FR2702493A1 - Process for converting cephalosporin C to glutaryl-7-aminocephalosporanic acid - Google Patents

Abstract

The invention relates to an improved enzymatic oxidation process for converting Cephalosporin C to glutaryl-7-amino cephalosporanic acid. The method according to the invention comprises the use of a mixture containing a D-amino acid oxidase whose esterase activity is 5% or less of the activity of D-amino acid oxidase. This process finds application in the conversion of cephalosporin C to glutaryl-7-amino cephalosporanic acid.

Description

The present invention relates to an enzymatic oxidation process

  improved to convert Cephalosporin C to glutaryl-7-aminocephalosporanic acid (hereinafter referred to as glutaryl-7-ACA) comprising the use of an amino acid oxidase-containing mixture (hereinafter referred to as a DAO-containing mixture). esterase in the mixture containing DAO used in

  the present process is selectively reduced.

  Glutaryl-7-ACA is an intermediate material for the preparation of 7-aminocephalosporanic acid (hereinafter referred to as 7-ACA) from cephalosporin C

  Cephalosporin C reacts with a D-amino acid oxidase (hereinafter

  after designated DAO) to produce a-keto adipoyl-7-

  aminocephalosporanic acid (hereinafter referred to as a-keto adipoyl-7-

  ACA) which is a thermally unstable intermediate and hydrogen peroxide, then the a-keto adipoyl-7-ACA product is oxidized by hydrogen peroxide to produce glutaryl-7-ACA in order to obtain a At a higher yield of glutaryl-7-ACA, various enzymatic processes for converting cephalosporin C to glutaryl-7-ACA using DAO derived from Trigonopsis variabilis (hereinafter referred to as T. variabilis) have been developed. No. 3,821,209, Japanese Patent Publications Nos. 35119/1980 and 15635/1984 disclose enzymatic oxidation processes carried out in the presence of catalase inhibitors such as inorganic azides and perborate and an excess of hydrogen peroxide. , respectively, to avoid decomposition of hydrogen peroxide by catalase US Patent No. 3,801,458 discloses enzymatic oxidation of cephalosporin C using activated cells of T variabilis with a cat inhibitor sodium azide alase to provide a good yield of glutaryl-7-ACA European Unexamined Patent Publication No. 409,521 discloses a method for inactivating a catalase in the presence of DAO by treating the enzymes with an aqueous basic solution of p H from about 11 to 12 before oxidation

  Enzymatic Cephalosporin C in glutaryl-7-ACA.

  However, these methods have disadvantages in yield reduction due to the esterase possessed by T. variabilis and in the deterioration of quality due to impurities such as catalase inhibitors when applied to an economical practice at Escherase is an enzyme that converts cephalosporin C, glutaryl-7-ACA, and keto adipoyl-7-ACA intermediate into their 3-desacetyl forms. These desacetyl derivatives not only reduce the yield of glutaryl-7 -ACA but also deteriorate the quality of glutaryl-7-ACA as impurities So, in order to improve the yield and the quality of glutaryl-7-ACA, it is very

  important to inactivate or inhibit esterase activity.

  European Unexamined Patent Publication No. 409,521 also discloses a method of activating esterase activity by treating T variabilis cells with acetone / water so as to avoid producing deacetyl cephalosporin C and deacetyl glutaryl. However, the process described requires a complicated and difficult step to remove acetone. Thus, there is a strong demand for a simple and more efficient method of esterase inactivation, applicable to

industrial practice.

  For this purpose, the invention provides an enzymatic oxidation process for converting cephalosporin C to 7-glutaryaminophalosporanic acid comprising the use of a mixture containing a D-amino acid oxidase having esterase activity of 5%. or less of the oxidase activity of

D-amino acid.

  According to a characteristic of a process of the invention, the mixture containing a D-amino acid oxidase is obtained from a catalase negative or deficient mutant capable of

  to produce a D-amino acid oxidase.

  In this case, the mutant is of the strain KC-103

  Trigonopsis variabilis or strain EL-17.

  According to another characteristic of the process of the invention, the mixture containing a D-amino acid oxidase is obtained by bringing the mixture into contact with compounds

of Cu.

  In this case, the mixture containing a D-amino acid oxidase is obtained by contacting the mixture with a Cu compound at a concentration of 10 to 2000 ppm. And the contact is implemented before or during

enzymatic oxidation process.

  According to yet another feature of the invention, the D-amino acid oxidase-containing mixture is obtained from a mutant showing 5% or less of esterase activity on the basis of its DAO activity, which is obtained by treatment. a microorganism capable of producing DAO with ultraviolet radiation, X-ray radiation,

or a mutagen.

  In this case, the mixture containing a D-amino acid oxidase is composed of partially destroyed cells, or permeabilized cells, of cell-free extracts.

  obtained from them, or an immobilized enzyme.

  And the mutant is negative or deficient in catalase.

  More particularly, the mutant is strain EL-17

Trigonopsis variabilis.

  The present invention also relates to this

  strain EL-17 Trigonopsis variabilis.

  Yet another object of the invention is a method

  to inactivate an esterase in a mixture containing a D-

  amino acid oxidase comprising contacting said

mixture with a Cu compound.

  The present invention relates to an improved enzymatic oxidation process for converting cephalosporin C to glutaryl-7-ACA comprising the use of a DAO containing mixture having esterase activity of about 5% or less of In the process of the present invention, the DAO-containing mixture whose esterase activity is lowered or inhibited was obtained by contacting the mixture with a Cu compound. In the process of the present invention, the mixture containing DAO above-mentioned is also obtained from a mutant showing 5% or less of esterase activity on the basis of its DAO activity, which is obtained by treatment of a microorganism capable of producing DAO with ultraviolet radiation, a In accordance with the method of the present invention, the excellent yield of glutaryl-7-ACA can be achieved at high quality. invention comprises simple and safe steps, so that

  it is also applicable on an industrial scale.

  The present invention relates to an enzymatic oxidation process for converting cephalosporin C to glutaryl-7-ACA comprising the use of a DAO-containing mixture whose esterase activity is 5% or less

of the activity of the DAO.

  Cephalosporin C used in the present invention is an aqueous solution of Cephalosporin C powder, or Cephalosporin C fermentation broths in which the cells and the solid are removed Cephalosporin C is produced by culturing Achremonium chrysogenum cells in the medium containing 2% sucrose, 3% corn starch, 5% beet molasses, 6% defatted soybean, 3% methyloleate, and 0.5% calcium carbonate, 1.25 calcium sulphate and 0.8% ammonium acetate and the like or in a

  nutrient fermentation medium.

  The DAO-containing mixture used in the present invention comprises DAO, eg, partially purified DAO and crude DAO, and shows 5% or less of esterase activity based on DAO activity. containing DAO can be used in any form such as intact cells, partially destroyed cells, cells made permeable, cell-free extracts, and immobilized enzyme. Of these, the cells partially destroyed, the cells permeated, cell free extracts and immobilized enzyme are preferred.

known methods.

  It is preferred that DAO be derived from T variabilis in the present invention. More preferably, a catalase deficient mutant such as T. variabilis strain KC-103 (deposited with the National Institute of Bioscience and Human-Technology, Agency of Industrial Science). and Technology under the deposit number FERM-BP-4359, deposit made under the Budapest Treaty) is used in order to avoid 1 '

a-keto adipoyl 7-ACA remaining.

  In the present invention, the DAO-containing mixture whose esterase activity is 5% or less of the DAO activity is obtained by contacting the mixture with a Cu compound. The esterase activity in the DAO-containing mixture is selectively reduced by contacting the mixture with the Cu compound at a concentration of 10 to 2000 ppm, based on the total amount of aqueous solution or suspension

  DAO containing mixture at 5 to 250 C for 2 to 24 hours.

  In order to inactivate the esterase without affecting the activity of the DAO, it is preferred that the concentration of the Cu compound be at 400 ppm. The esterase activity can be reduced by half or more by the treatment. Thus, when the mixture containing DAO is obtained from T variabilis strain KC-103 showing 9.1% esterase / DAO activity ratio, the ratio

  activity can be reduced to 4.5% or less.

  In the present invention, the Cu compound is contacted with a DAO-containing mixture prior to the enzymatic oxidation process, i.e., before Cephalosporin C is added Alternatively, the Cu compound can be in contact with the DAO-containing mixture during the enzymatic oxidation process, i.e., it is added to the reaction mixture containing Cephalosporin C and the DAO-containing mixture. In the latter case, the concentration of the Cu compound is from 10 to 2000 ppm based on the reaction mixture A representative example of

  Cu compound used in the present invention is Cu SO 4.

  In the present invention, the DAO-containing mixture whose esterase activity is 5% or less of the DAO activity is also obtained from a mutant showing 5% or less of esterase activity based on its DAO activity, which is obtained by treating a microorganism capable of producing DAO with ultraviolet radiation, a

  X-ray radiation, or a mutagen.

  Such a mutant is obtained by the following A parent strain capable of producing DAO is treated with ultraviolet radiation, X-ray radiation, or

  mutagen such as N-methyl-N'-nitro-N-nitrosoganisine (hereinafter

  after designated NTG), then is cultured in a fermentation medium The strain showing esterase activity

  reduced is chosen by measuring the esterase activity.

  In the present invention, it is preferred that as a parent strain, a catalase-negative or catalase-deficient mutant be used to prevent the formation of byproducts. A representative example of the mutant is strain EL-17 T variabilis (deposited with the National Institute of Bioscience). Human-Technology, Agency of Industrial Science and Technology under the registration number FERM BP-4467, deposit made according to the Budapest Treaty) It is obtained by treatment of the strain KC-103 of T variabilis (supra) with NTG. 3.0% esterase activity based on

CAD activity.

  The esterase activity is measured by the following An enzyme sample is added to a 0.2 M phosphate buffer (p H 7.5 =) containing 2 g / l of glutaryl-7-ACA, and they are then placed to react at 250 C for 30 minutes Methyl alcohol at the same amount as the reaction mixture is added to stop the reaction The reaction solution is subjected to HPLC to determine the amount of

  deacetyl glutaryl-7-ACA produced.

  The DAO activity is measured by the following: An enzyme sample is added to a 0.1 M phosphate buffer (pH 7.5) containing 10 g / L Cephalosporin C, and is reacted at 250 ° C. for 15 minutes. minutes Methyl alcohol to the same amount as the reaction mixture is added to stop the reaction The reaction solution is subjected to HPLC to determine the amount of

  glutaryl-7-ACA and OE-keto-adipoyl-7-ACA produced.

  When a mixture containing DAO is contacted with a Cu compound during the enzyme oxidation process, the total amount of deacetyl cephalosporin C and deacetyl glutaryl-7-ACA is measured before and after the oxidation process of Enzyme The increased amount of desacetyl derivatives based on the initial amount of Cephalosporin C is considered an activity ratio

esterase / CAM.

  The enzymatic oxidation is usually carried out by flushing oxygen through the reaction solution at 20 to 300 ° C. The reaction time depends on the Cephalosporin C concentration, the amount of the activity of the DAO-containing mixture, and the reaction temperature When the amount of glutaryl-7-ACA produced has reached a maximum, which can be followed by HPLC or TLC or analogs, the reaction can be stopped. The reaction is generally complete in 30 minutes to 4 hours. reduce the amount of remaining α-keto-adipoyl-7-ACA,

  Hydrogen peroxide can be added to the reaction mixture.

  Glutaryl-7-ACA is isolated from the reaction mixture

obtained by known methods.

  The present invention will be illustrated in greater detail in

  reference to the following non-limiting examples.

Example 1

  A KC-103 strain of T. variabilis was grown in medium containing 2% glucose, 2% macerated maize liquor and 0.2% DLmethionine at 250 C for 60 hours. 0.3 ml of toluene was added to 60 ml of the fermentation broth including 6 g of wet cells, and they were mixed at 251 C for 2 hours to make the cells permeable. 23.6 mg Cu SO 4 5H 20 were added to the fermentation broth obtained from the permeabilized cells and allowed to stand at room temperature for two hours. An appropriate amount of enzyme sample from the Cu-containing fermentation broth was added to a 0.2 M potassium phosphate buffer (pH 7.5) containing 2 g / l of glutaryl-7-ACA. The reaction was carried out at 25 WC for 30 minutes, and was stopped by addition of methyl alcohol in the same amount as the reaction mixture, and was then subjected to HPLC The deacetyl glutaryl-7-ACA produced in the reaction was not detected, indicating that the esterase in the enzyme sample was enough

inactivated.

  Cephalosporin C powder was dissolved in 1000 ml of water, and the pH was adjusted to 7.5. The Cephalosporin C solution obtained was mixed with the Cu broth obtained above, and reacted at 201 C for 180 minutes with an oxygen sweep at

  through the reaction mixture. The yield of glutaryl-7-

ACA was 93.2%.

Example 2 l

  A fermentation broth of T variabilis strain KC-103 permeabilized cells whose esterase activity was inactivated was obtained by the same method as described

in Example 1.

  A fermentation broth of A chrysogenum containing 8.5 g / l cephalosporin C was adjusted to pH 2.5, then the cells were removed from this broth The pH of the broth was adjusted to pH 7.5 1200 ml of the resulting broth was mixed with 60 ml of the above-obtained T variabilis fermentation broth while oxygen was swept through the reaction mixture and 23.9 ml of 3.5% hydrogen peroxide % were continuously added to the reaction mixture, the reaction was carried out at 201 ° C. for 240 minutes. The yield of glutaryl-7-ACA was

92.8%.

Example 3 l

  A fermentation broth of cells permeabilized strain KC-103 of T variabilis was obtained by the same

  method as described in Example 1.

  10 g of cephalosporin C powder were dissolved in 1000 ml of water, and the pH was adjusted to pH 7.5 The Cephalosporin C solution obtained was mixed with 60 ml of

  Fermentation broth of T variabilis above obtained.

  Then, 0.5 g of Cu SO 4 5H 2 O was added to the resulting mixture. The reaction was conducted at 20 ° C for about 180 minutes while oxygen was flushed through the reaction mixture. The yield of glutaryl 7-ACA was 92.2%. The total increased amount of deacetyl cephalosporin C and deacetyl glutaryl-7-ACA was 1% or less based on the amount of Cephalosporin C.

Example 4 l

  A fermentation broth of permeabilized cells of strain KC-103 of T variabilis was obtained by the same method as described in Example 1 60 ml of fermentation broth of T. variabilis and 1200 ml of the fermentation broth of A chrysogenum containing Cephalosporin C obtained by the same method as described in Example 2 were

been mixed.

  0.5 g of Cu SO 4 5H 2 O was added to the resulting mixture, and they were reacted at 20 ° C for about 240 minutes while the oxygen was swept through and 23.9 ml of hydrogen peroxide was added. 3.5% hydrogen were continuously added to the reaction mixture. The yield of glutaryl-7-ACA was 92.0%. The total increased amount of deacetyl cephalosporin C and deacetyl glutaryl-7-ACA was 1% or less on the base of the amount of cephalosporin C. Example 5 1 400 ml of a T variabilis fermentation broth containing 40 g of wet cells were obtained by the same method as described in Example 1, and were treated with 2 , 0 ml of toluene by the same method as described in

Example 1

  157.3 mg of Cu SO 4 5H 20 were added to the broth above

  obtained and the resulting mixture was allowed to stand at room temperature for 2 hours. The esterase activity of an enzyme sample from the resulting Cu-containing broth was measured by the same method as described in Example 1. esterase in the enzyme sample was

sufficiently inactivated.

  Cephalosporin C powder was dissolved in 1000 ml of water, and the pH was adjusted to pH 7.5. The solution obtained was mixed with 400 ml of the fermentation broth of T. variabilis obtained above. The reaction was conducted at 201 ° C for about 30 minutes with an oxygen sweep through the reaction mixture.

glutaryl-7-ACA was 99.0%.

Example 6 l

  400 ml of a fermentation broth of permeabilized cells of T variabilis strain KC-103 obtained by the same method as described in Example 1 and 1000 ml of aqueous Cephalosporin C solution obtained by the same method

  method as described in Example 1 were mixed.

  0.66 g of Cu SO 4 5H 2 O was added to the above reaction mixture, and the reaction was conducted at 20 ° C for 30 minutes while oxygen was flushed through.

through the reaction mixture.

  The yield of glutaryl-7-ACA was 98.1%. The total increased amount of deacetyl cephalosporin C and deacetyl glutaryl-7-ACA was 1% or less based on the amount of Cephalosporin C.

Example 7 l

  An EL-17 strain of T variabilis was grown in medium containing 2% glucose, 2% macerated maize liquor and 0.2% DLmethionine at 250 ° C for 60 hours. added to 25 ml of the fermentation broth having 2.5 g of wet cells, and they were mixed at 250 C for 2 hours to permeabilize the cells. An appropriate amount of enzyme sample from the fermentation mixture was added to a 0.2 M potassium phosphate buffer (pH 7.5) containing 2 g / L glutaryl-7-ACA, and they were The reaction was quenched by addition of methyl alcohol in the same amount as the reaction mixture, and then subjected to HPLC. The ratio of esterase activity to DAO activity was reduced to one third of that of the

  parent strain, that is, 3.0%.

  Cephalosporin C powder was dissolved in 1000 ml water, and the pH was adjusted to pH 7.5. Cephalosporin C solution obtained was mixed with the above fermentation broth of permeabilized cells. and the mixture was reacted at 200 ° C. for about 180 minutes with an oxygen sweep through the mixture

  The yield of glutaryl-7-ACA was 92.6%.

Example 8 l

  A fermentation broth of permeabilized cells of T variabilis strain EL-17 was obtained by the same

  method as described in Example 7.

  A chrysogenum fermentation broth containing 9 g / l Cephalosporin C was adjusted to pH 2.5, and then the cells were removed from it. The pH was adjusted to pH 7.5 with Na The resulting broth was mixed with 60 μl of the above-obtained T variabilis fermentation broth while oxygen was being swept through the reaction mixture and 13 μl of hydrogen peroxide was added. % was continuously added to the reaction mixture, the reaction was carried out at 201 ° C. for about 220 minutes. The yield of glutaryl-7-ACA was

92.2%.

he

Example 9 l

  ml of a fermentation broth of permeabilized cells of T variabilis strain EL17 obtained by the same method as described in Example 7 and 1000 ml of aqueous Cephalosporin C solution obtained by the same method as described in Example 1 were The mixture was then reacted at 200 ° C. for about 30 minutes with an oxygen sweep through the mixture.

  The yield of glutaryl-7-ACA was 96.2%.

  EXAMPLE 1 ml of a fermentation broth of permeabilized cells of strain KC-103 of T. variabilis obtained by the same method as described in Example 1 and 1000 ml of aqueous Cephalosporin C solution obtained by the same method as described in US Pat. Example 1 was mixed The mixture was then reacted at 200 ° C for about 180 minutes with a sweep of oxygen through the mixture

  The yield of glutaryl-7-ACA was 83.0%.

Claims (9)

  An enzymatic oxidation process for converting cephalosporin C to glutaryl-7-aminocephalosporanic acid characterized in that it comprises the use of a mixture containing an amino acid oxidase whose esterase activity is 5% or less the activity of D-amino acid oxidase. Process according to Claim 1, characterized in that the mixture containing a D-amino acid oxidase is obtained from a mutant which is negative or deficient in catalase.   capable of producing a D-amino acid oxidase.   3 Process according to claim 2, characterized in that the mutant is of the strain KC-103 Trigonopsis variabilis or strain EL-17.   4 Process according to claim 1, characterized in that the mixture containing a D-amino acid oxidase is obtained   by contacting the mixture with Cu compounds.   Process according to Claim 4, characterized in that the mixture containing a D-amino acid oxidase is obtained by bringing the mixture into contact with a Cu compound at a temperature of concentration of 10 to 2000 ppm.   Process according to Claim 4, characterized in that the contact is carried out before or during the process enzymatic oxidation.   Process according to claim 1, characterized in that the mixture containing a D-amino acid oxidase is obtained from a mutant showing 5% or less of esterase activity on the basis of its DAO activity, which is obtained by treatment of a microorganism capable of producing DAO with ultraviolet radiation, X-ray radiation, or a mutagen.   Process according to Claim 7, characterized in that the mixture containing a D-amino acid oxidase is composed of partially destroyed cells, or permeabilized cells, of cell-free extracts obtained from   these, or an immobilized enzyme. 14 2702493   Process according to Claim 7, characterized in that the mutant is negative or deficient in catalase.   Process according to Claim 7, characterized in that the mutant is strain EL-17 Trigonopsis variabilis.   11 strain EL-17 Trigonopsis variabilis. Process for inactivating an esterase in a mixture containing a D-amino acid oxidase, characterized in that   comprises contacting the mixture containing a D-amino acid oxidase with a Cu compound.