JPH0339676B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention utilizes intracellular enzymes of microorganisms to biochemically hydrolyze cystine bishydantoin, thereby producing N,N'-biscarbamoyl-
The present invention relates to a method for producing D-cysteine, and aims to industrially advantageously produce a D-cysteine precursor useful as a synthetic intermediate for semi-synthetic penicillin, semi-synthetic cephalosporin, or luciferins. The present inventors previously disclosed a method for producing 5-phenylhydantoin or its substituted derivatives in a patent application filed in 1983.
−11575 (Special Publication No. 11575-45195), Patent Application No. 1983-
No. 145748 and Patent Application No. 48717 (1983)
1910), and patent application No. 157713/1989 (1983), which describes a method for similarly producing N-carbamoyl-D-α-amino acids from 5-substituted hydantoins corresponding to natural amino acids or their substituted derivatives. The application was filed as (Kōsho 56-1909). The present invention, as one development of the above technology, has confirmed its application to cystine bishydantoin, which has two hydantoin rings in one molecule. The method of the present invention is expressed by the following formula. As described above, the present invention relates to a culture, a bacterial cell, or a microorganism having the ability to stereospecifically hydrolyze a hydantoin ring to cystine bishydantoin to produce N,N'-biscarbamoyl-D-cystine. N,N'-biscarbamoyl-D-, which is characterized in that the body-treated product is treated in an aqueous medium.
This is a method for producing cystine. The microbial enzyme used in the present invention is the D of each hydantoin ring of cystine bishydantoin.
It selectively hydrolyzes only the body and converts it to carbamoyl body. The produced carbamoyl body does not racemize under the present microbial conversion conditions, but on the other hand, each hydantoin ring of the substrate cystine bishydantoin has the property of racemizing in an aqueous medium with a pH of 7 to 9. There is. Therefore, even if the D-form hydantoin ring is consumed by the reaction, the D-form is always replenished to the reaction system due to racemization of the L-form. In other words, in the reaction system, enzymatic hydrolysis and racemization of the substrate proceed in parallel. Therefore, whether cystine bishydantoin in the DL-form (representing an optically inactive form, meaning DL- and meso-forms), L-form, or D-form is used as a raw material for the reaction, there is no substantial effect on cystine bishydantoin. are the same, and the product is N,N'-biscarbamoyl-D, which has both two hydantoin rings converted to the D-carbamoyl form.
- Cystine can be obtained. In the chemical synthesis of cystine and cysteine, due to the high reactivity and instability of the mercapto group,
The process is complicated and there are few industrially effective methods.
Therefore, there is no practical method to optically resolve these to obtain L- or D-cystine or cysteine. Conventionally, there is a method for producing D-cysteine that uses the action of acylase. For example, after converting easily available L-cystine into a diacetyl derivative, it is racemized with acetic anhydride, and then acylase is applied to this. A method in which bisacetyl-D-cystine, which is not subjected to enzyme action, is separated, acid hydrolyzed, and then reduced (Journal of
American Chemical Society Volume 79, 4538
However, in a compound such as cystine that theoretically has two asymmetric centers, D-cystine exists in only a small amount in the racemic compound, and the yield is low, making it impractical. do not have. Recently, due to microbial degradation of mercaptomethylhydantoin,
There are reports that D-cysteine and D-cystine are produced and accumulated (JP-A-54-2398, JP-A-54-2398).
52791, 54-89088, 55-11569, 55-
104890, 55-114291, etc.), due to the high reactivity of the mercapto group, there are many side reactions such as decomposition of the substrate, and the yield is low, making it impractical. However, by using the method of the present invention, cystine bishydantoin, which is relatively stable, can be used as a substrate using an inexpensive microbial enzyme, and not only DL-form but also L-form substrates can be used. D-form N,N'-biscarbamoyl- in high yield
It can be converted to cystine. N,
Since N'-biscarbamoyl-D-cystine is easily converted to D-cystine, for example by reaction with nitrous acid, the present invention provides
This provides an extremely advantageous means for industrially producing cysteine. The present invention will be explained in more detail below. As mentioned above, cystine bishydantoin used as a raw material in the present invention is DL-form,
Either L-form or D-form is good; for example, L-cystine bishydantoin obtained by reacting easily available L-cystine with an alkali metal cyanate salt and then cyclizing under acidic conditions is also good. It becomes a suitable substrate. The microorganism used in the present invention stereospecifically hydrolyzes the hydantoin ring of cystine bishydantoin to produce N,N'-biscarbamoyl-D-
Examining the presence or absence of the ability to produce cystine from wild strains existing in nature, strains stored in public microbial preservation institutions, or microorganisms artificially mutated from them. It is selected by For example, the following method is used to test this ability. First, 2 ml of the microbial culture is centrifuged to collect bacterial cells, washed with physiological saline, and then centrifuged again to collect the bacteria. This isolated bacterial body (wet weight 40-200
mg) to 2 ml of a buffer solution (PH7-9) containing L-cystine bishydantoin at a concentration of 0.5-2.0%, and react with gentle shaking at a temperature of 30-50°C for 10-40 hours. . After the reaction, a spot test (color developed by spraying a concentrated hydrochloric acid solution of p-dimethylaminobenzaldehyde) was performed on paper using a portion of the reaction solution, and for those that developed a yellow color,
High performance liquid column chromatography analysis is performed to determine the amount of N,N'-biscarbamoyl cystine produced. In this way, for strains that showed a relatively high conversion rate, the scale of the experiment was increased and the hydrolysis reaction of cystine bishydantoin was carried out again, the 3 reaction liquid was centrifuged, and the supernatant was lyophilized and then Using the residue, N,N'biscarbamoyl-cystine was isolated by silica gel column chromatography (developing solvent: n-propanol:water = 8:2), and a strain in which it was found to be in the D form was isolated. It is employed as a microorganism used in the present invention. The microorganisms used in the present invention are found in bacteria and actinomycetes. Those belonging to bacteria include the genus Bacillus and Pseudomonas, and those belonging to actinobacteria include the genus Nocardia and Mycobacterium. It is found inside. Specifically, the microorganisms of the present invention include the following. Bacillus species
KNK 108 (FERM P-6056) Microtechnical Research Institute
No. 6056 Pseudomonas striata
striata) IFO 12996 Nocardia coralina (Nocardia
corallina) IFO 3338 Mycobacterium smegmatis ATCC 607 The mycological properties of Bacillus sp. KNK108 are as follows. The experimental method is in the Manual of microbiology.
Methods (MJPelczav). () Morphology (1) Cell shape and size: Bacillus, 2-6 μm
×0.8μm (2) Presence or absence of cell pleomorphism: None (3) Presence or absence of motility, epiphytic state of flagella: None (4) Presence or absence of spores, shape, and size: OVal 1.0μm
×0.8μm (5) Gram staining: Positive () Growth status in each medium (1) Broth agar plate culture: Moderate growth, irregular, flat, lobed, translucent (2) Broth agar slant culture : Appropriate growth, filamentous to spreadable, no soluble pigments. (3) Meat juice liquid culture: Moderate growth Sediment (4) Meat juice gelatin puncture culture: Liquefied (layered) () Physiological properties (1) Nitrate reduction: Negative (2) MR test: Negative (3) VP test: Negative (4) Indole production: Negative (5) Starch hydrolysis: Negative (6) Citric acid utilization: Positive (7) Pigment production: No production (8) Urease: Positive (9) Catalase: Positive (10 ) Growth range: Grows at a temperature of 37℃, but does not grow at a temperature of 41℃. PH6-9.5 (11) Attitude towards oxygen: Aerobic (12) Presence or absence of acid production from sugar (peptone medium) L-arabinose: negative D-xylose: negative D-glucose: negative D-mannose: negative Trehalose: negative ( 13) Phenylalanine deaminase reaction:
Negative (14) Degradability of casein: Positive (15) Salt tolerance: Grows in 7% salt (16) Production of dihydroxyacetone: Negative This method utilizes the action of an enzyme, and this enzyme can be produced and accumulated within microbial cells by culturing the microorganism in a conventional medium containing an organic nutrient source. The culture medium usually contains an assimilable carbon source, a nitrogen source, and inorganic salts and nutrients necessary for the growth of each microorganism.
Furthermore, various pyrimidine nucleic acid bases or derivatives thereof, or various hydantoins are added at a concentration of 0.05 to
It is desirable to add 0.3% to adaptively enhance the desired enzyme. Examples of pyrimidine nucleic acid bases with high enzyme-inducing effects include uracil, cytosine, and thymine, and derivatives thereof include dihydrouracil and dihydrothymine. Also, among the hydantoins, hydantoin, DL-5-
Methylhydantoin and the like are relatively preferred. However, the most practically preferred enzyme-inducing substrate common to many microorganisms is uracil. Culture conditions range from a temperature of 20 to 85°C and a pH of 4 to 11, depending on the optimal growth conditions of the microorganism used, but generally the culture is carried out at a temperature of 20 to 40°C and a pH of 5 to 9. During cultivation, aeration and stirring may be performed to promote the growth of microorganisms. For the hydrolysis reaction of cystine bishydantoin, the microorganisms cultured as described above are used as a culture,
It is used in the form of bacterial cells or treated bacterial cells. Normally, a culture of microorganisms can be used as is for the reaction, but if components in the culture become a hindrance or if a large amount of enzyme is desired to be used, microbial cells isolated from the culture may be used. Although the bacterial cells can be used as viable cells, they can be used as dried cells such as freeze-dried cells or acetone-dried cells for convenience in storage and handling. In addition, it is also possible to use the microbial cells in the form of processed microbial products such as crushed microbial cells and microbial cell extracts, instead of the microbial cells themselves. Furthermore, it is also possible to use the above-mentioned bacterial cells or treated bacterial cells immobilized by a known method. In order to cause a microbial culture, bacterial cells, or treated bacterial cells to act on cystine bishydantoin, a method is generally used in which the two are mixed in an aqueous medium. There are no particular restrictions on the substrate concentration in the reaction solution, but at high concentrations of 1% or more, the substrate often does not dissolve completely, but as the reaction progresses, it gradually dissolves, so there is no problem. It won't be. The true substrate of the microbial enzyme used in the present invention is the D-form, and only the D-form is selectively hydrolyzed and converted to the carbamoyl form. However, since racemic equilibrium exists in the reaction system, the unhydrolyzed L form racemizes as the D form is consumed, and the D form is constantly supplied to the reaction system. Since the racemization reaction proceeds in parallel with the enzymatic reaction,
The L-form can also be considered as an indirect substrate. Therefore, the raw material cystine bishydantoin is in the DL form,
There is almost no difference in practical effects whether it is L-form or D-form. When carrying out the hydrolysis reaction of cystine bishydantoin in an aqueous medium, the practically preferred pH range is 7 to 9, particularly preferably 7.5 to 8.5. The reaction rate is extremely low below PH7;
Exceeding this amount causes undesirable side reactions and is therefore impractical. The reason why pH7-9 is preferable is that the optimum pH of the microbial enzyme used in the present invention is
is around 8, the solubility of the substrate increases as the pH increases, and the racemization reaction of the hydantoin ring is effectively promoted in alkaline conditions. , N'-biscarbamoyl-D-cystine. Since the pH of the medium decreases as the reaction progresses, it is desirable to continuously add a neutralizing agent to maintain the optimum pH during the reaction. Suitable neutralizing agents include ammonia, caustic soda, caustic potash, and soda carbonate. In addition, depending on the purpose, the reaction may be carried out by adding an organic solvent or a surfactant to the aqueous medium. The reaction temperature is determined to be suitable for the microorganism used, but is usually within the range of 25 to 55°C. N,N'-biscarbamoyl-D-cystine produced by the hydrolysis reaction can be used in the reaction for converting it into D-cystine, usually without being isolated. However, if isolation is desired, conventional methods such as silica gel column chromatography or anion exchange resin may be applied. Since N,N'-biscarbamoyl-D-cystine obtained by the method of the present invention can be easily converted to D-cystine by reacting with nitrous acid, for example, it is possible to synthesize pharmaceuticals by the method of the present invention. This makes it possible to industrially advantageously produce D-cysteine, which is useful as an intermediate, or D-cysteine easily derived therefrom. EXAMPLES The present invention will be specifically explained below with reference to Examples, but the present invention is not limited to these Examples. Example 1 A nutrient liquid medium having the following composition was prepared, and 500
Add 100 ml to a 120 ml shoulder shake flask.
Steam sterilization was performed at ℃ for 25 minutes. Meat extract 5g/, yeast extract 5g/, polypeptone 10g/, NaCl 3g/, uracil 1g/, MnCl ₂ 4H ₂ O 20mg/ (PH7.0) This was cultured for 24 hours at 33℃ on an agar medium with the same composition. One platinum loop of the microorganisms shown in the table was inoculated, and 33
Culture was carried out at ℃ for 18 hours with shaking. Bacterial cells were centrifuged from this culture solution, washed once with approximately the same amount of physiological saline as the culture solution, and then centrifuged again to collect the bacteria. The cells were dissolved in 0.1M phosphate buffer (PH7.8) containing DL-cystine bishydantoin 1g/dl for 50 minutes.
ml and kept at 33°C for 20 hours with gentle shaking to react. After the reaction, the supernatant was obtained by centrifuging the reaction solution, and the amount of N,N'-carbamoyl-cystine produced was separated and quantified by high performance liquid chromatography (column: Hitachi anion exchange resin #2632, 2.6× 500mm, containing 1mM NaCl
0.01N-HCl:methanol=3:1, detection 254n
m). Furthermore, this supernatant was adjusted to pH 3.0 with 3N-HCl, then freeze-dried, and the entire residue was used for silica gel column chromatography (developing solvent n).
-propanol:water=8:2). The effective liquid was concentrated under reduced pressure to isolate N,N'-biscarbamoyl-cystine in crystal form, and its specific optical rotation was measured. The above results are shown in the table for each microorganism used.

[Table] Example 2 Medium 18 having the following composition was prepared in a 30-volume jar fermenter and steam sterilized at 120°C for 25 minutes. Meat extract 30g/, glucose 10g/, salt 3g/, uracil 1g/, MnCl ₂ 4H ₂ O 20mg/(PH7.0) To this, in a 2-volume shoulder shake flask, add 250ml of the same composition medium at 33°C. The entire amount of Bacillus sp. KNK108 cultured for 24 hours was inoculated and cultured at 33° C. for 18 hours under aeration and stirring conditions of 2 N/min and 250 rpm to prepare a culture solution. To this culture solution 15, 300 g of L-cystine bishydantoin was added and under stirring conditions with nitrogen aeration, 2.5N-
The reaction was carried out at 37°C for 24 hours while maintaining the pH at 7.8 with NaOH. After the reaction, high performance liquid chromatography analysis of the reaction solution revealed that the amount of N,N'-biscarbamoyl-cystine produced was 181 g, and the conversion rate was 54%. Furthermore, while cooling and stirring this reaction solution, concentrated hydrochloric acid was added.
1750 ml was added, and while the temperature was maintained at around 5°C, 400 ml of an aqueous solution containing 76.6 g of sodium nitrite was added using a metering pump for 2 hours, and the reaction was continued for an additional 3 hours. This decarbamoyl reaction solution was centrifuged to remove insoluble matter, and then the IR-120B cation exchange resin (Na
It was passed through a column (8.5 x 70 cm) packed with mold) and washed with 6 portions of ion-exchanged water. Elution was performed with 5% ammonia water, and 2.5% of the effective solution was concentrated to 300 ml under reduced pressure, and the pH was adjusted to 4.8 using 6N-HCI to obtain 83.5 g of crude crystals of D-cystine. This crude crystal was recrystallized by neutralization crystallization and purified D-cystine 69.5%
g ([α] ²⁰ _D = +212 c = 0.1 N-HCl).

Claims (1)

[Scope of Claims] 1. Bacillus, Mycobacterium, and Nocardia having the ability to stereospecifically hydrolyze the hydantoin ring to cystine bishydantoin to produce N,N'-biscarbamoyl-D-cystine. Cultures of microorganisms of the genus Pseudomonas,
1. A method for producing N,N'-biscarbamoyl-D-cystine, which comprises reacting bacterial cells or a bacterial cell-treated product in an aqueous medium. 2. The production method according to claim 1, wherein cystine bishydantoin is in the DL form or the L form. 3. The manufacturing method according to claim 1, wherein the aqueous medium has a pH of 7 to 9. 4. The production method according to claim 1, wherein live or dried microbial cells are used as the microbial cells. 5. The manufacturing method according to claim 1, wherein a microbial cell crush product or a microbial cell extract is used as the microbial cell treatment product. 6. The production method according to claim 1, 4, or 5, in which an immobilized product of microbial cells or a processed product of microorganisms is used. 7. Claims 1, 4, and 5 that use microorganisms whose ability to stereospecifically hydrolyze hydantoin rings is enhanced by adding pyrimidine nucleobases or their derivatives to the culture medium. The manufacturing method according to item 6 or item 6.