JPH038760B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION BACKGROUND OF THE INVENTION TECHNICAL FIELD The present invention relates to an enzymatic process for the production of Ribavirin. The chemical name of ribavirin is 1-β-D-ribofuranosyl-1,2,4-triazole-3-carboxamide, also known as Virazole, which has a broad range of potent antiviral effects against DNA and RNA viruses. (Annals of the New York Academy of Sciences (Ann.
New York Acad. Sci.) 284, 272-292 (1977)). Prior Art Conventionally known methods for producing ribavirin include a synthesis method, a fermentation method, and an oxygen method. A typical synthesis method is to use 3-methoxycarbonyl-1,2,4-triazole and 1-O
-acetyl-2,3,5-tri-O-acyl-β
-D-ribofuranose (melting method) and the obtained 1-(2',3'-5'-tri-O-acyl-β-
D-ribofuranosyl)-3-methoxycarbonyl-1,2,4-triazole is treated with ammonia to amidate and deprotect
4469, JP-A-49-80070, and JP-A-49-80071), a method using an aralkyloxy group as a substituent at the 3-position of the triazole in the same method as above (JP-A-55-160793) (see publication),
3-Methoxycarbonyl-1,2,4-triazole was trimethylsilylated and reacted with a halide of 2,3,5-tri-O-benzoyl-β-D-ribofuranoside (silylation method), and then treated with ammonia. There are known methods to do this (see Japanese Patent Laid-Open Nos. 48-4469 and 86372-1987).
In all of these synthetic methods, it is necessary to protect the active groups of the raw material compounds before the reaction, and during the reaction, it may be necessary to activate ribose, or it may be necessary to heat to high temperatures. However, there are problems such as complicated reaction operations such as the need for deprotection and amidation after the reaction. Furthermore, the regioselectivity of the condensation reaction is not high. The fermentation method involves culturing and propagating microorganisms belonging to the genus Brevibacterium, Corynebacterium, Arthrobacter, Micrococcus or Bacillus, using carbon sources and nitrogen that are necessary for culturing the microorganisms. before or during cultivation,
Add 1,2,4-triazole-3-carboxamide all at once or intermittently, and add 1,2,4-triazole-3-carboxamide at once or intermittently, and
A method is known in which ribavirin is produced and accumulated in the medium by culturing for a long period of 8 days (Special Publication No. 17830/1983, Journal of the Japanese Society of Agricultural Chemistry,
50(9), 423-430 (1976)). However, this method seems to have the following drawbacks. That is, since the production of ribavirin is carried out in a nutrient medium during the growth of microorganisms, it is first necessary to prepare a medium containing various nutrient sources in order to grow the microorganisms, and these must be added before inoculating the inoculum. Pretreatment is complicated, as the culture medium must be sterilized. Cultivation involving the growth of microorganisms for the purpose of accumulating ribavirin is usually carried out at room temperature between 20 and 40°C, so not only must consideration be given to bacterial contamination, but also under these conditions ribavirin decomposes. Since activity is also present, the produced ribavirin is also degraded, reducing the yield of the target product.
Cultivation must be carried out over a long period of 2 to 8 days. Various nucleosides, phosphoric oxides of ribavirin, and other metabolites are produced as by-products.
In order to recover ribavirin from a culture solution, it is necessary to separate not only the raw material compound but also various by-products, making isolation and purification complicated. Microorganisms must be cultured each time ribavirin is manufactured. In addition, as an enzymatic production method, 1,2,4-triazole-3-carboxamide and ribose-1
A method is known in which phosphoric acid is reacted with phosphoric acid in the presence of nucleoside phosphorylase at a pH of 5 to 9 and a temperature of 0 to 50°C.
(see publication). This method also has drawbacks such as the ribose-1-phosphate used as the ribose donor is unstable, and the enzyme is not easy to prepare because it uses a purified enzyme that is not easy to obtain. I think that the. SUMMARY OF THE INVENTION The present inventors have discovered for the first time that ribavirin can be produced by an enzymatic reaction under conditions in which microorganisms do not proliferate, using a microbial culture, bacterial cells, or treated bacterial cells as an enzyme source. Based on this knowledge, the present invention was completed. That is, the present invention provides a method in which 1,2,4-triazole-3-carboxamide or a salt thereof and a ribose donor are reacted in an aqueous medium under the enzyme action of Brevibacterium acetylicum under conditions in which the microorganism does not grow. The present invention provides a method for producing ribavirin, which is characterized by producing ribavirin. In the present invention, "under the action of an enzyme" means in the presence of a culture, bacterial cells, or treated microbial cells of the microorganism used. The biggest difference between the method of the present invention and conventional fermentation methods is that in the present invention, microorganism cultures, microbial cells, or processed microbial cells are used as the enzyme agent, and the conditions are optimal for the enzymatic reaction so that microorganisms do not grow. Below, 1,2,4-triazole-3-carboxamide or a salt thereof, which is a reaction substrate, is reacted with a ribose donor. Effects Compared to the fermentation method, the method of the present invention has almost no bacterial contamination when the reaction is carried out under conditions where microorganisms do not grow, such as high temperature conditions, and the decomposition reaction of ribavirin is suppressed, resulting in a decrease in the yield of ribavirin. Since it is an enzymatic reaction, the reaction time is short, there are few by-products produced, and it is easy to isolate and purify ribavirin.The enzyme source can be used repeatedly, continuously, or continuously. It has the advantage that it can be stored and the enzyme source can be prepared and used at any time. Furthermore, compared to the enzymatic method, it has advantages such as easier preparation of the enzyme source, wider selection of ribose donors from nucleosides, nucleotides, etc., and easy availability of ribose donors. Furthermore, by selecting the most suitable enzyme source for the method of the present invention, ribavirin can be produced in a much higher yield than in these conventional methods. Detailed Description of the Invention Enzyme Source/Used Microorganisms The microorganisms used in the present invention have a culture, microbial cells, or processed microbial cells that react with 1,2,4-triazole-3-carboxamide and a ribose donor. It contains an enzyme system that catalyzes the production of ribavirin, specifically Brevibacterium acetylicum.
acetylicum). One of the representative strains with strong enzyme activity is the AT-6-7 strain, which was isolated from the sand of Koshien Stadium in Nishinomiya City, Hyogo Prefecture. The mycological properties of this strain are described below. A Morphology (1) Cell morphology and size: short rod-like, 0.8-1.0
×1.0-1.2μm (2) Spore formation: None (3) Gram staining: Positive B Growth status in various media (1) Culture on broth agar plate (28℃, 48 hours) Colony shape: Circular Colony Surface ridges: Flat, Smooth Size: 2-4 mm Color: Yellow to pink-yellow (2) Broth agar slant culture (28℃, 48 hours) Growth: Good Growth shape: Warty ( (3) Broth liquid culture (28℃, 48 hours) Growth: Forms a bacterial ring on the surface and produces a slight sediment. (4) Meat juice gelatin puncture culture (20°C, 6 days): Liquefies into a Straitiform. (5) Litmus milk medium (28°C, 4 days): Slight coagulation and peptonization is also observed. C Physiological properties (1) Reduction of nitrate (28°C, 5 days): No reducibility. (2) Hydrogen sulfide generation (28℃, 5 days): Not generated. (3) Hydrolysis of starch: Degradable. (4) Catalase: Positive (5) Indole production: Not produced. (6) Formation of ammonia from peptone and arginine: negative (7) Methylred test: negative (8) V-P test: positive (9) Attitude towards oxygen: aerobic (10) O-F test (Hugh (based on Leifson method): F
Fermention (11) Acid production from sugars Positive: glucose, mannose, fructose, maltose, sutucarose, trehalose Negative: arabinose, xylose, galactose, lactose, sorbitol, inosit, glycerin Growth PH range: PH6.0-9.0 (13) Optimum growth temperature: 25 to 37°C.
The search was conducted using the classification criteria of Bacteriology, 7th edition (1957). As a result, the AT-6-7 strain is a short bacillus that is almost like a coccus, is Gram-positive, does not form filaments, and produces acid rather than carbohydrates, so it is closely linked to strains belonging to the genus Brevibacterium. , and named Brevibacterium acetylicum AT-6-7. The identification and attribution of the AT-6-7 strain is based on Virgies Manual of Determinative Bacteriology, 7th edition, and due to changes in the classification criteria, the identification of this strain may have been based on different classification criteria. If attribution is made, the microorganisms named above may belong to other species or genera, but based on the deposit with the depository institution and the above-mentioned mycological properties, It can be uniquely identified. Regarding this strain, the 1981 Ministry of International Trade and Industry Notification No.
In accordance with No. 178, we filed an application for deposit with the Institute of Microbial Technology, Agency of Industrial Science and Technology, and the deposit was made on January 13, 1981, with the deposit number being FERM P-6305. has been granted. In addition, the above-mentioned strains may undergo induced mutations by general mutagenesis methods such as physical treatments such as irradiation with ultraviolet rays, The mutant strain thus derived can also be used in the present invention, as long as it does not lose the enzymatic activity involved in ribavirin production, which is the object of the present invention. Furthermore, the genes of the enzyme system involved in the production of ribavirin, which is the object of the present invention, obtained from the strains preferably used in the present invention as described above are of the genus Brevibacterium, Corynebacterium, Arthrobacter, When such a trait is expressed by being taken up by a microorganism other than the genus Micrococcus or Bacillus, the method of using the culture, cultured cells, or processed product of such a microorganism for the purpose of the present invention is as follows: Included in the present invention. Preparation/Culture of Enzyme Source When culturing these microorganisms to prepare the enzyme source used in the present invention, the medium and culture method used are not particularly limited as long as these microorganisms grow. The medium used contains appropriate amounts of carbon sources and nitrogen sources that can be assimilated by these microorganisms, and if necessary, inorganic salts, trace growth-promoting substances, antifoaming agents, etc. are added. Specifically, carbon sources include sugars such as glucose, fructose, maltose, galactose, ribose, sutucarose, starch, starch hydrolyzate, molasses, and waste molasses, or derivatives thereof such as fatty acid esters, wheat, 〓, natural carbohydrates such as rice, alcohols such as glycerol, mannitol, methanol, ethanol, fatty acids such as gluconic acid, pyruvic acid, acetic acid, citric acid, hydrocarbons such as normal paraffin, kerosene, glycine, glutamic acid, glutamine , alanine, asparagine, and other general carbon sources, one or more of them may be appropriately selected and used in consideration of the assimilation ability of the microorganisms used. Nitrogen sources include meat extract, peptone, yeast extract, dried yeast, soybean hydrolyzate, soybean flour, milk casein, casamino acids,
Various amino acids, cornstarch liquor, corn seed meal or its hydrolyzate, fruit meal or its hydrolyzate, other animals,
Organic nitrogen compounds such as hydrolysates of plants and microorganisms, ammonium salts such as ammonia, ammonium nitrate, ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate, ammonium acetate, nitrates such as sodium nitrate,
Among inorganic nitrogen compounds such as urea, one or more types are appropriately selected and used, taking into consideration the assimilation ability of the microorganisms used. In addition, trace amounts of magnesium, manganese, iron, zinc, copper, sodium,
phosphates and hydrochlorides such as calcium and potassium;
One or more of sulfates, carbonates, nitrates, acetates, etc. are added as appropriate, and vegetable oil,
Antifoaming agents such as surfactants, trace amounts of growth-promoting substances such as vitamins B ₁ and B ₂ , nicotinic acid, pantothenic acid, biotin, and p-aminobenzoic acid may be added. Furthermore, when using microorganisms that exhibit nutritional requirements, it is necessary to add to the medium a substance that satisfies the growth of the microorganisms. Cultivation is carried out in a liquid medium containing the above-mentioned medium components by selecting a culture method suitable for the microorganism used from among ordinary culture methods such as shaking culture, aerated agitation culture, static culture, and continuous culture. Culture conditions may be appropriately selected depending on the type of microorganism and medium used, but the pH at the start of culture is usually adjusted to about 6 to 8, and culture is carried out at a temperature of about 25 to 35°C. The culture period may be a period sufficient for the growth of the microorganism used, and is usually 1 to 3 days. Embodiments of enzyme source The enzyme source used in the method of the present invention includes: 1.
It contains an enzyme system that catalyzes the reaction of producing ribavirin from 2,4-triazole-3-carboxamide and a ribose donor. The main essential enzyme for the enzymatic reaction in the method of the present invention is nucleoside phosphorylase, and it is essential that the enzyme source used in the present invention has this enzyme activity. Furthermore, in embodiments in which a ribose donor that does not serve as a direct substrate for the present enzyme is used, it is preferable that the ribose donor contains enzyme system activity that leads from the ribose donor to the substrate. After culturing the microorganisms as described above, the obtained culture, viable bacterial bodies collected from the culture by conventional methods such as centrifugation, sedimentation, and flocculation, or appropriate treatment of viable bacterial bodies. A treated bacterial cell product obtained by subjecting the enzyme can be used as an enzyme source in the present invention. Here, the term "culture" refers to a state in which the culture medium and cultured bacterial cells are unseparated after cultivation. In addition, the bacterial cell treatment product is
Dried bacterial cells, cell membrane and/or wall-denatured bacterial cells, crushed bacterial cells, immobilized bacterial cells, bacterial cell extracts, protein fractions of bacterial cell extracts having enzyme activity involved in the production of ribavirin, which is the object of the present invention protein fraction or its purified product, protein fraction or its purified product, etc. The method for obtaining the treated bacterial cell product is illustrated below. That is, physical treatments such as freeze-thaw treatment, freeze-drying treatment, ventilation drying treatment, acetone drying treatment, heating treatment under acidic or alkaline conditions, grinding treatment, ultrasonic treatment, osmotic pressure difference treatment, etc. means or enzyme treatments such as lysozyme, cell wall lytic enzymes, toluene,
By subjecting the bacterial extract to chemical or biochemical treatments such as contact treatment with a solvent such as xylene or butyl alcohol (butanol) or a surfactant, alone or in combination, for example, salting out treatment, By applying enzymatic separation and purification methods such as isoelectric focusing treatment, organic solvent precipitation treatment, various chromatographic treatments, and dialysis treatment alone or in combination, live bacterial cells, bacterial cell extracts, or purified products thereof can be subjected to comprehensive treatment. A bacterial cell-treated product can be obtained by applying enzyme immobilization means such as crosslinking treatment, adsorption treatment to a carrier, etc. Reaction substrate The reaction substrate in the enzyme reaction of the present invention is 1, 2,
4-triazole-3-carboxamide and ribose donor. 1,2,4-triazole-3-carboxamide can be used in either a free form or a salt such as a sodium salt. The ribose donor may be ribonucleoside or D-ribose, or any of their various phosphoric acid esters. That is, the base portion of the ribonucleoside may be any purine-based or pyridine-based base, and can be used regardless of whether it is derived from a natural product or chemically synthesized. In addition, even if the hydroxyl group of the sugar moiety of ribonucleoside or D-ribose is unsubstituted, monophosphoric acid ester residues, di- It may have a phosphate ester residue or a triphosphate residue. Further, these phosphoric acid esters may be in a free form, or may be common alkali salts such as sodium, potassium, calcium, magnesium, ammonium, and triethylammonium. Specific examples of ribose donors include inosine, adenosine,
Ribonucleosides such as guanosine, xanthosine, uridine, cytidine, 5'-inosinic acid, 5'-
Adenylic acid, 5'-guanylic acid, 5'-xanthylic acid, 5'-uridic acid, 5'-cytidylic acid, 2'(3')-
Inosinic acid, 2'(3')-adenylic acid, 2'(3')-guanylic acid, 2'(3')-xanthylic acid, 2'(3')-uridylic acid, 2'(3')- Examples include ribonucleotides such as cytidylic acid, D-ribose, and D-ribose-1-phosphate. Reaction Substrate Solution The substrate solution used in the enzyme reaction of the present invention is basically an aqueous liquid in which the above-mentioned reaction substrate is dissolved or suspended in an aqueous medium. In addition to the above-mentioned reaction substrates, the aqueous liquid contains substances that promote enzyme reactions, such as phosphate ion donors, organic solvents, surfactants, metal salts, coenzymes, acids, bases, and sugars, as necessary. It may contain a substance that improves the solubility of the substrate, a substance that improves the contact between the enzyme and the reaction substrate, and the like. As the aqueous medium, water or various buffers suitable for enzyme reactions (phosphate buffer, imidazole-hydrochloric acid buffer, veronal-hydrochloric acid buffer, Tris-hydrochloric acid buffer, etc.) can be used. The enzyme reaction of the present invention is mainly based on the action of nucleoside phosphorylase, and therefore requires the presence of phosphate ions in the reaction system. If phosphate ions are not present in the enzyme reaction system, it is necessary to add a phosphate ion donor. As the phosphate ion donor, any substance that can be dissociated into phosphate ions in an aqueous medium may be used, such as free phosphoric acid itself, inorganic phosphates, alkali metals such as sodium and potassium, calcium, Salts with alkaline earth metals such as magnesium and ammonium are preferably used. Furthermore, as a phosphate ion donor, a system capable of releasing phosphate ions in the enzyme reaction solution, such as a combination of a ribose donor ribonucleotide and a phosphatase, or a combination of a nucleotide and a nucleotidase, etc. can be used. . The enzymes involved in the reaction in such a system may be mixed in the enzyme source used in the present invention, such as separately added enzymes, or bacterial cells or bacterial cell-treated products having the enzymatic activity. It may be. The above-mentioned phosphoric acid donor system may be added from outside the system during the oxygen reaction, or may be contained as a component in the enzyme source. That is, as long as it is in a form that can be used for enzymatic reactions, the above-mentioned substances alone or a combination of two or more of them, or microorganisms containing the above-mentioned substances or a processed product thereof, can be used as the enzyme of the present invention. These substances may be added separately to the reaction solution during the reaction, or these substances contained as bacterial cell components in the microorganism used may be used as they are. Examples of organic solvents include methanol, ethanol, propanol, butanol, pentanol, acetone, methyl ethyl ketone, ethyl acetate, toluene, toterahydrofuran, dioxane, dimethyl sulfoxide, dimethyl acetamide, dimethyl formamide, 2-methoxyethanol, 2-ethoxy ethanol. , 1,2-dimethoxyethane and the like. Contact Method The reaction of the present invention is achieved by contacting the enzyme source and the reaction substrate in an aqueous medium under conditions in which microorganisms do not grow. The contact method may be selected as appropriate depending on the form of the enzyme source, but it is usually a batch method in which the enzyme source is suspended or dissolved in a reaction substrate solution and preferably stirred or shaken while heating, or a contact method is used. Mix with appropriate carriers, auxiliaries, and adsorbents as necessary,
Alternatively, a column method may be applied in which the reaction substrate solution is passed through the reaction substrate by supporting it in a column. Concentrations or Added Amounts of Reaction Substrates and Enzyme Sources During the reaction, the substrate concentration of the reaction solution is not particularly limited, and a substrate concentration below the saturation concentration of the substrate in the aqueous medium used at the reaction temperature is usually adopted; The substrate concentration can also be increased by adding the aforementioned organic solvents, surfactants, etc. to the substrate solution. Alternatively, the substrates can be present in a suspended state at a saturation concentration or higher in the reaction solution, and each substrate can be dissolved as the reaction progresses. Alternatively, the substrate can be added sequentially during the reaction to maintain its concentration at an appropriate level. When dissolving the substrate,
The substrate concentration is usually 5 to 5 for 1,2,4-triazole-3-carboxamide or its salts.
The amount is about 200mM, preferably about 10-100mM. Regarding the ribose donor, if it is added separately, it is usually about 5 to 300mM, preferably 10mM.
~150mM. The amount of the enzyme source to be used can be easily determined by those skilled in the art through preliminary experiments, taking into account the type of microorganism, its mode of use, reaction efficiency, economics, etc.; For fresh (wet) bacterial cells, a 10-150 mg/ml substrate solution is sufficient; for dry bacterial cells, a 2-30 mg/ml substrate solution is sufficient; for column methods, use an appropriate amount according to the batch method. Can be set. Reaction Conditions The conditions for the reaction of the present invention are not particularly limited, except that the bacterial cells are subjected to the reaction under non-proliferation conditions, that is, in the state of resting or dead sterilized cells. As a method for conducting the reaction under conditions where microorganisms do not grow, the enzyme reaction temperature is set within a temperature range in which the microorganisms used do not grow (however, the temperature range does not inactivate the enzymes involved in the reaction of the present invention). ,
A method of subjecting the microorganisms to a state in which they cannot grow by physically, chemically or biochemically treating them as described above, and then subjecting them to a reaction; The method of adding a substance that inhibits the reaction to the reaction substrate solution may be used alone or in combination, but the method of manipulating the reaction temperature is particularly effective and simple. In the reaction of the present invention, the reaction temperature is an important condition as described above, and is a characteristic of the present invention. The reaction proceeds in the range of 37-80℃,
Considering practicality, the temperature range is preferably 40 to 70°C.
Note that optimal temperature conditions vary depending on the type of reaction substrate, but can be easily determined by those skilled in the art through preliminary experiments. By carrying out the enzymatic reaction at a temperature range of 40°C or higher, the growth of the microorganisms used is largely suppressed. For example, 1,2,4-triazole-3-carboxamide was obtained by using the same viable cells of the same microorganism as in Example 1 described later and reacting at each predetermined temperature of 28 to 60°C in the same manner as in Example 1. Table 1 below shows the relationship between the production rate (%) of ribavirin from and the survival rate (%) of the microorganisms used after the reaction. The survival rate of microorganisms is the percentage of the number of viable microorganisms/ml after the reaction to the number of viable microorganisms/ml before the start of the reaction. [Table] As mentioned above, this reaction must be carried out under conditions where the microorganisms used do not grow, that is, under conditions where most of the microorganisms are dormant or die. Furthermore, it has been experimentally confirmed that in the present invention, setting the reaction temperature within the above range not only increases the enzyme reaction rate but also suppresses the decomposition reaction of the produced ribavirin. As an example, suspension 1 of the same microorganism as in Example 1
Add ml to 1 ml of 20mM ribavirin solution and heat at 28-60°C.
Table 2 shows the residual rate (%) of ribavirin after incubation at each predetermined temperature for 20 hours. [Table] From the above results, it can be confirmed that a reaction temperature of 37°C or higher is suitable. The liquid properties of the reaction substrate solution should be generally maintained within the range of PH4 to 10, preferably PH6 to 8.
When the pH fluctuates, it may be corrected to a preferred pH range using an acid such as hydrochloric acid, sulfuric acid, or phosphoric acid, or an alkali such as sodium hydroxide, potassium hydroxide, aqueous ammonia, or ammonia gas. The reaction time should be determined while checking the conversion rate of the reaction substrate to the target product, but in the case of a batch method, it is usually sufficient to react for about 2 to 45 hours, preferably 24 to 36 hours. The reaction may be carried out by setting appropriate conditions according to the method. Separation and purification After the reaction, if necessary, isolate and remove the bacterial cells using conventional methods such as filtration, centrifugation, or aggregation.
Subjected to ribavirin separation and purification process. Separation and purification of ribavirin may be carried out using known methods or applications thereof, such as various chromatography methods such as ion exchange chromatography, adsorption chromatography, partition chromatography, and gel permeation methods, countercurrent distribution, General separation and purification methods such as methods that utilize distribution between two liquid phases such as countercurrent extraction, and methods that utilize differences in solubility such as concentration, cooling, and addition of organic solvents may be used alone or in appropriate combinations. . Analysis In Examples of the present invention, ribavirin and 1,2,4-triazole-3-carboxamide were analyzed by high performance liquid chromatography. When analyzed using the equipment and conditions shown below,
Ribavirin has a retention time of 1, 2, 4 around 3.50 minutes.
-triazole-3-carboxamide retention time
It is eluted around 2.65 minutes, and the amount of each can be calculated from the calibration curve. Equipment: Shimadzu high performance liquid chromatograph LC-3A model (manufactured by Shimadzu Corporation) Column: Micro Bondapak C ₁₈ , 4.6 mm x 250 mm (manufactured by Nippon Waters Limited) Eluent: Contains 2% acetonitrile 20mM Tris-HCl buffer (PH7.5) Flow rate: 1ml/min Measurement wavelength: 225nm Column operating temperature: room temperature Experimental Examples The present invention will be explained in more detail with experimental examples below, but none of these were carried out. This is one embodiment of the present invention, and is not intended to limit the scope of the present invention. Example 1 Brevibacterium acetylicum AT-6-
7 was inoculated into 5 liters of a 2% aqueous solution of powdered bouillon (manufactured by Kyokuto Pharmaceutical Industries, Ltd.) and cultured with shaking at 28°C for 24 hours. After culturing, the cells were collected by centrifugation, washed, and sterilized water was added to obtain 250 ml of cell suspension.
250 ml of the above bacterial cell suspension was added to 750 ml of an aqueous solution (PH 7.0) containing 66.7 mM 1,2,4-triazole-3-carboxamide, 66.7 mM inosine, and 100 mM potassium phosphate, and reacted at 60°C for 24 hours. (Ribavirin production rate 74.88%). After the reaction solution was centrifuged to remove bacterial cells, it was passed through a cation exchange resin (H ⁺ type), and the passed water washing solution was adsorbed on activated carbon. Ribavirin was eluted from the activated carbon column with an ethanol-ammonia solution, and after removing the ethanol after elution, it was passed through an anion exchange resin, and the washed water solution was concentrated under reduced pressure.
The volume was adjusted to 50 ml and cooled. After cooling, the precipitated crystals were separated and dried to obtain 6.5 g of ribavirin crystals. Comparative Example Using the same strain as in Example 1,
An attempt was made to produce ribavirin by adding a raw material triazole compound during the growth of microorganisms in the same manner as in Example 1 of Publication No. 17830. That is, the above strain was mixed with 130 g of glucose, 1 g of potassium phosphate, 3 g of dipotassium phosphate, 1 g of magnesium sulfate, 0.1 g of calcium chloride, 10 mg of iron sulfate, 5 mg of zinc sulfate, 10 mg of manganese sulfate, 5 mg of vitamin _B1 , and 10 mg of calcium pantothenate. , cystine 20mg, biotin 30μ
With a composition containing 10 g of meat extract, 2 g of ammonium sulfate, and 2 g of urea (separately sterilized) per liter, inoculate 10 ml of a medium adjusted to pH 7.6, and adjust the pH to 7.2 with aqueous ammonia every 12 hours. The cells were cultured with shaking at 28°C. 1,2,4- 24 hours after the start of culture
Triazole-3-carboxamide was added at a concentration of 2 mg/ml and cultured for an additional 4 days. After centrifuging the culture solution, the supernatant was analyzed, but no ribavirin production was observed. Example 2 The same bacterial strain as in Example 1 was cultured in the same manner as in Example 1 (however, each culture solution was 10 ml), and after culturing,
Bacteria were collected by centrifugation, and 1 ml of sterilized water was added to each to obtain a bacterial cell suspension. To this cell suspension, 1 ml each of an aqueous solution (PH7.0) containing 20 mM 1,2,4-triazole-3-carboxamide, 20 mM of various ribose donors shown in Table 3, and 25 mM monopotassium phosphate was added. 60
The reaction was allowed to take place at ℃ for 24 hours. After the reaction was completed, bacteria were removed by centrifugation, and the supernatant was analyzed, and the production rate of ribavirin was as shown in Table 3. Note that the ribavirin production rate refers to the conversion rate (%) of 1,2,4-triazole-3-carboxamide to ribavirin. [Table] Example 3 The same bacterial strain as in Example 1 was inoculated into each 100 ml of 2% powdered bouillon medium and cultured with shaking at 28°C for 22 hours to obtain cultured bacterial cells. A suspension of the processed material was obtained. 1 Acetone-dried bacterial cells: Add 50 ml of acetone to live bacterial cells, let stand for 15 minutes, centrifuge, add another 50 ml of acetone to the obtained bacterial cells, perform the same treatment, and then vacuum dry. Bacterial cells were obtained. Water was added to this to obtain 10 ml of a suspension of treated bacterial cells. 2. Freeze and thaw bacterial cells: Freeze live bacterial cells at -80°C overnight, thaw, and add water to make 10 ml of bacterial cell suspension.
I got it. 3 Osmotic pressure differential treatment of bacterial cells: Add 100 ml of saturated saline to live bacterial cells, cool on ice overnight, centrifuge to discard the supernatant, add water to isolated bacterial cells, and suspend the treated bacterial cells. 10 ml of liquid was obtained. 4. Ultrasonicated bacterial cells: Water was added to the live bacterial cells to make a total volume of 10 ml, and ultrasonication was performed for 20 minutes at an output voltage of 1.6 KV. 10 ml each of the above bacterial cell suspensions and Example 1
Add 10 ml of untreated bacterial cell suspension obtained in the same manner as
10 ml each of a reaction substrate solution (PH7.0) containing 20 mM 1,2,4-triazole-3-carboxamide, 20 mM inosine, and 25 mM potassium phosphate was added, and after reaction at 60°C for 24 hours, the production rate of ribavirin was analyzed. However, the results were as shown in Table 4. [Table] Example 4 The same bacterial strain as in Example 1 was inoculated into 25 ml of 2% bouillon medium, cultured with shaking at 28°C for 24 hours, and after culturing, the bacteria were collected, and water was added to each 2.5 ml of bacterial cells. A suspension was prepared. Add to this 2.5 ml each of the same reaction substrate solution as in Example 3.
was added and reacted for 20 hours at various temperatures from 28 to 70°C (Table 5), and the production rate of ribavirin was analyzed.
It was as shown in the table. [Table] Example 5 Using the same strain as in Example 1, add 2.5 ml each of the following reaction substrate solutions (A) or (B) to 2.5 ml each of the bacterial cell suspension prepared in the same manner as in Example 4. In addition, after reaction at 60° C. for 20 hours, the production rate of ribavirin was analyzed and was as shown in Table 6. Reaction substrate solution (A): 20mM 1,2,4-triazole-3-carboxamide and 20mM inosine Reaction substrate solution (B): Add 25mM monopotassium phosphate to the same amount of each substrate as in the above reaction substrate solution (A). [Table] Example 6 2.5 ml of the reaction substrate solution (B) of Example 5 was added to 2.5 ml of the same bacterial cell suspension as in Example 5, and after reaction at 60°C for 20 hours, the bacterial cells were separated. Add 10ml of water to this isolated bacterial body and use it for the next reaction, and repeat the same reaction as above for 10 minutes.
Repeated times. The first ribavirin production rate is 100
Table 7 shows the relative production rate of each reaction when [Table] Example 7 The same strain as in Example 1 was inoculated into 1 liter of 2% bouillon medium, and after culturing with shaking at 30°C for 22 hours,
Viable cells were obtained by centrifugation. Add solution A [24 ml of 1N hydrochloric acid, Tris
3.425g, aqueous solution prepared by dissolving 0.23ml of TEMED (N,N,N',N'-tetramethylenediamine) and diluting it to 100ml with water] 20ml, Solution B [acrylamide 30
g, BIS (N,N-methylenebis(acrylamide)) 0.8g dissolved in water to make 100ml]
20 ml and 40 ml of Solution C [an aqueous solution in which 0.3 g of ammonium persulfate was dissolved in water to make 200 ml] were added and allowed to stand to immobilize the bacterial cells. After immobilization, the cells were cut into small pieces using a homogenizer to obtain 180 ml of immobilized bacterial cells. 20ml of a substrate solution containing 20mM 1,2,4-triazole-3-carboxamide, 20ml inosine, and 25mM monopotassium phosphate to 10ml of these immobilized bacterial cells.
(PH7.0) and reacted at 60°C for 24 hours. Analysis of the reaction solution revealed that 62.89% ribavirin had been produced. In addition, when the reaction was carried out under the same conditions using viable cells, the production rate of ribavirin was 65.44%. Example 8 Brevibacterium acetylicum AT-6-
7 and the known strain Brevibacterium ammoniagenes (B.ammoniagenes) ATCC 6871
The strains were cultured under the same conditions and subjected to enzyme reaction under the same conditions, and the amount of ribavirin produced (production rate) was compared. That is, 1.5% yeast extract medium (PH7.0) each 9
Both strains were inoculated into ml and cultured with shaking at 28°C for 1 day. 10ml of a solution containing 40mM 1,2,4-triazole-3-carboxamide, 60mM disodium 5'-uridylate and 8mM monopotassium phosphate
Bacterial cells collected from the culture medium by centrifugation were added to the mixture, and the mixture was allowed to react at 45°C for 20 hours. After the reaction, the bacterial cells were removed by centrifugation, and the reaction solution was analyzed by high performance liquid chromatography. The results were as shown in Table 8. [Table] Example 9 Example 8 Using AT-6-7 strain and ATCC 6871 strain
100 ml of each culture was carried out in the same manner as above, and the cells were collected by centrifugation to obtain bacterial cells. The cells were treated with 40mM 1,2,4-triazole-3-carboxamide, 60mM uridine and
It was added to 100 ml of a solution containing 50 mM monopotassium phosphate, and reacted at 45°C for 20 hours. After the reaction, the production rate of ribavirin was measured and found to be 86.04% for the AT-6-7 strain.
In the case of ATCC 6871 stock, it was 2.30%. Example 10 Each bacterial cell of AT-6-7 strain and ATCC 6871 strain obtained by the same method as Example 8 (culture solution 20
ml min), 4mM 1,2,4-triazole-3-
A solution containing carboxamide, 4mM uridine and 5mM monopotassium phosphate (substrate solution A) and a solution containing 4mM 1,2,4-triazole-3-carboxamide, 4mM disodium 5'-uridate and 1mM monopotassium phosphate (substrate solution A). It was added to each of the substrate solutions B) and allowed to react for 24 hours. In addition, the reaction is performed at 45℃ in the case of AT-6-7 bacteria.
In the case of ATCC 6871 strain, it was carried out at 60°C. After the reaction, the production rate of ribavirin was measured and was as shown in Table 9. [Table] Example 11 Using the same method as in Example 8, strain AT-6-7 and
Bacterial cells obtained by culturing ATCC 6871 strain (5 ml of culture solution)
water was added to obtain 0.5 ml of bacterial cell suspension. Add 0.5 ml of the above cell suspension to 0.5 ml of a solution (PH7.0) containing 17.8 mM 1,2,4-triazole-3-carboxamide, 81.9 mM uridine, and 58.8 mM monopotassium phosphate, and incubate at 60°C for 10 Allowed time to react. After the reaction, the amount of ribavirin produced in the reaction solution was measured and the production rate of ribavirin was calculated.
It was as shown in the table. [Table] Example 12 Various strains of Brevibacterium acetylicum shown in Table 11 were cultured in the same manner as in Example 8,
Centrifuge the culture solution to remove bacterial cells (4 ml of each culture solution)
I got it. 4ml of a solution containing 40mM 1,2,4-triazole-3-carboxamide, 60mM disodium 5'-uridate and 10mM monopotassium phosphate
Each of the above bacterial cells was added to the solution and reacted at 45℃ for 22 hours. After the reaction, the production rate of ribavirin was measured.
It was as shown in Table 11. [Table] Example 13 The same operation as in Example 12 was performed except that 60 mM inosine was used as the ribose donor and the reaction temperature was 60° C., and the results shown in Table 12 were obtained. [Table] Reference Example In order to compare the phosphatase activities of the AT-6-7 strain and the ATCC 6871 strain, a cell suspension of each strain was added to a 5'-uridylate disodium solution and reacted. The conversion rate was measured. The bacterial cell suspension was prepared by suspending the bacterial cells (9 ml of culture solution) obtained by culturing in the same manner as in Example 8 in 1 ml of water, and 6 mM disodium 5'-uridylate was added as the substrate solution. 9 ml of the aqueous solution containing was used. The reaction was carried out at 45°C, and the conversion rate was measured at each time period from 0.5 to 24 hours. The conversion rate was measured by high performance liquid chromatography. The result is
It was as shown in Table 13. 【table】

Claims (1)

[Scope of Claims] 1 1,2,4-triazole-3-carboxamide or a salt thereof and a ribose donor are prepared from Brevibacterium acetylicum.
1. A method for producing ribavirin, which comprises producing ribavirin by reacting it in an aqueous medium under conditions in which the microorganism does not grow under the enzymatic action of M. acetylicum. 2. Claim 1, wherein Brevibacterium acetylicum is Brevibacterium acetylicum AT-6-7 (Feikoken Bibori No. 6305)
The method described in section. 3. A method in which the enzymatic reaction is carried out under conditions in which microorganisms do not grow, a method in which the reaction is carried out while the reaction solution is maintained at a temperature condition in which microorganisms cannot grow, and a method in which the microorganism cells are treated in advance by a method in which the microorganisms are suspended or killed. It is one type of method selected from the group consisting of a method of carrying out a reaction using this, and a method of carrying out a reaction by adding a substance that inhibits the growth of microorganisms to the reaction solution, or a method that is a combination of two or more types. , a method for producing ribavirin according to any one of claims 1 to 2. 4. Any one of claims 1 to 3, wherein the non-growth condition for microorganisms is a temperature condition of 37 to 70°C.
The method for producing ribavirin as described in section.