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 hi

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
Comparison scheme
Effect test

Example Embodiment

[0031] Example 1 Construction of a recombinant expression vector containing a gene encoding an aspartase mutant

[0032] Using 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 whole plasmid two-step PCR method (the reaction system is shown in Table 1, and the reaction conditions are shown in Table 2), that is, 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 enzyme was added to 20 μL of the PCR product to digest the template plasmid, and incubated at 25°C overnight or at 37°C for 3 to 4 hours. Pipette 5 μL of the digested product and transform it into E. coli BL21 (DE3) to ob...

Example Embodiment

[0039] Example 2 Construction of recombinant Escherichia coli engineering bacteria producing aspartase mutants

[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.

Example Embodiment

[0041] Example 3 Expression and purification of aspartase by recombinant bacteria pET21a-E427Q / E.coli BL21

[0042] The recombinant strain pET21a-E427Q / E.coli BL21 constructed in Example 2 was compared with 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). They were inoculated into 10 mL of LB medium containing ampicillin, cultured overnight at 37 °C with shaking, and transferred to 50 mL of LB medium containing ampicillin at 1% of the inoculum the next day. 0.5mM IPTG was induced at 16°C for 12-16h. Cells were collected and disrupted by centrifugation at 8000 rpm for 10 min at 4°C, and the cell-disrupted supernatant (crude enzyme solution) was collected for subsequent purification.

[0043] Purification of aspartase or aspartase mutant was carried out in a hot water bath at 60° C. for 30 min, followed by centrifugation at 12000 rmp for 90 min to obtain the purified enzyme. The r...

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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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