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Method for preparing cordyceps militaris polysaccharide through multi-gene combined expression

A technology of Cordyceps militaris polysaccharides and Cordyceps militaris, which is applied in the fields of genetic engineering and food and medicine, and can solve problems such as no inhibition of Escherichia coli and filamentous fungi, inability to meet the demand for polysaccharide products, and low polysaccharide production

Active Publication Date: 2020-08-11
ZHEJIANG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Cordyceps polysaccharides have inhibitory effects on Gram-positive bacteria such as Staphylococcus aureus, pneumococcus, and tetrazygous bacteria, but have no inhibitory effect on Escherichia coli and filamentous fungi
[0006] The existing polysaccharide extraction technology of Cordyceps is mainly extracted from the fermentation product of Cordyceps militaris, which involves Cordyceps militaris is a natural source of Cordyceps militaris, and its polysaccharide yield is not high, which makes the production cost of polysaccharides high and low in efficiency, which cannot satisfy the market Demand for polysaccharide products

Method used

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  • Method for preparing cordyceps militaris polysaccharide through multi-gene combined expression
  • Method for preparing cordyceps militaris polysaccharide through multi-gene combined expression
  • Method for preparing cordyceps militaris polysaccharide through multi-gene combined expression

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Embodiment 1 constructs the Cordyceps militaris strain that expresses pgm and ugdh in combination

[0032] Table 1 Sources of materials

[0033]

[0034] Table 2 Each medium component

[0035]

[0036]

[0037] The composition of the LB liquid medium: tryptone 10g / L, yeast extract 5g / L, NaCl 10g / L, solvent is distilled water, pH7.0.

[0038] The composition of the LB solid medium: tryptone 10g / L, yeast extract 5g / L, NaCl 10g / L, agar 15-20g / L, solvent is distilled water, pH 7.0.

[0039] The composition of the IM medium: 0.145% KH 2 PO 4 , 0.205%K 2 HPO 4 , 0.06% MgSO 4 ·7H 2 O, 0.03% NaCl, 0.01‰CaCl 2 , 0.001‰FeSO 4 , 0.05% NH 4 NO 3 , 5mL / L glycerin, 0.2% glucose, 5mL / L trace element stock solution, 40mL / L MES buffer solution (1mol / L, pH=5.5), solvent is distilled water; Described every 10ml trace element stock solution composition: ZnSO 4 ·7H 2 O 0.001g, CuSO 4 ·5H 2 O 0.001g, H 3 BO 4 0.001g, (NH 4 ) 2 SO 4 0.5g, MnSO 4 ·H 2 O0.001g, ...

Embodiment 2

[0066] Embodiment 2 constructs the Cordyceps militaris strain that expresses ugp and ugdh in combination

[0067] With the method of Example 1, the recombinant vector pCAMBIA-PgpdA-ugp-Tcbh1-hph-PtrpC was amplified by PCR using primers Insert-CobF&Insert-CobFR to obtain the insert fragment PgpdA-ugp-Tcbh1; using primers Plasmid-CobF&Plasmid-CobR to amplify the recombinant vector pCAMBIA -PgpdA-ugdh-Tcbh1-hph-PtrpC linearization.

[0068] use Seamless Cloning and Assembly Kit connects the insert to the linearized vector to obtain the combined expression recombinant vector PCAMBIA-PgpdA-ugp-ugdh-Tcbh1-hph-PtrpC (referred to as ugp-ugdh), such as figure 2 .

[0069] Other operations are the same as in Example 1, to obtain a genetically engineered strain of Cordyceps militaris expressing ugp and ugdh, and use forward primer F: 5'-CTATTCCTTTGCCCTCGGACGA-3' and reverse primer R: 5'-ATGCCTGAACTCACCGCGACGT-3' for PCR verification , to check whether the conversion is successful, t...

Embodiment 3

[0070] Embodiment 3 constructs the Cordyceps militaris bacterial strain that expresses pgm and ugp in combination

[0071] With the method of Example 1, the recombinant vector pCAMBIA-PgpdA-ugp-Tcbh1-hph-PtrpC was amplified by PCR using primers Insert-CobF&Insert-CobR to obtain the insert fragment PgpdA-ugp-Tcbh1; using primers Plasmid-CobF&Plasmid-CobR to amplify the recombinant vector pCAMBIA -PgpdA-pgm-Tcbh1-hph-PtrpC linearization.

[0072] use The Seamless Cloning and Assembly Kit connects the insert to the linearized vector to obtain the combined expression recombinant vector PCAMBIA-PgpdA-pgm-ugp-Tcbh1-hph-PtrpC (referred to as pgm-ugp), such as figure 2 .

[0073] Other operations are the same as in Example 1, obtain the Cordyceps militaris genetically engineered bacterial strain that expresses pgm and ugp in combination, and carry out PCR verification with forward primer F: 5'-CTATTCCTTTGCCCTCGGACGA-3' and reverse primer R: 5'-ATGCCTGAACTCACCGCGACGT-3' , to check ...

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Abstract

The invention discloses a method for preparing cordyceps militaris polysaccharide through multi-gene combined expression. The method comprises the following steps of introducing a combined gene into cordyceps militaris cells to construct recombinant cordyceps militaris genetically engineered bacteria, inoculating the recombinant cordyceps militaris genetically engineered bacteria into a fermentation culture medium, and carrying out fermentation culture at 22-25 DEG C at 100-150 rpm to obtain a fermentation liquor comprising cordyceps militaris polysaccharide; carrying out separating and purifying on the fermentation liquor to obtain the cordyceps militaris polysaccharide, wherein the combined gene is a combination of any two of a phosphoglucose mutase gene pgm, a pyrophosphorylase gene ugpor a UDP-glucose-6-dehydrogenase gene ugdh. The recombinant cordyceps militaris genetically engineered bacteria are constructed through a gene combination expression technology, and compared with a wild strain, the yield of cordyceps militaris exopolysaccharides in fermentation liquor of the genetically engineered bacteria is 1.78 times and is 5.713 g / L higher than the maximum yield of cordycepsmilitaris exopolysaccharides produced by fermentation reported in literatures at present.

Description

[0001] (1) Technical field [0002] The invention relates to the fields of genetic engineering and food medicine, in particular to a method for preparing polysaccharides from Cordyceps sinensis by using multi-gene combination expression technology. [0003] (2) Background technology [0004] Polysaccharide is a kind of macromolecular compound formed by the polymerization of sugar molecules connected by glycosidic bonds, which widely exists in animals, plants and microorganisms. Because of its good effects in pharmacological efficacy and biological activity, it has attracted the attention and research of a large number of scholars. The polysaccharides synthesized by Cordyceps militaris include intracellular polysaccharides and extracellular polysaccharides. The pure product of Cordyceps polysaccharide is white powder, easily soluble in water, but insoluble in high-concentration organic solvents. It is one of the main active ingredients in Cordyceps. exist in the fermentation s...

Claims

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Application Information

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IPC IPC(8): C12N15/80C12N15/61C12N15/54C12N15/53C12P19/04C12R1/645
CPCC12N15/80C12N9/90C12N9/0006C12N9/1241C12P19/04C12Y504/02002C12Y101/01022
Inventor 杨锡王逸峰孔潇慧季小康杨胜利
Owner ZHEJIANG UNIV OF TECH
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