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Recombinant bacillus subtilis of exocytosis PNAG polysaccharide and application of recombinant bacillus subtilis

A Bacillus subtilis, extracellular secretion technology, applied in the field of genetic engineering, can solve the problems of miscellaneous components and low PANG content, and achieve the effect of simple method, extensive culture conditions and good application prospects

Active Publication Date: 2019-08-30
杭州丰海生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the low content of PANG in the biofilm, the composition is heterogeneous, and the extraction and properties of PNAG are rarely involved.

Method used

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  • Recombinant bacillus subtilis of exocytosis PNAG polysaccharide and application of recombinant bacillus subtilis
  • Recombinant bacillus subtilis of exocytosis PNAG polysaccharide and application of recombinant bacillus subtilis
  • Recombinant bacillus subtilis of exocytosis PNAG polysaccharide and application of recombinant bacillus subtilis

Examples

Experimental program
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Effect test

Embodiment 1

[0056] Construction of recombinant plasmid

[0057] According to the PNAG gene PNS in Escherichia coli (E.coli, GenBank: WP_126317446.1) published on NCBI, through codon optimization, the gene synthesis sequence shown in SEQ ID NO.1, the specific design method is:

[0058] Design primer: PNS-F: 5’-atgaccgatcgcattattgctttcctgata-3’

[0059] PNS-R: 5’-tcacgattcgatcctcccaatgcca-3’

[0060] The primers designed above were used to amplify the PNS gene fragment using the synthetic PNAG synthetase encoding gene PNS as a template to express the plasmid pGrac-01 (such figure 1 Shown) After KpnI and HindIII double enzyme digestion and linearization, the amplified gene fragments are connected with T4 ligase to construct a recombinant plasmid. Double enzyme digestion verification and sequencing confirm that the recombinant plasmid is successfully constructed and named pGrac-01-PNS (Such as figure 2 Shown).

[0061] Specifically, Superpfu Mix reagent is used to amplify the target fragment of PNS g...

Embodiment 2

[0065] Construction of Recombinant Bacillus subtilis

[0066] Transform the constructed recombinant expression plasmid pGrac-01-PNS into Bacillus subtilis (Bacillus subtilis168), use PNS-F and PNS-R primers to select transformants for colony PCR, a 1200bp band appears, verifying the recombinant Bacillus subtilis The build was successful.

Embodiment 3

[0068] The production of PNAG polysaccharide by fermentation includes the following steps:

[0069] 1) The recombinant Bacillus subtilis successfully constructed in Example 2 was inoculated into a seed culture medium, and cultured at 37° C. and 220 rpm for 10 hours, for use. The seed culture medium includes the following components in mass concentration: tryptone 10g / L, yeast powder 5g / L and NaCl10g / L.

[0070] 2) Inoculating the above-mentioned cultured recombinant Bacillus subtilis seed solution in a liquid medium by volume 5%, fermenting at 37° C. and 220 rpm for 48 hours, separating the solid and liquid, and obtaining the supernatant of the fermentation broth. The fermentation medium contains the following components in mass concentration: sucrose 50g / L, peptone 10g / L, lactose 5g / L, ammonium sulfate 6g / L, yeast powder 20g / L, K 2 HPO 4 ·3H 2 O12.5g / L, KH 2 PO 4 2.5g / L and MgSO 4 ·7H 2 O 1.5g / L.

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Abstract

The invention discloses recombinant bacillus subtilis of exocytosis PNAG polysaccharide. A PNAG polysaccharide synthetase coding gene and expression plasmid are connected, recombinant expression plasmid is constructed, then the recombinant expression plasmid is transferred into bacillus subtilis, and the recombinant bacillus subtilis is obtained. The invention further discloses a construction method of the recombinant bacillus subtilis. A new metabolism synthetase gene is guided into bacillus subtilis, the recombinant bacillus subtilis is successfully constructed, PNAG polysaccharide can be produced through fermentation, the produced exocytosis PNAG polysaccharide can reach 136mg / L, and a base is established for further reforming the bacillus subtilis for producing the PNAG polysaccharidethrough metabolic engineering. The invention further discloses an application of the recombinant bacillus subtilis to preparation of products containing the PNAG polysaccharide through fermentation indifferent fields of foods, medicines and chemical industry. The PNAG polysaccharide produced from recombinant strains can promote growth of probiotics and restrain harmful bacteria, and has good application prospects in many fields.

Description

Technical field [0001] The present invention relates to the technical field of genetic engineering, in particular to a recombinant Bacillus subtilis that secretes PNAG polysaccharide extracellularly and its application. Background technique [0002] PNAG is a polysaccharide molecule composed of N-acetylglucosamine monomers connected by β-1,6 glycosidic bonds. This polysaccharide is usually produced by extracellular secretion of microorganisms and is a component of bacterial biofilm. The structure of the PNAG polysaccharide molecule is very similar to chitin, except that the chemical bond is replaced by 1,6 glycosidic bonds. Chitin has biological activities such as bactericidal and anti-inflammatory. The low molecular weight chitin oligosaccharides obtained after enzymatic hydrolysis of chitin have also found more anti-tumor, immune promotion, blood sugar regulation, and intestinal flora after recent years of research. Probiotics and other biological activities have very good app...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/75C12P19/18C12P19/04C12R1/125
CPCC12N9/1051C12N15/75C12P19/04C12P19/18
Inventor 李海峰
Owner 杭州丰海生物科技有限公司
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