Method for efficiently producing alpha-oxoglutarate by adopting whole-cell transformation

A technology of whole cell transformation and ketoglutaric acid, which is applied in the field of high-efficiency production of α-ketoglutaric acid by whole cell transformation, can solve the problems of pollution, low yield, high cost of α-ketoglutaric acid, etc. Step-by-step effect

Active Publication Date: 2014-07-09
JIANGNAN UNIV
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  • Abstract
  • Description
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  • Application Information

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Problems solved by technology

[0006] The technical problem to be solved by the present invention is to provide a method for efficiently producing α-ketoglutarate, which is to transform the second deaminase gene derived from P. Bacillus constructs a whole-cell catalyst to efficiently convert L-glutamic acid to produce α-ketoglutarate, thus solving the problems of high cost, low yield and serious pollution in industrial α-ketoglutarate production

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  • Method for efficiently producing alpha-oxoglutarate by adopting whole-cell transformation
  • Method for efficiently producing alpha-oxoglutarate by adopting whole-cell transformation

Examples

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Embodiment 1

[0022] Example 1 Error-prone PCR transforms L-amino acid deaminase

[0023] Using the constructed recombinant plasmid pET-pm1[1] as a template, Mutazyme II DNA polymerase low-fidelity enzyme amplifies the pm1 gene. After the PCR product was purified and digested, it was ligated with the vector pET-20b(+) to construct a recombinant plasmid, and transformed into Escherichia coli JM109 to construct a mutant library. Pick a single colony grown on the ampicillin-resistant plate and inoculate it into LB, culture it overnight in a 96-well plate with shaking at 37°C, and inoculate it into the TB medium of another 96-well plate, and induce it with IPTG for 5 hours. After screening, plasmids were extracted from strains with high enzyme activity, transformed into B.subtilis168, and whole-cell catalysts were constructed, and the transformation rate was further tested. For the strains with high transformation rate, extract the plasmid and use it as the template for the next round of error...

Embodiment 2

[0026] Example 2 Site-directed mutagenesis transforms L-amino acid deaminase

[0027] The mutation sites obtained in Example 1 were subjected to site-directed saturation mutation one by one, using the constructed recombinant plasmid pET-pm1 as a template, Prime-STAR HS DNA polymerase high-fidelity enzyme and designed mutation primers for one-step amplification, DpnI restriction endonuclease The template DNA was digested with enzymes, then phosphorylated, ligated into a complete plasmid, and transformed into Bacillus subtilis 168. The mutants with the highest yield per locus were: F110I, A255T, E31D, R228C, L249S, I351T. Combine these mutation sites to construct F110I / A255T / E31D / R228C / L249S / I351T.

Embodiment 3

[0028] Embodiment 3 preparation of whole cell catalyst and whole cell transformation process

[0029] The recombinant Bacillus subtilis 168 of Example 1 and Example 2 were inoculated with seed medium (chloramphenicol 10 mg / L), and cultivated overnight at 37°C and 200 rpm. Fermentation was carried out in a 3L NBS fermenter, 1% inoculum was added to 1.8L fermentation medium, the stirring speed, ventilation and temperature were 400rpm, 1.0vvm and 28°C, respectively, when OD 600 When 0.6 was reached, 0.4 mM IPTG was added to induce the expression of L-amino acid deaminase. After 5 hours of induction, centrifuge at 8,000 rpm for 10 minutes at low temperature, collect the bacterial cells, and wash the bacterial cells twice with 20 mM Tris-HCl (pH 8.0) buffer solution. The whole cell transformation system is: L-glutamic acid 15g / L, whole cell catalyst 20.0g / L, the reaction is carried out in 20mM Tris-HCl (pH8.0), 37°C, 200rpm transformation for 24h.

[0030] F110I / A255R / E31D / R228F / ...

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Abstract

The invention discloses a method for efficiently producing alpha-oxoglutarate by adopting whole-cell transformation. L-amino acid deaminase genes transformed by easily wrong PCR (Polymerase Chain Reaction) or site-saturation mutagenesis are expressed through bacillus subtilis, the transformation rate of the alpha-oxoglutarate produced by whole-cell transformation of L-glutamic acid is increased, and the gene sequence of the L-amino acid deaminase genes is shown as SEQ ID NO.1 or SEQ ID NO.2. A whole-cell transformation system in which the transformation rate is increased is constructed, the problem that a chemical method for synthesizing alpha-oxoglutarate is tedious in steps, low in yield and causes environmental pollution and the problem that the transformation rate during production of the alpha-oxoglutarate is low through enzymatic transformation are solved, the alpha-oxoglutarate is produced in a pollution-free mode at high yield through a one-step method, and a certain theoretical basis is laid for subsequent industrial production.

Description

technical field [0001] The invention relates to a method for efficiently producing α-ketoglutarate by whole-cell transformation, in particular to a method for transforming L-amino acid deaminase gene through error-prone PCR and site-directed saturation mutation, which improves whole-cell transformation of recombinant Bacillus subtilis Conversion of L-glutamic acid to α-ketoglutarate. Background technique [0002] α-ketoglutarate is an important intermediate in the tricarboxylic acid cycle, which plays an important role in coordinating intracellular carbon metabolism and nitrogen metabolism. α-Phenylpyruvate has many applications. It can be used to synthesize heterocyclic compounds of antineoplastic drugs, as an antioxidant and to promote wound healing. In biomedical diagnosis, α-ketoglutarate can be used as the substrate of ketoglutarate dehydrogenase, aspartate aminotransferase and alanine aminotransferase. α-ketoglutaric acid can be used as a raw material for the synthes...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P7/50C12N15/53C12N15/70C12R1/125
Inventor 陈坚刘龙堵国成李江华嘎子·侯赛因侯颖
Owner JIANGNAN UNIV
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