阅读说明：本技术 一种单唾液酸四己糖神经节苷脂的合成方法 (Synthesis method of monosialotetrahexosyl ganglioside ) 是由 王鹏 曹学峰 于 2020-04-23 设计创作，主要内容包括：本发明涉及一种工业合成单唾液酸四己糖神经节苷脂的方法,以氟代乳糖(Lac-F)为原料,经过10种酶催化的五步连续反应制备单唾液酸四己糖神经节苷脂,并达到由克级至公斤级的制备规模,从而满足了工业生产GM1的需求,取代了传统从猪脑提取的工艺路线。另外,本专利将制备单唾液酸四己糖神经节苷脂的工艺缩短为五步,并且在前三个步骤中未纯化中间产物,减少了因中间步骤纯化引起的产品损耗,节约了时间成本。(The invention relates to a method for industrially synthesizing monosialotetrahexosylganglioside, which takes fluorolactose (L ac-F) as a raw material, prepares the monosialotetrahexosylganglioside through five-step continuous reaction catalyzed by 10 enzymes, and reaches the preparation scale from gram level to kilogram level, thereby meeting the requirement of industrial production of GM1, and replaces the traditional process route of extracting from pig brain.)

1. A method for synthesizing monosialotetrahexosylganglioside is characterized by comprising the following steps:

A. catalyzing fluorolactose, sialic acid and cytidine triphosphate by adopting a first group of synthases to generate GM 3-F;

B. adopting N-acetylgalactosamine, adenosine triphosphate, uridine triphosphate and GM3-F to catalyze and generate GM2-F under the action of a second group of synthase;

C. galactose, adenosine triphosphate, uridine triphosphate and GM2-F are adopted to produce GM1-F under the action of a third group of synthase;

D. catalyzing GM1-F by adopting a fourth enzyme under the action of alcohol to generate GM 1-Sph;

E. using alkyl acyl chloride and GM1-Sph to synthesize monosialotetrahexosylganglioside.

2. The method of claim 1, wherein the step of synthesizing the monosialotetrahexosylganglioside comprises the steps of:

the first group of synthases in step A are NmCSS, PmST1 and PmPpA;

the second group of synthases in the step B are NaHK, AGX1, PmPpA and CgtA, the step B adopts N-acetylgalactosamine, uridine triphosphate and adenosine triphosphate to generate a first intermediate UDP-GalNac under the action of the three enzymes of NaHK, AGX1 and PmPpA, and then the UDP-GalNac and GM3-F are catalyzed to generate GM2-F under the action of the enzyme CgtA;

step C, using galactose, adenosine triphosphate and uridine triphosphate to generate a second intermediate UDP-Gal under the action of three enzymes of GalK, AtUSP and PmPpA; then UDP-Gal and GM2-F produce GM1-F under the action of enzyme CgtB;

the fourth enzyme in the step D is EGC II, and the enzyme EGC II is adopted in the step D to catalyze GM1-F and sphingosine to generate GM1-Sph in an ethanol-containing system;

and step E, synthesizing GM1-C18 by using octadecanoyl chloride and GM1-Sph, or synthesizing GM1-C20 by using eicosanoyl chloride and GM 1-Sph.

3. The method of synthesizing monosialotetrahexosylgangliosides according to claim 2, wherein in step a:

the molar concentration of the fluorolactose is 10-40 mM;

the molar concentration ratio of the fluorolactose to the sialic acid to the cytidine triphosphate is 1: (1.2-2): (1.3-2);

the dosage of the NmCSS is 80-140U;

the dosage of the PmST1 is 80-220U;

the reaction temperature is 10-40 ℃, and the pH value is 7-9.

4. The method of synthesizing monosialotetrahexosylgangliosides according to claim 2, wherein in step a:

the molar concentration of the fluorolactose is 30 mM;

the molar concentration ratio of the fluorolactose to the sialic acid to the cytidine triphosphate is 1:1.7: 1.6;

the dosage of the NmCSS is 100U;

the dosage of the PmST1 is 180U;

the reaction temperature was 30 ℃ and the pH was 9.

5. The method of synthesizing monosialotetrahexosylgangliosides according to claim 2, wherein in step B:

the concentration of the N-acetylgalactosamine is 20-50 mM;

the molar concentration ratio of the N-acetylgalactosamine, the adenosine triphosphate and the uridine triphosphate is 1: (1.2-1.3): (1.2-1.3);

the dosage of the NaHK is 200-260U;

the dosage of the AGX1 is 120-270U;

the reaction temperature of the first intermediate product is 30-42 ℃, and the pH value is 7-9;

the concentration of the GM3-F is 10-30 mM;

the molar concentration ratio of the GM3-F to the first intermediate product is 1: 1;

the CgtA addition amount is 100-300U;

the reaction temperature of the GM3-F and the first intermediate product is 25-40 ℃, and the pH value is 7-9.

6. The method of synthesizing monosialotetrahexosylgangliosides according to claim 2, wherein in step B:

the concentration of the N-acetylgalactosamine is 30 mM;

the molar concentration ratio of the N-acetylgalactosamine, the adenosine triphosphate and the uridine triphosphate is 1:1.3: 1.2;

the dosage of the NaHK is 240U;

the dosage of the AGX1 is 170U;

the reaction temperature of the first intermediate product is 37 ℃, and the pH value is 9;

the concentration of the GM3-F is 15 mM;

the CgtA addition amount is 100-300U;

the reaction temperature of GM3-F with the first intermediate product was 37 deg.C and the pH was 9.

7. The method of synthesizing monosialotetrahexosylgangliosides according to claim 2, wherein in step C:

the concentration range of the galactose is 20-80 mM;

the molar concentration ratio of the galactose, the adenosine triphosphate and the uridine triphosphate is 1: (1-1.2): (1-1.3);

the reaction temperature range for synthesizing the second intermediate product is 30-40 ℃, and the pH value range is 7-9;

the reaction for synthesizing the second intermediate product further comprises magnesium ions at a concentration of 5-20 mM;

the addition amount of the GalK is 40-120U;

the addition amount of the AtUSP is 20-80U;

the concentration of the GM2-F is 10 mM;

the molar concentration ratio of the GM2-F to the second intermediate product is 1: 1;

the reaction temperature range of the GM2-F and the second intermediate product is 25-40 ℃, and the pH value range is 7-9;

the reaction of GM2-F with the second intermediate further comprises manganese ions at a concentration of 5-20 mM;

the addition amount of the CgtB is 40-160U.

8. The method for synthesizing monosialotetrahexosylgangliosides according to claim 2 or 7, wherein in step C:

the concentration range of the galactose is 40 mM;

the molar concentration ratio of the galactose, the adenosine triphosphate and the uridine triphosphate is 1:1.1: 1.2;

the reaction temperature range for synthesizing the second intermediate product is 37 ℃, and the pH value range is 8;

synthesizing the magnesium ion concentration to be 5 mM;

the addition amount of the GalK is 100U;

the addition amount of the AtUSP is 40U;

the reaction temperature range of GM2-F and the second intermediate product is 37 ℃, and the pH value range is 7.5;

the concentration of the manganese ions is 10 mM;

the addition amount of CgtB is 120U.

9. The method of synthesizing monosialotetrahexosylganglioside according to claim 1 or 2, further comprising the purification process of GM1-Sph, said process being as follows:

1) adding 1:1 equivalent of ethanol into the mixed solution of GM1-Sph, adjusting the pH value, standing overnight at low temperature, and centrifuging to obtain supernatant;

2) carrying out ultrafiltration on the liquid obtained in the step A, and taking upper layer reflux liquid;

3) performing rotary evaporation to remove ethanol in the reflux liquid, and separating GM1-Sph by column chromatography;

4) removing methanol introduced in the step C by rotary evaporation, and then freeze-drying the liquid to obtain white solid powder, namely GM 1-Sph.

10. The purification process of GM1-Sph according to claim 9, wherein:

the pH value range in the step 1) is 5-9;

in the step 2), the ultrafiltration is performed by adopting one of a polyamide composite membrane or a polyether sulfone composite membrane, and the range of the molecular weight cut-off is 300-500D;

the column chromatography packing in the step 3) is a C18 material;

the column chromatography separation method in the step 3) adopts a water-methanol system;

the column chromatography process in the step 3) comprises the following steps: washing chromatographic column with water, then washing column volume with 50% methanol water solution until no impurity, then washing the lower product with 80% methanol water solution, and finally washing column with 100% methanol.

[ technical field ] A method for producing a semiconductor device

The invention relates to a method for industrially synthesizing monosialotetrahexosyl ganglioside, belonging to the technical field of biochemical catalytic synthesis.

[ background of the invention ]

Monosialotetrahexosyl ganglioside (GM1) is a substance extracted from pig brain and having an effect on neuronal function impairment. The structure of the compound comprises hydrophobic ceramide and hydrophilic oligosaccharide chain with sialic acid, and the two ends of the compound are connected together through glycosidic bonds. Hydrophobic ceramides contain mainly sphingosine and fatty acids, which are linked via imino groups. Sphingosine is composed of polyhydroxylated fatty amines, with a chain length of 18 or 20 carbon atoms, and has the formula:

[ summary of the invention ]

The invention aims to provide a method for synthesizing monosialotetrahexosyl ganglioside GM 1.

In order to achieve the above object, the synthesis of monosialotetrahexosylganglioside GM1 of the present invention comprises the steps of:

1. adopting NmCSS and PmST₁And PmPpA catalyzing fluorolactose (L ac-F), sialic acid (Neu5Ac) and Cytidine Triphosphate (CTP) to generate GM3-F；

Wherein, the molar concentration of the substrate fluorolactose is preferably 10-40mM, and more preferably 30 mM;

the molar concentration ratio of the substrates of the fluorolactose, the sialic acid and the cytidine triphosphate is preferably 1: (1.2-2): (1.3-2); more preferably 1:1.7: 1.6;

the dosage of the NmCSS is preferably 80-140U, and more preferably 100U;

the dosage of the PmST1 is preferably 80-220U, more preferably 180U;

the reaction temperature is preferably 10 to 40 ℃, more preferably 30 ℃;

the pH of the reaction is preferably 7 to 9, more preferably 9.

2. Under the action of two enzymes, namely NaHK and AGX1, N-acetylgalactosamine (GalNac), Uridine Triphosphate (UTP) and Adenosine Triphosphate (ATP) are adopted to generate an intermediate UDP-GalNac (step S1), and then the UDP-GalNac and a substrate GM3-F are catalyzed to generate GM2-F under the action of the enzyme CgtA (step S2).

Wherein, in the step S1 reaction:

the concentration of the GalNac is preferably 20 to 50mM, more preferably 30 mM;

the molar concentration ratio of the substrate GalNac, ATP and UTP is preferably 1: (1.2-1.3): (1.2-1.3), more preferably 1:1.3: 1.2;

the dosage of the NaHK is preferably 200-260U, and more preferably 240U;

the dosage of AGX1 is preferably 120-270U, and more preferably 170U;

the reaction temperature is preferably 30-42 ℃, more preferably 37 ℃;

the pH of the reaction is preferably 7 to 9, more preferably 9.

Further, in the step S2 reaction:

the concentration of GM3-F is preferably 10-30mM, more preferably 15 mM;

the molar concentration ratio of the substrate GM3-F, UTP-GalNac is preferably 1: 1;

the addition amount of CgtA is preferably 100-300U, and more preferably 200U;

the reaction temperature is preferably 25 to 40 ℃, more preferably 37 ℃;

the pH of the reaction is preferably 7 to 9, more preferably 9.

3. Generating an intermediate UDP-Gal by using galactose, ATP and UTP under the action of two enzymes of GalK and AtUSP (step S3); UDP-Gal and GM2-F produced GM1-F under the action of the enzyme CgtB (step S4).

Wherein, in the step S3:

the concentration of the substrate Gal is preferably in the range of 20 to 80mM, more preferably 40 mM;

the molar concentration ratio of the substrate Gal, ATP and UTP is preferably 1 (1-1.2) to (1-1.3), more preferably 1:1.1: 1.2;

the reaction temperature is preferably in the range of 30-40 ℃, more preferably 37 ℃;

the reaction pH value is preferably in the range of 7-9, more preferably 8;

the addition amount of the reaction enzyme GalK is preferably 40-120U, and more preferably 100U;

the addition amount of the reaction enzyme AtUSP is preferably 20-80U, and more preferably 40U;

the reaction further comprises the activator being a metal ion Mg⁺The amount of the surfactant added is preferably 5 to 20mM, more preferably 5 mM.

Further, in the step S4:

the concentration range of the substrate GM2-F is preferably 10 mM;

the molar concentration ratio of the substrate GM2-F, UDP-Gal is preferably 1: 1;

the reaction temperature is preferably in the range of 25 to 40 ℃, more preferably 37 ℃;

the reaction pH value is preferably in the range of 7-9, more preferably 7.5;

the addition amount of the reactive metal ion Mn + is preferably 5-20mM, more preferably 10 mM;

the amount of the reaction enzyme CgtB to be added is preferably 40 to 160U, more preferably 120U.

4. Catalyzing GM1-F and sphingosine (Sph) by adopting enzyme EGC II in an ethanol-containing system to generate GM 1-Sph;

respectively dissolving GM1-Sph and octadecanoyl chloride to synthesize GM1-C18, or respectively dissolving GM1-Sph and eicosanoyl chloride to synthesize GM 1-C20.

The enzyme used in the invention is the industry term English abbreviation, and the name and abbreviation comparison table is as follows:

the invention also aims to provide a purification process of a tetrahexosylgangliside intermediate GM1-Sph, which comprises the following steps:

GM1-Sph is purified by ion exchange column adsorption, ultrafiltration concentration and C18 column chromatography.

Further, the purification process of GM1-Sph is as follows:

1. adding 1:1 equivalent of ethanol into the mixed solution of GM1-Sph, adjusting the pH value, standing overnight, and centrifuging to obtain a supernatant;

2. carrying out ultrafiltration on the liquid obtained in the step 1, and taking upper layer reflux liquid;

3. performing rotary evaporation to remove ethanol in the reflux liquid, and separating GM1-Sph by column chromatography;

4. removing methanol introduced in the step 3) by rotary evaporation, and then lyophilizing the liquid to obtain a white solid powder, i.e., GM 1-Sph.

Further, the pH value in the step 1 is in a range of 5-9;

further, in the step 2, a polyamide composite membrane or a polyether sulfone composite membrane is adopted for ultrafiltration, and the range of the cut-off molecular weight is 300-500D;

further, in the step 3, the column chromatography packing is a C18 material;

further, the column chromatography separation method in the step 3 adopts a water-methanol system;

further, the column chromatography process in step 3 is as follows: washing chromatographic column with water, washing column with 50 vol% water solution of methanol until no impurity, washing the lower product with 80 vol% water solution of methanol, and washing column with 100 vol% methanol.

The purity of GM1 prepared by the above preparation method and purification process is above 99%, HP L C is shown in figure 1, and mass spectrum is shown in figure 9B.

Among these, HP L C results are as follows:

further, since the molecular weight of GM1 is known, the molecular weight obtained by primary mass spectrometry is 1590, and GM1 is the target product.

The invention has the beneficial effects that:

1. the method adopts the means of sedimentation, ultrafiltration, column chromatography and the like to improve the purity of the GM1-Sph to 99%. Compared with an untreated sample, the GM1-Sph product purified by the purification process of the invention does not contain salts, organic solvents, naked sugar and sugar nucleotides basically. Meanwhile, the scheme is also suitable for purifying GM1-Sph in the fields of food and medicine, and is beneficial to obtaining GM1-Sph in an industrial large scale.

2. The method takes the fluorolactose (L ac-F) as the raw material to generate the GM1 through the catalytic action of 10 enzymes, avoids the defect that each step of reaction needs to be purified compared with the prior art, and has short period of the whole preparation route, simple and convenient steps and low cost.

3. In the step 4, a proper amount of ethanol is added instead of ethylene glycol dimethyl ether, so that the use of an irritant organic solvent ethylene glycol dimethyl ether is avoided while the solubility of the sphingosine and GM1-Sph in the reaction is improved.

[ description of the drawings ]

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.

FIG. 1 is a high performance liquid chromatogram of GM1 synthesized in example 1 of the present invention.

FIG. 2 is a thin layer chromatogram of consumption and production of GM3-F of fluorolactose in example 1 of the present invention.

FIG. 3A is a thin layer chromatogram of consumption and generation of UDP-GalNac of N-acetylgalactosamine in example 1 of the present invention.

FIG. 3B is a high performance liquid chromatogram of UDP-GalNac produced in example 1 of the present invention.

FIG. 4 is a thin layer chromatogram of consumption and production of GM3-F in example 1 of the present invention.

FIG. 5A is a thin layer chromatogram of consumption of D-galactose and production of UDP-Gal in example 1 of the present invention.

FIG. 5B is a high performance liquid chromatogram for UDP-Gal production in example 1 of the present invention.

FIG. 6 is a thin layer chromatogram of consumption and production of GM2-F in example 1 of the present invention.

FIG. 7 is a thin layer chromatogram of the consumption and production of GM1-F in example 1 of the present invention, GM 1-sph.

FIG. 8 is a high performance liquid chromatogram of purified GM1-Sph of example 3 of the present invention.

FIG. 9A is a thin layer chromatogram of GM1-Sph consumption vs. GM1 production in example 4 of the invention.

FIG. 9B is a mass spectrum of GM1 generated in example 4 of the present invention.

[ detailed description ] embodiments

In order to make the objects, technical solutions and advantageous effects of the present invention more clearly apparent, the present invention is further described in detail below with reference to the accompanying drawings and the detailed description. It should be understood that the detailed description and specific examples, while indicating the preferred embodiment of the invention, are intended for purposes of illustration only and are not intended to limit the scope of the invention.