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A method for preparing cephalosporin c acylase immobilized enzyme

A technology for immobilizing enzymes and cephalosporins, applied in the downstream engineering pharmaceutical field of biotechnology, can solve problems such as low activity of cephalosporin C acylase, and achieve the effects of improving binding rate, improving enzyme activity and efficient operation

Active Publication Date: 2018-03-13
ANHUI BBCA FERMENTATION TECH ENG RES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the activity of wild-type cephalosporin C acylase is low, and it is urgent to provide a method for preparing immobilized enzyme of cephalosporin C acylase to improve the activity and stability of the enzyme, so as to meet the needs of industrial production

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] (1) 500L fermenter produces 400L E. coli fermentation liquid, solid content 12% (m / m), OD 600 The value is 110, and the enzyme activity is 10300U / L; the fermentation broth is concentrated to 80L with a 50nm ceramic membrane, the average flux is 69LMH, the operating temperature is <25°C, and 160L of phosphate buffer solution with a concentration of 0.01M and pH7.0 is added for dialysis. Obtain 80L of dense-phase bacterial liquid with a solid content of 34%; lower the temperature to below 24°C, and use a high-pressure homogenizer to crush 3 times under a pressure of 850Mpa to obtain a cell crushing liquid;

[0048] (2) Add 0.4g of aluminum sulfate, 0.4g of ferrous sulfate and 1.6g of neutral polyacrylamide to the cell disruption solution obtained in step (1), and let it stand for flocculation at room temperature for 50min; add 3.2kg of diatomite, of which clay The volume ratio to the laterite is 5:1, centrifuged after slight stirring, and the supernatant is collected;

...

Embodiment 2

[0055] (1) 500L fermenter produces 440L E. coli fermentation liquid, solid content 11.2% (m / m), OD 600 The value is 120, the enzyme activity is 9500U / L; the fermentation broth is concentrated to 90L with a 50nm ceramic membrane, the average flux is 76.6LMH, the operating temperature of the membrane is <30°C, and 180L of phosphate buffer solution with a concentration of 0.01M and pH7.0 is added for Dialyze to obtain 90L of dense-phase bacterial liquid with a solid content of 31.2%; cool down to 20°C, and use a high-pressure homogenizer to crush 4 times under a pressure of 880Mpa to obtain a cell crushing liquid;

[0056] (2) Add 0.45g of aluminum sulfate, 0.45g of ferrous sulfate and 1.8g of neutral polyacrylamide to the cell disruption solution obtained in step (1), and let it stand for flocculation at room temperature for 60 minutes; add 3.6kg of diatomite, of which clay The volume ratio to the laterite is 5:1, centrifuged after slight stirring, and the supernatant is collect...

Embodiment 3

[0062] (1) 10000L fermenter produces 7000L Escherichia coli fermentation liquid, solid content 11% (m / m), OD 600 The value is 105, the enzyme activity is 9000U / L; the fermentation broth is concentrated to 1400L with a 50nm ceramic membrane, the average flux is 95LMH, the operating temperature of the membrane is <35°C, and 180L of phosphate buffer solution with a concentration of 0.01M and pH7.0 is added for dialysis , to obtain 1400L of dense-phase bacterial liquid with a solid content of 29.4%; cool down to 25°C, and use a high-pressure homogenizer to crush 2 times under a pressure of 900Mpa to obtain a cell crushing liquid;

[0063] (2) Add 7kg of aluminum sulfate, 7kg of ferrous sulfate and 40g of neutral polyacrylamide to the cell disruption solution obtained in step (1), and let it stand for flocculation for 70min at room temperature; add 70kg of diatomite, wherein the volume of clay and laterite is The ratio is 5:1, centrifuged after slight stirring, and the supernatant ...

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Abstract

The invention provides a method for preparing a cephalosporin C acylase immobilized enzyme. The method comprises the following steps: (1) dialyzing with phosphate buffer liquor after concentrating escherichia coli fermentation liquor through a ceramic membrane, and performing high-pressure crushing to obtain cell crushing liquor; (2) adding a mixed flocculating agent consisting of aluminum sulfate, ferrous sulfate and neutral polyacrylamide into the cell crushing liquor, and flocculating at a room temperature; adding diatomite and centrifuging to collect liquid supernatant; (3) heating after adding composite sulfate into the liquid supernatant; after cooling to the room temperature, adding the diatomite and centrifuging to collect liquid supernatant; (4) concentrating through a hollow fiber membrane made of a polyacrylonitrile material and dialyzing with phosphate buffer liquor to obtain concentrated enzyme liquor; and (5) adding an immobilization carrier into the concentrated enzyme liquor, and performing suction filtration after sufficiently combining the enzyme with the carrier to obtain the cephalosporin C acylase immobilized enzyme. The invention further discloses a cephalosporin C acylase immobilized enzyme prepared by the method and an application thereof.

Description

technical field [0001] The invention relates to the field of downstream engineering pharmaceuticals of biotechnology, in particular to a preparation method of cephalosporin C acylase immobilized enzyme. Background technique [0002] In recent years, cephalosporin antibiotics have little allergic reaction, are relatively stable to β-lactamase, have broad antibacterial spectrum, good curative effect, low toxicity, no cross-resistance with other antibiotics, and less cross-allergy with penicillin Response and other advantages, has become the most important effective drug against infection. Studies have found that the antibacterial part of cephalosporins is its mother nucleus, 7-aminocephalosporanic acid (7-ACA). Therefore, as an important intermediate for the synthesis of semi-synthetic cephalosporins, the research and production of 7-ACA is of great interest. very important. Cephalosporin C (CPC) is cleaved chemically or enzymatically to obtain 7-ACA. The reaction condition...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N11/14C12N11/08C12N9/80A61K38/50A61P31/04
CPCY02A50/30
Inventor 尚海涛徐斌杨为华邓远德张毅潘声龙
Owner ANHUI BBCA FERMENTATION TECH ENG RES
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