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Mutant zymoprotein of D-amino acid oxidase and preparation method of mutant zymoprotein

An amino acid and oxidase technology, applied in the fields of genetic engineering and enzyme engineering, can solve the problems of high price and rising cost, and achieve the effect of improving activity

Active Publication Date: 2013-05-15
HEBEI NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There are very few commercial products related to D-amino acid oxidase. At present, only the D-amino acid oxidase pKDAAO from pig kidney has been well developed and applied. However, due to the high price of this product, the cost has increased

Method used

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  • Mutant zymoprotein of D-amino acid oxidase and preparation method of mutant zymoprotein
  • Mutant zymoprotein of D-amino acid oxidase and preparation method of mutant zymoprotein
  • Mutant zymoprotein of D-amino acid oxidase and preparation method of mutant zymoprotein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] Example 1 D-amino acid oxidase gene daao Acquisition and expression vector construction

[0061] (1) daao gene acquisition

[0062] According to the original Arthrobacter glass fly ( Arthrobacter protophormiae , DSM20168) to design primers for the D-amino acid oxidase gene (GenBank ID. JX855922.1) to A. protophormiae Genomic DNA was used as a template, and a specific fragment was obtained by PCR amplification ( figure 1 , lane1). daao The amplification primers are:

[0063] Sense: 5′-TCC CATATG CCCACAGCACCGTTGAG-3′ (underlined as Nde I restriction site);

[0064] Anti-sense: 5′-ATA CTCGAG GCTGGCCGGCTCGCCA-3′ (underlined as xho I restriction site);

[0065] The PCR amplification reaction conditions were: pre-denaturation at 94°C for 4 min; denaturation at 94°C for 45 sec, annealing at 55°C for 1 min, extension at 72°C for 2 min, and 25 cycles; extension at 72°C for 10 min.

[0066] The amplified PCR fragment was recovered by gel, ligated with the vec...

Embodiment 2

[0070] Example 2 Mutant E115A / N119D / T286A expression vector construction

[0071] (1) Design mutation primers

[0072] According to the nucleotide sequences around 115, 119 and 286 of the D-amino acid oxidase to be mutated in the original Arthrobacter glass fly, design the following three pairs of site-directed mutagenesis primers:

[0073] The 115-position mutation primer is:

[0074] 115 anti-sense: 5'-CCTCCCGGGCGGATCTGC-3';

[0075] 115-bit sense: 5'-GGCAGATCCGCCCGGGAG-3';

[0076] The 119-position mutation primer is:

[0077] 119 anti-sense: 5′-TCTGCCGGACGGCGCCCAC-3′;

[0078] 119-position sense: 5′-TGGGCGCCGTCCGGCAGAT-3′;

[0079] The 286 mutation primers are:

[0080] 286 anti-sense: 5′-GAGCACGTCGCGGGCCAC-3′;

[0081] 286-bit sense: 5′-GTGGCCCGCGACGTGCTC-3′;

[0082] (2) Construction of mutant E115A expression vector

[0083] Based on the Quick Change Site-Directed Mutagenesis (QuickChange Site-Directed Mutagenesis), the recombinant expression vector pE...

Embodiment 3

[0089] Example 3 Expression and Purification of Mutant Enzyme and Wild-type Enzyme Protein

[0090] The plasmids pET-DAAO, pET-ApDAAO and the expression vectors of each mutant were transformed into Escherichia coli BL21(DE3) competent cells, and the transformants were selected and cultured overnight at 37°C in LB medium (containing 100 μg / mL ampicillin); Inoculate in 100mL LB liquid medium (containing 100μg / mL ampicillin) at a ratio of 1:100, culture at 37°C with shaking at 180rpm until OD 600 is 0.5, add 0.5mM isopropylthiogalactoside (isopropyl- β -D-thiogalactopyranoside, IPTG), induced expression overnight at 30°C. Centrifuge at 8000rpm for 10min at 4°C to collect the cells and ultrasonically disrupt them. Purify with Ni-NTA affinity chromatography, and the imidazole concentration in the eluent is 250mM to obtain purified wild-type proteins DAAO, ApDAAO, mutant proteins E115A, E115A / N119D and E115A / N119D / T286A.

[0091] The purified enzyme protein was identified by SD...

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Abstract

The invention discloses a mutant zymoprotein of D-amino acid oxidase and preparation method of the mutant zymoprotein. The preparation method comprises the following steps: according to homologous sequence comparison result, respectively performing mutation on 115, 119 and 286 sites of D-amino acid oxidase in primary Arthrobacterprotophormiae (DSM15035) through site-specific mutagenesis technology to construct three-site mutation expression vector Pet-e115A / N119D / T286A; and carrying out prokaryotic expression and purification to obtain zymoprotein. An apparent secondary speed constant Kcat / Km of the obtained mutant protein to substrate D-Met is 1.39*10 to the power of 5s-1.M-1, is 5.03 times of wild type DAAO (D amino acid oxidase), and is 55.96 times of pKDAAO protein of pig kidney source. The detection efficiency of partial D-amino acid can be improved, and the good application value is realized.

Description

[0001] Technical field [0002] The invention involves a mutant enzyme protein of a prokaryotic biological source D-amino acid oxidase, which belongs to the field of genetic engineering and enzyme engineering technology. Background technique [0003] D-amino acid oxidase: OxidoreDuctase, DAAO, EC 1.4.3.3) is a typical lutein enzymes based on the auxin adenine gonadine (FAD) as the auxiliary base, oxidation D-Amino acid amino acids generate corresponding ketone and ammonia.The reaction is as follows: [0004] Rchnh 2 COOH + E-FAD → RC = NHCOOH + E-FADH 2 [0005] E-FADH 2 + O 2 → E-FAD + H 2 O 2 [0006] RC = nhcooh + H 2 O → rcocooh + nh 3 [0007] The D-amino acid oxidase has a high level of stereo and broad-spectrum on the catalytic reaction substrate, which can be widely used in the qualitative and quantitative analysis of D-amino acids, biosensor, L-amino acid, and α-ketone acid production.In the 1990s, DAAO was applied in terms of biotechnology enzyme catalysis. It was ...

Claims

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Application Information

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IPC IPC(8): C12N9/06C12N15/53C12N15/70C12N1/21C12Q1/26C12R1/06C12R1/19
Inventor 鞠建松徐书景赵宝华李辉欣冯利伟
Owner HEBEI NORMAL UNIV
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