A method for producing l-2-aminobutyric acid by a double-immobilized multi-enzyme system

A multi-enzyme system, aminobutyric acid technology, applied in the biological field, can solve the problems of increased separation and purification burden, low coenzyme regeneration efficiency, poor enzyme activity stability, etc., to reduce separation and purification steps, improve accessibility, and improve regeneration efficiency. Effect

Inactive Publication Date: 2016-04-06
李鑫
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This type of method has the disadvantages of using three free enzymes at one time, which cannot be recycled and reused, poor stability of enzyme activity, easy inactivation, low efficiency of coenzyme regeneration, mixing of free enzymes and products, increasing the burden of separation and purification, etc.

Method used

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  • A method for producing l-2-aminobutyric acid by a double-immobilized multi-enzyme system

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1: Preparation of co-immobilized multi-enzyme system of threonine deaminase and leucine dehydrogenase.

[0029] Dissolve EudragitS-100 with 0.05mol / L, pH8.0 phosphate buffer, the concentration of EudragitS-100 is 20g / L, and the volume ratio is 1:1, and the threonine deaminase containing 20IU / mL and Enzyme solution of 50 IU / mL leucine dehydrogenase was thoroughly mixed, and incubated at 25° C. and pH 8.0 for 0.2 hours. After incubation, adjust the pH of the solution to be lower than 6.0 with 0.1 mol / L phosphoric acid solution, let it stand for 2 hours to obtain a suspension, centrifuge at 8000 r / min for 30 minutes, and filter to obtain solid microspheres. According to the mass volume ratio of 1g:5mL, 0.05mol / L, pH6.0 phosphate buffer was added to wash the microspheres, and the suspension was centrifuged at 8000r / min for 30 minutes. The washing process was repeated three times to obtain a co-immobilized multi-enzyme system. Store at 4°C for later use.

Embodiment 2

[0030] Example 2: Preparation of co-immobilized multi-enzyme system of threonine deaminase and leucine dehydrogenase

[0031] Dissolve EudragitS-100 with 0.05mol / L, pH8.0 phosphate buffer, the concentration of EudragitS-100 is 20g / L, and the volume ratio is 1:5, and the threonine deaminase containing 80IU / mL and 90 IU / mL leucine dehydrogenase enzyme solution was thoroughly mixed, and incubated at 25° C. and pH 8.0 for 2 hours. After incubation, adjust the pH of the solution to be lower than 6.0 with 0.1 mol / L phosphoric acid solution, let it stand for 2 hours to obtain a suspension, centrifuge at 8000 r / min for 30 minutes, and filter to obtain solid microspheres. According to the mass volume ratio of 1g:5mL, 0.05mol / L, pH6.0 phosphate buffer was added to wash the microspheres, and the suspension was centrifuged at 8000r / min for 30 minutes. The washing process was repeated three times to obtain a co-immobilized multi-enzyme system. Store at 4°C for later use.

Embodiment 3

[0032] Example 3: Preparation of co-immobilized multi-enzyme system of threonine deaminase and leucine dehydrogenase

[0033] Dissolve EudragitS-100 with 0.05mol / L, pH8.0 phosphate buffer, the concentration of EudragitS-100 is 20g / L, and the volume ratio is 1:10, and the threonine deaminase containing 200IU / mL and The enzyme solution of 150 IU / mL leucine dehydrogenase was thoroughly mixed, and incubated at 25° C. and pH 8.0 for 6 hours. After incubation, adjust the pH of the solution to be lower than 6.0 with 0.1 mol / L phosphoric acid solution, let it stand for 2 hours to obtain a suspension, centrifuge at 8000 r / min for 30 minutes, and filter to obtain solid microspheres. According to the mass volume ratio of 1g:5mL, 0.05mol / L, pH6.0 phosphate buffer was added to wash the microspheres, and the suspension was centrifuged at 8000r / min for 30 minutes. The washing process was repeated three times to obtain a co-immobilized multi-enzyme system. Store at 4°C for later use.

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Abstract

The invention discloses a method for producing L-2-aminobutyric acid by double immobilized multi-enzyme systems. The method provided by the invention comprises the following steps that 1, threonine dehydrogenase and leucine dehydrogenase are fixed on reversely-dissoluble pH-sensitive polymer carriers so that a co-immobilized multi-enzyme system is obtained; and 2, alcohol oxidase, formaldehyde dehydrogenase and formate dehydrogenlyase are fixed on reversely-dissoluble pH-sensitive polymer carriers so that a co-immobilized coenzyme regeneration system is obtained. The method for producing L-2-aminobutyric acid by the double immobilized multi-enzyme systems utilizes dissolution reversibility of co-immobilized enzymes, realizes effective separation of the co-immobilized enzymes and products, improves accessibility between the co-immobilized enzymes and reactants, improves recovery and utilization rates of threonine dehydrogenase, leucine dehydrogenase, alcohol oxidase, formaldehyde dehydrogenase and formate dehydrogenlyase, improves coenzyme regeneration efficiency, reduces follow-up separation purification processes, simplifies a process flow and reduces a production cost.

Description

technical field [0001] The invention belongs to the field of biotechnology, and specifically relates to a biotransformation catalyst using L-threonine as a raw material, co-immobilized L-threonine deaminase and leucine dehydrogenase, and co-immobilized alcohol Oxidase, formaldehyde dehydrogenase and formate dehydrogenase are coenzyme regeneration catalysts, a method for producing L-2-aminobutyric acid and recovering immobilized enzyme. Background technique [0002] L-2-aminobutyric acid is a natural amino acid for human nerve information transmission, and is also an important chemical raw material and pharmaceutical intermediate, widely used in drug synthesis, such as: antiepileptic drug levetiracetam, anti-tuberculosis drug ethambutol hydrochloride etc. At present, L-2-aminobutyric acid is mainly used in the synthesis of levetiracetam. Levetiracetam is a new type of antiepileptic drug. It was certified by FDA in 2000 and sold in Europe and the United States. It is currentl...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P13/04C12N11/18
CPCY02P20/50
Inventor 李鑫
Owner 李鑫
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