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Eextraction and purification method of D-amino acid oxidase

A purification method and oxidase technology, applied in the directions of oxidoreductase, biochemical equipment and methods, enzymes, etc., can solve the problems of complex process, poor quality, serious pollution, etc., and achieve the effect of improving specific activity

Inactive Publication Date: 2006-03-08
HUNAN FLAG BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

And the key intermediate of semi-synthetic cephalosporin is 7-aminocephalosporanic acid (7-ACA), the method of traditional production in the past mainly is by chemical method, has four major steps, and process is complicated, and used reagent is expensive, and pollution is serious, And the yield is low, the quality is poor

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0012] The extraction and purification process of D-aminooxidase is as follows: first adjust the pH of the fermentation broth produced by fermentation to 7.0 with dilute hydrochloric acid, then lower the pH to 6.6 with 20% calcium chloride, and then add 2% chitosan , and finally adjust the pH to 7.6 with dilute alkali. Centrifuge with a high-speed centrifuge to obtain the centrifuged bacterial liquid. Control the activity of the bacterial liquid to 8u / ml, add the surfactant cetyltrimethylammonium bromide equivalent to 2% (g / l) of the bacterial liquid, adjust the pH to 9.0 with dilute alkali, heat up to 45°C with hot water, and stir Maintain for 20 minutes, naturally stir and soak for 3 hours. During the purification process, use 20% calcium chloride solution to lower the pH of the soaked bacterial solution to 5.8, collect the enzyme serum by plate and frame filtration, and use 10% sodium hydroxide solution to increase the pH of the enzyme serum to 7.6, and then collect the se...

Embodiment 2

[0014] The extraction and purification process of D-aminooxidase is as follows: first adjust the pH of the fermentation broth produced by fermentation with dilute hydrochloric acid to 7.2, then lower the pH to 6.8 with 20% calcium chloride, and then add 2% chitosan , and finally adjust the pH to 7.8 with dilute alkali. Use a high-speed centrifuge to separate the centrifuged bacterial liquid. Control the activity of the bacterial solution to 10u / ml, add the surfactant cetyltrimethylammonium bromide equivalent to 2% (g / l) of the bacterial solution, adjust the pH to 9.0 with dilute alkali, heat up to 45°C with hot water, and stir Maintain for 20 minutes, naturally stir and soak for 5 hours. During the purification process, use 20% calcium chloride solution to lower the pH of the soaked bacterial solution to 6.2, collect the enzyme serum by plate and frame filtration, and use 10% sodium hydroxide solution to increase the pH of the enzyme serum to 8.0, and then collect the serum b...

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PUM

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Abstract

The method for extracting and purifying D-amino acid oxidase includes the following steps: using dilute hydrochloric acid to regulate the pH value of fermented liquor produced by fermentation, then adding 20% calcium chloride, and adding chitosan, then using dilute alkali to regulate pH to 7.6-7.8, centrifuging to obtain centrifugal bacterial liquor, controlling activity of the bacteria liquor to 8-10 u / ml, adding surfactant, using dilute alkali to regulate pH, and using hot water to raise temp., stirirng and naturally stirring and soaking for 3-5 hr. Its purification process includes the following steps: using calcium chloride solution to downwards regulate pH to soaked bacterial liquor, plate-frame filtering and collecting enzyme clear liquor, using sodium hydroxide solution to upwards regulate pH of enzyme clear liquor to 7.6-8.0, and plate-frame filtering and collecting clear liquor.

Description

Technical field: [0001] The invention relates to a D-amino acid oxidase extraction and purification method for 7-aminocephalosporanic acid, a key intermediate in the synthesis of semi-synthetic cephalosporins. Background technique: [0002] Cephalosporins are antibiotics with important efficacy, while semi-synthetic cephalosporins have stronger antibacterial ability or can improve pharmacokinetic properties. And the key intermediate of semi-synthetic cephalosporin is 7-aminocephalosporanic acid (7-ACA), the method of traditional production in the past mainly is by chemical method, has four major steps, and process is complicated, and used reagent is expensive, and pollution is serious, And the yield is low and the quality is poor. The biotransformation method is a better method. Studies have shown that cephalosporin C can be oxidatively deaminated by D-amino acid oxidase, and further decarboxylated to generate GL-7-ACA, while the 7-side chain of GL-7-ACA can be oxidized by...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/02C12N9/06C07K1/14
Inventor 许岗朱敏莫章华曾红宇柳杏辉唐卫红胡爱红
Owner HUNAN FLAG BIOTECHNOLOGY CO LTD