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Method for preparing alpha-ketobutyric acid by using L-threonine as substrate

A technology of ketobutyric acid and threonine, which is applied in microorganism-based methods, biochemical equipment and methods, microorganisms, etc., achieves the effects of high conversion rate, simple product components, and favorable for subsequent separation and extraction.

Active Publication Date: 2013-11-27
上海肆芃科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

After searching, it was found that the method of producing α-ketobutyric acid using the whole cell of microorganism containing synthetic L-threonine dehydratase as a biocatalyst and L-threonine as a substrate has not been reported yet.

Method used

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  • Method for preparing alpha-ketobutyric acid by using L-threonine as substrate
  • Method for preparing alpha-ketobutyric acid by using L-threonine as substrate
  • Method for preparing alpha-ketobutyric acid by using L-threonine as substrate

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 2

[0061] (1) Preparation of suspension containing biocatalyst

[0062] Bacillus subtilis ATCC23857 was selected and aerobically cultured in LB medium in a conventional shake flask or fermenter; the bacteria were separated and collected, washed with pH 7.4 potassium phosphate buffer for 3 times, and the isolated complete microbial cells were obtained as Bio-catalyst. The biocatalyst is resuspended in potassium phosphate buffer or deionized water, so that the concentration of the biocatalyst reaches 200 g wet cells / liter, and the suspension containing the biocatalyst is obtained, and stored at 4°C for use;

[0063] (2) conversion

[0064] The biocatalyst-containing suspension prepared in step (1) is mixed with the L-threonine aqueous solution, and the concentration of L-threonine in the mixture is 10 grams per liter, and the biocatalyst concentration is 60 grams of wet cells / liter; under the conditions of 30°C and pH 8.0, the reaction was shaken at 200 rpm for 24 hours to obtai...

Embodiment 3

[0070] (1) Preparation of suspension containing biocatalyst

[0071] Select Pseudomonas stutzeri (P.stutzeri) SDM CCTCC NO: M206010, aerobic culture in LB medium with conventional shake flask or fermenter; separate and collect the bacteria, wash the bacteria with pH 7.4 potassium phosphate buffer 3 times, the isolated complete cells of microorganisms are biocatalysts. The biocatalyst is resuspended in potassium phosphate buffer or deionized water, so that the concentration of the biocatalyst reaches 200 g wet cells / liter, and the suspension containing the biocatalyst is obtained, and stored at 4°C for use;

[0072] (2) conversion

[0073] The biocatalyst-containing suspension prepared in step (1) is mixed with the L-threonine aqueous solution, and the concentration of L-threonine in the mixture is 60 grams per liter, and the biocatalyst concentration is 120 grams of wet cells / liter; under the conditions of 65°C and pH 7.0, the reaction was shaken at 200 rpm for 24 hours to ...

Embodiment 4

[0079](1) Preparation of suspension containing biocatalyst

[0080] Corynebacterium glutamicum ATCC13032 was selected and aerobically cultured in LB medium with conventional shake flasks or fermenters; the bacteria were separated and collected, washed with pH 7.4 potassium phosphate buffer for 3 times, and the obtained microbial complete cells were isolated is a biocatalyst. The biocatalyst is resuspended in potassium phosphate buffer or deionized water, so that the concentration of the biocatalyst reaches 200 g wet cells / liter, and the suspension containing the biocatalyst is obtained, and stored at 4°C for use;

[0081] (2) Conversion

[0082] The biocatalyst-containing suspension prepared in step (1) is mixed with the L-threonine aqueous solution, and the concentration of L-threonine in the mixture is 20 grams per liter, and the biocatalyst concentration is 40 grams of wet cells / liter; under the conditions of 60°C and pH 10.0, the reaction was shaken at 200 rpm for 20 ho...

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Abstract

The invention discloses a method for preparing alpha-ketobutyric acid by using L-threonine as a substrate, wherein a product, alpha-ketobutyric acid is prepared by using microbial complete cells containing synthetic L-threonine dehydratase as a biological catalyst, using the L-threonine as the substrate, and performing oscillating transformation at a rate of 50 to 250 turns / minute under the conditions that the temperature is between 20 and 65 DEG C and the pH is between 7.0 and 11.0. The method has the following characteristics that (1) a biological catalysis method is used, a reaction system is simple, reaction conditions are mild, steps are easy, and the operation is simple; and the biological catalyst can be easily removed, and the separation and purification of subsequent products are facilitated; (2) the transformation rate of the alpha-ketobutyric acid generated by the substrate, L-threonine can reach more than 99.6 percent, and the product, alpha-ketobutyric acid can be accumulated to reach a higher concentration; and (3) the substrate, L-threonine has a low price and is easy to obtain. According to the method, a basis of realizing high-efficient production of the alpha-ketobutyric acid is established.

Description

technical field [0001] The present invention relates to a kind of method of producing α-ketobutyric acid, specifically, relate to utilizing the whole cell of the microorganism that contains synthetic L-threonine dehydratase as biocatalyst, with L-threonine as substrate, Process for producing alpha-ketobutyric acid. Background technique [0002] α-Ketobutyric acid is an important intermediate that can be used in fields such as chemistry and medicine. For example, alpha-ketobutyric acid can be used to produce L-isoleucine 【1】 , 1-propanol 【2】【3】 , food spice components 【4】 and D-alpha-hydroxybutyrate 【5】 . Among them, D-α-hydroxybutyric acid can be used to prepare azinothricin family anticancer antibiotics and polymer poly α-hydroxybutyric acid [P(2HB) 【6】 . In addition, α-ketobutyric acid can be converted to L-α-aminobutyric acid, which is a chiral precursor for the synthesis of antiepileptic drugs such as levetiracetam 【7】【8】 . [0003] The production methods of α-k...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P7/40C12R1/38C12R1/15C12R1/125
Inventor 马翠卿高超张文许平
Owner 上海肆芃科技有限公司