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Method for preparing L-2-aminobutyric acid by enzyme method

A technology for aminobutyric acid and enzymatic preparation, applied in the direction of fermentation, etc., can solve the problems of low process yield, high cost, unsuitable process conditions, etc., and achieve the effects of low process cost, mild reaction conditions, and easy directional control.

Inactive Publication Date: 2010-09-01
SYNCOZYMES SHANGHAI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The enzyme-catalyzed preparation method has high efficiency and strong specificity, and is an ideal method for preparing pharmaceutical intermediates. However, there are not many examples of enzyme-catalyzed processes that can be truly industrialized. The main obstacle is the low yield and cost of the current process. high, and the process conditions are not suitable for industrial scale-up, so establishing an enzyme-catalyzed preparation process that is efficient, low-cost, and easy to industrialize large-scale production is still a field worth exploring, and it also has a good application prospect in the pharmaceutical industry

Method used

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  • Method for preparing L-2-aminobutyric acid by enzyme method

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Experimental program
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Effect test

Embodiment 1

[0014] Add 20mg L-threonine, 32mg ammonium formate (about 2.5 times equivalent), 0.2mgNAD to the 5ml reaction bottle + and 2ml phosphate buffered saline (Na 2 HPO 4 / NaH 2 PO 4 , 0.1M, pH 7.5), adjust the pH value of the solution to 7.5 with concentrated ammonia water, then add 1mg of threonine deaminase crude enzyme, 2mg of leucine dehydrogenase crude enzyme and 2mg of formate dehydrogenase crude enzyme, in After 3 hours of magnetic stirring reaction at 25°C, samples were taken for HPLC analysis, and the conversion rate>95% (C18 column; UV 200nm detection; mobile phase: 20mM phosphate buffer (pH 3.0) / acetonitrile=95 / 5, v / v ), ee value>99% (CR (+) column; UV 200nm detection; mobile phase: perchloric acid aqueous solution (pH 1.5)).

Embodiment 2

[0016] Add 50mg L-threonine, 80mg ammonium formate (about 2.5 times the equivalent), and 2.5mg NAD to a 25ml Erlenmeyer flask + and 5ml phosphate buffered saline (Na 2 HPO 4 / NaH 2 PO 4 , 0.1M, pH 8.0), adjust the pH value of the solution to 8.0 with concentrated ammonia water, then add 5mg of threonine deaminase crude enzyme, 10mg of leucine dehydrogenase crude enzyme and 20mg of formate dehydrogenase crude enzyme, in After reacting on a rotary shaker at 160 rpm at 37° C. for 20 hours, samples were taken for HPLC analysis. The conversion rate was >99%, and the ee value was >99%.

Embodiment 3

[0018] Add 150mg L-threonine, 480mg ammonium formate (about 2.5 times the equivalent), 2.5mgNAD in a 25ml Erlenmeyer flask + and 5ml phosphate buffered saline (Na 2 HPO 4 / NaH 2 PO 4 , 0.1M, pH 7.0), adjust the pH value of the solution to 7.0 with concentrated ammonia water, then add 2.5mg of threonine deaminase crude enzyme, 1mg of leucine dehydrogenase crude enzyme and 5mg of formate dehydrogenase crude enzyme, After reacting on a rotary shaker at 160 rpm at 30° C. for 24 hours, samples were taken for HPLC analysis. The conversion rate was about 65%, and the ee value was >99%.

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Abstract

The invention discloses a method for preparing L-2-aminobutyric acid by an enzyme method and mainly solves the problems that the yield is low, the cost is high and the process condition is not suitable for industrial production in the conventional enzyme catalysis preparation method. A technical scheme of the invention is that: the method for preparing the L-2-aminobutyric acid by the enzyme method is characterized by comprising the following steps of: converting L-threonine which is taken as a starting material into 2-ketobutyrate by using threonine deaminase; and then converting the 2-ketobutyrate into the L-2-aminobutyric acid by using leucine dehydrogenase, and adding hydrogenlyase used for coenzyme generation in the reaction. The method for preparing the L-2-aminobutyric acid by the enzyme catalysis preparation method has the advantages of high efficiency, low cost and easy implementation of industrial large-scale production.

Description

technical field [0001] The present invention relates to a method for preparing L-2-aminobutyric acid by enzymatic method, in particular, it relates to a method for preparing L-2-aminobutyric acid by using L-threonine as raw material and combining two enzymes . technical background [0002] 2-aminobutyric acid is a natural amino acid that inhibits the transmission of human nerve information, and is also an important chemical raw material and pharmaceutical intermediate, which has been widely used in drug synthesis, such as the synthesis of levetiracetam (WO 03014080, 2003). Levetiracetam is a new type of antiepileptic drug, which is mainly used to treat localized and secondary generalized epilepsy. Its molecular structure is shown in the figure, and the blue part is usually composed of L-2-aminobutyric acid or S-2-aminobutanamide as a precursor. [0003] [0004] The preparation methods of 2-aminobutyric acid mainly include chemical method and biological method at presen...

Claims

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Application Information

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IPC IPC(8): C12P13/04
Inventor 祝俊苏金环曾聪明陶军华文军
Owner SYNCOZYMES SHANGHAI
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