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Escherichia coli engineering bacteria for expressing pseudomonas putida creatininase and application thereof

A technology of Pseudomonas putida and Escherichia coli, applied in application, genetic engineering, enzymes, etc., can solve the problems of large demand for creatinine, difficulty in culturing wild bacteria, low specific enzyme activity, etc., and achieve temperature stability and pH stability. Good, easy to control induction conditions, fast and easy to cultivate

Inactive Publication Date: 2017-12-15
SOUTH CHINA UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, most of the creatinases that have been studied are from wild bacteria, but the cultivation of wild bacteria is difficult, the yield of creatinase is low, the specific enzyme activity is low, and there are difficulties in purification.
With the development of enzymatic creatinine detection kits, the demand for creatinase is increasing. The production of creatinase by wild bacteria is difficult to achieve industrial scale-up and the production cost is often too high.

Method used

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  • Escherichia coli engineering bacteria for expressing pseudomonas putida creatininase and application thereof
  • Escherichia coli engineering bacteria for expressing pseudomonas putida creatininase and application thereof
  • Escherichia coli engineering bacteria for expressing pseudomonas putida creatininase and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0031] Synthesis of Novel Creatinase Gene

[0032] Based on the genbank GI:34810367 gene sequence published on the National Center for Biotechnology Information (NCBI, http: / / www.ncbi.nlm.nih.gov / ), the sequence was optimized according to the codon preference of Escherichia coli , to obtain a new type of creatinase gene, its sequence is shown in SEQ ID NO.1, through biotechnology company (Shanghai Jierui Biological Engineering Co., Ltd.) whole gene synthesis, the synthetic gene clone in pGH plasmid (purchased from Jierui Bioengineering Co., Ltd. company) to get the plasmid pGH-cre.

Embodiment 2

[0034] Construction of the recombinant plasmid pET28a-cre containing the novel creatinase gene and the strain E.coliTop10 / pET28a-cre carrying the plasmid.

[0035] Plasmid pET-28a(+) has 6 codons encoding histidine behind the multiple cloning site region as a His tag, and a stop codon TGA behind the tag, and the stop codon was removed when designing the downstream primers. Design PCR primers according to the nucleotide sequence of the novel creatinase gene: forward primer cre-S: 5'-CGGCCATGGATGTCTAAGTCTGTT-3' (the underline is the NcoI restriction site) and reverse primer cre-A: 5'-ATACTCGAGAGTTGGTGGGAACTC- 3' (the underline is the XhoI restriction site, without stop codon).

[0036] PCR amplification was performed using the plasmid pGH-cre in Example 1 as a template, and cre-S and cre-A as primers. The gel-recovered and purified PCR product was double-digested with restriction endonucleases NcoI and XhoI, and ligated with the plasmid pET-28a(+) that had also been double-dige...

Embodiment 3

[0038] Construction of a Novel Creatinase-producing Strain Escherichia coliBL21 / pET28a-cre and Expression of Recombinant Creatinase

[0039]The recombinant plasmid pET28a-cre was transformed into competent cells of Escherichia coliBL21 (DE3) (purchased from Invitrogen Life Technologies Co., Ltd., USA), and positive transformants were screened on a kanamycin resistance plate. The transformants screened on the kanamycin resistance plate were used as templates, and cre-S and cre-A were used as primers for PCR identification, and the correct transformants identified by PCR were preliminarily identified as the new creatinase-producing strain Escherichia coliBL21 / pET28a-cre .

[0040] Pick 3 transformants identified correctly by PCR and inoculate them into BMGY medium, cultivate them at 37°C and 250rpm for 12h, then transfer the cultured bacterial solution to 100mL TB liquid medium (50μg / mL Kanamyces prime), 30°C, 250rpm and culture until OD600 is about 0.7-0.8. Add 1M IPTG to the...

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Abstract

An Escherichia coli engineering bacterium for expressing Pseudomonas putida creatinase, the Escherichia coli engineering bacterium is obtained by transforming an expression vector containing the following foreign DNA into Escherichia coli: the foreign DNA is putida The DNA coding sequence of Pseudomonas putida creatinase, the amino acid sequence of Pseudomonas putida creatinase is shown in Seq No.2. The present invention also provides a construction method of the Escherichia coli engineering bacterium and the application of the recombinant Pseudomonas putida creatinase obtained by fermentation of the Escherichia coli engineering bacterium of the present invention in creatinine detection. The Escherichia coli engineering bacterium of the present invention is fast and simple to cultivate, the induction conditions are easy to control, the production cost is low, and it has the potential of industrial production; meanwhile, the recombinant Pseudomonas putida creatinase prepared by fermentation thereof has good creatinase activity and exhibits Excellent temperature stability, heat resistance and pH stability.

Description

technical field [0001] The invention relates to the technical field of bioengineering, in particular to an Escherichia coli engineering bacterium for expressing Pseudomonas putida creatinase and application thereof. Background technique [0002] Creatininase (creatininase; EC 3.5.2.10) is a hydrolase that belongs to the urease-related amidohydrolase family and is distributed in a few species of bacteria. The active center of creatinase contains zinc ions, and the catalyzed hydrolysis synthesis reaction of creatinase catalyzes the reversible hydrolysis of cyclic amide creatinine to creatine. [0003] The determination of creatinine in serum and urine can provide objective indicators for clinical evaluation of glomerular filtration function, so it is a commonly used biochemical test item in clinical practice. The interference of heterosexual substances is gradually being replaced by enzymatic methods. The substrate of creatinase is specific, and the substrate can only be cre...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/70C12N9/78C12N15/55C12Q1/34
CPCC12Y305/0201C12N9/78C12N15/70C12Q1/34G01N2333/986
Inventor 林影梁书利侯赣生
Owner SOUTH CHINA UNIV OF TECH
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