JPS63129991A - Production of hyaluronic acid - Google Patents
Production of hyaluronic acidInfo
- Publication number
- JPS63129991A JPS63129991A JP27647286A JP27647286A JPS63129991A JP S63129991 A JPS63129991 A JP S63129991A JP 27647286 A JP27647286 A JP 27647286A JP 27647286 A JP27647286 A JP 27647286A JP S63129991 A JPS63129991 A JP S63129991A
- Authority
- JP
- Japan
- Prior art keywords
- hyaluronic acid
- molecular weight
- high molecular
- culture
- producing
- 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.)
- Pending
Links
- 229920002674 hyaluronan Polymers 0.000 title claims abstract description 45
- 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 title claims abstract description 44
- 229960003160 hyaluronic acid Drugs 0.000 title claims abstract description 44
- 238000004519 manufacturing process Methods 0.000 title claims description 8
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 18
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 18
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 claims abstract description 11
- 239000003795 chemical substances by application Substances 0.000 claims abstract description 11
- 235000019333 sodium laurylsulphate Nutrition 0.000 claims abstract description 11
- 244000005700 microbiome Species 0.000 claims abstract description 7
- 238000012258 culturing Methods 0.000 claims description 8
- 239000003398 denaturant Substances 0.000 claims description 7
- 241000194017 Streptococcus Species 0.000 claims description 5
- 229960000789 guanidine hydrochloride Drugs 0.000 abstract description 5
- PJJJBBJSCAKJQF-UHFFFAOYSA-N guanidinium chloride Chemical compound [Cl-].NC(N)=[NH2+] PJJJBBJSCAKJQF-UHFFFAOYSA-N 0.000 abstract description 5
- 108091005804 Peptidases Proteins 0.000 abstract description 4
- 239000004365 Protease Substances 0.000 abstract description 4
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 abstract description 3
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 abstract description 3
- 238000005273 aeration Methods 0.000 abstract description 3
- 229910052799 carbon Inorganic materials 0.000 abstract description 3
- 238000000354 decomposition reaction Methods 0.000 abstract description 3
- 150000003839 salts Chemical class 0.000 abstract description 3
- 241000264435 Streptococcus dysgalactiae subsp. equisimilis Species 0.000 abstract description 2
- 241000120569 Streptococcus equi subsp. zooepidemicus Species 0.000 abstract description 2
- 238000013019 agitation Methods 0.000 abstract description 2
- 239000002537 cosmetic Substances 0.000 abstract description 2
- 230000036425 denaturation Effects 0.000 abstract 4
- 238000004925 denaturation Methods 0.000 abstract 4
- 230000000813 microbial effect Effects 0.000 abstract 2
- 235000015097 nutrients Nutrition 0.000 abstract 1
- 238000000034 method Methods 0.000 description 19
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 8
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 7
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 6
- 229920002385 Sodium hyaluronate Polymers 0.000 description 6
- 238000000746 purification Methods 0.000 description 6
- 229940010747 sodium hyaluronate Drugs 0.000 description 6
- YWIVKILSMZOHHF-QJZPQSOGSA-N sodium;(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- Chemical compound [Na+].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 YWIVKILSMZOHHF-QJZPQSOGSA-N 0.000 description 5
- 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 4
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 239000004202 carbamide Substances 0.000 description 4
- 238000005119 centrifugation Methods 0.000 description 4
- 230000007423 decrease Effects 0.000 description 4
- 235000019441 ethanol Nutrition 0.000 description 4
- 239000008103 glucose Substances 0.000 description 4
- 241000894006 Bacteria Species 0.000 description 3
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 241000194048 Streptococcus equi Species 0.000 description 3
- 239000002253 acid Substances 0.000 description 3
- 229940041514 candida albicans extract Drugs 0.000 description 3
- 238000011033 desalting Methods 0.000 description 3
- 238000000855 fermentation Methods 0.000 description 3
- 230000004151 fermentation Effects 0.000 description 3
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 3
- 235000019341 magnesium sulphate Nutrition 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 3
- 235000019345 sodium thiosulphate Nutrition 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- 239000012138 yeast extract Substances 0.000 description 3
- FNEHAOQZWPHONV-UHFFFAOYSA-N 9h-carbazole;sulfuric acid Chemical compound OS(O)(=O)=O.C1=CC=C2C3=CC=CC=C3NC2=C1 FNEHAOQZWPHONV-UHFFFAOYSA-N 0.000 description 2
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 230000000052 comparative effect Effects 0.000 description 2
- 238000007796 conventional method Methods 0.000 description 2
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 2
- 235000019796 monopotassium phosphate Nutrition 0.000 description 2
- 239000003960 organic solvent Substances 0.000 description 2
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 2
- 238000001556 precipitation Methods 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- GEHJYWRUCIMESM-UHFFFAOYSA-L sodium sulfite Chemical compound [Na+].[Na+].[O-]S([O-])=O GEHJYWRUCIMESM-UHFFFAOYSA-L 0.000 description 2
- AKHNMLFCWUSKQB-UHFFFAOYSA-L sodium thiosulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=S AKHNMLFCWUSKQB-UHFFFAOYSA-L 0.000 description 2
- 230000001954 sterilising effect Effects 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
- 239000004094 surface-active agent Substances 0.000 description 2
- 238000000108 ultra-filtration Methods 0.000 description 2
- SQDAZGGFXASXDW-UHFFFAOYSA-N 5-bromo-2-(trifluoromethoxy)pyridine Chemical compound FC(F)(F)OC1=CC=C(Br)C=N1 SQDAZGGFXASXDW-UHFFFAOYSA-N 0.000 description 1
- 239000004254 Ammonium phosphate Substances 0.000 description 1
- 229920001287 Chondroitin sulfate Polymers 0.000 description 1
- 229920002261 Corn starch Polymers 0.000 description 1
- 229930091371 Fructose Natural products 0.000 description 1
- 239000005715 Fructose Substances 0.000 description 1
- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 0.000 description 1
- 230000005526 G1 to G0 transition Effects 0.000 description 1
- 241000287828 Gallus gallus Species 0.000 description 1
- 244000068988 Glycine max Species 0.000 description 1
- 235000010469 Glycine max Nutrition 0.000 description 1
- 229920002683 Glycosaminoglycan Polymers 0.000 description 1
- 102000035195 Peptidases Human genes 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- 150000007513 acids Chemical class 0.000 description 1
- WQZGKKKJIJFFOK-PHYPRBDBSA-N alpha-D-galactose Chemical compound OC[C@H]1O[C@H](O)[C@H](O)[C@@H](O)[C@H]1O WQZGKKKJIJFFOK-PHYPRBDBSA-N 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 229910000148 ammonium phosphate Inorganic materials 0.000 description 1
- 235000019289 ammonium phosphates Nutrition 0.000 description 1
- 239000003945 anionic surfactant Substances 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 239000002585 base Substances 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- 229940059329 chondroitin sulfate Drugs 0.000 description 1
- 239000000356 contaminant Substances 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 239000008120 corn starch Substances 0.000 description 1
- 229940099112 cornstarch Drugs 0.000 description 1
- 230000000593 degrading effect Effects 0.000 description 1
- 239000000645 desinfectant Substances 0.000 description 1
- MNNHAPBLZZVQHP-UHFFFAOYSA-N diammonium hydrogen phosphate Chemical compound [NH4+].[NH4+].OP([O-])([O-])=O MNNHAPBLZZVQHP-UHFFFAOYSA-N 0.000 description 1
- 229910000396 dipotassium phosphate Inorganic materials 0.000 description 1
- 235000019797 dipotassium phosphate Nutrition 0.000 description 1
- SMVRDGHCVNAOIN-UHFFFAOYSA-L disodium;1-dodecoxydodecane;sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O.CCCCCCCCCCCCOCCCCCCCCCCCC SMVRDGHCVNAOIN-UHFFFAOYSA-L 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 101150036577 fl11 gene Proteins 0.000 description 1
- 229930182830 galactose Natural products 0.000 description 1
- 238000012812 general test Methods 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 229910001385 heavy metal Inorganic materials 0.000 description 1
- 150000004688 heptahydrates Chemical class 0.000 description 1
- KIUKXJAPPMFGSW-MNSSHETKSA-N hyaluronan Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)C1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H](C(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-MNSSHETKSA-N 0.000 description 1
- 229940099552 hyaluronan Drugs 0.000 description 1
- 230000003301 hydrolyzing effect Effects 0.000 description 1
- -1 hydropotassium Chemical compound 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 238000009940 knitting Methods 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 230000002101 lytic effect Effects 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 239000012533 medium component Substances 0.000 description 1
- 230000003020 moisturizing effect Effects 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- WACRXVBKMRXTCA-UHFFFAOYSA-N platinum sodium Chemical compound [Na].[Pt] WACRXVBKMRXTCA-UHFFFAOYSA-N 0.000 description 1
- 230000001376 precipitating effect Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 235000010265 sodium sulphite Nutrition 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- DHCDFWKWKRSZHF-UHFFFAOYSA-N sulfurothioic S-acid Chemical compound OS(O)(=O)=S DHCDFWKWKRSZHF-UHFFFAOYSA-N 0.000 description 1
- 229940045136 urea Drugs 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 210000004127 vitreous body Anatomy 0.000 description 1
Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
Description
【発明の詳細な説明】
〔産業上の利用分野〕
本発明は、化粧品又は医薬品用として望まれる天然に存
在するヒアルロン酸に近い分子量２００〜４００万の高
分子量のヒアルロン酸を製造する方法に関する。DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a method for producing high molecular weight hyaluronic acid with a molecular weight of 2 to 4 million, which is close to naturally occurring hyaluronic acid and is desired for use in cosmetics or pharmaceuticals.
従来、ヒアルロン酸は、ニワトリのトサカ、牛の眼の硝
子体又は腰帯等よシ抽出によって得られていた。しかし
、生体からの分離精製は、夾雑タンパクやコンドロイチ
ン硫酸等のムコ多糖類の存在によシ、榛雑な工程全必要
とする上に、生体中のヒアルロニダーゼ等により、分離
−精製工程中に、元来高分子量であったヒアルロン酸が
分解さｎ、低分子量化され、粘度及び保湿性が低くなる
という欠点があった。そこで、ヒアルロン酸の分解を抑
制してより高分子量のヒアルロン酸全得る方法が種々提
案さｊているが、十分とは言い難く、またそのためにコ
ストアップの要因ともなってい念。Conventionally, hyaluronic acid has been obtained by extraction from chicken crests, cow eye vitreous bodies, waistbands, etc. However, separation and purification from living organisms requires complicated steps due to the presence of contaminant proteins and mucopolysaccharides such as chondroitin sulfate. Hyaluronic acid, which originally had a high molecular weight, was decomposed and reduced to a lower molecular weight, resulting in lower viscosity and moisture retention. Therefore, various methods have been proposed to obtain higher molecular weight hyaluronic acid by suppressing the decomposition of hyaluronic acid, but these methods cannot be said to be sufficient and are also a factor in increasing costs.
こｎらの欠点を解決するものとして、ヒアルコン酸生産
能を有するストレプトコツカス属の微生物を培養し、培
養液からヒアルロン酸を単離する方法が開示さｎている
（特開昭５８−５６６９２号公報、特開昭６０−１５′
５８９４号公報、特開昭６１−６３２９４号公報）。こ
れらの方法は、抽出法に比較して単離、精製が簡単でコ
ストが安いという利点がある。しかしながら、発酵法に
よシ得らｊるヒアルロン酸は分子量が低く、一般に１５
０万以下である。そのため、粘性、保湿効果が劣る。As a solution to these drawbacks, a method has been disclosed in which Streptococcus microorganisms capable of producing hyalconic acid are cultured and hyaluronic acid is isolated from the culture solution (Japanese Patent Laid-Open No. 58-56692). Publication No. 60-15'
5894, Japanese Unexamined Patent Publication No. 61-63294). These methods have advantages over extraction methods in that isolation and purification are easier and costs are lower. However, hyaluronic acid obtained by fermentation has a low molecular weight, generally 15
00,000 or less. Therefore, the viscosity and moisturizing effect are inferior.
また、培養液中に溶菌酵素や界面活性剤を添加してヒア
ルロン酸の収ｆｔヲ向上する方法（％開昭６１−２３９
８９８号公報）が開示さｎているが、分子量については
伺等ふｎらｎていない。In addition, a method for improving the yield of hyaluronic acid by adding lytic enzymes and surfactants to the culture solution (% 1986-239)
No. 898) has been disclosed, but the molecular weight has not been disclosed.
本発明者らは、発酵法で高分子量ヒアルロン酸を取得す
る方法について研究を行ない、培養液の…を７．５以上
９．０以下の範囲にコントロールすることにより、培養
液中に高分子量のヒアルロン酸を蓄積せしめる方法につ
いて提案した（％願昭６１−１７０９４３号明細省）。The present inventors conducted research on a method for obtaining high molecular weight hyaluronic acid by fermentation method, and by controlling the... of the culture solution within the range of 7.5 to 9.0, high molecular weight A method for accumulating hyaluronic acid was proposed (Department of Specification No. 170943/1983).
この方法により、一応高分子量のヒアルロン酸を含む培
養液が得られ、公矧のｎ鯛法により、高分子量のヒアル
ロン酸が得らする。By this method, a culture solution containing high molecular weight hyaluronic acid can be obtained, and by Koki's n-tai method, high molecular weight hyaluronic acid can be obtained.
〔発明が解決しようとする問題点〕
しかしながら、前記特願昭６１−１７０９４３号明細書
記載の方法では培養を長時間にわたって行なうと、ヒア
ルロン酸分子量の低下が見らｎｌまた精製に長時間を要
する大規模スケールで実施すると、精製工程での、ヒア
ルロン酸の分子量低下が著しく、高分子量ヒアルロン酸
が取得できないという問題があった。[Problems to be Solved by the Invention] However, in the method described in the specification of Japanese Patent Application No. 61-170943, when culture is carried out for a long time, a decrease in the molecular weight of hyaluronic acid is observed, and furthermore, a long time is required for purification. When carried out on a large scale, there is a problem that the molecular weight of hyaluronic acid decreases significantly during the purification process, making it impossible to obtain high molecular weight hyaluronic acid.
本発明者らは、かかる問題を解決すべく、検討を重ねた
結果、培養中又は培養終了後に、蛋白変性剤を添加する
ことによりヒアルロン酸の分子量低下を防止できること
を見い出した。すなわち、ヒアルロン酸生産菌株が、特
定の条件下で産生ずるヒアルロン酸分解酵素を蛋白変性
剤ｋｔｓ加することにより不活化し、ヒアルロン酸の分
解による分子量低下を妨げることを見い出し、本発明を
完成するに至った。In order to solve this problem, the present inventors have made repeated studies and found that it is possible to prevent a decrease in the molecular weight of hyaluronic acid by adding a protein denaturing agent during or after culturing. That is, we have discovered that adding the protein denaturant kts to a hyaluronic acid-producing bacterial strain can inactivate the hyaluronic acid degrading enzyme produced under specific conditions, thereby preventing the reduction in molecular weight due to the decomposition of hyaluronic acid, and have completed the present invention. reached.
すなわち、本発明は、ヒアルロン酸生成能を有する微生
物を培養して、高分子量ヒアルロン酸を採取するに際し
、培養中または培養終了後に、蛋白変性剤を添加するこ
とを特徴とする高分子量ヒアルロン酸の製法である。That is, the present invention provides a method for producing high molecular weight hyaluronic acid, which is characterized by adding a protein denaturing agent during or after culturing when culturing microorganisms capable of producing hyaluronic acid and collecting high molecular weight hyaluronic acid. It's the manufacturing method.
以下、本発明の詳細な説明する。The present invention will be explained in detail below.
本発明に用いる培養液は、ヒアルロン酸生産能を有する
微生物の培養液である。ヒアルロン酸生産菌としては、
ストレプトコッカス・エキ（５ｔｒｅｐｔｏｃｏｃｃｕ
ｓ ｅｑｕｉ ）、ストレプトコッカス−ズーエピデミ
カス（５ｔｒｅｐｔｏｃｏｃｃｕｓ ｚｏｏｅｐｌｅｍ
ｉｃｕｓ入ストレプトコッカス・エキシミリス（Ｓｔｒ
ｅｐｔｏｃｏｃｃｕｓｅｑｕｉｓｉｍｉｌｉｓ ） 、
ストレプトコッカス・デイスガラクテイアエ（５ｔｒｅ
ｐｔｏｃｏｃｃｕｓ ｄｙｓｇａｌａｃｔｉａｅ ）、
ストレプトコッカス・ビオケ９ネス（５ｔｒｅｐｔｏｃ
ｏｃｃｕθｐｙｏｇｅｎｅｓ ）などがあげらｊる。な
かでも、ストレプトコッカス−エキが好ましい。またこ
ｎらの菌株から藺導さｎた変異株も使用できる。The culture solution used in the present invention is a culture solution of a microorganism capable of producing hyaluronic acid. As hyaluronic acid producing bacteria,
Streptococcus equi (5treptococcus)
sequi), Streptococcus zooepidemicus (5treptococcus zooeplem)
Streptococcus excimilis (Str
eptococcusequisimilis),
Streptococcus daisgalactiae (5tre
ptococcus dysgalactiae),
Streptococcus bioceae 9nes (5treptoc
occuθpyogenes), etc. Among them, Streptococcus equi is preferred. Mutant strains derived from these strains can also be used.
本発明に用いる培地は通常の微生物の培養に用いるもの
で良く、グルコース、フラクトース、ガラクトース、シ
ュークロース等の炭素源、リン酸第１カリウム、リン酸
第２カリウム、硫酸マグネシウム、亜硫酸ソーダ、チオ
硫酸ソーダ、リン酸アンモニウム等の無機塩類、ポリペ
プトン、カブミノ酸、酵母エキス、コーンステイープリ
カー、大豆加水分解液等の有機栄養源の他、必要に応じ
て、各種アミノ酸、ビタミン類等が好適に用いらｎる。The medium used in the present invention may be one commonly used for culturing microorganisms, and includes carbon sources such as glucose, fructose, galactose, and sucrose, monopotassium phosphate, dipotassium phosphate, magnesium sulfate, sodium sulfite, and thiosulfate. In addition to organic nutritional sources such as inorganic salts such as soda and ammonium phosphate, polypeptone, cabumino acid, yeast extract, cornstarch liquor, and soybean hydrolyzate, various amino acids and vitamins are preferably used as needed. I don't need it.
こｎらの培地成分は、一括仕込、又は分割添加いずれで
も採用可能である。These medium components can be added all at once or added in portions.
本発明の培養は、通気攪拌培養等の公矧の方法でよく、
培ｆ編度は３０〜６５°Ｃが好ましい。The culture of the present invention may be carried out by any conventional method such as aerated agitation culture,
The culture f knitting degree is preferably 30 to 65°C.
培養液の…は菌の生育と共に低下するため、力性ソーダ
、力性カリ、アンモニア等の一調整剤を添加し、ＰＨ７
，５〜９．０好ましくは、ｐＨ８，０〜８．７にコント
ロールする。Since the pH value of the culture solution decreases as the bacteria grow, adjusters such as hydrolytic soda, hydropotassium, and ammonia are added to adjust the pH to 7.
, 5 to 9.0, preferably controlled to pH 8.0 to 8.7.
このように培養すると、ヒアルロン酸の生成と共に培養
液の粘度が次第に上昇してくる。使用炭素源が培養液中
で消費さねるまで培養を行なう。When cultured in this manner, the viscosity of the culture solution gradually increases as hyaluronic acid is produced. Cultivation is carried out until the carbon source used is consumed in the culture medium.
蛋白変性剤の添加は、培養中に適当な時点で行なうこと
ができるが、好ましくは、増殖が静止期に入ってから行
なう。培養終了後に添加するときは、Ｈ製工程中の適当
な時期に行なうことが出来るが、最も好ましいのは、培
養終了直後のぶ加である。The protein denaturing agent can be added at any appropriate time during the culture, but is preferably added after the growth has entered the stationary phase. When adding after culturing, it can be done at any appropriate time during the H production process, but it is most preferable to add it immediately after culturing.
培養終了後に蛋白変性剤を添加するときの、培養液の…
は３．５〜９６口、好ましくは４．０〜７．０である。When adding a protein denaturant after culturing, the culture solution...
is 3.5 to 96 ports, preferably 4.0 to 7.0.
温度は４０℃以下が好ましい。The temperature is preferably 40°C or less.
蛋白変性剤としては、塩酸グアニジン、尿素、界面活性
剤、酸、塩基、有機溶媒、重金属塩、プロテアーゼ等が
挙げらするが、製品への混入、ヒアルロン酸の用途等を
考慮し、好ましいもの全選択する必要がある。Examples of protein denaturing agents include guanidine hydrochloride, urea, surfactants, acids, bases, organic solvents, heavy metal salts, proteases, etc., but all of them are preferred in consideration of contamination with products, use of hyaluronic acid, etc. You need to choose.
好ましい蛋白変性剤は、プロテアーゼ、塩酸グアニジン
、尿素及びラウリル硫酸ナトリウム、ラウリルエーテル
硫條ナトリウム、のような陰イオン界面活性剤であるが
、少量で著しい効果があり、かつ安価であるラウリル硫
酸ナトリウムが最も好ましい。Preferred protein denaturants are protease, guanidine hydrochloride, urea and anionic surfactants such as sodium lauryl sulfate, sodium lauryl ether sulfate, but sodium lauryl sulfate is highly effective in small amounts and is inexpensive. Most preferred.
蛋白変性剤の添加量は、蛋白変性剤の種類によって異な
るが、例えば塩酸グアニジンでは、０．１％〜１０％、
尿素では、１〜５０％、ラウリル硫酸ナトリウムでは０
．００５〜１．０％である。The amount of protein denaturing agent added varies depending on the type of protein denaturing agent, but for example, for guanidine hydrochloride, it is 0.1% to 10%,
1-50% for urea, 0 for sodium lauryl sulfate
．． 0.005 to 1.0%.
蛋白変性剤を添加した培養液から、遠心分離による除菌
後、アルコール等の有機溶媒による析出、限外ろ過によ
る脱塩等の簡単な公知精製法により、高純度、高分子量
ヒアルロン酸として取得できる。High-purity, high-molecular-weight hyaluronic acid can be obtained from a culture solution containing a protein denaturant by simple known purification methods such as sterilization by centrifugation, precipitation with organic solvents such as alcohol, and desalting by ultrafiltration. .
また、使用可能な蛋白変性剤は、限外ろ過による脱塩等
の工程で除去さｎ１裂品には混入しないことが確認され
ている。Furthermore, it has been confirmed that usable protein denaturants do not contaminate N1-cleaved products that have been removed through desalting processes such as ultrafiltration.
次に実施例により本発明の詳細な説明するが本発明はこ
れに限定さｎるものではない。Next, the present invention will be explained in detail with reference to Examples, but the present invention is not limited thereto.
実施例１
グルコース２％、リン酸第１カリウム０．２％、硫酸マ
グネシウム７水墳０．［］ ５５％チオ硫酸ソーダ０．
１％、ポリペプトン１６０％、酵母エキス０．５優から
なるｐＨ８，５の培養液２５０ノに、同一培地からなる
ストレプトコッカス エキ（ｈＴｃａ ９５２７）の前
培養液２５０ｄを接種し、通気量ｉ、５ｖｖｍ。Example 1 Glucose 2%, monopotassium phosphate 0.2%, magnesium sulfate 7 water mounds 0. [ ] 55% Sodium Thiosulfate 0.
1% polypeptone, 160% polypeptone, and 0.5% yeast extract with a pH of 8.5 was inoculated with 250 d of a preculture of Streptococcus equi (hTca 9527) made of the same medium, and the aeration amount was i, 5 vvm.
攪拌６０回転／分、温度３６℃で力性ソーダで一ヲ８．
５にコントロールしながら１５時間培養した。Stir at 60 rpm, temperature 36°C, and mix with sodium hydroxide for 8.
The cells were cultured for 15 hours while controlling at 5.
グルコースが全量消費さ釘た時点で、ラウリル硫酸ナト
リウム１２５ｇを添加し、塩酸でｐ）Ｉ ４．５に調整
後、遠心分離によシ除菌金行なった。得らｔた除菌液ｆ
ｆ１ｌ Ｏｏｏ、、ｚ容のエチルアルコール中に添加し
、ヒアルロン酸ソーダを析出せしめる。こｊをろ別した
後、水に溶解して、分子量分画３万の限外ろ過器にかけ
、脱塩を行なう。さらに遠心分離を行なったのち、エチ
ルアルコールによる析ゾ
出を再度行なう。得らｎたヒアルロン酸ソーダを牢湛で
減圧乾燥して、４２０ｇの白金ヒアルロン酸ソーダを得
た。When the glucose was completely consumed, 125 g of sodium lauryl sulfate was added, the p)I was adjusted to 4.5 with hydrochloric acid, and the mixture was sterilized by centrifugation. The obtained disinfectant solution
f1l Ooo, , is added to z volume of ethyl alcohol to precipitate sodium hyaluronate. After filtering this product, it is dissolved in water and passed through an ultrafilter with a molecular weight fraction of 30,000 for desalting. After further centrifugation, precipitation with ethyl alcohol is performed again. The obtained sodium hyaluronate was dried under reduced pressure in a cellar to obtain 420 g of platinum sodium hyaluronate.
このヒアルロン酸ソーダを０．２ Ｍ ｔ＝化ナナトリ
ウム溶液し、極限粘度法によシ平均分子量を算出した。This sodium hyaluronate was dissolved in a 0.2 M sodium chloride solution, and the average molecular weight was calculated by the intrinsic viscosity method.
粘度測定法は日周一般試験法２８によシ、常法によって
極限粘度〔η〕を算出した。分子量はＷａｎｇＯ式［：
Ｒ，Ｃ１ｅｌ 、 、Ｔ、Ｌ、Ｗａｎｇ 、 Ｂｉｏ
ｐｏｌｙｍｅ１９ ７９９（１９７０ン 〕
分子量Ｍ−（〔η］ ／ ０．０００２２８ ）１・２
２５によシ算出した。The viscosity was measured according to the diurnal general test method 28, and the intrinsic viscosity [η] was calculated by a conventional method. The molecular weight is determined by the WangO formula [:
R,Cel, ,T,L,Wang,Bio.
Polyme19 799 (1970 tons) Molecular weight M-([η] / 0.000228) 1・2
It was calculated based on 25.
この結果、分子量の平均価は、２５０万であった。As a result, the average molecular weight was 2.5 million.
比較例１
ラウリル硫酸ナトリウムを添加しない以外は実施例１と
同様に行なった結果、４３０Ｆの白色ヒアルロン酸ソー
ダ會得、その分子量を測定したところ１００万であった
。Comparative Example 1 The same procedure as in Example 1 was carried out except that sodium lauryl sulfate was not added. As a result, a white sodium hyaluronate solution of 430 F was obtained, and its molecular weight was measured to be 1 million.
実施例２
実施例１において、蛋白変性剤として、表に示すとおり
ラウリル硫酸ナトリウム、塩酸グアニジン、尿素、プロ
テアーゼを各々添加して精製を行ない、分子量分画３万
の限外ろ過器にかけて、脱塩を行なった液につき析出さ
せることなく、そのまま、ｏ、２Ｍ塩化ナトリウム溶液
として、カルバゾール硫酸法によって、ヒアルロン岐濃
度を測定するとともに、極限粘度を測定し、実施例１と
同様に、分子量を算出した。結果を表に示す、実施例３
グルコース２％、リン酸第１カリウム０．２％、硫酸マ
グネシウムと７水塩０．０５％、チオ硫酸ソーダ０．１
％、ポリペプトン１．０％、酵母エキス０．５チからな
るｐＨ８，５の培養液１ノに、同一培地からなるストレ
プトコンカス エキ（ＡＴＣＣ９５２７）の前培養液１
Ｒ１を接種し、通気量１．５ｖｖｍ、攪拌２００回転／
分、編度３３℃で、力性ソーダで一ヲ８．５にコントロ
ールしながら培養した。培養１２時間目に０．５ｇのラ
ウリル硫酸ナトリウムを為加して後、さらに培養を１２
時時間上た。Example 2 In Example 1, purification was performed by adding sodium lauryl sulfate, guanidine hydrochloride, urea, and protease as protein denaturants as shown in the table, and desalted by passing through an ultrafilter with a molecular weight fraction of 30,000. The hyaluronan branch concentration was measured by the carbazole sulfuric acid method without precipitating the solution, and the intrinsic viscosity was measured, and the molecular weight was calculated in the same manner as in Example 1. . The results are shown in the table, Example 3 Glucose 2%, potassium monophosphate 0.2%, magnesium sulfate and heptahydrate 0.05%, sodium thiosulfate 0.1
%, polypeptone 1.0%, yeast extract 0.5%, pH 8.5 culture solution, and the same medium containing 1 pre-culture solution of Streptoconcus Eki (ATCC9527).
Inoculate R1, aeration rate 1.5vvm, stirring 200 rpm/
The cells were cultured at a temperature of 33° C. for 1 minute, controlling the temperature to 8.5 minutes with diluted soda. After 12 hours of culture, 0.5 g of sodium lauryl sulfate was added, and the culture was further continued for 12 hours.
Hours have passed.
培養終了時に、塩酸でＰＨ４，５に調整後、遠心分層に
より除菌を行なった。得られた除菌液全４ノ容のエチル
アルコール中に添加シ１、ヒアルロン酸ソーダを析出せ
しめた。こ−ｉ″Ｌをろ別した後、水に２解して、分子
量分画３万の限外ろ過器にかげ脱Ｆ２ ｋ行なった。At the end of the culture, after adjusting the pH to 4.5 with hydrochloric acid, bacteria were removed by centrifugation. The added sodium hyaluronate was precipitated in a total of 4 volumes of ethyl alcohol of the obtained sterilizing solution. After filtering off this product, it was dissolved in water and subjected to F2k removal using an ultrafilter with a molecular weight fraction of 30,000.
この液につき、各々０．２Ｍ塩化ナトリウム浴液にして
、カルバゾール硫酸法に二つ１、ヒアルロン酸濃度を１
ｆｌ１１定するとともに、僅限帖度と測定し、案３例１
と同様に分子量を算出したところ、２ＡＣ万であった。For this solution, make 0.2M sodium chloride bath solution, 1 for carbazole sulfuric acid method, and 1 for hyaluronic acid concentration.
In addition to determining fl11, it was also determined that the degree of clearance was slight, and the case 1 of plan 3 was determined.
When the molecular weight was calculated in the same manner as above, it was found to be 2 AC.
比較例２
実施例３において、ラウリル硫酸ナトリウム全添加しな
い以外は実施例３と同様に行なったも央、課外ろ過器の
液のヒアルロン酸分子ｆｆ１ｕ、１０５万であった。Comparative Example 2 Example 3 was carried out in the same manner as in Example 3 except that sodium lauryl sulfate was not added at all.The hyaluronic acid molecules ff1u of the liquid from the extracurricular filter were 1,050,000.
〔発明の効果〕
本発明に二り、発酵法によって、大規模スケールで、高
分子量のヒアルロン酸を得ることが出来る。[Effects of the Invention] Second to the present invention, high molecular weight hyaluronic acid can be obtained on a large scale by the fermentation method.
Claims (3)
高分子量ヒアルロン酸を採取するに際し、培養中または
培養終了後に、蛋白変性剤を添加することを特徴とする
高分子量ヒアルロン酸の製法。(1) Cultivating microorganisms capable of producing hyaluronic acid,
A method for producing high molecular weight hyaluronic acid, which comprises adding a protein denaturing agent during or after culturing when collecting high molecular weight hyaluronic acid.
許請求の範囲第（１）項記載の高分子量ヒアルロン酸の
製法。(2) The method for producing high molecular weight hyaluronic acid according to claim (1), wherein the protein denaturant is sodium lauryl sulfate.
トコッカス、エキである特許請求の範囲第（１）項記載
の高分子量ヒアルロン酸の製法。(3) The method for producing high molecular weight hyaluronic acid according to claim (1), wherein the microorganism having the ability to produce hyaluronic acid is Streptococcus or Equisum.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP27647286A JPS63129991A (en) | 1986-11-21 | 1986-11-21 | Production of hyaluronic acid |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP27647286A JPS63129991A (en) | 1986-11-21 | 1986-11-21 | Production of hyaluronic acid |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPS63129991A true JPS63129991A (en) | 1988-06-02 |
Family
ID=17569924
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP27647286A Pending JPS63129991A (en) | 1986-11-21 | 1986-11-21 | Production of hyaluronic acid |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPS63129991A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5529987A (en) * | 1993-08-04 | 1996-06-25 | Patent Biopharmaceutics, Inc. | Hyaluronic acid-urea pharmaceutical compositions and uses |
| US5550112A (en) * | 1992-12-30 | 1996-08-27 | Patent Biopharmaceutics, Inc. | Hyaluronic acid-urea pharmaceutical compositions and uses |
-
1986
- 1986-11-21 JP JP27647286A patent/JPS63129991A/en active Pending
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5550112A (en) * | 1992-12-30 | 1996-08-27 | Patent Biopharmaceutics, Inc. | Hyaluronic acid-urea pharmaceutical compositions and uses |
| US5529987A (en) * | 1993-08-04 | 1996-06-25 | Patent Biopharmaceutics, Inc. | Hyaluronic acid-urea pharmaceutical compositions and uses |
| US5583118A (en) * | 1993-08-04 | 1996-12-10 | Patent Biopharmaceutics, Inc. | Method of treating an anorectal disease using hyaluronic acid-urea pharmaceutical compositions |
| US5583120A (en) * | 1993-08-04 | 1996-12-10 | Patent Biopharmaceutics, Inc. | Hyaluronic acid-urea pharmaceutical compositions and uses |
| US5583119A (en) * | 1993-08-04 | 1996-12-10 | Patent Biopharmaceutics, Inc. | Hyaluronic acid-urea pharmaceutical compositions and uses |
| US5624915A (en) * | 1993-08-04 | 1997-04-29 | Patent Biopharmaceutics, Inc. | Hyaluronic acid-urea pharmaceutical compositions and uses |
| US5631242A (en) * | 1993-08-04 | 1997-05-20 | Patent Biopharmaceutics, Inc. | Hyaluronic acid-urea pharmaceutical compositions utilized for treatment of diseases of cutis |
| US5679655A (en) * | 1993-08-04 | 1997-10-21 | Patent Biopharmaceutics, Inc. | Method of treating lesions resulting from genital herpes with hyaluronic acid-urea pharmaceutical compositions |
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