A method for preparing unsaturated disaccharides of hyaluronic acid

Abstract

The present invention relates to a method for preparing unsaturated disaccharides of hyaluronic acid. The steps are as follows: (1) fermenting and culturing recombinant Kluyveromyces lactis containing a hyaluronic acid lyase gene for 72 to 84 hours to obtain a fermentation liquid A; (2) fermenting and culturing Streptococcus zooepidemicus for 12 to 18 hours to obtain a fermentation liquid B; mixing the fermentation liquid A and the fermentation liquid B evenly, and fermenting and culturing the mixed bacteria for 12 to 24 hours to obtain a fermentation liquid C; (3) first subjecting the fermentation liquid C to ceramic membrane filtration, ultrafiltration and nanofiltration membrane desalination and concentration, and then adsorption decolorization and spray drying to obtain unsaturated disaccharides of hyaluronic acid. The method ferments and cultures recombinant Kluyveromyces lactis containing a hyaluronic acid lyase gene and Streptococcus zooepidemicus mixed bacteria, and lyses the hyaluronic acid while the Streptococcus zooepidemicus produces hyaluronic acid to obtain unsaturated disaccharides of hyaluronic acid. The step of extracting and purifying hyaluronic acid lyase is omitted, which greatly saves costs, has low requirements for preparation conditions, and is suitable for large-scale production.

Description

Technical Field

[0001] The invention relates to a method for preparing unsaturated disaccharides of hyaluronic acid, belonging to the technical field of hyaluronic acid preparation. Background Art

[0002] Hyaluronic acid, also known as hyaluronic acid, is a non-branched high molecular glycosaminoglycan composed of N-acetylglucosamine and D-glucuronic acid with β-1,3 glycosidic bonds forming disaccharide units, and the disaccharide units are repeatedly connected with β-1,4 glycosidic bonds. The molecular weight of hyaluronic acid is generally 10 5 ~10 7 Dalton, according to the molecular weight, can be divided into high molecular, medium molecular, low molecular and oligomeric hyaluronic acid. Hyaluronic acid disaccharide is the smallest component unit. Due to the presence of unsaturated double bonds at the 4~5 glycosidic bonds of glucuronic acid, hyaluronic acid disaccharide is an unsaturated disaccharide. Related studies have found that unsaturated hyaluronic acid disaccharide has strong antioxidant properties and can significantly promote the proliferation of human umbilical vein endothelial cells and human corneal cells. It has a relatively broad application prospect in the fields of medicine and cosmetics. The preparation methods of medium, low molecular and oligomeric hyaluronic acid mainly include acid hydrolysis, alkaline hydrolysis, enzyme hydrolysis, etc.

[0003] Chinese patent literature CN110982862A discloses a method for preparing high-purity unsaturated hyaluronic acid disaccharides on a large scale, and unsaturated disaccharides are obtained by two-step enzymolysis, two-step purification and two-step drying. Specifically, high molecular weight hyaluronic acid or its salt is first enzymolyzed with hyaluronidase, and the resulting product is purified with a low organic solvent multiple to obtain a low molecular weight hyaluronic acid or its salt with higher purity, and then the low molecular weight hyaluronic acid or its salt is thoroughly enzymolyzed, and the resulting product is purified with a high organic solvent multiple, and the obtained hyaluronic acid unsaturated disaccharide purity can reach more than 99.5%. However, the preparation process of the method involves two enzymolysis, two purifications, one activated carbon decolorization, two dryings, and it is necessary to prepare hyaluronic acid lyase enzyme liquid before enzymolysis, and then use a high concentration of ethanol precipitation feed liquid to obtain unsaturated disaccharides, on the one hand, the organic solvent consumption is large, and alcohol precipitation is difficult, and the obtained unsaturated disaccharide molecular weight is 383, and the yield is very low; on the other hand, the material needs to be dried twice, and the energy consumption is high, that is, the process steps are cumbersome and complicated, and the cost of large-scale production preparation is high, and industrial production cannot be realized.

[0004] Chinese patent document CN101507733A discloses a nano-molecule hyaluronic acid, which uses hyaluronic acid with a molecular weight of tens of thousands to millions as raw material, and obtains nano-molecule hyaluronic acid through two-step reactions of acid degradation and enzyme degradation, with an average molecular weight below 1000 Daltons, and the content of hyaluronic acid monomer, dimer, trimer, and tetramer exceeds 90%. However, the average molecular weight of hyaluronic acid prepared by this method is 860-920, and no unsaturated disaccharide product of hyaluronic acid is obtained.

[0005] Chinese patent document CN104178539A discloses a method for large-scale preparation of small-molecule hyaluronic acid of specific molecular weight, using leech-derived hyaluronidase, water as the reaction medium, hydrolyzing high-molecular hyaluronic acid, and achieving efficient preparation of hyaluronic acid of specific molecular weight of 370000Da, 80000Da, 30000Da, 9000Da, 4000Da. However, the molecular weight of hyaluronic acid prepared by this method is at least 4000Da, and the end product obtained by enzymatic hydrolysis of hyaluronic acid by leech hyaluronidase is tetrasaccharide and hexasaccharide, and no unsaturated disaccharide of hyaluronic acid is obtained. Summary of the invention

[0006] In view of the shortcomings of the prior art, the present invention provides a method for preparing hyaluronic acid unsaturated disaccharide at low cost and high efficiency. The present invention adopts a one-step mixed bacteria fermentation method to obtain hyaluronic acid unsaturated disaccharide, uses a membrane technology concentration process to replace the traditional ethanol precipitation process, and uses a spray drying method to dry the material, and finally obtains high-purity hyaluronic acid unsaturated disaccharide.

[0007] The technical solution of the present invention is as follows:

[0008] A low-cost and high-efficiency method for preparing unsaturated hyaluronic acid disaccharides comprises the following steps:

[0009] (1) fermenting the recombinant Kluyveromyces lactis containing the hyaluronan lyase gene for 72 to 84 hours to obtain fermentation liquid A;

[0010] (2) fermenting the zooepidemic Streptococcus for 12 to 18 hours to obtain a fermentation liquid B; then evenly mixing the fermentation liquid A and the fermentation liquid B, and fermenting and culturing the mixed bacteria for 12 to 24 hours to obtain a fermentation liquid C containing unsaturated disaccharides of hyaluronic acid;

[0011] (3) The fermentation liquid C containing the hyaluronic acid unsaturated disaccharide is first filtered, ultrafiltered and desalted and concentrated, and then adsorbed, decolorized and spray-dried to obtain the hyaluronic acid unsaturated disaccharide.

[0012] According to the preferred embodiment of the present invention, in step (1), the nucleotide sequence of the hyaluronan lyase gene is shown in SEQ ID NO. 1, and the amino acid sequence is shown in SEQ ID NO. 2. The codons of the enzyme are optimized to make it more suitable for Kluyveromyces lactis.

[0013] Preferably, according to the present invention, in step (1), the method for constructing the recombinant Kluyveromyces lactis containing the hyaluronan lyase gene is as follows:

[0014] a. Artificially synthesize the hyaluronan lyase gene as shown in SEQ ID NO.1, and then connect the hyaluronan lyase gene to the pKLAC1 vector to obtain the recombinant plasmid pKLAC1-hyl; then transform the recombinant plasmid pKLAC1-hyl into Escherichia coli E. coli JM109, obtained recombinant E. coli E. coli JM109 / pKLAC1-hyl;

[0015] b. Recombinant Escherichia coli E. coli JM109 / pKLAC1-hyl was cultured overnight to extract the recombinant plasmid pKLAC1-hyl; the recombinant plasmid pKLAC1-hyl was linearized and digested with SacII restriction enzyme and then recovered to obtain the linearized recombinant plasmid pKLAC1-hyl;

[0016] c. Preparation of Kluyveromyces lactis Kluyveromyces lactis GG799 competent cells were used to transform the linearized recombinant plasmid pKLAC1-hyl into Kluyveromyces lactis by electroporation. Kluyveromyces lactis Among the GG799 competent cells, positive transformants were selected to obtain recombinant Kluyveromyces lactis containing the hyaluronan lyase gene.

[0017] According to the preferred embodiment of the present invention, in step (1), the fermentation culture temperature is 30°C, the fermentation time is 72h; the hyaluronan lyase activity in the fermentation liquid A is greater than or equal to 10 5 U / mL.

[0018] Preferably, according to the present invention, in step (2), the fermentation culture temperature of the zooepidemic streptococcus is 37° C., and the fermentation time is 16 h; the mixed bacteria fermentation culture temperature is 30° C., and the fermentation time is 20 h.

[0019] Preferably according to the present invention, in step (2), the volume ratio of the fermentation broth A to the fermentation broth B is 1:(3-5).

[0020] Preferably, according to the present invention, in step (2), the content of unsaturated disaccharides of hyaluronic acid in the fermentation broth C is greater than or equal to 30 g / L.

[0021] Preferably, in step (3), the filtration is to filter the fermentation broth C containing hyaluronic acid unsaturated disaccharide using a ceramic membrane with a pore size of 0.2-0.5 μm to remove bacterial cells and insoluble matter in the fermentation broth C.

[0022] Preferably, according to the present invention, in step (3), the ultrafiltration is to remove macromolecular protein substances by ultrafiltration with a molecular weight cutoff of 3000-5000Da.

[0023] Preferably, in step (3), the desalting and concentrating step is to concentrate the material using a nanofiltration membrane with a pore size of 1-2 nm, and the concentrated liquid has a solid content of 20-30%.

[0024] Preferably, in step (3), the adsorption decolorization is performed by adding 5-10‰ (m / V) of activated carbon to the concentrated solution, starting stirring, controlling the temperature to 70-85°C, maintaining decolorization for 30-60 minutes, and using a filter press for clarification filtration to obtain a clear liquid; the transmittance of the clear liquid at 600nm is not less than 99.5%, and the absorbance value at 280nm is not higher than 0.1.

[0025] Preferably, in step (3), the spray drying is carried out using a spray drying tower under the following conditions: an inlet air temperature of 180-220°C, an outlet air temperature of 60-80°C, and an atomizer speed of 15000-30000 r / min.

[0026] The beneficial effects of the present invention are:

[0027] 1. The method for preparing unsaturated disaccharides of hyaluronic acid provided by the present invention adopts a mixed bacteria fermentation process, wherein recombinant Kluyveromyces lactis containing a hyaluronate lyase gene and Streptococcus zooepidemicus are mixed and fermented, and hyaluronic acid is cracked while Streptococcus zooepidemicus produces hyaluronic acid to obtain unsaturated disaccharides of hyaluronic acid. The step of extracting and purifying hyaluronate lyase from the recombinant strain is omitted, which greatly saves costs compared to purchasing hyaluronate lyase, and the preparation conditions are low, which is suitable for industrialized large-scale production.

[0028] 2. After fermentation for 72 hours, the activity unit of hyaluronan lyase in the fermentation broth of the recombinant Kluyveromyces lactis containing the hyaluronan lyase gene constructed by the present invention is not less than 10 5 U / mL, so that the yield of hyaluronic acid unsaturated disaccharide prepared by the method of the present invention is significantly improved, reaching 35g / L.

[0029] 3. In the extraction and purification process of the unsaturated disaccharide of hyaluronic acid of the present invention, the membrane technology (ceramic membrane, ultrafiltration membrane, nanofiltration membrane) concentration process is used to replace the traditional ethanol precipitation process, thereby avoiding the use of a large amount of alcohol, reducing energy consumption, improving the extraction yield of the target product, and greatly reducing the cost, which is suitable for large-scale industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 The electrophoresis diagram of the hyaluronan lyase target gene in Example 1;

[0031] In the figure, lanes 1, 2 and 3 are all target genes.

[0032] Figure 2 This is a curve chart of the hyaluronic acid disaccharide content during the fermentation process of Example 2.

[0033] Figure 3 This is a curve chart of the hyaluronic acid content during the fermentation process of Comparative Example 1. DETAILED DESCRIPTION

[0034] In order to better understand the essence of the present invention, the present invention is further described below in conjunction with the embodiments, but they should not be regarded as limiting the present invention.

[0035] The Streptococcus zooepidemicus described in the present invention is a high-yielding hyaluronic acid strain obtained by compound mutagenesis using Streptococcus equi subspecies zooepidemicus ATCC39920 as the starting strain. For the specific compound mutagenesis method, please refer to the article "Liu Yiran. Mutagenesis and Optimization of Fermentation Conditions for Hyaluronic Acid Producing Bacteria [D]. Zhengzhou University, 2006".

[0036] Kluyveromyces lactis used in the present invention Kluyveromyces lactis GG799, Streptococcus equine subspecies zooepidemicus ATCC39920 and Escherichia coli JM109 are all common commercially available strains that can be purchased from microbial collection centers or strain sales companies.

[0037] Example 1

[0038] The recombinant Kluyveromyces lactis containing the hyaluronate lyase gene is constructed as follows:

[0039] a. Artificially synthesize the hyaluronan lyase gene as shown in SEQ ID NO.1, and then connect the hyaluronan lyase gene to the pKLAC1 vector to obtain the recombinant plasmid pKLAC1-hyl; then transform the recombinant plasmid pKLAC1-hyl into Escherichia coli E. coli JM109, obtained recombinant E. coli E. coli JM109 / pKLAC1-hyl;

[0040] b. Recombinant Escherichia coli E. coli JM109 / pKLAC1-hyl was cultured overnight, and the recombinant plasmid pKLAC1-hyl was extracted using a plasmid miniprep kit; the recombinant plasmid pKLAC1-hyl was linearized using the SacII restriction enzyme, and recovered using a DNA purification kit to obtain the linearized recombinant plasmid pKLAC1-hyl;

[0041] c. Prepare Kluyveromyces lactis GG799 competent cells using lithium acetate: DTT = 1:9 (volume ratio), and transform the linearized recombinant plasmid pKLAC1-hyl into Kluyveromyces lactis by electric pulse method (voltage 2500V) Kluyveromyces lactis GG799 competent cells; the yeast cells after electroporation were immediately added to 1 mL of pre-cooled 1 mol / L sorbitol solution, and the yeast solution after electroporation was collected and treated in a 30 ℃ water bath for 2 h. The bacteria were collected by centrifugation, resuspended in 1 mL of sterile water, and evenly spread on yeast basic carbon source medium (YCB) containing 5 mmol / L acetamide. The cells were cultured in a 30 ℃ incubator for 3-5 days, and the positive heavy transformants were selected. The yeast genome was extracted and the positive transformants were identified by PCR amplification to obtain the recombinant Kluyveromyces lactis containing the hyaluronan lyase gene. Kluyveromyces lactis GG799 / pKLAC1-hyl. The identification results are as follows Figure 1 As shown by Figure 1 It can be seen that the target band appears in the gel electrophoresis diagram and the band is single, indicating that the hyaluronan lyase gene is successfully recombinantly expressed.

[0042] Example 2

[0043] A low-cost and high-efficiency method for preparing unsaturated hyaluronic acid disaccharides comprises the following steps:

[0044] (1) The recombinant Kluyveromyces lactis containing the hyaluronan lyase gene prepared in Example 1 was Kluyveromyces lactis GG799 / pKLAC1-hyl was fermented at 30°C for 72 h to obtain fermentation liquid A;

[0045] (2) Fermenting the zooepidemic Streptococcus at 37° C. for 16 hours to obtain a fermentation liquid B; mixing the fermentation liquid A and the fermentation liquid B at a volume ratio of 1:3, and fermenting and culturing the mixed bacteria at 30° C. for 20 hours to obtain a fermentation liquid C containing hyaluronic acid unsaturated disaccharide;

[0046] (3) The fermentation liquid C containing hyaluronic acid unsaturated disaccharide was first filtered through a ceramic membrane with a pore size of 0.2 μm to remove bacterial cells and insoluble matter to obtain a clarified fermentation liquid; then, an ultrafiltration membrane with a molecular weight cutoff of 5000 Da was used to intercept and remove large molecular weight protein substances, and the permeate was collected to obtain a protein-free liquid; then, the material was concentrated using a nanofiltration membrane with a pore size of 1 nm, and the concentrated liquid was concentrated to a solid content of 25% to obtain a concentrated liquid; then, 10‰ (m / V, g / L) of activated carbon was added to the concentrated liquid, stirring was started, the temperature was controlled to 75°C, and decolorization was maintained for 30 minutes, and a filter press was used for clarification and filtration to obtain a clear liquid; finally, the clear liquid was pumped into a spray drying tower, and spray-dried under the conditions of an inlet air temperature of 200°C, an outlet air temperature of 70°C, and an atomizer speed of 20,000 r / min to obtain hyaluronic acid unsaturated disaccharide.

[0047] During the fermentation process in step (2) of this embodiment, the content of unsaturated disaccharides of hyaluronic acid was measured by high performance liquid chromatography every two hours. The results are as follows: Figure 1 As shown. Figure 1 It can be seen that after 20 hours of mixed fermentation, the fermentation yield of hyaluronic acid unsaturated disaccharide in fermentation liquid C reaches 35 g / L. After purification in step (3), the purity of hyaluronic acid unsaturated disaccharide can reach more than 95%.

[0048] Example 3

[0049] A low-cost and high-efficiency method for preparing unsaturated hyaluronic acid disaccharides comprises the following steps:

[0050] (1) The recombinant Kluyveromyces lactis containing the hyaluronan lyase gene prepared in Example 1 was Kluyveromyces lactis GG799 / pKLAC1-hyl was fermented at 30°C for 84 h to obtain fermentation liquid A;

[0051] (2) Fermenting the zooepidemic Streptococcus at 37° C. for 18 hours to obtain a fermentation liquid B; mixing the fermentation liquid A and the fermentation liquid B at a volume ratio of 1:5, and fermenting and culturing the mixed bacteria at 30° C. for 24 hours to obtain a fermentation liquid C containing hyaluronic acid unsaturated disaccharide;

[0052] (3) The fermentation liquid C containing hyaluronic acid unsaturated disaccharide was first filtered through a ceramic membrane with a pore size of 0.5 μm to remove bacterial cells and insoluble matter to obtain a clarified fermentation liquid; then, an ultrafiltration membrane with a molecular weight cutoff of 3000 Da was used to intercept and remove large molecular weight protein substances, and the permeate was collected to obtain a protein-free liquid; then, the material was concentrated using a nanofiltration membrane with a pore size of 1 nm, and the concentrated liquid was concentrated to a solid content of 30% to obtain a concentrated liquid; then, 5‰ (m / V, g / L) of activated carbon was added to the concentrated liquid, stirring was started, the temperature was controlled to 75°C, and decolorization was maintained for 30 minutes, and a filter press was used for clarification and filtration to obtain a clear liquid; finally, the clear liquid was pumped into a spray drying tower, and spray-dried under the conditions of an inlet air temperature of 200°C, an outlet air temperature of 70°C, and an atomizer speed of 20,000 r / min to obtain hyaluronic acid unsaturated disaccharide.

[0053] Comparative Example 1

[0054] The Streptococcus zooepidemicus was fermented and cultured at 37°C for 36 hours. The hyaluronic acid content was also measured by high performance liquid chromatography every two hours during the fermentation process. The results were as follows: Figure 2 As shown. Figure 2 It can be seen that the fermentation yield of hyaluronic acid in the fermentation broth is 12 g / L.

[0055] Comparative Example 2

[0056] The Streptococcus zooepidemicus was fermented and cultured at 37°C for 36 hours, and 5 volumes of recombinant Kluyveromyces lactis fermentation broth containing the hyaluronan lyase gene were added, and the yield of hyaluronic acid unsaturated disaccharide was 15 g / L.

[0057] By comparing Example 1 with Comparative Example 1, it can be seen that the fermentation culture of Streptococcus zooepidemicus can only produce hyaluronic acid, but cannot produce hyaluronic acid unsaturated disaccharides. By comparing Example 1 with Comparative Example 2, it can be seen that after the fermentation culture of Streptococcus zooepidemicus, the recombinant Kluyveromyces lactis fermentation liquid containing the hyaluronan lyase gene is added, the yield of hyaluronic acid unsaturated disaccharides is only 15 g / L, which is far less than 35 g / L in Example 2.

Claims

1. A method for preparing unsaturated disaccharides of hyaluronic acid, characterized in that: The steps include: (1) fermenting the recombinant Kluyveromyces lactis containing the hyaluronan lyase gene for 72 to 84 hours to obtain fermentation liquid A; The construction method of the recombinant Kluyveromyces lactis containing the hyaluronan lyase gene is as follows: Artificially synthesize the hyaluronan lyase gene as shown in SEQ ID NO.1, and then connect the hyaluronan lyase gene to the pKLAC1 vector to obtain the recombinant plasmid pKLAC1-hyl; then transform the recombinant plasmid pKLAC1-hyl into Escherichia coli E. coli JM109, obtained recombinant E. coli E. coli JM109 / pKLAC1-hyl; Recombinant Escherichia coli E. coli JM109 / pKLAC1-hyl was cultured overnight to extract the recombinant plasmid pKLAC1-hyl; the recombinant plasmid pKLAC1-hyl was linearized and digested with SacII restriction enzyme and then recovered to obtain the linearized recombinant plasmid pKLAC1-hyl; Preparation of Kluyveromyces lactis Kluyveromyces lactis GG799 competent cells were used to transform the linearized recombinant plasmid pKLAC1-hyl into Kluyveromyces lactis by electroporation. Kluyveromyces lactis In GG799 competent cells, positive transformants were selected to obtain recombinant Kluyveromyces lactis containing the hyaluronan lyase gene; (2) fermenting the zooepidemic Streptococcus for 12 to 18 hours to obtain a fermentation liquid B; then evenly mixing the fermentation liquid A and the fermentation liquid B, and fermenting and culturing the mixed bacteria for 12 to 24 hours to obtain a fermentation liquid C containing unsaturated disaccharides of hyaluronic acid; Wherein, the zooepidemic streptococcus is equine streptococcus zooepidemicus subspecies ATCC39920; (3) The fermentation liquid C containing the hyaluronic acid unsaturated disaccharide is first filtered, ultrafiltered and desalted and concentrated, and then adsorbed, decolorized and spray-dried to obtain the hyaluronic acid unsaturated disaccharide.

2. The method for preparing unsaturated disaccharides of hyaluronic acid according to claim 1, characterized in that: In step (1), the fermentation temperature is 30°C and the fermentation time is 72h; the hyaluronan lyase activity in the fermentation liquid A is greater than or equal to 10 5 U / mL.

3. The method for preparing unsaturated disaccharides of hyaluronic acid according to claim 1, characterized in that: In step (2), the fermentation temperature of the Streptococcus zooepidemicus is 37° C., and the fermentation time is 16 h; the mixed bacteria fermentation temperature is 30° C., and the fermentation time is 20 h.

4. The method for preparing unsaturated disaccharides of hyaluronic acid according to claim 1, characterized in that: In step (2), the volume ratio of the fermentation broth A to the fermentation broth B is 1:(3-5).

5. The method for preparing unsaturated disaccharides of hyaluronic acid according to claim 1, characterized in that: In step (3), the filtration is to filter the fermentation broth C containing hyaluronic acid unsaturated disaccharide using a ceramic membrane with a pore size of 0.2-0.5 μm to remove bacterial cells and insoluble matter in the fermentation broth C; the ultrafiltration is to remove macromolecular protein substances using an ultrafiltration membrane with a molecular weight cutoff of 3000-5000 Da.

6. The method for preparing unsaturated disaccharides of hyaluronic acid according to claim 1, characterized in that: In step (3), the desalting and concentrating step is to concentrate the material using a nanofiltration membrane with a pore size of 1-2 nm, and the concentrated liquid has a solid content of 20-30%.

7. The method for preparing unsaturated disaccharides of hyaluronic acid according to claim 1, characterized in that: In step (3), the adsorption decolorization is to add 5-10‰ activated carbon to the concentrated solution, start stirring, control the temperature to 70-85°C, maintain decolorization for 30-60 minutes, and use a filter press to clarify and filter to obtain a clear liquid; the transmittance of the clear liquid at 600nm is not less than 99.5%, and the absorbance value at 280nm is not higher than 0.

1.

8. The method for preparing unsaturated disaccharides of hyaluronic acid according to claim 1, characterized in that: In step (3), the spray drying is carried out using a spray drying tower, and the conditions are that the inlet air temperature is 180-220°C, the outlet air temperature is 60-80°C, and the atomizer speed is 15000-30000 r / min.

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