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Lysine decarboxylase mutant, encoding gene and expression thereof and application

A technology of lysine decarboxylase and mutants, which is applied in the field of lysine decarboxylase mutants, can solve the problem of weak tolerance of lysine decarboxylase, and achieves improved alkali resistance and catalytic performance, improved enzyme activity, The effect of alkali resistance and catalytic performance enhancement

Active Publication Date: 2018-11-13
NANJING UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Purpose of the invention: In order to solve the problem of weak tolerance of existing lysine decarboxylase at high pH, ​​the first aspect of the present invention provides a lysine decarboxylase mutant, and the second aspect provides lysine decarboxylation The coding gene of the enzyme mutant, the third aspect provides the expression and application of the lysine decarboxylase mutant

Method used

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  • Lysine decarboxylase mutant, encoding gene and expression thereof and application
  • Lysine decarboxylase mutant, encoding gene and expression thereof and application
  • Lysine decarboxylase mutant, encoding gene and expression thereof and application

Examples

Experimental program
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Embodiment 1

[0042] The construction of embodiment 1 recombinant plasmid pETDuet-1-CadA

[0043] Using the genome of Escherichia coli MG1655 (purchased from Wuhan Miaoling Biotechnology Co., Ltd.) as a template, the primers at both ends of the CadA gene were used for PCR amplification to obtain the CadA fragment (see Table 1), and then cloned into the vector pETDuet-1 (purchased from Wuhan Miaoling Biotechnology Co., Ltd. Between the NdeI and KpnI restriction sites of Ling Biotechnology Co., Ltd.) (see Table 2 and Table 3), the recombinant plasmid pETDuet-1-CadA was obtained. The nucleotide sequence of the CadA gene in Escherichia coli MG1655 is shown in SEQ ID No:1, and the amino acid sequence of the inducible lysine decarboxylase encoded by it is shown in SEQ ID No:2.

[0044] Wherein, the primers at both ends are as follows:

[0045] Primer1-F: 5'-GGAATTCCATATGAACGTTATTGCAATATTG-3'

[0046] Primer2-R: 5'-GGGGTACCTTTATTTTTTGCTTTCTTCTTTC-3';

[0047] Table 1 PCR reaction system and rea...

Embodiment 2

[0053] Example 2 Construction of site-directed mutagenesis plasmid

[0054] By analyzing the tertiary structure of the lysine decarboxylase decamer, excluding the amino acids located in the decarboxylase monomer and the conserved amino acids, three pairs of amino acids were selected and mutated into cysteines with stronger disulfide bonds. Improve the binding force of disulfide covalent bonds.

[0055] Using the plasmid pETDuet-1-CadA prepared in Example 1 as a template, the site-directed mutation plasmid pETDuet-1-CadA-M1(M2 / M3) was obtained by two rounds of PCR (polymerase chain reaction) amplification in vitro: M1(L89C / L442C) is about to mutate the 89th leucine / 442nd leucine of lysine decarboxylase such as the amino acid sequence shown in SEQ ID No: 2 into cysteine ​​respectively, and M2 (F102C / L547C) is about the amino acid sequence as shown in The 102nd phenylalanine / 547th leucine of the lysine decarboxylase shown in SEQ ID No: 2 are mutated into cysteine ​​respectively...

Embodiment 3

[0085] Embodiment 3 Verification amplification of mutant expression vector

[0086] The PCR stock solution after excising the template DNA obtained in Example 2 was transformed into E.coli Trans1-T1 (purchased from Beijing Quanshijin Biotechnology Co., Ltd.) competent cells, and coated with 100mg / L ampicillin-resistant On the LB plate, place the plate upside down in a 37°C incubator and incubate overnight. Pick several single colonies, extract the plasmid for sequencing, and after confirming that the mutation is correct, transform the correct plasmid into E.coli BL21 (DE3) (purchased from Beijing Quanshijin Biotechnology Co., Ltd.) Strain E. coli BL21(DE3) (pETDuet-1-CadA-M1(M2 / M3)) of lysine decarboxylase mutant with improved activity.

[0087] Among them, the formula of LB solid medium containing 100mg / L ampicillin is as follows: peptone 10g / L; yeast powder 5g / L; sodium chloride 5g / L; agar powder 25g / L; ampicillin 0.1g / L.

[0088] Wherein, the method for DNA conversion is:...

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Abstract

The invention discloses a lysine decarboxylase mutant. The lysine decarboxylase mutant is obtained by carrying out site-directed mutation on lysine decarboxylase with an amino acid sequence shown as SEQ ID No: 2 and is any one of (1) and (2): (1) valine at a 12th site of lysine decarboxylase with the amino acid sequence shown as SEQ ID No: 2 is mutated into cysteine and aspartic acid at a 41st site is mutated into the cysteine to obtain the lysine decarboxylase mutant V12C / D41C; (2) leucine at a 89th site of lysine decarboxylase with the amino acid sequence shown as SEQ ID No: 2 is mutated into the cysteine and leucine at a 442nd site is mutated into the cysteine to obtain the lysine decarboxylase mutant L89C / L442C. According to the lysine decarboxylase mutant provided by the invention, the alkali-resisting and catalytic performance is greatly improved and the lysine decarboxylase mutant is more suitable for industrialized production requirements and meets the requirements of social production.

Description

technical field [0001] The present invention relates to the mutation of lysine decarboxylase, in particular to a mutant of lysine decarboxylase, its coding gene and its expression and application. Background technique [0002] Nylon (PA), as the largest and most important variety of engineering plastics, has strong vitality, mainly because it achieves high performance after modification, followed by the automobile, electrical appliances, communications, electronics, machinery and other industries. demands are getting stronger. The rapid development of related industries has promoted the continuous increase in the demand for nylon in the global market. At present, traditional nylon materials are all derived from petroleum products, and it is undoubtedly an obvious defect that it is difficult to achieve sustainability. Due to concerns about global environmental issues and the increasing difficulty of waste disposal, bio-based nylon materials are the most ideal alternative ma...

Claims

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Application Information

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IPC IPC(8): C12N9/88C12N15/60C12N15/70C12N1/21C12P13/00C12R1/19
CPCC12N9/88C12P13/001C12Y401/01018
Inventor 陈可泉王璟许晟毛静文高思远欧阳平凯
Owner NANJING UNIV OF TECH
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