying out the culture by adding the organic compound having the mercapto group to the medium when culturing microorganisms in the fermentation medium, and collecting them. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

The present invention relates to a method for producing an S-position-substituted cysteine derivative and / or an S-position homocysteine derivative by fermentation. These are substances that are expected to be used as medicines, health and health drugs, or food additives.

As a method for producing S-substituted cysteine derivatives and / or S-substituted homocysteine derivatives by fermentation, at least L-methionine is grown in a fermentation medium containing a carbon source, nitrogen source, sulfur source and other nutrients necessary for the growth of microorganisms. S-substituted cysteine corresponding to the added organic compound having a mercapto group by culturing necessary microorganisms and adding an organic compound having a mercapto group (-SH group) to the fermentation medium at the beginning or during the fermentation There has been known a method according to the inventors' invention for producing and accumulating a derivative / or S-substituted homocysteine derivative in a fermented product and fermentation broth (Patent Document 1).

However, as an organic compound having a mercapto group, allyl mercaptan, propyl mercaptan, 3-mercapto-1-hexanol, or 3-mercapto-3-methylbutanol is added to the fermentation medium and the corresponding S-position substituted cysteine derivatives { S-allyl-L-cysteine, S-propyl-L-cysteine, S- (3-hydroxyhexyl) -L-cysteine, or S- (3-hydroxy-1,1-dimethylpropyl) -L-cysteine} and / Or S-substituted homocysteine derivatives {S-allyl-L-homocysteine, S-propyl-L-homocysteine, S- (3-hydroxyhexyl) -L-homocysteine, or S- (3-hydroxy-1 , 1-dimethylpropyl) -L-homocysteine} has not been known for producing and accumulating it in fermentation products and fermentation broth. Among these products, the former three S-substituted cysteine derivatives are substances that are considered to be physiologically active substances contained in genus plants such as garlic, onion, rakkyo, and leek, and the last one is called ferrinin, It is known as a sex attractant for cats, and any of them is a substance that is particularly expected to be used as a medicine, health care medicine or food additive.
JP2004-222518

S-substituted cysteine derivatives corresponding to allyl mercaptan, propyl mercaptan, 3-mercapto-1-hexanol or 3-mercapto-3-methylbutanol, which are expected to be used as pharmaceuticals, health care drugs or food additives {S- Allyl-L-cysteine, S-propyl-L-cysteine, S- (3-hydroxyhexyl) -L-cysteine, or S- (3-hydroxy-1,1-dimethylpropyl) -L-cysteine} and / or S-substituted homocysteine derivatives {S-allyl-L-homocysteine, S-propyl-L-homocysteine, S- (3-hydroxyhexyl) -L-homocysteine, or S- (3-hydroxy-1,1 It is an object of the present invention to provide an economically advantageous process for producing (dimethylpropyl) -L-homocysteine}.

In the present invention, when a microorganism suitable for a normal fermentation medium containing an inexpensive sugar source, nitrogen source, sulfur source, and other trace nutrients is cultured by a conventional method, allyl is added to the medium from the beginning of the fermentation or during the fermentation. Add organic compounds with mercapto groups such as mercaptan, propyl mercaptan, 3-mercapto-1-hexanol or 3-mercapto-3-methylbutanol to support each organic compound with mercapto groups in fermentation products and fermentation media S-substituted cysteine derivatives {S-allyl-L-cysteine, S-propyl-L-cysteine, S- (3-hydroxyhexyl) -L-cysteine, or S- (3-hydroxy-1,1-dimethylpropyl ) -L-cysteine} and / or S-substituted homocysteine derivatives {S-allyl-L-homocysteine, S-propyl-L-homocysteine, S- (3-hydroxyhexyl) -L- Mo cysteine or S- (3- hydroxy-1,1-dimethylpropyl) -L- homocysteine} is significant amount accumulated. And it is the fermentation method which extract | collects those S-position substituted cysteine derivatives and / or S-position substituted homocysteine derivatives from fermented material and fermentation medium.
Expensive L-amino acids, organic acids and others are not used as main raw materials.

The above S-substituted L-cysteine derivatives and S-positions corresponding to organic compounds having a mercapto group such as allyl mercaptan, propyl mercaptan, 3-mercapto-1-hexanol or 3-mercapto-3-methylbutanol by fermentation. Recombinant L-homocysteine derivatives can be produced economically advantageously.
Furthermore, the above S-substituted L-cysteine derivative or S-substituted L-homocysteine derivative may be selectively produced economically advantageously by examining the culture conditions and selecting strains.

According to the method of the present invention, when cultivating a microorganism suitable for a normal fermentation medium moderately containing an inexpensive sugar source, nitrogen source, sulfur source, and other trace nutrient sources, allyl mercaptan, propyl mercaptan, 3- When an organic compound having a mercapto group such as mercapto-1-hexanol or 3-mercapto-3-methylbutanol is added, the above S-substituted cysteine derivative and S-substituted homocysteine derivative corresponding to the fermentation product or fermentation medium, respectively. A significant amount is generated and accumulated.

When the general chemical formula of the organic compound having a mercapto group to be added is expressed as RSH, the general chemical formula of the S-substituted cysteine derivative produced and accumulated in the fermented product and fermentation broth is HOOCCH (NH ₂ ) CH ₂ SR, and the S-position substituted The general chemical formula of the homocysteine derivative is represented by HOOCCH (NH ₂ ) CH ₂ CH ₂ SR. Here, RSH is allyl mercaptan, propyl mercaptan, 3-mercapto-1-hexanol or 3-mercapto-3-methylbutanol.

As the microorganism used in the present invention, any microorganism can be used as long as it produces and accumulates the above S-substituted cysteine derivative and / or S-substituted homocysteine derivative under the above conditions. Can also be used. They may be wild strains or mutant strains, and typical examples include microorganisms belonging to Escherichia coli, Corynebacterium glutamicum, Bacillus spp., Saccharomyces cerevisiae, Aspergillus spp.

For example, a mutant strain of E. coli is suitable as the microorganism used. As the mutant strain, for example, a mutant strain requiring at least L-methionine for growth is suitable. This L-methionine requirement may be satisfied by DL-methionine and D-methionine. In addition to these methionines, it may be satisfied by any of L-homocysteine, DL-homocysteine, L-homocystine, DL-homocystine, and homocysteine thiolactone. In addition, the requirement may be satisfied by the above methionines, but may not be satisfied by homocysteines or homocystins. Further, this requirement may be a leakage type.

Furthermore, the microbial mutants used in the present invention are resistant or sensitive to various drugs (for example, structural analogs of amino acids, structural analogs of amino acid biosynthesis and nucleic acid biosynthesis intermediates, structural analogs of vitamins). Those with the properties of are used. A mutant strain having both of these drug resistance and drug sensitivity properties and the above auxotrophy properties may be used.

Furthermore, as a microbial mutant used in the present invention, enzymes of the cysteine biosynthetic pathway such as serine-O-acetyltransferase and O-acetylserine sulfhydrase are used for feedback control of L-cysteine. Or a mutant strain that has become resistant to the feedback control of L-methionine or its derivatives, such as homoserine succinyltransferase or cystathionine gamma synthase, or Mutants of both properties can be used. A mutant having both the property of resistance to feedback control and the above-described auxotrophy, drug resistance or drug sensitivity properties may be used.

Furthermore, a recombinant strain in which these enzymes are enhanced or deleted by genetic engineering techniques may be used.

A typical example of the microorganism used in the present invention is a mutant FERMP-18891 that belongs to Escherichia coli and requires at least L-methionine or L-homocysteine (or L-homocystine) for growth.

FERMP-18891 is a mutant strain that requires one of L-methionine, DL-methionine, D-methionine, DL-homocysteine or L-homocysteine for growth, as in the following experimental example. Yes, it is publicly known by being stored at the National Institute of Advanced Industrial Science and Technology.

E. coli mutant strain FERMP-18891 and E. coli wild-type strain K-12 were each inoculated on a broth agar medium and cultured overnight at 37 ° C. A part of the grown cells was collected and suspended in 2 ml of sterilized water to prepare cell suspensions containing about 10 ⁸ cells per ml. 0.1 ml of each of these cell suspensions was added to each of the four minimal agar media (glucose 1%, ammonium sulfate 0.2%, KH ₂ PO ₄ 0.15%, K ₂ HPO ₄₀ 0.05%, MgSO ₄ · 7H ₂ O 0.05%, FeSO ₄ · 7H ₂ O 0.01%, MnSO ₄ · 4H ₂ O 0.007%, NaCl 0.01%, inorganic salt mixture (in 1 L) 1 ml / L of CaCl ₂ · H ₂ O 5.3 g, CoCl ₂ 40 mg, CuSO ₄ · 5H ₂ O 40 mg, H ₃ Bo ₃ 30 mg, Na ₂ MoO ₄ 47 mg, ZnSO ₄ · 7H ₂ O 200 mg) Place a cylinder-cup in one corner of a plate medium containing 2% medium, pH 7} 30 ml, and an L-methionine (2.5 mg / ml) solution in each cup, 0.02 ml each of D-methionine (2.5 mg / ml) solution, DL-homocysteine (2.5 mg / ml) solution, or sterilized water (control) was injected, and the mixture was incubated at 30 ° C. for 48 hours. As a result, as shown in Table 1, in the wild strain, good growth was observed on the entire surface of the flat plate under any of four conditions.

On the other hand, in the mutant strain, the growth zone was observed only around the cylinder cup to which L-methionine, D-methionine or DL-homocysteine was given, and it was separated from or around the cup to which sterilized water (control) was given. No growth was observed in any area.

＊＋印は試薬を注入したシリンダ-カップの周辺に生育が認められたことを示
し、-印は生育が認められなかったことを示す。

* + Indicates that growth was observed around the cylinder-cup in which the reagent was injected, and − indicates that growth was not observed.

The fermentation medium used in the present invention may be any of a synthetic medium and a natural medium as long as it contains a carbon source, a nitrogen source, a sulfur source, inorganic salts and other nutrients and contains an organic compound having a mercapto group. Can be used.

Any carbon source may be used as long as the bacteria used can be used. For example, carbohydrates such as glucose, fructose, sucrose, lactose, mannose, maltose, molasses, starch hydrolysate, sugar alcohols such as glycerin, sorbitol, mannitol and xylitol -Amino acids such as aspartic acid, glutamic acid, lysine, cystine, cysteine, alanine, proline, glycine, tryptophan, serine, homoserine, threonine, lactic acid, pyruvic acid, acetic acid, malic acid, formic acid, succinic acid, fumar Organic acids such as acid, citric acid and various fatty acids, and alcohols such as ethanol, propanol, butanol and methanol can be used alone or in combination.

Nitrogen sources include ammonium sulfate, ammonium sulfite, ammonium sulfide, ammonium thiosulfate, ammonium chloride, ammonium nitrate, ammonium phosphate, ammonium carbonate and other inorganic ammonium salts, various organic acid ammonium salts, various nitrates, aqueous ammonia, urea, etc. Can be used alone or in combination.

Sulfur sources include sulfates such as ammonium sulfate, potassium sulfate, and sodium sulfate, sulfites such as ammonium sulfite, potassium sulfite, and sodium sulfite, sulfides such as ammonium sulfide, potassium sulfide, sodium sulfide, and hydrogen sulfide, thiosulfates, Mercapto compounds such as mercaptan and cytein (cystine), and other organic inorganic sulfur compounds can be used.

The required substances methionine and homocysteine can be used alone or in combination as long as they can be used in any of L-type, D-type and DL-type. In addition, natural or synthetic substances containing them, and substances that can be easily changed to these substances, such as hydantoin of methionine, homocystin, homocysteine thiolactone, etc., can be used. The amount used of these required substances varies depending on the composition of the medium and other culture conditions, but the growth is about 50-90% of the maximum growth when a sufficient amount of them is added under the culture conditions used. It is desirable to supply in a limited quantity so as to obtain an amount.
The above substances can be divided or added together in the initial stage of culture or in the middle of culture.

The culture is performed under aerobic conditions such as shaking culture and aeration and agitation culture. It is preferable that the pH of the culture solution is maintained at 5-9 during the culture. As the neutralizing agent, aqueous ammonia, sodium hydroxide, potassium hydroxide, calcium hydroxide, calcium carbonate, magnesium phosphate, potassium hydroxide, urea and the like are used.

The culture period is usually 3-7 days, and a significant amount of S-substituted cysteine derivatives and / or S-substituted homocysteine derivatives accumulate mainly in the culture solution.

The S-substituted cysteine derivative and / or S-substituted homocysteine derivative produced / accumulated varies depending on the type of organic compound having a mercapto group to be added to the medium, but the general chemical formula of the organic compound having a mercapto group to be added is The S-substituted cysteine derivative produced and accumulated when expressed as RSH can be represented by HOOCCH (NH ₂ ) CH ₂ SR, and the S-substituted homocysteine derivative can be represented by HOOCCH (NH ₂ ) CH ₂ CH ₂ SR.

Depending on the type of the organic compound having a mercapto group to be added to the medium, various surfactants or solvents may be used in combination with the medium at various concentrations, so that the above S-substituted cysteine derivative and / or S-position substituted The amount of homocysteine derivative produced may increase.

Examples of the organic compound having a mercapto group added to the medium and the S-substituted cysteine derivative and S-substituted homocysteine derivative produced and accumulated are shown in Table 2.

Isolation of the cysteine derivative and / or homocysteine derivative from the culture broth is performed by solvent extraction and various chromatographic methods by conventional methods.

Below, allyl mercaptan is added to the medium as an organic compound having a mercapto group, and S-allyl-L-cysteine is accumulated as an S-substituted cysteine derivative and S-allyl-L-homocysteine is simultaneously accumulated as an S-substituted homocysteine derivative. An example of the case will be described.

In Example 1, 10 ml of a pH 7 seed medium consisting of 10 g / l glucose, 10 g / l polypeptone, 5 g / l yeast extract, and 3 g / l urea was placed in a 300 ml Erlenmeyer flask at 120 ° C. for 10 minutes. -Sterilize the tray. This is inoculated with Escherichia coli mutant FERMP-18891 and cultured with shaking at a speed of 130 rpm on a reciprocating shaker at 30 ° C. for 24 hours. The obtained culture broth was inoculated to 0.7 ml in a 300 ml Erlenmeyer flask containing 7 ml of the sterilized fermentation medium having the following basic composition, and the final concentration in the medium of L-methionine separately sterilized is as shown in Table 3. Thus, it was added to each flask at 0-500 μg / ml and shake-cultured in the same manner as the seed culture.

The basic composition (per liter of medium) of the fermentation medium was as follows. Fractose 30 g, (NH ₄ ) ₂ SO ₄ 10 g, FeSO ₄ · 7H ₂ O 0.01 g, MnSO ₄ · 4H ₂ O 0.007 g, MgSO ₄ · 7H ₂ O 0.5 g, KH ₂ PO ₄ 1.5 g, _{K 2 HPO 4 0.5g, NaCl 0.1g} , yeast extract 1g, uracil _0.1g, CaCO 3 20g. However, (NH ₄ ) ₂ SO ₄ and CaCO ₃ were added after sterilization separately from other medium compositions. The pH of the medium was corrected to 7 with aqueous ammonia and then autoclaved at 120 ° C. for 10 minutes.

18% fructose solution sterilized at 18 hours of culture was added to each flask in 0.7 ml, and 20 mg / ml allyl mercaptan filtered and sterilized at the same time was added in 0.7 ml, and the cultivation was continued for a total of 40 Incubate for hours. As a result, the growth amount of the bacteria under each culture condition and the accumulation amount of S-allyl-L-cysteine and S-allyl-L-homocysteine in the culture solution were as shown in Table 3.

※）生育度は東京光電社製光電比色計モデルＡＮＡ-７Ａで、光路径２ｃｍの比色セルを用いて測定した。

*) The degree of growth was measured with a photoelectric colorimeter model ANA-7A manufactured by Tokyo Denshi Co., Ltd., using a colorimetric cell having an optical path diameter of 2 cm.

Separately, the same culture as described above was repeated under the condition of adding 125 μg / ml of L-methionine, and the fermentation broth was collected to 450 ml, frozen at −20 ° C., thawed, and centrifuged at 8,000 rpm at 4 ° C. The supernatant thus obtained contained 760 mg and 1120 mg of S-allyl-L-cysteine and S-allyl-L-homocysteine, respectively. This solution was passed through a column of H ⁺ type cation exchange resin Diaion SK # 1B having an inner diameter of 2.5 cm and a length of 30 cm at a rate of 153 ml per hour, and S-allyl-L-cysteine and S -Allyl-L-homocysteine was adsorbed.

259 ml of water was passed through the column in the same manner, washed with water, and then eluted in the same manner with 295 ml of 0.5N, 1N, 2N NH ₄ OH, and 77 ml fractions were collected.
Each fraction was tested by thin layer chromatography. As a result, S-allyl-L-homocysteine was eluted with 1NNH ₄ OH in the third and fourth fractions eluted with 0.5N NH ₄ OH. The second fraction was found to contain mainly S-allyl-L-homocysteine.

The respective fractions were collected, concentrated under reduced pressure, and dried in a desiccator to obtain 520 mg and 260 mg of crude products of S-allyl-L-cysteine and S-allyl-L-homocysteine, respectively. Dissolve a part of each product to 5 mg / ml, and perform high performance liquid chromatography using a preparative column of Develosil C30-UG5 (Nomura Chemical Co., Ltd.) to obtain S-allyl-L-cysteine and S -Allyl-L-homocysteine was fractionated, concentrated under reduced pressure, and dried in a desiccator to obtain each pure product.
As a result of NMR analysis, the respective data were consistent with S-allyl-L-cysteine and S-allyl-L-homocysteine. Further, as a result of LC-MS analysis, the molecular weight was consistent with the respective theoretical amount.

As Example 2, as an organic compound having a mercapto group, propyl mercaptan, 3-mercapto-1-hexanol or 3-mercapto-3-methylbutanol was used instead of allyl mercaptan in a medium so as to have a final concentration of 25 mM each. As a result of carrying out in the same manner as in Example 1 except for the addition, the S-position-substituted cysteine derivative and the S-position-substituted homocysteine derivative shown in Table 4 accumulated simultaneously.

S-substituted L-cysteine derivatives {S-allyl-L-cysteine, S-propyl-L-cysteine, S- (3-hydroxyhexyl) -L-cysteine, or S- (3-hydroxy-1) by fermentation , 1-dimethylpropyl) -L-cysteine} and S-substituted L-homocysteine derivatives {S-allyl-L-homocysteine, S-propyl-L-homocysteine, S- (3-hydroxyhexyl) -L- Homocysteine or S- (3-hydroxy-1,1-dimethylpropyl) -L-homocysteine} can be produced economically advantageously.

Claims (6)

When culturing a microorganism in a fermentation medium containing a carbon source, nitrogen source, sulfur source and other nutrients necessary for the growth of the microorganism, allyl mercaptan, propyl mercaptan, 3 as organic compounds having a mercapto group (-SH group) in the fermentation medium By adding -mercapto-1-hexanol or 3-mercapto-3-methylbutanol, the S-substituted cysteine derivative {S-allyl-L-cysteine, S-propyl corresponding to the added organic compound having a mercapto group -L-cysteine, S- (3-hydroxyhexyl) -L-cysteine, or S- (3-hydroxy-1,1-dimethylpropyl) -L-cysteine} and / or S-substituted homocysteine derivatives {S- Allyl-L-homocysteine, S-propyl-L-homocysteine, S- (3-hydroxyhexyl) -L-homocysteine, or S- (3-hydroxy-1,1-dimethylpropyl Pill) -L-homocysteine} is produced and accumulated in the fermented product and fermentation broth, and the S-substituted cysteine derivative and / or the S-substituted homocysteine derivative are collected from the fermented product and fermented solution. For producing these S-substituted cysteine derivatives and / or S-substituted homocysteine derivatives by fermentation The production method according to claim 1, wherein the microorganism used is Escherichia coli. The method according to claim 1, wherein the microorganism used is a mutant strain that requires at least methionine as a growth factor. 2. The method according to claim 1, wherein the microorganism used is a cystathionine / beta / lyase-deficient mutant. S-substituted cysteine derivatives produced are S-allyl-L-cysteine, S-propyl-L-cysteine, S- (3-hydroxyhexyl) -L-cysteine, or S- (3-hydroxy-1,1-dimethyl The production method according to claim 1, which is one of propyl) -L-cysteine or a combination thereof. The resulting S-substituted homocysteine derivative is S-allyl-L-homocysteine, S-propyl-L-homocysteine, S- (3-hydroxyhexyl) -L-homocysteine, or S- (3-hydroxy-1 The process according to claim 1, which is one or a combination of (1,1-dimethylpropyl) -L-homocysteine.