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Method for producing chiral pure (2S,3S)-2,3-butanediol

A butanediol and chiral technology, applied in the field of production of chiral pure (2S,3S)-2,3-butanediol, can solve problems such as inability to meet the needs of industrial production, and achieve convenient product separation and simple operation process , The effect of simple culture medium

Active Publication Date: 2013-03-06
上海肆芃科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the limitation of product concentration and optical purity, the above method cannot meet the needs of industrial production.

Method used

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  • Method for producing chiral pure (2S,3S)-2,3-butanediol
  • Method for producing chiral pure (2S,3S)-2,3-butanediol
  • Method for producing chiral pure (2S,3S)-2,3-butanediol

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Embodiment 1: 2S in E.cloacae ssp.dissolvens SDM, the application of 3S-BDH in producing 2S, 3S-BD

[0044] 1. Construction of recombinant genetically engineered bacteria

[0045] The genomic DNA of bacterial strain e.cloacae ssp.dissolvens SDM (CGMCC No.4230) is prepared by conventional methods, and this process can refer to the method for small-scale preparation of bacterial genomes in "Guidelines for Elaborate Molecular Biology" published by Science Press, The genomic DNA of E.cloacae ssp.dissolvens SDM was extracted; the 2S, 3S-BDH gene (budC) was amplified by PCR from the genomic DNA of E.cloacae ssp.dissolvens SDM using synthetic primers.

[0046] The source bacterium of budC gene, E.cloacae ssp.dissolvens SDM (CGMCC No.4230), is a high-yield 2,3-BD strain obtained from the preliminary screening of the applicant's laboratory, which has been preserved but not subjected to genome sequencing.

[0047] According to the sequenced Enterobacter sp.638 genome sequence, d...

Embodiment 2

[0064] Embodiment 2: 2S in K.pneumonia ATCC 49790, the application of 3S-BDH in producing 2S, 3S-BD

[0065] The method is the same as in Example 1, except that the source strain of 2S, 3S-BDH is changed to K.pneumonia ATCC 49790, and primers are designed according to the sequenced genome sequence of the bacterium as follows:

[0066] The upstream primer 5'-CCCGGATCCAATGAAAAAAGTCGCAC-3' carries a BamHI site;

[0067] The downstream primer 5'-CCCAAGCTTTTAGTTAAACACCATCCCGC-3' carries a HindIII site.

[0068] The length of the above 2S, 3S-BDH gene sequence is 771 bases, and its nucleotide sequence is shown in SEQ ID NO.2.

[0069] The whole cell of recombinant E.coli was obtained as a catalyst by the same operation as in Example 1, and the transformation reaction was carried out.

[0070] The supernatant was analyzed by gas chromatography to determine the concentration and optical purity of 2S, 3S-BD in the conversion liquid, and when the concentration of 2S, 3S-BD no longer i...

Embodiment 3

[0072] Example 3: Application of 2S in C.glutamicum ATCC 13032, 3S-BDH in the production of 2S, 3S-BD

[0073] The method is the same as in Example 1, except that the source strain of 2S, 3S-BDH is changed to C. glutamicum ATCC13032, and primers are designed according to the sequenced genome sequence of the bacterium as follows:

[0074] The upstream primer 5'-AAAGAATTCAATGAGCAAAGTTGCAATGGT-3' carries an EcoRI site;

[0075] The downstream primer 5'-TTTAAGCTTCTAGTTGTAGAGCATGCCGC-3' carries a HindIII site.

[0076] The length of the above 2S, 3S-BDH gene sequence is 777 bases, and its nucleotide sequence Genebank number is 3345609.

[0077] The whole cell of recombinant E.coli was obtained as a catalyst by the same operation as in Example 1, and the transformation reaction was carried out.

[0078] The supernatant was analyzed by gas chromatography to determine the concentration and optical purity of 2S, 3S-BD in the conversion liquid, and when the concentration of 2S, 3S-BD ...

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Abstract

The invention discloses a method for producing chiral pure (2S,3S)-2,3-butanediol, which is to express a (2S,3S)-2,3-butanediol dehydrogenase gene in Escherichia coli, and perform the conversion production of chiral pure (2S,3S)-2,3-butanediol by using the recombinant Escherichia coli full cell as a biocatalyst and diacetyl and glucose as a substrate. In the method disclosed by the invention, a culture medium required by the strain is simple and low in cost, and the production process and product separation are convenient. The highest yield of the produced chiral pure (2S,3S)-2,3-butanediol reaches over 26gL<-1>, the ee value is more than 99 percent, and the method has a promotion and application value and can create considerable economic benefit.

Description

technical field [0001] The invention relates to a method for producing chiral pure 2,3-butanediol (2,3-BD), in particular to a method for producing chiral pure (2S,3S)-2,3-butanediol (2S, 3S-BD) method, specifically, (2S, 3S)-2,3-butanediol dehydrogenase (2S, 3S-BDH) gene is expressed in Escherichia coli (E.coli), and with this A method for efficiently producing chiral pure 2S, 3S-BD with recombinant E. coli whole cells as biocatalysts using diacetyl (DA) and glucose as substrates. Background technique [0002] 2,3-BD is a colorless, odorless and transparent liquid at room temperature, with three isomers: meso-2,3-butanediol (meso-BD), (2R,3R)-2,3 - Butanediol (2R, 3R-BD) and 2S, 3S-BD. 2,3-BD is widely used in many fields such as chemical industry, food, fuel and aerospace (Syu M J., Appl. Microbiol. Biotechnol., 2001, 55: 10-18). Optically active 2R, 3R-BD and 2S, 3S-BD can also be used as antifreeze, and because of its chiral carbon atoms, it also has important uses in...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P7/18C12R1/19
Inventor 马翠卿许平李理想王钰
Owner 上海肆芃科技有限公司
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