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Recommbined D-amino acid oxidase

一种氨基酸、氧化酶的技术,应用在生物工程领域,能够解决比活低等问题

Inactive Publication Date: 2007-10-10
BIORIGHT WORLDWIDE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But the specific activity of these two kinds of enzymes catalyzing cephalosporin C is all low (Simonetta, et al., Biochimica et Biophysica Acta, 914:136-142 (1987); U.S.Pat.No.5,453,374; U.S.Pat.No.5,208,155 )

Method used

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  • Recommbined D-amino acid oxidase

Examples

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Effect test

example 1

[0021] Example 1 Construction of D-amino acid oxidase gene recombinant plasmid pRSET-A-DAO

[0022] According to the known 5' and 3' end sequences of the Trichomona trichomona D-amino acid oxidase gene (Gonzalez, F.J., Montes, J., Martin, F., Lopez, M.C., Ferminan, E., Catalan, J., Galan, M.A. Dominguez, A. Molecular cloning of TvDAO1, a gene encoding aD-amino acid oxidase from Trigonopsis variabilis and its expression in Saccharomyces cerevisiae and Kluyveromyces lactis. Yeast 13: 1399-1408; 1997) designed primers as follows:

[0023] 5'-NdeI (introduced NdeI restriction site):

[0024] 5’-TAGGGCTGA CATATG GCTAAAATCGTTGTTATTGGTGC-3'

[0025] (Sequence 7)

[0026] 3'-BglII (BglII restriction site introduced):

[0027]5’-TAGGGCTGA AGATCT CTAAAGGTTTGGACGAGTAAGAGC-3' (SEQ ID NO: 8)

[0028] Using the plasmid pJL (Yang Yunliu et al., Chinese patent application publication number: CN 1371999A) as a template, using the above two primers, under the action of Pfu DNA polymerase...

example 2

[0034] Construction of example 2 pRSET-kan vector

[0035] In order to remove the ampicillin resistance gene from pRSET-A, primers were designed according to the sequence of the vector pRSET-A as follows:

[0036] VET-F: 5'-CTGTCAGACCAAGTTTACTCATATATACTTTAG-3'

[0037] (Sequence 9)

[0038] VET-R: 5'-ACTCTTCCTTTTTCAATATTATTGAAGC-3' (SEQ ID NO: 10)

[0039] For amplifying the kanamycin resistance gene from the carrier pET-28b (Novogen), the primers are designed as follows according to the sequence of the carrier pET-28b (Novogen):

[0040] KAN-F: 5'-ATGAGTCATATTCAACGGGAAAC-3' (SEQ ID NO: 11)

[0041] KAN-R: 5'-TTAGAAAAACTCATCGAGCATCAAATG-3' (SEQ ID NO: 12)

[0042] The PCR conditions for amplifying the pRSET-A fragment that removes the ampicillin resistance gene are: 50ng pRSET-A, 0.4μM VET-F, 0.4μM VET-R, 50μM dATP, 50μM dTTP, 50μM dCTP, 50μM dGTP, 20mM Tris-HCl (pH 8.8), 10mM KCl, 10mM (NH 4 ) 2 SO 4 , 2mM MgSO 4 , 0.1% Triton X-100, 2.5U Pfu DNA polymerase, adjust t...

example 3

[0047] Example 3 Construction of recombinant D-amino acid oxidase GHA

[0048] The recombinant D-amino acid oxidase GHA was constructed by site-directed mutagenesis. The site-directed mutagenesis technique mainly refers to the description in the book PCR Protocols (Editors: John M.S. Bartlett and David Stirling. Totowa, N.J.: Humana Press, 2003.).

[0049] According to the sequence (sequence 1) of the Trichomona trichomona D-amino acid oxidase gene cloned in Example 1, the primers are designed as follows:

[0050] Primer A: 5'TAGGGCTGA CATATG GCTAAAATCGTTGTTATTG-3' (SEQ ID NO: 14)

[0051] Primer B: 5'TAGGGCTGA AGATCT CTAAAGGTTTGGACGAG-3' (SEQ ID NO: 15)

[0052] Primer C 1: 5'-GCAGGTGCCAACTGGCTC CCG TTTTACGATGGAGGCAAG-3' (SEQ ID NO: 16)

[0053] Primer D: 5'-GAGCCAGTTGGCACCTGCCCAAGG-3' (SEQ ID NO: 17)

[0054] Primers A and B are a pair of outer primers. Primer A contains an NdeI restriction site, and some bases overlap with the 5' end sequence of the D-amino acid o...

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Abstract

A recombination D- amino acid oxidase that is 25% higher than parents in property of catalysis rhzomorph C.

Description

technical field [0001] The invention relates to the technical field of bioengineering, in particular to the preparation and application of a new D-amino acid oxidase. The two high-activity recombinant D-amino acid oxidases provided by the invention can be used to convert cephalosporin C (cephalosporin C) into glutaryl-7-aminocephalosporanic acid (glutaryl-7-aminocephalosporanic acid). Background technique [0002] The mother nucleus of semi-synthetic cephalosporins, 7-aminocephalosporanic acid (7-aminocephalosporanic acid), can be obtained by chemically cracking cephalosporin C. However, the chemical method uses a large amount of toxic chemical reagents, which pollutes the environment, and the reaction steps are complicated and the yield is low. In recent years, enzymatic method has become another main process for preparing 7-aminocephalosporanic acid. Enzymatic conversion of cephalosporin C includes two steps: (1) D-amino acid oxidase oxidizes cephalosporin C (cephalospor...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/53C12N9/02C12N15/63C12P21/02C12N9/06
CPCC12N9/0024
Inventor 王骏萧游龙叶康坚曾实现曾伟基游明翰吕旭新
Owner BIORIGHT WORLDWIDE
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