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Immobilized microbial cell method for converting ganglioside

A technology for immobilizing microorganisms and gangliosides, applied in the direction of microorganism-based methods, biochemical equipment and methods, microorganisms, etc., can solve problems such as high cost of hydrolysis, difficulty in separation and purification, and short production run time, so as to improve product quality. The effect of yield and running time, stable product quality, and improved conversion efficiency

Active Publication Date: 2011-08-17
SHANDONG NEWTIME PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There are some deficiencies and shortcomings in the existing technical methods of hydrolysis conversion. For example, acid hydrolysis will produce asialic acid GM1, a by-product with no physiological activity, and the conversion rate is low; commercial sialid enzymatic hydrolysis costs are high, and it is not suitable for large-scale production. ;Compared with acid and enzymatic conversion, microbial conversion hydrolysis has the advantages of low cost and high conversion rate, but impurities such as culture medium, microorganisms and their metabolites will inevitably be introduced during the operation itself, which will bring difficulties to downstream separation and purification. Difficulty increases the instability of the product and makes the product quality uncontrollable
[0008] Using the cell immobilization method to prepare monosialotetrahexosyl ganglioside will overcome the defects of the above methods, but the current cell immobilization method has problems such as low survival of immobilized cells, short production run time, and low product yield.

Method used

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  • Immobilized microbial cell method for converting ganglioside

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] (1) Cell culture

[0028] Transfer the mature Micromonas viridans cultured on the slant medium into four 1L Erlenmeyer flasks containing 250ml of seed medium (glucose 20 g / L, peptone 15 g / L, yeast powder 10 g / L, pH 7.2), culture at 28°C, 250rpm for 48 hours, and then insert 10 L of fermentation medium (polyacetylneuraminic acid 2 g / L, starch 30 g / L, ammonium sulfate 15 g / L , Potassium dihydrogen phosphate 1 g / L, magnesium sulfate 0.5 g / L, zinc sulfate 0.3 g / L) in a fermenter, stirred and aerated at 28°C for 72 hours (aeration ratio: 0.4~1.0 vvm), continuous flow The cells were collected by centrifugation, and a total of about 2000 g of wet cells were obtained. Wash twice with normal saline (0.9%) and suspend in transformation reaction buffer with a pH value of 6.1 to prepare 4 L of bacterial suspension.

[0029] (2) Preparation of porous silk fibroin gel

[0030] Weigh about 4kg of waste silk scraps, shear and pulverize them, soak them in 90°C alkaline aqueous solution...

Embodiment 2

[0036] (1) Cell culture

[0037] Transfer the mature Micromonospora viridans cultured on the slant medium into four 1L Erlenmeyer flasks containing 250ml of seed medium (glucose 20 g / L, peptone 15 g / L, yeast powder 10 g / L, pH 7.2), culture at 28°C, 250rpm for 48 hours, and then insert 10L of fermentation medium (polyacetylneuraminic acid 5 g / L, starch 30 g / L, ammonium sulfate 15 g / L, Potassium dihydrogen phosphate 1 g / L, magnesium sulfate 0.5 g / L, zinc sulfate 0.3 g / L) in a fermenter, stirred and aerated at 28°C for 36 hours (aeration ratio: 0.4~1.0 vvm), continuous flow centrifugation The cells were collected, and a total of about 1500 g of wet bacteria was obtained. Wash twice with normal saline (0.9%) and suspend in transformation reaction buffer with a pH value of 6.4 to prepare 3 L of bacterial suspension.

[0038] (2) Preparation of porous silk fibroin gel

[0039] Weigh about 4kg of waste silk scraps, shear and pulverize them, soak them in 90°C alkaline aqueous solut...

Embodiment 3

[0045] (1) Cell culture

[0046] Transfer the mature Micromonas viridans cultured on the slant medium into four 1L Erlenmeyer flasks containing 250ml of seed medium (glucose 20 g / L, peptone 15 g / L, yeast powder 10 g / L, pH 7.2), culture at 28°C, 250rpm for 48 hours, and then insert 10L of fermentation medium (polyacetylneuraminic acid 8 g / L, starch 30 g / L, ammonium sulfate 15 g / L, Potassium dihydrogen phosphate 1 g / L, magnesium sulfate 0.5 g / L, zinc sulfate 0.3 g / L) in a fermenter, stirred and aerated at 28°C for 72 hours (aeration ratio: 0.4~1.0 vvm), continuous flow centrifugation The cells were collected, and a total of about 2300 g of wet bacteria was obtained. Wash twice with normal saline (0.9%) and suspend in transformation reaction buffer with a pH value of 6.2 to prepare 4.6 L of bacterial suspension.

[0047] (2) Preparation of porous silk fibroin gel

[0048] Weigh about 4kg of waste silk scraps, shear and pulverize them, soak them in 90°C alkaline aqueous solutio...

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Abstract

The invention discloses an immobilized microbial cell method for converting ganglioside, which can specifically convert ganglioside so as to generate monosialoganglioside (GM1) and solves the problem that acid hydrolysis has low conversion rate and easily generates inactive monosialoganglioside and simultaneously solves the problem that in an non immobilized microbial cell method, medium components or microbial metabolite impurities are easily mixed into the monosialoganglioside. After cells are fixed with fibroin gel, a columnar type reaction bed is filled; the ganglioside to be converted reversely flows through a packed column and is converted into GM1.

Description

technical field [0001] The invention belongs to the field of biopharmaceuticals, and in particular relates to a method for preparing monosialotetrahexosyl ganglioside (GM1) by transforming gangliosides with immobilized microbial cells. Background technique [0002] Ganglidosides (GLS) are amphiphilic macromolecules formed by linking sialic acid-containing hydrophilic oligosaccharides with hydrophobic ceramides through β-1,1 glycosidic bonds. The GLS containing a sialic acid is called Monosialotetrahexosylganglioside (GM1). GM1 can pass through the blood-brain barrier, gather damaged brain regions and embed in the cell membrane, and imitate the role of endogenous GM1, which can promote Neural reconstruction of nerve cell survival, axon growth and synapse growth, restore the function of central nervous system damage caused by various reasons, and be used for the treatment of craniocerebral injury, neurodegeneration, cerebral edema, Parkinson's syndrome and other brain diseases...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P19/44C12R1/13C12N11/04C12R1/29
Inventor 赵志全董惠钧
Owner SHANDONG NEWTIME PHARMA
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