Arginine deiminase producing strain and construction method thereof

A technology of arginine deiminase and construction method, applied in the field of arginine deiminase production bacteria and its construction, to achieve the effect of simple composition

Pending Publication Date: 2022-02-01
XINTAI JIAHE BIOTECH CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are few reports on the expression of ADI by Bacillus subtilis

Method used

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  • Arginine deiminase producing strain and construction method thereof
  • Arginine deiminase producing strain and construction method thereof
  • Arginine deiminase producing strain and construction method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1: Codon optimization

[0038] The nucleotide sequence of the arcA gene obtained by the inventor from the existing database is shown in SEQ ID NO.1. In order to make the arcA gene more suitable for the expression system of Bacillus subtilis, the present invention optimizes the codons of the nucleotide sequence composition of the arcA gene.

[0039] The nucleotide sequence of the arcA gene after codon optimization is shown in SEQ ID NO.2; the codon relative fitness figure before optimization is shown in figure 1 Shown; the codon relative fitness map after optimization is shown in figure 2 shown.

[0040] Depend on figure 2 It can be seen that the codon relative fitness of the arcA gene after codon optimization can reach 1.0, which significantly improves the adaptability of the protein-coding gene in the Bacillus subtilis expression system.

Embodiment 2

[0041] Embodiment 2: Construction of recombinant expression vector

[0042] Plasmid pWB980 ( image 3 shown) treated with EcoRI and KpnⅠ double enzyme digestion ( Figure 4 ), the Pg3 strong promoter sequence (shown in SEQ ID NO.3) was integrated into the plasmid pWB980 after the double enzyme digestion treatment to obtain the plasmid pWB980-Pg3 ( Figure 5), its nucleotide sequence is shown in SEQ ID NO.4.

[0043] The plasmid pWB980-Pg3 was digested with BamHI and SphⅠ, and the codon-optimized arcA gene (shown in SEQ ID NO.2) was integrated into the digested plasmid pWB980-Pg3 ( Image 6 ) to obtain the recombinant expression vector (pWB980-Pg3-arcA).

[0044] The constructed recombinant expression vector was digested with two enzymes BamHI and SphⅠ, and the results were as follows: Figure 7 shown. The results showed that the arcA gene (shown in SEQ ID NO.2) had been successfully integrated into the plasmid pWB980-Pg3.

Embodiment 3

[0045] Embodiment 3: Construction of arginine deiminase-producing bacteria

[0046] The recombinant expression vector (pWB980-Pg3-arcA) constructed in Example 2 was introduced into Bacillus subtilis 168 to obtain a transformant. Spread the transformant on an LB plate containing 30 μg / ml kanamycin (kan), pick out a single colony capable of growing, and use it as a positive transformant.

[0047] Inoculate positive transformants into glycerol agar medium containing 30 μg / ml kanamycin and culture at 37°C until OD 600 =0.6, slowly lower the temperature to 28°C, add IPTG (to make the final concentration of IPTG 0.2mmol / L), and induce culture for 16h. After the induction culture was completed, the bacteria were disrupted by ultrasonication, centrifuged, and the supernatant was separated, and detected by Western bolt. The results were as follows: Figure 8 shown. There is an expression band at 50.4KDa, which is consistent with the theoretically calculated molecular weight of the p...

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Abstract

The invention discloses an arginine deiminase producing strain and a construction method thereof. The method comprises the following steps: carrying out double enzyme digestion treatment on a plasmid pWB980 by using EcoR I and KpnI, integrating a Pg3 strong promoter sequence onto the plasmid pWB980 subjected to the double enzyme digestion treatment to obtain a plasmid pWB980-Pg3, carrying out enzyme digestion treatment on the plasmid pWB980-Pg3 by using BamH I and SphI, and integrating an arcA gene onto the plasmid pWB980-Pg3 subjected to the enzyme digestion treatment to obtain a recombinant expression vector; and introducing the obtained recombinant expression vector into bacillus subtilis, and constructing to obtain the arginine deiminase producing strain. According to the invention, arginine deiminase is prepared by fermentation of the producing strain provided by the invention, and the enzyme activity of the arginine deiminase can be remarkably improved.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to an arginine deiminase-producing bacterium and a construction method thereof. Background technique [0002] Arginine deiminase (Arginine deiminase, EC3.5.3.6) is referred to as ADI. ADI widely exists in bacteria, archaea and some eukaryotic cells, and the properties of ADI from different sources are also different. The specific enzyme activity ranges from 5.4 to 140.3 IU / mg, and the optimum pH ranges from 5.6 to 7.6. ADI has important value in medicine. ADI has been proved to be an inhibitor of cell proliferation, which can prevent the expansion of cells in the inner wall of blood vessels; moreover, ADI has no obvious toxic and side effects, and can be used to treat leukemia and malignant tumors. ADI can also be applied to the enzymatic production of citrulline, which has broad market prospects. [0003] For the cloning and expression of ADI, researchers have express...

Claims

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Application Information

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IPC IPC(8): C12N9/78C12N15/75C12N1/21C12N15/113C12P13/10C12R1/125
CPCC12N9/78C12N15/75C12Y305/03006C07K14/32C12P13/10C12N2800/22
Inventor 岳明瑞谢沛曹华杰郭永胜
Owner XINTAI JIAHE BIOTECH CO LTD
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