CN110964771A

CN110964771A - Process for preparing 7-aminocephalosporanic acid

Info

Publication number: CN110964771A
Application number: CN201811147580.1A
Authority: CN
Inventors: 罗晖; 常雁红; 仝双明; 秦波; 刘子嘉; 朱琳琳; 孙宏旭; 于慧敏; 沈忠耀
Original assignee: University of Science and Technology Beijing USTB
Current assignee: University of Science and Technology Beijing USTB
Priority date: 2018-09-29
Filing date: 2018-09-29
Publication date: 2020-04-07

Abstract

The invention discloses a method for preparing 7-aminocephalosporanic acid, in particular to a method for carrying out catalytic reaction on cephalosporin C acylase and cephalosporin C in ammonium bicarbonate and/or ammonium carbonate aqueous solution. The method can obviously improve the catalytic stability of the cephalosporin C acylase and prolong the service life of the cephalosporin C acylase.

Description

Process for preparing 7-aminocephalosporanic acid

Technical Field

The invention relates to a method for preparing 7-aminocephalosporanic acid, in particular to a method for preparing 7-aminocephalosporanic acid by catalyzing cephalosporin C with cephalosporin C acylase.

Background

7-aminocephalosporanic acid (hereinafter referred to as "7-ACA") having a molecular formula of C₁₀H₁₂N₂O₅S is a parent nucleus for synthesizing various cephalosporin antibiotics in the pharmaceutical industry, and has very important market development value.

At present, 7-ACA is produced by a one-step enzyme method mainly in industry, namely under the action of cephalosporin C acylase (hereinafter referred to as CPC acylase), substrate cephalosporin C (CPC) is directly hydrolyzed to α -aminoadipic acid and a product 7-ACA.

At present, pure water systems are generally used in industrially applied enzyme catalysis. Since acidic substances are generated in the process of catalyzing CPC to generate 7-ACA by CPC acylase, alkali is continuously added to adjust pH so as to maintain smooth catalytic reaction, however, the operation of adding strong alkali causes the pH value of local solution to be too high, which may lead to enzyme inactivation, so that the catalytic stability of CPC acylase is reduced.

Phosphate buffer systems are also used, but the buffer capacity of the buffer solution with the general concentration is often insufficient to maintain the pH value of the reaction system, and the subsequent separation of the product is affected by adding the buffer solution (salt components in the buffer solution are crystallized along with the 7-ACA, so that the purity of the 7-ACA product is reduced), thereby reducing the quality of the product and increasing the cost.

Therefore, there is a need in the industry to further improve the catalytic stability of CPC acylase and simplify the operation process.

Disclosure of Invention

The invention aims to improve the catalytic stability of CPC acylase in the process of preparing 7-ACA by a one-step enzyme method, thereby prolonging the service life of the enzyme and reducing the cost. To this end, the present invention proposes the use of ammonium bicarbonate and/or ammonium carbonate to improve the catalytic stability of CPC acylase.

Unlike commonly used buffers (e.g., phosphates, acetates, borates, Tris-HCl, sodium carbonate, and the like), ammonium bicarbonate and/or ammonium carbonate are themselves very unstable and are not typically used to formulate buffers. However, the present inventors have unexpectedly found that the addition of ammonium bicarbonate and/or ammonium carbonate to the catalytic system can significantly improve the catalytic stability of CPC acylase, thereby prolonging the service life of the enzyme.

In addition, ammonium bicarbonate and/or ammonium carbonate can be easily removed in the subsequent separation of the catalytic products (7-ACA is generally precipitated by adjusting the pH to acidity with hydrochloric acid, during which the ammonium bicarbonate and/or ammonium carbonate will generate carbon dioxide to be removed) without affecting the quality of the 7-ACA product. Ammonium bicarbonate and ammonium carbonate are very cheap in terms of production cost.

Drawings

FIG. 1 shows the pH change in the microenvironment of CPC acylase for carrying out catalytic reactions in pure water, phosphate buffer and aqueous ammonium bicarbonate, respectively.

Detailed Description

A process for the one-step enzymatic preparation of 7-ACA using CPC acylase with CPC as a substrate is known. The reaction process is simply illustrated as follows:

as a reaction substrate CPC, in practical use, a carboxyl group in the CPC structure is ionized into-COO in an aqueous solution^-It may bind a wide variety of cations, however, whatever the cation bound, the resulting CPC salt is encompassed within the term "cephalosporin C" or "CPC" of the invention. Therefore, in the context of the present invention, the term "cephalosporin C" or "CPC" is to be understood in a broad sense and includes not only CPC itself but also salts of CPC such as sodium and zinc salts of CPC and the like, and CPC fermentation broth, which is a solution obtained by preliminary purification of fermentation broth obtained by fermentative production of CPC, and the like. In the present invention, CPC sodium salt is used as an example.

In the present invention, the aqueous solution of ammonium bicarbonate and/or ammonium carbonate may be added to the reaction system in various ways, for example, by dissolving the CPC acylase in the aqueous solution of ammonium bicarbonate and/or ammonium carbonate or by adding the CPC acylase and the substrate CPC to the aqueous solution of ammonium bicarbonate and/or ammonium carbonate at the same time. By way of example, the practice of the invention employs dissolving CPC in an aqueous solution of ammonium bicarbonate and/or ammonium carbonate to produce a substrate solution for use. Specifically, the preparation of the substrate solution may be carried out as follows: firstly, dissolving ammonium bicarbonate and/or ammonium carbonate in aqueous solution, and then adding CPC; or dissolving CPC in water solution, and adding ammonium bicarbonate and/or ammonium carbonate; it is also possible to dissolve CPC and ammonium bicarbonate and/or ammonium carbonate separately in aqueous solutions and then mix the two aqueous solutions together while maintaining the pH in the range of 7-9. The pH value is kept in the range of 7-9, and the pH value can be adjusted by dissolving CPC and ammonium bicarbonate and/or ammonium carbonate in an aqueous solution and then using dilute hydrochloric acid or ammonia water, or during the mixing process, or by estimating and adjusting the pH value before mixing the CPC and the ammonium bicarbonate and/or the ammonium carbonate so that the pH value after mixing the CPC and the ammonium bicarbonate and/or the ammonium carbonate is in the range.

Preferably, the concentration of ammonium bicarbonate and/or ammonium carbonate in the aqueous solution is from 10mmol/L to 2mol/L, such as from 10mmol/L to 500mmol/L, from 50mmol/L to 400mmol/L, from 80mmol/L to 300mmol/L, from 100mmol/L to 280 mmol/L.

As the catalyst CPC acylase used in the present invention, those skilled in the art know that CPC acylases capable of catalyzing CPC can be used in one-step enzyme catalytic preparation processes, and in the catalytic process, free cell catalysis can be adopted, and immobilized enzyme catalysis can also be adopted. In the present invention, an immobilized CPC acylase is used as an example.

As an example for describing the present invention in detail, there is exemplified CPC acylase (Amin, Chimin, Rahui, Song Wen, Shen.) produced using genetically engineered Escherichia coli BL21(DE3)/pET28-acy or BL21(DE3)/pET28-CPCacy, artificial synthesis and recombinant expression of CPC acylase gene, university of Qinghua, 2008,48(9):119-123, Chimin, Song Wen, Anmin, Luhui, Shen-A cephalosporin C acylase, and vector and use thereof, patent No. ZL: 200810102219.7).

In the process of the present invention, the catalytic reaction is carried out by mixing CPC with CPC acylase in an aqueous solution of ammonium bicarbonate and/or ammonium carbonate, catalyzing at 10 ℃ to 37 ℃, and adjusting the pH of the reaction by adding an alkali solution such as aqueous ammonia during the catalysis, the pH being usually controlled at 7 to 9, such as 8 to 9, for example 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, in particular 8.5. And (3) carrying out HPLC detection on the remaining CPC of the reaction by timing sampling until the reaction is finished to obtain a 7-ACA reaction solution.

The temperature is usually controlled to be 10 ℃ to 37 ℃, for example, 10 ℃, 12 ℃, 15 ℃,20 ℃, 25 ℃, 30 ℃, the higher the temperature is, the higher the enzyme activity is, but the catalytic stability of the enzyme is reduced, and the CPC substrate is easy to degrade.

In the context of the present invention, the term "reaction system" or "catalytic system" has the usual meaning in the art, including the substrates, enzymes and the resulting products and by-products involved in the reaction during the catalytic performance, and also including the solvents and various auxiliaries added thereto; in practice, this is understood to mean the contents of the vessel in which the catalytic reaction is carried out, with the exception of the reactor apparatus.

Filtering the reaction solution of 7-ACA to obtain supernatant (the filtered immobilized CPC acylase or free cells can be reused), crystallizing by conventional method to obtain 7-ACA crystal, for example, adding hydrochloric acid to adjust pH to 3.5, standing at 4 deg.C to precipitate 7-ACA crystal, centrifuging, washing with acetone, and drying to obtain 7-ACA.

As described above, acid is generated during the preparation of 7-ACA by CPC acylase catalyzing CPC, so that the pH of the reaction system is continuously decreased, and a base is required to increase the pH so that the reaction is performed under an optimal condition. However, the addition of a strong base causes a local pH to be too high, which may result in inactivation of CPC acylase, decreasing enzyme stability.

The inventor has unexpectedly found in practice that the catalytic stability of the CPC acylase can be greatly improved by using ammonium bicarbonate and/or ammonium carbonate in the reaction system, thereby prolonging the service life of the enzyme. Without wishing to be bound by any theory, the present inventors speculate that the use of ammonium bicarbonate and/or ammonium carbonate allows the pH of the microenvironment around the CPC acylase to be stably adjusted, thereby maintaining the stability of the CPC acylase.

Referring to fig. 1, pH changes in the CPC acylase microenvironment for catalytic reactions in pure water, phosphate buffer and aqueous ammonium bicarbonate, respectively. Wherein the pH of the main phase of the reaction system was maintained at 8.5 during the reaction. However, in the reaction system using pure water, although the pH of the host phase solution is controlled to be 8.5 by dropwise adding ammonia water, the pH of the microenvironment of the immobilized enzyme is greatly reduced, and the lowest pH of the microenvironment is even about 7.2, which is far lower than the optimal pH of 8.5. In the catalysis process of adding 0.1M phosphate buffer, the pH of the immobilized enzyme microenvironment is controlled to a certain degree, and the lowest microenvironment pH is above 8. In the catalysis process of adding 0.1M ammonium bicarbonate as a buffer solution, the pH of the microenvironment of the immobilized enzyme is always controlled to be about 8.5. The method for obtaining the data in figure 1 is given in example 4.

Examples

The process of the present invention is illustrated in detail by the examples below.

Measurement or calculation methods:

the conversion rate of CPC was calculated as follows:

CPC conversion (CPC concentration at reaction zero time-CPC concentration at reaction completion)/CPC concentration at reaction zero time

The calculation method of the molar yield of 7-ACA comprises the following steps:

molar yield of 7-ACA (7-ACA molar concentration at the end of reaction/(CPC molar concentration at reaction zero time-CPC molar concentration at the end of reaction)

The detection method of CPC concentration or 7-ACA concentration comprises the following steps:

in the reaction process, 20 microliter of reaction solution is added into 180 microliter of stop solution (formed by mixing 50mmol/L NaOH solution and 20% glacial acetic acid solution according to the volume ratio of 1: 2), 20 microliter of the reaction solution is added into 180 microliter of ultrapure water, namely the reaction solution is diluted by 100 times, 40 microliter of the reaction solution is subjected to liquid chromatography sampling, and the concentrations of 7-ACA and CPC are obtained according to the integrated peak areas of 7-ACA and CPC peaks and a standard curve.

The liquid chromatographic analysis conditions were: the chromatographic column is Phenomenex Luna C-18 (4.6X 150mm), the mobile phase is 15% methanol-7.5% acetonitrile-1% acetic acid, the flow rate is 0.8mL/min, and the detection wavelength is 254 nm.

Half-life of the enzyme: refers to a use batch when the catalysis takes twice as much time as the initial catalysis to achieve the same CPC conversion (e.g., 95% conversion).

Preparation of immobilized CPC acylase

Cultivation of genetically engineered Escherichia coli BL21(DE3)/pET 28-CPCacy:

LBK medium (tryptone 1%, yeast powder 0.5%, NaCl 1%, kanamycin 50. mu.g/mL, pH7.0)

Corn steep liquor medium (5% corn steep liquor, 0.017M KH)₂PO₄，0.072M K₂HPO₄0.4% glucose, 20mM lactose, 50. mu.g/mL kanamycin, pH7.5)

Culturing: genetically engineered Escherichia coli BL21(DE3)/pET28-CPCacy was cultured in LBK medium at 37 ℃ and 200r/min for 10 hours, inoculated in a corn steep liquor medium at 4% inoculum size, and cultured at 30 ℃ and 200r/min for 12 hours.

Collecting: the cells were harvested by centrifugation at 10000rpm for 5 min.

Weighing 1g of the harvested thallus, resuspending the thallus with 100ml of deionized water, and crushing the thallus by ultrasonic waves. The conditions of the ultrasound were: carrying out ultrasonic treatment for 3 seconds at intervals of 3 seconds, carrying out 99 cycles of ultrasonic treatment at the ultrasonic power of 200W, and crushing twice. Centrifuging at 10000rpm for 10min at 4 deg.C to obtain supernatant as enzyme solution of CPC acylase.

The enzyme solution obtained above was adjusted to pH8.5, and covalently coupled immobilized enzyme carrier LX1000-EP (science and technology Co., Ltd., West An blue) was added thereto, followed by shaking in a shaker at 20 ℃ for 24 hours. Washing with 0.1mol/L sodium phosphate buffer solution with pH of 8.5 for three times, vacuum filtering to obtain the required immobilized CPC acylase, and storing at 4 ℃ for later use.

Example 1:

preparation of CPC solution: 3g of CPC sodium salt was weighed, dissolved in 250mM of 100ml of ammonium carbonate aqueous solution, and the solution was adjusted to pH9 with aqueous ammonia, wherein the concentration of CPC was 70 mmol/L.

Catalyzing: 3g of the immobilized CPC acylase obtained in the above preparation example was added to 30ml of the above CPC solution, and the reaction was catalyzed at 20 ℃ with adjusting the reaction system pH to 8.5 with 2M aqueous ammonia. The change of the content of CPC in the reaction system is monitored while the catalytic reaction is carried out, and the conversion rate of CPC reaches 99.2 percent and the molar yield of 7-ACA reaches 95.2 percent after 40min of catalysis.

Example 2:

preparation of CPC solution: 3g of CPC sodium salt was weighed, dissolved in 200mM of 100ml of an aqueous ammonium bicarbonate solution, and the solution was adjusted to pH8 with aqueous ammonia, wherein the concentration of CPC was 70 mmol/L.

Catalyzing: 1g of the immobilized CPC acylase obtained in the foregoing production example was added to 10ml of the above CPC solution, and the reaction was catalyzed at 37 ℃ with adjusting the reaction system pH8.5 with 2M aqueous ammonia. The change of the content of CPC in the reaction system is monitored while the catalytic reaction is carried out, and the conversion rate of CPC reaches 98.1 percent and the molar yield of 7-ACA reaches 92.5 percent after 30min of catalysis.

After obtaining the immobilized enzyme by suction filtration, catalyzing the immobilized enzyme under the same reaction conditions, and after 13 times of catalysis, when the immobilized enzyme catalyzes CPC to ensure that the conversion rate reaches 95%, 63min is consumed, namely the half-life period of the immobilized enzyme reaches the activity. Indicating that the half-life of the immobilized CPC acylase catalyzed under this condition was 13 batches.

Example 3:

preparation of CPC solution: 3g of CPC sodium salt was weighed, dissolved in 100mM of 100ml of an aqueous ammonium bicarbonate solution, and the solution was adjusted to pH8.5 with ammonia water to have a CPC concentration of 70 mmol/L.

Catalyzing: 1g of the immobilized CPC acylase obtained in the foregoing production example was added to 10ml of the above CPC solution, and the reaction was catalyzed at 25 ℃ with adjusting the reaction system pH8.5 with 2M aqueous ammonia. The change of the content of CPC in the reaction system is monitored while the catalytic reaction is carried out, and the conversion rate of CPC reaches 98.5 percent and the molar yield of 7-ACA reaches 95.5 percent after 50min of catalysis.

After the immobilized enzyme is obtained by suction filtration, the CPC is catalyzed under the same reaction conditions, and the conversion rate of the CPC reaches 99.5 percent and the molar yield of the 7-ACA reaches 96.3 percent after continuous catalysis for 50 min.

After the immobilized enzyme is obtained by suction filtration, the catalysis of CPC is carried out under the same reaction conditions, and after 60 batches of catalysis, 105min is consumed when the immobilized enzyme catalyzes CPC to ensure that the conversion rate of CPC reaches 95%, namely the half-life period of the activity of the immobilized enzyme is reached. At this time, the conversion of CPC was 95.2%, and the molar yield of 7-ACA was 92.3%.

Separation and purification of 7-ACA: taking 30ml of the obtained catalytic reaction liquid, cooling to 10 ℃, slowly adding 15% hydrochloric acid into the bottom of the reaction liquid to adjust the pH value to 3.5, standing at 4 ℃ for 5 hours, filtering, and leaching with acetone. Drying at 4 ℃ gave a product powder with a purity of 98.3% by liquid chromatography and a yield of 91.1% during the purification.

Comparative example 1

Preparation of CPC solution: 3g of CPC sodium salt was weighed, dissolved in 100ml of purified water, and the solution was adjusted to pH8.5 with ammonia water to a CPC concentration of 70 mmol/L.

Catalyzing: 1g of the immobilized CPC acylase obtained in the previous preparation example was added to 10ml of the CPC solution, the reaction was catalyzed at 37 ℃, the pH of the catalytic system was adjusted to 8.5 with 2M ammonia water, and the change in the content of cephalosporin C in the reaction system was monitored, and the conversion of cephalosporin C reached 95% after 30min of catalysis.

After the immobilized enzyme is obtained by suction filtration, catalysis of cephalosporin C is carried out under the same reaction conditions, and after 3 batches of catalysis, 62min is consumed when the immobilized enzyme catalyzes cephalosporin C to ensure that the conversion rate of cephalosporin C reaches 95%, namely the half-life period of the activity of the immobilized enzyme is reached. This shows that the half-life of the immobilized CPC acylase is only 3 batches when catalyzed by substrates formulated in pure water.

Comparative example 2

Catalyzing: 1g of the immobilized CPC acylase obtained in the previous preparation example was added to 10ml of the above CPC solution, and the reaction was catalyzed at 25 ℃ with 2M ammonia water to adjust the pH of the catalyst system to 8.5 while monitoring the change in the CPC content in the reaction system, and the conversion of CPC reached 96% after 50min of catalysis.

After the immobilized enzyme is obtained by suction filtration, the catalysis of CPC is carried out under the same reaction conditions, and after 24 batches of catalysis, 105min is consumed when the immobilized enzyme catalyzes CPC to ensure that the conversion rate of CPC reaches 95%, namely the half-life period of the activity of the immobilized enzyme is reached. It is shown that the half-life of the immobilized CPC acylase is 24 batches when the substrate formulated with pure water is catalyzed at 25 ℃.

Cooling the obtained catalytic reaction solution 100ml to 10 ℃, slowly adding 15% hydrochloric acid into the bottom of the reaction solution to adjust the pH to 3.5, standing at 4 ℃ for 5h, filtering, and leaching with acetone. Drying at 4 ℃ gave a product powder with a purity of 98.1% by liquid chromatography and a yield of 91.3% during the purification.

Comparative example 3

Preparation of CPC solution: 3g of CPC sodium salt was weighed, dissolved in 100ml of 100mmol/L sodium phosphate buffer solution, and the solution was adjusted to pH8.5 with ammonia water to have a CPC concentration of 70 mmol/L.

Catalyzing: 1g of the immobilized CPC acylase obtained in the previous preparation example was added to 10ml of the above CPC solution, and the reaction was catalyzed at 37 ℃ with 2M ammonia water to adjust the pH of the catalyst system to 8.5 while monitoring the change in the CPC content in the reaction system, and the conversion of CPC reached 95% after 30min of catalysis.

After the immobilized enzyme is obtained by suction filtration, the catalysis of CPC is carried out under the same reaction conditions, and after 5 batches of catalysis, 65min is consumed when the immobilized enzyme catalyzes CPC to ensure that the conversion rate of CPC reaches 95%, namely the half-life period of the activity of the immobilized enzyme is reached. It is shown that the half-life of the immobilized CPC acylase is only 5 batches when catalyzed by a substrate formulated in 100mmol/L sodium phosphate buffer.

Cooling the obtained catalytic reaction solution 100ml to 10 ℃, slowly adding 15% hydrochloric acid into the bottom of the reaction solution to adjust the pH to 3.5, standing at 4 ℃ for 5h, filtering, and leaching with acetone. Drying at 4 ℃ gave a product powder with a purity of 82.5% by liquid chromatography and a yield of 90.8% during purification.

Comparative example 4

Preparation of CPC solution: 3g of CPC sodium salt was weighed, dissolved in 100ml of 200mmol/L sodium phosphate buffer solution, and the solution was adjusted to pH8.5 with ammonia water to have a CPC concentration of 70 mmol/L.

After the immobilized enzyme is obtained by suction filtration, the catalysis of CPC is carried out under the same reaction conditions, and after 6 batches of catalysis, 63min is consumed when the immobilized enzyme catalyzes CPC to ensure that the conversion rate of CPC reaches 95%, namely the half-life period of the activity of the immobilized enzyme is reached. It is shown that the half-life of the immobilized CPC acylase is only 6 batches when catalyzed by a substrate formulated in 200mmol/L sodium phosphate buffer.

Example 4: detection of microenvironment pH in immobilized enzyme catalysis process

Preparation of CPC acylase:

the preserved strain E. coli BL21(DE3)/pET28-CPCAcy is inoculated into an LBK culture medium (tryptone 1%, yeast powder 0.5%, NaCl 1%, kanamycin 50 mu g/mL, pH7.0), subjected to shake cultivation at 37 ℃ and 160rpm for 12h, transferred into a fresh LBK culture medium, and cultivated for 6h to ensure that the strain reaches the logarithmic phase. Inoculating the obtained strain into culture medium (peptone 10g/L, yeast powder 5g/L, Na) at an inoculation amount of 2.5%₂HPO₄·12H₂O 8.95g/L，KH₂PO₄3.4g/L，NH₄Cl2.67g/L，Na₂SO₄0.7g/L，MgSO₄0.24g/L, 5g/L of glycerol, 0.5g/L of glucose and 2g/L of lactose) in a tank, wherein the specific fermentation conditions are as follows: stirring speed 300rpm at 28 ℃, volume of feed liquid 3L, and fermenting for 24 h. The obtained mycelia were resuspended in 0.1mol/L pH8.5 sodium phosphate buffer, and the cells were disrupted by means of a high-pressure homogenizer at a disruption pressure of 800 bar. The disruption solution was centrifuged at 7000rpm at 4 ℃ for 10min to obtain the supernatant, which was the enzyme solution of the desired CPC acylase.

Preparation of immobilized CPC acylase labeled with 5-aminofluorescein:

100mL of the CPC acylase enzyme solution obtained above was weighed, 200mL of 1.25mol/L sodium phosphate buffer solution (pH8.0) was added, and the mixture was mixed well. 3g of covalently coupled immobilized enzyme carrier LX 1000-EPC (science and technology Co., Ltd., West Ann blue) was added, and 0.015g of 5-aminofluorescein sodium was added and mixed well. Shaking the mixture in a shaker at 25 ℃ for 24 hours. Vacuum filtration and washing with a large amount of deionized water. Washing with 0.1mol/L sodium phosphate buffer solution with pH of 8.5 for three times, vacuum filtering to obtain the required immobilized CPC acylase, and storing at 4 ℃ for later use.

Preparation of CPC solution:

(1) CPC sodium salt 2.5g was weighed, dissolved in 100ml of 100mM ammonium bicarbonate solution, and the solution was adjusted to pH8.5 with aqueous ammonia.

(2) CPC sodium salt 2.5g was weighed, dissolved in 100ml of 100mM sodium phosphate buffer solution, and the solution was adjusted to pH8.5 with aqueous ammonia.

(3) CPC sodium salt 2.5g was weighed, dissolved in 100ml of water, and the solution was adjusted to pH8.5 with aqueous ammonia.

Catalyzing: 2.5g of the immobilized CPC acylase was added to 30ml of the 3 CPC solutions, and the pH of the main catalyst solution was controlled to 8.5 by adding ammonia water to control the reaction at 37 ℃ while monitoring the pH of the microenvironment of the immobilized enzyme by using a fiber pH sensor pH-1 mini v2 (Presense Precision Sensing GmbH). And (3) detecting pH-1 mini v2 to obtain the fluorescence intensity of the catalytic system, and converting the pH value of the microenvironment of the immobilized enzyme by using the detected fluorescence intensity through a fluorescence intensity relation curve corresponding to the standard pH. The obtained curves are shown in fig. 1.

Finally, it should be noted that: although the present invention has been described in detail with reference to the above embodiments, it should be understood by those skilled in the art that: the invention may be modified and equivalents substituted; any modification or partial replacement without departing from the spirit and scope of the present invention should be covered within the scope of the present invention.

Claims

1. A process for preparing 7-aminocephalosporanic acid, characterized in that cephalosporin C acylase and cephalosporin C are subjected to a catalytic reaction in an aqueous solution of ammonium bicarbonate and/or ammonium carbonate.

2. The method of claim 1, wherein the catalytic reaction is carried out at a pH of 7-9, such as pH8-9, in particular pH 8.5.

3. The process of claim 1 or 2, wherein the pH is adjusted using aqueous ammonia.

4. A process according to any one of claims 1 to 3, wherein the catalytic reaction is carried out at a temperature of from 10 ℃ to 37 ℃, for example from 10 ℃ to 25 ℃.

5. A process according to any one of claims 1 to 4 wherein the aqueous solution of ammonium bicarbonate and/or ammonium carbonate has a concentration of from 10 to 2mol/L, for example from 10 to 500 mmol/L.

6. The method as claimed in any one of claims 1 to 5, wherein the cephalosporin C acylase is a cephalosporin C acylase produced by genetically engineering Escherichia coli BL21(DE3)/pET28-acy or BL21(DE3)/pET 28-CPCacy.

7. The method of any one of claims 1 to 6, wherein the cephalosporin C acylase is an immobilized cephalosporin C acylase.

8. Use of ammonium bicarbonate and/or ammonium carbonate for increasing the stability of a cephalosporin C acylase in the preparation of 7-aminocephalosporanic acid using the cephalosporin C acylase as an enzyme.

9. Use of ammonium bicarbonate and/or ammonium carbonate for maintaining the stability of the micro-environmental pH of an immobilized cephalosporin C acylase.