GB2275925A

GB2275925A - Converting cephalosporin C to glutaryl-7-aminocephalosporanic acid

Info

Publication number: GB2275925A
Application number: GB9404103A
Authority: GB
Inventors: Kenji Matsuyama; Satoshi Soga
Original assignee: Asahi Chemical Industry Co Ltd; Asahi Kasei Kogyo KK
Current assignee: Asahi Kasei Corp; Asahi Chemical Industry Co Ltd
Priority date: 1993-03-08
Filing date: 1994-03-03
Publication date: 1994-09-14
Anticipated expiration: 2014-03-03
Also published as: GB2275925B; FR2702493B1; FR2702493A1; GB9404103D0; AT401269B; ATA47694A; ES2097080B1; ES2097080A1; ITMI940418A1; JPH06315396A; DE4407699C2; DE4407699A1; IT1269301B; ITMI940418A0

Description

2275925 A PROCESS FOR CONVERTING CEPHALOSPORIN C TO GLUTARYL-7-

AMINOCEPHALOSPORANIC ACID

Field of the Invention

The present invention relates to an improved enzymatic oxidation process for converting Cephalosporin C to glutaryl-7-aminocephalosporanic acid (hereinafter glutaryl7-ACA) comprising the use of a D-amino acid oxidasecontaining mixture (hereinafter DAO-containing mixture). More particularly, an esterase activity in the DAO-containing mixture employed in the present process is selectively reduced. Description of Related Art

Glutaryl-7-ACA is an intermediate material for the preparation of 7aminocephalosporanic acid (hereinafter 7-ACA) from Cephalosporin C. Cephalosporin C reacts with D-amino acid oxidase (hereinafter DAO) to produce a-keto adipoyl7-aminocephalosporanic acid (hereinafter a -keto adipoyl-7-ACA) which is a thermally unstable intermediate and hydrogen peroxide, then the produced a -keto adipoyl-7-ACA is oxidized by the hydrogen peroxide to produce glutaryl-7-ACA. For the purpose of obtaining a higher yield of glutaryl-7-ACA, various enzymatic processes for converting Cephalosporin C to glutaryl-7-ACA using DAO derived from Trigonopsis varlabills (hereinafter T. varlabills) have been developed. For example, U.S. Patent No. 3,821,209, Japanese Patent Publication 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 1 hydrogen peroxide, respectively, to avoid the decomposition of hydrogen peroxide by catalase. U.S. Patent No. 3,801,458 discloses an enzymatic oxidation of Cephalosporin C using activated cells of T. var-iabilis with a catalase inhibitor of sodium azide to provide a good yield of glutaryl- 7-ACA. European Patent Unexamined Publication No. 409,521 discloses a process for inactivating catalase in the presence of DAO by treating the enzymes with an aqueous basic solution of pE about 11 to 12 prior to the enzymatic oxidation of Cephalosporin C toglutaryl-7-ACA.

However, these processes have disadvantages in the reduction of the yield due to the esterase possessed by T. var-iab-ills and in the deterioration of quality due to impurities such as catalase inhibitors when they are applied to economical practice on an industrial scale. The esterase is an enzyme which converts Cephalosporin C, glutaryl7-ACA, and a -keto adipoyl7-ACA intermediate to their 3-desacetyl forms. These desacetyl derivatives not only reduce the yield of glutaryl-7-ACA but also deteriorate the quality of the glutaryl-7-ACA as impurities. Therefore, in order to improve the yield and quality of glutaryl-7-ACA, it is very important to inactivate or inhibit the esterase activity.

European Patent Unexamined Publication No. 409,521 also discloses a process for inactivating esterase activity by treating the cells of T. varlab-ills with acetone/water in order to avoid producing desacetyl Cephalosporin C and desacetyl 2 glutary]-7-ACA. However, the described process requires a complicated and difficult step to remove the acetone. Thus, there has been a qreat need for a simple and more efficient process for inactivating esterase, applicable to industrial practice.

SUMMARY OF THE INVENTION

In one aspect, the present invention relates to an improved enzymatic oxidation process for converting CeDhalosporin C to qlutary]-7-ACA comprisinq the use of a DAO-containinq mixture whose esterase activity is about 5% or less of the DAO activity.

The DAO-containing mixture whose esterase activity is lowered or inhibited may be obtained by contacting the mixture with a Cu-compound. This forms another aspect of the invention.

The above-mentioned DAO-containing mixture may also be obtained from a microorganism showing 5% or less of esterase activity based on its DAO activity. A suitable catalasedeficient or negative mutant microorganism may be obtained by treating a microorganism capable of producing DAO with ultraviolet radiation, X-ray radiation, and a mutagent.

The microorganism may be a catalase deficient strain of Trigonopsis variabi7is or a negative mutant thereof, such as T. variabi7is KC-103 (FERM BP-4359) or T. variabi7is EL-17 (FERM BP-4467). This forms a further aspect of the this invention.

Using the process of the present invention, an excellent yield of glutary]-7-ACA can be attained in hiqh quality. Moreover, the process of the Present invention comprises simple and safe steps, so it is applicable to industrial scale.

Detailed Description of the Invention

The present invention relates to an enzymatic oxidation process for converting Cephalosporin C to 3 glutaryl-7-ACA comprising the use of a DAO-containing mixture whose esterase activity is % or less of the DAO activity.

Cephalosporin C used in the present invention is an aqueous solution of Cephalosporin C powder, or Cephalosporin C fermentation broths wherein cells and solid are removed. Cephalosporin C is produced by cultivating cells of Achremonium chxysogenum in the culture medium containing 2 % of sucrose, 3 % of corn starch, 5 % of beetmolasses, 6 % of defatted soybean, 3 % of methyloleate, 0.5 % of calcium carbonate, 1.25 % of calcium sulfate and 0.8 % of ammonium acetate and the like or in a nutrient fermentation medium.

The DAO-containing mixture used in the present invention comprises DAO, e. g., partly purified DAO and crude DAO, and shows 5 % or less of esterase activity based on the DAO activity. The DAO-containing mixture may be used in any form such as intact cells, partly destroyed cells, permeabilized cells, cell-free extracts, and immobilized enzyme. Of these, partly destroyed cells, permeabilized cells, cell-free extracts, and immobilized enzyme are preferred. They are obtained by known methods.

It is preferred that DA0 be derived from T. varlabills in the present invention. More preferably, a catalasedeficient mutant such as T. varlabills KC-103 strain (deposited in National institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology as Deposit No. FERM BP-4359) are used in order to avoid remaining a -keto adipoyl 4 7-ACA.

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 Cucompound at a concentration of 10 to 2000 ppm, based on the total amount of aqueous solution or suspension of the DA0-containing mixture at 50 to 250 c for 2 to 24 hours. In order to inactivate the esterase without affecting on the DA0 activity, it is preferred that the concentration of the Cu-compound be 10 to 400 ppm. The esterase activity can be reduced to half or more by the treatment. Therefore, when the DA0-containing mixture Is obtained from T. varlabills KC-103 strain showing 9.1 % of activity ratio of esterase/DA0, the activity ratio can be reduced to 4.5 % or less.

In the present invention, the Cu-compound is contacted with a DA0containing mixture before the enzymatic oxidation process, i.e., before Cephalosporin C is added. Alternatively, the Cu-compound may be contacted with the DAO-containing mixture during the enzymatic oxidation process, i. e., it is added to the reaction mixture containing Cephalosporin C and the DA0-containing mixture. In the latter case, the concentration of the Cu-compound is 10 to 2000 ppm based on the reaction mixture. Representative example of the Cu-compound used in the present invention is CuS04.

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 DA0 activity, which is obtained by treating a microorganism capable of producing DA0 with ultraviolet radiation, 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 a mutagen such as N-methyl-N'-nitro-N-nitrosoganisine (hereinafter.NTG), then is cultured in a fermentation medium. The strain showing reduced esterase activity is selected by measuring esterase activity.

In the present invention, it is preferred that as a parent strain, a catalase-deficient or negative mutant be used to avoid by-products formation. A representative example of the mutant is T. varlabills EL-17 strain (deposited in National Institute of Bioscience and HumanTechnology, Agency of Industrial Science and Technology as Deposit No. FERM BP-4467). It is obtained by treating T. varlabills KC-103 strain (supra) with NTG. The mutant has 3.0 % of the esterase activity based on the DAO activity.

Esterase activity is measured by the following. Enzyme sample is added to 0.2 M phosphate buffer (pH 7.5) containing 2 g/1 of glutaryl-7-ACA, and they are reacted at 25 C for 30 minutes. Methyl alcohol in the same amount as the 6 reaction mixture is added to stop the reaction. The reaction solution is subjected to HPLC to determine the amount of the produced desacetyl glutaryl-7-ACA.

DAO activity is measured by the following. Enzyme sample is added to 0.1 M phosphate buffer (pH 7.5) containing 10 g/1 of Cephalosporin C, and they are reacted at 250 C for 15 minutes. Methyl alcohol in 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 produced glutaryl7-ACA and a -keto adipoyl-7-ACA.

When a DAO-containing mixture is contacted with a Cu-compound during the enzyme oxidation process. the total amount of desacetyl Cephalosporin C and desacetyl glutaryl-7-ACA is measured before and after the enzyme oxidation process. The increased amount of the desacetyl-derivatives based on the initial amount of Cephalosporin C is regarded as an activity ratio of esterase/DAO.

The enzymatic oxidation is usually carried out by blowing oxygen through the reaction solution at 20 to 300 C. The reaction time is dependent on the concentration of Cephalosporin C, the amount and activity of a DAOcontaining mixture, and the reaction temperature. When the amount of produced glutaryl-7-ACA has reached a maximum, which can be traced with HPLC or TLC or the like, the reaction may be stopped. The reaction is generally complete in 30 minute to 4 7 hours. In order to reduce an amount of remaining a -keto adipoyl-7-ACA, hydrogen peroxide may be added to the reaction mixture.

Glutaryl-7-ACA is isolated from the obtained reaction mixture by known methods. DESCRIPTION OF PREFERRED EMBODIMENTS

The present invention will be illustrated in more detail with reference to the following non-limiting Examples. [Example 1] T. varlabills KC-103 strain was grown in a medium containing 2 % of glucose, 2 % of cornsteepliquor and 0.2 % of M-methionine 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 250 C for 2 hours to permeabilize the cells.

23.6 mg of CuSO 4.5H20 was added to the obtained fermentation broth of permeabilized cells and left to stand at room temperature for two hours. An appropriate amount of enzyme sample from the Cu-containing fermentation broth was added to 0.2 M potassium phosphate buffer (pH 7.5) containing 2 g/1 of glutaryl-7-ACA. The reaction was carried out at 250 C for 30 minutes, and was stopped by adding methyl alcohol in the same amount as the reaction mixture, and then subjected to HPLC. Desacetyl glutaryl-7-ACA produced in the reaction was not detected, indicating the esterase in the enzyme sample was sufficiently inactivated.

8 g of Cephalosporin C powder was dissolved in 1000 ml of water, and the pH was adjusted to pH 7.5. The obtained Cephalosporin C solution was mixed with the above-obtained Cu-containing broth, and they were reacted at 200 C for about 180 minutes with oxygen blowing through the reaction mixture. The yield of glutaryl-7-ACA was 93.2 [Example 2] A fermentation broth of the permeabilized cells of T. variab-ilis KC-103 strain whose esterase activity was inactivated was obtained by the same method as described in Example 1.

A A. chrysogenum fermentation broth containing 8.5 g/1 of Cephalosporin C was adjusted to pE 2.5, then cells were removed therefrom. The pH of the broth was adjusted to pH 7.5. 1200 ml of the obtained broth was mixed with 60 ml of the above-obtained T. varlabills fermentation broth. while oxygen was blown through the reaction mixture and 23.9 ml of 3.5 % hydrogen peroxide was continuously added to the reaction mixture, the reaction was conducted at 200 C for about 240 minutes. The yield of glutaryl-7-ACA was 92.8 %. [Example 3] A fermentation broth of the permeabilized cells of T. varlabills KC-103 strain was obtained by the same method as described in Example 1.

g of Cephalosporin C powder was dissolved in 1000 mi of water, and the pH was adjusted to pH 7.5. The obtained 9 Cephalosporin C solution was mixed with 60 ml of the above-obtained T. variab-ilis fermentation broth. Then, 0.5 g of CUS04. 5H20 was added to the resulting mixture. The reaction was conducted at 20 C for about 180 minutes while oxygen was blowing through the reaction mixture. The yield of glutaryl-7-ACA was 92.2 %. The total increased amount of desacetyl Cephalosporin C and desacetyl glutaryl-7-ACA was 1 % or less based on the amount of Cephalosporin C. [Example 4] A fermentation broth of the permeabilized cells of T. varlabills KC-103 strain was obtained by the same method as described in Example 1. 60 ml of the T. varlabills fermentation broth and 1200 ml of the A. chxysogenum fermentation broth containing Cephalosporin C obtained by the same method as described in Example 2 were mixed.

0.5 g of CUSO..5H.0 was added to the resulting mixture, and they were reacted at 200 C for about 240 minutes while oxygen was blown through and 23.9 ml of 3.5 % hydrogen peroxide was continuously added to the reaction mixture. The yield of glutaryl-7-ACA was 92.0 The total increased amount of desacetyl Cephalosporin C and desacetyl glutaryl-7-ACA was 1 % or less based on the amount of Cephalosporin C.

[Example 51

400 ml of a T. varlabills fermentation broth containing 40 g of wet cells was obtained by the same method as described in Example 1, and was treated with 2.0 ml of toluene by the same method as described in Example 1.

157.3 mg of CuSO 4. 5H 2 0 was added to the above-obtained broth, and the resulting mixture was left to stand at room temperature for 2 hours. The esterase activity of an enzyme sample from the obtained Cu-containing broth was measured by the same method as described in Example 1. The esterase in the enzyme sample was sufficiently inactivated.

g of Cephalosporin C powder was dissolved in 1000 ml of water, and the pH was adjusted to pH 7.5. The obtained solution was mixed with 400 ml of the above-obtained T. varlabills fermentation broth. The reaction was conducted at 200 C for about 30 minutes with oxygen blowing through the reaction mixture. The yield of glutaryl-7-ACA was 99.0 [Example 6] 400 ml of a fermentation broth of the permeabilized cells of T. varlabilis KC-103 strain obtained by the same method as described in Example 1 and 1000 ml of Cephalosporin C aqueous solution obtained by the same method as described in Example 1 were mixed.

0.66 g of CuSO 4 - 5H20 was added to the above-obtained reaction mixture, and the reaction was conducted at 200 C for about 30 minutes while oxygen was blowing through the reaction mixture.

The yield of glutaryl-7-ACA was 98.1 %. The total increased amount of desacetyl Cephalosporin C and desacetyl glutaryl-7-ACA was 1 % or less based on the amount of 11 Cephalosporin C. [Example 7] T. varlabills EL-17 strain was grown in a medium containing 2 % of glucose, 2 % of cornsteepliquor and 0.2 % of M-methionine at 250 C for 60 hours. 0.2 ml of toluene was 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 broth was added to 0.2 M potassium phosphate buffer (pH 7.5) containing 2 g/1 of glutaryl-7-ACA, and they were reacted at 250 C for 30 minutes. The reaction was stopped by adding methyl alcohol in the same amount as the reaction mixture, and then subjected to EPLC. The ratio of the estefase activity to the DAO activity was reduced to one third of that of the parent strain, i.e., 3.0 %.

g of Cephalosporin C powder was dissolved in 1000 mi of water, and the pH was adjusted to pH 7.5. The obtained Cephalosporin C solution was mixed with the above-obtained fermentation broth of permeabilized cells, and the mixture was reacted at 20' C for about 180 minutes with oxygen blowing through the reaction mixture. The yield of glutaryl-7-ACA was 92. 6 %. [Example 8] A fermentation broth of the permeabilized cells of T. va-r.iab-il-is EL- 17 strain was obtained by the same method as 12 described in Example 7.

A A. chrysogenum fermentation broth containing 9 g/1 of Cephalosporin C was adjusted to pH 2.5, and then cells were removed therefrom. The pH was adjusted to pH 7.5 with 6N NaOH. 6 & of the obtained broth was mixed with 60 1 of the above-obtained T. varlabIlis fermentation broth. While oxygen was blown through the reaction mixture and 13 1 of 35 % hydrogen peroxide was continuously added to the reaction mixture, the reaction was conducted at 200 C for about 220 minutes. The yield of glutaryl-7-ACA was 92.2 [Example 91 ml of a fermentation broth of the permeabilized cells of T. varlabills EL- 17 strain obtained by the same method as described in Example 7 and 1000 ml of Cephalosporin C aqueous solution obtained by the sjame method as described in Example 1 were mixed. The mixture was then reacted at 200 C for about 30 minutes with oxygen blowing through the reaction mixture. The yield of glutaryl-7-ACA was 96.2 %. [Comparative Example] ml of a fermentation broth of permeabilized cells of T. var-iab-il-is KC- 103 strain obtained by the same method as described in Example 1 and 1000 ml of Cephalosporin C aqueous solution obtained by the same method as described in Example 1 were mixed. The mixture was then reacted at 20 C for about 180 minutes with blowing oxygen-through the reaction mixture. The yield of glutaryl-7-ACA was 83.0 13

Claims

CLAIMS:

1 An enzymatic oxidation process for converting Cephalosporin C to glutary]-7-aminocephalosporanic acid comprising the use of a D-amino acid oxidase-containing mixture whose esterase activity is 5% or less of the Damino acid oxidase activity.

2. A process according to Claim 1, wherein the D-amino acid oxidasecontaining mixture is obtained from a catalasedeficient or negative mutant microorganism capable of producing D-amino acid oxidase.

3. A process according to Claim 2, wherein the microorganism is, or is derived from, the Trigonopsis variabi7is KC-103 strain or the EL-17 strain.

4. A process according to Claim 1, wherein the D-amino acid oxidasecontaining mixture is obtained by contacting the mixture with a Cucompound,

5. A process according to Claim 4, wherein the D-amino acid oxidase- containing mixture is obtained by contacting the mixture with a Cu- compound at a concentration of 10 to 2000 PPM.

6. A process accordinq to Claim 4 or 5, wherein the contact 14 is carried out before or during the enzymatic oxidation process.

7. A process accordinq to Claim 1. wherein the D-amino acid oxidasecontaininq mixture is obtained form a mutant microorqanism showinq 5% or less of esterase activity based on i t s DAO activity, which is obtained by treating a microorqanism capable of producing DAO with ultraviolet radiation, X-ray radiation, or a mutagen.

8. A process accordinq to Claim 7, wherein the D-amino acid oxidasecontaining mixture is partly destroyed cells, permeabilized cells. cellfree extracts obtained therefrom, or immobilised enzyme.

9. A Process according to Claim 7 or 8, wherein the mutant is catalasedeficient or negative.

10. A process accordinq to Claim 7, 8 or 9, wherein the mutant is the Trigononsis variabi7is EL17 strain.

11. A Process accordinq to Claim 1. subst ant i a] 1 y as described in any one of the Examples herein.

12. A process for preparing a D-amino acid oxidasecontaining mixture whose esterase activity is 5% or less of the D-amino acid oxidase activity comprising contacting a D- amino acid oxidase-containinq mixture with a Cu-compound.

13. A process according to Claim 12. in which the Cucompound is used at a concentration of 10 to 2000 ppm.

14. A microorganism which is a catalase deficient strain of Triponoosis variabi7is or a negative mutant thereof.

15. A microorganism according to Claim 14 which is T. variabi7is KC103 (FERM BP-4359) or a catalase-deficient mutant thereof.

16. A microorganism accordinq to Claim 15 which is T. variabi7is EL-17 (FERM BP-4467) 16