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Pyranose oxidase gene constructed from molecular chaperone, protein, phchia pastoris and preparation and application of pyranose oxidase gene

A molecular chaperone, Pichia pastoris technology, applied in oxidoreductase, biochemical equipment and methods, applications, etc., can solve the problems of low efficiency of protein folding and secretion, and achieve the effect of ensuring genetic stability and improving expression.

Pending Publication Date: 2019-12-31
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

[0007] Inefficient protein folding and secretion are important bottlenecks in improving exogenous protein production in P. pastoris

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] The pyranose oxidase gene constructed by molecular chaperone, its gene sequence is:

[0048] GAATTCATGTCTACTTCCTCCTCCGATCCATTCTTCAACTTTACCAAGTCCTCCTTCAGATCCGCT GCTGCTCAAAAGGCTTCTGCTACTTCTTTGCCACCATTGCCAGGTCCAGACAAGAAAGTTCCAGGT ATGGACATCAAGTACGACGTGGTTATCGTTGGTTCCGGTCCAATCGGTTGTACTTACGCTAGAGAA TTGGTTGAGGCCGGTTACAAGGTTGCCATGTTCGACATTGGTGAGATCGACTCCGGTTTGAAGATT GGTGCTCACAAGAAGAACACCGTCGAGTACCAGAAGAACATCGACAAGTTCGTCAACGTCATCCAG GGTCAGTTGATGTCTGTTTCCGTTCCAGTTAACACCCTGGTCATCGATACTTTGTCTCCAACTTCT TGGCAGGCCTCCTCATTCTTCGTTAGGAACGGTTCTAACCCAGAGCAGGACCCATTGAGAAACTTG TCTGGTCAGGCTGTTACCAGAGTTGTTGGTGGTATGTCTACTCACTGGACTTGTGCTACTCCAAGA TTCGACAGAGAGCAAAGACCATTGCTGGTTAAGGATGACACTGATGCTGATGACGCTGAATGGGAC AGACTGTACACTAAGGCTGAGTCCTACTTCAAGACTGGTACTGACCAGTTCAAAGAGTCCATCAGA CACAACCTGGTCTTGAACAAGTTGGCCGAAGAGTACAAGGGTCAGAGAGACTTCCAACAGATTCCA TTGGCTGCCACTAGAAGATCCCCAACTTTTGTTGAATGGTCCTCCGCCAACACCGTTTTCGACTTG CAAAACAGACCAAACACTGACGCTCCAAACGAGAGATTCAACTTGTTTCCAGCTGTTGCCTGTGAG AGAGTCGTTAGAAACACTTCCAA...

Embodiment 2

[0050] Utilize the protein encoded by the above-mentioned pyranose oxidase gene gene, its amino acid sequence is:

[0051]EFMSTSSSDPFFNFTKSSFRSAAAQKASATSLPPLPGPDKKVPGMDIKYDVVIVGSGPIGCTYARE LVEAGYKVAMFDIGEIDSGLKIGAHKKNTVEYQKNIDKFVNVIQGQLMSVSVPVNTLVIDTLSPTS WQASSFFVRNGSNPEQDPLRNLSGQAVTRVVGGMSTHWTCATPRFDREQRPLLVKDDTDADDAEWD RLYTKAESYFKTGTDQFKESIRHNLVLNKLAEEYKGQRDFQQIPLAATRRSPTFVEWSSANTVFDL QNRPNTDAPNERFNLFPAVACERVVRNTSNSEIESLHIHDLISGDRFEIKADVFVLTAGAVHNAQL LVNSGFGQLGRPDPANPPRLLPSLGSYITEQSLVFCQTVMSTELIDSVKSDMIIRGNPGDPGYSVT YTPGASTNKHPDWWNEKVKNHMMQHQEDPLPIPFEDPEPQVTTLFQPSHPWHTQIHRDAFSYGAVQ QSIDSRLIVDWRFFGRTEPKEENKLWFSDKITDTYNMPQPTFDFRFPAGRTSKEAEDMMTDMCVMS AKIGGFLPGSLPFMEPGLVLHLGGTHRMGFDQEDKCCVNTDSRVFGFKNLFLGGCGNIPTAYGANP TLTAMSLAIKSCEYIKNNFTPSPFTDQAQHHHHHH*AA。

Embodiment 3

[0053] The preparation method of the pyranose oxidase gene constructed by molecular chaperone is characterized in that it comprises the following process steps:

[0054] A. Construction of plasmids and strains

[0055] Prepare Pichia pastoris CGMCC No.16552 into a competent state;

[0056] B. Molecular chaperone gene cloning

[0057] Design oligonucleotide chains according to the gene sequences of chaperone proteins Ero1, PDI, CNE1 and SEC53, synthesize primers, use the genome of Pichia pastoris CGMCC No.16552 as a template, use primers to Pichia pastoris by PCR technology The yeast genome was used as a template to amplify the gene sequences of Ero1, PDI, CNE1 and SEC53 respectively, and the primers were as follows:

[0058] ERoI-F: 5′-ctat ttcgaa acgatgaggatagtaaggagcgtagctatc-3′, where the underline is the BstBI restriction site;

[0059] ERoI-R: 5′-ataagaat gcggccgc ttacaagtctactctatatgtggtatc-3', where the underline is the NotI restriction site;

[0060] PDI-F: 5′-c...

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Abstract

The invention belongs to preparation of a new gene, construction of engineering bacteria, and particularly relates to a pyranose oxidase gene constructed from molecular chaperone, protein, phchia pastoris and preparation and application of the pyranose oxidase gene. Genes Ero I, PDI, CNE1, SEC53 and the like in pichia pastoris are respectively cloned and connected to series derivative plasmids ofpichia pastoris expression vectors pPICZ and the like for connection, and the genes and P20 are subjected to coexpression to realize conversion into Pichia pastoris GS115 bacterial strains to be coated to YPD flats being different in bleomycin concentration gradients for inspection of effects. Through overexpression of molecular chaperone and optimization of the fermentation condition, and throughscreening and identifying, a bacterial strain with enhanced secretory expression pyranose oxidase than an original bacterial strain is obtained. For the pyranose oxidase expressed by the bacterial strain, the enzyme activity in a 10L fermenter can achieve 405U / mL, and compared with the bacterial strain without overexpression of the molecular chaperone, the enzyme activity is improved by twice, sothat a favorable foundation is established for large-scale production of the pyranose oxidase.

Description

technical field [0001] The invention belongs to the preparation of new genes and the construction of engineering bacteria, in particular to a pyranose oxidase gene, protein, Pichia yeast and its preparation and application constructed by using molecular chaperones. Background technique [0002] Pyranose oxidase (pyranose oxidase, PROD, referred to as P2O) is also known as pyranose-2-oxidase or glucose-2-oxidase (pyranose-2-oxidase, glucose-2-oxidase), system number EC 1.1. 3.10, oxidoreductases with flavoprotein as coenzyme and six-membered ring pyranose compound as substrate, with multi-substrate catalytic characteristics. The enzyme belongs to the family of glucose-methanol-choline oxidoreductases (GMC), which can catalyze the oxidation of glucose and several other pyranose C-2 positions to generate corresponding 2-ketose sugars, and at the same time convert O 2 Revert to H 2 o 2 . Pyranose oxidase is widely used in industries such as glycochemistry, analytical chemist...

Claims

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Application Information

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IPC IPC(8): C12N15/53C12N9/04C12N15/81C12N1/19C12R1/84
CPCC12N9/0006C12N15/815C12Y101/0301
Inventor 董聪王玥高庆华王庆庆罗同阳马清河马金国
Owner 河北省微生物研究所有限公司
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