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Recombinant pichiapastoris for producing FAD-dependent glucose dehydrogenase as well as construction method and application thereof

A technology of glucose dehydrogenase and construction method, which can be used in microorganism-based methods, recombinant DNA technology, biochemical equipment and methods, etc., and can solve problems such as not obtaining enzyme activity.

Inactive Publication Date: 2017-12-12
河北省微生物研究所有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

According to the eukaryotic vector expression of other enzymes in this field, the FAD-dependent glucose dehydrogenase expressed by eukaryotic vector recombinant expression does not obtain ideal enzyme activity

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] FAD-dependent synthesis of the glucose dehydrogenase gene

[0056] Based on the Aspergillus terreus NIH2624 gene with the accession number XM_001216916 on NCBI, the codons of the gene were optimized according to the codon preference of Pichia pastoris, and the codon optimization software http: / / www.jcat.de / was used for analysis. It was found that there are many rare codons of Pichia pastoris in the protease gene. The codon optimization software http: / / www.jcat.de / was used to optimize the codon of the protease gene, and the optimization was obtained without changing the amino acid sequence. The gene sequence of the protease. An EcoRI restriction enzyme cutting site was added to the 5' end of the sequence, and a NotI restriction enzyme cutting site was added after six histidine tags were added to the 3' end. Then the optimized gene sequence was sent to Anhui General Biology Company for synthesis. The synthetic gene was ligated into the pUC57T vector.

[0057] The seq...

Embodiment 2

[0061] Construction of recombinant plasmid pMD-GDH

[0062] Due to the introduction of EcoRI and NotI restriction enzyme sites at both ends of the target gene fragment, the PMD-AOX plasmid provided by the Institute of Microbiology, Chinese Academy of Sciences also has these two restriction enzyme sites. The pUC57-GDH prepared in Example 1 Carry out EcoRI and NotI double digestion with the pMD-AOX plasmid stored in the laboratory, respectively, and separate the digested products by 1% agarose gel electrophoresis, and recover by cutting the gel to obtain the target gene GDH and the carrier pMD with the same sticky ends; The recovered product was ligated with T4 ligase, and the ligated pMD-GDH was transformed into Escherichia coli DH5α, and the plasmids extracted from the identified positive clones were sequenced, and the recombinant plasmid pMD-GDH was successfully constructed by comparison and analysis.

Embodiment 3

[0064] Construction of recombinant Pichia pastoris producing FAD-GDH strain

[0065] The recombinant plasmid pMD-GDH was linearized with SacI and then electroporated to express the host Pichiapastoris X-33 to construct the recombinant strain X33 / pMD-GDH. Electroporation conditions: 4Kv, 3ms, quickly add 1ml of pre-cooled YPDS liquid medium, electroporated competent cells Place in an incubator at 30°C for 4 hours, centrifuge at 4000rpm for 5 minutes, discard the supernatant, wash 3 times with 1ml filter-sterilized saturated saline, then take 500μL and spread it on a YPD plate with a final concentration of 250μg / mLG418, 3 Positive transformants were obtained 2 days later.

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Abstract

The invention provides recombinant pichiapastoris for producing FAD-dependent glucose dehydrogenase as well as a construction method and application thereof and belongs to the technical field of genetic engineering. The invention provides a recombinant plasmid pMD-GDH containing FAD-dependent glucose dehydrogenase genes. After the FAD-dependent glucose dehydrogenase genes are subjected to codon preference adjustment, EcoRI restriction sites and NotI restriction sites are added at two ends of the sequence, and six histidine tags are added at the 3' end. The recombinant pichiapastoris takes Pichia pastoris as an expression host and comprises the recombinant plasmid. The invention further provides application of the recombinant pichiapastoris for producing FAD-dependent glucose dehydrogenase in fermentation production of the FAD-dependent glucose dehydrogenase. The embodiments show that when the recombinant pichiapastoris is subjected to induction culture for 136 hours during culture in a 10L of fermentation tank, the enzyme activity reaches 257600U / L and is 5.3 times that of the enzyme activity reported in the prior art.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and specifically relates to a recombinant Pichia pastoris producing FAD-dependent glucose dehydrogenase and its construction method and application. Background technique [0002] FAD-dependent glucose dehydrogenase (FAD-dependent glucose dehydrogenase, referred to as FAD-GDH, EC 1.1.99.10), and glucose oxidase (glucose oxidase, referred to as GOD), pyranose dehydrogenase, choline dehydrogenase and Methanol oxidase belongs to the GMC oxidoreductase (glucose-methanol-choline-oxidoreductase) family. It is based on FAD as a prosthetic group that can be used in NAD(P) + In the presence of β-D-glucose, it catalyzes the formation of D-glucono-δ-lactone, and D-glucono-δ-lactone will spontaneously form gluconic acid. [0003] Among the currently used enzymes for blood glucose detection, glucose oxidase (GOD) can use oxygen as an electron acceptor, and the change of oxygen partial pressure in...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/19C12N9/04C12N15/81C12R1/84
CPCC12N9/0006C12N15/815C12N2800/22C12Y101/9901
Inventor 董聪高庆华王玥王庆庆罗同阳刘蕾
Owner 河北省微生物研究所有限公司
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