JPS6231919B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for the enzymatic production of amoxicillin, and more particularly to a method for the enzymatic production of amoxicillin and 5-aminopenicillanic acid.
The present invention relates to an enzymatic method for producing amoxicillin directly from (p-hydroxyphenyl)hydantoin. Amoxicillin (6-[D-(-)-α-amino-D-hydroxyphenylacetamide] penicillanic acid) is an excellent antibiotic substance with strong antibacterial activity against a wide range of Gram-positive and Gram-negative bacteria. In recent years, they have come to occupy a particularly important position as chemotherapeutic agents for various infectious diseases of humans and animals caused by contamination with these bacteria. Chemical synthesis methods and enzymatic production methods are known for the production of amoxicillin, but the chemical synthesis methods require steps such as introduction and removal of a protecting group, and conversion of an acyl donor into an active derivative. Since the entire reaction process is complex and diverse, enzymatic production methods have been developed as an alternative. However, in the conventionally known enzymatic production method, the substrate 6-aminopenicillanic acid and the active derivative of D-p-hydroxyphenylglycillin are mixed with the cells of microorganisms belonging to the genus Pseudomonas, Achromobacter, Bacillus, etc. This is an excellent method for producing amoxicillin in one step by enzymatic reaction in the presence of the treated product. is generally a step of hydrolyzing 5-(p-hydroxyphenyl)hydantoin,
Since it is produced through complicated steps such as an optical resolution step to obtain D-form and an esterification or amidation step to obtain an active derivative, it cannot be said to be necessarily satisfactory. As a result of extensive research by the present inventors to develop a method for enzymatically producing amoxicillin efficiently in a simple process, in the coexistence of a culture of a microorganism belonging to the genus Flavobacterium or a processed product thereof,
The inventors discovered that amoxicillin can be directly produced in one step by reacting 6-aminopenicillanic acid and 5-(p-hydroxyphenyl)hydantoin, leading to the completion of the present invention. That is, the present invention relates to a microorganism that belongs to the genus Flavobacterium and has the ability to synthesize amoxicillin from 6-aminopenicillanic acid and 5-(p-hydroxyphenyl)hydantoin in the presence of a culture or a processed product thereof. , 6-aminopenicillanic acid and 5-(p-hydroxyphenyl)hydantoin are reacted to produce amoxicillin, which is an enzymatic method for producing amoxicillin. The microorganisms used in the method of the present invention belong to the genus Flavobacterium and have the ability to produce an enzyme that synthesizes amoxicillin from 5-(p-hydroxyphenyl)hydantoin and 6-aminopenicillanic acid, such as Flavo. Bacterium hydantoinophilum AJ 11322, FERM−
P4819 can be used, but it is not limited to this strain; any strain, whether natural or mutant, that produces a synthetic enzyme can be used in the present invention. The mycological properties of the above microorganism strain AJ11322 are described below. Mycological properties of strain AJ11322 (a) Morphology (1) Cell shape and size: coccoid to short bacillus
0.4-1×1-2.5μ (2) Presence or absence of cell pleomorphism: None (3) Presence or absence of motility, epiphytic status of flagella: None (4) Presence or absence of spores: None (5) Gram staining: Negative (6) Acid-fast: Negative (b) Growth status in each medium (1) Broth agar plate culture: Moderate growth, circular, convex circular to ridged, full edges, translucent, moist light, homogeneous, smooth, Buff color to straw color (2) Juicy agar slant culture: Moderate growth, thin film-like,
Stringy, buff to straw color (3) Meat juice liquid culture: uniformly cloudy (4) Meat juice gelatin puncture culture: does not liquefy. (5) Litmus milk: does not liquefy, weakly alkaline (c) Physiological properties (1) Nitrate reduction: + (2) Denitrification reaction: - (3) MR test: - (4) VP test: - (5 ) Production of indole: - (6) Production of hydrogen sulfide: - (7) Hydrolysis of starch: - (8) Utilization of citric acid: Not used in Koser medium
Used in Christensen medium (9) Inorganic nitrogen source: Do not use nitrates. Do not use ammonium salts. (10) Pigment production: No production. (11) Urease: - (12) Oxidase: + (13) Catalase: + (14) ) Growth range: Grows at a temperature of 37℃, but does not grow at 41℃ PH6-9 (15) Attitude towards oxygen: Aerobic (16) O-F test (by Hugh & Leifson method): O (17) From sugars to acids and Presence or absence of gas generation [Peptone medium]

[Table] (18) Oxidation of gluconic acid (Haynes method):
- (19) Deaminase reaction of phenylalanine (method of Ewing et al): - (20) Decarboxylase reaction (method of Mφller): Lysine - Ornithine - Arginine - (21) Arginine dihydrolase reaction (method of Stanier et al): NT (22) Degradability of casein: - (23) Degradability of DNA: - (24) Accumulation of poly-β-hydroxybutyrate: None (25) Auxotrophy: Yes (26) Degradability of cellulose: - (27) Salt tolerance: Does not grow in 3% salt (d) DNA GO content: 69.5% This bacterium is a Gram-negative bacillus, non-motile, aerobic,
It belongs to the genus Flavobacterium because it is catalase positive, oxidase positive, gradually decomposes sugar oxidatively to produce acid, and the color tone of the colony is buff to straw color. When searching for species, the known bacterial species described in the Bergey Manual 8th edition and this bacteria are motile, salt tolerant,
They do not match in important properties such as growth at 37℃, gelatin decomposition property, cellulose decomposition property, GO content of DNA, etc. Therefore, this bacterium is recognized as a new bacterial species.
It was named Flavobacterium hydantoinophilum. Some of the microorganisms used in the method of the present invention produce harmful enzymes, such as β-lactamase and amidase, which act on and convert the produced amoxicillin; It is desirable to breed mutant strains that do not. To produce the desired amoxicillin using microorganisms that produce amoxicillin synthase, these microorganisms are usually first cultured in a nutrient medium, and 6-aminopenicillanic acid and 5-(p-hydroxyphenyl)hydantoin may be reacted with 6-aminopenicillanic acid and 5-(p-
Add hydroxyphenyl) hydantoin,
Amoxicillin can also be produced by culturing microorganisms and carrying out an enzyme reaction simultaneously. The medium used for culturing microorganisms contains carbon sources, nitrogen sources,
A conventional nutrient medium containing inorganic ions and, if necessary, organic micronutrients such as vitamins, amino acids, and yeast extract is used. As the carbon source, carbohydrates such as glucose, sucrose, maltose, starch hydrolyzate, acetic acid, ethanol, etc. are used as appropriate. Further, as the nitrogen source, ammonia gas, aqueous ammonia, ammonium salt, peptone, meat extract, yeast extract, hydrolyzate of animal and plant proteins, etc. are used. As the inorganic ions, magnesium ions, potassium ions, sodium ions, calcium ions, iron ions, manganese ions, etc. are used as appropriate. Furthermore, in order to increase the activity of amoxicillin synthase, 5-substitutions shown in Table 1 such as DL-5-methylmercaptoethylhydantoin, DL-5-indolylmethylhydantoin, DL-5-(p-hydroxyphenyl)hydantoin, etc. It is desirable to add a hydantoin derivative to the medium as appropriate, and by adding 0.1 to 0.5 g/dl to the medium, the enzyme activity can be increased 4 to 5 times. The culture temperature may be within a temperature range suitable for the growth of microorganisms, and is 15 to 40°C, preferably 25 to 37°C. The culture time varies depending on the type of strain used, culture conditions, etc., but it is sufficient to terminate the culture when the enzymatic activity of the synthetic enzyme reaches its maximum, which is usually 16 to 48 hours.
The time is appropriate. The culture obtained in this way or its treated product is applied to the production reaction of amoxicillin, but the culture here refers to the culture solution itself, as well as viable bacterial bodies isolated from the culture solution, or bacteria from the culture solution. This refers to the culture fluid (supernatant) from which insoluble substances such as body fluids have been removed, and the treated product refers to the culture fluid that has been subjected to appropriate treatments to increase the enzymatic activity of amoxicillin synthase or maintain its activity stably for a long period of time. It refers to amoxicillin that has been processed to a state more suitable for manufacturing amoxicillin. For example, if the present synthetic enzyme is an extracellular enzyme, it can be purified from the above-mentioned culture solution by using known enzyme purification methods such as salting out, dialysis, adsorption/ion exchange chromatography, and gel filtration as appropriate, alone or in combination; It refers to a partially purified enzyme, or an immobilized enzyme obtained by adsorbing or comprehensively immobilizing the above enzyme on a water-insoluble carrier, and if the synthetic enzyme is an intra-strain enzyme, it refers to a live bacterial cell isolated from a culture solution. Organic solvent-treated bacterial cells obtained by washing with water and treatment with an organic solvent such as acetone, methanol, or ethanol, or physical treatment such as grinding, ultrasonication, cell wall lytic enzyme treatment, or surfactant treatment to viable bacterial cells. , chemically treated bacterial strains, cell-free extracts obtained by extracting these treated bacterial strains with water, buffer solutions, etc., and their purified enzymes, immobilized enzymes,
Or immobilized microorganisms, etc. The enzymatic reaction for producing amoxicillin from 6-aminopenicillanic acid and 5-(p-hydroxyphenyl)hydantoin using these cultures and their treated products is carried out in an aqueous solution, but from the viewpoint of yield. Hydrophilic organic solvents such as methanol, ethanol, isopropanol, acetone, methylcellosolve, dioxane, t-butanol, etc.
It is more desirable to carry out in an aqueous solution containing %. The enzyme reaction is carried out at a reaction pH of 4.0 to 9.0 and a reaction temperature of 20 to 50°C. When the enzyme is soluble in water, the enzymatic reaction is carried out in a solution, but when the enzyme is insoluble in water or is an immobilized enzyme, the reaction is carried out in suspension or in a column. In addition, if 5-(p-hydroxyphenyl)hydantoin and 6-aminopenicillanic acid are added to the medium or culture for culturing microorganisms, microbial culture, that is, enzyme production and amoxicillin synthesis, can occur at the same time. It is convenient because it can be done. Enzyme reaction time depends on reaction conditions such as substrate concentration, enzyme activity, pH, type and amount of contaminants, and reaction temperature, but in short, the reaction should be terminated when the maximum amount of amoxicillin produced is reached. Usually 1~
10 hours is enough. 6-Aminopenicillanic acid used in the present invention may be used in free form or in the form of sodium salt, potassium salt, etc., and 5-(p-hydroxyphenyl)hydantoin is in the D, L, and DL forms. Either method may be used, and since the DL form can be used directly, it is extremely convenient as an industrial production method. Amoxicillin can be collected from the reaction solution obtained in this way by following a known method.For example, unreacted 5-(p-hydroxyphenyl)hydantoin is a substance that is difficult to dissolve in water. Most of the unreacted 5-(p-hydroxyphenyl)hydantoin and insoluble substances such as bacterial strains are removed by centrifuging the reaction solution. Therefore, this solution is passed through an adsorption resin column such as HP-20 to adsorb amoxicillin and eluted with an appropriate solvent such as an organic solvent or water.The fraction containing amoxicillin is concentrated and crystallized by adding an appropriate hydrophilic organic solvent. Amoxicillin can be collected by doing this. As described above, the present invention provides a new enzymatic method for producing amoxicillin directly from 6-aminopenicillanic acid and 5-(p-hydroxyphenyl)hydantoin. The present invention will be specifically explained below with reference to Examples. Example 1 Glucose 2.0g/dl, (NH ₄ ) ₂ SO ₄ 0.5g/dl,
KH ₂ PO ₄ 0.1g/dl, K ₂ HPO ₄ 0.3g/dl,
MgSO ₄・7H ₂ O0.05g/dl, FeSO ₄・7H ₂ O1mg/
dl, MnSO ₄・4H ₂ O1mg/dl, yeast extract 1.0g/
1.0 g/dl of peptone, 0.2 g/dl of DL-5-methylcaptoethylhydantoin, and 50 ml of a medium (PH7.0) containing calcium carbonate (separately sterilized) was poured into a 500 ml flask and sterilized at 120°C for 15 minutes. . Flavobacterium hydantoinophilum was cultured on bouillon agar medium at 30°C for 24 hours.
One platinum loop of AL 11322FERM-P4819 was inoculated at 30°C.
The cells were cultured for 20 hours. Bacterial cells were collected from this culture solution by centrifugation, and diluted with the same amount of physiological saline as the culture solution.
The cells were washed twice and collected. 6-aminopenicillanic acid 0.5g/dl
and DL-5-(p-hydroxyphenyl)hydantoin was added to 0.05 M/dl phosphate buffer (final pH 7.0, 5 ml) containing 1 g/dl to give a concentration of 5 g/dl, and the mixture was heated at 30°C for 3 hours. Retention reaction was performed. After the reaction, the bacterial cells were removed by centrifugation, and the amoxicillin produced in the reaction solution was quantified by high performance liquid chromatography using a cation exchange resin (DuPont's "ZIPAX-SCX"), and it was found to be 54.6 μg/ml. of amoxicillin was obtained. In addition, when the amoxicillin produced by the reaction was compared by thin layer chromatography using n-butanol:acetic acid:water = 4:1:2 and n-propanol:water = 7:3, it was completely compared with the standard amoxicillin. Agreed. Furthermore, when the production amount was confirmed by a bioassay method using Proteus mirabilis, almost the same results were obtained. Example 2 Glucose 2.0g/dl, (NH ₄ ) ₂ SO ₄ 0.5g/dl,
KH ₂ PO ₄ 0.1g/dl, K ₂ HPO ₄ 0.3g/dl,
MgSO ₄・7H ₂ O0.05g/dl, FeSO ₄・7H ₂ O1mg/
dl, MnSO ₄・4H ₂ O1mg/dl, yeast extract 1.0g/
50ml of medium (PH7.0) containing peptone 1.0g/dl and calcium carbonate (separately sterilized) in a 500ml flask.
ml and sterilized at 120°C for 15 minutes. Flavobacterium hydantoinophilum was cultured on bouillon agar medium at 30°C for 24 hours.
FERM-P4819 was inoculated and cultured at 30°C for 16 hours. 5-substituted cydantoin (separately sterilized) shown in Table 1 was added to the culture solution at a concentration of 0.2 g/dl, and the culture was continued for an additional 6 hours. Bacterial strains were collected from this culture solution by centrifugation, washed once with physiological saline in the same amount as the culture solution, and collected. 6-aminopenicillanic acid 0.5g/dl
and 0.05M phosphate buffer (terminated) containing DL-5-(p-hydroxyphenyl)hydantoin 1g/dl
PH7.0 5ml) to a concentration of 5g/dl,
The reaction was maintained at 30°C for a period of time. After the reaction, the bacterial cells were removed by centrifugation, and amoxicillin produced in the reaction solution was quantified in the same manner as in Example 1. The amount of coagulation is shown in Table 1.

【table】

[Table] Example 3 The medium shown in Example 2 was grown on bouillon agar at 30°C.
Flavobacterium hydantoinophyllum FERM-P4819 cultured for 24 hours was inoculated at 30℃.
After culturing for 16 hours, add DL-5-p-(hydroxyphenyl)hydantoin (separately sterilized) to the culture solution.
It was added at a concentration of 0.4 g/dl and cultured for 4 hours. To this culture solution, 6-aminopenicillanic acid and DL-5-(p-hydroxyphenyl)hydantoin were added to a sterile solution at a concentration of 0.5 g/dl and 1.0 g/dl, respectively, and the culture was continued for an additional 3 hours. Ta. The bacterial strain in this culture solution is removed by centrifugation,
Example 1 Amoxicillin produced in the culture supernatant
The amount was determined in the same manner as above, and the result was 45 μg/ml. Example 4 Flavobacterium hydantoinophyllum FERM-P4819 cells prepared in the same manner as in Example 1 were added to a 0.5M phosphate buffer solution (final 10ml) to a concentration of 5g/dl. 5 with 20KC ultrasound
Processed for minutes. The treated product was centrifuged to obtain a supernatant. 6-aminopenicillanic acid and DL-(5-p-hydroxyphenyl)hydantoin were added to 5 ml of this supernatant at a concentration of 0.5 g/dl and 1.0 g/dl, respectively (final pH 7.0). The reaction was carried out at ℃ for 3 hours. When the supernatant of this reaction solution was measured, it was 73.5μ.
g/ml of amoxicillin was produced. Example 5 1 g of Flavobacterium hydantoinophyllum FERM-P4819 cells prepared in the same manner as in Example 1 was suspended in 4 ml of deionized water, cooled on ice, and then 750 mg of acrylamide and 45 mg of methylenebisacrylamide were added. 3.5 mg of ammonium persulfate and 8 μl of N·N'-dimethylaminopropionitrile were added, and the mixture was allowed to stand still under ice-cooling. After 1 hour, the resulting gel containing bacterial cells was strained through a 50-mesh wire mesh and washed with physiological saline to prepare an immobilized gel. 2 g of this immobilized product was added to 5 ml of 0.05 phosphate buffer solution (PH7.0) containing 1 g/dl of DL-5-(p-hydroxyphenyl)hydantoin and 0.5 g/dl of 6-aminopenicillanic acid. ℃ for 3 hours. When the supernatant of this reaction solution was measured, it was 17.8μg/
ml of amoxicillin was produced. Example 6 The cells of Flavobacterium hydantoinophyllum FERM-P4819 prepared by the method of Example 1 were treated with 0.5 g/dl of 6-aminopenicillanic acid and DL-5-
(p-hydroxyphenyl)hydantoin 1.0
g/dl, and 5.0 in 0.05 M phosphate buffer (final pH 7.0, 5.0 ml) containing ethanol 5-20% (V/V).
g/dl, and the enzymatic reaction was carried out at 30°C for 3 hours. As shown in Table 2, the amount of amoxicillin produced was approximately doubled by adding ethanol.

【table】

Claims (1)

[Claims] 1 Belongs to the genus Flavobacterium and contains 6-aminopenicillanic acid and 5-(p-hydroxyphenyl)
Amoxicillin, characterized in that 6-aminopenicillanic acid and 5-(p-hydroxyquiphenyl)hydantoin are reacted in the presence of a culture of a microorganism having the ability to synthesize amoxicillin from hydantoin or a treated product thereof. manufacturing method.