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A kind of l-aspartic acid β-decarboxylase mutant and its application

A technology of aspartic acid and decarboxylase, which is applied in the field of bioengineering, can solve the problems of long time, low specific enzyme activity, and low catalytic activity, and achieve good enzymatic properties and improved acid stability of mutants

Active Publication Date: 2021-01-29
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Most of the L-aspartate β-decarboxylases discovered so far have poor acid stability and low catalytic activity under neutral conditions (pH 7.0)
Xu Hong et al obtained a strain of Pseudomonas with L-aspartic acid β-decarboxylase activity through mutagenesis, the catalytic conversion rate of the substrate L-aspartic acid is close to 100%, but it takes 5 days to catalyze The reaction time is too long (cited literature: Xu Hong, Wang Xuegen, Liu Jirui, etc. Utilize Pseudomonas aspartate_decarboxylase to efficiently produce L_alanine [J]. Journal of Nanjing Institute of Chemical Technology, 1995, 17( 1).). Wang et al expressed the Asd enzyme derived from Pseudomonas sp.ATCC 19121 in Escherichia coli heterologously. cloning of the aspartate 4-decarboxylase gene from Pseudomonas sp.ATCC 19121 and characterization of the bifunctional recombinant enzyme[J].Appl Microbiol Biotechnol,2006,73(2):339-348.)

Method used

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  • A kind of l-aspartic acid β-decarboxylase mutant and its application
  • A kind of l-aspartic acid β-decarboxylase mutant and its application
  • A kind of l-aspartic acid β-decarboxylase mutant and its application

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Experimental program
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Effect test

Embodiment 1

[0039] The construction of embodiment 1 recombinant escherichia coli

[0040] The Asd gene sequence was artificially synthesized (positions 1415236 to 1416837 of the gene whose NCBI accession number is AP019740.1), and specific primers P1 and P2 were designed (the underlined parts are the restriction endonuclease sites of EcoRI and XhoI, respectively).

[0041] Table 1 Primer list

[0042] P1 5'-CCG GAATTC ATGGGGAATGTAGATTATTCTAAAT-3'

P2 5'-CCG CTCGAG TCAGGACTCATCTTTTTTTAGTTCCC-3'

[0043] The L-aspartic acid β-decarboxylase gene Asd was digested by EcoRI and XhoI and connected to the expression vector pET 28a(+) digested with the same restriction enzyme to obtain the recombinant plasmid pET 28a-ArAsd.

[0044] Existing studies have shown that changing the charged amino acid residues on the surface of the enzyme molecule has a certain effect on the optimum reaction pH of the enzyme (Alan J. Russell A R F. Rational modification of enzyme catalysi...

Embodiment 2

[0053] Example 2 Expression and purification of L-aspartate β-decarboxylase

[0054] BL21 / pET28a-N35D, BL21 / pET28a-A179E recombinant Escherichia coli were inoculated in 5 mL of LB medium with a kanamycin concentration of 50 μg / mL, and cultured overnight at 37°C with shaking at 200 rpm. The above overnight culture was inoculated into 2YT medium containing 50 μg / mL of kanamycin at an inoculum size of 1%, and cultured with shaking at 37°C and 200 rpm until the bacterial liquid OD 600 To 0.6-0.8, add IPTG to a final concentration of 0.2mmol / L, induce culture at 20°C for about 20h to obtain bacteria. After collecting the bacteria by centrifugation at 6000rpm, they were ultrasonically crushed, and the protein was purified using a His Trap HP affinity column, and the target protein was detected by SDS-PAGE. The results were as follows: figure 1 shown. The specific enzyme activity of the pure enzyme was measured, and the specific enzyme activity of the enzyme mutants N35D and A179E ...

Embodiment 3

[0055] Example 3 pH Stability Determination

[0056] like figure 2 As shown, the enzymatic activity of L-aspartic acid β-decarboxylase mutants N35D and A179E increased by 238% and 244% respectively under the condition of pH 7.0.

[0057] After 12 hours of treatment at pH 4.5, the residual enzyme activities of N35D and A179E mutants remained 83.9% and 85.5%, respectively, compared with 24.8% of the wild-type enzyme, and the acid stability of the mutants was significantly improved.

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Abstract

The invention discloses a L-aspartic acid β-decarboxylase mutant and application thereof, belonging to the technical field of bioengineering. For the L-aspartic acid β-decarboxylase mutants N35D and A179E provided by the present invention, the catalytic enzyme activity is increased by 238% and 244% respectively under the condition of pH 7.0. After 12 hours of treatment at pH 4.5, 83.9% and 85.5% of the remaining enzyme activity remained, compared with 24.8% of the remaining enzyme activity of the wild-type enzyme in the control, the acid stability of the mutant was significantly improved. Therefore, the L-aspartic acid β-decarboxylase mutants N35D and A179E provided by the present invention have better enzymatic properties.

Description

technical field [0001] The invention relates to a mutant of L-aspartic acid β-decarboxylase and application thereof, belonging to the technical field of bioengineering. Background technique [0002] L-alanine is an amino acid with great value, which is widely used in daily chemical, food and pharmaceutical industries. In the field of daily chemicals, L-alanine is the raw material for the synthesis of amino acid surfactants; in the field of food, L-alanine has a sweet taste and can be made into mixed condiments with sodium glutamate, which can be prepared and significantly improve food Flavor without destroying the original flavor of food; in the field of medicine, L-alanine is the main raw material for the synthesis of vitamin B6 and L-aminopropanol (the precursor of levofloxacin hydrochloride synthesis); at the same time, L-alanine is the compound The main component of amino acid injection and infusion is also a myocardial function enhancer. [0003] In the production of ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/88C12N15/60C12N15/70C12N1/21C12P13/06C12R1/19
CPCC12N9/88C12N15/70C12P13/06C12Y401/01011
Inventor 周哲敏刘中美于佳印赵庭周丽崔文璟郭军玲
Owner JIANGNAN UNIV
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