Immobilized whole-cells, preparation method of immobilized whole-cells, and application in production of 3-aminopropionic acid

A whole cell and cell technology, applied in the direction of immobilization on or in inorganic carrier, immobilization on/in organic carrier, biochemical equipment and methods, etc., can solve the problem of low bacterial tolerance, low yield, etc. problem, to achieve the effect of improving catalytic activity, good biocompatibility, and strong designability

Pending Publication Date: 2020-06-23
南京凯诺生物科技有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

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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 an immobilized whole cell for the deficiencies of the prior art, the low yield and low bacterial tolerance of Escherichia coli in the process of biocatalyzing the production of 3-alanine A method for preparing 3-aminopropionic acid, the method has significantly improved the yield of 3-aminopropionic acid, and significantly improved the tolerance of bacteria in the biocatalytic process

Method used

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  • Immobilized whole-cells, preparation method of immobilized whole-cells, and application in production of 3-aminopropionic acid
  • Immobilized whole-cells, preparation method of immobilized whole-cells, and application in production of 3-aminopropionic acid
  • Immobilized whole-cells, preparation method of immobilized whole-cells, and application in production of 3-aminopropionic acid

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Embodiment 1 produces the construction of L-aspartic acid decarboxylase bacterial strain and cultivation process [1～3]

[0038] 1.1 Construction of strains

[0039] Extract the genomes of C.glutamicum, E.coli MG1655 and B.subtilis, use these three genomes as templates, and amplify the ADC gene panD by PCR (see Table 1). I double digestion, and then use T4 DNA ligase to connect to the pET28a vector that has also undergone double digestion, construct the recombinant plasmid pET28a-panDC.g, transform it into E.coli BL21 (DE3), and screen a single colony on the Kanna plate .

[0040] Table 1

[0041]

[0042] 1.2 Medium

[0043] Plate medium (L -1 ): peptone 10g, yeast powder 5g, sodium chloride 5g, agar 20g, kanamycin 0.05g, solvent is water.

[0044] LB medium (L -1 ): peptone 10g, yeast powder 5g, sodium chloride 5g, kanamycin 0.05g, and the solvent is water.

[0045] Sterilization method: the antibiotics were sterilized by filtration, and the rest were steril...

Embodiment 2

[0055] Example 2 Preparation process of immobilized whole cells

[0056] 2.1 Construction of strains

[0057] Refer to the construction of 1.1 bacterial strain in embodiment 1

[0058] 2.2 Medium

[0059] Refer to 1.2 medium in embodiment 1

[0060] 2.3 Plate culture

[0061] Refer to 1.3 plate culture in embodiment 1

[0062] 2.4 Seed liquid culture

[0063] Refer to 1.4 seed liquid culture in embodiment 1

[0064] 2.5 Shake flask fermentation culture

[0065] Refer to 1.5 shake flask fermentation culture in embodiment 1

[0066] 2.6 Preparation of immobilized whole cells

[0067] Resuspend the collected bacteria sludge with water to obtain a cell suspension with a concentration of OD600=15, mix 10 mL of a cell suspension with a concentration of OD600=15 with 5 mL of 45 mmol / L cobalt ion solution for 15 minutes, then add 5 mL of 80 mmol / L The dimethylimidazole solution was stirred at room temperature at a speed of 500 rpm for 30 to 60 minutes, and then washed with wa...

Embodiment 3

[0068] Embodiment 3 analytical method

[0069] 3.1 Catalyzed method for producing 3-aminopropionic acid

[0070] In 10mL of PBS buffer solution, add the whole cells prepared in Example 1 and the whole cells immobilized by the metal-organic framework material prepared in Example 2, so that the cell concentration is OD 600 =5, 35°C and 200rpm vibration reaction for 10h. After the decarboxylation reaction, the cells were removed by centrifugation, and the supernatant was taken for liquid phase detection.

[0071] 3.2 Method for detecting 3-aminopropionic acid

[0072] Take 100 μL of the sample, add 100 μL of 0.5mol / L sodium bicarbonate solution (pH 9.0), 100 μL of 1% 2,4-dinitrofluorophenylacetonitrile solution in turn, put it in a shaker at 60°C for 1 hour in the dark, and then add 700 μL 0.05mol / L PBS buffer (pH 7.0). 12000rpm high-speed centrifugation for 10min, and the supernatant was taken for liquid phase analysis.

[0073] Liquid phase analysis conditions: the chromat...

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Abstract

The invention discloses immobilized whole-cells, a preparation method of the immobilized whole-cells, and application in production of 3-aminopropionic acid. The preparation method of the immobilizedwhole-cells comprises the steps of: (1), culturing an L-aspartate decarboxylase strain, and centrifuging, so that thallus is obtained; (2), re-suspending the thallus obtained in the step (1) by usingwater, so that cell re-suspension solution is obtained; and (3), mixing the cell re-suspension solution obtained in the step (2) with ion solution, adding ligand solution, stirring, washing, and precipitating the solid part after being washed, so that precipitates, namely immobilized whole-cells, are obtained. Compared with the prior art, the method in the invention has the advantages that: the catalytic activity, the tolerance and the repeated utilization rate of cells in a catalytic system are improved.

Description

technical field [0001] The invention belongs to the field of whole cell catalysis and microorganism immobilization, and in particular relates to an immobilized whole cell, a preparation method thereof and an application in producing 3-alanine. Background technique [0002] 3-Alanine, also known as β-alanine, is the only natural β-type amino acid, which has very important physiological functions in organisms and is an indispensable medium in the synthesis of pantothenic acid. As one of the most potential three-carbon compounds, 3-alanine is widely used in medicine, construction, food and other fields. At present, the problems of harsh reaction conditions, serious pollution, and low conversion rate in the process of chemical synthesis of 3-alanine make the enzymatic synthesis of 3-alanine receive widespread attention. Using L-aspartic acid as a substrate to synthesize 3-alanine by whole-cell catalysis is a wise choice, which has many advantages, such as high stability, cost s...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C12N15/60C12N11/14C12N11/02C12P13/04
CPCC12N9/88C12N11/02C12N11/14C12N15/70C12P13/04C12Y401/01011
Inventor 陈可泉许晟王昕冯娇
Owner 南京凯诺生物科技有限公司
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