Gene for coding glutamine dipeptide biosynthetic enzyme and application thereof

A glutamate dipeptide and biosynthesis technology, applied in the biological field, can solve the problems of inability to be widely used in the market, high cost of enzyme separation and purification, unsuitable for industrial production, etc., and achieve obvious market competitiveness, fast reaction rate, equipment operation and control. easy effect

Active Publication Date: 2017-05-31
INNOBIO CORP LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The production of CG dipeptide by chemical synthesis has many reaction steps, many by-products, highly toxic reagents and is not environmentally friendly (Tang Guo. Synthesis and reaction research of N(2)-L-alanyl-L-glutamine dipeptide. Xiamen University ,2004; Chinese Patent, Synthetic Method of C-Gludipeptide, CN1392156A)
[0004] However, the biological enzyme catalysis method also has the disadvantages of large amount of enzyme, low peptide productivity, and can only catalyze the synthesis of some highly hydrophobic amino acids.
In recent years, foreign researchers have obtained an amino acid ligase (Lal) from Bacillus subtilis, and developed a patented technology for fermenting and producing dipeptides. The production cost is still high, and it cannot be widely used in the market (Tabata K, Hashimoto S. Fermentative production of L-alanyl-L-glutamine by a metabolically engineered Escherichia coli strain expressing L-amino acid α-ligase. Applied and environmental microbiology, 2007, 73 (20):6378-6385.)
[0005] CN104480075A and CN105274174A disclose two kinds of bioenzyme-catalyzed methods for synthesizing glutamic acid dipeptide, which adopts bioenzyme freeze-dried powder or bioenzyme buffer to carry out catalytic reaction, but because the cost of enzyme separation and purification is high, and it is difficult to recycle and reuse after the reaction, it is not suitable for actual industrial production

Method used

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  • Gene for coding glutamine dipeptide biosynthetic enzyme and application thereof
  • Gene for coding glutamine dipeptide biosynthetic enzyme and application thereof
  • Gene for coding glutamine dipeptide biosynthetic enzyme and application thereof

Examples

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preparation example Construction

[0025] Next, the present invention provides a biosynthetic method based on the above-mentioned recombinant Escherichia coli to synthesize dipeptide. The method at least includes the process of fermenting and producing the recombinant Escherichia coli obtained by the above method according to the prior art, which involves the step of transformation reaction of the recombinant Escherichia coli to the substrate.

[0026] The biosynthesis of CG dipeptide is a relatively mature technology, and its substrate can be described as a substrate comprising a carboxyl component and an amine component according to the prior art. In the present invention, the carboxyl component is selected from amino acid esters and amino acid Amide, most preferably L-alanine methyl ester hydrochloride; said amine component is selected from amino acids, C-protected amino acids and amines, most preferably L-glutamine. The substrate concentration in the reaction system is set according to the proportional reac...

Embodiment 1

[0048] Construction of biological enzyme expression vector and recombinant Escherichia coli:

[0049] According to the known nucleotide sequence (SEQ ID NO.5) of related biological enzymes, primers (SEQ ID NO.3 / 4) were designed to derive from Sphingobacter siyangii ( Sphingobacterium siyangensis, strain number: 1.6855) genome is used as a template to amplify the biological enzyme gene SEQ ID NO.1.

[0050] Among them, the PCR reaction system (50μL):

[0051]

[0052] PCR reaction conditions:

[0053]

[0054] After the PCR product was recovered and purified by gel, it was combined with the plasmid pMD TM 19-T connection, positive clones were sequenced after extracting plasmids. The bioenzyme gene in the positive clone obtained by enzyme digestion was connected to the expression vector pET29a that had been treated in the same way, and the connection product was transformed into E.coli DH5α competent cells, cultivated on LB-resistant medium, picked a single colony, and ...

Embodiment 2

[0056] Fermentation culture of recombinant Escherichia coli:

[0057] The recombinant Escherichia coli was inoculated into LB liquid medium: yeast extract powder 5g / L, tryptone 10g / L, NaCl 10g / L), and the cells were activated at 37°C and 200rpm. Then transferred to the seed medium, 37 ° C, 200 rpm overnight shaking culture. Inoculate 1.5L fermentation medium with 1% inoculum amount, culture cells to OD at 37°C with 200rpm aeration 620 After = 0.6-0.8, the inducer IPTG was added to induce overnight, and the bacteria were collected by centrifugation. As the recombinant Escherichia coli used in the following examples of the present invention.

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Abstract

The invention discloses a gene for coding a glutamine dipeptide biosynthetic enzyme and application thereof. The nucleotide sequence of the gene is shown as SEQ ID NO.1. The invention further discloses an amino acid sequence coded by the gene, and provides a recombinant vector containing the gene, recombinant escherichia coli, and a method for performing biotransformation to synthesize the glutamine dipeptide by using the gene. The gene and the application of a recombinant bacterial strain of the gene have the advantages of high mol conversion rate, high reaction speed, easy separation, low cost and the like. In the synthesis method, the maximum mol conversion rate of the glutamine dipeptide can reach 83.3 percent; meanwhile, the thalli can be applied to the catalytic synthesis of the glutamine dipeptide again after the circulation recovery; in addition, the catalytic activity is stable. Therefore high market competitive power and application values are realized; a foundation is laid for the industrial production of the glutamine dipeptide.

Description

technical field [0001] The invention relates to a biosynthetic method of dipeptide, in particular directly using recombinant Escherichia coli as a whole-cell catalyst to efficiently synthesize dipeptide, which belongs to the field of biotechnology. Background technique [0002] Glutamine dipeptide (L-Ala-Gln), also known as N(2)-L-alanyl-L-glutamine, has the advantages of high solubility, water solubility and strong thermal stability and has gradually replaced glutamine (L -Gln) has become the main parenteral nutrition drug. [0003] At present, the synthesis of CG dipeptide mainly adopts chemical synthesis and biological enzyme catalysis. The production of CG dipeptide by chemical synthesis has many reaction steps, many by-products, highly toxic reagents and is not environmentally friendly (Tang Guo. Synthesis and reaction research of N(2)-L-alanyl-L-glutamine dipeptide. Xiamen University , 2004; Chinese patent, C-glutin dipeptide synthesis method, CN1392156A). [0004] ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/52C12N15/70C12N1/21C12N9/00C12P21/02C12R1/19
CPCC12N9/93C12N15/70C12N2800/101C12P21/02C12Y603/02009
Inventor 袁文杰范超李益民吴文忠
Owner INNOBIO CORP LTD
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