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Method of preparing N-acetylneuraminic acid with immobilized double enzymes

A technology for acetylneuraminic acid and neuraminic acid aldolase, which is applied in the field of preparing N-acetylneuraminic acid, can solve the problems of low conversion rate, stable enzyme without immobilized enzyme, low efficiency, etc. The effect of increasing conversion rate and stable conversion rate

Inactive Publication Date: 2019-08-16
浙江正硕生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the current enzymatic method for preparing N-acetylneuraminic acid generally uses free enzymes for conversion. The enzymes in this method are not as stable as immobilized enzymes and cannot be reused. Moreover, the reaction takes a long time and is inefficient.
In addition, enzymatic conversion using the free state is not conducive to the separation of products
The patent application number CN200510025745.4 proposes "a method for preparing N-acetyl-D-neuraminic acid", using the metal of N-acetyl-D-neuraminic acid aldolase with a histidine residue at the N-terminus The chelating affinity carrier immobilizes the enzyme for catalysis, but after the immobilized enzyme is added to the carrier, the carrier may react with the reaction system, easily causing pollution, low conversion rate, the enzyme and the carrier must be firmly combined, and the stability of the enzyme-catalyzed reaction is poor

Method used

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  • Method of preparing N-acetylneuraminic acid with immobilized double enzymes

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Effect test

Embodiment 1

[0019] A method for preparing N-acetylneuraminic acid by immobilizing double enzymes, the immobilization process of immobilized N-acetylglucosamine-2-epimerase and immobilized N-acetylneuraminic acid aldolase is as follows: The total enzyme activity ratio of N-acetylglucosamine-2-epimerase enzyme solution numbered E.C.5.1.3.8 and N-acetylneuraminic acid aldolase enzyme solution numbered E.C.4.1.3.3 is 1:2 Mix, after cooling in an ice-water bath, add ammonium sulfate with a mass fraction of 75% while stirring to form a precipitate, collect the precipitate after centrifugation, then add a glutaraldehyde solution with a mass fraction of 25% while stirring, and react at 15°C for 3 hours Cross-linking and immobilization to form immobilized N-acetylglucosamine-2-epimerase and immobilized N-acetylneuraminic acid aldolase.

[0020] The process of preparing N-acetylneuraminic acid includes the following steps: at a pH of 7 and a temperature of 30°C, N-acetylglucosamine and sodium pyruv...

Embodiment 2

[0022] A method for preparing N-acetylneuraminic acid by immobilizing double enzymes, the immobilization process of immobilized N-acetylglucosamine-2-epimerase and immobilized N-acetylneuraminic acid aldolase is as follows: N-acetylglucosamine-2-epimerase enzyme solution and N-acetylneuraminic acid aldolase enzyme solution are mixed according to the total enzyme activity ratio of 1:3. After cooling in an ice-water bath, add mass fraction while stirring. 50% ammonium sulfate forms a precipitate, centrifuges to collect the precipitate, then adds a glutaraldehyde solution with a mass fraction of 25%, and reacts at 18°C ​​for 3 hours for cross-linking and fixation. When cross-linking and fixing, the glutaraldehyde solution in the enzyme solution The mass fraction was maintained at 0.2%, and immobilized N-acetylglucosamine-2-epimerase and immobilized N-acetylneuraminic acid aldolase were formed.

[0023] The process of preparing N-acetylneuraminic acid includes the following steps:...

Embodiment 3

[0025] A method for preparing N-acetylneuraminic acid by immobilizing double enzymes, the immobilization process of immobilized N-acetylglucosamine-2-epimerase and immobilized N-acetylneuraminic acid aldolase is as follows: N-acetylglucosamine-2-epimerase enzyme solution and N-acetylneuraminic acid aldolase enzyme solution are mixed according to the total enzyme activity ratio of 1:4, after cooling in an ice-water bath, add mass fraction while stirring 60% ammonium sulfate forms a precipitate, centrifuges to collect the precipitate, then adds its own glutaraldehyde solution with a mass fraction of 25%, and reacts at 18°C ​​for 3 hours for cross-linking and fixation. When cross-linking and fixing, the glutaraldehyde solution in the enzyme solution The mass fraction was maintained at 0.5%, forming immobilized N-acetylglucosamine-2-epimerase and immobilized N-acetylneuraminic acid aldolase.

[0026] The process of preparing N-acetylneuraminic acid includes the following steps: at...

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Abstract

The invention relates to a method of preparing N-acetylneuraminic acid with immobilized double enzymes. The method herein comprises the steps of under PH of 7-9 and the temperature of 22-32 DEG C, mixing N-acetylglucosamine and pyruvic acid or pyruvate having a concentration of 0.2-0.6 mol / L relative to the total reaction system, with immobilized N-acetylglucosamine-2-epimerase and immobilized N-acetylneuraminic acid aldolase to obtain the N-acetylneuraminic acid; conversion is quick and stable; the immobilized enzymes are not immobilized via a vector, so that preparing is convenient.

Description

technical field [0001] The invention relates to a method for preparing N-acetylneuraminic acid by enzymatic reaction, a method for preparing N-acetylneuraminic acid by using immobilized double enzymes. Background technique [0002] Sialic acid is an acidic amino sugar with 9 carbon atoms and a pyranose structure. N-acetylneuraminic acid is the main representative of sialic acid. This kind of compound can bind to the terminal positions of glycoproteins and glycolipids, and play a very important role in the process of biological recognition. For example, during the process of influenza virus infecting host cells, the sialidase of influenza virus can recognize N-acetylneuraminic acid and its derivatives, and bind to sialic acid to infect cells and release the replicated virus. If its sialidase is inhibited, then the disease caused by the virus can be treated. According to the structure of sialic acid, design its analogue, use this analogue to bind the sialidase of the virus,...

Claims

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Application Information

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IPC IPC(8): C12P19/26C12N11/18
CPCC12N9/88C12N9/90C12N11/18C12P19/26C12Y401/03003C12Y501/03008
Inventor 吴黎诚郭小雷章权刘建峰章云燕
Owner 浙江正硕生物科技有限公司
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