CN111876402A - Preparation method of immobilized cephalosporin C acylase - Google Patents
Preparation method of immobilized cephalosporin C acylase Download PDFInfo
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- CN111876402A CN111876402A CN202010615993.9A CN202010615993A CN111876402A CN 111876402 A CN111876402 A CN 111876402A CN 202010615993 A CN202010615993 A CN 202010615993A CN 111876402 A CN111876402 A CN 111876402A
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
- C12N9/80—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5) acting on amide bonds in linear amides (3.5.1)
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- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
- C12N11/08—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer
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- C12Y305/00—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5)
- C12Y305/01—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5) in linear amides (3.5.1)
- C12Y305/01093—Glutaryl-7-aminocephalosporanic-acid acylase (3.5.1.93)
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Abstract
The invention provides a preparation method of immobilized cephalosporin C acylase, which comprises the following steps: (1) breaking the thallus; (2) clarifying and concentrating; (3) salting out; (4) desalting; (5) adsorption and desorption; (6) loading and curing; (7) and (4) storing. The preparation method of the immobilized cephalosporin C acylase reduces the equipment cost and the production cost required by extraction. Reduces the content of impurity esterase, shortens the reaction time and improves the reaction batch.
Description
Technical Field
The invention belongs to the technical field of medical biology, and relates to a method for immobilizing cephalosporin C acylase.
Background
Cephalosporin C Acylase (CCA) is a key enzyme for producing 7-aminocephalosporanic acid (7-ACA) by a one-step enzyme method, and can directly catalyze raw material cephalosporin C (CPC) to remove a 7-position D-alpha aminoadipoyl side chain to generate the 7-ACA.
The action molecular formula of Cephalosporin C Acylase (CCA) is shown as follows:
during the production process of Cephalosporin C Acylase (CCA), the requirements of macroporous resin adsorption and ion exchange on filtrate are high, and the filtrate contains a large amount of inorganic ions to influence the resin adsorption and exchange; a large amount of soluble protein exists in the filtrate, so that the resin is polluted, the adsorption effect is poor, and the service life of the resin is shortened; the immobilized CPC acylase produced by the existing method for extracting the CPC acylase has higher D value, fast attenuation of enzyme activity and less reaction batches; the method for extracting CPC acylase reported in the literature can meet the requirements of 7-ACA production that the reaction time is longer when the CPC residue is less than or equal to 200, the average residue of the D value is more than or equal to 0.4.
Disclosure of Invention
In order to solve the above problems, the present invention provides a method for preparing an immobilized cephalosporin C acylase, which comprises the following steps:
(1) and (3) crushing thalli: centrifuging a cephalosporin C acylase fermentation liquid, collecting fermentation thalli, adding phosphate buffer solution, uniformly mixing, homogenizing, adding a flocculating agent into the homogenized liquid, stirring, centrifuging, and collecting a light phase to obtain a crude liquid enzyme;
(2) clarification and concentration: adding a calcium chloride solution into the crude liquid enzyme obtained in the step (1), centrifuging, collecting supernate and concentrating to obtain concentrated solution;
(3) salting out: adding ammonium sulfate into the concentrated solution obtained in the step (2), dissolving, uniformly mixing, cooling to 15-20 ℃, stirring, centrifuging to collect heavy-phase protein, adding phosphate buffer solution for dissolving, centrifuging, and collecting light phase to obtain liquid enzyme;
(4) desalting: cooling the liquid enzyme in the step (3) to 8-15 ℃, and desalting to obtain desalted liquid;
(5) adsorption and desorption: loading the desalted solution obtained in the step (4) into a resin column, analyzing by using a phosphate buffer solution containing 0.03-0.05mol/l NaCl, and collecting an analysis solution;
(6) loading and fixing: adding macroporous adsorption resin into the analysis solution obtained in the step (5), and stirring and curing to obtain immobilized cephalosporin C acylase;
(7) and (3) storage: and (5) rinsing the immobilized enzyme in the step (6), drying, adding a biological enzyme bactericide, and stirring to obtain the biological enzyme bactericide.
Further, adding phosphate buffer solution to 2.5 times of the original volume in the step (1); the homogenizing pressure is 80-120 Mpa; adjusting the pH value of the homogenized solution to 6.8-7.2 by using 12% potassium hydroxide; the volume ratio of the homogeneous liquid to the flocculating agent is 1000: 23; the stirring time is 1 h.
Further, the flocculant is polyquaternium-37.
Further, the volume ratio of the crude liquid enzyme to the calcium chloride solution in the step (2) is 1000: 18; the concentration of the calcium chloride solution is 25%; the concentration is performed by using an ultrafiltration membrane with the molecular weight cutoff of 10000.
Further, adding ammonium sulfate to a concentration of 1.5-2.2mol/L in the step (3); the stirring speed is 20-30rpm, and the time is 2 h; the mass-volume ratio of the heavy-phase protein to the phosphate buffer salt is 1: 7.5.
Further, the desalting in the step (4) is desalting by using a desalting membrane group capable of removing small-molecule anions.
Further, the dosage of the phosphate buffer solution in the step (5) is 1.5 BV.
Further, the adding amount of the macroporous adsorption resin in the step (6) is that 1500u enzyme activity is loaded on each 1g of macroporous adsorption resin; the curing temperature is 25-28 ℃ and the curing time is 24 h.
Further, the rinsing in the step (7) is rinsing with 10 times of volume of purified water; the drying is centrifugal drying; the volume-mass ratio of the biological enzyme bactericide to the dried immobilized enzyme is 5: 1; the biological enzyme bactericide is prepared by dissolving the biological enzyme bactericide in phosphate buffer solution according to the proportion of 12g to 1000ml
Further, the phosphate buffer is 0.1M potassium phosphate buffer pH 7.50.
According to the preparation method of the immobilized cephalosporin C acylase, the average value of the produced immobilized enzyme activity is 153u/ml, the average reaction time is 40min when the cracking verification CPC residual titer is less than or equal to 200, and the average value D is 0.36, so that the content of impurity esterase is reduced, the reaction time is shortened, the cracking reaction batch of the immobilized cephalosporin C acylase is improved, the equipment cost and the production cost required by extraction are reduced, and the preparation method has popularization and application values.
Obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical spirit of the invention, as defined by the following claims.
The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.
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FIG. 1 is a process flow diagram
Detailed Description
The raw materials and equipment used in the embodiment of the present invention are known products and obtained by purchasing commercially available products.
EXAMPLE 1 preparation of immobilized cephalosporin C acylase of the invention
(1) Centrifuging cephalosporin C acylase fermentation liquor, collecting fermentation thallus, adding 0.1M potassium phosphate buffer solution with pH of 7.50 into the collected fermentation thallus to dilute to 2.5 times of the original volume, mixing uniformly to obtain cell sap, pumping the uniformly mixed cell sap into a high-pressure homogenizer, homogenizing under 80-120Mpa to obtain homogenized liquor, adjusting pH of the homogenized liquor to 6.8-7.2 with 12% potassium hydroxide, and mixing at 23L/M3Adding polyquaternium-37 for flocculation, stirring for 1h, centrifuging, and collecting supernatant to obtain crude liquid enzyme.
(2) Adding the crude liquid enzyme obtained in the step (1) into a 25% calcium chloride solution of 18L/m3And centrifuging to collect supernatant.
(3) And (3) concentrating the supernatant obtained in the step (2) in an ultrafiltration membrane with the molecular weight cutoff of 10000, and collecting to obtain a concentrated solution.
(4) Taking the concentrated solution in the step (3), adding ammonium sulfate until the concentration of the ammonium sulfate in the concentrated solution is 1.5-2.2mol/L, dissolving and uniformly mixing, cooling to 15-20 ℃, stirring at the rotating speed of 20-30rpm, salting out for 2h, centrifuging to collect heavy-phase protein, adding 7.5 times (v/w, ml/g) of 0.1M potassium phosphate buffer solution with the pH value of 7.50, dissolving the protein, centrifuging, and collecting light phase to obtain purified liquid enzyme;
(5) cooling the liquid enzyme in the step (4) to 8-15 ℃, and desalting by using a desalting membrane group capable of removing micromolecular anions to obtain desalted liquid;
(6) slowly adding the desalting solution obtained in the step (5) into a resin column for adsorption, after the desalting solution is completely adsorbed, analyzing by using 1.5BV of 0.03-0.05mol/L NaCl-containing potassium phosphate buffer (the potassium phosphate buffer is 0.1M potassium phosphate buffer with the pH value of 7.50), and collecting an analysis solution;
(7) putting the analysis solution obtained in the step (6) into a macroporous adsorption resin carrier according to 1500U/g, slowly stirring, controlling the temperature to be 25-28 ℃, and immobilizing for 24 hours to obtain immobilized cephalosporin C acylase;
(8) and (3) storage: and (3) rinsing the immobilized cephalosporin C acylase obtained in the step (7) by using 10 times volume (v/w) of purified water, then feeding the washed enzyme into a centrifugal machine for centrifugation for 40min to obtain an immobilized cephalosporin C finished product, adding 5 times volume (v/w) of 12 per thousand biological enzyme bactericide (the biological enzyme bactericide is dissolved in phosphate buffer solution according to the proportion of 12g to 1000 ml) into the finished product, and stirring the mixture for 30min to obtain the immobilized cephalosporin C acylase.
A total of 15 batches of immobilized cephalosporin C acylase were produced according to the above procedure (scheme FIG. 1) and the production assay is reported in Table 1.
TABLE 1 production assay record for immobilized cephalosporin C acylase
As can be seen from the above table: by using the preparation method of the immobilized cephalosporin C acylase, the average value of the produced immobilized enzyme activity is 153u/ml, the average reaction time is 40min when the CPC residual titer is verified to be less than or equal to 200 by cracking, and the average value D is 0.36.
The invention can obviously improve the quality of the CPC acylase and reduce the content of the impurity D by comprehensively considering the reaction time, the reaction batch and the D value of the finished product of the immobilized CPC acylase.
In conclusion, according to the preparation method of the immobilized cephalosporin C acylase, the average value of the enzyme activity of the produced immobilized enzyme is 153u/ml, the average reaction time is 40min when the CPC residual titer is less than or equal to 200 through cracking verification, the average value of D is 0.36, the content of impurity esterase is reduced, the reaction time is shortened, the cracking reaction batch of the immobilized cephalosporin C acylase is improved, the equipment cost and the production cost required by extraction are reduced, and the preparation method has popularization and application values.
Claims (10)
1. A method for preparing immobilized cephalosporin C acylase, which is characterized in that: it comprises the following steps:
(1) and (3) crushing thalli: centrifuging a cephalosporin C acylase fermentation liquid, collecting fermentation thalli, adding phosphate buffer solution, uniformly mixing, homogenizing, adding a flocculating agent into the homogenized liquid, stirring, centrifuging, and collecting a light phase to obtain a crude liquid enzyme;
(2) clarification and concentration: adding a calcium chloride solution into the crude liquid enzyme obtained in the step (1), centrifuging, collecting supernate and concentrating to obtain concentrated solution;
(3) salting out: adding ammonium sulfate into the concentrated solution obtained in the step (2), dissolving, uniformly mixing, cooling to 15-20 ℃, stirring, centrifuging to collect heavy-phase protein, adding phosphate buffer solution for dissolving, centrifuging, and collecting light phase to obtain liquid enzyme;
(4) desalting: cooling the liquid enzyme in the step (3) to 8-15 ℃, and desalting to obtain desalted liquid;
(5) adsorption and desorption: loading the desalted solution obtained in the step (4) into a resin column, analyzing by using a phosphate buffer solution containing 0.03-0.05mol/l NaCl, and collecting an analysis solution;
(6) loading and fixing: adding macroporous adsorption resin into the analysis solution obtained in the step (5), and stirring and curing to obtain immobilized cephalosporin C acylase;
(7) and (3) storage: and (5) rinsing the immobilized enzyme in the step (6), drying, adding a biological enzyme bactericide, and stirring to obtain the biological enzyme bactericide.
2. The method of claim 1, further comprising: adding phosphate buffer solution to 2.5 times of the original volume in the step (1); the homogenizing pressure is 80-120 Mpa; adjusting the pH value of the homogenized solution to 6.8-7.2 by using 12% potassium hydroxide; the volume ratio of the homogeneous liquid to the flocculating agent is 1000: 23; the stirring time is 1 h.
3. The method of claim 2, further comprising: the flocculant is polyquaternium-37.
4. The method of claim 1, further comprising: the volume ratio of the crude liquid enzyme to the calcium chloride solution in the step (2) is 1000: 18; the concentration of the calcium chloride solution is 25%; the concentration is performed by using an ultrafiltration membrane with the molecular weight cutoff of 10000.
5. The method of claim 1, further comprising: adding ammonium sulfate to the solution in the step (3) until the concentration of the ammonium sulfate is 1.5-2.2 mol/L; the stirring speed is 20-30rpm, and the time is 2 h; the mass-volume ratio of the heavy-phase protein to the phosphate buffer salt is 1: 7.5.
6. the method of claim 1, further comprising: and (4) desalting by using a desalting membrane group capable of removing small-molecule anions.
7. The method of claim 1, further comprising: the dosage of the phosphate buffer solution in the step (5) is 1.5 BV.
8. The method of claim 1, further comprising: the adding amount of the macroporous adsorption resin in the step (6) is that 1500u enzyme activity is loaded on each 1g of macroporous adsorption resin; the curing temperature is 25-28 ℃ and the curing time is 24 h.
9. The method of claim 1, further comprising: the rinsing of the step (7) is rinsing with 10 times volume of purified water; the drying is centrifugal drying; the volume-mass ratio of the biological enzyme bactericide to the dried immobilized enzyme is 5: 1; the biological enzyme bactericide is prepared by dissolving the biological enzyme bactericide in phosphate buffer solution according to the proportion of 12g to 1000 ml.
10. The method of claim 1, further comprising: the phosphate buffer was 0.1M potassium phosphate buffer, pH 7.50.
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| Application Number | Priority Date | Filing Date | Title |
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| CN202010615993.9A CN111876402A (en) | 2020-06-30 | 2020-06-30 | Preparation method of immobilized cephalosporin C acylase |
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| CN202010615993.9A CN111876402A (en) | 2020-06-30 | 2020-06-30 | Preparation method of immobilized cephalosporin C acylase |
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1999001566A1 (en) * | 1997-07-03 | 1999-01-14 | Dsm N.V. | PREPARATION OF ENZYME WITH REDUCED β-LACTAMASE ACTIVITY |
| CN1428423A (en) * | 2002-12-30 | 2003-07-09 | 湖南福来格生物技术有限公司 | Extraction and purification method of glutary-7-aminocefaphytanic acid acyltransferase |
| CN101173264A (en) * | 2007-10-31 | 2008-05-07 | 天津大学 | Beta-mannase composite preparations and method for producing the same |
| CN104593351A (en) * | 2014-12-30 | 2015-05-06 | 安徽丰原发酵技术工程研究有限公司 | Method for preparing cephalosporin C acylase immobilized enzyme |
| CN105087537A (en) * | 2015-09-16 | 2015-11-25 | 山东鲁抗立科药业有限公司 | Preparation and application method for 7-ACA immobilized enzyme LK118 |
| CN107177583A (en) * | 2017-07-27 | 2017-09-19 | 河北华石生物科技有限公司 | A kind of immobilization is acylated enzyme stabilizers and the method for improving immobilization acylase storage stability |
-
2020
- 2020-06-30 CN CN202010615993.9A patent/CN111876402A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1999001566A1 (en) * | 1997-07-03 | 1999-01-14 | Dsm N.V. | PREPARATION OF ENZYME WITH REDUCED β-LACTAMASE ACTIVITY |
| CN1428423A (en) * | 2002-12-30 | 2003-07-09 | 湖南福来格生物技术有限公司 | Extraction and purification method of glutary-7-aminocefaphytanic acid acyltransferase |
| CN101173264A (en) * | 2007-10-31 | 2008-05-07 | 天津大学 | Beta-mannase composite preparations and method for producing the same |
| CN104593351A (en) * | 2014-12-30 | 2015-05-06 | 安徽丰原发酵技术工程研究有限公司 | Method for preparing cephalosporin C acylase immobilized enzyme |
| CN105087537A (en) * | 2015-09-16 | 2015-11-25 | 山东鲁抗立科药业有限公司 | Preparation and application method for 7-ACA immobilized enzyme LK118 |
| CN107177583A (en) * | 2017-07-27 | 2017-09-19 | 河北华石生物科技有限公司 | A kind of immobilization is acylated enzyme stabilizers and the method for improving immobilization acylase storage stability |
Non-Patent Citations (4)
| Title |
|---|
| INMACULADA等: "Characterization of cross-linked immobilized arylesterase from Gluconobacter oxydans 621H with activity toward cephalosporin C and 7-aminocephalosporanic acid", 《BIOCATALYSTS AND BIOREACTOR DESIGN》 * |
| KLEI等: "Purification and preliminary crystallographic studies of penicillin G acylase from Providencia rettgeri", 《PROTEIN SCIENCE》 * |
| 徐文玲: "头孢菌素C酰化酶提取及固定化工艺研究", 《中国优秀硕士学位论文全文数据库》 * |
| 高孔荣等: "《食品分离技术》", 30 November 1998 * |
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Application publication date: 20201103 |