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Construction and application of recombinant and expression system of CPC acylation enzyme gene

An acylase and recombinant plasmid technology, applied in the biological field, can solve problems such as low CPC activity

Inactive Publication Date: 2015-08-19
SHENZHEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Because wild strains have low activity on CPC and cannot meet the needs of industrial production, scholars at home and abroad have used genetic engineering techniques to artificially modify and chemically synthesize the CPC acylase gene.

Method used

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  • Construction and application of recombinant and expression system of CPC acylation enzyme gene
  • Construction and application of recombinant and expression system of CPC acylation enzyme gene
  • Construction and application of recombinant and expression system of CPC acylation enzyme gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] [Example 1] Optimization of CPC Acylase Gene Sequence and Selection of Transgenic Recipient Strains

[0032] According to GenBank accession number AAA25690, based on the protein sequence of Pseudomonas sp.SE83cephalosporin acylase (its wild-type nucleotide sequence is shown in SEQ ID NO.7), based on the principle of codon preference of Escherichia coli, through Two codon-optimized gene sequences were obtained by codon translation, named S1 gene (SEQ ID NO.1) and S2 gene (SEQ ID NO.2), respectively. The GC content of the optimized S1 gene was 62.3%, and the GC content of the S2 gene was 59.9%. Both the S1 gene and the S2 gene described in this example were artificially synthesized in full sequence by Sangon Bioengineering (Shanghai) Co., Ltd., and the synthesized SEQ ID NO.1 and SEQ ID NO.2 were respectively present in Escherichia coli containing the plasmid PUC57 In the medium (DH5α), by amplifying Escherichia coli, extracting plasmids PUC57-S1 and PUC57-S2 to obtain o...

Embodiment 2

[0035] [Example 2] Construction of the Escherichia coli genetic expression vector of CPC acylase through codon optimization

[0036] The present invention uses Escherichia coli genetically transformed plasmid PUC57. Take 4 mL of overnight cultured bacterial liquid, and use Omega Plasmid Mini Kit plasmid extraction kit to extract plasmid PUC57 from E. coli DH5α bacteria. According to the S1 and S2 gene sequences in the plasmid PUC57, PCR primers introducing EcoR Ⅰ and Xho Ⅰ restriction sites were designed and PCR reactions were carried out, wherein

[0037] S1 upstream primer: 5'-CCGGAATTCATGACCATGGCGGCGAAAACTG-3'

[0038] S1 downstream primer: 5'-CCGCTCGAGTTACGCCGGCACCAGTTCCT-3'

[0039] S2 upstream primer: 5'-CCGGAATTCATGACCATGGCAGCAAAAACCG-3'

[0040] S2 downstream primer: 5'-CCGCTCGAGTTACG CAGGCACCAGTTCCT-3'

[0041] Use the Omega Agarose Gel DNA Purification Kit to recover and purify the PCR products (S1 and S2 genes). The expression vector pET-28a was digested with Eco...

Embodiment 3

[0042] [Example 3] Expression and enzyme activity of CPC acylase gene after optimization

[0043] The expression vectors PET-28a-S1 and PET-28a-S2 were respectively transformed into the four host Escherichia coli described in Example 1, and the successfully constructed transformants were fermented and expressed, specifically, the strains were inoculated into 25mL cultured overnight in LB medium. Take 250 μL of the overnight culture solution and inoculate it into a 250 mL shake flask containing 25 mL of LB medium. When the OD value reached 0.6, IPTG (isopropyl-β-D-thiogalactoside) with a final concentration of 0.05 mM was added for induction. 28°C, 150r / min, after induction for 20h, the bacterial liquid was taken to measure the enzyme activity. The method for measuring enzyme activity is to measure enzyme activity by colorimetry, take 0.5mL bacterial liquid, centrifuge for 2min, and discard the supernatant; use 0.5mL 0.1mol / L pH8. g and potassium dihydrogen phosphate 0.41g, ...

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Abstract

The invention discloses a CPC acylation enzyme gene after codon optimization. The CPC acylation enzyme gene contains expression vector and host bacteria of the gene, and also amino acid sequence obtained by translating the CPC acylation enzyme gene. The invention builds a system which stably expresses colibacillus of CPC acylation enzyme gene via genetic engineering, and activity of CPC acylation enzyme gene is increased by optimizing culture medium.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to the construction and application of an expression system for expressing CPC acylase. Background technique [0002] Cephalosporins are a family of broad-spectrum semi-synthetic antibiotics. Like penicillin antibiotics, they all have β-lactam rings in their molecular structure, so they are collectively called β-lactam antibiotics. They all destroy bacterial cell walls. Antibiotics synthesized into mechanisms. In recent years, due to its small allergic reaction, stability to acid and β-lactamase, and no cross-resistance with other antibiotics, it has received increasing attention from researchers in the antibiotic industry. [0003] The structure of cephalosporin antibiotics is composed of a mother nucleus and side chains. The mother nucleus 7-aminocephalosporanic acid (7-ACA) is the antibacterial part of cephalosporins and is a variety of semi-synthetic cephalosporin antib...

Claims

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Application Information

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IPC IPC(8): C12N15/55C12N15/70C12N1/21C12N9/80
Inventor 余少文孙勇汤新胡萍
Owner SHENZHEN UNIV
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