Trigenic coexpression vector for synthesizing DL-alanine and application

A technology of co-expression vectors and expression vectors, which is applied in the fields of enzyme engineering and compound biosynthesis, can solve the problems of unsuitable industrial production, poor stability of enzyme protein, difficulty in meeting market demand, etc., and achieve the effect of good promotion and application value

Active Publication Date: 2018-03-13
HEBEI NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the prior art, the preparation methods of DL-alanine mainly include microbial fermentation method, chemical synthesis method and biological enzyme method. Among them, the reaction mechanism of chemical synthesis method is complex, the process is long, and the production cost is high; the production cycle of fermentation method is long, Large equipment investment, complicated separation, and high cost; the biological enzyme method uses the enzyme produced by microorganisms to catalyze the conversion of substrates into DL-alanine. This method has good regio and stereoselectivity, mild reaction conditions, and is easy to operate. Less pollution, etc., but due to the poor stability, low activity and high production cost of the required enzyme protein
It is precisely because of the existence of these shortcomings that the above three methods do not meet the requirements of industrial production and are difficult to meet the needs of the market.

Method used

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  • Trigenic coexpression vector for synthesizing DL-alanine and application
  • Trigenic coexpression vector for synthesizing DL-alanine and application
  • Trigenic coexpression vector for synthesizing DL-alanine and application

Examples

Experimental program
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Effect test

Embodiment 1

[0049] Example 1 Construction of three gene co-expression vectors

[0050] (1) Transformation vector pET-22b(+)

[0051] a, Primer design: According to the nucleotide sequence of the region to be mutated near the T7 promoter and terminator in the commercial vector pET-22b(+), design PCR amplification reaction primers:

[0052] Nhe-F01: 5'-GAGATCTCGATGCTAGCAAATTAATACGACTC-3';

[0053] Spe-F01: 5'-AGGAGGAACTAGTTCCGGATTGGC-3';

[0054] Spe-R01: 5'-GCCAATCCGGAACTAGTTCCTCCT-3';

[0055] b. Increase the SpeI recognition site: use site-directed mutagenesis (Site-Directed Mutagenesis), use the plasmid pET-22b(+) as a template, and use Spe-F01 and Spe-R01 as primers to perform site-directed mutagenesis PCR. The PCR reaction conditions are: : Pre-denaturation at 94°C for 4min; denaturation at 94°C for 35sec, annealing at 55°C for 1min, extension at 72°C for 7min, cycle 16 times; full extension at 72°C for 10min.

[0056] After the PCR reaction product was digested with restriction e...

Embodiment 2

[0079] Embodiment 2 biosynthetic ectoine

[0080] (1) Co-expression of three genes

[0081] The three-gene co-expression vector pET-22bNS-G / A / A was transformed into Escherichia coli BL21(DE3) competent cells, and the transformants were selected and cultured overnight in LB medium containing 100 μg / mL ampicillin at 37°C; The culture solution was inoculated into 100 mL of LB culture solution containing 100 μg / mL ampicillin at a ratio of 1:100, and cultured at 37°C with shaking at 180 rpm until OD 600When the temperature is 0.5 to 0.6, induce at 30°C for 15 hours, collect the bacterial cells by centrifugation at 8000 rpm, and wash the bacterial cells with 0.8% NaCl solution;

[0082] (2) Whole-cell catalytic synthesis of ectoine

[0083] Weigh 1 g of bacteria and resuspend in 20 mL of reaction solution (20 mM Na 2 CO 3 -NaHCO 3 Buffer, pH 10; 200mM sodium pyruvate, 200mM ammonium chloride, 200mM glucose), shake culture at 37°C and 180rpm for 3h, then centrifuge to remove bac...

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Abstract

The invention discloses construction of a trigenic coexpression vector for synthesizing DL-alanine and application. The construction is characterized in that encoding alanine dehydrogenase (ald), alanine racemase (alr) and glucose dehydrogenase (gdh) genes in pseud bacillus firmus are connected in series and then are inserted into reconstructed plasmid pET-22bNS, so as to construct the trigenic coexpression vector pET-22bNS-G / A / A. After constructed trigenic coexpression vector is transformed into escherichia coli BL21(DE3) and is subjected to whole-cell catalytic reaction for 3h through recombination strain, the output of L-alanine and the output of D-alanine are the highest and are correspondingly 7.0mg / mL and 6.5mg / mL, and the maximum synthesizing efficiency of L-alanine and the maximumsynthesizing efficiency of D-alanine are correspondingly 56.4mg / mL / d and 51.9mg / mL / d. The constructed trigenic coexpression vector has a capacity of efficiently synthesizing DL-alanine and is high inapplication value.

Description

technical field [0001] The invention relates to a construction method and application of a three-gene co-expression vector for synthesizing DL-alanine, and belongs to the technical fields of enzyme engineering and compound biosynthesis. Background technique [0002] DL-alanine is an unnatural amino acid and an important chiral intermediate. It is widely used in food, cosmetics and pharmaceuticals, and its demand in domestic and foreign markets is increasing day by day. In the prior art, the preparation methods of DL-alanine mainly include microbial fermentation method, chemical synthesis method and biological enzyme method. Among them, the reaction mechanism of chemical synthesis method is complex, the process is long, and the production cost is high; the production cycle of fermentation method is long, Large equipment investment, complicated separation, and high cost; the biological enzyme method uses the enzyme produced by microorganisms to catalyze the conversion of subst...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C12N1/21C12P13/06C12N9/04C12N9/06C12N9/90C12R1/19
CPCC12N9/0006C12N9/0016C12N9/90C12P13/06C12Y101/9901C12Y104/01001C12Y501/01001
Inventor 鞠建松徐书景王珊珊孙晴晴蔡晓赵宝华
Owner HEBEI NORMAL UNIV
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