CN106399428A - 一种高效分离制备单一分子量透明质酸寡糖的方法 - Google Patents
一种高效分离制备单一分子量透明质酸寡糖的方法 Download PDFInfo
- Publication number
- CN106399428A CN106399428A CN201610865128.3A CN201610865128A CN106399428A CN 106399428 A CN106399428 A CN 106399428A CN 201610865128 A CN201610865128 A CN 201610865128A CN 106399428 A CN106399428 A CN 106399428A
- Authority
- CN
- China
- Prior art keywords
- hyaluronic acid
- buffer
- molecular
- weight hyaluronic
- molecular weight
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 229920002674 hyaluronan Polymers 0.000 title claims abstract description 37
- 229960003160 hyaluronic acid Drugs 0.000 title claims abstract description 37
- 229920001542 oligosaccharide Polymers 0.000 title claims abstract description 21
- -1 hyaluronic acid oligosaccharides Chemical class 0.000 title claims abstract description 16
- 238000000034 method Methods 0.000 title claims abstract description 16
- 238000000926 separation method Methods 0.000 title abstract description 8
- 102000004190 Enzymes Human genes 0.000 claims abstract description 8
- 108090000790 Enzymes Proteins 0.000 claims abstract description 8
- 239000000872 buffer Substances 0.000 claims description 22
- KIUKXJAPPMFGSW-DNGZLQJQSA-N (2S,3S,4S,5R,6R)-6-[(2S,3R,4R,5S,6R)-3-Acetamido-2-[(2S,3S,4R,5R,6R)-6-[(2R,3R,4R,5S,6R)-3-acetamido-2,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-2-carboxy-4,5-dihydroxyoxan-3-yl]oxy-5-hydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-3,4,5-trihydroxyoxane-2-carboxylic acid Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@H](O3)C(O)=O)O)[C@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](C(O)=O)O1 KIUKXJAPPMFGSW-DNGZLQJQSA-N 0.000 claims description 21
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 16
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 12
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 claims description 8
- 239000000203 mixture Substances 0.000 claims description 8
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 7
- 229940088598 enzyme Drugs 0.000 claims description 7
- 239000003480 eluent Substances 0.000 claims description 6
- 238000000855 fermentation Methods 0.000 claims description 6
- 230000004151 fermentation Effects 0.000 claims description 6
- 238000002360 preparation method Methods 0.000 claims description 6
- 239000011780 sodium chloride Substances 0.000 claims description 6
- 239000008367 deionised water Substances 0.000 claims description 5
- 229910021641 deionized water Inorganic materials 0.000 claims description 5
- 238000010828 elution Methods 0.000 claims description 5
- 239000004471 Glycine Substances 0.000 claims description 4
- 108010003272 Hyaluronate lyase Proteins 0.000 claims description 4
- 102000001974 Hyaluronidases Human genes 0.000 claims description 4
- 229960002773 hyaluronidase Drugs 0.000 claims description 4
- 238000005342 ion exchange Methods 0.000 claims description 4
- 239000007788 liquid Substances 0.000 claims description 4
- 239000000243 solution Substances 0.000 claims description 4
- 230000002779 inactivation Effects 0.000 claims description 3
- 241000237903 Hirudo Species 0.000 claims description 2
- 239000007986 glycine-NaOH buffer Substances 0.000 claims description 2
- 230000007062 hydrolysis Effects 0.000 claims description 2
- 238000006460 hydrolysis reaction Methods 0.000 claims description 2
- 230000001934 delay Effects 0.000 claims 1
- 150000002482 oligosaccharides Chemical class 0.000 abstract description 5
- 238000001819 mass spectrum Methods 0.000 description 13
- 230000000694 effects Effects 0.000 description 6
- 239000002253 acid Substances 0.000 description 5
- 239000002537 cosmetic Substances 0.000 description 5
- 239000000047 product Substances 0.000 description 5
- 239000003643 water by type Substances 0.000 description 4
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
- 238000005119 centrifugation Methods 0.000 description 3
- 239000003814 drug Substances 0.000 description 3
- 238000005227 gel permeation chromatography Methods 0.000 description 3
- 230000014759 maintenance of location Effects 0.000 description 3
- 238000001742 protein purification Methods 0.000 description 3
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 238000005349 anion exchange Methods 0.000 description 2
- 150000001450 anions Chemical class 0.000 description 2
- 230000004071 biological effect Effects 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 238000010612 desalination reaction Methods 0.000 description 2
- 238000009792 diffusion process Methods 0.000 description 2
- 239000011521 glass Substances 0.000 description 2
- 238000005461 lubrication Methods 0.000 description 2
- 229920002521 macromolecule Polymers 0.000 description 2
- 238000005374 membrane filtration Methods 0.000 description 2
- 230000003020 moisturizing effect Effects 0.000 description 2
- 238000006384 oligomerization reaction Methods 0.000 description 2
- 230000035699 permeability Effects 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 235000011121 sodium hydroxide Nutrition 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 230000001629 suppression Effects 0.000 description 2
- 230000029663 wound healing Effects 0.000 description 2
- 241000283690 Bos taurus Species 0.000 description 1
- 102000004127 Cytokines Human genes 0.000 description 1
- 108090000695 Cytokines Proteins 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- OVRNDRQMDRJTHS-FMDGEEDCSA-N N-acetyl-beta-D-glucosamine Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O OVRNDRQMDRJTHS-FMDGEEDCSA-N 0.000 description 1
- MBLBDJOUHNCFQT-LXGUWJNJSA-N N-acetylglucosamine Natural products CC(=O)N[C@@H](C=O)[C@@H](O)[C@H](O)[C@H](O)CO MBLBDJOUHNCFQT-LXGUWJNJSA-N 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- LEHOTFFKMJEONL-UHFFFAOYSA-N Uric Acid Chemical compound N1C(=O)NC(=O)C2=C1NC(=O)N2 LEHOTFFKMJEONL-UHFFFAOYSA-N 0.000 description 1
- TVWHNULVHGKJHS-UHFFFAOYSA-N Uric acid Natural products N1C(=O)NC(=O)C2NC(=O)NC21 TVWHNULVHGKJHS-UHFFFAOYSA-N 0.000 description 1
- 239000012190 activator Substances 0.000 description 1
- VZTDIZULWFCMLS-UHFFFAOYSA-N ammonium formate Chemical compound [NH4+].[O-]C=O VZTDIZULWFCMLS-UHFFFAOYSA-N 0.000 description 1
- 230000033115 angiogenesis Effects 0.000 description 1
- 239000011324 bead Substances 0.000 description 1
- 210000004204 blood vessel Anatomy 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 210000000988 bone and bone Anatomy 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 150000001720 carbohydrates Chemical group 0.000 description 1
- 239000003054 catalyst Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 239000003086 colorant Substances 0.000 description 1
- 238000011082 depyrogenation Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000002086 displacement chromatography Methods 0.000 description 1
- 238000004821 distillation Methods 0.000 description 1
- 229920001971 elastomer Polymers 0.000 description 1
- 239000000806 elastomer Substances 0.000 description 1
- 239000003792 electrolyte Substances 0.000 description 1
- 238000002330 electrospray ionisation mass spectrometry Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 239000003889 eye drop Substances 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 238000002523 gelfiltration Methods 0.000 description 1
- 102000034356 gene-regulatory proteins Human genes 0.000 description 1
- 108091006104 gene-regulatory proteins Proteins 0.000 description 1
- 239000003292 glue Substances 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 150000002460 imidazoles Chemical class 0.000 description 1
- 230000002519 immonomodulatory effect Effects 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 230000001050 lubricating effect Effects 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000004949 mass spectrometry Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000006199 nebulizer Substances 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 229920002401 polyacrylamide Polymers 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 231100000075 skin burn Toxicity 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 238000004704 ultra performance liquid chromatography Methods 0.000 description 1
- 238000000108 ultra-filtration Methods 0.000 description 1
- 229940116269 uric acid Drugs 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/26—Preparation of nitrogen-containing carbohydrates
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
Landscapes
- Organic Chemistry (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Health & Medical Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Microbiology (AREA)
- General Chemical & Material Sciences (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Molecular Biology (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
本发明公开了一种高效分离制备单一分子量透明质酸寡糖的方法,属于生物工程技术领域。本发明采用生物酶法催化降解HA,将制备的寡糖规模化分离和纯化,实现了工业化生产制备单一分子量的透明质酸寡糖。本发明将单一分子量的透明质酸寡聚糖进行分离,在工业上用于制备单一寡聚透明质酸及其衍生体具有潜在而非常广泛的价值。
Description
技术领域
本发明涉及一种高效分离制备单一分子量透明质酸寡糖的方法,属于生物工程技术领域。
背景技术
透明质酸(hyaluronic acid,简称HA),俗称玻璃尿酸,是一种由D-葡萄糖醛酸和N-乙酰氨基葡萄糖为重复双糖单位,通过β(1-3)和β(1-4)糖苷键交替连接而成的大分子酸性黏性多糖,1934年由Meyer等人从牛玻璃球眼中首次提取获得,广泛用于医药、化妆品、食品等领域。研究表明,分子量对透明质酸的活性有很大影响,不同分子量的透明质酸甚至表现出截然相反的活性。
透明质酸以其独特的分子结构和理化性质在机体内显示出多种重要的生理功能,如润滑关节,调节血管壁的通透性,调节蛋白质,水电解质扩散及运转,促进创伤愈合等。尤为重要的是,透明质酸具有特殊的保水作用,是目前发现的自然界中保湿性最好的物质,被称为理想的天然保湿因子,由于HA具有良好的保湿性、粘弹性、渗透性和延展性,同时无任何免疫原性和毒性,被广泛应用于化妆品、食品和医药等行业领域。
根据文献研究表明,分子量大小对HA的生物活性影响较大,不同分子量范围的HA表现出截然不同的生理学功能。高分子量的HA(Mr>2×106)由于具有较好的粘弹性、保湿性、抑制炎性反应、润滑等功能,可应用于高端化妆品行业、眼科手术黏弹剂和关节腔内注射治疗。中等分子量的HA(介于1×105-106)具有良好的保湿性、润滑和药物缓释作用,可广泛用于化妆品、滴眼液、皮肤烧伤愈合及术后防粘连。低分子量的HA和寡聚透明质酸,表现出非常强的生物活性,具有抑制肿瘤扩散、促进创伤愈合、促进骨和血管生成、免疫调节等作用,且易于渗透到真皮中,免疫细胞、细胞因子的激活剂。因此,小分子透明质酸在食品保健、化妆品以及临床医疗领域具有广阔的应用前景。
透明质酸经过酶解得到的产物一般为多种O-HA的混合物,如果要制备不同糖残基数的O-HA,必须经过分离。Tawada等用阴离子交换色谱柱进行分离,将冻干的样品溶于蒸馏水,通过阴离子交换色谱柱将不同残基数的寡糖分离开,不同的O-HA片段用凝胶过滤脱盐,然后用离心超滤装置除菌除热原,得到不同大小的O-HA。Ikegami-Kawai等用聚丙烯酰胺凝胶色谱进行分离,此外还有凝胶渗透色谱法(GPC)、HPLC法等分离方法。O-HA经凝胶层析分离只能得到一定Mr范围的混合物,不能制得单一分子量的O-HA。O-HA经Bio-gel P6凝胶层析分离只能够将大分子HA分离出来。Dowex1×2强阴离子交换层析不能完全将不同Mr的O-HA分离出来。
发明内容
本发明公开了一种利用离子交换柱实现单一分子量透明质酸寡糖的制备方法。
所述方法包括以下步骤:
(1)以透明质酸酶水解透明质酸得到透明质酸寡糖混合物;
(2)使用离子交换柱HiPrep Q FF 16/10对透明质酸寡糖混合物进行分离纯化;
所述步骤(2)中,先使用10倍柱体积的缓冲液A冲洗柱子,再通过10倍柱体积的洗脱剂,以2mL/min的流速对色谱柱进行线性洗脱,;所述洗脱剂由缓冲液A、缓冲液B组成,其中NaCl浓度于100min内从0提高到0.2mol/L,收集、合并同一峰对应的洗脱液;所述缓冲液A是50mmol/L的pH 8.5的甘氨酸-氢氧化钠缓冲液,所述缓冲液B是pH 8.5的0.5mol/L的NaCl缓冲液。
在本发明的一种实施方式中,缓冲液A的制备是称取3.75g甘氨酸溶解于500mL去离子水,用5MNaOH溶液调节pH至8.5,加去离子水定容至1L;缓冲液B的制备是称取29.22gNaCl溶于缓冲液A中,并定容至1L。
在本发明的一种实施方式中,步骤(1)是在发酵罐中加入900mL的水,并加入透明质酸酶3.5×107U和透明质酸40g,并加水至1L,在45℃、转速为700rpm条件下反应,反应11小时后高温灭活。
本发明为解决规模化制备单一分子量透明质酸寡糖的的问题,采用生物酶法催化降解HA,将制备的寡糖规模化分离和纯化,实现了工业化生产制备单一分子量的透明质酸寡糖。本发明将单一分子量的透明质酸寡聚糖进行分离,在工业上用于制备单一寡聚透明质酸及其衍生体具有潜在而非常广泛的价值。
附图说明
图1是罐中酶活3.5×104U/mL的分子量变化趋势图;
图2是水解产物的分离图;
图3是纯化出的HA4的质谱图;
图4是纯化出的HA6的质谱图;
图5是纯化出的HA8的质谱图;
图6是纯化出的HA10的质谱图;
图7是纯化出的HA12的质谱图;
图8是纯化出的HA14的质谱图。
具体实施方式
水解产物分离方法:
水解产物15000r/min离心20min后取上清,缓冲液A:50mmol/L,pH 8.5的甘氨酸-氢氧化钠缓冲液(称取3.75g甘氨酸溶解于500mL去离子水,用5M NaOH溶液调节pH至8.5,加去离子水定容至1L);缓冲液B:pH 8.5,0.5mol/LNaCl缓冲液(称取29.22gNaCl溶于800mL缓冲液A中,并定容至1L),使用柱子HiPrep 16/10Q FF(柱体积:20mL)美国GE公司。通过10倍色谱柱体积(CV)的洗脱剂,以2mL/min的流速对色谱柱进行线性洗脱,洗脱剂由缓冲液A、缓冲液B组成,其中NaCl浓度于100min内从0提高到0.2mol/L,AKTA蛋白纯化系统美国Amersham Biosciences公司。AKTA蛋白纯化系统配备有96孔板收集装置,每个孔收集1mL的洗脱液。分离结束后,将96孔板从AKTA中取出,将收集的溶液放入冰中保存。
水解产物的质谱检测方法:
洗脱样品15000r/min离心20min后取上清,稀释至1mg/mL上样。BEHAmide(100mm×2.1mm,1.7μm)美国Waters公司;Waters Maldi synpat Q-TOF MS美国Waters公司,色谱条件:ESI-MS条件;色谱仪:Waters Acquity UPLC;分析色谱柱:BEHAmide 2.1×100mm1.7μm,柱温:45℃,流速0.3mL/min,进样量1μL,流动相:A:甲酸铵,B:乙腈,离子方式:ESI。质谱条件:离子源参数ESI,雾化器压力:30psi;脱溶剂气体流速50L/hour;温度400℃;负离子模式。可检测出分离产物的成分。
实施例1规模化制备特定分子量透明质酸寡糖
以水蛭来源的透明质酸酶对大分子量透明质酸进行水解,大分子量透明质酸的起始分子量大小为1.21×106Da。在发酵罐中加入900mL的水,并加入透明质酸酶3.5×107U和透明质酸40g,最后加水至1L,在45℃、转速为700rpm条件下反应。每隔1小时取样并将样品在沸水中煮沸10分钟以将酶灭活,然后采用0.22μm的滤膜过滤,使用凝胶柱进行分子量测定,绘制出分子量变化趋势图。如图1所示的是,发酵罐中初始酶活3.5×104U/mL条件下透明质酸分子量随时间的变化趋势图。
发酵罐在11小时高温灭活,水解产物进行分离纯化。将发酵罐中的水解样品离心15000rpm、20min,并通过0.22μm的水系膜过滤,取10ml样品,待缓冲液A将柱子冲洗10倍柱体积后,将样品注入AKTA系统。通过10倍色谱柱体积(CV)的洗脱剂,以2mL/min的流速对色谱柱进行线性洗脱,洗脱剂由缓冲液A、缓冲液B组成,其中NaCl浓度于100min内从0提高到0.2mol/L,AKTA蛋白纯化系统配备有96孔板收集装置,每个孔收集1mL的洗脱液。分离结束后,将96孔板从AKTA收集器中取出,如图2所示水解产物的离子交换图,将每个峰对应的96孔板中的洗脱液收集起来。将96孔板中收集的同一峰的洗脱液进行合并,合并后每一个峰的样品通过透析袋进行脱盐,之后将收集的样品在-80℃进行冷冻,在冷冻干燥机中进行冻干。
冻干后的样品使用咪唑硫酸法测定寡糖的纯度,其中HA4、HA6、HA8、HA10、HA12、HA14的产物得率分别为75%、64.5%、55%、43.5%、43%、40.5%。HA4、HA6、HA8、HA10、HA12、HA14的产物得率分别为94%、91%、88%、87.5%、85%、82%、80.5%。对纯化的样品进行质谱检测,如图3所示,是纯化出HA4的质谱图;如图4所示,是纯化出HA6的质谱图;如图5所示,是纯化出HA8的质谱图;如图6所示,是纯化出HA10的质谱图;如图7所示,是纯化出HA12的质谱图;如图8所示,是纯化出HA14的质谱图。
Claims (5)
1.一种制备单一分子量透明质酸寡糖的方法,其特征在于,包括以下步骤:
(1)以透明质酸酶水解透明质酸得到透明质酸寡糖混合物;
(2)使用离子交换柱HiPrep Q FF 16/10对透明质酸寡糖混合物进行分离纯化;
所述步骤(2)中,先使用10倍柱体积的缓冲液A冲洗柱子,再通过10倍柱体积的洗脱剂,以2mL/min的流速对色谱柱进行线性洗脱,;所述洗脱剂由缓冲液A、缓冲液B组成,其中NaCl浓度于100min内从0提高到0.2mol/L,收集、合并同一峰对应的洗脱液;所述缓冲液A是50mmol/L的pH 8.5的甘氨酸-氢氧化钠缓冲液,所述缓冲液B是pH 8.5的0.5mol/L的NaCl缓冲液。
2.根据权利要求1所述的方法,其特征在于,缓冲液A的制备是称取3.75g甘氨酸溶解于500mL去离子水,用5M NaOH溶液调节pH至8.5,加去离子水定容至1L;缓冲液B的制备是称取29.22gNaCl溶于缓冲液A中,并定容至1L。
3.根据权利要求1所述的方法,其特征在于,步骤(1)是在发酵罐中加入900mL的水,并加入透明质酸酶3.5×107U和透明质酸40g,并加水至1L,在45℃、转速为700rpm条件下反应,反应11小时后高温灭活。
4.根据权利要求1所述的方法,其特征在于,所述透明质酸酶是水蛭来源的透明质酸酶。
5.根据权利要求1所述的方法,其特征在于,所述透明质酸的分子量是1.21×106Da。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201610865128.3A CN106399428B (zh) | 2016-09-29 | 2016-09-29 | 一种高效分离制备单一分子量透明质酸寡糖的方法 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201610865128.3A CN106399428B (zh) | 2016-09-29 | 2016-09-29 | 一种高效分离制备单一分子量透明质酸寡糖的方法 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN106399428A true CN106399428A (zh) | 2017-02-15 |
CN106399428B CN106399428B (zh) | 2019-08-06 |
Family
ID=59228139
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN201610865128.3A Active CN106399428B (zh) | 2016-09-29 | 2016-09-29 | 一种高效分离制备单一分子量透明质酸寡糖的方法 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN106399428B (zh) |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN109517012A (zh) * | 2018-11-13 | 2019-03-26 | 华熙福瑞达生物医药有限公司 | 一种高纯度透明质酸寡糖的制备方法 |
CN111040048A (zh) * | 2019-12-21 | 2020-04-21 | 南京汉欣医药科技有限公司 | 一种超低分子量透明质酸及其制备方法 |
CN114288308A (zh) * | 2021-12-02 | 2022-04-08 | 华熙生物科技股份有限公司 | 一种以四糖为主的透明质酸寡糖组合物及其制备方法和应用 |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN103484513A (zh) * | 2013-10-22 | 2014-01-01 | 江南大学 | 一种酶法制备小分子寡聚透明质酸的方法 |
CN104610467A (zh) * | 2015-01-27 | 2015-05-13 | 江南大学 | 一种透明质酸四糖和六糖的分离方法 |
-
2016
- 2016-09-29 CN CN201610865128.3A patent/CN106399428B/zh active Active
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN103484513A (zh) * | 2013-10-22 | 2014-01-01 | 江南大学 | 一种酶法制备小分子寡聚透明质酸的方法 |
CN104610467A (zh) * | 2015-01-27 | 2015-05-13 | 江南大学 | 一种透明质酸四糖和六糖的分离方法 |
Non-Patent Citations (1)
Title |
---|
原攀红等: "《体外酶法水解制备小分子透明质酸》", 《食品科学技术学报》 * |
Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN109517012A (zh) * | 2018-11-13 | 2019-03-26 | 华熙福瑞达生物医药有限公司 | 一种高纯度透明质酸寡糖的制备方法 |
CN109517012B (zh) * | 2018-11-13 | 2020-05-22 | 华熙生物科技股份有限公司 | 一种透明质酸寡糖的制备方法 |
CN111040048A (zh) * | 2019-12-21 | 2020-04-21 | 南京汉欣医药科技有限公司 | 一种超低分子量透明质酸及其制备方法 |
CN114901701A (zh) * | 2019-12-21 | 2022-08-12 | 南京汉欣医药科技有限公司 | 一种超低分子量透明质酸及其制备方法 |
CN114288308A (zh) * | 2021-12-02 | 2022-04-08 | 华熙生物科技股份有限公司 | 一种以四糖为主的透明质酸寡糖组合物及其制备方法和应用 |
Also Published As
Publication number | Publication date |
---|---|
CN106399428B (zh) | 2019-08-06 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Deniaud et al. | Structural studies of the mix-linked β-(1→ 3)/β-(1→ 4)-D-xylans from the cell wall of Palmaria palmata (Rhodophyta) | |
Liu et al. | A rhamnan-type sulfated polysaccharide with novel structure from Monostroma angicava Kjellm (Chlorophyta) and its bioactivity | |
US20210155720A1 (en) | Method for Preparing Hyaluronan Odd-numbered Oligosaccharides by Double Enzyme Hydrolysis | |
CN106399428B (zh) | 一种高效分离制备单一分子量透明质酸寡糖的方法 | |
CN103360439B (zh) | 制备肝素五糖的新中间体及其制备方法 | |
CN109400753B (zh) | 大鲵软骨制剂 | |
CN110357983B (zh) | 一种海参岩藻聚糖硫酸酯和硫酸软骨素低聚糖的制备方法 | |
CN111234044B (zh) | 一种低分子量金耳葡糖醛酸-木甘聚糖及其制备方法和应用 | |
CN105399848B (zh) | 一种岩藻聚糖硫酸酯、其制备方法和用途 | |
Liu et al. | Strategy for the sequence analysis of heparin | |
CN113201081A (zh) | 具免疫活性的平卧菊三七多糖及其制备方法和应用 | |
AU2021103870A4 (en) | Low-molecular-weight tremella aurantialba glucuronoxylomannan as well as preparation method and application thereof | |
CN106467561B (zh) | 一种快速分离、纯化人参精氨酸糖苷的方法 | |
EP0475383B1 (en) | Polysaccharide composition or polysaccharide having heparinoid activity, process for producing the same, and anticoagulant containing the same as active ingredient | |
CN101698683B (zh) | 从林蛙油中提取多糖的方法 | |
EP1024147B1 (en) | Process for preparing (1-3)-beta-d-glucan from fungi | |
CN105949335B (zh) | 一种具有抗血小板聚集活性的寡糖类组分及其制备方法 | |
CN105331655B (zh) | 一种茶籽低聚糖及其制备方法 | |
CN115386013B (zh) | 一种具有强效抗凝活性的海带多糖及其制备方法和应用 | |
CN109251255B (zh) | 一种岩藻糖化硫酸软骨素FCShm及其制备方法和应用 | |
CN116003645A (zh) | 一种泡叶藻岩藻多糖及其制备和纯化方法 | |
CN109439709A (zh) | 一种新Iota卡拉胶寡糖的制备方法 | |
JP3939592B2 (ja) | オリゴ糖(塩)の製造方法 | |
CN111057115B (zh) | 一种从贵妃蚌中提取的抗血栓类肝素及其制备方法和应用 | |
CN113307893A (zh) | 一种高纯度灰树花多糖提取物的制备与纯化方法 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
C06 | Publication | ||
PB01 | Publication | ||
C10 | Entry into substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
GR01 | Patent grant | ||
GR01 | Patent grant | ||
EE01 | Entry into force of recordation of patent licensing contract | ||
EE01 | Entry into force of recordation of patent licensing contract |
Application publication date: 20170215 Assignee: Shuiyang Cosmetics Manufacturing Co.,Ltd. Assignor: Jiangnan University Contract record no.: X2024980001574 Denomination of invention: An efficient method for separating and preparing single molecular weight hyaluronic acid oligosaccharides Granted publication date: 20190806 License type: Exclusive License Record date: 20240130 |