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Lysine decarboxylase strain and application thereof in production of pentamethylenediamine

A technology of lysine decarboxylase and pentamethylenediamine, which is applied in the direction of application, microbial-based methods, enzymes, etc., can solve the problems of low production efficiency of pentamethylenediamine and achieve the effect of improving production efficiency

Inactive Publication Date: 2019-04-19
SHANDONG SHOUGUANG JUNENG GOLDEN CORN CO LTD +3
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] In order to solve the problem of low production efficiency of existing pentamethylenediamine, one of the technical solutions provided by the present invention: a lysine decarboxylase strain, said strain is to introduce the lysine decarboxylase gene into The recombinant bioengineering bacteria after Escherichia coli is the initial strain, and the lysine decarboxylase strain with high enzymatic activity obtained through mutagenesis screening

Method used

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  • Lysine decarboxylase strain and application thereof in production of pentamethylenediamine
  • Lysine decarboxylase strain and application thereof in production of pentamethylenediamine
  • Lysine decarboxylase strain and application thereof in production of pentamethylenediamine

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Embodiment 1

[0038] Example 1: Acquisition of lysine decarboxylase initial strain E.coli BL21(DE3) / pET24a-cadA

[0039] According to the gene sequence (NCBI accession number NC_000913.3) (SEQ ID NO: 1) of the lysine decarboxylase cadA (NCBI accession number NC_000913.3) (SEQ ID NO: 1) of E. 2) and cadA-BamHI-R (SEQ ID NO: 3): cadA-NdeI-F: 5'-GGAATTCCATATGaacgttattgcaatattg-3' cadA-BamHI-R: 5'-CGGGATCCttattttttgctttcttctttc-3'

[0040]The PCR reaction system includes: 50 pmol each of cadA-NdeI-F and cadA-BamHI-R, 1 μL of E.coli MG1655 bacterial solution, 1× KOD FX buffer, 0.2 mM dNTPs, 2% DMSO, 1 μL of each 10 μM primer, KOD FX neo 0.5 U, add water to 50 μL of the total system.

[0041] The PCR reaction conditions are: 94°C, 2min; 98°C for 10s, 55°C for 30s, 68°C for 1min15s, 30 cycles; 68°C for 7min.

[0042] After the PCR reaction, analyze with agarose gel electrophoresis, and detect a target-specific band of about 2148bp, which is required. The PCR amplified product was recovered with...

Embodiment 2

[0044] Embodiment 2: BL21(DE3) / pET24a-cadA engineering bacteria culture preparation

[0045] The lysine decarboxylase strain stored in the glycerol tube was inoculated in the LB medium added with kanamycin, activated at 37° C. at 220 rpm for 12 hours. The strain activated for 12 hours was inoculated into a Erlenmeyer flask with a liquid volume of 150 mL of seed medium, with an inoculum volume of 5%, and cultured in a constant temperature incubator at 37° C. with shaking at 140 rpm. The bacterial cells cultured for 14 h were collected by centrifugation at 4000 rpm, and washed once with sterile saline. Use pH 6.5 phosphate buffer to make 108 / mL bacterial suspension;

Embodiment 3

[0046] Embodiment 3: mutagenesis treatment

[0047] Take 5 mL of the bacterial suspension and add it to a 15 mm sterile test tube, add 0.5% NTG diluted culture solution and shake for 2 h, dilute a large amount to terminate the mutagenesis, spread on the screening plate and culture for 1 d.

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Abstract

The invention discloses a lysine decarboxylase strain and an application method thereof in the production of pentamethylenediamine. The lysine decarboxylase strain adopts recombinant bioengineering bacteria obtained by introducing a lysine decarboxylase gene into escherichia coli by a genetic engineering method as a starting strain, a high enzyme activity lysine decarboxylase strain is obtained bymutagenesis screening, the preservation number is CGMCC No. 16762, and compared with original engineering bacteria, the lysine decarboxylase activity is increased by 3.93 times. The invention furtherdiscloses the application of the lysine decarboxylase strain in the production of the pentamethylenediamine, the lysine decarboxylase strain is subjected to fermentation culture to obtain a fermentation broth containing the lysine decarboxylase, the fermentation broth is added in an amount of 5% in the lysine hydrochloride and a coenzyme solution, the conversion is carried out for 3 h at 40 DEG C, the highest content of the pentanediamine is 326.3 g / L, and the molar conversion of the lysine hydrochloride reaches 99.98%.

Description

technical field [0001] The invention relates to a lysine decarboxylase strain and its application in the production of pentamethylenediamine, belonging to the technical field of pentamethylenediamine production. Background technique [0002] Pentylenediamine, also known as cadaverine, can be polymerized with dibasic acid to synthesize high molecular polyamide material (namely nylon). About 7 million tons of polyamide materials are produced globally every year, consuming a lot of petrochemical resources. Petrochemical resources are non-renewable resources. Therefore, pentamethylenediamine, an important constituent monomer of polyamide, is the development trend of future technology and has important economic and ecological significance. [0003] Patent application number 201510767145.9 discloses a pentamethylenediamine-producing strain and its high-efficiency production process of pentamethylenediamine. During the construction of the patented strain, the promoter ldcCp is rep...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/01C12N15/60C12P13/00C12R1/19
CPCC12N9/88C12N15/01C12P13/001C12Y401/01018
Inventor 高世军刘象刚吴泽华马光王志强孙敬善张杰褚玉强
Owner SHANDONG SHOUGUANG JUNENG GOLDEN CORN CO LTD
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