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Preparation method of immobilized cephalosporin C acylase

A cephalosporin and acylase technology, applied in the field of preparation of immobilized cephalosporin C acylase, can solve the problems of unsuitable catalysis of macromolecular substrate reaction, diffusion restriction, easy loss of immobilized enzyme, etc. In industrial production, the process is simple and the cost is low

Active Publication Date: 2013-10-09
UNIV OF SCI & TECH BEIJING
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Encapsulation immobilized enzymes are easy to leak, often have problems such as diffusion limitations, and the catalytic reaction is affected by mass transfer resistance, so it is not suitable for catalyzing the reaction of macromolecular substrates

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Immobilization of Epoxy Carrier LX-1000EP and CPC Acylase in the Presence of Sodium Acetate

[0054] (1) Mix the epoxy carrier LX-1000EP with 0.1M, pH 8.0 sodium phosphate buffer at a ratio of 1:4 (w / v), shake at room temperature, then dry the carrier with a vacuum pump and store at 4°C Save for later.

[0055] (2) Add 0, 0.123, 0.369, and 1.23 g of sodium acetate into 20 mL of sodium phosphate buffer (1.25 M, pH 8.0), shake well and adjust the final pH of the immobilization system to 8.0 with 1 M HCl after the sodium acetate is completely dissolved. , add 10mL of CPCC acylase enzyme solution, make the final concentration of sodium phosphate buffer solution in the immobilization system be 0.83M, make the final concentration of added sodium acetate be 0, 0.05, 0.15, 0.5M respectively. Add 1 g of the carrier activated in step (1) into the system, and stir at a low speed for 24 hours at 25°C.

[0056] (3) The immobilized enzyme obtained after the above immobilization was...

Embodiment 2

[0059] Immobilization of Epoxy Carrier LX-1000EP and CPC Acylase in the Presence of Sodium Bicarbonate

[0060] (1) Same as Example 1, first activate the carrier.

[0061] (2) Add 0, 0.062, 0.185, and 0.615 g of sodium bicarbonate to 10 mL of sodium phosphate buffer (1.25 M, pH 8.0), shake well and adjust the final pH of the immobilization system with 1 M HCl after the sodium bicarbonate is completely dissolved. 8.0, add 5mL of CPCC acylase enzyme solution, so that the final concentration of sodium phosphate buffer in the immobilization system is 0.83M, so that the final concentration of added sodium bicarbonate is 0, 0.05, 0.15, 0.5M respectively. Add 0.5 g of the carrier activated in step (1) into the system, and stir at a low speed for 24 hours at 25°C.

[0062] (3) Same as Example 1, wash the obtained immobilized enzyme.

[0063] The enzyme activity of the treated immobilized enzyme was measured, and the enzyme activity of the immobilized CPC acylase prepared by adding 0...

Embodiment 3

[0065] Immobilization of Epoxy Carrier LX-1000EPC4 and CPC Acylase in the Presence of Sodium Acetate

[0066] (1) Same as Example 1, first activate the carrier.

[0067] (2) Add 0 and 2.46 g of sodium acetate to 40 mL of sodium phosphate buffer (1.25 M, pH 8.0), shake well and adjust the final pH value of the immobilization system to 8.0 with 1 M HCl after the sodium acetate is completely dissolved, add 20 mL of CPCC The final concentration of the sodium phosphate buffer in the immobilization system is 0.83M, and the final concentration of the added sodium acetate is 0 and 0.5M respectively. Add 2 g of the carrier activated in step (1) into the system, and stir at a low speed for 24 hours at 25°C.

[0068] (3) Same as Example 1, wash the obtained immobilized enzyme.

[0069] The enzyme activity of the treated immobilized enzyme was measured, and the enzyme activity of the immobilized CPC acylase prepared by adding 0 and 0.5 M sodium acetate was 78 and 113 U / g respectively. ...

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Abstract

The invention discloses a preparation method of immobilized cephalosporin C acylase and belongs to the field of biotechnology. According to the preparation method disclosed by the invention, cephalosporin C acylase is immobilized on a carrier in the presence of acetate and / or bicarbonate to obtain the immobilized cephalosporin C acylase. The immobilized cephalosporin C acylase which is prepared by using the preparation method disclosed by the invention has high activity and thermal stability, and the preparation method disclosed by the invention is simple, stable in process and low in production cost.

Description

technical field [0001] The invention belongs to the field of biotechnology and relates to a preparation method of immobilized cephalosporin C acylase, in particular adding acetate and / or bicarbonate during the immobilization process. Background technique [0002] CPC acylase is an acylase for the one-step enzymatic production of 7-aminocephalosporanic acid (7-ACA), which can directly catalyze the substrate cephalosporin C (CPC) and remove D-α-aminoadipate Side chain, one-step production of 7-ACA. Compared with the traditional two-step enzymatic method, the one-step enzymatic method is more simple, convenient, and low-cost, and has been paid more and more attention. CPC acylase is similar to GL-7-ACA acylase in the two-step enzymatic process, and both belong to cephalosporin acylase, but the catalytic substrate specificity of the two is different (Li Y et al. Eur J Biochem, 1999, 262(3):713-719). Strains producing cephalosporin C acylase are usually screened from soil or s...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N11/14
Inventor 罗晖韩凯华常雁红魏艳梅何华于慧敏李强沈忠耀
Owner UNIV OF SCI & TECH BEIJING
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