Cordyceps militaris membrane protein beta-1, 3-glucan synthetase as well as preparation method and application thereof

A technology of glucan synthase and Cordyceps militaris, which is applied in the fields of fungal biology and food biology, and can solve the problems of enzymatic characteristics and preparation methods that have not been reported.

Pending Publication Date: 2022-08-05
JIANGSU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Although Zhu Zhenyuan of Tianjin University of Science and Technology has studied that fermentation conditions can affect the production of mycelium polysaccharides and exopolysaccharides by affecting the transcription level and enzyme activity of enzymes related to polysaccharide synthesis, but the glucans related to Cordyceps militaris glucan synthesis There are still no relevant reports on the enzymatic properties, preparation methods and related applications of glycan synthase

Method used

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  • Cordyceps militaris membrane protein beta-1, 3-glucan synthetase as well as preparation method and application thereof
  • Cordyceps militaris membrane protein beta-1, 3-glucan synthetase as well as preparation method and application thereof
  • Cordyceps militaris membrane protein beta-1, 3-glucan synthetase as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1 Cloning of Cordyceps militaris β-1,3-glucan synthase CMGLS gene

[0041] 1. Extraction of Cordyceps militaris genomic DNA

[0042] Centrifugal collection of Cordyceps militaris CICC 14015 (purchased from China Industrial Microorganism Culture Collection and Management Center) culture mycelium, add liquid nitrogen to fully grind, add 15mL containing 500mmol / L NaCl, 100mmol / L tris-HCl, pH 8; 50mmol / LEDTA , pH 8; 10mmol / L β-mercaptoethanol extract, then add 0.5mL pH 7.2 10% SDS, 65 ℃ water bath for 30min, add chloroform / isoamyl alcohol, centrifuge to get the supernatant, add 2 volumes of ice ethanol, centrifuge The collected precipitate is the genomic DNA of Cordyceps militaris, and 1 mL of LTE is added to dissolve and preserve.

[0043] 2. Cordyceps militaris total RNA extraction and cDNA synthesis

[0044] Centrifugal collection of Cordyceps militaris CICC 14015 (purchased from China Industrial Microorganism Culture Collection and Management Center) culture m...

Embodiment 2

[0053] Example 2 Preparation of Cordyceps militaris β-1,3-glucan synthase CMGLS

[0054] 1. Culture of bacteria

[0055] First, the strains of Cordyceps militaris were cultured in a flat medium at a constant temperature of 25°C for 7 days. Then cut the plane strain into small pieces and inoculate it into the seed culture solution (glucose 20g / L, peptone 10g / L, MgSO 4 ·7H 2 O 1g / L, KH 2 PO 4 1g / L, pH is natural), the shaker speed is 150rpm for 3 days and then inoculated into the fermentation medium (glucose 30g / L, peptone 6g / L, KH 2 PO 4 3g / L, MgSO 4 ·7H 2 O 1.5g / L, pH natural) to expand the culture. After 3 days of fermentation, the bacterial cells were collected for extraction.

[0056] 2. Crude extraction of Cordyceps militaris β-1,3-glucan synthase CMGLS

[0057] Detergent solubilization of membrane components was used for extraction, and the specific operation was as follows: after the Cordyceps militaris mycelium was fermented in a shake flask, centrifuge at 60...

Embodiment 3

[0063] Example 3 Determination of the enzymatic properties of Cordyceps militaris β-1,3-glucan synthase CMGLS

[0064] The purified CMGLS is mixed with the substrate UDP-glucose to release nucleotide diphosphate (UDP) after a certain period of time, and then specific phosphatase is added to react. The phosphatase can quantitatively remove inorganic phosphate from the UDP generated by the reaction, and then pass Released inorganic phosphates were detected by the malachite green phosphate method. The amount of inorganic phosphate was calculated by making a phosphate standard curve with different concentrations of phosphate standard buffer. The production rate of inorganic phosphate reflects the activity of β-1,3-glucan synthase. Therefore, the amount of 1 pmol of phosphate released per microgram of β-1,3-glucan synthase protein (μg) per unit time (min) is defined as 1 unit of enzyme activity (pmol·min -1 ·μg -1 ).

[0065] The enzyme activity calculation formula is:

[006...

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Abstract

The invention discloses cordyceps militaris beta-1, 3-glucan synthetase as well as a preparation method and application thereof, and belongs to the technical field of fungal biology and food biology. And a gene sequence for coding the cordyceps militaris beta-1, 3-glucan synthetase is as shown in SEQ ID No.3. The invention also discloses a preparation method of the cordyceps militaris beta-1, 3-glucan synthetase. Cordyceps militaris is used as a raw material, a detergent solubilizing membrane component extraction mode is combined with product trapping and Superdex10/300GL chromatographic column purification to obtain the high-purity cordyceps militaris beta-1, 3-glucan synthetase CMGLS, the enzyme catalyzes glucan chain synthesis by using UDP-glucose as a unique substrate, and the invention systematically provides the application of the cordyceps militaris beta-1, 3-glucan synthetase CMGLS in the preparation of the high-purity cordyceps militaris beta-1, 3-glucan synthetase CMGLS in the preparation of the high-purity cordyceps militaris beta-1, 3-glucan synthetase CMGLS. The catalytic characteristics of the 1, 3-glucan synthetase and the structural characteristics of the synthesized product provide important theoretical basis and necessary reference basis for the preparation of multiple transmembrane structures derived from other fungi and high-molecular-mass membrane proteins and the synthesis of glucan with different structures.

Description

technical field [0001] The invention relates to the technical fields of fungal biology and food biology, in particular to a Cordyceps militaris membrane protein β-1,3-glucan synthase and a preparation method and application thereof. Background technique [0002] At present, edible and medicinal fungal polysaccharides have been confirmed to have functions and activities such as immune regulation, anti-tumor, anti-aging, anti-viral and hypolipidemic, and have received extensive attention at home and abroad. Glucan is a large class of macromolecular multimers formed by α / β-1,3, 1,4 or 1,6-glycosidic bonds linking glucose monomers. It is one of the important structural components of fungal cell walls. It plays a key role in maintaining cell shape and integrity, protecting cells from internal swelling pressures and the external environment. Common fungal glucans are mainly derived from yeast, shiitake mushrooms, Yunzhi and Grifola frondosa, etc., and have been developed as comme...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/10C12N15/54C12P19/08C12P19/18
CPCC12N9/1051C12Y204/01034C12P19/08C12P19/18
Inventor 崔凤杰付鑫昝新艺孙雷孙文敬梁英英
Owner JIANGSU UNIV
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