阅读说明：本技术 一种基于褐藻胶裂解酶的褐藻胶寡糖测序方法及其应用 (Alginate oligosaccharide sequencing method based on alginate lyase and application thereof ) 是由 李福川 路丹荣 张庆冬 关靖雯 王淑敏 于 2019-10-22 设计创作，主要内容包括：本发明涉及一种基于褐藻胶裂解酶的褐藻胶寡糖测序方法及其应用。本发明首次建立了一种基于褐藻胶裂解酶的褐藻胶寡糖的测序方法,尤其是利用外切型褐藻胶裂解酶AlyPB2在降解多糖及寡糖底物时,是从非还原端逐个切割糖醛酸的特点,利用外切型褐藻胶裂解酶AlyPB2对褐藻胶寡糖进行部分酶切,通过凝胶过滤色谱分离制备不同聚合度的部分酶切寡糖,再利用<Sup>1</Sup>H-NMR技术对酶切产物的还原端和非还原端的糖基结构进行测定,从而简便快速的确定单一组分或聚合度均一多组分褐藻胶寡糖的精确组成和结构,该方法所需样品量较少,且大大简化了后期分析过程,彻底解决了高分子量(DP≥4)褐藻胶寡糖组分的结构鉴定问题。(The invention relates to an alginate oligosaccharide sequencing method based on alginate lyase and application thereof. The invention firstly establishes a sequencing method of alginate oligosaccharides based on alginate lyase, and particularly utilizes the characteristic that when exo-type alginate lyase AlyPB2 is used for degrading polysaccharide and oligosaccharide substrates, uronic acid is cut one by one from a non-reducing end, partial enzyme digestion is carried out on the alginate oligosaccharides by utilizing exo-type alginate lyase AlyPB2, partial enzyme digestion oligosaccharides with different polymerization degrees are prepared by gel filtration chromatography separation, and then partial enzyme digestion oligosaccharides with different polymerization degrees are utilized 1 The H-NMR technology is used for measuring the glycosyl structures of the reducing end and the non-reducing end of the enzyme digestion product, so that the accurate composition and structure of single-component or multi-component alginate-derived oligosaccharide with uniform polymerization degree can be simply, conveniently and quickly determined, the required sample amount is small, the later analysis process is greatly simplified, and the problem of structural identification of the high-molecular-weight (DP is more than or equal to 4) alginate-derived oligosaccharide component is thoroughly solved.)

1. An alginate oligosaccharide sequencing method based on alginate lyase is characterized by comprising the following steps:

(1) adding alginate oligosaccharide into heavy water (D)₂O), repeated freeze-drying to complete replacement of hydrogen and deuterium, and then carrying out¹H-NMR analysis is carried out to determine the glycosyl structures of the reducing end and the non-reducing end of the alginate oligosaccharide;

(2) partially degrading the alginate oligosaccharides in the same batch as the alginate oligosaccharide in the step (1) by using an externally tangent type alginate lyase, and collecting degradation products for multiple times and respectively concentrating to obtain intermediate product oligosaccharides with different polymerization degrees; the circumscribed type alginate lyase is alginate lyase AlyPB 2;

(3) freeze drying and desalting the intermediate product oligosaccharide obtained in step (2) repeatedly, and adding heavy water (D)₂O), repeatedly freezing and drying to complete the replacement of hydrogen and deuterium,and performing (a) on¹H-NMR analysis to determine the glycosyl structure of the reducing end and the non-reducing end of each intermediate product oligosaccharide, and the combination of the alginate oligosaccharides in the step (1)¹H-NMR results showed that the sugar chain structure of the alginate oligosaccharides was determined.

2. The alginate oligosaccharide sequencing method of claim 1, wherein the alginate oligosaccharide is a single-component or multi-component alginate oligosaccharide with uniform polymerization degree, which is formed by mixing beta-D-1, 4-mannuronic acid (M) and alpha-L-1, 4-guluronic acid (G).

3. The alginate oligosaccharide sequencing method of claim 1, wherein the polymerization degree of the alginate oligosaccharide is not less than 4.

4. The alginate oligosaccharide sequencing method of claim 1, wherein the desalting step (3) is repeated freeze-drying for more than 3 times.

5. The alginate oligosaccharide sequencing method of claim 1, wherein the desalting in step (3) is performed by repeated freeze-drying for 3-6 times.

6. The alginate oligosaccharide sequencing method of claim 1, wherein the deuterium-hydrogen replacement is performed by repeated freeze-drying for 3-5 times.

7. The alginate oligosaccharide sequencing method of claim 1, wherein the deuterium-hydrogen substitution is performed by repeated freeze-drying 3 times.

8. Use of the alginate oligosaccharide sequencing method based on alginate lyase according to claim 1 in sugar chain structure determination of alginate oligosaccharides.

Technical Field

The invention relates to an alginate oligosaccharide sequencing method based on alginate lyase and application thereof, belonging to the technical field of analytical chemistry.

Background

Algin (alginate) is the most abundant carbohydrate in the cell wall and matrix of brown algae such as kelp, gulfweed, and kelp, and accounts for about 40% of the dry weight of brown algae. Algin is a linear anionic acidic polysaccharide formed by β -D-mannuronic acid (β -D-Mannuronate, M) and its C5 epimer α -L-guluronic acid (α -L-Guluronate, G) linked by β -1,4 glycosidic linkages, and is classified into polymannuronic acid segments (PolyM), polyguluronic acid segments (PolyG), and alternating blocks of mannuronic and guluronic acids (PolyMG/GM) depending on the order of arrangement of the two uronic acids. In recent years, algin is widely applied to industries such as food, chemical industry, medicine, textile and the like due to characteristics such as high viscosity, gel property and the like. However, the application of algin is greatly limited due to the defects of large molecular weight, poor water solubility, difficult absorption and utilization and the like. The algin oligosaccharide is a functional oligomer obtained by degrading algin, and has received great attention due to the advantages of strong stability, easy absorption and utilization, no toxicity and no harm. More and more reports show that the alginate oligosaccharide has a plurality of important biological activities, such as plant growth promotion, tumor resistance, anticoagulation, bacteria resistance, oxidation resistance, cell apoptosis regulation, neuroinflammation, blood sugar and blood fat regulation, and the like, and has important research value and development potential.

The preparation method of the alginate oligosaccharide mainly comprises a physical degradation method, a chemical degradation method and an enzymatic hydrolysis method, wherein the enzymatic hydrolysis method is a common oligosaccharide preparation method and has the advantages of mild reaction conditions, no pollution, high yield, strong specificity and the like. The algin lyase is a polysaccharide degrading enzyme which catalyzes the breakage of the glycosidic bond in the algin molecule through beta-elimination reaction, and forms C4 ═ C5 unsaturated double bond at the non-reducing end of the newly generated algin oligosaccharide, and the unsaturated uronic acid at the non-reducing end has a structure different from that of the M and G saccharide units, so that the algin lyase is called delta monosaccharide. According to different degradation modes of the alginate lyase, the degradation mode is divided into an endo-lyase and an exo-lyase; according to the preference of alginate lyase to substrate, the degrading enzyme can be divided into M-specific lyase, G-specific lyase and bifunctional lyase, the unsaturated oligosaccharides obtained by degrading algin with different preference algin lyase have different structures, and related reports show that the structure of oligosaccharide and the activity thereof have great relevance, such as: (1) compared with guluronic acid oligosaccharide, mannuronic acid oligosaccharide can better stimulate cytokine production; in addition, 971 prepared from mannuronic acid oligosaccharide can be used for treating Alzheimer disease; (2) the oxidation resistance of the alginate oligosaccharide is related to the relative molecular mass and the M/G ratio of the alginate oligosaccharide; (3) oligosaccharides with a G-terminus can significantly increase the growth rate of keratinocytes. Therefore, the preparation of the functional alginate-derived oligosaccharide and the identification of the structure of the functional alginate-derived oligosaccharide have important significance for the exploration of the structure and the functional relationship of the alginate.

At present, the sugar structure identification mainly adopts spectral analysis such as mass spectrometry, nuclear magnetic resonance technology and the like, wherein the difficulty of the sugar structure identification is increased by the problems of expensive equipment, large required sample amount, complex analysis process and the like of the nuclear magnetic resonance technology. The structural identification of the enzymolysis product of the alginate lyase is commonly adopted¹H and¹³c NMR, sugar end group protons at reducing end and non-reducing end of alginate unsaturated oligosaccharide¹Has characteristic absorption peaks in H-NMR, so that the reducing end and the non-reducing end of the oligosaccharide only pass through the glycosyl structure¹H-NMR was confirmed. However, when the molecular weight of the unsaturated oligosaccharide is larger (greater than or equal to UDP4), the structure of the unsaturated oligosaccharide can be determined only by comprehensive analysis of one-dimensional and two-dimensional nuclear magnetic resonance technologies, the two-dimensional nuclear magnetic resonance technology requires a large amount of samples, the requirement of the two-dimensional spectrum analysis process on specialized knowledge is high, and the analysis difficulty of non-nuclear magnetic resonance technology professionals is high, so that the structure identification of the alginate oligosaccharide with larger molecular weight is difficult, and the structure-activity relationship research of the alginate oligosaccharide and the development of related biomedical products are greatly hindered.

In 2019, chinese patent document (application No. 201910705619.5) describes an exo-type alginate lyase AlyPB2 from photobacterium sp.fc615. The exo-type alginate lyase AlyPB2 is a bifunctional lyase that exhibits strong exonuclease activity for both the polyM and polyG segments. This patent application also describes that AlyPB2 cleaves uronic acid one by one from the non-reducing end of the alginate sugar chain. Based on the important characteristics, the invention establishes an alginate oligosaccharide sequencing method based on alginate lyase.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides an alginate oligosaccharide detection method based on alginate lyaseSequencing method and application thereof. The method utilizes circumscribed algin lyase to perform partial enzyme digestion on algin oligosaccharide, prepares algin oligosaccharide with different polymerization degrees through gel filtration chromatographic separation, and then utilizes¹H-NMR analysis identifies the end group oligosaccharide of the enzyme digestion product, thereby simply, conveniently and rapidly determining the accurate composition and structure of single component and multi-component algin oligosaccharide with uniform polymerization degree, the method not only saves samples, but also greatly reduces the resolution difficulty of the algin oligosaccharide structure, and thoroughly solves the problem of structural identification of high molecular weight (DP is more than or equal to 4) algin oligosaccharide components.

The technical scheme of the invention is as follows:

an alginate oligosaccharide sequencing method based on alginate lyase comprises the following steps:

(1) adding alginate oligosaccharide into heavy water (D)₂O), repeated freeze-drying to complete replacement of hydrogen and deuterium, and then carrying out¹H-NMR analysis is carried out to determine the glycosyl structures of the reducing end and the non-reducing end of the alginate oligosaccharide;

(2) partially degrading the alginate oligosaccharides in the same batch as the alginate oligosaccharide in the step (1) by using an externally tangent type alginate lyase, and collecting degradation products for multiple times and respectively concentrating to obtain intermediate product oligosaccharides with different polymerization degrees; the circumscribed type alginate lyase is alginate lyase AlyPB 2;

(3) freeze drying and desalting the intermediate product oligosaccharide obtained in step (2) repeatedly, and adding heavy water (D)₂O), repeated freeze-drying to complete replacement of hydrogen and deuterium, and carrying out¹H-NMR analysis to determine the glycosyl structure of the reducing end and the non-reducing end of each intermediate product oligosaccharide, and the combination of the alginate oligosaccharides in the step (1)¹H-NMR results showed that the sugar chain structure of the alginate oligosaccharides was determined.

According to the present invention, the alginate oligosaccharide is preferably a single component composed of beta-D-1, 4-mannuronic acid (M) and alpha-L-1, 4-guluronic acid (G) or a multi-component alginate oligosaccharide with uniform polymerization degree.

According to the invention, the polymerization degree of the algin oligosaccharide is preferably more than or equal to 4.

In the present invention, the alginate lyase AlyPB2 in step (2) is derived from Photobacterium sp FC 615. The alginate lyase AlyPB2 cleaves uronic acid one by one from the non-reducing end when degrading polysaccharide and oligosaccharide substrates. Alginate lyase AlyPB2 is described in patent application 201910705619.5.

According to the invention, the desalting in the step (3) needs to be repeated freeze-dried for more than 3 times; further preferably, the desalting is performed by repeated freeze drying for 3-6 times.

According to the optimization of the invention, the replacement of deuterium and hydrogen needs to be carried out for 3-5 times by repeated freeze drying; further preferably, the deuterium/hydrogen substitution is performed by repeating the freeze-drying 3 times.

In the present invention, the sequence of step (1) and step (2) of the alginate oligosaccharide sequencing method based on alginate lyase is not limited.

The method is applied to the sugar chain structure determination of the algin oligosaccharide.

In the preferred technical scheme of the invention, after the algin polysaccharide is thoroughly degraded by an endo-type algin lyase, the obtained degradation products comprise algin disaccharide, algin trisaccharide, algin tetrasaccharide, algin pentasaccharide and algin hexasaccharide, and the molar ratio is 23:52:18.6:4: 2.4; subjecting the enzymolysis product to¹H-NMR analysis, partial degradation of alginate lyase AlyPB2, and then¹H-NMR analysis is carried out, and the sugar chain structure determination of the alginate oligosaccharides is completed;

wherein the algin disaccharide is mainly a disaccharide unit at the non-reducing end of deltaG and contains a small amount of disaccharide unit at the non-reducing end of deltaM, and the molar ratio of the disaccharide unit at the non-reducing end of deltaG to the disaccharide unit at the non-reducing end of deltaM is as follows: 3.4: 1;

the algin trisaccharide is a trisaccharide unit containing two non-reducing ends of delta G and delta M, and the molar ratio of the trisaccharide unit at the non-reducing end of the delta G to the trisaccharide unit at the non-reducing end of the delta M is as follows: 6.7: 1; the reducing end terminal of the algin trisaccharide is G, so the algin trisaccharide contains two structures delta GG and delta MG, and the molar ratio is as follows: 6.7: 1;

the algin tetrasaccharide is a tetrasaccharide unit containing two kinds of non-reducing ends of delta G and delta M, and the molar ratio of the tetrasaccharide unit at the non-reducing end of delta G to the tetrasaccharide unit at the non-reducing end of delta M is as follows: 1: 3.2; the reducing end of the algin tetrasaccharide and the glycosyl closest to the reducing end are both M, so the algin tetrasaccharide contains two structures of delta GMM and delta MMM, and the molar ratio is as follows: 1: 3.2;

the algin pentasaccharide is a pentasaccharide unit containing two non-reducing ends of delta G and delta M, and the molar ratio is as follows: 3.5: 1; the central sugar unit of the algin pentasaccharide chain is G and M, and the molar ratio is: 1.5: 1; the reducing end of the algin pentasaccharide and the glycosyl group closest to the reducing end are both M, so that the unsaturated pentasaccharide contains four structures of delta MMMM, delta MGMM, delta GMMM and delta GGMM, and the molar ratio is as follows: 1:1.5:3.5: 5.25;

the algin hexaose is a hexaose unit containing two non-reducing ends of delta G and delta M, and the molar ratio is as follows: 1.6: 1; all the algin hexaose are M reducing end terminal.

The invention has the technical characteristics that:

in the invention, algin oligose (UDPn, n is more than or equal to 4) is added¹H-NMR analysis can determine the glycosyl structures of the reducing end and the non-reducing end of the alginate oligosaccharide; partially degrading algin oligosaccharide (UDPn) with algin lyase AlyPB2 to obtain intermediate oligosaccharides UDP (n-1), UDP (n-2) · · UDP3, UDP2, and subjecting the intermediate oligosaccharides UDP (n-1), UDP (n-2) · · UDP3 to¹H-NMR analysis can determine the glycosyl structure of the reducing end and the non-reducing end of the intermediate product oligosaccharide, and the combination of the intermediate product oligosaccharide and the alginate oligosaccharide (UDPn)¹As a result of H-NMR analysis, the sugar chain structure of the alginate oligosaccharides can be determined.

Advantageous effects

The invention firstly establishes a sequencing method of alginate oligosaccharides based on alginate lyase, and particularly utilizes the characteristic that when exo-type alginate lyase AlyPB2 is used for degrading polysaccharide and oligosaccharide substrates, uronic acid is cut one by one from a non-reducing end, partial enzyme digestion is carried out on the alginate oligosaccharides by utilizing exo-type alginate lyase AlyPB2, partial enzyme digestion oligosaccharides with different polymerization degrees are prepared by gel filtration chromatography separation, and then partial enzyme digestion oligosaccharides with different polymerization degrees are utilized¹H-NMR technology is used for measuring the glycosyl structures of the reducing end and the non-reducing end of the enzyme digestion product, thereby simply, conveniently and rapidly determining single component or multiple homogeneous polymerization degreesThe method has the advantages of less required sample amount, greatly simplified later analysis process, and completely solved the problem of structural identification of high molecular weight (DP is more than or equal to 4) alginate oligosaccharide components, and has important significance for the research of the structure and functional relationship of the alginate oligosaccharide.

Drawings

FIG. 1 is a schematic diagram of the principle of alginate oligosaccharide sequencing method based on exo-type alginate lyase AlyPB 2;

FIG. 2 shows the synthesis of fucoidan disaccharide and fucoidan trisaccharide¹An H-NMR spectrum; in the figure, UDP2 is algin disaccharide, UDP3 is algin trisaccharide;

FIG. 3 is of fucoidan tetrasaccharide¹An H-NMR spectrum; in the figure, UDP4 is algin tetrasaccharide;

FIG. 4 shows the degradation of the intermediate oligosaccharide alginotriose by AlyPB2 of algintetraose¹An H-NMR spectrum; in the figure, UDP3-UDP4 is an oligosaccharide algin trisaccharide as an intermediate product of algin tetrasaccharide;

FIG. 5 is of fucoidan pentasaccharide¹An H-NMR spectrum; in the figure, UDP5 is algin pentasaccharide;

FIG. 6 shows the degradation of intermediate oligosaccharide by AlyPB2 of alginate pentasaccharide¹An H-NMR spectrum; in the figure, UDP3-UDP5 is an oligosaccharide algin trisaccharide which is an intermediate product of algin pentasaccharide, and UDP4-UDP5 is an oligosaccharide algin tetrasaccharide which is an intermediate product of algin pentasaccharide.

Detailed Description

The following examples are set forth so as to provide a thorough disclosure of some of the commonly used techniques of how the present invention may be practiced, and are not intended to limit the scope of the invention. The inventors have made the best effort to ensure accuracy with respect to parameters (e.g., amounts, temperature, etc.) used in the examples, but some experimental errors and deviations should be accounted for. Unless otherwise indicated, molecular weight in the present invention refers to average molecular weight and temperature to degrees celsius.