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Immobilized L-aspartic acid-alpha-decarboxylase and preparation method and application thereof

A technology of aspartic acid and decarboxylase, applied in the field of bioengineering, can solve problems such as air pollution, environmental pollution, and high production costs, and achieve the effect of improving biological activity

Active Publication Date: 2017-09-15
NANJING UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There are three processes in chemical synthesis: (1) Acrylonitrile method: In this method, acrylonitrile is reacted with ammonia in diphenylamine and tert-butanol solution to produce β-aminopropionitrile, which is then obtained by alkaline hydrolysis; the disadvantages of this method There are more than 40% NaCl and impurities in the product, which need repeated purification, and the NH produced in the reaction process pollutes the atmosphere; (2) β-aminopropionitrile method: β-aminopropionitrile reacts with barium hydroxide to produce β-amino Barium propionate and nitrogen, with CO 2 , the barium salt is precipitated out to produce β-alanine, and the barium ion is removed with a resin. The disadvantage of this method is that the production cost is high; (3) succinimide degradation method: succinimide is generated in alkaline sodium chlorate solution For β-alanine, adjust the pH of the reaction solution with hydrochloric acid, precipitate the inorganic salt with 3 times the amount of 95% ethanol, dilute the filtrate with 4 times the amount of distilled water, exchange with ion exchange resin, decolorize the exchange liquid, concentrate and crystallize, this method requires A large amount of ethanol, high production costs, and high safety requirements for the production site
In a word, the raw materials, intermediates and by-products of the chemical synthesis method are poisonous and seriously pollute the environment.

Method used

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  • Immobilized L-aspartic acid-alpha-decarboxylase and preparation method and application thereof
  • Immobilized L-aspartic acid-alpha-decarboxylase and preparation method and application thereof
  • Immobilized L-aspartic acid-alpha-decarboxylase and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0071] Example 1: Construction of expression plasmid vector containing L-aspartic acid-α-decarboxylase gene.

[0072] According to the sequence of Escherichia coli (Escherichia coli str.K-12substr.MG1655) gene reported on Genbank, the primers were designed using Vector NTI software. The primer sequences are shown in Table 1:

[0073] Table 1 Primer Sequence

[0074]

[0075]

[0076]According to the instructions provided by the manufacturer, the genomic DNA of Escherichia coli strain N (Escherichia coli str. / L) Agarose gel electrophoresis to detect the obtained genomic DNA, respectively using the extracted Escherichia coli genomic DNA as a template for PCR amplification.

[0077] The PCR (polymerase chain reaction) amplification system is: 2 μL of genomic DNA, 2 μL of primers panD-up and primer panD-down, 4 μL of dNTP, 5 μL of 10×Taq buffer, 1 μL of Taq enzyme, ddH 2 O 34 μL;

[0078] The PCR reaction program was: pre-denaturation at 94°C for 2 minutes; denaturation ...

Embodiment 2

[0089] Example 2: Induced expression of genetically engineered bacteria.

[0090] Two ways are used to induce expression of the genetically engineered bacteria obtained in Example 1:

[0091] (1) Prepare 1L of seed solution, the medium is LB liquid medium (peptone 10g / L, yeast powder 5g / L, NaCl 10g / L), after sterilizing at 121℃ for 30min, put it into several 500mL wide-mouth triangles in the bottle. Inject a loop of the genetically engineered bacteria in Example 1 into the seed solution with an inoculation needle, and place it on a shaker at 37° C. at a speed of 200 rpm for overnight cultivation. Prepare 1000 mL of LB medium containing 10 g / L of peptone, 5 g / L of yeast powder, and 10 g / L of NaCl, and distribute it in a wide-mouthed Erlenmeyer flask with a capacity of 500 mL. The volume of each bottle is 100 mL; ℃ autoclave for 30 minutes. After the culture medium is cooled, add 1mL of the overnight cultured seed solution, place the Erlenmeyer flask on a shaker at 37°C at a ...

Embodiment 3

[0093] Embodiment 3: Ni column pure enzyme and ultrafiltration concentrate

[0094] Take 500mL of genetically engineered bacterial fermentation broth, centrifuge at 8500rpm for 15min, wash with normal saline and centrifuge again, and ultrasonically crush to obtain the supernatant, connect to a peristaltic pump, pass through two 5mL Ni columns connected in series, and then filter the obtained eluate through a 3000Da ultrafiltration Tube, 5500rpm, 30min The upper layer is the concentrate. Detected by SDS-PAGE, the results are as follows Figure 4 As shown, there are characteristic proteins of corresponding size in whole cells and supernatants, but not in the permeate and washing liquid, and there are also characteristic proteins in the eluate, and the amount is very large, and it is more obvious in the concentrated solution, so the enzyme can be used Purification was carried out by any of the two methods of Ni column adsorption and ultrafiltration tube concentration.

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Abstract

The invention discloses an immobilizing method of L-aspartic acid-alpha-decarboxylase, and belongs to the technical field of biological engineering, TiO2 modified by a poly amino acid is used as a carrier, the L-aspartic acid-alpha-decarboxylase is bonded onto the surface of the carrier, and the poly amino acid is gamma-polyglutamic acid or epsilon-polylysine. Immobilization studies on the L-aspartic acid-alpha-decarboxylase by respective use of the modified carrier and an unmodified carrier find that the enzyme carrier content of the modified carrier is about 8 times of that of the unmodified carrier.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and specifically relates to an immobilized L-aspartic acid-α-decarboxylase and a preparation method and application thereof. Background technique [0002] L-aspartate-α-decarboxylase (L-aspartate-α-decarboxylase, EC4.1.1.11, abbreviated as PanD, ADC), also known as L-aspartate-1-decarboxylase, can catalyze the removal of The α-carboxyl group of L-aspartic acid produces β-alanine. PanD is an unusual enzyme that relies on its own processing of acetonephthalein for its catalytic ability. Ramjee et al. proposed that during the synthesis of tetramer enzymes, each monomer first forms an inactive zymogen (π-protein), in which three monomers are cleaved on a special Gly-Ser bond to form a C-terminal containing The β-subunit of the hydroxyl group and the α-subunit containing the pyruvyl group at the N-terminal, after the zymogen is processed, are labeled with fluorescein thiosemicarbazide, and it...

Claims

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Application Information

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IPC IPC(8): C12N11/14C12N9/88C12P13/06C12R1/19
CPCC12N9/88C12N11/14C12P13/06C12Y401/01011Y02P20/50
Inventor 徐虹詹伊婧梁金丰殷文峰李莎徐铮
Owner NANJING UNIV OF TECH
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