Preparation of novel microbial source N-sulfoheparin precursor polysaccharide

Abstract

The invention relates to the technical field of biological medicines, in particular to a preparation method of novel microbial source N-sulfoheparin precursor polysaccharide, which comprises the following specific steps: fermenting an EcN8 strain to obtain a heparin precursor fermentation liquor, centrifuging, carrying out low-temperature alcohol precipitation, dissolving and precipitating to obtain a crude heparin precursor solution, carrying out N-deacetylation reaction, and purifying to obtain the novel microbial source N-sulfoheparin precursor polysaccharide. The preparation method comprises the following steps: precipitating protein impurities by adjusting the pH value of a reaction system, centrifuging to remove the protein impurities, dialyzing supernatant to remove small molecular impurities, adding PAPS and N-sulfotransferase to complete an N-sulfonylation reaction, and carrying out purification, dialysis desalination and vacuum freeze drying on a product to obtain the N-sulfoheparin precursor. Toxic organic reagents are not used in purification of the heparin precursor prepared through microbial fermentation, N-deacetylation structure modification of the heparin precursor is achieved in the purification and impurity removal process, and after enzymatic N-sulfo modification is conducted, fine purification is further conducted to obtain the high-purity N-sulfo heparin precursor polysaccharide. And efficient purification and structural modification of the heparin precursor are realized.

Description

Technical Field

[0001] The invention relates to the technical field of biomedicine, and in particular to the preparation of a novel microbial-derived N-sulfoheparin precursor polysaccharide. Background Art

[0002] Heparin precursor (Heparosan) is a macromolecular anionic glycosaminoglycan composed of repeated disaccharide units of [→4)β-D-glucuronic acid (GlcA)(1→4)N-acetyl-α-D-glucosamine (GlcNAc)(1→]n. Since heparin precursor has a high structural similarity with the commonly used clinical anticoagulant drug heparin (Heparin), the structure of heparin precursor can be modified by chemical enzymatic methods, biosynthesis methods, etc. to convert it into a heparin-like polysaccharide. Therefore, microbial heparin precursor can be used as a raw material for synthetic bioengineering heparin to replace traditional animal-derived heparin, avoiding potential product contamination and adulteration risks.

[0003] At present, heparin precursors are mainly produced by microbial biosynthesis and fermentation. For example, the yield of heparin precursors can reach 15 g / L by batch fermentation using E. coli K5 with glucose as the carbon source under sufficient oxygen conditions. However, since E. coli K5 is a human pathogen and can easily cause urinary tract infections, more and more studies are using non-pathogenic bacteria to achieve the biosynthesis of heparin precursors. At present, through different methods of bacterial strain modification, heparosan has been biosynthesized in strains such as Bacillus subtilis, Bacillus megaterium, and Corynebacterium glutamicum, and fermentation production has been achieved. The fermentation broth of heparin precursors biosynthesized by microorganisms usually contains a variety of impurities, which affect the purity of the final product. Among them, protein is the most important contaminant in the purification process. At present, in the process of extraction and purification of microbial fermentation heparin precursors and separation and purification of polysaccharides from other sources, commonly used methods for removing proteins include trichloroacetic acid (TCA) method, hydrochloric acid method, Sevage method and methods using other organic solvents to extract and separate proteins; except for the hydrochloric acid method, these methods use toxic organic reagents such as trichloroacetic acid, chloroform, phenol, sodium dodecyl sulfate (SDS), isopropanol, etc., which will have adverse effects on the human body and the environment; although the hydrochloric acid method does not use toxic organic reagents, is simple to operate, and has a high protein removal rate, the polysaccharide recovery rate is low, and the overly acidic purification environment will also affect the structure of the polysaccharide.

[0004] Therefore, in view of the fact that the existing heparin precursor purification process derived from microbial fermentation must use a large amount of toxic organic reagents to achieve product extraction and impurity separation, and the low product recovery rate caused by the traditional fermentation product separation and purification method, it is urgent to develop a method that can remove protein impurities without using toxic organic reagents, and can also modify the structure of heparin precursors during the purification process, so as to simplify the process steps and reduce the polysaccharide loss during the purification process. Summary of the invention

[0005] In view of the above-mentioned shortcomings of the prior art, the present invention provides a novel preparation method of microbial-derived N-sulfoheparin precursor polysaccharide, which can effectively solve the problems mentioned in the prior art.

[0006] To achieve the above objectives, the present invention is implemented through the following technical solutions:

[0007] The present invention provides a novel preparation method of microbial-derived N-sulfoheparin precursor polysaccharide, comprising the following steps:

[0008] The fermentation broth rich in heparin precursor is prepared by fermentation with Escherichia coli EcN8 strain;

[0009] The heparin precursor fermentation supernatant prepared by microbial fermentation is obtained by centrifugation, mixed with anhydrous ethanol 3 times the volume of the supernatant, and subjected to low-temperature alcohol precipitation to obtain a crude heparin precursor polysaccharide precipitate;

[0010] After the crude heparin precursor polysaccharide precipitate is fully dissolved with ultrapure water, a crude heparin precursor solution is obtained, and then urea and sodium hydroxide are added to the crude heparin precursor solution, and the solution is placed in a 60° C. water bath for 18 h to perform an N-deacetylation reaction;

[0011] After the reaction is finished, the reaction solution is obtained, and after cooling, the pH value of the reaction solution is adjusted to be greater than or equal to the isoelectric point of the impurity protein in the reaction solution to precipitate the impurity protein;

[0012] The reaction solution was allowed to stand at 4°C for 6-8 hours, and then centrifuged to obtain a supernatant, and the supernatant was placed in a dialysis bag for dialysis to remove small molecular impurities, thereby obtaining an N-deacetylheparin precursor reaction solution;

[0013] After the polysaccharide concentration of the dialyzed N-deacetylheparin precursor reaction solution is determined, reagents are added thereto to prepare an N-sulfonation reaction system environment, and then PAPS and N-sulfotransferase are added according to the amount of polysaccharide involved in the reaction. The reaction is carried out at 25° C. for 24 hours to complete the N-sulfonation modification and obtain the final product;

[0014] The final product was purified by DEAE anion exchange resin column, dialyzed for desalting and vacuum freeze-dried to obtain a high-purity N-sulfoheparin precursor.

[0015] The specific technical solution steps are as follows:

[0016] (1) The microorganism described in the present invention is a non-pathogenic Escherichia coli EcN8, which is based on Escherichia coli Nissle1917, and uses antibiotic-driven chromosome evolution to amplify the kps gene site for synthesizing and exporting heparin precursors, thereby achieving genetic engineering transformation of multi-copy kps gene sites and becoming a high-yield heparin precursor strain; fermentation is performed using a 5L fermenter in a batch culture stage and a fed-batch culture stage to obtain a fermentation product; when the OD of the seed culture with a total volume of 220mL is 600 When the value reaches 2.0, it is added to a 5L fermenter containing 2.2L sterile M9VB1 medium to start fermentation; the M9VB1 medium used in the batch culture stage consists of 8.0g / L glucose, 6.8g / LNa2HPO4, 3.0g / LKH2PO4, 1.0g / LNH4Cl, 0.5g / LNaCl, 300mg / L thiamine, 2.0mmol / LMgSO4·7H2O and 0.1mmol / LCa Cl2·2H2O, the culture environment was maintained at 37°C, pH7.0, and the kanamycin concentration was 50 mg / L; under automatic monitoring, the pH value was adjusted by adding 5 mol / L hydrochloric acid or 25% ammonia water, and the fermentation tank stirring speed was set to 700 rpm; when the dissolved oxygen value first dropped and then rose sharply, it entered the fed-batch culture stage, at which time the glucose in the culture had been exhausted, and the feed composition of the fed-batch culture stage included 400 g / L glucose, 20.0 g / L MgSO4·7H2O and 250 mg / L thiamine; a feed pump was used to add the feed solution to the fermentation tank, and the growth rate μ was maintained at 0.10 h -1 ; After 40 hours of fermentation, the heparin precursor fermentation liquid was obtained for treatment;

[0017] (2) centrifuging the heparin precursor fermentation broth obtained in step (1) at 4° C. at a speed of not less than 6000 rpm for 40 min, and repeating the centrifugation 2-3 times until no obvious bacterial precipitation occurs, thereby obtaining a fermentation supernatant;

[0018] (3) The fermentation supernatant obtained in step (2) is fully mixed with 3 volumes of anhydrous ethanol, and allowed to stand at -20°C for 8-12 hours to precipitate heparin precursor polysaccharide; the mixture is then centrifuged at 8000 rpm for 35 minutes at 4°C to recover the crude heparin precursor polysaccharide precipitate; the polysaccharide precipitate is dissolved in ultrapure water of the same volume as the fermentation supernatant used initially to obtain a crude heparin precursor polysaccharide solution;

[0019] (4) taking an appropriate volume of the crude heparin precursor polysaccharide solution obtained in step (3), first adding urea to the crude heparin precursor polysaccharide solution until the urea concentration in the solution reaches 2.0-10.0 mol / L, and after the urea is completely dissolved, adding sodium hydroxide (other strong bases such as potassium hydroxide can also achieve similar effects) solid until the sodium hydroxide concentration in the solution reaches 0.5-3.0 mol / L; preferably, the urea concentration is 6.0 mol / L and the sodium hydroxide concentration is 2.0 mol / L; the reaction system is reacted at 60° C. for 18 h to complete the N-deacetylation reaction of the heparin precursor and prepare an N-deacetylheparin precursor reaction liquid. After the reaction is completed, the N-deacetylheparin precursor reaction liquid is rapidly cooled;

[0020] (5) Using concentrated hydrochloric acid (dilute hydrochloric acid, sulfuric acid, acetic acid, etc. can also achieve similar effects) to adjust the pH value of the cooled N-deacetylheparan precursor reaction solution to 6.0-9.0; preferably, using concentrated hydrochloric acid to adjust the pH value of the reaction solution to 8.0; the reaction solution is divided into centrifuge tubes and allowed to stand at 4°C for 6-8 hours to allow protein impurities to settle; then centrifuged at 10,000 rpm for 20 minutes at 4°C to remove precipitated protein impurities and solids, and the obtained N-deacetylheparan precursor supernatant is placed in a dialysis bag with a molecular weight cutoff of 3.5 kDa, and dialyzed in ultrapure water to remove small molecule impurities;

[0021] (6) taking the dialyzed N-deacetylheparin precursor reaction liquid obtained in step (5), and quantifying the total amount of polysaccharides in the dialyzed N-deacetylheparin precursor reaction liquid; taking the quantified N-deacetylheparin precursor reaction liquid, adding MES thereto until its concentration reaches 50 mmol / L, adding MnCl2 and MgCl2 until their concentrations both reach 1 mmol / L, adding 1% (v / v) Triton X-100, adjusting the pH value to 7.0, and preparing an N-sulfonation reaction environment; according to the quantified total amount of N-deacetylheparin precursor polysaccharides, adding PAPS and N-sulfotransferase at a ratio of 1:1.2 to the mass ratio of polysaccharide to PAPS and 2:1 to the mass ratio of polysaccharide to N-sulfotransferase; incubating the reaction system at 25° C. for 24 h to complete the N-sulfonation reaction, converting the N-deacetylheparin precursor into the N-sulfoheparin precursor, and obtaining the N-sulfoheparin precursor reaction liquid;

[0022] (7) The N-sulfoheparin precursor reaction liquid after the reaction in step (6) was placed in a boiling water bath for 3 minutes to terminate the reaction, cooled, and centrifuged at 8500 rpm for 25 minutes at 4°C; after centrifugation, the N-sulfoheparin precursor supernatant was taken and mixed with two volumes of UPAS buffer (50 mmol / L NaAcO, 150 mmol / L NaCl, 1 mmol / L EDTA, 6 mol / L urea, 0.01% [v / v] Triton X-100, pH 5.5), and incubated at 4°C for 30 minutes; the mixed reaction liquid was loaded into a DEAE anion exchange resin column equilibrated with 10 column volumes of UPAS buffer using a peristaltic pump at a flow rate of 1.0 mL / min; then 3 column volumes of UPAS buffer and 5 column volumes of NaCl buffer I (50 mmol / L NaAcO, 150 mmol / L NaCl, 0.001% [v / v] Triton X-100) were used in sequence. X-100, pH 5.5), and finally, NaCl buffer II (50 mmol / L NaAcO, 1 mol / L NaCl, 0.001% [v / v] Triton X-100, pH 5.5) was used to elute the N-sulfoheparin precursor from the column at a flow rate of 3.0 mL / min.

[0023] (8) The eluate after elution in step (7) is transferred to a dialysis bag with a molecular weight cutoff of 3.5 kDa, and dialyzed in ultrapure water for desalination. The dialyzed solution is subjected to vacuum freeze drying to obtain a high-purity N-sulfoheparin precursor.

[0024] Compared with the known prior art, the technical solution provided by the present invention has the following beneficial effects:

[0025] (1) The method used in the present invention does not require the use of a large amount of toxic and harmful organic reagents, and the reaction process is mild, without the risk of causing harm to the environment and personnel; the waste water after the reaction contains only urea and inorganic salts, without toxic and harmful organic compounds, and the process waste liquid is easy to handle and is not likely to cause damage to the environment and human body;

[0026] (2) The present invention simplifies the process flow, ensures the purity of the purified product, effectively removes protein and small molecule impurities, and realizes the structural modification of the heparin precursor. By reducing the number of purification times and changing the process method, the loss caused in the processing process is effectively reduced, and the recovery rate of the final product is improved;

[0027] (3) The present invention effectively affects and destroys the non-covalent interaction between anionic polysaccharides and impurity proteins by controlling the urea concentration and adjusting the pH value after the deacetylation reaction is completed, and achieves a better heparin-like polysaccharide recovery effect by exploring the optimal process parameters through experiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the prior art descriptions are briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention, and for ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0029] Figure 1 The heparin-like polysaccharide rate and protein residual rate in the supernatant obtained by centrifugation after the deacetylation reaction is completed;

[0030] Figure 2 is the proportion of each component in the heparin precursor deacetylation reaction solution after dialysis;

[0031] Figure 3 UV-visible analysis of impurities in the heparin precursor deacetylation reaction solution after dialysis;

[0032] Figure 4 is the recovery rate of heparin-like polysaccharide in each purification stage;

[0033] Figure 5 This is a comparison of the H NMR spectra of the heparin precursor and the product after the N-deacetylation reaction;

[0034] Figure 6 is the mass spectrum of the N-sulfonated product;

[0035] Figure 7 is the S / N ratio of heparin precursor, N-deacetylheparan precursor, and N-sulfoheparin precursor;

[0036] Figure 8 This is a graph showing the comparison of the molecular weights of the polysaccharide gel electrophoresis of N-deacetylheparan precursor and N-sulfoheparan precursor;

[0037] Fig. 9 This is a molecular weight distribution curve of N-sulfoheparin precursor determined by gel permeation chromatography;

[0038] Fig.10 The effect of adding urea on the polysaccharide recovery rate and protein residual rate in a 2 mol / L sodium hydroxide reaction system was shown. The final pH value after the reaction was 7.0. The total amount of polysaccharides and total amount of protein in the supernatant was determined by centrifugation.

[0039] Fig.11 To determine the polysaccharide recovery rate and protein residual rate in the supernatant after adjusting the reaction system of 2 mol / L sodium hydroxide and 6 mol / L urea to different pH values;

[0040] Fig.12The polysaccharide recovery rate and protein residual rate in the supernatant obtained by centrifugation were analyzed by adding different concentrations of sodium hydroxide to a reaction system containing 6 mol / L urea and adjusting the final pH value to 8.0;

[0041] Fig.13 Polysaccharide gel electrophoresis analysis of heparin-like polysaccharides obtained after treatment with different concentrations of sodium hydroxide. DETAILED DESCRIPTION

[0042] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0043] The present invention will be further described below in conjunction with the embodiments.

[0044] Embodiment 1:

[0045] 1.1 Purification and structural modification of E. coli fermented heparin precursor:

[0046] Heparin precursor was prepared by microbial fermentation using strain EcN8 in a 5L fermenter; when the OD of the seed culture with a total volume of 220mL was 600 When the value reached 2.0, it was added into a 5L fermenter containing 2.2L sterile M9VB1 medium for fermentation. The fermentation included a batch culture stage and a fed-batch culture stage. The M9VB1 medium in the batch culture stage included 8.0g / L glucose, 6.8g / L Na2HPO4, 3.0g / L KH2PO4, 1.0g / L NH4C l, 0.5g / LNaCl, 300mg / L thiamine, 2.0mmo l / L MgSO4·7H2O and 0.1mmo l / L CaCl2·2H2O. The culture environment was maintained at 37°C, pH 7.0, and the kanamycin concentration was 50mg / L. The pH value was adjusted using 5mo l / L l / L hydrochloric acid or 25% ammonia water, the fermentation tank stirring speed is set to 700rpm; when the dissolved oxygen value first decreases and then rises sharply, it enters the fed-batch culture stage; the feed composition includes 400g / L glucose, 20.0g / L MgSO4·7H2O and 250mg / L thiamine, and the growth rate μ is maintained at 0.10h by controlling the feed addition amount -1 ; After 40 hours of fermentation, the heparin precursor fermentation liquid was obtained;

[0047] The heparin precursor fermentation liquid was centrifuged at 6000 rpm for 40 min at 4°C using a refrigerated centrifuge, and the centrifugation was repeated twice until there was no obvious bacterial precipitation; 200 mL of the heparin precursor fermentation supernatant was taken, mixed with 3 times the volume of anhydrous ethanol, and dispensed into 250 mL centrifuge bottles, and placed in a -20°C refrigerator for 12 h; then centrifuged at 8000 rpm for 35 min at 4°C to recover the crude heparin precursor polysaccharide precipitate, and the precipitate was dissolved with 200 mL of ultrapure water to obtain a crude heparin precursor polysaccharide solution;

[0048] Urea is added to the crude heparin precursor polysaccharide solution until the concentration reaches 6.0 mol / L, and after the urea is completely dissolved, sodium hydroxide solid is added until the concentration reaches 2.0 mol / L; the reaction mixture is reacted in a water bath at 60° C. for 18 hours to perform a deacetylation reaction to prepare an N-deacetylheparin precursor reaction solution; after the reaction is completed, the N-deacetylheparin precursor reaction solution is rapidly cooled, and the pH value of the reaction system is adjusted to 8.0 using concentrated hydrochloric acid; then the solution is dispensed into centrifuge tubes, allowed to stand at 4° C. for 6 hours, and centrifuged at 10,000 rpm for 20 minutes at 4° C. to remove protein impurities and solids, and the obtained N-deacetylheparin precursor supernatant is placed in a dialysis bag with a molecular weight cutoff of 3.5 kDa, and dialyzed in ultrapure water to remove small molecular impurities. Before dialysis, the heparin polysaccharide content and protein impurity content in the N-deacetylheparin precursor reaction solution can be measured;

[0049] After the dialyzed N-deacetylheparin precursor reaction solution was quantified, the reaction solution containing 20 mg of N-deacetylheparin precursor was taken and prepared into a reaction system containing 50 mmol / L MES (pH 7.0), 1 mmol / L MnCl2, 1 mmol / L MgCl2, 1% (v / v) Triton X-100, 880 μmol / L PAPS and 10 mg of N-sulfotransferase according to the volume, and incubated at 25°C for 24 hours to prepare N-sulfoheparin precursor; after the reaction was completed, the reaction was terminated by boiling water bath for 3 minutes, and after cooling, the reaction was centrifuged at 8500 rpm for 25 minutes at 4°C; after centrifugation, the N-sulfoheparin precursor supernatant was taken and mixed with two volumes of UPAS buffer (50 mmol / L NaAcO, 150 mmol / L NaCl, 1 mmol / L EDTA, 60 mol / L urea, 0.01% [v / v] Triton X-100, pH5.5) and incubated at 4°C for 30 min; the mixed reaction solution was pumped into a DEAE anion exchange resin column (8 mL) equilibrated with UPAS buffer using a peristaltic pump at a flow rate of 1.0 mL / min; then, 20 mL of UPAS buffer and 45 mL of NaCl buffer I (50 mmol / L NaAcO, 150 mmol / L NaCl, 0.001% [v / v] Triton X-100, pH5.5) were used to wash the impurities at a flow rate of 3.0 mL / min, and finally NaCl buffer II (50 mmol / L NaAcO, 1 mol / L NaCl, 0.001% [v / v] Triton X-100, pH 5.5) to elute the N-sulfoheparin precursor from the column; the collected eluate is transferred to a dialysis bag with a molecular weight cutoff of 3.5 kDa, dialyzed in ultrapure water for desalination, and the dialyzed solution is vacuum freeze-dried to obtain the final product, a high-purity N-sulfoheparin precursor solid.

[0050] 1.2 Result analysis:

[0051] In the experiment, the quantification of heparin-like polysaccharides was carried out by ultrafiltration combined with sulfuric acid-anthrone method. 500 μL of sample was loaded into a 3 kDa ultrafiltration tube (0.5 ml, Millipore) and centrifuged at 14000 g for 25 min. Ultrapure water was then added to the ultrafiltration tube for rinsing 3 times, each time at 14000 g for 20 min. Finally, the ultrafiltration tube was inverted and centrifuged to collect the sample. Subsequently, 300 μL of ultrapure water was added to rinse the ultrafiltration tube and the rinsing solution was recovered by inverting. The rinsing solution was mixed with the sample to reduce the volume to 500 μL. 100 μL of sample was mixed with 300 μL of sulfuric acid-anthrone reagent (containing 0.1% anthrone and 80% concentrated sulfuric acid), and the mixture was placed in a boiling water bath for 15 min and an ice bath for 15 min. The OD was measured. 620Absorbance value; protein impurity amount was determined using Bradford reagent; DNA impurity quantification was determined by NanoDrop micro-spectrophotometer (ThermoFisher); UV-visible analysis of impurities was completed using Cary5000UV-Vis-NIR spectrophotometer (Agilent);

[0052] The samples include the crude heparin precursor polysaccharide solution obtained in this example, the centrifugal supernatant after the deacetylation reaction, the heparin precursor deacetylation reaction solution after dialysis, and the N-sulfoheparin precursor solution obtained by purification and elution of a DEAE anion exchange resin column, wherein the crude heparin precursor polysaccharide solution obtained by ethanol precipitation is used as the control group;

[0053] like Figure 1 As shown, with the crude heparin precursor polysaccharide solution obtained by ethanol precipitation as a control, a higher polysaccharide recovery rate can be achieved in the supernatant obtained by centrifugation after the deacetylation reaction is completed, and the protein residue in the supernatant obtained by centrifugation after the deacetylation reaction does not exceed 5% of the total protein impurities in the crude heparin precursor polysaccharide solution, indicating that this method can effectively remove protein impurities;

[0054] like Figure 2 As shown, in the heparin precursor deacetylation reaction solution after dialysis, heparin-like polysaccharides accounted for more than 95.4%, DNA accounted for 1.82%, and protein accounted for 2.78%; Figure 3 As shown, by UV-visible light analysis, the dialyzed heparin precursor deacetylation reaction solution sample was scanned in the wavelength range of 185-500nm, and it can be seen that there are no obvious UV absorption peaks of nucleic acids and proteins at 260nm and 280nm; Figure 2 , Figure 3 All of these indicate that this method achieves effective removal of impurities, and the purified heparin-like polysaccharide has a high purity, which can meet the needs of subsequent structural modification and production;

[0055] like Figure 4 As shown, compared with the total heparin-like polysaccharide amount in the crude heparin precursor polysaccharide solution obtained by ethanol precipitation as a control, the heparin-like polysaccharide in the heparin precursor deacetylation reaction solution after removing protein impurities obtained by centrifugation before dialysis can reach a recovery rate of 97.1%; the recovery rate of the heparin-like polysaccharide in the heparin precursor deacetylation reaction solution after dialysis can reach 93.6%; after completing the N-sulfonation reaction, the recovery rate of the N-sulfonated heparin precursor eluted after purification by DEAE anion exchange resin column is 76.0%. This process can achieve a higher recovery rate and reduce the loss during purification and structural modification.

[0056] Embodiment 2:

[0057] 2.1 Provide H NMR analysis to compare the structure of the product after the N-deacetylation reaction of the heparin precursor and the method of Example 1. The specific steps are as follows:

[0058] (1) Purification and preparation of heparin precursors The heparin precursors were purified according to the heparin precursor purification method using phenol-chloroform organic solvent to remove proteins in the literature Yu YY, Gong BX, Wang HL, et al. Chromosome evolution of Escherichia colli Nissle 1917 for high-level production of heparosan [J]. Biotechnology and Bioengineering, 2023, 120(4): 1081-1096;

[0059] (2) The quantitative heparin precursor N-deacetylation reaction solution after dialysis was taken and mixed with 2 column volumes of buffer A (20mmol / LNaAcO, 50mmol / LNaCl, pH4.0), and incubated at 4°C for 30 minutes. The mixed reaction solution was injected into a DEAE anion exchange resin column equilibrated with 10 column volumes of buffer A through a peristaltic pump, and the flow rate was set to 1.0mL / min; after the column was loaded, 5 column volumes of buffer A were used for washing, and finally buffer B (20mmol / LNaAcO, 1mol / LNaCl, pH4.0) was used for elution, and the washing and elution flow rates were both set to 3.0mL / min; the eluate was placed in a dialysis bag with a molecular weight cutoff of 3.5kDa and dialyzed in ultrapure water for desalination; the heparin precursor and the product of the heparin precursor N-deacetylation reaction were prepared into solid samples by vacuum freeze drying after dialysis.

[0060] (3) Take 10 mg of solid sample, dissolve it in 500 μL of D2O, and transfer it to a 5 mm standard NMR tube; the H NMR spectrum was measured using an Agilent VNMRS 600 MHz NMR spectrometer, and each sample was scanned 256 times. The H NMR spectrum results were obtained using Agilent VnmrJ3.2A software, and the results were analyzed and processed using MestReNova software.

[0061] 2.2 Result analysis:

[0062] like Figure 5As shown in the figure, the structures corresponding to the proton signals in the hydrogen spectrum of the heparin precursor are as follows: the H-1 atom of GlcNAc (5.32 ppm), the H-1 atom of GlcA (4.43 ppm), the H-2, H-3, H-5 and H-6 atoms of GlcNAc (3.79 ppm), the H-5 atom of GlcNAc (3.68 ppm), the H-4 atom of GlcNAc and the H-3 and H-4 atoms of GlcA (3.62 ppm), the H-2 atom of GlcA (3.30 ppm), and the methyl H atom on the acetyl group of GlcNAc (1.9 8ppm); the structures corresponding to the proton signals of the hydrogen spectrum of the product after the N-deacetylation reaction are as follows: the H-1 atom of GlcNH2 (5.41ppm), the H-1 atom of GlcA (4.51ppm), the H-2 and H-6 atoms of GlcNH2 (3.88ppm), the H-3 and H-5 atoms of GlcNH2 (3.82ppm), the H-5 atom of GlcA (3.75ppm), the H-4 atom of GlcNH2 and the H-3 and H-4 atoms of GlcA (3.67ppm), and the H-2 atom of GlcA (3.37ppm);

[0063] The H NMR spectrum of the heparin-like polysaccharide obtained after the N-deacetylation reaction is basically consistent with the H spectrum of the heparin precursor, except for the disappearance of the methyl H atom on the acetyl group of GlcNAc near 1.98 ppm. This process can achieve the N-deacetylation structural modification of the heparin precursor and realize the green and efficient preparation of the N-deacetylated heparin precursor.

[0064] Embodiment 3:

[0065] 3.1 The final product of Example 1 was characterized by time-of-flight mass spectrometry, elemental analysis, polysaccharide gel electrophoresis and gel permeation chromatography. The specific steps are as follows:

[0066] (1) Time-of-flight mass spectrometry characterization of the product: 2 mg of the final product prepared in Example 1 was dissolved in 3 mL of PBS solution containing 0.9 mg of heparin lyase III, incubated at 37°C for 12 h, then terminated the reaction in a boiling water bath for 3 min, centrifuged at 12000 rpm for 2 min, and the supernatant was dialyzed for desalting. After filtering through a 0.22 μm filter head, the molecular weight was scanned in the range of 50-1500 m / z using an Agilent 6230 time-of-flight liquid chromatography-mass spectrometry (LC / TOF) system, and the data were analyzed and processed using Agilent MassHunter software;

[0067] (2) Characterization of products by elemental analysis: The degree of N-sulfonation of the N-sulfoheparin precursor can be determined based on the S / N ratio data of sulfur and nitrogen elements; 10 mg of solid samples were taken from each of the heparin precursor and the N-deacetylheparin precursor and N-sulfoheparin precursor prepared in Example 1, and the CHNS element was detected using a varioELcube elemental analyzer (Elementar);

[0068] (3) Characterization of products by polysaccharide gel electrophoresis: To analyze and compare the molecular weight of polysaccharides, 22.5 μL of sample solution containing 0.2 mg of polysaccharide was mixed with 2.5 μL of 10× polysaccharide loading buffer (65% [w / v] sucrose, 0.05% [v / v] phenol red, 450 mmol / L Tris, 450 mmol / L boric acid, 10 mmol / L ETA, pH 8.3) and prepared to be added to the gel spotting wells; the vertical plate electrophoresis gel of polysaccharide was composed of 7.02 mL of deionized water, 1.08 mL of 5× TBE buffer (450 mmol / L Tris, 450 mmol / L boric acid, 10 mmol / L ETA, pH 8.3), 2.7 mL of acrylamide / methylenebisacrylamide 40% solution, 32.4 μL of 10% ammonium persulfate (APS) and 10.8 μL of TEMED; after loading, electrophoresis was performed at 100 V for 1 h using a vertical electrophoresis instrument. Use destaining solution (25% [v / v] ethanol, 10% [v / v] acetic acid) for pre-decolorization for 30 minutes, use staining solution (0.5% [w / v] Alcian blue, 2% [v / v] acetic acid) for staining for 45 minutes, then use destaining solution to decolorize until the gel background is transparent, and use gel imager to observe and take pictures of polysaccharide gel electrophoresis results;

[0069] (4) Gel chromatography characterization of products: The molecular weight distribution of the N-sulfoheparin precursor polysaccharide of the final product was determined using an Agilent 1260 Infinity II Multidetector GPC / SEC system, with a sample concentration of 2.5 mg / mL, a sample volume of 50 μL, a PL aquagel-OHSEC MIXED-H column (8 μm, 300×7.5 mm, Agilent), and a column temperature of 45° C. The molecular weight distribution of the sample was fitted and calculated using the Agilent GPC / SEC software.

[0070] 3.2 Result analysis:

[0071] like Figure 6As shown, the disaccharide composition of the N-sulfonated product finally obtained by cleavage of heparin lyase III is analyzed by time-of-flight mass spectrometry; heparin lyase III can cleave the α(1-4) glycosidic bond between GlcNAc-GlcA or GlcNS-GlcA, undergo an elimination reaction, and produce a disaccharide product containing unsaturated uronic acid residues, and then use mass spectrometry to analyze the disaccharide composition and determine the structure of the product after the N-sulfonation reaction. The disaccharide composition of the product after the N-sulfonation reaction was analyzed by time-of-flight mass spectrometer in negative ionization mode, and it can be seen that at m / z416.05[MH] - The peak at is the disaccharide of N-sulfoheparin precursor, which proves that this process can achieve the preparation of N-sulfoheparin precursor;

[0072] Figure 7 The S / N ratios of the heparin precursor, N-deacetylheparin precursor and N-sulfoheparin precursor obtained by elemental analysis are 0.05, 0.00 and 0.84, respectively. This result shows that the N-sulfonation rate of the N-sulfoheparin precursor obtained by this method can reach 84%, and the preparation of the N-sulfoheparin precursor can be effectively realized. The N-sulfonation rate is basically consistent with the N-sulfonation rate of medical heparin, and meets the raw material requirements of synthetic bioengineering heparin.

[0073] Figure 8 The molecular weights of the prepared N-sulfoheparin precursors were compared by polysaccharide gel electrophoresis; Figure 8 As shown, the molecular weight distribution of the N-sulfoheparin precursor prepared by the method is slightly lower than that of heparin sodium, higher than that of enoxaparin sodium, and significantly lower than that of the heparin precursor, and there is no significant change in the molecular weight distribution before and after the N-sulfonation reaction; the molecular weight of heparin sodium is mainly distributed between 3 and 30 kDa, and the most concentrated distribution is between 14 and 20 kDa; enoxaparin sodium is a low molecular weight heparin, and its molecular weight is mainly between 3500 and 5500 Da; according to the electrophoresis results, it is inferred that the molecular weight of the product should be between 4000 and 15000 Da; the results show that the process simultaneously undergoes a β-elimination reaction during the deacetylation reaction by sodium hydroxide, thereby reducing the molecular weight of the polysaccharide;

[0074] like Fig. 9 As shown, the molecular weight distribution of the prepared N-sulfoheparin precursor was specifically measured by gel permeation chromatography; the average weight average molecular weight (MWavg) of the N-sulfoheparin precursor was measured to be 5.6 kDa by fitting, which is within the molecular weight distribution range of low molecular weight heparin; the data results measured by GPC are basically consistent with the electrophoresis results, so the product prepared by this method can be used as a raw material for further synthesis of bioengineering low molecular weight heparin.

[0075] Embodiment 4:

[0076] 1. To explore the effect of adding or not adding urea on the recovery rate of heparin-like polysaccharides and the residual amount of protein impurities.

[0077] Urea is a common protein denaturant that can affect non-covalent interactions by destroying hydrogen bonds and reducing hydrophobic interactions. This example investigates the effect of adding urea to a reaction system with a sodium hydroxide concentration of 2 mol / L on the recovery rate of heparin-like polysaccharides and the total amount of protein impurities. The urea concentration of the reaction system in the urea group is 6.0 mol / L. The experimental steps are consistent with those in Example 1, with the only differences being whether urea is added and whether the pH value is adjusted to 7.0 at the end of the deacetylation reaction.

[0078] 2. Results Analysis

[0079] The experimental results are as follows Fig.10 As shown, compared with the polysaccharide recovery rate of the group without adding urea before the N-deacetylation reaction, the recovery rate of the group with added urea was significantly improved, which was 2.1 times that of the group without adding urea. Specifically, the recovery rate exceeded 39.5%; this result shows that the addition of urea promotes the separation of heparin-like polysaccharides from impurity proteins and destroys the non-covalent interaction between them; from the perspective of protein removal effect, compared with the control group of the crude heparin precursor polysaccharide solution obtained by ethanol precipitation and pure water dissolution, the protein residual rates of the group with added urea and the group without added urea are very low; therefore, it is chosen to add urea before the N-deacetylation reaction in order to better ensure the recovery of polysaccharides.

[0080] Embodiment 5:

[0081] 1. To investigate the effect of adjusting the pH value of the reaction system after N-deacetylation on the recovery rate of heparin-like polysaccharides and the amount of residual protein impurities

[0082] The pH value of the solution environment will affect the charge carried by the protein. When the pH value is lower than the pI (isoelectric point) of the protein, the protein will be positively charged, attracting anionic polysaccharides with opposite charges to form coacervates; therefore, strictly controlling the pH value of the system after the deacetylation reaction plays a key role in achieving the separation of impurity proteins and heparin-like polysaccharides; in this embodiment, the effect of adjusting to different pH values ​​on the polysaccharide recovery rate and protein residue was studied; the N-deacetylation reaction of the heparin precursor was completed in a reaction system containing 2 mol / L sodium hydroxide and 6 mol / L urea. After reacting in a 60°C water bath for 18 hours, the pH was adjusted to 5.0, 6.0, 7.0, 8.0, and 9.0 with concentrated hydrochloric acid, and the recovery rate of heparin-like polysaccharides and the residual rate of impurity proteins in the supernatant obtained by centrifugation were measured and compared;

[0083] 2. Results Analysis

[0084] The experimental results are as follows Fig.11 As shown in the figure, as the pH value decreases, the recovery rate of polysaccharides shows a downward trend. In particular, in the comparison of the two groups with pH values ​​of 8.0 and pH values ​​of 7.0, the recovery rate decreases significantly. The main reason for this phenomenon is that the p I value of the impurity protein in the mixed system may be between pH values ​​of 7.0 and 8.0. When the pH value reaches 7.0, which is lower than the p I value, the protein carries a positive charge and attracts the anionic polysaccharide to form a coagulation. Therefore, after the pH value is adjusted to 7.0 and below, white floccules are clearly visible in the solution, which are mainly complexes formed by the mutual attraction of impurity proteins and polysaccharides. After the reaction solution system is adjusted to pH 8.0, the supernatant polysaccharide recovery rate can reach more than 96%, which is 2.3 times the polysaccharide recovery rate of the system at pH 7.0. At the same time, it was also found that as the pH value of the system decreases, the amount of protein residue also shows a downward trend until it remains basically unchanged. The main reason for this phenomenon is that the attraction of polysaccharides to proteins also promotes the precipitation of impurity proteins, causing the proteins to separate into two phases from the aqueous solution. When the pH value was 8.0, the residual protein content in the supernatant accounted for 4.7% of the original impurity protein content in the crude heparin precursor polysaccharide solution, which basically met the requirements of preliminary purification.

[0085] Embodiment 6:

[0086] 1. To investigate the effects of different concentrations of sodium hydroxide on the recovery rate of heparin-like polysaccharides, the amount of residual protein impurities and the molecular weight distribution of polysaccharides

[0087] This example investigates the polysaccharide recovery rate, protein residue, and polysaccharide molecular weight distribution change trend in the supernatant of the reaction system after the heparin precursor completes the N-deacetylation reaction in a reaction system containing 6 mol / L urea and different concentrations of sodium hydroxide. After the reaction, the pH value of the supernatant is adjusted to 8.0.

[0088] 2. Results Analysis

[0089] The experimental results are as follows Fig.12 As shown in the figure, all reaction groups with sodium hydroxide addition can obtain a high recovery rate of heparin-like polysaccharides in the supernatant, and the protein residual rate gradually decreases with the increase of sodium hydroxide concentration. Adding urea alone cannot help remove impurity proteins, while adding sodium hydroxide is the key to removing impurity proteins; in the 2 mol / L sodium hydroxide group, the protein residual amount is less than 5%, which can effectively remove impurity proteins; the heparin-like polysaccharides obtained after treatment with different concentrations of sodium hydroxide were subjected to polysaccharide gel electrophoresis to analyze the molecular weight changes, as shown in the figure. Fig.13As shown in the electrophoresis bands, the molecular weight of the polysaccharide decreases with the increase of the sodium hydroxide concentration in the reaction system; these molecular weights are significantly lower than the molecular weight of the heparin precursor purified and extracted by the phenol-chloroform method in lane 1; at the same time, the molecular weight distribution of the 2 mol / L sodium hydroxide group (lane 7) is slightly lower than that of the commercial heparin sodium (lane 2); a higher concentration of sodium hydroxide reaction will lead to a more extensive depolymerization of the polysaccharide, generating a heparin-like polysaccharide with a lower molecular weight; polysaccharides with too low molecular weight are not conducive to the recovery of polysaccharides in the later and subsequent processes, and are prone to cause higher losses; therefore, when preparing N-deacetylheparan precursor, a reaction system with a sodium hydroxide concentration of 2 mol / L was determined.

[0090] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements will not cause the essence of the corresponding technical solutions to deviate from the protection scope of the technical solutions of the embodiments of the present invention.

Claims

1. Preparation of a novel microbial-derived N-sulfoheparin precursor polysaccharide, characterized in that: The following steps are involved: The fermentation broth rich in heparin precursor is prepared by fermentation with Escherichia coli EcN8 strain; The heparin precursor fermentation supernatant prepared by microbial fermentation is obtained by centrifugation, mixed with anhydrous ethanol 3 times the volume of the supernatant, and subjected to low-temperature alcohol precipitation to obtain a crude heparin precursor polysaccharide precipitate; After the crude heparin precursor polysaccharide precipitate is fully dissolved with ultrapure water, a crude heparin precursor solution is obtained, and then urea and sodium hydroxide are added to the crude heparin precursor solution, and the solution is placed in a 60° C. water bath for 18 h to perform an N-deacetylation reaction; After the reaction is finished, the reaction solution is obtained, and after cooling, the pH value of the reaction solution is adjusted to be greater than or equal to the isoelectric point of the impurity protein in the reaction solution to precipitate the impurity protein; The reaction solution was allowed to stand at 4°C for 6-8 hours, and then centrifuged to obtain a supernatant, and the supernatant was placed in a dialysis bag for dialysis to remove small molecular impurities, thereby obtaining an N-deacetylheparin precursor reaction solution; After the polysaccharide concentration of the dialyzed N-deacetylheparin precursor reaction solution is determined, reagents are added thereto to prepare an N-sulfonation reaction system environment, and then PAPS and N-sulfotransferase are added according to the amount of polysaccharide involved in the reaction. The reaction is carried out at 25° C. for 24 hours to complete the N-sulfonation modification and obtain the final product; The final product was purified by DEAE anion exchange resin column, dialyzed for desalting and vacuum freeze-dried to obtain a high-purity N-sulfoheparin precursor.

2. The preparation method of a novel microbial-derived N-sulfoheparin precursor polysaccharide according to claim 1, characterized in that: The microorganism is non-pathogenic Escherichia coli EcN8.

3. The preparation method of a novel microbial-derived N-sulfoheparin precursor polysaccharide according to claim 1, characterized in that: The heparin precursor fermentation supernatant is a fermentation product obtained by using a fermenter in a manner consisting of a batch culture stage and a fed-batch culture stage; the fermenter is filled with a sterile M9VB1 culture medium.

4. The preparation method of a novel microbial-derived N-sulfoheparin precursor polysaccharide according to claim 3, characterized in that: The M9VB1 medium used in the batch culture stage comprises glucose, Na2HPO4, KH2PO4, NH4Cl, NaCl, thiamine, MgSO4·7H2O and CaCl2·2H2O, the culture environment is maintained at 37°C, pH 7.0, and the kanamycin concentration is 50 mg / L; the feed composition of the fed batch culture stage comprises glucose, MgSO4·7H2O and thiamine, a feed pump is used to add the feed solution to the fermentation tank, and the growth rate is maintained at 0.10h -1 .

5. The preparation method of a novel microbial-derived N-sulfoheparin precursor polysaccharide according to claim 1, characterized in that: The concentration of added urea is 6.0 mol / L, and the concentration of sodium hydroxide is 2.0 mol / L.

6. The preparation method of a novel microbial-derived N-sulfoheparin precursor polysaccharide according to claim 1, characterized in that: The solution used to adjust the pH value of the reaction solution is concentrated hydrochloric acid, and the pH value is adjusted to 8.

0.

7. The preparation method of a novel microbial-derived N-sulfoheparin precursor polysaccharide according to claim 1, characterized in that: The N-sulfonation reaction system environment is prepared by adding MES to the N-deacetylheparin precursor reaction solution until its concentration reaches 50mmol / L, then adding MnCl2 and MgCl2 until their concentrations both reach 1mmol / L, and finally adding 1% (v / v) Triton X-100, and the pH value of the reaction system is 7.

0.

8. The preparation method of a novel microbial-derived N-sulfoheparin precursor polysaccharide according to claim 1, characterized in that: The mass ratios of the amount of polysaccharide involved in the reaction and the added PAPS and N-sulfotransferase are 1:1.2 and 2:1 respectively.

9. The preparation method of a novel microbial-derived N-sulfoheparin precursor polysaccharide according to claim 1, characterized in that: The DEAE anion exchange resin column was equilibrated with 10 column volumes of UPAS buffer at a flow rate of 1.0 mL / min; then, 3 column volumes of UPAS buffer and 5 column volumes of NaCl buffer I were used for washing, and finally, the N-sulfoheparin precursor was eluted from the column with NaCl buffer II at a flow rate of 3.0 mL / min; UPAS buffer includes 50mmol / L NaAcO, 150mmol / L NaCl, 1mmol / L EDTA, 6mol / L urea and 0.01% [v / v] Triton X-100, with a pH value of 5.5; NaCl buffer I includes 50mmol / L NaAcO, 150mmol / L NaCl and 0.001% [v / v] Triton X-100, with a pH value of 5.5; NaCl buffer II includes 50mmol / L NaAcO, 1mol / L NaCl and 0.001% [v / v] Triton X-100, with a pH value of 5.

5.

10. The preparation method of a novel microbial-derived N-sulfoheparin precursor polysaccharide according to claim 1, characterized in that: The molecular weight cut-off of the dialysis bag is 3.5 kDa.

Images