CN104342469A - Method for preparing single sialic acid four-hexose gangliosides through biotransformation - Google Patents
Method for preparing single sialic acid four-hexose gangliosides through biotransformation Download PDFInfo
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- CN104342469A CN104342469A CN201310332516.1A CN201310332516A CN104342469A CN 104342469 A CN104342469 A CN 104342469A CN 201310332516 A CN201310332516 A CN 201310332516A CN 104342469 A CN104342469 A CN 104342469A
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- monostalotetrahexosylgangliside
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Abstract
The invention discloses a method for biologically transforming polysialic acid four-hexose gangliosides into single sialic acid four-hexose gangliosides (GM1). A polysialic acid four-hexose ganglioside mixture is simply and efficiently hydrolyzed through bacteria for preparing the GM1. The using bacteria belong to the fiber genus, are named as Cellulosimicrobium sp.21, and are preserved in a common microorganism center of the China Committee for Culture Collection of Microorganisms; and the preservation serial number is CGMCC No.7587. The method has the beneficial effects of being good in selectivity, high in activity, simple in process, short in production path, high in product purity and the like; and the possibility is provided for mass production of the GM1.
Description
Technical field
The present invention relates to a kind of biotransformation method and prepare Monostalotetrahexosylgangliside, belong to field of biological pharmacy.
Background technology
Sphingolipids,sialo are the one in glycosphingolipid, be made up of, be mainly present in brain and nervous tissue the ceramide of a part long-chain, the lipid acid of a part long-chain and one containing sialic oligonucleotide chain.Sphingolipids,sialo have multiple biological activity, play an important role in nerve generation, growth, atomization, CO2 laser weld after damage also played an important role, promotes neural regeneration, promote neural axon growth and Synaptic formation, recovery innervation function, nerve conduction of improving, promote the recovery of electrical activity of brain and other Electrophysiology indexs; The effects such as Cell protection film, the various enzyme recoveries of primary cell film.Monostalotetrahexosylgangliside has been used for the treatment of neurological disorders disease as clinical medicine in recent years, as Alzheimer's disease, and parkinsonism, Spinal injury and apoplexy etc.Current research shows that Monostalotetrahexosylgangliside (GM1) directly can act on neurocyte by hemato encephalic barrier; its mechanism of action mainly the Na+-K+-ATP enzyme of Cell protection film and Ca2+-Mg2+-ATP active; improve intraor extracellular ion imbalance; active oxygen radical can be reduced simultaneously; prevent the damage of neurocyte with dead, promote the regeneration of neurocyte.But GM1 content be cerebral tissue ten thousand/, traditional extracting method is from animal brain, carry out Extraction and separation by the extraction of organic reagent, and method is complicated, and yield is low.And the biotransformation method announced at present prepares the GM1(patent No. 2004100182805), the GM1 removed before transforming in original Sphingolipids,sialo is required in its method, although improve total output but still need separating for several times, increase workload, too increase the risk of product pollution simultaneously.
Summary of the invention
The invention provides and a kind ofly utilize bacterium that the composite nerve joint glycosides fat in animal brain is converted into the method for GM1.
Technical solution of the present invention is: utilize bacterium simple, efficiently many ganglioside sialic acids are converted into GM1.
The bacterium that the present invention relates to is called Cellulosimicrobium sp.21, and be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on May 14th, 2013, deposit number is CGMCC No.7587.
Strain morphology feature: this bacterial strain Gram-positive, elongated rod shape, late stage of culture gradually becomes rod-short.The size of thalline is 0.4 ~ 0.6 micron × 1.6 ~ 3.0 microns.Somatic cells is that the moderate-length of marshalling is shaft-like, and fence arranges, and afterbody is blunt end.
Bacterial strain colony characteristics: bacterium colony is circular, and at solid culture primary surface, thalline forms translucent oyster white bacterium colony, and bacterium colony is round and smooth.
The present invention is in accordance with the following steps:
A) preparation of fermented bacterium: get preserve in cryopreservation tube bacteria culture, inoculating strain in LB substratum, 37 DEG C of shake-flask culture 1 day.
B) bio-transformation: bacterial strain is joined in bioconversion medium according to volume fraction 1%-5%, 15-40 DEG C of constant temperature culture 3-7 days.
C) reclaim GM1: centrifugal removing thalline, dry, Extraction buffer dissolves, and carries out column chromatography for separation and prepares GM1 sterling.Wherein, bioconversion medium is the ganglioside mixture that the animal brain containing volume fraction 5%-30% extracts, inorganic salt, adjust ph 5-9.Extraction buffer dissolves by ethyl acetate, ethanol, calcium chloride, and water forms.
By the present invention, after transforming, the ratio of GM1 accounts for more than 80% of total Sphingolipids,sialo, and step is simply efficient, can prepare GM1 by rapid, high volume.
Accompanying drawing explanation
Fig. 1, HPLC result figure.Wherein, 1 is the ganglioside mixture after bio-transformation; 2 is the ganglioside mixture before bio-transformation; 3 is standard model.
Embodiment
The preparation of embodiment 1 fermented bacterium
Get preserve in cryopreservation tube bacteria culture, inoculation bacterial classification is in LB substratum, and 37 DEG C of shake-flask culture 1 day, treat that spawn culture is to finite concentration, is inoculated in bioconversion medium.
Embodiment 2
Get ganglioside mixture, join 1L bioconversion medium (inorganic salt, pH7.0) in, make its concentration be 10%, add the fermented bacterium 50ml that embodiment 1 is obtained, in 37 DEG C of shake-flask culture, take out after 3 days, adopt document Journal of Pharmaceutical and Biomedical Analysis.Vol.8,1063-1066, high performance liquid chromatography inspection disclosed in nineteen ninety, the ratio that the rear GM1 of conversion accounts for total Sphingolipids,sialo reaches 70%.
Embodiment 3
Get ganglioside mixture, join 1L bioconversion medium (inorganic salt, pH7.0) in, make its concentration be 20%, add the fermented bacterium 50ml that embodiment 1 is obtained, in 37 DEG C of shake-flask culture, take out after 3 days, adopt document Journal of Pharmaceutical and Biomedical Analysis.Vol.8,1063-1066, high performance liquid chromatography inspection disclosed in nineteen ninety, the ratio that the rear GM1 of conversion accounts for total Sphingolipids,sialo reaches 80%.
Embodiment 4
Get ganglioside mixture, join 1L bioconversion medium (inorganic salt, pH7.0) in, make its concentration be 10%, add the fermented bacterium 30ml that embodiment 1 is obtained, in 37 DEG C of shake-flask culture, take out after 3 days, adopt document Journal of Pharmaceutical and Biomedical Analysis.Vol.8,1063-1066, high performance liquid chromatography inspection disclosed in nineteen ninety, the ratio that the rear GM1 of conversion accounts for total Sphingolipids,sialo reaches 60%.
Claims (3)
1. one kind utilizes bacterium that the composite nerve joint glycosides fat in animal brain is converted into the method for Monostalotetrahexosylgangliside, it is characterized in that using fiber to belong to bacterium Cellulosimicrobium sp.21, numbering CGMCC No.7587 bacterial strain carries out bio-transformation, and its step is as follows:
A) prepare fermented bacterium: get preserve in cryopreservation tube bacteria culture, inoculating strain in LB substratum, 37 DEG C of shake-flask culture 1 day;
B) bio-transformation: bacterial strain is joined in bioconversion medium according to volume fraction 1%-5%, 15-40 DEG C of constant temperature culture 3-7 days;
C) reclaim: centrifugal removing thalline, dry; Extraction buffer dissolves, and column chromatography for separation prepares Monostalotetrahexosylgangliside sterling.
2. the method for Monostalotetrahexosylgangliside is prepared in bio-transformation according to claim 1, it is characterized in that in step b), and bioconversion medium is the ganglioside mixture that the animal brain containing volume fraction 5%-30% extracts, inorganic salt.
3. the method for Monostalotetrahexosylgangliside is prepared in bio-transformation according to claim 1, it is characterized in that in step c), and Extraction buffer dissolves by ethyl acetate, ethanol, and calcium chloride forms.
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| CN201310332516.1A CN104342469B (en) | 2013-08-01 | 2013-08-01 | A kind of method that bioconversion prepares Monostalotetrahexosylgangliside |
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| CN201310332516.1A CN104342469B (en) | 2013-08-01 | 2013-08-01 | A kind of method that bioconversion prepares Monostalotetrahexosylgangliside |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| RU2632709C1 (en) * | 2016-07-27 | 2017-10-09 | Лонг Шенг Фарма Лимитед | Neuroprotective agent from pig brain based on sodium monosial ganglioside |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1570080A (en) * | 2004-05-12 | 2005-01-26 | 华东理工大学 | Single sialic acid tetrahexose ganglioside preparation method |
| WO2007101862A1 (en) * | 2006-03-09 | 2007-09-13 | Centre National De La Recherche Scientifique (Cnrs) | Method of producing sialylated oligosaccharides |
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2013
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1570080A (en) * | 2004-05-12 | 2005-01-26 | 华东理工大学 | Single sialic acid tetrahexose ganglioside preparation method |
| WO2007101862A1 (en) * | 2006-03-09 | 2007-09-13 | Centre National De La Recherche Scientifique (Cnrs) | Method of producing sialylated oligosaccharides |
Non-Patent Citations (2)
| Title |
|---|
| JIANGUO ZHANG ET AL.: ""Efficient conversion from polysialogangliosides to monosialotetrahexosylganglioside using Oerskovia xanthineolytica YZ-2"", 《BIOPROCESS BIOSYST ENG》 * |
| 陈燕红等: ""一株纤维化纤维细菌的生物学特性及其对几种苯环类化合物的利用研究"", 《微生物学通报》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| RU2632709C1 (en) * | 2016-07-27 | 2017-10-09 | Лонг Шенг Фарма Лимитед | Neuroprotective agent from pig brain based on sodium monosial ganglioside |
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Inventor after: Ji Li Inventor after: Zhou Yifa Inventor after: Leng Jiayi Inventor after: Sun Chengxin Inventor after: Tai Guihua Inventor before: Zhou Yifa Inventor before: Ji Li Inventor before: Leng Jiayi Inventor before: Sun Chengxin Inventor before: Tai Guihua |
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