Enzyme catalyst and preparation method and application thereof in production of 3-aminopropionic acid

An enzyme catalyst and reaction technology, which is applied in the direction of biochemical equipment and methods, enzymes, lyases, etc., can solve the problems of poor controllability of the catalytic effect of immobilized enzyme catalysts, cumbersome operation process of enzyme catalysts, etc., and achieve good reuse rate, The preparation process is simple and convenient, and the effect of low equipment demand

Inactive Publication Date: 2020-06-19
南京凯诺生物科技有限公司
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  • Summary
  • 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 the cumbersome operation process of preparing the enzyme catalyst and the poor controllability of the catalytic effect of the metal-organic framework material immobilized enzyme catalyst, and provides a method for preparing 3-alanine by immobilized whole cells method, the method has significantly improved the yield of 3-aminopropionic acid, and significantly improved the performance of the immobilized enzyme catalyst

Method used

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  • Enzyme catalyst and preparation method and application thereof in production of 3-aminopropionic acid
  • Enzyme catalyst and preparation method and application thereof in production of 3-aminopropionic acid
  • Enzyme catalyst and preparation method and application thereof in production of 3-aminopropionic acid

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Embodiment 1 produces the construction of L-aspartic acid decarboxylase bacterial strain and cultivation process [1～3]

[0041] 1.1 Construction of strains

[0042]Extract the genomes of C.glutamicum ATCC13032, E.coli MG1655, and B.subtilis, and use these three genomes as templates to amplify the ADC gene panD by PCR. The PCR product is recovered from agarose and digested with Nco I and Xho I. , and then use T4 DNA ligase to connect to the pET28a vector that has also undergone double digestion to construct the recombinant plasmid pET28a-panD C.g , Transformed into E.coli BL21(DE3), a single colony was screened out on the Kanna plate.

[0043] 1.2 Medium

[0044] Plate medium (L -1 ): peptone 10g, yeast powder 5g, sodium chloride 5g, agar 20g, kanamycin 0.05g.

[0045] LB medium (L -1 ): peptone 10g, yeast powder 5g, sodium chloride 5g, kanamycin 0.05g.

[0046] Sterilization method: the antibiotics were sterilized by filtration, and the rest were sterilized by hi...

Embodiment 2

[0056] The preparation process of embodiment 2 two-step method immobilized L-aspartic acid decarboxylase

[0057] 2.1 Preparation of L-aspartate decarboxylase and surfactant mixture

[0058] The thalli that embodiment 1 is collected is formulated into 20mL OD with water 600 =10 resuspension, add 5mL of TX-100 to the resuspension, stir at room temperature at a stirring speed of 700rpm for 45 minutes, then centrifuge at 6000rpm for 10 minutes and take the supernatant to obtain L-aspartic acid Decarboxylase and Surfactant Mixture A.

[0059] 2.2 Preparation of surfactant-modified immobilized L-aspartate decarboxylase

[0060] Measure 20mL of L-aspartate decarboxylase and surfactant mixed solution A, add 10mL 25mmol / L cobalt ion solution and mix for 10 minutes, then add 17mL 100mmol / L dimethylimidazole solution, Stir at 700rpm for 30 minutes, then wash with water and centrifuge to leave the precipitate to obtain surfactant-modified immobilized L-aspartate decarboxylase, which i...

Embodiment 3

[0061] The preparation of embodiment 3L-aspartic acid decarboxylase

[0062] The thalli that embodiment 1 is collected is formulated into 20mL OD with water 600 = 1-10 resuspension liquid, sonicate for 15 minutes, and centrifuge at 3000-6000 rpm to keep the supernatant, which is the L-aspartate decarboxylase solution.

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Abstract

The invention discloses an enzyme catalyst and a preparation method and application thereof in production of 3-aminopropionic acid. The preparation method comprises the following steps: (1) culturingan L-aspartate decarboxylase strain, and performing centrifuging to obtain thalli; (2) re-suspending the thalli obtained in the step (1) with water to obtain a resuspension, adding a surfactant into the resuspension, and performing stirring and centrifuging to obtain a supernatant; and (3) adding metal ions into the supernatant obtained in the step (2), performing mixing, adding a ligand solution,and performing stirring, washing and centrifuging to obtain a precipitate, namely the enzyme catalyst. Compared with the prior art, the method simplifies the operation process of extracting enzyme from cells, prepares the nano-material immobilized enzyme catalyst in a two-step rapid synthesis form, is simple and convenient in preparation process, and has low requirement on equipment; and the method realizes recycling and good repeated utilization rate of L-aspartate decarboxylase.

Description

technical field [0001] The invention belongs to the field of enzyme catalysis and immobilized enzymes, and specifically relates to an enzyme catalyst and its preparation method and application in the production of 3-aminopropionic acid. Background technique [0002] 3-Alanine, also known as β-alanine, as one of the most potential three-carbon compounds, can be used to synthesize vitamin B5, calcium pantothenate, balsalazide, carnosine, poly-β-alanine, etc. Products are widely used in medicine, chemical industry, beauty, food, environment and so on. At present, most of the chemical synthesis of 3-aminopropionic acid requires relatively harsh process conditions, and there are many by-products and environmental pollution problems, so that the biocatalytic synthesis of 3-aminopropionic acid has attracted extensive attention. Using L-aspartic acid as a substrate to synthesize 3-alanine in a whole-cell catalyzed way is a green and environmentally friendly way. However, problems ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/88C12P13/04
CPCC12N9/88C12P13/04C12Y401/01011
Inventor 陈可泉冯娇王昕
Owner 南京凯诺生物科技有限公司
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