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Cephalosporin C acylase mutant as well as coding gene and application thereof

A technology of acylase mutants and cephalosporins, which is applied in the fields of application, genetic engineering, and plant gene improvement, and can solve problems such as incompetence for industrial production and low activity of cephalosporin acylases

Inactive Publication Date: 2013-04-24
ANHUI BBCA GENETIC ENG TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the activity of wild-type cephalosporin acylase is low, and it cannot meet the needs of industrial production.

Method used

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  • Cephalosporin C acylase mutant as well as coding gene and application thereof
  • Cephalosporin C acylase mutant as well as coding gene and application thereof
  • Cephalosporin C acylase mutant as well as coding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] The acquisition of embodiment 1 cephalosporin C acylase gene

[0041] Using 1 μg of Pseudomonas sp. SE83 genomic DNA as a PCR reaction template, design the forward primer CPC-F as 5′-AAA according to the sequence of SEQ ID NO.1 ACGATGGCGGCCA-3'; reverse primer: 5'-AAA TCAATGATGATGATGATGATGATGGGCCGGGACGAGTTCCTGCGAG-3'. The parts in italics are the enzyme cutting sites NdeI and XhoI respectively. The PCR reaction was carried out in a total volume of 50 μL. The reaction conditions were 30 cycles of denaturation at 94°C for 5 min, denaturation at 94°C for 50 s, annealing at 58°C for 1 min, and extension at 72°C for 2 min. Take 3 μL of PCR amplification products for agarose gel electrophoresis verification, the results are as follows figure 1 shown. Take 100 μL of the PCR product for agarose gel electrophoresis, and recover the target fragment according to the steps of the gel recovery kit.

Embodiment 2

[0042] Embodiment 2 Construction of wild-type cephalosporin C acylase gene expression vector

[0043]After the PCR product in Example 1 was digested by restriction endonucleases NdeI and XhoI, it was ligated with the pET-30a plasmid (purchased from Novagen) digested with the same endonuclease, and the constructed vector was called pET -CPCA, then transform Escherichia coli DH5-a (purchased from Promega) with the ligation product pET-CPCA mixture, and perform sequence determination to extract the plasmid of the transformed cell library, and transform Escherichia coli BL (DE3) strain (purchased from Novagen) .

Embodiment 3

[0044] Example 3 Error-prone PCR amplification of cephalosporin C acylase gene

[0045] Using the property that TaqDNA polymerase does not have 3′-5′ proofreading function, under high magnesium ion concentration (5mmol / L-9mmol / L) and different concentrations of dNTP (1.5mmol / L-3.5mmol / L), to Control the frequency of random mutations, introduce random mutations into the target gene, and construct a mutation library. The optimal mutation rate in the experiment is about 0.6%.

[0046] Error-prone PCR reaction system (100μl):

[0047]

[0048]

[0049] The PCR program was: pre-denaturation at 96°C for 4 min, denaturation at 94°C for 1 min, annealing at 56°C for 1 min, extension at 75°C for 1 min and 40 s, 45 cycles of reaction, and finally extension at 75°C for 15 min. The PCR product was recovered by gel recovery method. Take 5 μl of the product for 1% agarose gel electrophoresis, and store it at -20°C for later use.

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Abstract

The invention relates to a cephalosporin C acylase mutant as well as a coding gene and the application thereof. An amino acid sequence is shown as SEQ ID NO. 4; or after one or more amino acids are replaced, deleted or added, the sequence forms an amino acid sequence having an equivalent function and derived from the SEQ ID NO.4. The cephalosporin C acylase mutant provided by the invention has the advantages as follows: in the enzymatic reaction process of converting CPC into 7-ACA by using the mutant, the conversion rate reaches 99% and the yield reaches 96%.

Description

technical field [0001] The invention relates to the field of biotechnology and pharmacy, in particular to a cephalosporin C acylase mutant, its coding gene and its application. Background technique [0002] In recent years, cephalosporin antibiotics have less allergic reactions, are more stable to β-lactamase, have a broad antibacterial spectrum, good curative effect, low toxicity, no cross-resistance with other antibiotics, and less cross-allergic reactions with penicillin And other advantages, has become the most important effective drug against infection. Studies have found that the antibacterial part of cephalosporins is its mother nucleus 7-aminocephalosporanic acid (7-ACA). Therefore, as an important intermediate for the synthesis of semi-synthetic cephalosporins, the research and production of 7-ACA is very important. important. [0003] Cephalosporin C (CPC) is cleaved chemically or enzymatically to obtain 7-ACA. The reaction conditions of the chemical method are ...

Claims

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Application Information

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IPC IPC(8): C12N9/80C12N15/55C12N15/63C12N1/21C12N15/70C12P35/02C12R1/38
Inventor 徐斌郑辉汪本助
Owner ANHUI BBCA GENETIC ENG TECH CO LTD
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