Preparation method of hyaluronic acid oligosaccharide with controllable polymerization degree and derivative of hyaluronic acid oligosaccharide

Abstract

The invention relates to a preparation method of hyaluronic acid oligosaccharide with controllable polymerization degree and a derivative thereof. The invention firstly provides a method for preparing the hyaluronic acid oligosaccharides and the derivatives thereof through cooperation of chondroitin synthase AuCS, hyaluronic acid synthase PmHAS and chondroitin lyase ODV-E66, the hyaluronic acid oligosaccharides or the derivatives thereof with any polymerization degree can be obtained, the defects of traditional physical or chemical degradation and enzymolysis methods are overcome, the diversity of oligosaccharide structures is enriched, and the application prospect is wide. And a foundation is laid for research on functions and characteristics of the hyaluronic acid oligosaccharides with different polymerization degrees and the derivatives of the hyaluronic acid oligosaccharides. Then, on the basis of the method, the hyaluronic acid oligosaccharide and the derivative thereof are prepared by utilizing a chondroitin synthase AuCS mutant, a hyaluronic acid synthase PmHAS mutant and chondroitin lyase ODV-E66, so that the synthesis efficiency and the yield of the product are greatly improved, and rapid, safe and large-scale preparation of the hyaluronic acid oligosaccharide and the derivative thereof is realized.

Description

Technical Field

[0001] The invention relates to a preparation method of hyaluronic acid oligosaccharide with controllable polymerization degree and derivatives thereof, belonging to the field of biotechnology. Background Art

[0002] Hyaluronic acid (HA) is an acidic mucopolysaccharide composed of repeating disaccharide units of D-β-glucuronic acid and N-acetylglucosamine, which provides compressive strength, lubricity and hydration in the extracellular matrix. HA can regulate cell adhesion and mobility and mediate cell proliferation and differentiation. It is not only a structural component of tissues, but also an active signaling molecule. HA is widely used in clinical treatment and beauty care due to its unique viscoelasticity, hygroscopicity, and biocompatibility.

[0003] The biological function of HA is closely related to its molecular weight. HA oligosaccharides can stimulate fibroblast proliferation and collagen synthesis, and selectively kill cancer cells by destroying the interaction between macromolecular HA and cell receptors; they can penetrate the dermis of the skin, replenish the HA content in the body, regulate skin metabolism, and delay aging, and have important application prospects in the fields of medical care and medicine.

[0004] The currently disclosed methods for preparing HA oligosaccharides mainly include physical or chemical degradation and enzymatic hydrolysis. Among them, the physical degradation method mainly uses physical treatment methods such as thermal degradation and ultrasound, which can only reduce the molecular weight of HA to about 10,000, and most of the obtained products are complex mixtures, which are difficult to purify. Existing chemical degradation is mostly acid hydrolysis. For example, Chinese patent document CN102304193A discloses a method for preparing oligomeric hyaluronic acid and its use, but this method is not environmentally friendly, and the reaction may affect the structure of HA after degradation, reducing product quality.

[0005] In recent years, enzymatic hydrolysis has been widely concerned because of mild reaction conditions and non-toxicity. The advantage of this technology is high reproducibility and controllable degradation, such as the preparation method of unsaturated hyaluronic acid odd-numbered oligosaccharides disclosed in Chinese patent document CN106518934A, but the HA oligosaccharides obtained by the enzymatic hydrolysis of this patent are usually tetrasaccharides to octasaccharides, and the oligosaccharide polymerization degree is an even number and the polymerization degree is small, and the product obtained by enzymatic hydrolysis may have unsaturated bonds. Chinese patent document CN109097421A discloses a method for preparing hyaluronic acid odd-numbered oligosaccharides by double enzyme hydrolysis, and the patent uses bovine testicular hyaluronidase (BTH) and leech type hyaluronidase (LHase) two hydrolases to prepare odd-numbered polymerization degree HA oligosaccharides, but is subject to the limited source of bovine testicles, expensive, and in the long run, it is still difficult to obtain HA oligosaccharides with odd-numbered polymerization degrees on a large scale. Moreover, the research on HA oligosaccharide derivatives with uniform structure is very scarce, and no effective preparation means is found. Therefore, there is an urgent need for an efficient and economical method for preparing HA oligosaccharides and their derivatives, which can stably and effectively obtain HA oligosaccharides and their derivatives with either even or odd degrees of polymerization. Summary of the invention

[0006] In view of the shortcomings of the prior art, the present invention provides a method for preparing hyaluronic acid oligosaccharides and their derivatives with controllable polymerization degree. The purpose of the present invention is to provide a general method for preparing HA oligosaccharides and their derivatives with controllable polymerization degree. The present invention first prepares a series of chimeric oligosaccharides of chondroitin and hyaluronic acid by chemical enzyme method, and then the chimeric oligosaccharides are cleaved by chondroitin cleaving enzyme ODV-E66 to remove the chondroitin structure at the reducing end to obtain hyaluronic acid oligosaccharides or their derivatives with natural structure of any polymerization degree. The product structure is clear, the purification method is simple, and it is easy to realize industrialization.

[0007] Terminology Note:

[0008] GlcA-pNP: The full name in Chinese is 4-nitrophenyl-β-D-glucuronic acid, which serves as the starting receptor substrate for the synthesis of hyaluronic acid and chondroitin chimeric oligosaccharides;

[0009] UDP-GalNAc: The full name in Chinese is uridine diphosphate-N-acetylgalactosamine, which is used to provide acetylgalactosamine donor for the synthesis of hyaluronic acid and chondroitin chimeric oligosaccharides;

[0010] UDP-GlcNAc: The full name in Chinese is uridine diphosphate-N-acetylglucosamine, which is used to provide acetylglucosamine donor for the synthesis of hyaluronic acid and chondroitin chimeric oligosaccharides;

[0011] UDP-GlcA: The full name in Chinese is uridine diphosphate-N-glucuronic acid. Its function is to provide a glucuronic acid donor for the synthesis of hyaluronic acid and chondroitin chimeric oligosaccharides.

[0012] The technical solution of the present invention is as follows:

[0013] A hyaluronic acid oligosaccharide and its derivatives, the structure of which is shown in the following formula (1) or formula (2):

[0014]

[0015] Wherein, in the structural formula, m=1, 2, 3; n=1, 2, 3; R1 is H or Na or K or NH4; R2 is any one of NHCOCH3, NHCOCF3, NHCOCH2N3, NHCOCH2CH3, NHCOCH(CH3)2, OH, NH2, NHSO3H; formula (1) represents an oligosaccharide structure with an even degree of polymerization, and formula (2) represents an oligosaccharide structure with an odd degree of polymerization;

[0016] When R2 is NHCOCH3, the structure shown is a hyaluronic acid oligosaccharide with a natural structure, and the degree of polymerization (dp) is 2m or 2n+1; when R2 is NHCOCF3, NHCOCH2N3, NHCOCH2CH3, NHCOCH(CH3)2, OH, NH2 or NHSO3H, the structure shown is a hyaluronic acid oligosaccharide derivative with a natural structure, and the degree of polymerization (dp) is 2m or 2n+1.

[0017] The preparation method of the above hyaluronic acid oligosaccharide and its derivatives comprises the following steps:

[0018] (1) GlcA-pNP was used as the initial acceptor substrate, UDP-GalNAc and UDP-GlcA were used as donor substrates in turn, and the chondroitin synthase AuCS and Mn 2+ The reaction was carried out under the catalysis of for 3 to 24 hours to obtain chondroitin trisaccharide;

[0019] (2) The chondroitin trisaccharide obtained in step (1) is used as the acceptor substrate, and the sugar donor corresponding to the UDP-GlcNAc / oligosaccharide derivative and UDP-GlcA are alternately used as the donor substrate, and the hyaluronan synthase PmHAS and Mn 2+ The reaction is carried out under the catalysis of for 3 to 24 hours to obtain a series of chimeric oligosaccharides or their derivatives with a chondroitin trisaccharide at the reducing end and a hyaluronic acid chain at the non-reducing end;

[0020] (3) The chimeric oligosaccharide or its derivative obtained in step (3) is separated and purified, eluted, concentrated and freeze-dried, and then dissolved in double distilled water, the pH value is adjusted to 7.5-8.5, and then chondroitin lyase ODV-E66 is added, and cleaved at 35-40° C. for 0.5-12 h. After enzyme inactivation, separation and purification, and elution, the components with absorption at 210 nm but no absorption at 310 nm are collected, and then concentrated and purified again to obtain hyaluronic acid oligosaccharide or its derivative.

[0021] Preferably, according to the present invention, in step (1), the chondroitin synthase AuCS is derived from Actinobacillus Ureae, its amino acid sequence is shown in SEQ ID NO.2, the nucleotide sequence of the encoding gene is shown in SEQ ID NO.1, and the final concentration of the addition is 1 to 3 mg / mL.

[0022] Preferably according to the present invention, in step (2), the sugar donor corresponding to the oligosaccharide derivative is UDP-GlcNTFA, UDP-GlcNAz, UDP-GlcNPr, UDP-Glc-i-Bu, UDP-Glc, UDP-GlcNH2 or UDP-GlcNS.

[0023] Preferably according to the present invention, in step (2), the hyaluronan synthase PmHAS is derived from Pasteuella multocida, its amino acid sequence is shown in SEQ ID NO.4, the nucleotide sequence of the encoding gene is shown in SEQ ID NO.3, and the final added concentration is 1 to 3 mg / mL.

[0024] Preferably according to the present invention, in step (3), the chondroitin lyase ODV-E66 is derived from Autographa californica nucleopolyhedrovirus, and its amino acid sequence is shown in SEQ ID NO.6, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.5.

[0025] The present invention optimizes the tool enzyme used in the synthesis on the basis of the above preparation method, and provides a preparation method of hyaluronic acid oligosaccharides and their derivatives with improved synthesis efficiency and yield and controllable polymerization degree, which specifically comprises the following steps:

[0026] 1) Using GlcA-pNP as the initial acceptor substrate and UDP-GalNAc as the donor substrate, the chondroitin synthase AuCS mutant AuCS (D517N / D519N) and Mn 2+ The reaction was carried out under the catalysis of for 3 to 24 hours to obtain chondroitin disaccharide;

[0027] 2) Using chondroitin disaccharide as the acceptor substrate and UDP-GlcA as the donor substrate, the chondroitin synthase AuCS mutant AuCS (D237N / D239N) and Mn 2+ The reaction was carried out under the catalysis of for 3 to 24 hours to obtain chondroitin trisaccharide;

[0028] 3) Using chondroitin trisaccharide as the acceptor substrate and the sugar donor corresponding to UDP-GlcNAc / oligosaccharide derivative as the donor substrate, the hyaluronan synthase PmHAS mutant PmHAS (D527N / D529N) and Mn 2+ The reaction was carried out under the catalysis of 3 to 24 hours, and a sugar group was extended at the non-reducing end of the sugar chain to obtain a chimeric tetrasaccharide or its derivative; the chimeric tetrasaccharide or its derivative was used as an acceptor substrate and UDP-GlcA was used as a donor substrate in the presence of hyaluronan synthase PmHAS mutant PmHAS (D247N / D249N) and Mn 2+ The reaction is carried out under the catalysis of 3 to 24 hours to extend a sugar group at the non-reducing end of the sugar chain; repeating this step, the hyaluronic acid and chondroitin chimera tetrasaccharide, pentasaccharide, hexasaccharide, heptasaccharide, octasaccharide, nonasaccharide, decasaccharide or their derivatives can be obtained respectively;

[0029] 4) The hyaluronic acid and chondroitin chimera pentasaccharide or its derivative obtained in step 3) is purified, eluted, concentrated and freeze-dried, and then dissolved in double distilled water, and the pH value is adjusted to 7.5-8.5, and then chondroitin lyase ODV-E66 is added, and the lysis is carried out at 35-40° C. for 0.5-12 hours. After enzyme inactivation, separation and purification, and elution, the components with absorption at 210 nm but no absorption at 310 nm are collected, and then concentrated and purified again to obtain hyaluronic acid disaccharide or its derivative with natural structure; hyaluronic acid and chondroitin chimera hexasaccharide, heptasaccharide, octasaccharide, nonasaccharide, decasaccharide or its derivatives are treated in the same way to obtain hyaluronic acid trisaccharide or its derivatives, hyaluronic acid tetrasaccharide or its derivatives, hyaluronic acid pentasaccharide or its derivatives, hyaluronic acid hexasaccharide or its derivatives, and hyaluronic acid heptasaccharide or its derivatives.

[0030] Preferably according to the present invention, in step 1), the chondroitin synthase AuCS mutant AuCS (D517N / D519N) is obtained by mutating the aspartic acid at positions 517 and 519 in the amino acid sequence of the chondroitin synthase AuCS to asparagine, so as to allow the enzyme to retain only the N-acetylgalactosamine transferase activity.

[0031] Preferably according to the present invention, in step 2), the chondroitin synthase AuCS mutant AuCS (D237N / D239N) is obtained by mutating the aspartic acid at positions 237 and 239 in the amino acid sequence of the chondroitin synthase AuCS to asparagine, so as to allow the enzyme to retain only the glucuronyl transferase activity.

[0032] Preferably according to the present invention, in step 3), the hyaluronan synthase PmHAS mutant PmHAS (D527N / D529N) is a mutant of the hyaluronan synthase PmHAS, wherein the aspartic acid at positions 527 and 529 in the amino acid sequence of the hyaluronan synthase PmHAS is mutated to asparagine, the purpose of which is to make the enzyme retain only the N-acetylglucosamine transferase activity, and to add an N-acetylglucosamine group or a derivative corresponding to the sugar group at the non-reducing end of the chimeric oligosaccharide or its derivative;

[0033] The hyaluronan synthase PmHAS mutant PmHAS (D247N / D249N) is a mutant in which the aspartic acid at positions 247 and 249 in the amino acid sequence of the hyaluronan synthase PmHAS is mutated into asparagine, with the purpose of making the enzyme retain only the glucuronyl transferase activity and adding a glucuronic acid group to the non-reducing end of the chimeric oligosaccharide or its derivative.

[0034] Preferably, in step 1) to step 3), the molar ratio of the donor substrate to the acceptor substrate is 1.2:1; the Mn 2+ The final concentration is 10 mM.

[0035] Preferably, according to the present invention, the final concentrations of the chondroitin synthase AuCS mutant AuCS (D517N / D519N) in step 1), the chondroitin synthase AuCS mutant AuCS (D237N / D239N) in step 2), and the hyaluronan synthase PmHAS mutant PmHAS (D527N / D529N) and mutant PmHAS (D247N / D249N) in step 3) are all added at 1-3 mg / mL.

[0036] Preferably according to the present invention, in step 4), the final concentration of the chondroitin lyase ODV-E66 added is 0.1-2.5 mg / mL.

[0037] In the method provided by the present invention, separation and purification is carried out by C18-AQ separation column, methanol gradient elution; secondary purification uses a low-pressure gel chromatography column, the low-pressure gel chromatography column filler is Superdex 30, Bio Gel P2 or Bio GelP4, and pure water is the mobile phase; sodium hydroxide, potassium hydroxide, sodium carbonate, sodium bicarbonate or ammonium hydroxide is used to adjust the pH; an ultraviolet detector is used to detect the absorbance and collect the product.

[0038] The technical features of the present invention are as follows:

[0039] like Figure 1As shown, the present invention uses GlcA-pNP as a starting substrate, and utilizes chondroitin synthase AuCS and hyaluronic acid synthase PmHAS to prepare a series of chondroitin and hyaluronic acid chimeric oligosaccharides or their derivatives with uniform structures. The introduction of the pNP group allows the prepared oligosaccharides or their derivatives to be easily purified in one step through a C18 separation column; subsequently, the chimeric oligosaccharides or their derivatives with different chain lengths are cleaved by chondroitin cleavage enzyme ODV-E66 to obtain hyaluronic acid oligosaccharides or their derivatives with different polymerization degrees. However, AuCS and PmHAS are both bifunctional enzymes, and product mixing will occur during the oligosaccharide preparation process, resulting in reduced preparation efficiency and yield; while AuCS mutants AuCS (D517N / D519N), AuCS (D237N / D239N) and PmHAS mutants PmHAS (D527N / D529N), PmHAS (D247N / D249N) only retain single transferase activity, which can effectively avoid product mixing, improve preparation efficiency, and facilitate promotion to industrial applications. Unlike most chondroitin lyases, chondroitin lyase ODV-E66 has strong degradation activity for chondroitin sulfate, but weak activity for hyaluronic acid, and can be used to cleave the prepared chimeric oligosaccharides of chondroitin and hyaluronic acid or their derivatives, thereby removing the pNP group introduced at the reducing end to obtain hyaluronic acid oligosaccharides or their derivatives.

[0040] Beneficial effects:

[0041] 1. The present invention provides a hyaluronic acid oligosaccharide and its derivatives. The hyaluronic acid oligosaccharide and its derivatives do not have unsaturated bonds, which not only enriches the diversity of oligosaccharide structures, but also has very important significance for further exploring the structure-activity relationship of hyaluronic acid oligosaccharides and guiding their application in food, cosmetics, disease occurrence and development, etc. The present invention also provides a preparation method of the hyaluronic acid oligosaccharide and its derivatives. By cooperating with chondroitin synthase AuCS, hyaluronic acid synthase PmHAS and chondroitin lyase ODV-E66, hyaluronic acid oligosaccharides or their derivatives with any polymerization degree can be prepared, which makes up for the deficiencies of traditional physical or chemical degradation and enzymatic hydrolysis, enriches the diversity of oligosaccharide structures, and lays a foundation for the study of the functions and characteristics of hyaluronic acid oligosaccharides and their derivatives with different polymerization degrees.

[0042] 2. Based on the preparation method of hyaluronic acid oligosaccharides and their derivatives, the present invention optimizes the tool enzymes used in the preparation process, and improves the synthesis efficiency, yield and output. Specifically, by using chondroitin synthase AuCS mutant AuCS (D517N / D519N), chondroitin synthase AuCS mutant AuCS (D237N / D239N), hyaluronic acid synthase PmHAS mutant PmHAS (D527N / D529N) and hyaluronic acid synthase PmHAS mutant PmHAS (D247N / D249N) with single transferase activity, the glycosyl structure and glycosyl linking bond of the non-reducing end of the sugar chain are effectively controlled, and the mixing of products in the oligosaccharide preparation process is avoided, and then with the chondroitin lyase ODV-E66, hyaluronic acid oligosaccharides or their derivatives with any degree of polymerization can be prepared. At the same time, compared with the initial method of the present invention, this method effectively improves the synthesis efficiency, yield and output. The purity of each hyaluronic acid oligosaccharide reaches more than 95%, the output of hyaluronic acid oligosaccharides reaches 6.04-28.9 mg, and the yield reaches more than 83%, realizing the rapid, safe and large-scale preparation of hyaluronic acid oligosaccharides and their derivatives. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 The invention provides a preparation scheme for hyaluronic acid oligosaccharides with controllable degree of polymerization and their derivatives.

[0044] Figure 2 The figure is the SDS-PAGE detection result of chondroitin synthase AuCS and its mutants, hyaluronan synthase PmHAS and its mutants;

[0045] In the figure: M is a marker; the other bands are purified recombinant proteins.

[0046] Figure 3 This is the SDS-PAGE detection result of chondroitin cleaving enzyme ODV-E66.

[0047] Figure 4 HPLC chromatogram of each chimeric oligosaccharide prepared in Example 2;

[0048] In the figure: (a) is a chimeric pentasaccharide; (b) is a chimeric hexasaccharide; (c) is a chimeric heptasaccharide.

[0049] Figure 5 ESI-MS results of each hyaluronic acid oligosaccharide prepared in Example 2;

[0050] In the figure: (a) is HA disaccharide; (b) is HA trisaccharide; (c) is HA tetrasaccharide.

[0051] Figure 6HPLC chromatogram of the products obtained when PmHAS and PmHAS (D247N / D249N) were used to prepare the hyaluronic acid and chondroitin chimera heptasaccharide in Example 3.

[0052] Figure 7 HPLC chromatogram of each chimeric oligosaccharide prepared in Example 3;

[0053] In the figure: (a) is chondroitin disaccharide; (b) is chondroitin trisaccharide; (c) is hyaluronic acid and chondroitin chimera tetrasaccharide; (d) is hyaluronic acid and chondroitin chimera pentasaccharide; (e) is hyaluronic acid and chondroitin chimera hexasaccharide; (f) is hyaluronic acid and chondroitin chimera heptasaccharide; (g) is hyaluronic acid and chondroitin chimera octasaccharide; (h) is hyaluronic acid and chondroitin chimera nonasaccharide; (i) is hyaluronic acid and chondroitin chimera decasaccharide.

[0054] Figure 8 The ESI-MS results of each chimeric oligosaccharide prepared in Example 3;

[0055] In the figure: (a) is chondroitin disaccharide; (b) is chondroitin trisaccharide; (c) is hyaluronic acid and chondroitin chimera tetrasaccharide; (d) is hyaluronic acid and chondroitin chimera pentasaccharide; (e) is hyaluronic acid and chondroitin chimera hexasaccharide; (f) is hyaluronic acid and chondroitin chimera heptasaccharide; (g) is hyaluronic acid and chondroitin chimera octasaccharide; (h) is hyaluronic acid and chondroitin chimera nonasaccharide; (i) is hyaluronic acid and chondroitin chimera decasaccharide.

[0056] Fig. 9 HPLC chromatogram of each hyaluronic acid oligosaccharide prepared in Example 3;

[0057] In the figure: (a) is HA disaccharide; (b) is HA trisaccharide; (c) is HA tetrasaccharide; (d) is HA pentasaccharide; (e) is HA hexasaccharide; (f) is HA heptasaccharide.

[0058] Fig.10 The ESI-MS characterization results of the hyaluronic acid oligosaccharides prepared in Example 3;

[0059] In the figure: (a) is HA disaccharide; (b) is HA trisaccharide; (c) is HA tetrasaccharide; (d) is HA pentasaccharide; (e) is HA hexasaccharide; (f) is HA heptasaccharide;

[0060] Fig.11 HPLC chromatographic results of each hyaluronic acid derivative prepared in Example 4;

[0061] In the figure: (a) is a hyaluronic acid disaccharide derivative; (b) is a hyaluronic acid tetrasaccharide derivative; (c) is a hyaluronic acid hexasaccharide derivative.

[0062] Fig.12 HPLC chromatographic results of each hyaluronic acid derivative prepared in Example 5;

[0063] In the figure: (a) is a hyaluronic acid disaccharide derivative; (b) is a hyaluronic acid tetrasaccharide derivative; (c) is a hyaluronic acid hexasaccharide derivative. DETAILED DESCRIPTION

[0064] The technical solution of the present invention is further described below in conjunction with the accompanying drawings and embodiments, but the protection scope of the present invention is not limited thereto. Unless otherwise specified, the technical means used in the present invention are methods well known to those skilled in the art.

[0065] The GlcA-pNP, UDP-GalNAc, UDP-GlcA, and UDP-GlcNAc used in the present invention are all purchased from Sigma. The plasmids of chondroitin synthase AuCS, chondroitin synthase AuCS mutant AuCS (D517N / D519N), chondroitin synthase AuCS mutant AuCS (D237N / D239N), hyaluronan synthase PmHAS, hyaluronan synthase PmHAS mutant PmHAS (D527N / D529N), hyaluronan synthase PmHAS mutant PmHAS (D247N / D249N) and chondroitin lyase ODV-E66 are all entrusted to Nanjing GenScript Biological Co., Ltd. to produce. The Escherichia coli BL21 (DE3) competent cells used are purchased from Nanjing Nuowei Zan Biotechnology Co., Ltd. HPLC detection is carried out using the amino column of YMC, and the liquid phase system is produced by Shimadzu Corporation of Japan, and the ultraviolet detection system is SPD-20A.

[0066] Example 1. Obtaining chondroitin synthase and its mutants, hyaluronan synthase and its mutants, and chondroitin lyase

[0067] 1. The chondroitin synthase gene AuCS, the hyaluronan synthase gene PmHAS and the chondroitin lyase gene ODV-E66 were used as the target genes, and pET-28a(+) was used as the vector plasmid to construct the recombinant vector plasmids pET28a(+)-AuCS, pET28a(+)-PmHAS and pET28a(+)-ODV-E66. Then, the recombinant vector plasmids pET28a(+)-AuCS, pET28a(+)-PmHAS and pET28a(+)-ODV-E66 were transformed into Escherichia coli BL21(DE3) competent cells by 42°C heat shock, spread on LB solid medium (containing kanamycin, 100 μg / mL), and cultured at 37°C overnight to obtain recombinant Escherichia coli AuCS, recombinant Escherichia coli PmHAS and recombinant Escherichia coli ODV-E66, and then the positive transformants were screened.

[0068] The construction of the above recombinant vector plasmids and recombinant Escherichia coli was completed by Nanjing GenScript Biotechnology Co., Ltd. The chondroitin synthase AuCS is derived from Actinobacillus Ureae, and its amino acid sequence is shown in SEQ ID NO.2, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.1; the hyaluronan synthase PmHAS is derived from Pasteuella multocida, and its amino acid sequence is shown in SEQ ID NO.4, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.3; the chondroitin lyase ODV-E66 is derived from Autographa californica nucleopolyhedrovirus, and its amino acid sequence is shown in SEQ ID NO.6, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.5.

[0069] According to the same method, Nanjing GenScript Biotechnology Co., Ltd. constructed recombinant vector plasmids and recombinant Escherichia coli containing chondroitin synthase AuCS mutant AuCS (D517N / D519N), chondroitin synthase AuCS mutant AuCS (D237N / D239N), hyaluronan synthase PmHAS mutant PmHAS (D527N / D529N), and hyaluronan synthase PmHAS mutant PmHAS (D247N / D249N), respectively.

[0070] The chondroitin synthase AuCS mutant AuCS (D517N / D519N) is a chondroitin synthase AuCS amino acid sequence (SEQ ID NO.2) wherein the aspartic acid at positions 517 and 519 is mutated to asparagine, the gat at 1549-1551 bp of the chondroitin synthase AuCS nucleotide sequence (SEQ ID NO.1) is mutated to aac, and the gat at 1555-1557 bp is mutated to aac;

[0071] The chondroitin synthase AuCS mutant AuCS (D237N / D239N) is obtained by mutating the aspartic acid at positions 237 and 239 in the chondroitin synthase AuCS amino acid sequence (SEQ ID NO.2) to asparagine, and mutating the gat at 709-711 bp to aac and the gat at 715-717 bp to aac in the chondroitin synthase AuCS nucleotide sequence (SEQ ID NO.1);

[0072] The hyaluronan synthase PmHAS mutant PmHAS (D527N / D529N) is obtained by mutating the aspartic acid at positions 527 and 529 in the hyaluronan synthase PmHAS amino acid sequence (SEQ ID NO.4) to asparagine, and mutating the gac at 1579-1581 bp to aac, and the gac at 1585-1587 bp to aac in the hyaluronan synthase PmHAS nucleotide sequence (SEQ ID NO.3);

[0073] The hyaluronan synthase PmHAS mutant PmHAS (D247N / D249N) is a mutant in which the aspartic acid at positions 247 and 249 in the hyaluronan synthase PmHAS amino acid sequence (SEQ ID NO.4) is mutated into asparagine, the gac at positions 739 to 741 bp of the hyaluronan synthase PmHAS nucleotide sequence (SEQ ID NO.3) is mutated into aac, and the gat at positions 745 to 747 bp is mutated into aac.

[0074] 2. Pick a single colony of the recombinant E. coli AuCS positive transformant and place it in 50 mL of sterilized LB liquid medium (containing kanamycin, 100 μg / mL), and culture it at 37°C, 225 r / min overnight; inoculate the activated bacterial solution into 5 L of LB liquid medium (containing kanamycin, 100 μg / mL) at a 1% inoculum volume, and culture it at 37°C, 225 r / min until OD 600 value is 0.6~0.8, add IPTG with a final concentration of 0.2mM, induce at 22℃, 225r / min for 16~18h, and then collect the bacteria; use equilibrium buffer (20mM imidazole; 0.5M NaCl; 20mM Tris-HCl, pH=7.5) to resuspend, ultrasonically disrupt on ice (working 15s, rest 45s, amplitude 33%, energy 1500KJ, 4℃) for 30min, centrifuge the disrupted bacteria at 12000rpm for 20min, and collect the supernatant; filter the supernatant with 0.22μm filter membrane, use nickel column for protein purification, wash with equilibrium buffer after loading, and then use washing buffer (40mM imidazole; 0.5M NaCl; 20mM Tris-HCl, pH=8.00) to wash away the impurities, and finally use elution buffer (250mM imidazole; 0.5M NaCl; 20mM The target protein chondroitin synthase AuCS was eluted with Tris-HCl, pH=8.00, and the purified chondroitin synthase AuCS was stored in 20% glycerol and stored in a -80°C refrigerator.

[0075] The heterologous recombinant proteins chondroitin synthase AuCS mutant AuCS (D517N / D519N), chondroitin synthase AuCS mutant AuCS (D237N / D239N), hyaluronan synthase PmHAS mutant PmHAS (D527N / D529N), hyaluronan synthase PmHAS mutant PmHAS (D247N / D249N) and chondroitin lyase ODV-E66 were obtained according to the same method as above.

[0076] The purified chondroitin synthase AuCS and its mutants and hyaluronan synthase PmHAS and its mutants were identified by SDS-PAGE. Figure 2 shown.

[0077] Depend on Figure 2 It can be seen that the chondroitin synthase AuCS mutant AuCS (D517N / D519N), the chondroitin synthase AuCS mutant AuCS (D237N / D239N), the hyaluronan synthase PmHAS mutant PmHAS (D527N / D529N), and the hyaluronan synthase PmHAS mutant PmHAS (D247N / D249N) were all successfully heterologously expressed and purified, and the relative purity reached more than 95%.

[0078] The purified chondroitin lyase ODV-E66 was identified by SDS-PAGE. Figure 3 shown.

[0079] Depend on Figure 3 It can be seen that the heterologous expression and purification of chondroitin cleaving enzyme ODV-E66 were successful, and the relative purity reached more than 98%.

[0080] Example 2. Preparation and purification of hyaluronic acid oligosaccharides with controllable degree of polymerization

[0081] 1. Preparation and purification of chondroitin and hyaluronic acid chimeric oligosaccharides

[0082] 1) Using commercial GlcA-pNP as the initial acceptor substrate, UDP-GalNAc and UDP-GlcA as donor substrates, the chondroitin synthase AuCS and Mn 2+ The reaction was carried out in a water bath at 37°C for 12 h under the catalysis of to obtain chondroitin trisaccharide;

[0083] 2) Chondroitin trisaccharide is used as the acceptor substrate and UDP-GlcNAc is used as the donor substrate. 2+The reaction was carried out in a 37°C water bath under the catalysis of , and a sugar moiety was extended at the non-reducing end of the sugar chain to obtain a chimeric tetrasaccharide of hyaluronic acid and chondroitin. Then, the chimeric tetrasaccharide was used as the acceptor substrate and UDP-GlcA as the donor substrate. In the presence of hyaluronan synthase PmHAS and Mn 2+ The mixture was reacted in a water bath at 37°C for 6 hours under the catalysis of , and a GlcA group was added to the non-reducing end of the sugar chain to obtain a hyaluronic acid and chondroitin chimera pentasaccharide; the above steps were repeated to obtain a hyaluronic acid and chondroitin chimera hexasaccharide and heptasaccharide; in this step, HPLC was used to monitor the preparation process of the chimera oligosaccharide in real time;

[0084] The acceptor substrate, donor substrate, enzyme and Mn in step 1) to step 2) 2+ The reaction systems are shown in Table 1.

[0085] Table 1. Synthesis system of chondroitin oligosaccharides

[0086]

[0087] The HPLC detection method in step 1) to step 2) is shown in Table 2, and the mobile phase flow rate is 0.5 mL / min.

[0088] Table 2. HPLC analysis method used for oligosaccharide detection

[0089]

[0090] The purification method in step 1) to step 2) is:

[0091] use C18-AQ separation column, AKTA for purification. The reaction solution was terminated with 0.1% TFA, and the system pH was adjusted to about 3.0. Before purification, the reaction solution with adjusted pH was centrifuged (room temperature, 12000rpm, 10min) to take the supernatant, filtered with a 0.22μm filter membrane, and then loaded onto a C18 separation column that had been equilibrated with double distilled water (0.1% TFA) in advance. The specific purification procedure is shown in Table 3, and the flow rate is 3ml / min.

[0092] Table 3. Oligosaccharide C18 purification procedure

[0093]

[0094] Depend on Figure 4 It can be seen that the hyaluronic acid and chondroitin chimera pentasaccharide, hexasaccharide, and heptasaccharide were successfully obtained.

[0095] 2. Preparation and purification of hyaluronic acid oligosaccharides with controllable degree of polymerization

[0096] 3) 14.6 mg of the chimeric pentasaccharide obtained in step 2) was dissolved in double distilled water to a concentration of 5 mM, the pH was adjusted to neutral, and the purified chondroitin lyase ODV-E66 was added according to the volume of the oligosaccharide solution to a final concentration of 10 μg / mL. The oligosaccharide solution was lysed at 37°C for 0.5 to 12 h, and the reaction progress was detected by HPLC (the same method as above). After the cleavage was complete, the enzyme was removed: the reaction solution was boiled for 5 min, and then centrifuged at 12000 rpm for 10 min; the pH of the system was adjusted to about 3.0 for purification (the same method as above), and all components that absorbed at 210 nm but not at 310 nm were collected. The product was rotary evaporated to a volume of about 2 ml, transferred to a Bio-Gel P-2 chromatography column (Φ2cm*80cm) balanced with double distilled water for chromatography purification, and eluted isocratically with pure water. The product with a purity of >90% was collected into a collection tube, and centrifuged or freeze-dried to obtain pure hyaluronic acid disaccharide; the chimeric hexasaccharide and heptasaccharide were treated according to the above method to obtain hyaluronic acid trisaccharide and hyaluronic acid tetrasaccharide. The above products were characterized, and the results are shown in Table 4 and Figure 5 shown.

[0097] Table 4. Summary of preparation of hyaluronic acid oligosaccharides

[0098]

[0099] From Table 4 and Figure 5 It can be seen that the mass spectrometry molecular weight of each hyaluronic acid oligosaccharide is consistent with the expected molecular weight, indicating that the present invention successfully prepared hyaluronic acid disaccharide, hyaluronic acid trisaccharide, and hyaluronic acid tetrasaccharide, and achieved controllable polymerization degree of hyaluronic acid oligosaccharides during the preparation process.

[0100] Example 3. Preparation and purification of hyaluronic acid oligosaccharides with high synthesis efficiency and yield

[0101] Although the method provided in Example 2 successfully prepared hyaluronic acid oligosaccharides with different polymerization degrees, product mixing occurred in the process of preparing the chimeric heptasaccharide, and changing the reaction conditions could not effectively eliminate this phenomenon. This is because PmHAS has two domains with transferase functions. The UDP produced during the synthesis of the chimeric heptasaccharide undergoes an enzymatic reaction reverse reaction with the substrate hexasaccharide in the GlcNAc-T domain to generate a chimeric pentasaccharide and UDP-GlcNAc. The HPLC chromatogram of the products obtained by PmHAS and PmHAS (D247N / D249N) in the preparation of the chimeric heptasaccharide of hyaluronic acid and chondroitin is shown in FIG. Figure 6As shown in the literature (Deng JQ, Lu Z, Liu J, et al. Heparosan oligosaccharide synthesis using engineered single-function glycosyltransferases [J]. Catalysis Science & Technology, 2022, 12. DOI: 10.1039 / D1CY02061G.), the yield of chimeric oligosaccharides was reduced. In order to improve the synthesis efficiency and yield of hyaluronic acid oligosaccharides, the present invention further proposes the following preparation method.

[0102] 1. Preparation and purification of chimeric oligosaccharides with controllable degree of polymerization

[0103] 1) Using commercial GlcA-pNP as the starting acceptor substrate and UDP-GalNAc as the donor substrate, the chondroitin synthase AuCS mutant AuCS (D517N / D519N) and Mn 2+ The reaction was carried out in a water bath at 37°C for 6 hours under the catalysis of , and the progress of the reaction was detected by HPLC. After the reaction was complete, the chondroitin disaccharide was purified;

[0104] 2) Using chondroitin disaccharide as the acceptor substrate and UDP-GlcA as the donor substrate, the chondroitin synthase AuCS mutant AuCS (D237N / D239N) and Mn 2+ The reaction was carried out in a water bath at 37°C for 6 hours under the catalysis of , and the progress of the reaction was detected by HPLC. After the reaction was complete, the chondroitin trisaccharide was purified;

[0105] 3) Using chondroitin trisaccharide as the acceptor substrate and UDP-GlcNAc as the donor substrate, the hyaluronan synthase PmHAS mutant PmHAS (D527N / D529N) and Mn 2+ The reaction was carried out in a 37°C water bath under the catalysis of , and a sugar moiety was extended at the non-reducing end of the sugar chain to obtain a chimeric tetrasaccharide of hyaluronic acid and chondroitin. Then, the chimeric tetrasaccharide was used as the acceptor substrate and UDP-GlcA as the donor substrate. In the presence of the hyaluronan synthase PmHAS mutant PmHAS (D247N / D249N) and Mn 2+The reaction was carried out in a water bath at 37°C for 6 hours under the catalysis of , and a hyaluronic acid and chondroitin chimera pentasaccharide was obtained; UDP-GlcNAc and UDP-GlcA were used alternately as donor substrates, and hyaluronic acid and chondroitin chimera hexasaccharide, heptasaccharide, octasaccharide, nonasaccharide and decasaccharide were obtained under the alternating catalysis of PmHAS (D527N / D529N) and PmHAS (D247N / D249N); in this step, HPLC was required to detect the progress of the reaction for each extension of a sugar group, and the product was purified after the reaction was complete to ensure that the purity of the product was greater than 90% before the next extension step could be carried out;

[0106] The acceptor substrate, donor substrate, enzyme and Mn in step 1) to step 3) 2+ The reaction systems are all as shown in Table 1 in Example 2.

[0107] The HPLC detection method in step 1) to step 3) is as shown in Table 2 of Example 2, and the mobile phase flow rate is 0.5 mL / min.

[0108] The purification method described in step 1) to step 3) is:

[0109] use C18-AQ separation column, AKTA for purification. After the reaction progress reaches more than 99%, 0.1% TFA is used to terminate the reaction and the pH of the system is adjusted to about 3.0. Before purification, the reaction solution with adjusted pH is centrifuged (room temperature, 12000rpm, 10min) to take the supernatant, filter it with a 0.22μm filter membrane and load it onto a C18 separation column that has been equilibrated with double distilled water (0.1% TFA) in advance. The specific purification procedure is shown in Table 3 in Example 2, and the flow rate is 3ml / min.

[0110] The target product position was roughly determined based on the elution peak, and further confirmed by HPLC. The pH of the collected product solution was adjusted back to about 7.0, and the methanol in the reactant was removed by rotary evaporation. The solid obtained after rotary evaporation was redissolved in double distilled water, and the purity and yield were tested by HPLC. Figure 7 As shown, HPLC showed that the purity of each oligosaccharide prepared according to this scheme reached more than 95%. To further confirm the prepared oligosaccharides, electrospray ionization mass spectrometry (ESI-MS) analysis was performed. All samples for MS were prepared by dissolving in 50% methanol. The experiment was performed in negative ion mode with a spray voltage of 5 kV and a capillary temperature of 275°C. ESI-MS results are shown in Figure 8 .

[0111] Depend on Figure 7 and Figure 8 It can be seen that the molecular weight of each oligosaccharide is consistent with the calculated molecular weight, indicating that the present invention has successfully obtained a series of chimeric oligosaccharides with controllable polymerization degree, wherein the reducing end is chondroitin trisaccharide and the non-reducing end is hyaluronic acid chain.

[0112] 2. Preparation and purification of hyaluronic acid oligosaccharides with controllable degree of polymerization

[0113] 4) Dissolve 18 mg of the hyaluronic acid and chondroitin chimera pentasaccharide obtained in step 3) in double distilled water to a concentration of 5 mM, adjust the pH to neutral, add the purified chondroitin lyase ODV-E66 according to the volume of the oligosaccharide solution to a final concentration of 10 μg / mL, and lyse at 37°C for 0.5 to 12 h. Detect the progress of the reaction by HPLC (the same method as above). After the lysis is complete, remove the enzyme: boil the reaction solution for 5 min, then centrifuge at 12000 rpm for 10 min; adjust the pH of the system to about 3.0 for purification (the same method as above), and collect all components that absorb at 210 nm but not at 310 nm. The product was rotary evaporated to a volume of about 2 ml, and transferred to a Bio-Gel P-2 chromatography column (Φ2cm*80cm) balanced with double distilled water for chromatography purification, and isocratically eluted with pure water. The product with a purity of >90% was collected into a collection tube, and centrifuged or freeze-dried to obtain pure hyaluronic acid disaccharide;

[0114] 5) treating the hyaluronic acid and chondroitin chimera hexasaccharide, heptasaccharide, octasaccharide, nonasaccharide and decasaccharide according to the method described in step 4) to obtain hyaluronic acid trisaccharide, hyaluronic acid tetrasaccharide, hyaluronic acid pentasaccharide, hyaluronic acid hexasaccharide and hyaluronic acid heptasaccharide.

[0115] The above products were tested by HPLC to determine the purity, and then further confirmed by ESI-MS. The results are shown in Table 5 Fig. 9 and Fig.10 shown.

[0116] Table 5. Summary of preparation of hyaluronic acid oligosaccharides

[0117]

[0118] From Table 5, Fig. 9 and Fig.10 It can be seen that the mass spectrometry molecular weight of each hyaluronic acid oligosaccharide is consistent with the expected molecular weight, indicating that the present invention successfully prepared hyaluronic acid disaccharide, hyaluronic acid trisaccharide, hyaluronic acid tetrasaccharide, hyaluronic acid pentasaccharide, hyaluronic acid hexasaccharide and hyaluronic acid heptasaccharide, the polymerization degree of hyaluronic acid oligosaccharides prepared by this method is controllable, and theoretically, hyaluronic acid oligosaccharides with any polymerization degree can be successfully prepared according to this method, the purity of each hyaluronic acid oligosaccharide is more than 95%, the output of hyaluronic acid oligosaccharides reaches 6.04-28.9 mg, and the yield reaches more than 83%.

[0119] Example 4. Preparation and purification of hyaluronic acid oligosaccharide derivative 1 with controllable degree of polymerization

[0120] A hyaluronic acid oligosaccharide derivative, the structure of which is shown in the following formula:

[0121]

[0122] Wherein, in the structural formula, n=1, 2, 3; R1 is H or Na or K or NH4; the acetylamino group at the C2 position of acetylglucosamine in the natural structure hyaluronic acid is replaced by trifluoroacetylamino group; in the structural formula, when n=1, it is a hyaluronic acid disaccharide derivative; when n=2, it is a hyaluronic acid tetrasaccharide derivative; when n=3, it is a hyaluronic acid hexasaccharide derivative.

[0123] The preparation and purification scheme of the above hyaluronic acid oligosaccharide derivatives are as follows:

[0124] 1) obtaining chondroitin trisaccharide by referring to the method described in step 1) to step 2) in Example 3; then using chondroitin trisaccharide as an acceptor substrate and UDP-GlcNTFA as a donor substrate, in the presence of a hyaluronan synthase PmHAS mutant PmHAS (D527N / D529N) and Mn 2+ The reaction was carried out in a water bath at 37°C for 6 h under the catalysis of , and a UDP-GlcNTFA group was added to the non-reducing end of the sugar chain to obtain a chimeric tetrasaccharide. Then, the chimeric tetrasaccharide was used as an acceptor substrate and UDP-GlcA as a donor substrate in the presence of a hyaluronan synthase PmHAS mutant PmHAS (D247N / D249N) and Mn 2+ The reaction was carried out in a water bath at 37°C for 6 hours under the catalysis of Mn to obtain a chimeric pentasaccharide of a hyaluronic acid derivative and chondroitin. The chimeric heptasaccharide of a hyaluronic acid derivative and chondroitin was obtained after one round of two extensions, and a chimeric nonasaccharide of a hyaluronic acid derivative and chondroitin was obtained after two rounds of extension. The acceptor substrate, donor substrate, enzyme and Mn in the above reaction system 2+ The addition amount is shown in Table 1; the purification method is also consistent with the method described in Example 2, using C18-AQ separation column, AKTA for purification;

[0125] 2) Dissolve 12 mg of the hyaluronic acid derivative and chondroitin chimera pentasaccharide obtained in step 1) in double distilled water to a concentration of 5 mM, adjust the pH to neutral, add chondroitin lyase ODV-E66 to a final concentration of 10 μg / mL, and lyse at 37°C for 0.5 to 3 h. C18-AQ separation column, methanol gradient elution; secondary purification using Bio-Gel P-2 chromatography column (Φ2cm*80cm), pure water as mobile phase, collecting products with purity > 90% into a collection tube, centrifugation concentration or freeze drying, thus obtaining a pure hyaluronic acid disaccharide derivative with the structure of GlcA-GlcNTFA;

[0126] 3) The hyaluronic acid derivative and chondroitin chimera hepta-saccharide prepared in step 1) are cleaved by ODV-E66 according to the method described in step 2), and purified in two steps to obtain a pure hyaluronic acid tetrasaccharide derivative with a structure of GlcA-GlcNTFA-GlcA-GlcNTFA; similarly, the hyaluronic acid derivative and chondroitin chimera nonasaccharide prepared in step 1) can finally obtain a pure hyaluronic acid hexa-saccharide derivative with a structure of GlcA-GlcNTFA-GlcA-GlcNTFA-GlcA-GlcNTFA;

[0127] The hyaluronic acid oligosaccharide derivatives prepared in this example were identified, and the results were as follows: Fig.11 As shown. Fig.11 It can be seen that the present invention uses UDP-GlcNTFA as a donor substrate to successfully prepare hyaluronic acid disaccharide derivatives, hyaluronic acid tetrasaccharide derivatives and hyaluronic acid hexasaccharide derivatives.

[0128] Example 5. Preparation and purification of hyaluronic acid oligosaccharide derivative 2 with controllable degree of polymerization

[0129] A hyaluronic acid oligosaccharide derivative, the structure of which is shown in the following formula:

[0130]

[0131] Wherein, in the structural formula, n=1, 2, 3; R1 is H or Na or K or NH4; the acetylamino group at the C2 position of acetylglucosamine in the natural structure hyaluronic acid is replaced by azidoacetylamino group; in the structural formula, when n=1, it is a hyaluronic acid disaccharide derivative; when n=2, it is a hyaluronic acid tetrasaccharide derivative; when n=3, it is a hyaluronic acid hexasaccharide derivative.

[0132] The preparation and purification scheme of the above hyaluronic acid oligosaccharide derivatives are as follows:

[0133] 1) Chondroitin trisaccharide was obtained by referring to the method described in step 1) to step 2) of Example 3. Chondroitin trisaccharide was used as the acceptor substrate and UDP-GlcNAz was used as the donor substrate. In the presence of hyaluronan synthase PmHAS mutant PmHAS (D527N / D529N) and Mn 2+ The reaction was carried out in a water bath at 37°C for 6 h under the catalysis of , and a UDP-GlcNAz group was added to the non-reducing end of the sugar chain to obtain a chimeric tetrasaccharide. Then, the chimeric tetrasaccharide was used as an acceptor substrate and UDP-GlcA as a donor substrate in the presence of hyaluronan synthase PmHAS mutant PmHAS (D247N / D249N) and Mn 2+The reaction was carried out in a water bath at 37°C for 6 hours under the catalysis of to obtain a hyaluronic acid derivative and chondroitin chimera pentasaccharide; two extensions were considered as one round, and after one round, a hyaluronic acid derivative and chondroitin chimera hepta-saccharide was obtained, and after two rounds, a hyaluronic acid derivative and chondroitin chimera nonasaccharide was obtained; the purification of the chimera oligosaccharide was consistent with the purification scheme in step 1) of Example 4;

[0134] 2) The hyaluronic acid derivative and chondroitin chimeric oligosaccharide obtained in step 1) was cleaved by ODV-E66, and the cleavage product was The purification was carried out in two steps using a C18-AQ separation column and a Bio-Gel P-2 chromatography column (Φ2cm*80cm); the specific implementation scheme was consistent with step 2) to step 3) in Example 4.

[0135] The hyaluronic acid oligosaccharide derivatives prepared in this example were identified, and the results were as follows: Fig.12 As shown. Fig.12 It can be seen that the chimeric pentasaccharide can ultimately obtain a pure hyaluronic acid disaccharide derivative with a structure of GlcA-GlcNAz after cleavage; the chimeric heptasaccharide can ultimately obtain a pure hyaluronic acid tetrasaccharide derivative with a structure of GlcA-GlcNAz-GlcA-GlcNAz; the chimeric nonasaccharide can ultimately obtain a pure hyaluronic acid hexasaccharide derivative with a structure of GlcA-GlcNAz-GlcA-GlcNAz-GlcA-GlcNAz, that is, the present invention uses UDP-GlcNAz as a donor substrate to successfully prepare hyaluronic acid disaccharide derivatives, hyaluronic acid tetrasaccharide derivatives and hyaluronic acid hexasaccharide derivatives.

Claims

1. A hyaluronic acid oligosaccharide and its derivatives, whose structure is shown in the following formula (1) or formula (2): in, In the structural formula, m=1, 2, 3; n=1, 2, 3; R1 is H or Na or K or NH4; R2 is any one of NHCOCH3, NHCOCF3, NHCOCH2N3, NHCOCH2CH3, NHCOCH(CH3)2, OH, NH2, NHSO3H; formula (1) represents an oligosaccharide structure with an even degree of polymerization, and formula (2) represents an oligosaccharide structure with an odd degree of polymerization; When R2 is NHCOCH3, the structure shown is a hyaluronic acid oligosaccharide with a natural structure, and the degree of polymerization (dp) is 2m or 2n+1; when R2 is NHCOCF3, NHCOCH2N3, NHCOCH2CH3, NHCOCH(CH3)2, OH, NH2 or NHSO3H, the structure shown is a hyaluronic acid oligosaccharide derivative with a natural structure, and the degree of polymerization (dp) is 2m or 2n+1.

2. The method for preparing hyaluronic acid oligosaccharides and derivatives thereof according to claim 1, characterized in that: The following steps are involved: (1) GlcA-pNP was used as the initial acceptor substrate, UDP-GalNAc and UDP-GlcA were used as donor substrates in turn, and the chondroitin synthase AuCS and Mn 2+ The reaction was carried out under the catalysis of for 3 to 24 hours to obtain chondroitin trisaccharide; (2) The chondroitin trisaccharide obtained in step (1) is used as the acceptor substrate, and the sugar donor corresponding to the UDP-GlcNAc / oligosaccharide derivative and UDP-GlcA are alternately used as the donor substrate, and the hyaluronan synthase PmHAS and Mn 2+ The reaction is carried out under the catalysis of for 3 to 24 hours to obtain a series of chimeric oligosaccharides or their derivatives with a chondroitin trisaccharide at the reducing end and a hyaluronic acid chain at the non-reducing end; (3) The chimeric oligosaccharide or its derivative obtained in step (3) is separated and purified, eluted, concentrated and freeze-dried, and then dissolved in double distilled water, the pH value is adjusted to 7.5-8.5, and then chondroitin lyase ODV-E66 is added, and cleaved at 35-40° C. for 0.5-12 h. After enzyme inactivation, separation and purification, and elution, the components with absorption at 210 nm but no absorption at 310 nm are collected, and then concentrated and purified again to obtain hyaluronic acid oligosaccharide or its derivative.

3. The method for preparing hyaluronic acid oligosaccharides and derivatives thereof according to claim 2, characterized in that: In step (1), the chondroitin synthase AuCS is derived from Actinobacillus Ureae, the amino acid sequence of which is shown in SEQ ID NO.2, the nucleotide sequence of the encoding gene is shown in SEQ ID NO.1, and the final concentration of the added enzyme is 1 to 3 mg / mL.

4. The method for preparing hyaluronic acid oligosaccharides and derivatives thereof according to claim 2, characterized in that: In step (2), the sugar donor corresponding to the oligosaccharide derivative is UDP-GlcNTFA, UDP-GlcNAz, UDP-GlcNPr, UDP-Glc-i-Bu, UDP-Glc, UDP-GlcNH2 or UDP-GlcNS; The hyaluronan synthase PmHAS is derived from Pasteuella multocida, and its amino acid sequence is shown in SEQ ID NO.4, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.

3. The final added concentration is 1 to 3 mg / mL.

5. The method for preparing hyaluronic acid oligosaccharides and derivatives thereof according to claim 2, characterized in that: In step (3), the chondroitin lyase ODV-E66 is derived from Autographacalifornica nucleopolyhedrovirus, and its amino acid sequence is shown in SEQ ID NO.6, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.

5.

6. A method for preparing hyaluronic acid oligosaccharides and their derivatives according to claim 1 with improved synthesis efficiency and yield and controllable polymerization degree, characterized in that: The specific steps are as follows: 1) Using GlcA-pNP as the initial acceptor substrate and UDP-GalNAc as the donor substrate, the chondroitin synthase AuCS mutant AuCS (D517N / D519N) and Mn 2+ The reaction was carried out under the catalysis of for 3 to 24 hours to obtain chondroitin disaccharide; 2) Using chondroitin disaccharide as the acceptor substrate and UDP-GlcA as the donor substrate, the chondroitin synthase AuCS mutant AuCS (D237N / D239N) and Mn 2+ The reaction was carried out under the catalysis of for 3 to 24 hours to obtain chondroitin trisaccharide; 3) Using chondroitin trisaccharide as the acceptor substrate and the sugar donor corresponding to UDP-GlcNAc / oligosaccharide derivative as the donor substrate, the hyaluronan synthase PmHAS mutant PmHAS (D527N / D529N) and Mn 2+ The reaction was carried out under the catalysis of 3 to 24 hours, and a sugar group was extended at the non-reducing end of the sugar chain to obtain a chimeric tetrasaccharide or its derivative; the chimeric tetrasaccharide or its derivative was used as an acceptor substrate and UDP-GlcA was used as a donor substrate in the presence of hyaluronan synthase PmHAS mutant PmHAS (D247N / D249N) and Mn 2+ The reaction is carried out under the catalysis of 3 to 24 hours to extend a sugar group at the non-reducing end of the sugar chain; repeating this step, the hyaluronic acid and chondroitin chimera tetrasaccharide, pentasaccharide, hexasaccharide, heptasaccharide, octasaccharide, nonasaccharide, decasaccharide or their derivatives can be obtained respectively; 4) The hyaluronic acid and chondroitin chimera pentasaccharide or its derivative obtained in step 3) is purified, eluted, concentrated and freeze-dried, and then dissolved in double distilled water, and the pH value is adjusted to 7.5-8.5, and then chondroitin lyase ODV-E66 is added, and the lysis is carried out at 35-40° C. for 0.5-12 hours. After enzyme inactivation, separation and purification, and elution, the components with absorption at 210 nm but no absorption at 310 nm are collected, and then concentrated and purified again to obtain hyaluronic acid disaccharide or its derivative with natural structure; hyaluronic acid and chondroitin chimera hexasaccharide, heptasaccharide, octasaccharide, nonasaccharide, decasaccharide or its derivatives are treated in the same way to obtain hyaluronic acid trisaccharide or its derivatives, hyaluronic acid tetrasaccharide or its derivatives, hyaluronic acid pentasaccharide or its derivatives, hyaluronic acid hexasaccharide or its derivatives, and hyaluronic acid heptasaccharide or its derivatives.

7. The method for preparing hyaluronic acid oligosaccharides and derivatives thereof according to claim 6, characterized in that: In step 1), the chondroitin synthase AuCS mutant AuCS (D517N / D519N) is obtained by mutating the aspartic acid at positions 517 and 519 in the amino acid sequence of the chondroitin synthase AuCS to asparagine; In step 2), the chondroitin synthase AuCS mutant AuCS (D237N / D239N) is obtained by mutating the aspartic acid at positions 237 and 239 in the amino acid sequence of the chondroitin synthase AuCS to asparagine; In step 3), the hyaluronan synthase PmHAS mutant PmHAS (D527N / D529N) is obtained by mutating the aspartic acid at positions 527 and 529 in the amino acid sequence of the hyaluronan synthase PmHAS to asparagine; The hyaluronan synthase PmHAS mutant PmHAS (D247N / D249N) is obtained by mutating the aspartic acid at the 247th and 249th positions in the amino acid sequence of the hyaluronan synthase PmHAS to asparagine.

8. The method for preparing hyaluronic acid oligosaccharides and derivatives thereof according to claim 6, characterized in that: In step 1) to step 3), the molar ratio of the donor substrate to the acceptor substrate is 1.2:1; 2+ The final concentration is 10 mM.

9. The method for preparing hyaluronic acid oligosaccharides and derivatives thereof according to claim 6, characterized in that: The final concentrations of the chondroitin synthase AuCS mutant AuCS (D517N / D519N) in step 1), the chondroitin synthase AuCS mutant AuCS (D237N / D239N) in step 2), and the hyaluronan synthase PmHAS mutant PmHAS (D527N / D529N) and mutant PmHAS (D247N / D249N) in step 3) are all 1 to 3 mg / mL.

10. The method for preparing hyaluronic acid oligosaccharides and derivatives thereof according to claim 6, characterized in that: In step 4), the final concentration of the chondroitin lyase ODV-E66 added is 0.1-2.5 mg / mL.

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