JPH06225794A - Production of deacetylcephalosporin c and related compound - Google Patents

Abstract

PURPOSE:To efficiently obtain deacetylcephalosporin C useful as an intermediate, etc., for synthesizing beta-lactam antibiotics by reacting an enzyme source derived from a microorganism which belongs to Pseudomonas and capable of deacetylating cephalosporin C, etc., and then harvesting the reactional product from the reaction mixture. CONSTITUTION:A microorganism which belongs to the Henus Pseudomonas [e.g. Pseudomonas sp.-14-2-2 strain (FERM P-3939)] is inoculated into a sterilized medium and subjected to shake culture at 26 deg.C at 150rpm for 96hr and the cultured mixture is centrifuged and the bacterial cell is harvested to afford an enzyme source having activity capable of deacetylating cephalosporin C or 7-aminocephalosporanic acid. The enzyme source is added to a reactional liquid containing cephalosporin C or 7-aminocephalosporanic acid and subjected to shake culture reaction therewith at 26 deg.C for 72hr to produce deacetylcephalosporin C or deacetyl-7-aminocephalosporanic acid in the reactional liquid and then the product is harvested from the reactional liquid to provide the objective deacetylcephalosporin C and its related compound.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to an industrial process for producing deacetylcephalosporin C or deacetyl-7-aminocephalosporanic acid, which is useful as an intermediate for the synthesis of various β-lactam antibiotics.

[0002]

Deacetyl cephalosporin C [3-hydroxymethyl-7β- (D-5-amino-5-carboxypentamido) -3-cephem-4-carboxylic acid], deacetyl-7-aminocephalosporan Acid (3
-Hydroxymethyl-7β-aminoce-3-em-4-
Carboxylic acid) is produced by deacetylation reaction of cephalosporin C or 7-aminocephalosporanic acid, respectively, and various natural products and microorganisms have been known as enzyme sources for catalyzing this deacetylation. That is, citrus fruits, staphylococci or cholera (Japanese Patent Publication No. 39-989)
4), wheat germ, Rhizobium sp. (Japanese Patent Publication No. 42-7553), Bacillus sp. (Japanese Patent Publication No. 49-35993), various mold yeasts (Japanese Patent Publication No.
54-43598, Japanese Examined Patent Publication 1-47158, Japanese Examined Patent Publication No. 61-11600), Streptomyces sp. (Japanese Patent Publication No. 55-39375), Bran (Japanese Patent Publication No. 61
-70999) is known.

Regarding the method using these enzyme sources,
From an industrial point of view, various problems to be improved still remain. With natural products such as wheat germ, citrus fruits, and bran, it is difficult to uniformly take in raw materials, and the enzyme must be extracted and purified for use, and the operation is complicated. Although microorganisms are a promising enzyme source, contamination with β-lactamase may reduce yield and purity.

[0004]

Means for Solving the Problems The present inventors have conducted extensive research on a method for efficiently performing enzymatic deacetylation of cephalosporin C, 7-aminocephalosporanic acid, and Pseudomonas that has not been known so far. The present invention has been completed as a result of discovering a new method using a genus bacterium and conducting further research based on this.

DETAILED DESCRIPTION OF THE INVENTION According to the present invention, an enzyme source derived from a microorganism belonging to the genus Pseudomonas and having an activity of deacetylating cephalosporin C or 7-aminocephalosporanic acid is cephalosporin. C or 7-aminocephalosporanic acid is allowed to act in the reaction solution to give deacetylcephalosporin C or deacetyl-
7-aminocephalosporanic acid is produced, and deacetylcephalosporin C or deacetyl-
Deacetyl cephalosporin C or deacetyl- characterized by collecting 7-aminocephalosporanic acid
A method for producing 7-aminocephalosporanic acid can be provided.

As the enzyme source used in the present invention, any enzyme source derived from a microorganism belonging to the genus Pseudomonas and having an activity of deacetylating cephalosporin C or 7-aminocephalosponic acid belongs to the genus Pseudomonas. , Cephalosporin C or 7-aminocephalosporanic acid deacetylating microbial cells, a culture solution, a culture solution supernatant or a processed product thereof, an enzyme extracted from the microorganism, or the like.

Microorganisms belonging to the genus Pseudomonas and having the activity of deacetylating cephalosporin C or 7-aminocephalosporanic acid specifically belong to the genus Pseudomonas and include cephalosporin C or 7-aminocephalosporanic acid. It has an enzymatic activity for eliminating the acetyl group at the 3-position, and, for example, Pseudomonas sp.
-2C stock can be raised. The taxonomic characteristics of this strain are as follows, based on the Budapest Treaty.
As of July 23, 2013, it has been deposited at the Institute of Microbial Science and Technology of the Institute of Industrial Science and Technology as Micromachine Research Article No. 3939 (FERM BP-3939).

[0008] The 14-2-2C strain is a bacillus of 0.9 to 1.0 × 2.0 to 4.2 microns, has no polymorphism, is motile, and has a single polar flagella. It does not sporulate, is Gram-negative and has no acid resistance. In the broth agar plate culture, yellow colonies with a diameter of 1.8 to 2.8 mm were formed.
The edges are gold edges. The surface is smooth, shiny and buttery. It grows well in broth agar slope culture and has a yellow color.
Grows on the surface in broth liquid culture. No liquefaction of gelatin in broth gelatin stab culture and no peptone formation was observed in litmus milk medium.

The physiological properties are as follows. Nitrate is not reduced, denitrification reaction is negative, MR test is negative, VP test is negative, indole is not generated, hydrogen sulfide is not generated, and starch is not decomposed. Utilizes citric acid and an inorganic nitrogen source. A water-insoluble yellow pigment is produced in both KingA and KingB media. Urease negative, oxidase negative, catalase positive. grows at pH 5-9,
It grows well at 20-40 ° C and does not grow at 46 ° C. Aerobic,
The OF test is oxidative. L-arabinose, D-
Xylose, D-glucose, D-mannose, D-fructose, D-galactose, maltose, sucrose, lactose and trehalose are used, and D-sorbit, D-mannitol, inosit, glycerin and starch are not used. It does not decompose arginine and accumulates polyhydroxybutyric acid. There is no clear positive reaction in the test of aromatic ring cleavage mode, it is difficult to determine whether ortho or meta cleavage, and there is a possibility that it will not be cleaved.

The above-mentioned various properties are described in the Vergie's Manual
Of Systematic Bacteriology, Volume 2, (1
986), the 14-2-2C strain is considered to belong to the genus Pseudomonas, but no bacterial species with the same characteristics is found.

In the present invention, the above-mentioned enzyme source is allowed to act on cephalosporin C or 7-aminocephalosporanic acid, which has the above-mentioned properties in order to obtain a sufficient amount of the enzyme source. The microorganisms are grown in the medium normally used for the cultivation of trace substances. As the medium, a medium such as a carbon source, a nitrogen source, and an inorganic salt used for culturing ordinary microorganisms is used. Examples of the carbon source may be sugars such as glucose, organic acids such as citric acid, and natural products such as molasses used by microorganisms. As the nitrogen source, ammonium sulfate, ammonium chloride and natural products such as peptone, meat extract and yeast extract are usually used. As the inorganic salt, a small amount of so-called trace element, which is effective for growth, such as potassium phosphate and magnesium sulfate is used.

The culture is preferably carried out under aerobic conditions such as shaking culture and aeration and stirring culture. Culture is 15-40 ℃
It is preferably carried out at 20 to 37 ° C., pH 5 to 9 and preferably 6 to 8 for usually 24 to 120 hours.

The thus obtained microorganism cells, culture solution, culture solution supernatant or a processed product thereof, an enzyme extracted from the microorganism and the like are used as an enzyme source. As the treated product, a dried product of bacterial cells, a surfactant and / or an organic solvent-treated product, a lytic enzyme-treated product, a fixed enzyme or an extracted enzyme preparation from bacterial cells, a culture solution, a concentrate of a culture supernatant, Examples thereof include dried products, surfactants and / or organic solvent additives, and lysed enzyme-treated products.

Further, a microorganism introduced with the activity of deacetylating from the above microorganism by a technique such as recombination can also be used. The enzyme source thus obtained is suspended in a reaction solution containing cephalosporin C or 7-aminocephalosporanic acid, and the substrate is substantially deacetylated to deacetylcephalosporin C or deacetylase, respectively. The reaction is continued until it is converted to -7-aminocephalosporanic acid.

The aqueous reaction solution used for the reaction is usually a buffer solution for reducing the fluctuation of the pH of the reaction solution during the reaction, but the reaction solution containing an organic solvent may be used if it also contains water. it can. Reaction pH 4-10, temperature 10-60
Perform at ℃. The reaction is usually carried out by shaking the reaction solution to accelerate the progress of the reaction, but the reaction may be carried out by allowing it to stand.

Cephalosporin C or 7- in the reaction solution
When the amount of aminocephalosporanic acid decreased and the amount of each deacetylated product reached a substantial amount, the enzyme source was removed from the reaction solution by centrifugation, etc., and then carbon treatment, concentration, salting out, ionization Deacetyl cephalosporin C or deacetyl-7-aminocephalosporanic acid can be separated and recovered from the reaction solution by applying a known method such as a treatment with an exchange resin (for example, the method described in Japanese Patent Publication No. 39-9894). .

[0017]

The present invention will be described in more detail with reference to the following examples. In the examples, analysis of cephalosporin C, 7-aminocephalosporanic acid and deacetylated 3-positions thereof was performed by high performance liquid chromatography. The conditions are as follows. Column: CAPC ELL-PAK C18 SG 120 (4.6 × 150 mm) (manufactured by Shiseido Co., Ltd.) Mobile phase: 10 mM KH ₂ PO ₄ , 5 mM sodium 1-octanesulfonate, 7.5% methanol (pH 2.8) Flow rate: 1 ml / min Detection : UV 254nm

Example 1 Pseudomonas sp. 14-2-2C strain was treated with meat extract 0.4
%, Peptone 0.4%, yeast extract 0.2% and dipotassium phosphate 0.1% (pH 7.0) in a 300 ml Erlenmeyer flask containing 50 ml of a sterilized medium, and shake-cultured at 26 ° C. for 72 hours. 50 ml of this seed culture was inoculated into a 2 liter Erlenmeyer flask containing 500 ml of a medium having the same composition as the seed culture, and shake-cultured at 26 ° C. and 150 rpm for 96 hours. The cells were collected from this culture solution by centrifugation, and the cells were washed twice with 0.1 M phosphate buffer (pH 7.0) and used for the reaction.
The reaction was performed by adding 500 ml of the reaction solution prepared by adding cephalosporin C to a concentration of 3.6 mg / ml and adding the cells to 10 mM phosphate buffer (pH 7.0) so that the concentration was 5 times the concentration in the growth culture. The mixture was placed in a liter Erlenmeyer flask and reacted with shaking at 26 ° C. 72 hours after the reaction, deacetylcephalosporin C was produced in the reaction solution at a concentration of 2.69 mg / ml, and (conversion molar yield
The residual concentration of cephalosporin C was 90 μg / ml. To 1,200 ml of this reaction solution, 12 g of activated carbon was added and stirred for 10 minutes, then the activated carbon was removed and the mixture was concentrated to dryness under reduced pressure at 40 ° C.
After adding 20 ml of 75% alcohol to remove the melted portion, wash the solids with ethanol, ether, and acetone successively,
Vacuum drying was carried out at 0 ° C. to obtain 3.0 g of a yellow powder. The purity of this product was 55%.

Example 2 In Example 1, the inoculation amount of the main culture of the seed culture was set to 15 ml, 7-aminocephalosporanic acid was used instead of cephalosporin C in the reaction solution composition, and a phosphate buffer solution was used instead. Example 1 was repeated, except that distilled water was used as the reaction solution and the reaction solution was adjusted to pH 4.5 with hydrochloric acid and sodium hydroxide. After 43 hours of reaction, 7-aminocephalosporanic acid was almost completely converted to deacetyl-7-aminocephalosporanic acid. After 45 hours of the reaction, the liquid obtained by removing the bacterial cells from the reaction solution was concentrated to dryness under reduced pressure at 40 ° C., and the obtained solid content was dissolved in 25 ml of water, and the pH was adjusted to 4.0 with hydrochloric acid for isoelectric precipitation. The precipitate was collected by centrifugation and dissolved in 50 ml water. After adjusting the pH to 7.0 with sodium hydroxide, three times the amount of alcohol was added to remove a slight amount of insoluble matter, and the mixture was dried under reduced pressure.
Dissolve this in water to make 100 ml solution, add 1 g of activated carbon to decolorize, concentrate again to dryness, add 5 ml of alcohol, collect the precipitate, wash with acetone and vacuum dry to obtain 1.2 g of brown powder. Got The purity of deacetyl-7-aminocephalosporanic acid of this sample was 71%.

[0020]

INDUSTRIAL APPLICABILITY According to the present invention, deacetylcephalosporin C or deacetyl-7-aminocephalosporanic acid useful as a synthetic intermediate for various β-lactam antibiotics can be efficiently produced.

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[Procedure amendment]

[Submission date] October 12, 1992

[Procedure Amendment 1]

[Document name to be amended] Statement

[Name of item to be corrected] 0002

[Correction method] Change

[Correction content]

[0002]

Deacetyl cephalosporin C [3-hydroxymethyl-7β- (D-5-amino-5-carboxypentamido) -3-cephem-4-carboxylic acid], deacetyl-7-aminocephalosporan Acid (3
-Hydroxymethyl-7β-amino-3-cephem-4
-Carboxylic acid) is produced by deacetylation reaction of cephalosporin C or 7-aminocephalosporanic acid, respectively, and various natural products and microorganisms have been known as enzyme sources for catalyzing this deacetylation. . That is, citrus fruits, staphylococci or cholera (Japanese Patent Publication No. 39-989)
4), wheat germ, Rhizobium sp. (Japanese Patent Publication No. 42-7553), Bacillus sp. (Japanese Patent Publication No. 49-35993), various mold yeasts (Japanese Patent Publication No. 5
4-43598, Japanese Examined Patent Publication 1-47158, Japanese Examined Patent Publication No. 61-11600), Streptomyces spp. (Japanese Patent Publication No. 55-39375), Bran (Japanese Patent Publication No. 61
-70999) is known.

[Procedure Amendment 2]

[Document name to be amended] Statement

[Correction target item name] 0008

[Correction method] Change

[Correction content]

[0008] The 14-2-2C strain is a bacillus of 0.9 to 1.0 × 2.0 to 4.2 microns, has no polymorphism, is motile, and has a single polar flagella. It does not sporulate, is Gram-negative and has no acid resistance. In the broth agar plate culture, yellow colonies with a diameter of 1.8 to 2.8 mm were formed.
The margin is the whole margin. The surface is smooth, shiny and buttery. It grows well in broth agar slope culture and has a yellow color.
Grows on the surface in broth liquid culture. No liquefaction of gelatin in broth gelatin stab culture and no peptone formation was observed in litmus milk medium.

[Procedure 3]

[Document name to be amended] Statement

[Correction target item name] 0010

[Correction method] Change

[Correction content]

The above-mentioned various properties are described in the Vergie's Manual
Of Systematic Bacteriology, Volume 1, (1
(984), the 14-2-2C strain is considered to belong to the genus Pseudomonas, but no bacterial species with the same properties is found.

[Procedure amendment 4]

[Document name to be amended] Statement

[Correction target item name] 0011

[Correction method] Change

[Correction content]

In the present invention, the above-mentioned enzyme source is allowed to act on cephalosporin C or 7-aminocephalosporanic acid, which has the above-mentioned properties in order to obtain a sufficient amount of the enzyme source. The microorganism is grown in a medium commonly used in the culture of microorganisms. As the medium, a medium such as a carbon source, a nitrogen source, and an inorganic salt used for culturing ordinary microorganisms is used. Examples of the carbon source may be sugars such as glucose, organic acids such as citric acid, and natural products such as molasses used by microorganisms. As the nitrogen source, ammonium sulfate, ammonium chloride and natural products such as peptone, meat extract and yeast extract are usually used. As the inorganic salt, a small amount of so-called trace element, which is effective for growth, such as potassium phosphate and magnesium sulfate is used.

Claims (2)

[Claims] 1. A reaction solution of an enzyme source derived from a microorganism belonging to the genus Pseudomonas and having an activity of deacetylating cephalosporin C or 7-aminocephalosporanic acid to cephalosporin C or 7-aminocephalosporanic acid. In the reaction solution to produce deacetylcephalosporin C or deacetyl-7-aminocephalosporanic acid, and deacetylcephalosporin C or deacetyl-7-aminocephalosporanic acid is produced from the reaction solution. A method for producing deacetylcephalosporin C or deacetyl-7-aminocephalosporanic acid, which comprises collecting. 2. A bacterial cell, a culture solution, a culture solution supernatant, or a treated product thereof, wherein the enzyme source belongs to the genus Pseudomonas and has the ability to deacetylate cephalosporin C or 7-aminocephalosporanic acid. The method according to claim 1, wherein the enzyme is an enzyme extracted from the microorganism.