CN1415756A

CN1415756A - Method for preparing ganglioside GMI of monosialic acid tetrahexose

Info

Publication number: CN1415756A
Application number: CN 01132052
Authority: CN
Inventors: 张丽莉; 李红英
Original assignee: QILU PHARMACEUTICAL FACTORY
Current assignee: QILU PHARMACEUTICAL FACTORY
Priority date: 2001-10-30
Filing date: 2001-10-30
Publication date: 2003-05-07
Anticipated expiration: 2021-10-30
Also published as: CN1182253C

Abstract

A process for preparing monosialotetrahexaglyco-ganglioside GM, features that the Brevibacterium QL-1 is used to decompose and transform the other components in the ganglioside by fermenting to become GM1. Its advantages are high output rate and low cost.

Description

Monostalotetrahexosylgangliside GM 1The preparation method

Technical field

The present invention relates to biological technical field, be specifically related to Monostalotetrahexosylgangliside GM ₁The preparation method.

Background technology

(gangliosides is that a class contains sialic acid sphingoglycolipid GIS) to Sphingolipids,sialo, is made up of sphingosine, lipid acid and oligonucleotide chain three parts, and different according to saccharide residue number in the molecule and sialic acid number and link position are named as GM respectively ₁, GM ₂, GM ₃, GD _1a, GD _1b, GT _1bOr the like.These compounds are distributed widely in animal tissues, particularly content is abundanter in the brain cell membrane, they play requisite effect in nerve generation, growth, atomization, also extremely important for the nerve reparation after the damage, have the neurotization of promotion, promote neural axon growth and cynapse to form, recover the innervation function; Improve the recovery of nerve conduction, promotion electrical activity of brain and other neuroelectricity physical signs; Effects such as protection cytolemma, the various enzymic activity recoveries of promotion cytolemma.

GM ₁Be one of most important Sphingolipids,sialo, in the treatment of central nervous system pathological change, play an important role.GM ₁Except that acting in conjunction with above-mentioned Sphingolipids,sialo, can also be by keeping the Na on the axoneure film ⁺-K ⁺-ATP enzyme and Ca ²⁺-Mg ²⁺The activity of-ATP enzyme plays and keeps the inside and outside ionic equilibrium of cell, alleviates the neurocyte oedema, prevents Ca in the cell ²⁺The effect of gathering.So GM ₁Have the effect that promotes neural reconstruct, promptly by promoting the improvement of various forms, biological chemistry, histological chemistry, nervous physiology and behavior parameter, reparation that finally can the promoting nerve cell farthest recovers original neural function.The clinical central nervous system acute injury or the vascular injury of (comprising brain and spinal cord) of being used for.

Sphingolipids,sialo are mixtures, and with animal brain's separation preparation is to contain GM ₁The mixture Sphingolipids,sialo.Traditional separation GM ₁Method is to separate from composite nerve joint glycosides fat with silicagel column, complex operation step, length consuming time, and consume a large amount of organic solvents, the wherein GM of Ti Quing ₁Content only be ten thousand of cerebral tissue content/, yield is low, the cost height.

Summary of the invention

Technical problem to be solved by this invention is by microbial conversion process, and other component fermentation decomposition and inversion in the Sphingolipids,sialo extract is become GM ₁, provide a kind of yield height, the GM that cost is low ₁The preparation method.

Sphingolipids,sialo GM disclosed by the invention ₁The preparation method comprise the following steps:

1. fermented bacterium preparation

Get the freeze-drying pipe and hide bacterial classification, be inoculated on the nutrient agar slant medium, cultivated 1 day in 37 ℃, take out in refrigerator and place, take a morsel in the nutrient agar medium liquid nutrient medium, in 37 ℃ ± 1 ℃ shake-flask culture 1 day, it was standby to take out in refrigerator storage.

2.GM ₁Preparation

Getting concentration is that 0.1%-10% separates GM ₁After other ganglioside fat raw material, soluble in water, add the 2-5% fermented bacterium, transfer pH5-8, in 32 ℃ ± 5 ℃ cultivations, after 3-5 days, tlc detects the GM that promptly obtains fermentation finished thoroughly1 ₁Solution, purity reaches 20%-65%, gets pure product through chromatography.

The present invention prepares GM ₁Fermented bacterium be brevibacterium sp (Brevibacterium sp.) QL-1 strain, on December 11st, 2000 by China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation, deposit number is CGMCC No.0522.

This bacterial classification is to separate from Qilu Pharmaceutical Factory's soil, obtains after screening and mutagenesis.

GM disclosed by the invention ₁Preparation method's extraction yield than existing methods improves 100%.This method is easy and simple to handle, with low cost, is GM ₁Scale operation provides may.

EmbodimentThe preparation of embodiment 1 fermented bacterium

Get the freeze-drying pipe hide bacterial classification a little, be inoculated on the nutrient agar slant medium, cultivated 1 day in 37 ℃, take out in refrigerator and place, take a morsel in the nutrient agar medium liquid nutrient medium, in 37 ℃ ± 1 ℃ shake-flask culture 1 day, it was standby to take out in refrigerator storage.

Embodiment 2

Get no GM ₁Composite nerve joint glycosides fat, be mixed with 2% the aqueous solution, transfer PH7.0, add 5 milliliters of the fermented bacteriums that embodiment 1 makes, in 32 ℃ ± 5 ℃ shake-flask culture, take out purity 60% after 3 days.

Embodiment 3

Learn from else's experience and separate GM ₁After composite nerve joint glycosides fat, be mixed with 4% the aqueous solution, transfer PH6.8, add 5 milliliters of the fermented bacteriums that embodiment 1 makes, in 32 ℃ ± 5 ℃ shake-flask culture, take out after 3 days, collect pure product GM through chromatography ₁

Embodiment 4

Learn from else's experience and separate GM ₁Composite nerve joint glycosides fat, be mixed with 4% the aqueous solution, transfer PH7.0, add 5 milliliters of the fermented bacteriums that embodiment 1 makes, leave standstill cultivation in 32 ℃ ± 5 ℃ triangular flasks, during the control pH value after 7.0,5 days, takes out, through the pure product GM of chromatography collection ₁

Embodiment 5

Learn from else's experience and separate GM ₁Other composite nerves joint glycosides fat, be mixed with 10% the aqueous solution, transfer PH7.2, add 10 milliliters of the fermented bacteriums that embodiment 1 makes, leave standstill cultivation in 32 ℃ ± 5 ℃ triangular flasks, during the control pH value after 7.2,5 days, takes out, through the pure product GM of chromatography collection ₁

Claims

1, a kind of Monostalotetrahexosylgangliside GM ₁The preparation method, it is characterized in that this method comprises the following steps:

(1) fermented bacterium preparation

(2) GM ₁Preparation

Getting concentration is that 0.1%-10% separates GM ₁After other ganglioside fat raw material, soluble in water, add the 2-5% fermented bacterium, transfer PH6-8, cultivate 3-5 days promptly in 32 ℃ ± 5 ℃, purity reaches 20%-65%, gets pure product through chromatography.

2, a kind of Monostalotetrahexosylgangliside GM as claimed in claim 1 ₁The preparation method, it is characterized in that wherein used fermented bacterium is brevibacterium sp QL-1 strain.