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
CN103103167AActive Publication Date: 2013-05-15HEBEI NORMAL UNIV
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Patent Information

Authority / Receiving Office
CN · China
Current Assignee / Owner
HEBEI NORMAL UNIV
Publication Date
2013-05-15

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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.
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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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