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Preparation method of L-2-aminobutyric acid

An aminobutyric acid and amino acid technology, applied in the biological field, can solve the problems of increased separation and purification burden, poor stability of enzyme activity, low coenzyme regeneration efficiency, etc., and achieves mild enzyme catalytic reaction characteristics, simple steps, and continuous controllability. Effect

Pending Publication Date: 2021-03-19
LIYANG WEIXIN CHEM
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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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Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0027] The invention provides a kind of preparation method of L-2-aminobutyric acid, comprises the following steps:

[0028] (1) Preparation of co-immobilized multi-enzyme system of threonine deaminase, alcohol dehydrogenase, threonine aldehyde dehydrogenase and L-amino acid dehydrogenase:

[0029] Dissolve Eudragit S-100 in ethanol solution, the concentration of Eudragit S-100 is 20g / L, and the volume ratio of 1:1~5 is mixed with threonine deaminase containing 20~50IU / mL, 40~80IU / mL Alcohol deaminase, 40-90IU / mL threonine aldehyde dehydrogenase and 30-70IU / mL L-amino acid dehydrogenase enzyme solution are fully mixed, at 250C, pH6.0-8.0, Incubate for 3 hours; after incubation, adjust the pH of the solution to <6.0 with phosphoric acid solution, let it stand for 2 hours to obtain a suspension, centrifuge at 9000r / min for 30 minutes, and filter to obtain solid microspheres: according to the mass volume ratio of 1g:6mL, add The microspheres were washed with ethanol solution, an...

Embodiment 1

[0042] The present embodiment provides a kind of preparation method of L-2-aminobutyric acid, comprises the following steps:

[0043] (1) Preparation of co-immobilized multi-enzyme system of threonine deaminase, alcohol dehydrogenase, threonine aldehyde dehydrogenase and L-amino acid dehydrogenase:

[0044] Dissolve Eudragit S-100 in ethanol solution, the concentration of Eudragit S-100 is 20g / L, and mix with 50IU / mL threonine deaminase, 70IU / mL alcohol deaminase, Mix 90IU / mL threonine aldehyde dehydrogenase and 70IU / mL L-amino acid dehydrogenase enzyme solution thoroughly, incubate for 3 hours at 250C, pH8.0; after incubation, rinse with phosphoric acid solution Adjust the pH of the solution to <6.0, let it stand for 2 hours to obtain a suspension, centrifuge at 9000r / min for 30 minutes, and filter to obtain solid microspheres: according to the mass volume ratio of 1g:6mL, add ethanol solution to wash the microspheres, and the suspension is then 9000r / min Centrifuge for 30 m...

Embodiment 2

[0053] The invention provides a kind of preparation method of L-2-aminobutyric acid, comprises the following steps:

[0054] (1) Preparation of co-immobilized multi-enzyme system of threonine deaminase, alcohol dehydrogenase, threonine aldehyde dehydrogenase and L-amino acid dehydrogenase:

[0055] Dissolve Eudragit S-100 with ethanol solution, the concentration of Eudragit S-100 is 20g / L, and mix with threonine deaminase containing 40IU / mL, alcohol deaminase 60IU / mL, Mix 60IU / mL threonine aldehyde dehydrogenase and 60IU / mL L-amino acid dehydrogenase enzyme solution thoroughly, incubate for 3 hours at 250C, pH7.0; after incubation, rinse with phosphoric acid solution Adjust the pH of the solution to <6.0, let it stand for 2 hours to obtain a suspension, centrifuge at 9000r / min for 30 minutes, and filter to obtain solid microspheres: according to the mass volume ratio of 1g:6mL, add ethanol solution to wash the microspheres, and the suspension is then 9000r / min Centrifuge for ...

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Abstract

The invention provides a preparation method of L-2-aminobutyric acid. The method comprises the following steps of immobilizing threonine deaminase, alcohol dehydrogenase, threonine aldehyde dehydrogenase and L-amino acid dehydrogenase on a reversible soluble pH-sensitive polymer carrier to form a co-immobilized multienzyme system; immobilizing NAD or NADP and pyridoxal phosphate cofactors on the reversible soluble pH-sensitive polymer carrier to form a co-immobilized coenzyme system; and producing the L-2-aminobutyric acid by utilizing the double immobilized multienzyme systems. The effectiveseparation of immobilized enzymes and a product is realized by utilizing the reversible dissolution characteristics of co-immobilized enzymes, and in combination with a purification and separation method comprising the steps of performing concentration and crystallization, performing one or more of ion exchange resin separation, membrane separation and chromatographic separation, performing separation liquid re-concentration and crystallization and finally performing mixing and re-crystallization, the high-purity L-2-aminobutyric acid is obtained with high yield, the product purity reaches 99.68% or above, the enzyme recycling rate is high and reaches 99.8% or above, and the reactant conversion rate reaches 99.5% or above.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a preparation method of L-2-aminobutyric acid. Background technique [0002] L-2-aminobutyric acid is a non-natural chiral amino acid, which can inhibit the transmission of human nerve information, enhance the activity of glucose phosphatase and promote the metabolism of brain cells. It is an important chemical raw material and pharmaceutical intermediate. It has been widely used in drug synthesis, such as antibacterial and anti-tuberculosis drug ethambutol hydrochloride, antiepileptic drugs levetiracetam and brivaracetam, etc. [0003] At present, the production methods of L-2-aminobutyric acid at home and abroad are divided into chemical method and biological enzymatic method. The chemical method is a relatively classic method, mainly including chemical synthesis and chemical resolution. The chemical synthesis is mainly based on n-butyric acid and bromine as the starting raw mater...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P13/00C12N11/087C12N9/02C12N9/04C12N9/06C12N9/88C07C227/40C07C227/42C07C229/08
CPCC12P13/005C12N11/087C12N9/88C12N9/0006C12N9/0016C12N9/0008C07C227/40C07C227/42C12Y403/01019C12Y101/01C12Y104/01005C12Y102/01C07B2200/07C07C229/08
Inventor 楼志华张培东
Owner LIYANG WEIXIN CHEM