Molecular modification method for improving activity and stability of lysine decarboxylase

A technology of lysine decarboxylase and molecular transformation, which is applied in the field of molecular transformation to improve the activity and stability of lysine decarboxylase, which can solve the problems of poor stability and alkali resistance of lysine decarboxylase, and achieve the improvement of catalytic activity , avoid uncertainty and tedious screening process, good stability and catalytic activity effect

Inactive Publication Date: 2019-07-12
TIANJIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The present invention aims at the problems of lysine decarboxylase not being resistant to alkali and having poor stability, and first proposes a method for EK modification of lysine decarboxylase (LDC) molecules

Method used

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  • Molecular modification method for improving activity and stability of lysine decarboxylase
  • Molecular modification method for improving activity and stability of lysine decarboxylase
  • Molecular modification method for improving activity and stability of lysine decarboxylase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] 1. Obtain the gene fragment encoding lysine decarboxylase molecule, gene sequence (SEQ ID NO.1, GenBank Accession number: NC_000913).

[0041] 2. Link the gene fragment (SEQ ID NO.2) encoding the EK polypeptide to the 5' end of the gene fragment encoding the lysine decarboxylase molecule (SEQ ID NO.1, GenBank Accession number: NC_000913). This embodiment uses PCR method, the specific operation method is as follows:

[0042] (1) Primer design: CadA-F-EK: SEQ ID NO.6; CadA-R: SEQ ID NO.7

[0043] (2) Using SEQ ID NO.1 as a template and using SEQ ID NO.6 and SEQ ID NO.7 as primers to perform a PCR reaction to obtain SEQ ID NO.3;

[0044] PCR cycling process (Taq enzyme):

[0045] Step1: 94°C for 3 minutes

[0046] Step2: 94℃30s

[0047] Step3: 55℃30s

[0048] Step4: 128s at 72°C (to step2, 30 cycles)

[0049] Step5: 5min at 72°C

[0050] Detection of PCR products: Take 5 μL of PCR products and detect them by agarose gel electrophoresis.

[0051] 3. Ligate the SEQ I...

Embodiment 2

[0054] 1. Obtain the gene fragment encoding lysine decarboxylase molecule, gene sequence (SEQ ID NO.1, GenBank Accession number: NC_000913).

[0055] 2. Link the gene fragment (SEQ ID NO.2) encoding the EK polypeptide to the 3' end of the gene fragment (SEQ ID NO.1, GenBank Accession number: NC_000913) encoding the lysine decarboxylase molecule. This embodiment uses PCR method, the specific operation method is as follows:

[0056] (1) Primer design: CadA-F: ​​SEQ ID NO.8; CadA-R-EK: SEQ ID NO.9

[0057] (2) Using SEQ ID NO.1 as a template and using SEQ ID NO.8 and SEQ ID NO.9 as primers to perform a PCR reaction to obtain SEQ ID NO.4;

[0058] The PCR cycle process is the same as in Example 1.

[0059] 3. Connect the SEQ ID NO.4 obtained in step 2 to the pET-32a(+) vector, and transform it into the host Escherichia coli BL21(ED3), the specific operation method is as follows:

[0060] Use Bam HI and Hind III to double-digest plasmid pET-32a(+) and SEQ ID NO.4, and use T4 DNA...

Embodiment 3

[0062] 1. Obtain the gene fragment encoding lysine decarboxylase molecule, gene sequence (SEQ ID NO.1, GenBank Accession number: NC_000913).

[0063] 2. The gene fragment (SEQ ID NO.2) encoding the EK polypeptide is simultaneously connected to the 5' end and the 3' end of the gene fragment encoding the lysine decarboxylase molecule (SEQ ID NO.1, GenBank Accession number: NC_000913), This embodiment adopts PCR method, and concrete operation method is as follows:

[0064] (1) Primer design: CadA-F-EK: SEQ ID NO.6; CadA-R-EK: SEQ ID NO.9

[0065] (2) Using SEQ ID NO.1 as a template and using SEQ ID NO.6 and SEQ ID NO.9 as primers to perform a PCR reaction to obtain SEQ ID NO.5;

[0066] The PCR cycle process is the same as in Example 1.

[0067] 3. Link the SEQ ID NO.5 obtained in step 2 to the pET-32a(+) vector, and transform it into the host Escherichia coli BL21(ED3), the specific operation method is as follows:

[0068] Use Bam HI and Hind III to double digest plasmid pET-...

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Abstract

The invention relates to a molecular modification method for improving the activity and stability of lysine decarboxylase. One end of a lysine decarboxylase (LDC) molecule is connected with a polypeptide segment (EK). The method includes the steps of firstly, obtaining a gene segment of the lysine decarboxylase molecule; secondly, connecting a gene segment for encoding an EK polypeptide to one endof a lysine decarboxylase gene; thirdly, connecting the lysine decarboxylase gene connected with an EK polypeptide gene to an expression carrier, transferring the gene into a host, and expressing themodified lysine decarboxylase (LDC-EK). By rationally modifying the enzyme molecule, the random mutation uncertainty and the complicated screening process are avoided; the modified lysine decarboxylase has high stability and catalytic activity. The catalytic activity of the LDC-EK is tripled, the high activity can be kept within the wide temperature range, and various organic solvents can be tolerated. The method is easier to operate, and protein is easier to prepare after modification.

Description

technical field [0001] The invention relates to a method for modifying lysine decarboxylase (LDC) protein molecule. More specifically, the present invention relates to a polypeptide fragment EK fragment which is alternately arranged by linking lysine (K) and glutamic acid (E) at the C-terminus and (or) N-terminus of lysine decarboxylase. Methods of modifying lysine decarboxylase to increase its activity and stability. Background technique [0002] 1,5-pentanediamine, also known as cadaverine, is a linear five-carbon diamine. It can be widely used in a variety of commercial purposes, such as synthetic polyamides, fungicides, petroleum and fuel additives, resins, chelating agents, asphalt, etc., and is an important petrochemical product. The chemical synthesis of pentamethylenediamine relies on petroleum and other non-renewable resources, and the pollution is relatively serious, with many disadvantages. In contrast, biosynthetic methods can utilize environmentally friendly ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/88C12N9/96C07K19/00C12N15/70C12N15/62
CPCC07K2319/00C12N9/88C12N9/96C12N15/62C12N15/70C12Y401/01018
Inventor 张雷齐海山杜岩
Owner TIANJIN UNIV
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