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Construction and application of an engineered bacterium that secretes and expresses chitobiose deacetylase

A technology of deacetylase and succinobiose, which is applied in the field of fermentation engineering, can solve the problems of reducing the production cost of pure enzymes, and achieve the effects of simplifying the steps of enzyme separation and purification, mild production conditions, and simple purification process steps

Active Publication Date: 2020-10-09
JIANGNAN UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no report on the secretion and expression of chitobiose deacetylase. The extracellular production of the enzyme can greatly simplify the subsequent protein purification process, without the need for complex extraction processes such as cell disruption and protein renaturation, and chitobiose deacetylase The special heat resistance and thermal stability can simplify the downstream purification process and significantly reduce the production cost of pure enzyme

Method used

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  • Construction and application of an engineered bacterium that secretes and expresses chitobiose deacetylase
  • Construction and application of an engineered bacterium that secretes and expresses chitobiose deacetylase
  • Construction and application of an engineered bacterium that secretes and expresses chitobiose deacetylase

Examples

Experimental program
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Effect test

Embodiment 1

[0057] Example 1 Construction of chitobiose deacetylase secreted expression recombinant plasmid

[0058] The gene (nucleotide sequence shown in SEQ ID NO.1) of coding signal peptide yncM and the gene (nucleotide sequence shown in SEQ ID NO.2) of coding chitobiose deacetylase Dac, utilize Insert the two target genes into the expression vector pP43NMK (the insertion sites are KpnI and PstI) by seam cloning method to construct the recombinant plasmid pP43NMK-yncM-Dac (such as figure 1 ).

Embodiment 2

[0059] Example 2 Obtaining of 5' untranslated region mutants

[0060] In the process of constructing the recombinant plasmid pP43NMK-yncM-Dac in Example 1, the 5' untranslated region mutant was obtained, and after sequencing analysis, it was found that there was a base insertion of 73bp at the 5' end (such as figure 2 ), the mutated recombinant plasmid was named pP43mut-yncM-Dac (the nucleotide sequence is shown in SEQ ID NO.4).

Embodiment 3

[0061] The construction of embodiment 3 recombinant Bacillus subtilis

[0062] Transform the recombinant plasmid pP43mut-yncM-Dac obtained in Example 2 into the cloning host E.coli JM109, pick a single colony grown on the LB Kanna resistance plate, perform colony PCR verification, and cultivate positive clones to extract the plasmid Perform double enzyme digestion verification, pick out the correct restriction bands for sequencing, and transform the recombinant plasmid with correct sequencing results into the expression host B. subtilis WB600.

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Abstract

The invention discloses the construction and application of an engineering bacterium that secretes and expresses chitobiose deacetylase, and belongs to the technical field of fermentation engineering. The present invention first constructs a recombinant Bacillus subtilis strain heterologously secreting and expressing the chitobiose deacetylase gene, and adds a signal peptide fragment yncM to the recombinant vector for the first time, and the signal peptide can convert the target protein chitobiose deacetylase It is secreted outside the cells of the recombinant Bacillus subtilis and obtains a mutant of the 5' untranslated region, which significantly increases the expression of the target protein and greatly simplifies the steps of subsequent enzyme separation and purification. The obtained chitobiose deacetylase has the highest enzyme activity of 1548.7U / mL when fermented and cultivated in the fermentation medium for 50-60 hours, and the highest chitobiose deacetylase yield is about 620mg / L. At the same time, the method has the advantages of low production cost, mild production conditions, simple purification process steps, safe production operation and the like.

Description

technical field [0001] The invention relates to the construction and application of an engineering bacterium that secretes and expresses chitobiose deacetylase, and belongs to the technical field of fermentation engineering. Background technique [0002] Chitobiose deacetylase (Diacetylchitobiose Deacetylase) is derived from the extreme thermophilic archaea (Pyrococcus horikoshii OT3), which is involved in the process of biodegrading chitin and chitosan and producing chitosan oligosaccharides and chitosan monosaccharides key enzymes. Among them, chitobiose deacetylase also has high activity on acetylglucosamine monomer and can be used to produce glucosamine. Glucosamine plays an important role in repairing and maintaining cartilage and joint tissue functions. It is a highly recognized drug in the world today that can effectively reduce the incidence of osteoarthritis. When used as a dietary supplement, it can effectively slow down osteoarthritis. . In addition, chitobiose...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12N9/80C12P19/26A61K35/742A23L33/135C12R1/125
CPCA61K31/7008A23L33/125C12N9/80C12N15/75C12P19/26A23L29/30C12N1/20C12N2500/12C12N2500/74
Inventor 刘龙陈坚堵国成吕雪芹李江华姜竹卢伟张弘治卢健行刘长峰
Owner JIANGNAN UNIV
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