Method for producing R-alpha-hydroxybutyrate by using 1, 2-butanediol as substrate

Abstract

The invention discloses a method for producing 1,2-butanediol by using R-alpha-hydroxybutyrate as a substrate. The method comprises steps of: culturing Gluconobacter oxydans DSM 2003 by a conventional method to prepare a biological catalyst; mixing the biological catalyst with a substrate 1,2-butylene glycol aqueous solution; adding ethylene diamine tetraacetic acid and oscillating for 1-30 h under condition of 20-50 DEG C and pH of 4.0-10.0 to obtain a transformation liquid; and preparing a solution containing R-alpha-hydroxybutyrate through the transformation liquid. The method provided by the invention has the following characteristics: (1) the method employs biological catalysis, has simple reaction system, mild reaction conditions, short steps and simple operation; and the biological catalyst can be easily removed, so as to facilitate subsequent separation and purification; (2) the method has a short reaction period, and the product R-alpha-hydroxybutyrate can accumulate to a high concentration; (3) the substrate 1,2-butanediol has low price, and is easy to acquire; and (4) product enantiomer has high excessive rate reaching higher than 99%, so as to lay foundation for the efficient production of R-alpha-hydroxybutyrate.

Description

technical field

[0001] The present invention relates to a method for producing R-alpha-hydroxybutyric acid, in particular to a method for producing R-alpha-hydroxybutyric acid by using the intact cells of Gluconobacter oxidans DSM 2003 as a biocatalyst to catalyze 1,2-butanediol sour method. Background technique

[0002] α-Hydroxybutyric acid is an important industrial intermediate, which can be used to synthesize isoleucine and some drugs. α-Hydroxybutyric acid is divided into two configurations, R and S. High-purity chiral R-α-hydroxybutyric acid can be used to synthesize biodegradable polymer polyα-hydroxybutyric acid [P(2HB)] 【1】 . In addition, R-α-hydroxybutyric acid can be used to prepare azinothricin family anticancer antibiotics 【2，3】 .

[0003] After literature search, there are usually two starting substrates for the production of high-purity R-α-hydroxybutyric acid. One is α-ketobutyrate, which is produced by stereoselective enzymatic reduction to R-α-hydrox...

Examples

Embodiment 1

[0042] (1) Preparation of biocatalyst

[0043] Gluconobacter oxidans DSM 2003 (purchased from the German Microorganism Collection Center) was selected, cultivated by conventional methods, separated and collected, washed with pH 7.4 potassium phosphate buffer for 3 times, resuspended in deionized water, and the concentration of the bacteria was increased. When it reaches 200 grams of wet cells / liter, the obtained complete cell suspension is the biocatalyst, which is stored at 4°C for later use;

[0044] Among them, the above-mentioned Gluconobacter oxidans is cultivated using the following medium formula: D-sorbitol 80 g / L; yeast powder 24 g / L; ammonium sulfate 5 g / L; potassium dihydrogen phosphate 2 g / L; magnesium sulfate 5 g / L liter; calcium carbonate 10 g / l.

[0045] (2) conversion

[0046] The biocatalyst prepared in the step (1), that is, the complete cell, is mixed with the substrate 1,2-butanediol aqueous solution, so that the concentration of the biocatalyst in the mi...

Embodiment 2

[0052] (1) Preparation of biocatalyst

[0053] Select Gluconobacter oxydans DSM 2003, cultivate it by conventional methods, separate and collect the bacteria, wash the bacteria three times with pH 7.4 potassium phosphate buffer, resuspend the bacteria in deionized water, and make the bacteria concentration reach 200 g wet cells / liter, The obtained complete cell suspension is the biocatalyst and stored at 4°C for later use;

[0054] Among them, the above-mentioned Gluconobacter oxidans is cultivated using the following medium formula: D-sorbitol 80 g / L; yeast powder 24 g / L; ammonium sulfate 5 g / L; potassium dihydrogen phosphate 2 g / L; magnesium sulfate 5 g / L liter; calcium carbonate 10 g / l.

[0055] (2) conversion

[0056] The biocatalyst prepared in the step (1), that is, the complete cell, is mixed with the substrate 1,2-butanediol aqueous solution, so that the concentration of the biocatalyst in the mixture is 100 grams of wet cells / liter, 1,2-butanediol The concentration...

Embodiment 3

[0062] (1) Preparation of biocatalyst

[0063] Select Gluconobacter oxydans DSM 2003, cultivate it by conventional methods, separate and collect the bacteria, wash the bacteria three times with pH 7.4 potassium phosphate buffer, resuspend the bacteria in deionized water, and make the bacteria concentration reach 200 g wet cells / liter, The obtained complete cell suspension is the biocatalyst and stored at 4°C for later use;

[0064] Among them, the above-mentioned Gluconobacter oxidans is cultivated using the following medium formula: D-sorbitol 80 g / L; yeast powder 24 g / L; ammonium sulfate 5 g / L; potassium dihydrogen phosphate 2 g / L; magnesium sulfate 5 g / L liter; calcium carbonate 10 g / l.

[0065] (2) conversion

[0066] The biocatalyst prepared in the step (1), that is, the complete cell, is mixed with the substrate 1,2-butanediol aqueous solution, so that the concentration of the biocatalyst in the mixture is 120 grams of wet cells / liter, 1,2-butanediol The concentration...

technical field

[0001] The present invention relates to a method for producing R-alpha-hydroxybutyric acid, in particular to a method for producing R-alpha-hydroxybutyric acid by using the intact cells of Gluconobacter oxidans DSM 2003 as a biocatalyst to catalyze 1,2-butanediol sour method. Background technique

[0002] α-Hydroxybutyric acid is an important industrial intermediate, which can be used to synthesize isoleucine and some drugs. α-Hydroxybutyric acid is divided into two configurations, R and S. High-purity chiral R-α-hydroxybutyric acid can be used to synthesize biodegradable polymer polyα-hydroxybutyric acid [P(2HB)] 【1】 . In addition, R-α-hydroxybutyric acid can be used to prepare azinothricin family anticancer antibiotics 【2，3】 .

[0003] After literature search, there are usually two starting substrates for the production of high-purity R-α-hydroxybutyric acid. One is α-ketobutyrate, which is produced by stereoselective enzymatic reduction to R-α-hydrox...

Examples

Embodiment 1

[0042] (1) Preparation of biocatalyst

[0043] Gluconobacter oxidans DSM 2003 (purchased from the German Microorganism Collection Center) was selected, cultivated by conventional methods, separated and collected, washed with pH 7.4 potassium phosphate buffer for 3 times, resuspended in deionized water, and the concentration of the bacteria was increased. When it reaches 200 grams of wet cells / liter, the obtained complete cell suspension is the biocatalyst, which is stored at 4°C for later use;

[0044] Among them, the above-mentioned Gluconobacter oxidans is cultivated using the following medium formula: D-sorbitol 80 g / L; yeast powder 24 g / L; ammonium sulfate 5 g / L; potassium dihydrogen phosphate 2 g / L; magnesium sulfate 5 g / L liter; calcium carbonate 10 g / l.

[0045] (2) conversion

[0046] The biocatalyst prepared in the step (1), that is, the complete cell, is mixed with the substrate 1,2-butanediol aqueous solution, so that the concentration of the biocatalyst in the mi...

Embodiment 2

[0052] (1) Preparation of biocatalyst

[0053] Select Gluconobacter oxydans DSM 2003, cultivate it by conventional methods, separate and collect the bacteria, wash the bacteria three times with pH 7.4 potassium phosphate buffer, resuspend the bacteria in deionized water, and make the bacteria concentration reach 200 g wet cells / liter, The obtained complete cell suspension is the biocatalyst and stored at 4°C for later use;

[0054] Among them, the above-mentioned Gluconobacter oxidans is cultivated using the following medium formula: D-sorbitol 80 g / L; yeast powder 24 g / L; ammonium sulfate 5 g / L; potassium dihydrogen phosphate 2 g / L; magnesium sulfate 5 g / L liter; calcium carbonate 10 g / l.

[0055] (2) conversion

[0056] The biocatalyst prepared in the step (1), that is, the complete cell, is mixed with the substrate 1,2-butanediol aqueous solution, so that the concentration of the biocatalyst in the mixture is 100 grams of wet cells / liter, 1,2-butanediol The concentration...

Embodiment 3

[0062] (1) Preparation of biocatalyst

[0063] Select Gluconobacter oxydans DSM 2003, cultivate it by conventional methods, separate and collect the bacteria, wash the bacteria three times with pH 7.4 potassium phosphate buffer, resuspend the bacteria in deionized water, and make the bacteria concentration reach 200 g wet cells / liter, The obtained complete cell suspension is the biocatalyst and stored at 4°C for later use;

[0064] Among them, the above-mentioned Gluconobacter oxidans is cultivated using the following medium formula: D-sorbitol 80 g / L; yeast powder 24 g / L; ammonium sulfate 5 g / L; potassium dihydrogen phosphate 2 g / L; magnesium sulfate 5 g / L liter; calcium carbonate 10 g / l.

[0065] (2) conversion

[0066] The biocatalyst prepared in the step (1), that is, the complete cell, is mixed with the substrate 1,2-butanediol aqueous solution, so that the concentration of the biocatalyst in the mixture is 120 grams of wet cells / liter, 1,2-butanediol The concentration...

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