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Use of recommbined D-amino acid oxidase

A technology of amino acid and oxidase, applied in the field of bioengineering, can solve the problem of low specific activity

Active Publication Date: 2008-05-07
BIORIGHT WORLDWIDE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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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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  • Use of recommbined D-amino acid oxidase
  • Use of recommbined D-amino acid oxidase

Examples

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example 1

[0022] Example 1 Construction of D-amino acid oxidase gene recombinant plasmid pRSET-A-DAO

[0023] 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 a D-aminoacid oxidase from Trigonopsis variabilis and its expression in Saccharomyces cerevisiae and Kluyveromyces lactis. Yeast 13: 1399-1408; 1997) designed primers as follows:

[0024] 5'-Ndel (introduce Ndel restriction site):

[0025] 5’-TAGGGCTGA CATATG GCTAAAATCGTTGTTATTGGTGC-3' (SEQ ID NO: 7)

[0026] 3'-BgIII (BgIII 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 (Promega...

example 2

[0031] Construction of example 2 pRSET-kan vector

[0032] 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:

[0033] VET-F: 5'-CTGTCAGACCAAGTTTACTCATATATACTTTAG-3' (SEQ ID NO: 9)

[0034] VET-R: 5'-ACTCTTCCTTTTTCAATATTATTGAAGC-3' (SEQ ID NO: 10)

[0035] 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):

[0036] KAN-F: 5'-ATGAGTCATATTCAACGGGAAAC-3' (SEQ ID NO: 11)

[0037] KAN-R: 5'-TTAGAAAAACTCATCGAGCATCAAATG-3' (SEQ ID NO: 12)

[0038] The PCR conditions for amplifying the pRSET-A fragment that removes the ampicillin resistance gene are: 50ngpRSET-A, 0.4μM VET-F, 0.4μM VET-R, 50μM dATP, 50μM dTTP, 50μM dCTP, 50μM dGTP, 20mM Tris-HCl (pH8.8), 10mM KCl, 10mM (NH 4 ) 2 SO 4 , 2mM MgSO 4 , 0.1% Triton X-100, 2.5U Pfu DNA polymerase, adjust the reactio...

example 3

[0041] Example 3 Construction of recombinant D-amino acid oxidase GHA

[0042] 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.).

[0043] 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:

[0044] Primer A: 5'-TAGGGCTGA CATATG GCTAAAATCGTTGTT ATTG-3' (SEQ ID NO: 14)

[0045] Primer B: 5'-TAGGGCTGA AGATCT CTAAAGGTTTGGACGAG-3' (SEQ ID NO: 15)

[0046] Primer C1: 5'-GCAGGTGCCAACTGGCTC CCG TTTTACGATGGAGGCAAG-3' (SEQ ID NO: 16)

[0047] Primer D: 5'-GAGCCAGTTGGCACCTGCCCAAGG-3' (SEQ ID NO: 17)

[0048] Primers A and B are a pair of outer primers. Primer A contains an Ndel restriction site, and some bases overlap with the 5' end sequence of the D-amino aci...

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Abstract

A recombinative D- amino acid oxidase and its DNA code sequence. Activity to catalysis rhzomorphC of the enzyme is 25% higher than that of its parents at least.

Description

[0001] This application is a divisional application of the invention patent application with the application number 200410030842.8 submitted by the applicant Bairui Global Co., Ltd. on April 8, 2004. technical field [0002] 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 [0003] 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 met...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P35/00
Inventor 王骏叶康坚曾伟基吕旭新萧游龙曾实现游明翰
Owner BIORIGHT WORLDWIDE
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