Engineered yeast strain for expressing pseudomonas putida creatininase and application thereof

A technology of Pseudomonas putida and yeast engineering, applied in microorganism-based methods, enzymes, enzymes, etc., can solve the problems of large demand for creatinine, difficult cultivation of wild bacteria, low specific enzyme activity, etc., to achieve temperature stability and pH. Good stability, easy control of induction conditions, fast and simple cultivation

Inactive Publication Date: 2018-02-06
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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  • Engineered yeast strain for expressing pseudomonas putida creatininase and application thereof
  • Engineered yeast strain for expressing pseudomonas putida creatininase and application thereof
  • Engineered yeast strain for expressing pseudomonas putida creatininase and application thereof

Examples

Experimental program
Comparison scheme
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 carried out according to the codon preference of Pichia pastoris Optimized to obtain a novel creatinase gene, its sequence is shown in SEQ ID NO.1, through biotechnology company (Shanghai Jierui Bioengineering Co., Ltd.) whole gene synthesis, the synthetic gene clone in pGH plasmid (purchased from Jierui Bioengineering Co., Ltd. Co., Ltd.) to obtain the plasmid pGH-cre.

Embodiment 2

[0034] Construction of Recombinant Plasmid pHKFA1-cre Containing Novel Creatinase Gene and E.coliTop10 / pHKFA1-cre Carrying the Plasmid

[0035] Design PCR primers according to the nucleotide sequence of the novel creatinase gene: Forward primer cre-F: 5'-CGGGAATTCATGCATCATCATCATCATCATTCTAAGTCTGT

[0036] TTTT-3' (the underline is the EcoRI restriction site, which also contains a 6×His tag) and the reverse primer cre-R: 5'-GATGCGGCCGCTTAAGTTGGTGGGAAC-3' (the underline is the NotI restriction site, which also contains a stop codon) .

[0037] The plasmid pGH-cre in Example 1 was used as a template, and cre-F and cre-R were used as primers for PCR amplification. The gel-recovered and purified PCR product was double-digested with restriction endonucleases EcoRI and NotI, and ligated with the plasmid pHKFA1 that had also been double-digested with EcoRI and NotI with T4 ligase overnight at 16°C, and the ligated product was chemically transformed into E.coliTop10 (purchased from In...

Embodiment 3

[0039] Construction of a Novel Creatinase-producing Strain PichiapastorisGS115 / pHKFA1-cre and Expression of Recombinant Creatinase

[0040] The recombinant plasmid pHKFA1-cre was linearized and purified by Kpn2I single enzyme digestion, and then electrotransformed into Pichiapastoris GS115 (purchased from Invitrogen Life Technologies Co., Ltd., USA) competent cells, and positive transformants were screened on MD plates. The transformants screened on the MD plate were used as templates, and cre-F and cre-R were used as primers for PCR identification. The correct transformants identified by PCR were preliminarily identified as the new creatinase-producing strain PichiapastorisGS115 / pHKFA1-cre.

[0041]Pick 3 transformants identified by PCR and inoculate them into BMGY medium, culture at 30°C, 250rpm until OD600 is close to 6.0, collect cells by centrifuging at 6000rpm, 4°C for 5min, and then resuspend the collected cells in BMMY medium to start The OD600 is close to 1.0, culture...

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Abstract

A yeast engineering bacterium for expressing Pseudomonas putida creatinase, which is obtained by transforming an expression vector containing the following foreign DNA into Pichia pastoris: the foreign DNA is Pseudomonas 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 the construction method of the yeast engineering bacterium and the application of the recombinant Pseudomonas putida creatinase fermented by the yeast engineering bacterium of the present invention in creatinine detection. The yeast engineering bacterium of the present invention is quick and easy 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 Excellent temperature stability, heat resistance and pH stability.

Description

technical field [0001] The invention relates to the technical field of bioengineering, in particular to a yeast engineering bacterium for expressing Pseudomonas putida creatinase and its application. 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 creatinine or creat...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/19C12N15/81C12N9/86C12R1/84
CPCC12N9/86C12N15/815C12N2800/22C12Y305/0201
Inventor 林影梁书利侯赣生
Owner SOUTH CHINA UNIV OF TECH
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