Method for producing glutaric acid by double-cell adhesion coupling method

A technology of glutaric acid and cells, which is applied in the field of double-cell adhesion coupling method to produce glutaric acid, which can solve the problems of poor catalytic effect and low mass transfer efficiency, so as to improve permeability, improve catalytic efficiency and reduce use cost Effect

Pending Publication Date: 2020-06-19
南京凯诺生物科技有限公司
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

[0004] Purpose of the invention: the technical problem to be solved by the present invention is to provide a double-cell adhesion coupling method for the production of pentadiol for the poor catalytic effect caused by the low mass transfer efficiency between the two cells in the prior art. acid method, this method has significantly improved the production of glutaric acid, and significantly improved the performance of the double-cell biocatalyst

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  • Method for producing glutaric acid by double-cell adhesion coupling method
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  • Method for producing glutaric acid by double-cell adhesion coupling method

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Embodiment 1

[0037] Construction and cultivation process of embodiment 1 bacterial strain [1]

[0038] 1.1 Construction of strains

[0039] The preparation process of E.coli BL21 (DE3) competent refers to the operation of the E. coli competent preparation kit of Baori Medical Biotechnology Company;

[0040] Recombinant plasmids pET22b-DavAB and pACYC-GabTD were provided by our laboratory, and the plasmids were extracted using the plasmid mini-extraction kit from TIANGEN Company;

[0041] The recombinant plasmid pET22b-DavAB was introduced into the competence of E.coli BL21(DE3), and the recombinant strain E.coli BL-22AB overexpressing DavAB was obtained;

[0042] The recombinant plasmid pACYC-GabDT was introduced into the competence of E.coli BL21(DE3), and the recombinant strain E.coli BL-YDT overexpressing GabDT was obtained.

[0043] 1.2 Medium

[0044] LB medium: tryptone 10g / L, yeast extract 5g / L, sodium chloride 5g / L, solvent is water, pH 7.0.

[0045] Solid LB medium: tryptone ...

Embodiment 2

[0054] Example 2 Preparation process of double-cell adhesion coupling

[0055] 2.1 Permeability modification of recombinant strain E.coli BL-22AB

[0056] Resuspend the recombinant bacterial strain E.coli BL-22AB bacterium sludge obtained in Example 1 with PBS buffer at pH 7.0 to obtain OD 600 =5 concentration of the recombinant bacterial strain E.coli BL-22AB cell solution, add 0.5% (volume concentration) TritonX-100 surfactant in the solution, stir 3 hours under the condition of 200r / min, 37 ℃, promptly obtain pass Cell solution of recombinant strain E.coli BL-22AB with increased permeability.

[0057] 2.2 Preparation of adhesive coating on the surface of recombinant strain E.coli BL-22AB after permeability modification

[0058] Take 10mL of the recombinant bacterial strain E.coli BL-22AB cell solution with increased permeability obtained in 2.1, mix it with 10mL of 2g / L dopamine solution in pH 7.5 Tris-HCl buffer, and mix it under the conditions of 200r / min and 18℃ After...

Embodiment 3

[0063] Embodiment 3 analytical method

[0064] 3.1 Catalytic method

[0065] (1) Traditional two-cell coupling method: resuspend the microbial cells collected in Example 1 with PBS at pH 7.0 to the cell solution concentration OD of E.coli BL-22AB and E.coli BL-YDT in the system 600 =5, and add the prepared L-lysine mother liquor and α-ketoglutaric acid mother liquor with pH 7.0, so that the final concentrations of L-lysine and α-ketoglutaric acid in the system are both 10g / L , catalyzed at 37°C for 30 hours. After the reaction, the microbial cell pellet was removed by centrifugation, and the supernatant was taken for liquid phase detection.

[0066] (2) Two-cell adhesion coupling method: Resuspend the coupling adhesion sludge C collected in Example 2 with PBS of pH 7.0 to the cell solution concentration OD of E.coli BL-22AB and E.coli BL-YDT in the system 600 =5, and add the prepared L-lysine mother liquor and α-ketoglutaric acid mother liquor with pH 7.0, so that the final...

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Abstract

The invention discloses a method for producing glutaric acid by a double-cell adhesion coupling method. The method comprises the following steps of: (1) resuspending recombinant strain E.coli BL-22ABbacterial sludge by using a PBS buffer solution, then adding a surfactant, and stirring; (2) mixing the material obtained in the step (1) with a dopamine solution, stirring, then adding a mannose solution, stirring, and centrifuging to obtain bacterial sludge; (3) resuspending the recombinant strain E.coli BL-YDT bacterial sludge by using the PBS buffer solution to obtain a cell solution A; resuspending the bacterial sludge obtained in the step (2) by using the PBS buffer solution to obtain a cell solution B; mixing the cell solution A with the cell solution B, stirring, adding the dopamine solution, stirring, and centrifuging to obtain coupled adhesion bacterial sludge; and (4) resuspending the coupled adhesion bacterial sludge by using the PBS buffer solution, then adding an L-lysine solution and an alpha ketoglutaric acid solution, reacting, and centrifuging to obtain the glutaric acid.

Description

technical field [0001] The invention belongs to the technical field of microbial cell catalysis and glutaric acid production, and in particular relates to a method for producing glutaric acid by a double-cell adhesion coupling method. Background technique [0002] Glutaric acid is an important C5 platform chemical, which is widely used in the field of polyamide materials, and can also be used as plasticizers, corrosion inhibitors, rigid-flexible lanthanide coordination polymers and other material fields. The process of chemical synthesis of glutaric acid has problems such as expensive raw materials, serious pollution, and low conversion rate. Biosynthesis of glutaric acid has attracted extensive attention in recent years. In microorganisms, glutaric acid can be catabolized by L-lysine through the 5-aminovaleric acid (5-AMV) pathway. The intermediate metabolite 5-aminovaleric acid accumulates seriously in the process of catalyzing the synthesis of glutaric acid from L-lysine...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P7/44C12R1/19
CPCC12P7/44
Inventor 陈可泉王昕许晟
Owner 南京凯诺生物科技有限公司
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