A method for preparing oligosaccharides by combining glycoside phosphorylase with monosaccharide kinase and polyphosphate kinase

Abstract

The present invention relates to a method for preparing oligosaccharides by a glycoside phosphorylase combined with monosaccharide kinase and polyphosphate kinase. The method comprises the steps of using donor monosaccharide, acceptor sugar, polyphosphate, and low equivalent adenosine phosphate as substrates, adding magnesium chloride to the substrate, and then co-catalyzing the reaction for 1.5 to 22 hours at 25 to 40 ° C by polyphosphatase, monosaccharide kinase and glycoside phosphorylase to generate crude oligosaccharide products, and obtaining oligosaccharides after purification. The present invention provides a method for preparing important oligosaccharides by using monosaccharides, acceptor substrates, polyphosphate, and low equivalent adenosine phosphate as substrates, and realizing ATP regeneration by using polyphosphate kinase as substrate, effectively solving the problem that a large amount of expensive sugar nucleotide donors and auxiliary factor ATP need to be added in the process of synthesizing oligosaccharides by bioenzymatic method, resulting in the problem that the cost is too high and cannot be mass-produced. The method of the present invention was successfully used to synthesize lactoyl-N-disaccharide, galactoyl-N-disaccharide, chitosan, and protein N-sugar chain core trisaccharide derivatives.

Description

Technical Field

[0001] The invention relates to a method for preparing oligosaccharides by combining glycoside phosphorylase with monosaccharide kinase and polyphosphate kinase, and belongs to the technical field of biosynthesis. Background Art

[0002] Carbohydrates are the most abundant biopolymers in nature. As part of most living organisms, they are widely found in plants, animals and microorganisms. These carbohydrates are polymerized from monosaccharides and their derivatives to form oligosaccharides and polysaccharides. They can also form glycoconjugates such as glycoproteins and glycolipids through covalent bonds with proteins and fat chains. Sugar chains such as oligosaccharides and polysaccharides with specific structures have important biological functions and have been and continue to be used in the fields of medicine, population health, etc.

[0003] In addition to extraction from natural products, chemical synthesis and enzymatic synthesis are important methods for preparing sugar chains. Chemical synthesis requires multiple steps such as protection, deprotection and selective modification, and has problems such as cumbersome operation, low yield and environmental pollution. The bioenzymatic method uses enzyme catalysis to synthesize oligosaccharides or polysaccharides with specific structures. It is simple to operate, has a relatively higher yield and is environmentally friendly. In the enzymatic synthesis of oligosaccharides, there are mainly three commonly used enzymes: glycosyltransferases, glycoside hydrolases and their engineered mutant transglycosylases, and glycoside phosphorylases (GPs).

[0004] The most commonly used tool enzyme in enzymatic synthesis is glycosyltransferase, which catalyzes the formation of glycosidic bonds between activated glycosyl donor sugar nucleotides and unactivated glycosyl acceptors. Glycosyltransferase reactions require the use of sugar nucleotides as donor substrates, but sugar nucleotides are expensive, the glycosylation process is subject to feedback inhibition by the byproduct nucleoside diphosphate, and the limited source of glycosyltransferases. These factors limit the application of glycosyltransferases in large-scale sugar synthesis.

[0005] Compared with glycosyltransferases, glycoside phosphorylases (GPs) use monosaccharide-1-phosphate (monosaccharide-1-P) as a donor substrate, which has the advantages of easy availability of donor substrates and low cost. At the same time, GPs generally have a broader substrate specificity. Glucoside phosphorylase was discovered to be the earliest and most widely used enzyme. Typical applications include the preparation of glycerol glucoside used as a cosmetic additive, cellulose oligosaccharides / cellulose dextrins as functional foods, etc. Glucoside phosphorylase uses glucose-1-phosphate as a donor substrate to synthesize oligosaccharides, and glucose-1-phosphate can be obtained by sucrose phosphorylase using sucrose as a substrate.

[0006] In addition to synthesizing glucosides, GPs are also used as biocatalysts to synthesize other important sugar chains, mainly in the discovery of GPs related to galactose (Gal), N-acetylglucosamine (GlcNAc) and mannose (Man) and their applications in oligosaccharide synthesis. For example, galactosyl-beta1,3-N-acetyl-D-hexosamine phosphorylase (BiGHNP) from Bifidobacterium infantis is used to synthesize human milk oligosaccharide components lacto-N-biose (Gal-beta1,3-GlcNAc, LNB), galacto-N-biose (Gal-beta1,3-GalNAc, GNB), tumor-associated carbohydrate antigen T-antigen, sialylated T-antigen, Lewis a, etc. with galactose-1-phosphate (Gal-1-P). Chitobiose phosphorylase (VpCPase) from Vibrio proteolyticus and other sources synthesizes chitobiose from GlcNAc-1-P, and beta1,4-mannose phosphorylase BtMPase from Bacteroides thetaiotaomicron VPI-5482 synthesizes 1,2-cis mannosides, including protein N-sugar chain core trisaccharide Man-beta1,4-GlcNAc-beta1,4-GlcNAc, from mannose-1-phosphate (Man-1-P).

[0007] The synthesis of oligosaccharides by Gal, GlcNAc and Man-related GPs requires the corresponding monosaccharide-1-P, namely Gal-1-P, GlcNAc-1-P and Man-1-P. These three monosaccharide-1-P are mainly synthesized by ATP (adenosine triphosphate)-dependent monosaccharide kinase (GlyK), such as galactokinase GalK from Bifidobacterium infantis and Escherichia coli, and NahK from Bifidobacterium longum. The synthesis of GlcNAc-1-P and Man-1-P. Monosaccharide kinase and GPs can be combined in a one-pot reaction to prepare glycoside products (galactoside, GlcNAc glycoside, mannoside) from ATP, monosaccharides (Gal, GlcNAc, Man), and acceptor sugar substrates, and produce by-products ADP and phosphate. The specific process is shown in the following formula.

[0008]

[0009] The combined reaction of monosaccharide kinase and GPs can promote the reversible reaction catalyzed by GPs to the direction of oligosaccharide synthesis, so that the synthesis yield of oligosaccharides LNB and GNB catalyzed by BiGHNP can reach more than 90%, and it also provides a method for the convenient preparation of protein N-glycan core trisaccharide Man-beta1,4-GlcNAc-beta1,4-GlcNAc. However, these synthesis reactions require a large amount of ATP, resulting in high costs, which seriously limits the large-scale production of oligosaccharides. Summary of the invention

[0010] In view of the shortcomings of the prior art, the present invention provides a method for preparing oligosaccharides by combining glycoside phosphorylase with monosaccharide kinase and polyphosphate kinase. The oligosaccharides are synthesized by combining glycoside phosphorylase GP with monosaccharide kinase GlyK and polyphosphate kinase PPK, which solves the problem of adding a large amount of expensive sugar nucleotide donors and auxiliary factor ATP in the process of enzymatic synthesis of oligosaccharides. The present invention provides a method for synthesizing oligosaccharides by catalysis of polyphosphate kinase EbPPK, monosaccharide kinase GalK or NahK, glycoside phosphorylase BiGHNP or VpCPase or BtMPase from microorganisms, and is applied to the preparation of oligosaccharides, realizing the simple and efficient enzymatic synthesis preparation of four oligosaccharide derivatives of LNB, GNB, chitobiose, and protein N-sugar chain core trisaccharide derivatives.

[0011] The technical solution of the present invention is as follows:

[0012] A method for preparing oligosaccharides by using glycoside phosphorylase in combination with monosaccharide kinase and polyphosphate kinase comprises the following steps:

[0013] With donor monosaccharide, acceptor sugar, polyphosphate and low equivalent adenosine phosphate as substrates, magnesium chloride is added to the substrates, and then polyphosphatase, monosaccharide kinase and glycoside phosphorylase jointly catalyze the reaction at 25-40°C for 1.5-22h to generate crude oligosaccharide products, which are then purified to obtain oligosaccharides.

[0014] Preferably, the donor monosaccharide includes galactose (Gal), N-acetyl-D-glucosamine (GlcNAc) and mannose (Man). The monosaccharide-1-phosphate corresponding to the donor monosaccharide can be synthesized by an adenosine triphosphate ATP-dependent kinase using monosaccharide and ATP, and the monosaccharide-1-phosphate can be used as a donor substrate for the corresponding glycoside phosphorylase.

[0015] Preferably according to the present invention, the final concentration of the donor monosaccharide is 40-100 mM.

[0016] Preferably according to the present invention, the acceptor sugar includes N-acetyl-D-glucosamine (GlcNAc), N-acetyl-D-galactosamine (GalNAc), and a chitobiose derivative (GlcNAc-beta1,4-GlcNAc-beta-EthN3).

[0017] Preferably according to the present invention, the final concentration of the acceptor sugar is 40-70 mM.

[0018] Preferably according to the present invention, the oligosaccharides include lactoyl-N-disaccharide, galactoyl-N-disaccharide, chitosan and protein N-sugar chain core trisaccharide derivatives, corresponding to the substrate.

[0019] Preferably, according to the present invention, the polyphosphate is a metal salt of a phosphate compound having a degree of polymerization of 2 phosphoric acids or more;

[0020] More preferably, the polyphosphate is sodium hexametaphosphate or sodium tripolyphosphate.

[0021] Preferably according to the present invention, the final concentration of the polyphosphate is 20-40 mM.

[0022] Preferably according to the present invention, the low equivalent adenosine phosphate is adenosine monophosphate (AMP) or adenosine diphosphate (ADP) or adenosine triphosphate (ATP).

[0023] Preferably according to the present invention, the final concentration of the low equivalent adenosine phosphate is 2-3 mM.

[0024] Preferably according to the present invention, the final concentration of magnesium chloride is 40-60 mM.

[0025] Preferably according to the present invention, the monosaccharide kinase (GlyK) is the monosaccharide kinase GalK or the monosaccharide kinase NahK.

[0026] The monosaccharide kinase GalK is derived from Bifidobacterium infantis, the amino acid sequence is shown in SEQ ID NO.1, and the coding gene sequence is shown in SEQ ID NO.2; the coding gene sequence is codon-optimized and suitable for recombinant expression in Escherichia coli.

[0027] The monosaccharide kinase NahK is derived from Bifidobacterium longum, the amino acid sequence is shown in SEQ ID NO.7, and the coding gene sequence is shown in SEQ ID NO.8; the coding gene sequence is codon-optimized and suitable for recombinant expression in Escherichia coli.

[0028] Preferably according to the present invention, the final concentration of the monosaccharide kinase is 0.3-1.5 mg / mL.

[0029] Preferably according to the present invention, the polyphosphate kinase (PPK) is polyphosphate kinase EbPPK, which is derived from Erysipelotrichaceae bacterium, has an amino acid sequence as shown in SEQ ID NO.3, and a coding gene sequence as shown in SEQ ID NO.4; its coding gene sequence is codon optimized and suitable for recombinant expression in Escherichia coli.

[0030] Preferably according to the present invention, the final concentration of the polyphosphate kinase is 0.1-0.7 mg / mL.

[0031] Preferably according to the present invention, the glycoside phosphorylase (GP) is glycoside phosphorylase BiGHNP, glycoside phosphorylase VpCPase or glycoside phosphorylase BtMPase;

[0032] The glycoside phosphorylase BiGHNP is derived from Bifidobacterium infantis, the amino acid sequence is shown in SEQ ID NO.5, and the coding gene sequence is shown in SEQ ID NO.6; the coding gene sequence is codon-optimized and suitable for recombinant expression in Escherichia coli.

[0033] The glycoside phosphorylase VpCPase is derived from Vibrio proteolyticus, the amino acid sequence is shown in SEQ ID NO.9, and the coding gene sequence is shown in SEQ ID NO.10; the coding gene sequence is codon-optimized and suitable for recombinant expression in Escherichia coli.

[0034] The glycoside phosphorylase BtMPase is derived from Bacteroides thetaiotaomicron VPI-5482, the amino acid sequence is shown in SEQ ID NO.11, and the coding gene sequence is shown in SEQ ID NO.12; the coding gene sequence is codon-optimized and suitable for recombinant expression in Escherichia coli.

[0035] Preferably according to the present invention, the final concentration of the glycoside phosphorylase is 0.2-2 mg / mL.

[0036] Preferably according to the present invention, the preparation method of the monosaccharide kinase GalK, the monosaccharide kinase NahK, the polyphosphate kinase EbPPK, the glycoside phosphorylase BiGHNP, the glycoside phosphorylase VpCPase and the glycoside phosphorylase BtMPase comprises the following steps:

[0037] (1) using the coding gene of monosaccharide kinase GalK as the target gene and pET-22b as the vector plasmid to construct a recombinant expression vector; then using the coding genes of monosaccharide kinase NahK, polyphosphate kinase EbPPK, glycoside phosphorylase BiGHNP, glycoside phosphorylase VpCPase and glycoside phosphorylase BtMPase as the target genes and pET-30a as the vector plasmid to construct recombinant expression vectors, and then transforming the recombinant expression vectors into Escherichia coli to obtain recombinant Escherichia coli;

[0038] (2) selecting the successfully transformed recombinant E. coli for expansion culture, adding IPTG with a final concentration of 0.1 to 1 mM or adding 0.1 to 1% lactose for induction, collecting the bacterial liquid, crushing and centrifuging, and obtaining the supernatant of the recombinant E. coli;

[0039] (3) The supernatant was purified by immobilized metal ion affinity chromatography to obtain monosaccharide kinases GalK and NahK, polyphosphate kinase EbPPK, and glycoside phosphorylases BiGHNP, VpCPase, and BtMPase, respectively.

[0040] According to the preferred embodiment of the present invention, when the generated oligosaccharide is lactoyl-N-disaccharide (LNB) or galactoyl-N-disaccharide (GNB), the specific preparation method is as follows:

[0041] Galactose as a donor monosaccharide, N-acetyl-D-glucosamine (GlcNAc) or N-acetyl-D-galactosamine (GalNAc), AMP, polyphosphate (PolyPn), and magnesium chloride as an acceptor sugar are mixed in water, the pH is adjusted to 8.0, monosaccharide kinase GalK, glycoside phosphorylase BiGHNP, and polyphosphate kinase EbPPK are added, the reaction is carried out at 37° C. and 110 rpm for 1.5 h, the reaction is terminated, and lactoyl-N-disaccharide or galactoyl-N-disaccharide is separated and purified;

[0042] The final concentration of galactose is 60-80 mM; the final concentration of N-acetyl-D-glucosamine (GlcNAc) or N-acetyl-D-galactosamine (GalNAc) is 40-50 mM; the final concentrations of AMP, polyphosphate and magnesium chloride are 2 mM, 20 mM and 40 mM respectively; the final concentrations of monosaccharide kinase GalK, glycoside phosphorylase BiGHNP and polyphosphate kinase EbPPK are 0.3-0.7 mg / mL, 0.2-0.5 mg / mL and 0.1-0.2 mg / mL respectively; the termination reaction is to add an equal volume of cold ethanol and terminate the reaction at 4°C for 30 min.

[0043] According to the preferred embodiment of the present invention, when the generated oligosaccharide is chitosan (GlcNAc-beta1,4-GlcNAc), the specific preparation method is as follows:

[0044] N-acetyl-D-glucosamine, AMP, polyphosphate, magnesium chloride, and Tris-HCl solution were mixed in water, the pH was adjusted to 7.5, monosaccharide kinase NahK, glycoside phosphorylase VpCPase, and polyphosphate kinase EbPPK were added, the reaction was carried out at 37°C for 6 hours, the reaction was terminated, and chitobiose was separated and purified;

[0045] The final concentration of N-acetyl-D-glucosamine is 90-100 mM; the final concentrations of AMP, polyphosphate, magnesium chloride and Tris-HCl solution are 3 mM, 30 mM, 60 mM and 200 mM respectively; the final concentrations of monosaccharide kinase NahK, glycoside phosphorylase VpCPase and polyphosphate kinase EbPPK are 0.5-1.5 mg / mL, 0.5-2 mg / mL and 0.3-0.7 mg / mL respectively; the termination reaction is to add an equal volume of cold ethanol, and terminate the reaction at 4°C for 30 minutes. The N-acetyl-D-glucosamine is both a donor monosaccharide and an acceptor sugar.

[0046] According to the preferred embodiment of the present invention, when the generated oligosaccharide is a protein N-sugar chain core trisaccharide derivative (Man-beta1,4-GlcNAc-beta1,4-GlcNAc-beta-EthN3), the specific preparation method is as follows:

[0047] Mix mannose, chitobiose derivatives, AMP, polyphosphate, magnesium chloride, Tris-HCl solution in water, adjust the pH to 7.5, monosaccharide kinase NahK, glycoside phosphorylase BtMPase and polyphosphate kinase EbPPK, react at 37°C for 22 hours, terminate the reaction, separate and purify to obtain protein N-sugar chain core trisaccharide derivatives;

[0048] The final concentration of mannose is 60-80 mM; the final concentration of the chitobiose derivative is 40-50 mM; the final concentrations of AMP, polyphosphate, magnesium chloride and Tris-HCl solution are 2 mM, 20 mM, 40 mM and 100 mM, respectively; the final concentrations of monosaccharide kinase NahK, glycoside phosphorylase BtMPase and polyphosphate kinase EbPPK are 0.7-1.5 mg / mL, 0.7-1.5 mg / mL and 0.3-0.6 mg / mL, respectively; and the termination reaction is to add an equal volume of cold ethanol and terminate the reaction at 4°C for 30 min.

[0049] The chitosan derivative (GlcNAc-beta1,4-GlcNAc-beta-EthN3) is obtained by terminal chemical derivatization of chitosan, and the derivatization method is referred to Beilstein J. Org. Chem. 2013, 9, 1867-1872. The purpose of terminal derivatization is to facilitate chemical structure analysis, and the method can be applied to the synthesis of Man-beta1,4-GlcNAc-beta1,4-GlcNAc trisaccharide and its terminal derivatives.

[0050] Preferably, the purification is specifically as follows: removing the precipitate by centrifugation, purifying by activated carbon adsorption, molecular exclusion chromatography, or ion exchange chromatography;

[0051] Specifically, when the oligosaccharide product is lactoyl-N-disaccharide or galactoyl-N-disaccharide, the reaction solution is purified by selecting a Bio-Gel P2 chromatographic column to separate the product disaccharide from the raw material monosaccharide to obtain a crude product; the crude product is purified by DEAE-Cellulose 52 to obtain a high-purity product; wherein the steps of purifying the crude product by DEAE-Cellulose 52 are: using deionized water as a mobile phase at a flow rate of 5 to 6 mL / min, collecting fractions and detecting the product by TLC, concentrating the fractions containing the product by using a rotary evaporator, and then freeze-drying to obtain lactoyl-N-disaccharide or galactoyl-N-disaccharide;

[0052] When the oligosaccharide product is chitobiose or a protein N-glycoside core trisaccharide derivative, the reaction solution is desalted by activated carbon adsorption; the reaction treatment solution or the eluate after desalting by activated carbon adsorption is purified by a Bio-Gel P2 chromatographic column to obtain a crude product; the crude product is purified by DEAE-Cellulose 52 to obtain a high-purity product; wherein the steps of crude purification by activated carbon are: first, three column volumes are flushed with deionized water as a mobile phase; then, impurities are eluted with 2wt% ethanol as a mobile phase; 30wt% ethanol aqueous solution is eluted to collect the chitobiose or protein N-glycoside core trisaccharide derivative.

[0053] The technical features of the present invention are as follows:

[0054] The present invention uses low-equivalent adenosine phosphate and polyphosphate (PolyPn) as substrates, polyphosphate kinase catalyzes the generation of ATP, monosaccharide kinase utilizes the generated ATP to catalyze monosaccharide to generate monosaccharide-1-phosphate and ADP, ADP can be catalyzed by polyphosphate kinase to regenerate ATP, and glycoside phosphorylase GP uses monosaccharide-1-phosphate as a donor to catalyze the generation of corresponding glycosides. The specific process is shown in formula (I).

[0055]

[0056] Beneficial effects:

[0057] 1. The present invention provides a method for preparing important oligosaccharides using monosaccharides, receptor substrates, polyphosphates, and low-equivalent adenosine phosphate (AMP or ADP) as substrates, and uses polyphosphate kinase to regenerate ATP using polyphosphate as a substrate, effectively solving the problem of high costs and inability to scale up production in the process of synthesizing oligosaccharides by bioenzymatic methods due to the need to add a large amount of expensive sugar nucleotide donors and auxiliary factor ATP. The present invention greatly reduces the amount of adenosine phosphate used, which is only equivalent to 1 / 20 of the prior art.

[0058] 2. The present invention uses the oligosaccharide synthesis method to prepare lactoyl-N-disaccharide (LNB, Gal-beta1,3-GlcNAc), galactoyl-N-disaccharide (GNB, Gal-beta1,3-GalNAc), chitosan (GlcNAc-beta1,4-GlcNAc), protein N-sugar chain core trisaccharide derivative (Man-beta1,4-GlcNAc-beta1,4-GlcNAc-beta-EthN3). The bioenzymatic method of the present invention has simple steps, is economical and efficient, is suitable for large-scale preparation of important oligosaccharides or polysaccharides, and has great application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] Figure 1 is the SDS-PAGE electrophoresis diagram of the prepared monosaccharide kinase GalK;

[0060] Among them, 1 is the intracellular protein sample of recombinantly expressed Escherichia coli, 2 is the supernatant after centrifugation of the cell lysate, 3 is the precipitate resuspension after centrifugation of the cell lysate; 4 is the flow-through in the nickel ion affinity chromatography purification process, 5 is the impurity protein elution liquid, and 6 is the target protein elution liquid.

[0061] Figure 2 is the SDS-PAGE electrophoretogram of the prepared glycoside phosphorylase BiGHNP;

[0062] Among them, 1 is the intracellular protein sample of recombinantly expressed Escherichia coli, 2 is the supernatant after centrifugation of the cell lysate, 3 is the precipitate resuspension after centrifugation of the cell lysate; 4 is the flow-through in the nickel ion affinity chromatography purification process, 5 is the impurity protein elution liquid, and 6 is the target protein elution liquid.

[0063] Figure 3 is the SDS-PAGE electrophoretogram of the prepared polyphosphate kinase EbPPK;

[0064] Among them, 1 is the supernatant after centrifugation of the cell lysate, 2 is the flow-through during the nickel ion affinity chromatography purification process, 3 is the impurity protein eluate, and 4 is the target protein eluate.

[0065] Figure 4is the SDS-PAGE electrophoretogram of the prepared monosaccharide kinase NahK;

[0066] Among them, 1 is the intracellular protein sample of recombinantly expressed Escherichia coli, 2 is the supernatant after centrifugation of the cell lysate, 3 is the precipitate resuspension after centrifugation of the cell lysate; 4 is the flow-through in the nickel ion affinity chromatography purification process, 5 is the impurity protein elution liquid, and 6 is the target protein elution liquid.

[0067] Figure 5 is the SDS-PAGE electrophoretogram of the prepared glycoside phosphorylase VpCPase;

[0068] Among them, 1 is the supernatant after centrifugation of the cell lysate, 2 is the precipitate resuspension after centrifugation of the cell lysate; 3 is the flow-through in the nickel ion affinity chromatography purification process, 4 is the impurity protein elution solution, and 5 is the target protein elution solution.

[0069] Figure 6 is the SDS-PAGE electrophoretogram of the prepared glycoside phosphorylase BtMPase;

[0070] Among them, 1 is the intracellular protein sample of recombinantly expressed Escherichia coli, 2 is the supernatant after centrifugation of the cell lysate, 3 is the precipitate resuspension after centrifugation of the cell lysate; 4 is the flow-through in the nickel ion affinity chromatography purification process, 5 is the impurity protein elution liquid, and 6 is the target protein elution liquid.

[0071] Figure 7 is a diagram of the TLC analysis results of the enzyme reaction in Example 2;

[0072] Among them, 1 is Gal standard, 2 is GlcNAc standard, 3 is LNB standard, and 4 is the reaction system.

[0073] Figure 8 It is the result of high-resolution mass spectrometry detection of lactoyl-N-disaccharide (LNB, Gal-beta1,3-GlcNAc).

[0074] Fig. 9 It is the NMR of lactoyl-N-disaccharide (LNB, Gal-beta1,3-GlcNAc) 1 H spectrum.

[0075] Fig.10 It is the NMR of lactoyl-N-disaccharide (LNB, Gal-beta1,3-GlcNAc) 13 C spectrum.

[0076] Fig.11 This is a TLC analysis result diagram for exploring the optimal polyphosphate (PolyPn) concentration;

[0077] Among them, 1 is a galactose standard, 2 is a GlcNAc standard, 3 is an AMP standard, 4 is an ATP standard, 5 is an LNB standard, 6 is a reaction solution containing 5 mM PolyPn, 7 is a blank control containing 5 mM PolyPn, 8 is a reaction solution containing 10 mM PolyPn, 9 is a blank control containing 10 mM PolyPn, 10 is a reaction solution containing 15 mM PolyPn, 11 is a blank control containing 15 mM PolyPn, 12 is a reaction solution containing 20 mM PolyPn, and 13 is a blank control containing 20 mM PolyPn;

[0078] Fig.12 This is a TLC analysis result diagram for exploring the optimal AMP concentration;

[0079] Among them, 1 is Gal standard, 2 is GlcNAc standard, 3 is AMP standard, 4 is ATP standard, 5 is LNB standard, 6 is reaction solution containing 2 mM AMP, 7 is blank control containing 2 mM AMP, 8 is reaction solution containing 5 mM AMP, 9 is blank control containing 5 mM AMP, 10 is reaction solution containing 8 mM AMP, 11 is blank control containing 8 mM AMP, 12 is reaction solution containing 11 mM AMP, and 13 is blank control containing 11 mM AMP;

[0080] Fig.13 This is a TLC analysis result chart for exploring the optimal magnesium chloride (MgCl2) concentration;

[0081] Among them, 1 is a Gal standard, 2 is a GlcNAc standard, 3 is an AMP standard, 4 is an ATP standard, 5 is an LNB standard, 6 is a reaction solution containing 20 mM MgCl2, 7 is a blank control containing 20 mM MgCl2, 8 is a reaction solution containing 30 mM MgCl2, 9 is a blank control containing 30 mM MgCl2, 10 is a reaction solution containing 40 mM MgCl2, 11 is a blank control containing 40 mM MgCl2, 12 is a reaction solution containing 50 mM MgCl2, and 13 is a blank control containing 50 mM MgCl2;

[0082] Fig.14 This is a graph of TLC analysis results to explore the optimal pH;

[0083] Among them, 1 is Gal standard, 2 is GlcNAc standard, 3 is AMP standard, 4 is ATP standard, 5 is LNB standard, 6 is pH 4 reaction solution, 7 is pH 4 blank control, 8 is pH 5 reaction solution, 9 is pH 5 blank control, 10 is pH 6 reaction solution, 11 is pH 6 blank control, 12 is pH 7 reaction solution, 13 is pH 7 blank control, 14 is pH 8 reaction solution, 15 is pH 8 blank control;

[0084] Fig.15 is a diagram of the TLC analysis results of the enzyme reaction in Example 4;

[0085] Among them, 1 is galactose standard, 2 is GalNAc standard, 3 is GNB standard, and 4 is the reaction system.

[0086] Fig.16 It is the result of high-resolution mass spectrometry detection of galactoyl-N-disaccharide (GNB, Gal-beta1,3-GalNAc).

[0087] Fig.17 It is the NMR of galactoyl-N-disaccharide (GNB, Gal-beta1,3-GalNAc) 1 H spectrum.

[0088] Fig.18 It is the NMR of galactoyl-N-disaccharide (GNB, Gal-beta1,3-GalNAc) 13 C spectrum.

[0089] Fig.19 is a diagram of the TLC analysis results of the enzyme reaction in Example 5;

[0090] Among them, 1 is a GlcNAc standard, 2 is a N-acetyl-D-glucosamine-1-phosphate (GlcNAc-1-P) standard, 3 is a GlcNAc-beta1,4-GlcNAc standard, and 4 is a reaction system;

[0091] Fig. 20 This is the result of high-resolution mass spectrometry detection of chitosan (GlcNAc-beta1,4-GlcNAc).

[0092] Fig.21 It is the NMR of chitosan (GlcNAc-beta1,4-GlcNAc) 1 H spectrum.

[0093] Fig. 22 It is the NMR of chitosan (GlcNAc-beta1,4-GlcNAc) 13 C spectrum.

[0094] Fig.23 is a diagram of the TLC analysis results of the enzyme reaction in Example 6;

[0095] Among them, 1 is Man standard, 2 is mannose-1-phosphate (Man-1-P) standard, 3 is GlcNAc-beta1,4-GlcNAc-beta-EthN3 standard, 4 is product trisaccharide standard, and 5 is reaction system;

[0096] Fig.24 It is the result of high-resolution mass spectrometry detection of protein N-sugar chain core trisaccharide derivative (Man-beta1,4-GlcNAc-beta1,4-GlcNAc-beta-EthN3).

[0097] Fig.25 It is the nuclear magnetic resonance of protein N-glycan core trisaccharide derivative (Man-beta1,4-GlcNAc-beta1,4-GlcNAc-beta-EthN3) 1 H spectrum.

[0098] Fig.26 It is the nuclear magnetic resonance of protein N-glycan core trisaccharide derivative (Man-beta1,4-GlcNAc-beta1,4-GlcNAc-beta-EthN3) 13 C spectrum. DETAILED DESCRIPTION

[0099] To facilitate the application of the present invention, the technical solution of the present invention is further described below with reference to embodiments, but the protection scope of the present invention is not limited to the following embodiments.

[0100] In the embodiments, nickel ion agarose gel was purchased from Changzhou Tiandirenhe Biotechnology Co., Ltd., mannose and N-acetyl-D-glucosamine were purchased from TCI (Shanghai) Chemical Industry Development Co., Ltd., galactose was purchased from Shanghai Dibai Biotechnology Co., Ltd., N-acetyl-D-galactosamine was purchased from Shanghai Bid Pharmaceutical Technology Co., Ltd., adenine nucleoside monophosphate was purchased from Shanghai Jizhi Biochemical Technology Co., Ltd., activated carbon was Activated charcoal sold by McLean, Bio-Gel P2 was from Bio-Rad, DEAE-Cellulose 52 was purchased from Shanghai Dibai Biotechnology Co., Ltd., and other chemical reagents were commercially available analytical grade or above reagents.

[0101] Example 1: Preparation of monosaccharide kinases GalK, NahK, glycoside phosphorylases BiGHNP, VpCPase, BtMPase and polyphosphate kinase EbPPK

[0102] The amino acid sequence of the monosaccharide kinase GalK in the present invention is shown as SEQ ID NO.1. The monosaccharide kinase GalK encoding gene is designed according to the amino acid sequence of the monosaccharide kinase GalK. The monosaccharide kinase GalK encoding gene is artificially synthesized. The nucleotide sequence is shown as SEQ ID NO.2. The nucleotide sequence is codon-optimized and suitable for recombinant expression in Escherichia coli.

[0103] The artificially synthesized monosaccharide kinase GalK encoding gene was inserted into the vector pET-22b to obtain a recombinant expression vector; the recombinant expression vector was transformed into E. coli BL21 (DE3) competent cells, spread on LB plates containing 50 μg / mL antibiotics, and cultured at 37°C overnight; a single colony was picked, and the recombinant E. coli was preserved after being cultured in LB liquid culture medium overnight.

[0104] Take the stored recombinant E. coli and inoculate it into 50mL LB liquid medium, activate it at 37℃ overnight; take 10mL of the activated bacterial solution and inoculate it into 1L LB liquid medium, and culture it at 37℃, 180-220rpm until OD 600 The pH value was about 0.6, and the mixture was taken out and cooled in an ice water bath; IPTG was added at a final concentration of 0.2 mM, and the expression was induced at 16°C for 20 hours; the bacteria were collected using a vertical centrifuge (8000 rpm, 10 min, 4°C), and the bacteria were resuspended in a buffer solution (20 mM Tris-HCl, pH 7.5, 500 mM NaCl), and the resuspended bacteria were placed in an ice-water mixture for ultrasonic disruption (power 40 W, ultrasound 3 s, interval 2 s, disruption 30 min); the disrupted bacterial solution was centrifuged (4°C, 12000 rpm, 30 min), and the supernatant was collected.

[0105] The preparation methods of monosaccharide kinase NahK, glycoside phosphorylase BiGHNP, VpCPase, BtMPase and polyphosphate kinase EbPPK are the same as those of monosaccharide kinase GalK. The vector plasmid used is pET-30a, the amino acid sequence of the monosaccharide kinase NahK is shown in SEQ ID NO.7, and the coding gene sequence is shown in SEQ ID NO.8. The amino acid sequence of the glycoside phosphorylase BiGHNP is shown in SEQ ID NO.5, and the coding gene sequence is shown in SEQ ID NO.6. The amino acid sequence of the glycoside phosphorylase VpCPase is shown in SEQ ID NO.9, and the coding gene sequence is shown in SEQ ID NO.10. The amino acid sequence of the glycoside phosphorylase BtMPase is shown in SEQ ID NO.11, and the coding gene sequence is shown in SEQ ID NO.12. The amino acid sequence of the polyphosphate kinase (PPK) is the polyphosphate kinase EbPPK, and the coding gene sequence is shown in SEQ ID NO.3.

[0106] The proteins expressed by the six enzyme encoding genes have six histidines and can be purified using immobilized metal ion affinity chromatography. Therefore, the supernatant collected above was purified using nickel ion affinity chromatography. The purification steps were carried out according to the product instructions of nickel ion agarose gel. The impurities were thoroughly washed away with a buffer containing 20mM imidazole, and the target protein was eluted with a buffer containing 500mM imidazole. The protein was desalted and concentrated by ultrafiltration, and the protein concentration was determined by the Bradford method. 20% glycerol was added to the obtained protein, stored in aliquots at -20°C, and verified by SDS-PAGE. The verification results were as follows: Figures 1 to 6 shown.

[0107] Depend on Figures 1 to 6 It can be seen that the band of the purified target protein sample is single and bright, indicating that monosaccharide kinase GalK, NahK, glycoside phosphorylase BiGHNP, VpCPase, BtMPase and polyphosphate kinase EbPPK are successfully synthesized. The sizes of enzymes GalK, BiGHNP, NahK, VpCPase, BtMPase and EbPPK are approximately 43kDa, 86.5kDa, 41.4kDa, 91.4kDa, 38.0kDa and 36.8kDa respectively. The above operation steps can be operated with reference to the relevant description in the "Succinct Molecular Biology Operation Experiment Guide" (Science Press, 2005.1, ISBN: 9787030147257).

[0108] Example 2: Preparation of lactoyl-N-disaccharide (LNB) using monosaccharide kinase GalK, glycoside phosphorylase BiGHNP and polyphosphate kinase EbPPK

[0109] Galactose (1.009 g, 5.60 mmol) and N-acetyl-D-glucosamine (0.797 g, 3.60 mmol) were added to the container, and 2 mM AMP, 20 mM polyphosphate (PolyPn), and 40 mM MgCl2 were added according to the final concentration of the reaction system. The pH was adjusted to 8.0 by adding NaOH solution, and 46 mg of purified monosaccharide kinase GalK, 30 mg of purified glycoside phosphorylase BiGHNP, and 10 mg of purified polyphosphate kinase EbPPK were added. The volume of the reaction solution was fixed to 80 mL by adding water, and the reaction was carried out at 37 ° C and 110 rpm for 1.5 hours. After the substrate conversion was completed, TLC detection was performed ( Figure 7 ), add an equal volume of 80 mL of cold ethanol and place at 4°C for 30 min to terminate the reaction, and centrifuge at 12000 rpm for 30 min to obtain the supernatant.

[0110] A 10 mm × 85 cm Bio-Gel P2 chromatographic column was used to purify the reaction solution, and the product disaccharide was separated from the raw material monosaccharide to obtain a crude product: deionized water was used as the mobile phase at a flow rate of 0.5 to 1 mL / min, the fractions were collected and the product was detected by TLC, and the fractions containing the product were concentrated using a rotary evaporator to obtain a crude product.

[0111] The crude product was further purified using DEAE-Cellulose 52 to remove charged impurities (such as PolyPn, ATP, ADP, Gal-1-P, etc.) in the crude product: deionized water was used as the mobile phase at a flow rate of 5 to 6 mL / min, fractions were collected and the product was detected by TLC, the fractions containing the product were concentrated using a rotary evaporator, and then freeze-dried to obtain white powdery lactoyl-N-disaccharide (LNB), weighing 1.35 g, with a calculated yield of approximately 92%; the chromatographic column was rinsed for 1 hour using 1 M NaCl as the mobile phase to elute the impurities adsorbed on the column; then deionized water was rinsed for 1 hour; and the column was stored in 20% ethanol at 4°C for a long term.

[0112] The purified LNB was prepared into a 10mM solution and analyzed for purity by TLC. Only one color spot of the target product was observed. A 1mM LNB solution was prepared for high-resolution mass spectrometry analysis. The mass spectrometry results were as follows: Figure 8 As shown. Figure 8 It can be seen that the characteristic ion peak is m / z[M+Na] + 406.1355, which is consistent with the theoretical value [M+Na] + 406.1428 agree.

[0113] Take 30 mg of LNB and dissolve it in 500 μL D2O. Use 600 MHz NMR to analyze it. The results are as follows: Fig. 9 and Fig.10As shown, the spectrum analysis results are: 1 H NMR(600MHz,D2O)d 5.21 (d, J=3.6Hz, 0.6H), 4.51 (d, J=7.9Hz, 0.6H), 4.48 (d, J=7.8Hz, 0.4H), 4.10 (d, J=7.0Hz, 0.4H), 3.99–3.50 (m, 12H), 2.07 (s, 3H). 13 C NMR(151MHz,D2O)δ174.90,174.63,103.60,103.47,94.78,91.13,82.67,80.21,75.50,75.33,75.29,72 .60,72.57,71.31,70.81,70.76,68.79,68.74,68.64,61.10,60.81,60.66,55.66,52.97,22.41,22.16.

[0114] The above data show that the present invention successfully prepared lactoyl-N-disaccharide (LNB).

[0115] Example 3: Optimization and expansion of the reaction system for preparing lactoyl-N-disaccharide (LNB)

[0116] (1) Polyphosphate (PolyPn) concentration

[0117] 70 mM Gal, 45 mM GlcNAc, 7 mM AMP, 40 mM magnesium chloride, different concentrations of PolyPn (5 mM, 10 mM, 15 mM, 20 mM), 58 μg purified monosaccharide kinase GalK, 38 μg purified glycoside phosphorylase BiGHNP, 14 μg purified polyphosphate kinase EbPPK were mixed in aqueous solution; the reaction was carried out at 37°C, pH 6.5, 110 rpm for 1 hour, and the product formation and substrate consumption were detected by thin layer chromatography (TLC); the detection results are shown in FIG. Fig.11 As shown. Fig.11 It can be seen that the reaction effect is best when the concentration of PolyPn is 20mM.

[0118] (2) Adenosine monophosphate (AMP) concentration

[0119] 70 mM Gal, 45 mM GlcNAc, different concentrations of AMP (2 mM, 5 mM, 8 mM, 11 mM), 40 mM magnesium chloride, 20 mM PolyPn, 58 μg purified monosaccharide kinase GalK, 38 μg purified glycoside phosphorylase BiGHNP, and 14 μg purified polyphosphate kinase EbPPK were mixed in aqueous solution; the reaction was carried out at 37°C, pH 6.5, and 110 rpm for 1 hour, and the product generation and substrate consumption were detected by TLC; the detection results are shown in FIG. Fig.12 As shown. Fig.12 It can be seen that the most economical and effective reaction is achieved when the AMP concentration is 2 mM.

[0120] (3) Concentration of magnesium chloride (MgCl2)

[0121] 70 mM Gal, 45 mM GlcNAc, 2 mM AMP, different concentrations of MgCl2 (20 mM, 30 mM, 40 mM, 50 mM), 20 mM PolyPn, 58 μg purified monosaccharide kinase GalK, 38 μg purified glycoside phosphorylase BiGHNP, 14 μg purified polyphosphate kinase EbPPK were mixed in aqueous solution, and the reaction was carried out at 37°C, pH 6.5, 110 rpm for 1 hour, and TLC was used to detect the product generation and substrate consumption. The detection results are shown in FIG. Fig.13 As shown. Fig.13 It can be seen that the reaction effect is best when the MgCl2 concentration is 40mM.

[0122] (4) Determination of pH of reaction system

[0123] 70 mM Gal, 45 mM GlcNAc, 2 mM AMP, 40 mM magnesium chloride, 20 mM PolyPn, 58 μg purified monosaccharide kinase GalK, 38 μg purified glycoside phosphorylase BiGHNP, and 14 μg purified polyphosphate kinase EbPPK were mixed in aqueous solution, and the reaction was carried out at different pH (pH 4.0, 5.0, 6.0, 7.0, 8.0) at 37°C and 110 rpm for 1 hour. The product formation and substrate consumption were detected by TLC, and the change of pH of the reaction system was detected by pH precision test paper; the detection results are shown in FIG. Fig.14 As shown. Fig.14 It can be seen that the reaction effect is best when the pH of the reaction system is 8.0.

[0124] Example 4: Preparation of galactoyl-N-disaccharide (GNB) using monosaccharide kinase GalK, glycoside phosphorylase BiGHNP and polyphosphate kinase EbPPK

[0125] Using a one-pot three-enzyme catalytic synthesis method, galactose (0.126 g, 0.70 mmol) and N-acetyl-D-galactosamine (0.099 g, 0.45 mmol) were added to the container, 2 mM AMP, 20 mM polyphosphate (PolyPn), and 40 mM MgCl2 were added according to the final concentration of the reaction system, and the pH was adjusted to 8.0 by adding NaOH solution; 5.8 mg of purified monosaccharide kinase GalK, 3.8 mg of purified glycoside phosphorylase BiGHNP, and 1.4 mg of purified polyphosphate kinase EbPPK were added, and the volume of the reaction solution was fixed to 10 mL by adding water; the reaction was carried out at 37 ° C and 110 rpm for 1.5 hours, and TLC detection was performed after the substrate conversion was completed ( Fig.15 ), add an equal volume of 10 mL of cold ethanol and place at 4°C for 30 min to terminate the reaction, and centrifuge at 12000 rpm for 30 min to obtain the supernatant.

[0126] The reaction solution was purified using a Bio-Gel P2 column and DEAE-Cellulose 52, and the treatment method was the same as in Example 2; white powdery galactoyl-N-disaccharide (GNB) was obtained, weighing 172 mg, with a calculated yield of about 93%.

[0127] The purified GNB was prepared into a 10 mM solution. Only one color spot of the target product was observed by TLC analysis. A 1 mM GNB solution was prepared for high-resolution mass spectrometry analysis. The mass spectrometry results were as follows: Fig.16 As shown. Fig.16 It can be seen that the characteristic ion peak of the product is m / z[M+Na] + 406.1347, which is consistent with the theoretical value [M+Na] + 406.1428 agree.

[0128] 30 mg of GNB was dissolved in 500 μL D2O and analyzed using 600 MHz NMR. The results are as follows: Fig.17 and 18 As shown, the spectrum analysis results are: 1H NMR(600MHz,D2O)d 5.25(d,J=3.6Hz,0.6H),4.73(d,1J=8.5Hz,0.4H),4.54(d,J=7.9Hz,0.6H),4.49(d,J=7.8Hz,0.4H),4.32(dd,J=3 .7and11.1Hz,0.4H),4.28(d,J=2.9Hz,0.6H),4.21(d,J=3.3Hz,0.4H),4.17(t,J=6.1Hz,0.6H),4.07(dd,J=3.2and 11.1Hz, 0.6H), 4.03 (t, J=10.7Hz, 0.4H), 3.96–3.63 (m, 8H), 3.56 (t, J=9.6Hz, 1H), 2.06 (s, 3H). 13 C NMR(151MHz,D2O)δ175.09,174.78,104.90,104.73,95.26,91.29,80.15,77.12,75.04,74.93,72.63 ,72.58,70.76,70.71,70.30,68.84,68.69,68.17,61.31,61.09,61.07,52.51,49.09,22.43,22.20.

[0129] The above data show that the present invention successfully prepared galactoyl-N-disaccharide (GNB).

[0130] Example 5: Preparation of chitobiose (GlcNAc-beta1,4-GlcNAc) using monosaccharide kinase NahK, glycoside phosphorylase VpCPase and polyphosphate kinase EbPPK

[0131] N-acetyl-D-glucosamine (1.99 g, 9.0 mmol) was added to the container, and 200 mM Tris-HCl (pH 8.0), 3 mM AMP, 30 mM PolyPn, and 60 mM MgCl2 were added according to the final concentration of the reaction system to adjust the pH to 7.5; 100 mg of purified monosaccharide kinase NahK, 100 mg of purified glycoside phosphorylase VpCPase, and 50 mg of purified polyphosphate kinase EbPPK were added, and the volume of the reaction solution was fixed to 90 mL with water; the reaction was carried out at 37°C for 6 hours, and after the substrate conversion was completed by TLC detection ( Fig.19 ), add an equal volume of 90 mL of cold ethanol and place at 4°C for 30 min to terminate the reaction, and centrifuge at 12000 rpm for 30 min to obtain the supernatant.

[0132] The reaction solution was crudely purified by activated carbon: first, three column volumes were rinsed with deionized water as the mobile phase; then, 2 wt% ethanol was used as the mobile phase to separate GlcNAc-1-P until all GlcNAc-1-P was collected, and then eluted with 30 wt% ethanol aqueous solution to collect the crude product.

[0133] A 10 mm × 85 cm Bio-Gel P2 column was used to further purify the crude product, with deionized water as the mobile phase at a flow rate of 0.5 to 1 mL / min. The fractions were collected and the product was detected by TLC.

[0134] DEAE-Cellulose 52 was used for further purification to remove charged impurities: deionized water was used as the mobile phase at a flow rate of 5-6 mL / min, the fractions were collected and the product was detected by TLC, the fractions containing the product were concentrated using a rotary evaporator and then freeze-dried to obtain white powdered chitosan (GlcNAc-beta1,4-GlcNAc), weighing 760 mg, with a calculated yield of approximately 40%.

[0135] A 20 mM solution of GlcNAc-beta1,4-GlcNAc was prepared and analyzed for purity by TLC. Only one color spot of the target product was observed. A 1 mM solution of GlcNAc-beta1,4-GlcNAc was prepared for high-resolution mass spectrometry analysis. The mass spectrometry results are as follows: Fig. 20 As shown. Fig. 20 It can be seen that the characteristic ion peak is m / z[M+H] + 425.1778, same as theoretical value [M+H] + 425.4110 matches.

[0136] 10 mg of GlcNAc-beta1,4-GlcNAc was dissolved in 500 μL D2O and analyzed using 600 MHz NMR. The results are as follows: Fig.21 and Fig. 22 As shown, the spectrum analysis results are: 1 H NMR(600MHz,D2O)δ5.20(d,J=2.8Hz,0.6H),4.70(d,J=8.1Hz,0.4H),4.59(dd,J=8.5,5.3H z,1H),3.95–3.73(m,6H),3.71–3.60(m,3H),3.59–3.45(m,3H),2.07(s,3H),2.04(s,3H). 13C NMR(151MHz,D2O)δ174.54,174.40,101.44,101.42,94.76,90.37,79.80,75.85,74 .51,73.40,72.48,69.97,69.64,62.44,60.48,59.78,56.02,53.57,22.07,21.84.

[0137] The above data indicate that the present invention successfully prepared chitosan (GlcNAc-beta1,4-GlcNAc).

[0138] Example 6: Preparation of protein N-sugar chain core trisaccharide derivative (Man-beta1,4-GlcNAc-beta1,4-GlcNAc-beta-EthN3) using monosaccharide kinase NahK, glycoside phosphorylase BtMPase and polyphosphate kinase EbPPK

[0139] The acceptor sugar GlcNAc-beta1,4-GlcNAc-beta-EthN3 is obtained from chitosan by terminal chemical derivatization. The derivatization method is described in Beilstein J. Org. Chem. 2013, 9, 1867-1872. The purpose of terminal derivatization is to facilitate chemical structure analysis. The method of this embodiment can be applied to the synthesis of Man-beta1,4-GlcNAc-beta1,4-GlcNAc trisaccharide and its terminal derivatives.

[0140] A one-pot three-enzyme catalytic synthesis method was used. Mannose (0.054 g, 0.3 mmol) and GlcNAc-beta1,4-GlcNAc-beta-EthN3 (0.099 g, 0.2 mmol) were added to the container. 100 mM Tris-HCl (pH 8.0), 2 mM AMP, 20 mM polyphosphate (PolyPn), and 40 mM MgCl2 were added according to the final concentration of the reaction system, and the pH was adjusted to 7.5. 5 mg of purified monosaccharide kinase NahK, 5 mg of purified glycoside phosphorylase BtMpse, and 2.5 mg of purified polyphosphate kinase EbPPK were added, and the volume of the reaction solution was fixed to 5 mL with water. The reaction was carried out at 37°C for 22 hours. After the substrate conversion was completed, TLC was detected ( Fig.23 ), add an equal volume of 5 mL of cold ethanol and place at 4°C for 30 min to terminate the reaction, and centrifuge at 12000 rpm for 30 min to obtain the supernatant.

[0141] The reaction mixture was crudely purified by activated carbon; the crude product was further purified by Bio-Gel P2 column; DEAE-Cellulose 52 was used for further purification, and the treatment method was the same as that in Example 5; a white powdery protein N-sugar chain core trisaccharide derivative (Man-beta1,4-GlcNAc-beta1,4-GlcNAc-beta-EthN3) was obtained, weighing 62 mg, with a yield of about 47%.

[0142] The purified product was prepared into a 10 mM solution. Only one color spot of the target product was observed by TLC analysis. A 1 mM solution was prepared for high-resolution mass spectrometry analysis. The mass spectrometry results were as follows: Fig.24 As shown, the characteristic ion peak of the product is m / z[M+H] + 656.2634, same as theoretical value [M+H] + 656.2627 agree.

[0143] 10 mg of the product was dissolved in 500 μL of D2O and analyzed using 600 MHz NMR. The results are as follows: Fig.25 and 26 As shown, the spectrum analysis results of the protein N-sugar chain core trisaccharide derivative (Man-beta1,4-GlcNAc-beta1,4-GlcNAc-beta-EthN3) are: 1 H NMR(600MHz,D2O)δ4.78(s,1H),4.61(d,J=8.0Hz,1H),4.59(d,J=8.3Hz,1H),4.07-4.03(m,2H),3. 95–3.65(m,14H),3.65–3.57(m,3H),3.55–3.47(m,2H),3.45–3.4(m,2H),2.08(s,3H),2.05(s,3H). 13 C NMR(151MHz,D2O)δ174.54,174.44,101.25,100.80,99.94,79.15,78.49,76.29,74.46,74.43,72 .62,72.35,71.84,70.37,68.65,66.48,60.79,59.94,59.92,54.90,54.79,50.23,22.12,22.00.

[0144] The above data indicate that the present invention successfully prepared the protein N-sugar chain core trisaccharide derivative (Man-beta1,4-GlcNAc-beta1,4-GlcNAc-beta-EthN3).

Claims

1. A method for preparing oligosaccharides using glycoside phosphorylase in combination with monosaccharide kinase and polyphosphate kinase, characterized in that: The steps include: Using donor monosaccharide, acceptor sugar, polyphosphate, and low equivalent adenosine phosphate as substrates, magnesium chloride is added to the substrates, and then polyphosphatase, monosaccharide kinase, and glycoside phosphorylase are used to jointly catalyze the reaction at 25-40°C for 1.5-22 hours to generate crude oligosaccharide products, which are then purified to obtain oligosaccharides; The donor monosaccharide is galactose, N -acetyl-D-glucosamine or mannose; the final concentration of the donor monosaccharide is 40-100 mM; The acceptor sugar is N -Acetyl-D-glucosamine, N -acetyl-D-galactosamine or chitosan derivatives; the final concentration of the acceptor sugar is 40-70 mM; The polyphosphate is a metal salt of a phosphate compound having a degree of polymerization of 2 phosphates or more; the final concentration of the polyphosphate is 20-40 mM; The low equivalent adenosine phosphate is adenosine monophosphate, adenosine diphosphate or adenosine triphosphate; the final concentration of the low equivalent adenosine phosphate is 2-3 mM; the final concentration of the magnesium chloride is 40-60 mM; The monosaccharide kinase is a monosaccharide kinase GalK or a monosaccharide kinase NahK; The monosaccharide kinase GalK is derived from Bifidobacterium infantis , the amino acid sequence is shown in SEQ ID NO.1 and the coding gene sequence is shown in SEQ ID NO.2; The monosaccharide kinase NahK is derived from Bifidobacterium longum , the amino acid sequence is shown in SEQ ID NO.7, and the coding gene sequence is shown in SEQ ID NO.8; The final concentration of the monosaccharide kinase is 0.3-1.5 mg / mL; The polyphosphate kinase is polyphosphate kinase EbPPK, derived from Erysipelotrichaceae bacteria , the amino acid sequence is shown in SEQ ID NO.3, and the coding gene sequence is shown in SEQ ID NO.4; The final concentration of the polyphosphate kinase is 0.1-0.7 mg / mL; The glycoside phosphorylase is glycoside phosphorylase BiGHNP, glycoside phosphorylase VpCPase or glycoside phosphorylase BtMPase; The glycoside phosphorylase BiGHNP is derived from Bifidobacterium infantis The amino acid sequence is shown in SEQ ID NO.5, and the coding gene sequence is shown in SEQ ID NO.6; the glycoside phosphorylase VpCPase is derived from Vibrio proteolyticus The amino acid sequence is shown in SEQ ID NO.9, and the coding gene sequence is shown in SEQ ID NO.10; the glycoside phosphorylase BtMPase is derived from In Bacteroides thetaiotaomicron VPI-5482 , the amino acid sequence is shown in SEQ ID NO.11, and the coding gene sequence is shown in SEQ ID NO.12; The final concentration of the glycoside phosphorylase is 0.2-2 mg / mL.

2. The method for preparing oligosaccharides according to claim 1, characterized in that: The polyphosphate is sodium hexametaphosphate or sodium tripolyphosphate.

3. The method for preparing oligosaccharides according to claim 1, characterized in that: The preparation method of the monosaccharide kinase GalK, the monosaccharide kinase NahK, the polyphosphate kinase EbPPK, the glycoside phosphorylase BiGHNP, the glycoside phosphorylase VpCPase and the glycoside phosphorylase BtMPase comprises the following steps: (1) The coding gene of monosaccharide kinase GalK was used as the target gene and the pET-22b vector plasmid was used to construct a recombinant expression vector; then the coding genes of monosaccharide kinase NahK, polyphosphate kinase EbPPK, glycoside phosphorylase BiGHNP, glycoside phosphorylase VpCPase and glycoside phosphorylase BtMPase were used as the target genes and the pET-30a vector plasmid was used to construct a recombinant expression vector, and then the recombinant expression vectors were transformed into Escherichia coli to obtain recombinant Escherichia coli; (2) Select the successfully transformed recombinant E. coli for expansion culture, add IPTG with a final concentration of 0.1~1mM or add 0.1~1% lactose for induction, collect the bacterial liquid, crush and centrifuge to obtain the supernatant of the recombinant E. coli; (3) The supernatant was purified by immobilized metal ion affinity chromatography to obtain monosaccharide kinases GalK and NahK, polyphosphate kinase EbPPK, and glycoside phosphorylases BiGHNP, VpCPase, and BtMPase, respectively.

4. The method for preparing oligosaccharides according to claim 1, characterized in that: When the generated oligosaccharide is lactoyl-N-disaccharide or galactoyl-N-disaccharide, the specific preparation method is as follows: Mix galactose as the donor monosaccharide and N -Acetyl-D-glucosamine or N -acetyl-D-galactosamine, AMP, polyphosphate, magnesium chloride, adjust the pH to 8.0, add monosaccharide kinase GalK, glycoside phosphorylase BiGHNP and polyphosphate kinase EbPPK, react at 37°C, 110 rpm for 1.5 h, terminate the reaction, separate and purify lactoyl-N-disaccharide or galactoyl-N-disaccharide; Wherein, the final concentration of galactose is 60-80 mM; N -Acetyl-D-glucosamine or N The final concentration of -acetyl-D-galactosamine is 40-50 mM; the final concentrations of AMP, polyphosphate, and magnesium chloride are 2 mM, 20 mM, and 40 mM, respectively; the final concentrations of monosaccharide kinase GalK, glycoside phosphorylase BiGHNP, and polyphosphate kinase EbPPK are 0.3-0.7 mg / mL, 0.2-0.5 mg / mL, and 0.1-0.2 mg / mL, respectively; and the termination reaction is terminated by adding an equal volume of cold ethanol at 4°C for 30 minutes.

5. The method for preparing oligosaccharides according to claim 1, characterized in that: When the generated oligosaccharide is chitosan, the specific preparation method is as follows: Mix in water N -acetyl-D-glucosamine, AMP, polyphosphate, magnesium chloride, Tris-HCl solution, adjust the pH to 7.5, add monosaccharide kinase NahK, glycoside phosphorylase VpCPase and polyphosphate kinase EbPPK, react at 37°C for 6 hours, terminate the reaction, separate and purify chitobiose; Among them, the N The final concentration of -acetyl-D-glucosamine is 90-100 mM; the final concentrations of the AMP, polyphosphate, magnesium chloride, and Tris-HCl solution are 3 mM, 30 mM, 60 mM, and 200 mM, respectively; the final concentrations of the monosaccharide kinase NahK, glycoside phosphorylase VpCPase, and polyphosphate kinase EbPPK are 0.5-1.5 mg / mL, 0.5-2 mg / mL, and 0.3-0.7 mg / mL, respectively; and the termination reaction is terminated by adding an equal volume of cold ethanol at 4°C for 30 min.

6. The method for preparing oligosaccharides according to claim 1, characterized in that: When the generated oligosaccharide is a protein N-sugar chain core trisaccharide derivative, the specific preparation method is as follows: Mix mannose, chitobiose derivatives, AMP, polyphosphate, magnesium chloride, Tris-HCl solution in water, adjust the pH to 7.5, monosaccharide kinase NahK, glycoside phosphorylase BtMPase and polyphosphate kinase EbPPK, react at 37°C for 22 h, terminate the reaction, separate and purify to obtain protein N-sugar chain core trisaccharide derivatives; The final concentration of mannose is 60-80 mM; the final concentration of chitobiose derivative is 40-50 mM; the final concentrations of AMP, polyphosphate, magnesium chloride and Tris-HCl solution are 2 mM, 20 mM, 40 mM and 100 mM, respectively; the final concentrations of monosaccharide kinase NahK, glycoside phosphorylase BtMPase and polyphosphate kinase EbPPK are 0.7-1.5 mg / mL, 0.7-1.5 mg / mL and 0.3-0.6 mg / mL, respectively; and the termination reaction is to add an equal volume of cold ethanol and terminate the reaction at 4°C for 30 min.

7. The method for preparing oligosaccharides according to claim 1, characterized in that: The purification is specifically as follows: removing the precipitate by centrifugation, purifying by activated carbon adsorption, molecular exclusion chromatography, and ion exchange chromatography; When the oligosaccharide product is lactoyl-N-disaccharide or galactoyl-N-disaccharide, the reaction solution is purified by selecting a Bio-Gel P2 chromatographic column to separate the product disaccharide from the raw material monosaccharide to obtain a crude product; the crude product is purified by DEAE-Cellulose 52 to obtain a high-purity product; wherein the steps of purifying the crude product by DEAE-Cellulose 52 are: using deionized water as a mobile phase at a flow rate of 5-6 mL / min, collecting fractions and detecting the product by TLC, concentrating the fractions containing the product by a rotary evaporator, and then freeze-drying to obtain lactoyl-N-disaccharide or galactoyl-N-disaccharide; When the oligosaccharide product is chitobiose or a protein N-glycoside core trisaccharide derivative, the reaction solution is desalted by activated carbon adsorption; the reaction treatment solution or the eluate after activated carbon adsorption and desalting is purified by a Bio-Gel P2 chromatographic column to obtain a crude product; the crude product is purified by DEAE-Cellulose 52 to obtain a high-purity product; wherein the steps of crude purification by activated carbon are: first, three column volumes are flushed with deionized water as the mobile phase; then, impurities are eluted with 2wt% ethanol as the mobile phase; 30wt% ethanol aqueous solution is eluted to collect the chitobiose or protein N-glycoside core trisaccharide derivative.

Images