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Aspartase mutant, recombinant expression vector containing aspartase mutant, recombinant bacterium and application thereof

A technology of aspartase and expression vector, which is applied in the field of genetic engineering, can solve the problems of harsh requirements, low enzyme activity, and high production cost of β-amino acids, and achieve the effects of strengthening capacity, improving enzyme activity, and improving catalytic efficiency

Active Publication Date: 2020-02-14
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the current aspartase has strict requirements on the pH value and temperature of the reaction, relies on the reaction environment of high temperature and strong alkali, and has low enzyme activity under mild conditions. This defect leads to high production costs of β-amino acids

Method used

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  • Aspartase mutant, recombinant expression vector containing aspartase mutant, recombinant bacterium and application thereof
  • Aspartase mutant, recombinant expression vector containing aspartase mutant, recombinant bacterium and application thereof
  • Aspartase mutant, recombinant expression vector containing aspartase mutant, recombinant bacterium and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0031] Embodiment 1 Contains the construction of the recombinant expression vector of the gene encoding aspartase mutant

[0032] With the pET-21a recombinant plasmid containing the nucleotide sequence shown in SEQ ID NO: 4 as a template, Fprimer (sequence shown in SEQ ID NO: 5) and Rprimer (sequence shown in SEQ ID NO: 6) as primers, The mutant plasmid was constructed by the two-step PCR method of the whole plasmid (the reaction system is shown in Table 1, and the reaction conditions are shown in Table 2), and the gene E427Q shown in SEQ ID NO: 3 was obtained.

[0033] Table 1 PCR reaction system

[0034]

[0035] Table 2 PCR reaction conditions

[0036]

[0037]

[0038] The PCR product was checked by gel electrophoresis, and then 1 μL of Dpn I restriction endonuclease was added to 20 μL of the PCR product to digest the template plasmid, and incubated overnight at 25° C. or 37° C. for 3 to 4 hours. Pipette 5 μL of the digested product and transform it into Escheri...

Embodiment 2

[0039] Embodiment 2 produces the recombinant escherichia coli engineering bacterium construction of aspartase mutant

[0040] The strain containing the correct recombinant plasmid pET21a-E427Q obtained in Example 1 is the recombinant strain pET21a-E427Q / E.coli BL21 of the present invention.

Embodiment 3

[0041] Example 3 Expression and Purification of Aspartase from Recombinant Bacteria pET21a-E427Q / E.coli BL21

[0042] The recombinant strain pET21a-E427Q / E.coli BL21 constructed in Example 2 and the control strain pET21a-AspB / E.coli BL21 expressing the unmutated original enzyme BsAspB (wild type, amino acid sequence shown in SEQ ID NO:3) Inoculate in 10mL ampicillin-containing LB medium respectively, culture overnight at 37°C with shaking, transfer to 50mL ampicillin-containing LB medium the next day at 1% inoculum size, culture at 37°C for 2-3 hours, then add 0.5mM IPTG was induced at 16°C for 12-16h. The cells were collected by centrifugation at 8000 rpm for 10 min at 4° C. and crushed, and the cell crushed supernatant (crude enzyme liquid) was collected for subsequent purification.

[0043] Purification of aspartase or aspartase mutants is carried out in a hot water bath at 60° C. for 30 minutes, and then centrifuged at 12000 rpm for 90 minutes to obtain the purified enzym...

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Abstract

The invention provides an aspartase mutant, a recombinant expression vector containing the aspartase mutant, recombinant bacteria containing the aspartase mutant and an application thereof, which belong to the technical field of gene engineering. The amino acid sequence of the aspartase mutant is shown as SEQ ID NO: 1. According to the aspartase mutant disclosed by the invention, glutamic acid atthe 427th site is mutated into glutamine on the basis of aspartase (an amino acid sequence is shown as SEQ ID NO: 3). The 427th amino acid residue of aspartase is mutated into glutamine, the polar environment near an active site is changed, and ammonia supply during a substrate reaction is facilitated, so that the enzyme activity is improved, the beta-amino acid synthesis capacity of the aspartaseis enhanced, and an actual effective strategy is provided for industrial production of the aspartase.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to an aspartase mutant, a recombinant expression vector containing the aspartase mutant, recombinant bacteria and applications. Background technique [0002] β-amino acids are widely used target molecules in the pharmaceutical industry and are the structural units of biologically active compounds or natural products and drugs. Among them, β-aminobutyric acid is a potent elicitor that confers broad-spectrum disease protection in at least 40 plant species. In addition, β-aminobutyric acid can be used as the precursor of pharmaceutical intermediate β-aminobutanol. β-aminobutanol is a key intermediate of the AIDS drug Dolutegravir. [0003] The natural substrate of aspartase is aspartic acid, which was selected for the preparation of β-amino acids with high substrate specificity and the characteristics of the secondary carboxylate binding pocket. However, the current aspa...

Claims

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Application Information

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IPC IPC(8): C12N9/88C12N15/60C12N15/70C12N1/21C12P13/04C12R1/19
CPCC12N9/88C12P13/04C12Y403/01001C12N15/70
Inventor 饶志明王雅玲陈佳敏杨套伟徐美娟张显邵明龙张佳宁彭安褀徐淑萍吴美琪
Owner JIANGNAN UNIV
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