CN107384990B - Method for preparing heparin by catalyzing heparin precursor through in vitro enzyme method - Google Patents
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Abstract
The invention discloses a method for preparing heparin by catalyzing heparosan through an in vitro enzyme method, belonging to the field of biological medicine. The invention provides a method for synthesizing heparin pentasaccharide with biological function by pure biological enzyme method. Maltose fusion protein or amphiphilic short peptide sequence is fused at the 5' end of the gene to improve the expression quantity and enzyme activity of corresponding sulfate transferase or allosteric enzyme, so that the blood coagulation medicine with stable structure and single function is obtained.
Description
Technical Field
The invention relates to a method for preparing heparin by catalyzing heparosan through an in vitro enzyme method, belonging to the field of biological medicine.
Background
Heparin (HP) is a mucopolysaccharide formed by alternating between glucuronic acid and N-acetylglucosamine in α 1 → 4, β 1 → 4 glycosidic bonds and modified with glucuronic acid to a certain degree of allosterization, sulfation and N-acetylglucosamine sulfation, wherein Heparan Sulfate (HS) is a special form of heparin, HP and HS are widely present on cell surfaces and extracellular matrices and have significant effects on maintaining neural stem cell stability, neuronal migration, neurite elongation, dendritic regeneration, etc. and are widely used clinically as anticoagulant drugs.
Based on the above limitations of animal-derived HS, enzymatic synthesis of HS has been proposed, which has the advantage of obtaining heparin pentasaccharide with specificity and biological activity. The enzymatically synthesized HS may be formed from heparosan by sulfation and allosterization with 5 enzymes such as N-deacetyl-phosphotransferase (NDST), glucuronic acid C5-allosteric enzyme (C5epi), heparan sulfate 2-phosphotransferase (HS2ST), heparan sulfate 6-phosphotransferase (HS6ST), and heparan sulfate 3-phosphotransferase (HS3 ST). However, the enzymes are almost all derived from animals, and the preparation cost of the enzymes is high. Thus, currently, laboratory-scale HS is not completely prepared by a biological enzyme method, but is mainly synthesized by a chemical enzyme method, such as deacetylation with NaOH instead of deacetylation function in NDST, and the introduction of chemical reagents is liable to cause influence on the activity of other sulfate transferases.
Disclosure of Invention
In order to solve the defects in the prior art, the invention provides a method for synthesizing heparin pentasaccharide with biological functions by a pure biological enzyme method. Maltose fusion protein or amphiphilic short peptide sequence is fused at the 5' end of the gene to improve the expression quantity and enzyme activity of corresponding sulfate transferase or allosteric enzyme, so that the blood coagulation medicine with stable structure and single function is obtained.
The first object of the present invention is to provide a method for preparing heparin having biological activity, which is to prepare heparin by using heparosan as a substrate, and by sulfating heparin N-deacetylated phosphotransferase, heparan sulfate 2-phosphotransferase, heparan sulfate 6-phosphotransferase and heparan sulfate 3-phosphotransferase in a PAPS regeneration system and allosterizing the heparosan by glucuronic acid C5-allosteric enzyme.
In one embodiment of the invention, the N-deacetyl phosphotransferase, glucuronic acid C5-allosteric enzyme, heparan sulfate 2-phosphotransferase, heparan sulfate 6-phosphotransferase and heparan sulfate 3-phosphotransferase are derived from an animal.
In one embodiment of the invention, the N-deacetyl phosphotransferase, glucuronic acid C5-allosteric enzyme, heparan sulfate 2-phosphotransferase, heparan sulfate 6-phosphotransferase and heparan sulfate 3-phosphotransferase are heterologously expressed and produced by a microorganism.
In one embodiment of the invention, the gene encoding heparin N-deacetyl phosphotransferase (NDST) is GeneID: NP-077337.1 gene.
In one embodiment of the invention, the gene encoding glucuronic acid C5-allosteric enzyme (C5epi) is GeneID: NP-056369.1 gene.
In one embodiment of the invention, the Gene encoding heparan sulfate 2-phosphotransferase (HS2ST) is Gene ID: NP-989812.1 gene.
In one embodiment of the present invention, the Gene encoding heparan sulfate 6-phosphotransferase (HS6ST) is Gene ID: NP-989813.1 gene.
In one embodiment of the present invention, the Gene encoding heparan sulfate 3-phosphotransferase (HS3ST) is Gene ID: NP-034604.1 gene.
In one embodiment of the invention, the N-deacetyl phosphotransferase, glucuronic acid C5-allosteric enzyme, heparan sulfate 2-phosphotransferase, heparan sulfate 6-phosphotransferase and heparan sulfate 2-phosphotransferase are produced by a microorganism.
In one embodiment of the present invention, the NDST, C5epi, HS2ST, HS6ST, and HS3ST are the Gene ID: the gene NP-077337.1, NP-056369.1, NP-989812.1, NP-989813.1, or NP-034604.1 was obtained by fusing a maltose fusion protein or an amphipathic short peptide nucleotide sequence to the 5' -end of the gene, and expressing the resulting fusion protein or amphipathic short peptide nucleotide sequence in E.coli using a pET-series vector as an expression vector.
In one embodiment of the present invention, the NDST, C5epi, HS2ST, HS6ST, and HS3ST are the Gene ID: the gene of NP-077337.1, NP-056369.1, NP-989812.1, NP-989813.1 or NP-034604.1 is obtained by fusing maltose fusion protein or amphiphilic short peptide nucleotide sequence at 5' end and expressing in Pichia pastoris with pPIC series vector as expression vector.
In one embodiment of the invention, the pET series vectors include, but are not limited to, pET20b, pET22b, pET26b, pET28a, pET32 a.
In one embodiment of the invention, the escherichia coli comprises e.coli BL21, e.coli JM109, e.coli DH5 α, or e.coli TOP 10.
In one embodiment of the present invention, the pichia pastoris includes p.pastoris JC233, p.pastoris JC234, p.pastoris JC247, p.pastoris JC248, p.pastoris GS190, p.pastoris JC235, p.pastoris JC236, p.pastoris JC237, p.pastoris JC252, p.pastoris JC251, p.pastoris JC239, p.pastoris GS115, p.pastoris JC240, p.pastoris JC241, p.pastoris JC242, p.pastoris JC220, p.pastoris JC221, p.pastoris JC222, p.pastoris JC223, p.pastoris JC224, p.pastoris JC225, p.pastoris JC226, p.pastoris JC254, p.pastoris JC255, and the like.
In one embodiment of the present invention, the PAPS regeneration system is prepared by adding 0.1-50mM p-nitrobenzenesulfonic acid (PNPS), 1-200. mu.M 3 '5' -diphosphonic acid adenosine (PAP), and 0.1-100. mu.g ASST IV to 1-200mM Tris-HCl solution with pH 5-9.
In one embodiment of the present invention, the acyl sulfotransferase (ASST IV) is an acyl sulfotransferase prepared by reacting the amino acid sequence of NCBI: 83783 is obtained by fusing maltose fusion protein or amphiphilic short peptide nucleotide sequence at 5' end and then expressing in Escherichia coli by using pET series vector as expression vector.
In one embodiment of the invention, when heparin is prepared, 0.1-100 μ g NDST, C5epi, HS2ST, HS6ST, and HS3ST are added to a 1mL PAPS regeneration system.
In one embodiment of the invention, the enzyme activity of ASST IV is 0.1-1000. mu. mol/mg protein, and the enzyme activity of NDST, C5epi, HS2ST, HS6ST and HS3ST is 0.1-10000nmol/mg protein.
In one embodiment of the invention, the temperature of the enzymatic reaction is 10-50 ℃ when preparing heparin.
In one embodiment of the invention, the enzymatic reaction time is 1-50h when preparing heparin.
In one embodiment of the invention, the enzymatic reaction is at a pH of 5-9 when preparing heparin.
The second purpose of the invention is to provide the application of the method in preparing heparin and food and medicines containing heparin.
Has the advantages that: the invention firstly adopts the sulfate transferase, the allosteric enzyme and the like obtained by pure microbial fermentation to integrate the heparosan and the ASST, the enzyme activity reaches 0.1-200 mu mol/mg, the heparin and the heparan sulfate with biological activity are obtained, the conversion rate reaches 20-80 percent, and the invention has great industrial application value.
Drawings
FIG. 1 SDS-PAGE patterns of recombinantly expressed ASST IV, wherein M: 200kDa Marker (corresponding to bands of 192, 96, 62, 49, 38, 28, 14, 6, 3kDa, respectively); 1: an intracellular whole cell SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) map of the recombinant strain constructed by the pET series empty vector; 2: an intracellular whole-cell SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) map of the E.coli ASST recombinant strain constructed by pET series vectors; 3: an intracellular whole cell SDS-PAGE map of the E.coli MBP-ASST recombinant strain constructed by pET series vectors; 4: an intracellular whole cell SDS-PAGE map of the E.coli AP1-ASST recombinant strain constructed by pET series vectors; 4: the purified AP1-ASSTSDS-PAGE pattern.
FIG. 2 is a SDS-PAGE pattern of recombinant expression of NDST, C5epi, HS2ST, HS6ST, and HS3ST, wherein M: marker; 1: carrying out no-load transformation on pPET series vectors to form an extracellular supernatant SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) map of the recombinant bacteria; 2: an extracellular supernatant SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) map of a recombinant strain expressing AP1-NDST constructed by pPET series vectors; 3: an extracellular supernatant SDS-PAGE pattern of a recombinant strain expressing AP1-C5epi constructed by pPET series vectors; 4: an extracellular supernatant SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) map of the recombinant strain expressing AP1-HS2ST constructed by pPET series vectors; 5: an extracellular supernatant SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) map of the recombinant strain expressing AP1-HS6ST constructed by pPET series vectors; 6: an extracellular supernatant SDS-PAGE pattern of the recombinant strain expressing AP1-HS3ST constructed by pPET series vectors.
FIG. 3 Western blot maps of recombinant expression of NDST, C5epi, HS2ST, HS6ST and HS3ST, where M: marker; 1: an extracellular supernatant SDS-PAGE map of the MBP-NDST expressing recombinant strain constructed by the pPIC series vector; 2: an extracellular supernatant SDS-PAGE map of the MBP-C5 epi-expressing recombinant strain constructed by pPIC series vectors; 3: an extracellular supernatant SDS-PAGE map of the MBP-HS2 ST-expressing recombinant strain constructed by pPIC series vectors; 4: SDS-PAGE pattern of extracellular supernatant of MBP-HS6 ST-expressing recombinant strain constructed with pPIC series vector, 5: SDS-PAGE pattern of extracellular supernatant of MBP-HS3 ST-expressing recombinant strain constructed with pPIC series vector, 6: and (3) carrying out no-load transformation on the formed extracellular supernatant SDS-PAGE map of the recombinant bacteria by using pPIC series vectors.
FIG. 4 shows the enzyme activities of NDST, C5epi, HS2ST, HS6ST, and HS3ST, wherein the enzyme activities of C5epi and HS2ST are characterized by p-nitrophenol (PNP) formed in the catalytic process of HS2 ST.
FIG. 5 is a mass spectrum of heparin disaccharide unit, wherein OS (GlcNAc-GlcUA), NS (GlcNS-GlcUA), 2S (GlcNAc-IdoUA2S), NS6S (GlcNS6S-IdoUA), NS2S (GlcNS6S-IdoUA2S), Tri (GlcNS6S3S-IdoUA2S)
Detailed Description
Abbreviations and their corresponding chinese meanings referred to in the detailed description:
ASST IV: acyl-phosphotransferase IV, the Gene sequence encoding this enzyme is described in NCBI by Gene ID: 83783.
NDST: heparin N-deacetyl-phosphotransferase, the Gene sequence encoding this enzyme is described in NCBI by Gene ID: NP _ 077337.1.
C5 epi: glucuronic acid C5-allosteric enzyme, the Gene sequence encoding this enzyme is found in Gene ID in NCBI: NP _ 056369.1.
HS2 ST: heparan sulfate 2-sulfate transferase, the Gene sequence encoding this enzyme is described in NCBI by Gene ID: NP _ 989812.1.
HS6 ST: heparan sulfate 6-sulfate transferase, the Gene sequence encoding this enzyme is described in NCBI by Gene ID: NP _ 989813.1.
HS3 ST: heparan sulfate 3-sulfate transferase, the Gene sequence encoding this enzyme is described in NCBI by Gene ID: NP _ 034604.1.
HS: heparan sulfate; HP: heparin; PNPS: p-nitrobenzenesulfonic acid; PNP: p-nitrophenol; PAP: 3 ', 5' -adenosine diphosphate;
the enzyme activity determination method of NDST comprises the following steps: a catalytic reaction system is constructed by taking heparosan as a raw material, and the catalytic reaction system comprises 20mM Tris-HCl (pH7.0), 3mM PNPS, 20 mu M PAP, 10mg ASST IV, 5mg/mL heparosan and 20 mu g NDST, the light absorption value is measured at the wavelength of 400nm after the reaction is stopped by heating at 100 ℃ for 5min after 20h reaction at 37 ℃.
The enzyme activity determination method of C5epi and HS2ST comprises the following steps: a catalytic reaction system is constructed by taking deacetylated heparan precursors as raw materials, and comprises 20mM Tris-HCl (pH7.0), 3mM PNPS, 20 mu M PAP, 10mg ASST IV, 5mg/mL deacetylated heparan, 20 mu g C5epi and 20 mu g HS2ST, wherein the absorbance value is measured at the wavelength of 400nm after the reaction is stopped by heating at 100 ℃ for 5min after 20h at 37 ℃.
The enzyme activity determination method of HS6ST comprises the following steps: non-sulfated heparin was used as a raw material to construct a catalytic reaction system comprising 20mM Tris-HCl (pH7.0), 3mM PNPS, 20. mu.M PAP, 10mg ASST IV, 5mg/mL non-sulfated heparin and 20. mu. gHS6ST, at 37 ℃ after 20 hours, the reaction was terminated by heating at 100 ℃ for 5min, and then the absorbance was measured at 400 nm.
The enzyme activity determination method of HS3ST comprises the following steps: heparin is used as a raw material to construct a catalytic reaction system, which comprises 20mM Tris-HCl (pH7.0), 3mM PNPS, 20 mu M PAP, 10mg ASST IV, 5mg/mL heparin and 20 mu g HS3ST, the temperature is 37 ℃, after the reaction is performed for 20 hours, the temperature is 100 ℃, the reaction is heated for 5min, and the absorbance is measured at the wavelength of 400nm after the reaction is stopped.
The calculation formula of the product yield is as follows: y is 10-3*(18.83*(AC-AASST IV) + 0.38); wherein A isCThe absorbance value of NDST enzyme activity is measured; a. theASST IVAs control absorbance values.
Detection of HS: the detection conditions for LC were as follows: mobile phase a (4mM acetic acid), mobile phase B (4mM acetic acid, 70% methanol/water solution), column: c18Reverse phase column, 0.3mm 250mm, elution conditions are shown in Table 1. The conditions for MS are as follows: the speed of the atomization carrier gas is 0.75L/min, the atomization temperature is 140 ℃, and the drying carrier gas N is2The speed is 1.2L/min, the range of M/Z is 40-2000 by anion mode scanning, and a characteristic peak is obtained (figure 5).
TABLE 1 HPLC elution conditions
EXAMPLE 1 formulation of PAPS regeneration System
The PAPS regeneration system comprises the following components: PNPS concentration of 3mM, PAP concentration of 20. mu.M, ASST IV of 10mg/L, with 1-200mM Tris-HCl pH 5-9 as the base. Adding 1-100mg/L C4ST or C6ST as chondroitin sulfate radical donor.
Example 2 preparation of the tool enzymes ASST IV, C4ST, C6ST
(1) Shake flask expression of recombinant E.coli
By using an inductive vector pET26b series vector as a vector, pET26b-ASST IV, pET26b-NDST, pET26b-C5epi, pET26b-HS2ST, pET26b-HS6ST and pET26b-HS3ST are respectively constructed, and then the transformant is obtained by transforming Escherichia coli. As a control, a transformant obtained by transforming E.coli with pET26b, which is a pET-series vector having no gene ligated thereto, was used.
A single colony of the recombinant strain constructed using the pET series vector was cultured overnight at 37 ℃ and 200rpm in 3mL of LB medium to which ampicillin was added to a final concentration of 50. mu.g/mL. 50mL of LTB medium (to which ampicillin was added to a final concentration of 50. mu.g/mL) was transferred at a ratio of 1% (V/V), and cultured at 37 ℃ and 200rpm for 2 hours to OD600nmAbout 0.6-0.8, IPTG was added to a final concentration of 0.1mM and induction was carried out for 48 h. Centrifuging the culture at 8000rpm and 4 deg.C for 5min to collect thallus, washing with precooled Tris-HCl (pH7.0), resuspending thallus to 50mL, and performing ultrasonic treatment in ice bath under the conditions of: 400W, 2s of work, 3s of pause, 200 cycles until the bacterial liquid is clarified, 14000rpm, 20min of centrifugation at 4 ℃, and collecting supernatant, namely the crude enzyme liquid (figure 1 and figure 2).
As can be seen from FIG. 1, the recombinant strain constructed with pET26b vector had significant ASST IV bands in intracellular whole cells. As can be seen from FIG. 2, the recombinant strains constructed with pET26b vector expressing NDST, C5epi, HS2ST, HS6ST and HS3ST, respectively, showed the corresponding bands of interest in the extracellular supernatant, but not in the control.
(2) Shake-flask expression of recombinant Pichia pastoris
Constructing pPIC9K-mbp-ASST IV, pPIC9K-mbp-NDST, pPIC9K-mbp-C5epi, pPIC9K-mbp-HS2ST, pPIC9K-mbp-HS6ST and pPIC9K-mbp-HS3ST by using an inducible promoter pPIC series vector, and transforming Pichia pastoris to obtain a transformant; pichia pastoris was transformed with pPIC9K to obtain transformants as controls. Single colonies of the recombinant strain constructed with pPIC series vectors were picked up and cultured overnight at 30 ℃ and 200rpm in 3mLYPD medium. The mixture was transferred to 50mLBMMY medium (added with methanol to a final concentration of 0.5 g/L) at a ratio of 1% (V/V) and induced at 200rpm at 20 ℃ for 5 days. The above culture was centrifuged at 8000rpm at 4 ℃ for 5min to collect the supernatant, which was the crude enzyme solution (FIG. 3).
As can be seen from FIG. 3, the recombinant strain constructed with the pPIC series vector achieved secretory expression of the enzyme, whereas the control had no corresponding band.
(3) Purification of ASST-IV, NDST, C5epi, HS2ST, HS6ST, and HS3ST tool enzymes
After the Ni-NTA agarose Fast Flow column was equilibrated with Buffer A (20mM Tris-HCl, 0.5M NaCl, 20mM imidazole, pH 7.4), the crude enzyme solution was passed through a 0.22 μ M filter, and the supernatant was passed through the Ni-NTA agarose Fast Flow column at a rate of 3mL/min, and after binding for 10min, the enzyme active fraction was collected by elution with Buffer B (20mM Tris-HCl, 0.5M NaCl, 500mM imidazole, pH 7.4), concentrated, freeze-dried, and the enzyme activity was measured (FIG. 4). As shown in FIG. 4, the enzyme activities of NDST, C5epi, HS2ST, HS6ST and HS3ST expressed by Escherichia coli are respectively 18, 25, 142, 113 and 169U/L, and the enzyme activities of NDST, C5epi, HS2ST, HS6ST and HS3ST expressed by Pichia pastoris are respectively 1420, 1935, 2892, 2038 and 2788U/L.
Example 3 preparation of HS
A catalytic reaction system is constructed by taking heparosan as a raw material, and the catalytic reaction system comprises 20mM Tris-HCl (pH7.0), 3mM PNPS, 20 mu M PAP, 10mg ASST IV (enzyme activity unit is 0.1-100 nmol/min. mg. protein), 5mg/mL heparosan and 20 mu g NDST, C5epi, HS2ST, HS6ST and HS3ST (enzyme activity unit is 0.1-100 pmol/min. mg. protein), and the reaction is stopped by heating at 100 ℃ for 5min after 20h reaction at 37 ℃.
Adding heparin lyase I, II and III into the reaction solution after the reaction is ended, reacting for 5-20h at 37 ℃, heating at 100 ℃ for 5min to end the reaction, and performing LC-MS detection after a filter membrane with the diameter of 0.22 mu m is penetrated. As shown in FIG. 5, the conversion of NDST was 55.88%, the conversion of C5 epi-conjugated HS2ST was 65.23%, the conversion of HS6ST was 78.02%, the conversion of HS3ST was 78.98%, the conversion of the finally synthesized biologically active pentasaccharide unit was 22.06%, and the yield was 1.1 mg/mL.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (5)
1. A method for preparing heparin with biological activity is characterized in that heparin is prepared by using heparosan as a substrate and using heparin N-deacetylated phosphotransferase, heparan sulfate 2-phosphotransferase, heparan sulfate 6-phosphotransferase, heparan sulfate 3-phosphotransferase and glucuronic acid C5-allosteric enzyme expressed by microbial cells to catalyze the heparosan; the coding Gene of the heparin N-deacetylation phosphotransferase is shown as Gene ID: NP-077337.1; genes encoding the glucuronic acid C5-allosteric enzyme are as described by Gene ID: NP-056369.1; genes encoding the heparan sulfate 2-phosphotransferase include Gene ID: NP-989812.1; genes encoding the heparan sulfate 6-phosphotransferase include, for example, GeneID: NP-989813.1; genes encoding the heparan sulfate 3-phosphotransferase include Gene ID: NP-034604.1; the method is to prepare heparosan in a PAPS regeneration system; the PAPS regeneration system comprises 0.1-50mM p-nitrobenzenesulfonic acid, 1-200 mu M3 '5' -diphosphonidine and 0.1-100 mu g acyl-thiotransferase, which are all dissolved in 1-200mM Tris-HCl solution with pH value of 5-9;
the expression is (1) or (2):
(1) expressing N-deacetylation phosphotransferase, glucuronic acid C5-allosteric enzyme, heparan sulfate 2-phosphotransferase, heparan sulfate 6-phosphotransferase or heparan sulfate 3-phosphotransferase by taking an escherichia coli cell as a host and taking a pET series vector as an expression vector, wherein the escherichia coli cell is any one of E.coli BL21, E.coli JM109, E.coli DH5 α or E.coli TOP 10;
(2) uses pichia pastoris cells as a host and pPIC series vectors as expression vectors to express N-deacetylate phosphotransferase, glucuronic acid C5-allosteric enzyme, heparan sulfate 2-phosphotransferase, heparan sulfate 6-phosphotransferase or heparan sulfate 3-phosphotransferase.
2. The method of claim 1, wherein the N-deacetyl-phosphotransferase, glucuronic acid, are present
C5-allosteric enzyme, heparan sulfate 2-sulfate transferase, heparan sulfate 6-sulfate transferase and heparan sulfate 3-sulfate transferase from animal sources.
3. The method according to claim 1, wherein 0.1-100 μ g N-deacetyl phosphotransferase, glucuronic acid C5-allosteric enzyme, heparan sulfate 2-phosphotransferase, heparan sulfate 6-phosphotransferase and heparan sulfate 3-phosphotransferase are added per 1mL of PAPS regeneration system, respectively; the enzyme activity of the acyl-thiotransferase is 0.1-1000 mu mol/mg protein, and the enzyme activity of the N-deacetyl-thiotransferase, glucuronic acid C5-allosteric enzyme, heparan sulfate 2-thiotransferase, heparan sulfate 6-thiotransferase and heparan sulfate 3-thiotransferase is 0.1-10000nmol/mg protein.
4. The method of any one of claims 1 to 3, wherein the catalysis is carried out at a temperature of 10 to 50 ℃ and a pH of 5 to 9 for 1 to 50 hours.
5. Use of the method according to any one of claims 1 to 4 for the preparation of heparin and heparin-containing food and pharmaceutical products.
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| CN107384990A (en) | 2017-11-24 |
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Application publication date: 20171124 Assignee: NANJING HANXIN PHARMACEUTICAL TECHNOLOGY Co.,Ltd. Assignor: Jiangnan University Contract record no.: X2021980014491 Denomination of invention: A method for preparing heparin before enzymatic catalysis of heparin in vitro Granted publication date: 20200508 License type: Exclusive License Record date: 20211214 |