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A kind of recombinant escherichia coli producing mannan and its application

A technology for recombining Escherichia coli and mannan, which is applied in the field of bioengineering, can solve the problems of low yield, complex extraction process, and high molecular weight of konjac mannan, and achieves a technology with less extracellular secretion impurities, strong growth efficiency, and easy separation and purification. Effect

Active Publication Date: 2021-11-16
ZHEJIANG FORESTRY UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The extraction and separation process of konjac mannan is complicated, and the yield and purity are low; and konjac mannan has high molecular weight and high viscosity, and the purified product needs to be further degraded to be used as mannose oligosaccharides, and the product quality is not stable enough
Although brewer's yeast has been used for fermentation in recent years to extract mannan from the cell wall, it is difficult to achieve industrial scale production due to its low yield and complicated extraction process.

Method used

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  • A kind of recombinant escherichia coli producing mannan and its application
  • A kind of recombinant escherichia coli producing mannan and its application
  • A kind of recombinant escherichia coli producing mannan and its application

Examples

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Effect test

Embodiment 1

[0024] Example 1: Cloning and recombinant construction of mannan synthase gene

[0025] According to the data query of NCBI genome information database, the cDNA sequence of the mannan synthase gene derived from guar was optimized for nucleic acid sequence, and the ctManS gene was artificially synthesized (the nucleotide sequence is shown in SEQ ID NO.1).

[0026] According to the plasmid pET21a, primers E / ctManS-F and E / ctManS-R were designed; the primer pair E / ctManS-F and E / ctManS-R were used for PCR amplification of the ctManS gene sequence, and the recovered product was restricted by BamHI and SalI. Dicer enzymes were used for double-digestion; plasmid pET21a was double-digested with BamHI and SalI restriction endonucleases.

[0027] The fragment ctManS and the pET21a vector were recombined, and T4 DNA ligase was used to ligate overnight at 16°C; the ligation product was transformed into E. monoclonal), using primers E / ctManS-F and E / ctManS-R to carry out PCR identificat...

Embodiment 2

[0032] Example 2: Construction of the gene operon of the mannan synthesis pathway

[0033] In order to further enhance the synthesis efficiency of mannan from the metabolic pathway, the manC-manB operon ( figure 2 ).

[0034] According to the genome information of E.coli BL21(DE3), primers were designed for the manC and manB genes to be cloned for PCR amplification. In order to recombine the manC-manB fragment into the pET21a-ctmanS constructed in Example 1, SalI and XhoI restriction enzyme sites were introduced into the primer pair E / manC-F and E / manB-R, respectively.

[0035] Using the E.coli BL21 (DE3) genome as a template, using primers E / manC-F and E / manB-R to amplify the manC-manB fragment (the nucleotide sequence is shown in SEQ ID NO.4), the recovered DNA The fragment SalI and XhoI restriction endonucleases were used for double cutting, and the recombinant plasmid pET21a-ctmanS was used for double cutting with SalI and XhoI restriction endonucleases; then the recove...

Embodiment 3

[0039] Example 3: Shake Flask Fermentation of Recombinant Bacillus subtilis Strains

[0040] Pick the recombinant bacterial strain E.coli BL21 (DE3) / pET21a-ctmanS among the embodiment 1 and the E.coli BL21 (DE3) / pET21a-ctmanS-manCB monoclonal in the embodiment 2 and inoculate in 5mL LB medium (containing 100ug / mL ampicillin), placed at 200rpm and cultured overnight at 37°C. After 16 hours, inoculate 1 mL of the bacterial solution into a 250 mL Erlenmeyer shaker flask containing 100 mL of fermentation medium, and culture the shaker flask at 200 rpm at 37°C until OD 600 0.6-0.8, the final concentration of 0.1mM inducer IPTG was added to induce the expression of the operon gene, and then the shake flask was placed at 30°C and 200rpm for 48h.

[0041] After the fermentation, collect the fermentation broth, centrifuge at 10,000rpm at 4°C for 10min, collect the supernatant of the fermentation broth, use 3 times the volume of absolute ethanol to precipitate the mannan product, and ce...

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Abstract

The invention discloses a mannan-producing recombinant escherichia coli and an application thereof, belonging to the technical field of bioengineering. The present invention adopts the mannan synthase gene ManS derived from the plant guar, integrates it into the pET21a plasmid controlled by the T7 promoter, and realizes the synthesis of mannan in the recombinant plasmid in Escherichia coli E.coli BL21 (DE3). Heterogeneous synthesis, the yield can reach 0.68g / L. The invention lays a certain foundation for the high-efficiency fermentation and preparation of mannan by microorganisms using cheap carbon sources, and is suitable for industrial production and application.

Description

technical field [0001] The invention relates to a mannan-producing recombinant Escherichia coli and an application thereof, belonging to the technical field of bioengineering. Background technique [0002] Mannan is a hemicellulose polysaccharide widely present in various life forms. As an excellent low-calorie, high-cellulose water-soluble dietary fiber, mannan is widely used in food and medicine industries. Mannan has unique biological activity due to its unique molecular structure and physical and chemical properties, and can participate in some very important physiological processes in the body, such as stimulating the body's immune response, promoting the vegetative growth of beneficial bacteria in the intestine, and reducing serum cholesterol levels Wait. [0003] Mannan is widely found in plants such as konjac flour, guar gum, field gum and dendrobium, as well as in the cell walls of various microorganisms. The current commercialized mannan is mainly extracted from ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12P19/04C12R1/19
CPCC12N9/2488C12P19/04
Inventor 金鹏杜琪珍陈美霞王园园
Owner ZHEJIANG FORESTRY UNIVERSITY
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