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A kind of production method of optically pure r-acetoin

A production method and technology of acetoin, applied in the field of high-efficiency production of optically pure R-acetoin, can solve the problems of low R-acetoin yield, low optical purity, and high cost, and achieve simple operation process and simple culture medium , low-cost effect

Active Publication Date: 2017-01-04
GUANGXI ACAD OF SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0008] Aiming at the shortcomings of low R-acetoin yield, low optical purity, difficult product extraction, high cost, and difficulty in large-scale and low-cost production in the above-mentioned existing methods, the present invention provides a production method for optically pure R-acetoin method

Method used

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  • A kind of production method of optically pure r-acetoin
  • A kind of production method of optically pure r-acetoin
  • A kind of production method of optically pure r-acetoin

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Embodiment 1

[0038] Construction of recombinant Escherichia coli BL21 / pETDuet-bdh-gdh

[0039] Genomic DNA of the strain Paenibacillus polymyxa DSM365 was prepared by conventional methods, the genome of the strain was sequenced and metabolic network analysis was carried out, and related genes were annotated and functionally identified, and the (R,R)-2 of the strain was obtained, Coding sequence of 3-butanediol dehydrogenase gene; (R,R)-2,3-butanediol dehydrogenase was amplified by PCR using synthetic primers P1 and P2, using genomic DNA of P.polymyxa DSM365 as a template Hydrogenase gene (bdh). The genome sequence of the Bacillus subtilis168 strain was obtained from the NCBI database. The glucose dehydrogenase gene (gdh) was amplified by PCR using the synthetic primers P3 and P4 and using the genomic DNA of B. subtilis168 as a template.

[0040] The sequences of P1, P2, P3 and P4 are respectively:

[0041] P1: 5'-GATGCCATGGAAGCATTGAGATGGCATGG-3';

[0042] P2: 5'-TCGCGAGCTCTTAAGCTTGCGGAG...

Embodiment 2

[0049] Preparation of Recombinant Escherichia coli Resting Cell Biocatalyst

[0050] (1) Plate culture: Streak the recombinant Escherichia coli BL21 / pETDuet-bdh-gdh onto an LB plate containing 1.8% agar by mass volume ratio and containing 100 μg / mL ampicillin, and culture at 37° C. for 12 hours;

[0051] (2) Seed culture: under sterile conditions, use a toothpick to pick a single colony on the plate of step (1), and then inoculate it into 20 mL of LB liquid medium containing 100 μg / mL ampicillin, shake it at 37 ° C Shake culture for 12 hours;

[0052] (3) Fermentation tank culture: under sterile conditions, take the bacterial solution obtained in step (2) and inoculate it into 2 L of LB liquid medium containing 100 μg / mL ampicillin at an inoculum size of 1% by volume, and cultivate at 37 ° C 3 hours, then add IPTG with a final concentration of 0.5mM, induce for 10 hours at 16°C, and stop the culture;

[0053] Wherein, the LB medium formula described in the above steps (1) to...

Embodiment 3

[0056] Preparation of Optically Pure R-Acetoin Using Recombinant Escherichia coli Resting Cells as Biocatalyst

[0057]Select the recombinant Escherichia coli resting cells obtained above as a catalyst to carry out the following transformation process: at 30°C and pH 7, shake on a 180rpm shaker, convert diacetyl with an initial concentration of 20g / L, and add 20g / L of glucose to supplement Consumption of cofactors required for the reaction; samples were taken every 3 hours, and the samples were centrifuged at 8,000rpm for 10 minutes to remove the added whole-cell biocatalyst, and the concentration of diacetyl in the supernatant was detected by gas chromatography. According to the concentration of diacetyl Diacetyl was added to maintain the diacetyl concentration between 10 and 20 g / L; the supernatant was analyzed by gas chromatography to determine the concentration and optical purity of R-acetoin in the conversion solution; after 16 hours, the test results showed: The concentr...

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Abstract

A production method of optically pure R-acetoin, comprising the steps of: (R, R)-2,3-butanediol dehydrogenase gene (bdh) and glucose dehydrogenase gene (gdh) in Escherichia coli Co-expressed, and the recombinant E. coli resting cells were used as biocatalysts, and diacetyl was transformed to produce optically pure R-acetoin. Glucose dehydrogenase can provide the coenzyme NADH required by (R,R)‑2,3‑butanediol dehydrogenase, and (R,R)‑2,3‑butanediol dehydrogenase can It has the characteristics of catalyzing diacetyl to generate optically pure R-acetoin, the output of R-acetoin can reach 26g / L, and the optical purity is 99.6%. The method of the invention has simple culture medium, convenient operation process, convenient product separation, no complicated chiral resolution steps, and the regeneration of intracellular coenzymes is realized, with low cost and great industrial application prospect.

Description

technical field [0001] The invention relates to a production method of optically pure R-acetoin, which comprises (R,R)-2,3-butanediol dehydrogenase gene (bdh) and glucose dehydrogenase gene (gdh) in the large intestine Bacillus expression, and using recombinant E. coli resting cells as biocatalysts, using diacetyl and glucose as substrates to efficiently produce optically pure R-acetoin. Background technique [0002] Acetoin, namely 3-hydroxy-2-butanone (3-hydroxy-2-butanone), has two optical isomers (R-acetoin and S-acetoin), naturally occurring In various foods such as dairy products, butter, cheese, wine, corn, grapes, apples, strawberries, cocoa, etc., the national standard GB2760-86 stipulates that it is a food flavor that is allowed to be added. Acetoin is widely used. It has obvious characteristics of cheese and fat. It is mainly used in the production of cream, dairy products, coffee, yogurt and other spices. In addition, acetoin is an important chemical synthesis ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/53C12P7/26C12R1/19
Inventor 谢能中黄日波李检秀郭铃黄艳燕杜奇石王青艳李亿
Owner GUANGXI ACAD OF SCI
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