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A method for whole-cell biocatalytic production of glutaric acid

A technology of biocatalysis and glutaric acid, applied in the field of biology, can solve problems such as time-consuming, increased production costs, and difficult product separation

Active Publication Date: 2019-08-20
NANJING TECH UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

This method requires exogenous addition of expensive amino acceptor 2-ketoglutarate, which greatly increases the production cost. In addition, the molar yield of glutaric acid produced by the fermentation method is low, takes a long time, the reaction system is more complicated, and the products are separated. more difficult
[0005] Jake Adkins et al. used recombinants that overexpressed davAB and davDT Escherichia coli The strain uses glucose as a substrate and produces 0.82g / L glutaric acid after 48 hours of fermentation. This method has a complicated reaction system and a low molar yield of glutaric acid
[0006] After searching, a cost-effective two-cell coupling production method of glutaric acid has not been reported yet

Method used

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  • A method for whole-cell biocatalytic production of glutaric acid
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  • A method for whole-cell biocatalytic production of glutaric acid

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Experimental program
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Effect test

Embodiment 1

[0030] Construction of overexpressed DavBA and gabDT strains:

[0031] (1) Recombinant strains overexpressing davBA E. coli BL-22AB is provided by our laboratory, the recombinant strain E. coli BL-22AB was made into competent cells.

[0032] (2) gabT was synthesized by Jinweizhi, and the restriction sites were NdeI and XhoI, and it was connected to the pACYC vector to obtain the recombinant plasmid pACYC-gabT,

[0033] (3) gabD was synthesized by Jinweiweizhi, with restriction sites NcoI and HindIII, and connected to the pACYC vector to obtain the recombinant plasmid pACYC-gabD.

[0034] (4) After the recombinant plasmid pACYC-gabD was recovered by treatment with restriction endonucleases NcoI and HindIII, the fragment gabD with restriction sites of NcoI and HindIII was obtained, and the fragment was combined with the recombinant plasmid treated with the same restriction endonuclease Plasmid pACYC-gabT was ligated using T4 DNA ligase at 25°C for 30min.

[0035] (5) Trans...

Embodiment 2

[0041] Optimum temperature screening for inducing LGOX expression

[0042] Pick recombinant strains from the plate E. coli A single colony of 28LGOX was placed in a 5MLLB shake tube containing 50mg / L kanamycin resistance, cultured for 6-8h, then transferred to 100ml LB containing 50mg / L kanamycin resistance, until OD 600 =0.6, add 0.5mmol of IPTG, culture in a shaker at 20°C, 25°C, 30°C, and 37°C respectively, after 4h, centrifuge at 6000g for 5min to obtain cells overexpressing LGOX.

[0043] The obtained cells were resuspended with 5ml of 100mmol PBS, crushed using a cell disruptor, the crushing conditions were super 2s, stop 2s, the temperature was 4°C, the power was 30%, the crushing time was 10min, and then centrifuged at 7000g for 20min.

[0044] After measuring the protein concentration of supernatants and precipitates induced under different temperature conditions, the protein concentration was 20ug, and then analyzed by SDS-PAGE to obtain the protein expression afte...

Embodiment 3

[0046] The whole cell catalyzed process of glutamate to produce 2-oxoglutarate is coupled with the process of whole cell catalyzed conversion of L-lysine to glutarate:

[0047] Cultivate and collect the recombinant bacteria that have overexpressed LGOX according to the mode of Example 2 E. coli 28LGOX cells as a catalyst.

[0048] At the same time, pick the recombinant strains from the plate E. coli A single colony of BL-22AB-YDT was inoculated into a 5ml LB shaking tube containing 100mg / L ampicillin resistance and 35mg / L chloramphenicol resistance. and 35mg / L chloramphenicol resistance in a 100ml shake flask to OD 600 =0.6, add 0.5mmol of IPTG, induce culture at 20°C for 12h, then centrifuge at 7000g for 5min to obtain the recombinant strain E. coli BL-22AB-YDT cells and use it as a catalyst.

[0049] Use 100mmolPBS to resuspend the recombinant bacteria E. coli 28LGOX and E. coli BL-22AB-YDT cells.

[0050] In the reaction system, add recombinant bacteria E. col...

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Abstract

The invention discloses a method of whole-cell-biocatalytically producing glutaric acid. The method includes the steps of: performing induced expression and collecting cells of a recombinant strain E.coli BL-22AB-YDT and a recombinant strain E.coli 28LGOX, and mixing the cells of the recombinant strain E.coli 28LGOX and the recombinant strain E.coli BL-22AB-YDT according to the ratio of 1:1-5; adding substrates including L-glutamic acid and L-lysine according to the molar ratio of 1:0.5-4; and adding a surfactant to perform the whole-cell-catalytic production to obtain the glutaric acid. The method is free of addition of 2-ketoglutaric acid, so that production cost is reduced, and problems of long production period, complex metabolic products, low substrate conversion rate, difficulty in separation and extraction of products, and high energy consumption in a fermentation method are solved. The method also solves a defect that a cascade catalytic process in enzymic catalysis is difficult to achieve, so that the method can improve catalytic efficiency and is free of an enzyme purifying process.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for producing glutaric acid by whole cell biocatalysis. Background technique [0002] Glutaric acid, alias: gum acid, α,γ-propanedicarboxylic acid, 1,3-propanedicarboxylic acid, can be used as an important chemical raw material and organic intermediate, and has a wide range of applications in various fields. In industry, glutaric acid can be used to synthesize polyvinyl chloride, polyester, polyamide and polyester plasticizer of polyvinyl chloride, etc. It can also be used to synthesize liquid polyester (improving the molecular structure of PET fiber, improving PET Fiber dyeability, improve dye uptake rate). In medicine, glutaric acid can be used to synthesize various sterilization lotions and medicines. In addition, in daily life, glutaric acid can be used in the formulation of detergents, the preparation of adhesives, and the preparation of sulfur-containing flue gases. ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P7/44C12N15/53C12N15/70C12N1/21C12R1/19
Inventor 陈可泉蔡沛沛王昕应晗笑王璟欧阳平凯
Owner NANJING TECH UNIV
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