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Cephalosporin C acrylase and its vector and application

A technology of acylase and acylase encoding, applied in the field of cephalosporin C acylase, can solve the problem of inability to catalyze CPC substrates, and achieve the effects of high expression activity, high catalytic activity, and high product tolerance

Inactive Publication Date: 2010-06-02
TSINGHUA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Natural GL-7-ACA acylase can only catalyze GL-7-ACA to generate 7-ACA, but cannot catalyze CPC substrate; while natural CPC acylase can catalyze CPC to generate 7-ACA in one step, but it has no effect on CPC The catalytic activity of GL-7-ACA is only 2-4% of the catalytic activity of GL-7-ACA (Li Y et al. Eur JBiochem, 1999,262 (3): 713-719.Saito Y et al. Appl Envir Microb, 1996, 62 (8) : 2919-2925)

Method used

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  • Cephalosporin C acrylase and its vector and application
  • Cephalosporin C acrylase and its vector and application
  • Cephalosporin C acrylase and its vector and application

Examples

Experimental program
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Effect test

Embodiment 1

[0043] Design and Synthesis of Cephalosporin C Acylase Gene Sequence

[0044] (1) CPC acylase gene design of the present invention: the protein sequence of Pseudomonas sp.SE83cephalosporin acylase II in Genebank (SEQ ID NO: 1, Genbank AAA25690) was used, and six mutation sites in SEQ ID NO: 3 were introduced at the same time: Val122Ala-Gly140Ser-Phe58βArg-Ile75βThr-Ile176βVal-Ser471βCys (WO2005 / 014821A1), using the online server DNAWorks (URL: http: / / helixweb.nih.gov / dnaworks) to reverse design the gene sequence of CPC acylase. According to the codon degeneracy and preference of E. coli, the above-mentioned CPC acylase gene sequence redesigned for high expression in E. coli hosts is SEQ ID NO: 4, in which E. coli generally rare codons (low frequency of use) 20%) decreased from 76 of the wild gene SEQ ID NO: 2 to 43, accounting for 5.6% of the whole gene; highly rare codons (use frequency less than 10%) decreased from 46 of SEQ ID NO: 2 to 0; GC content is reduced from 68.99% ...

Embodiment 2

[0050] Construction of Inducible Expression Vector pET28-CPCacy and Transformant BL21(DE3) / pET28-CPCacy

[0051](1) Construction of plasmid pET28-CPCacy: the plasmid vector pUC18-acy obtained in Example 1 was digested with BamHI / HindIII double enzymes. The enzyme digestion reaction volume is 20 μL, the amount of plasmid is 4 μL, the amount of each enzyme is 1 μL, the buffer is 2 μL, and 20 μL is made up with sterile water. React overnight at 37°C to obtain the digested product CPC acylase gene. The plasmid vector pET28a (Novagen Company) was cut with the same method to obtain the linear plasmid backbone of pET28a. After purifying the digested product with a PCR product recovery kit (Tiangen Biochemical Technology (Beijing) Co., Ltd.), the CPC acylase gene and the linearized plasmid backbone of pET28a were ligated at 4°C for 14 hours with T4 DNA ligase. The ligation reaction was transformed into competent cells of the host strain E.coli JM109, spread on LB plates (kanamycin r...

Embodiment 3

[0054] Construction of Constitutive CPC Acylase Expression Vector and Transformant

[0055] The construction method of the plasmid pMKC-CPCacy is the same as that in Example 2 (1), and the whole gene of CPC acylation enzyme is obtained by double digestion with BamHI / HindIII. At the same time, the plasmid vector pMKC-Acy (YUH.M. et al. Journal of Molecular Biocatalysis B: Enzymatic, 2006, 43, 118-123) was digested with the same method to obtain the linear plasmid backbone of pMKC. After purification with the PCR product recovery kit, the CPC acylase gene and the linearized plasmid backbone of pMKC were ligated at 4°C for 14 hours with T4 DNA ligase to construct the constitutively expressed recombinant plasmid pMKC-CPCacy (see figure 2 ). The transformation method was the same as in Example 2 (2). The electroporation transformation method was used to transform pMKC-CPCacy into Escherichia coli E. coli JM105 (Tiangen Biochemical Technology (Beijing) Co., Ltd.) to obtain the tra...

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Abstract

The invention discloses a CPC acylation enzyme whose encode gene is DNA sequence shown as SEQ IN NO: 4 and DNA sequence having at least 80154000mology with the above DNA sequence. The invention also discloses carriers and transformants containing coding CPC acylation enzyme and the use of the enzyme. The enzyme has high expressing activity, high catalytic activity and high outcome tolerance to catalysis bottom object in high efficiency to prepare outcome 7-ACA.

Description

technical field [0001] The invention belongs to the field of bioengineering, in particular to a cephalosporin C acylase, an expression vector containing the cephalosporin C acylase gene; and a method for preparing 7-aminocephalosporanic acid with the enzyme. Background technique [0002] Cephalosporins, like penicillin, are a family of β-lactam broad-spectrum antibiotics, which play a bactericidal role by interfering with the synthesis of bacterial cell walls and accelerating the destruction of cell walls. The antibacterial part of cephalosporins is its mother nucleus 7-aminocephalosporanic acid (7-ACA), which is formed by the fusion of dihydrothiazide ring and β-lactam ring. It is a semi-synthetic cephalosporin Important intermediates of mycocin antibiotics. 7-ACA is mainly obtained from cephalosporin C (Cephalosporin C, CPC) by enzymatic or chemical cleavage and removal of the 7-position D-α-aminoadipyl side chain. The enzymatic method has the advantages of simple proces...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/60C12N9/88C12N15/63C12N1/21C12P35/00C12R1/19
Inventor 于慧敏宋文斯安明罗晖沈忠耀
Owner TSINGHUA UNIV
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