JPS63245671A - Modified superoxide dismutase - Google Patents
Modified superoxide dismutaseInfo
- Publication number
- JPS63245671A JPS63245671A JP8069887A JP8069887A JPS63245671A JP S63245671 A JPS63245671 A JP S63245671A JP 8069887 A JP8069887 A JP 8069887A JP 8069887 A JP8069887 A JP 8069887A JP S63245671 A JPS63245671 A JP S63245671A
- Authority
- JP
- Japan
- Prior art keywords
- superoxide dismutase
- modified
- polyalkylene glycol
- glycol
- produced
- 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
- 102000019197 Superoxide Dismutase Human genes 0.000 title claims abstract description 53
- 108010012715 Superoxide dismutase Proteins 0.000 title claims abstract description 53
- OUUQCZGPVNCOIJ-UHFFFAOYSA-M Superoxide Chemical class [O-][O] OUUQCZGPVNCOIJ-UHFFFAOYSA-M 0.000 title claims abstract description 27
- 229920001515 polyalkylene glycol Polymers 0.000 claims abstract description 18
- 239000002202 Polyethylene glycol Substances 0.000 claims abstract description 10
- 229920001223 polyethylene glycol Polymers 0.000 claims abstract description 10
- 241000193830 Bacillus <bacterium> Species 0.000 claims abstract description 8
- 229920001577 copolymer Polymers 0.000 claims abstract description 3
- 229920001451 polypropylene glycol Polymers 0.000 claims abstract description 3
- 230000000694 effects Effects 0.000 abstract description 16
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 abstract description 6
- 125000003277 amino group Chemical group 0.000 abstract description 5
- 239000003814 drug Substances 0.000 abstract description 5
- 241000193385 Geobacillus stearothermophilus Species 0.000 abstract description 4
- 239000002537 cosmetic Substances 0.000 abstract description 4
- NXLNNXIXOYSCMB-UHFFFAOYSA-N (4-nitrophenyl) carbonochloridate Chemical compound [O-][N+](=O)C1=CC=C(OC(Cl)=O)C=C1 NXLNNXIXOYSCMB-UHFFFAOYSA-N 0.000 abstract description 3
- 241000894006 Bacteria Species 0.000 abstract description 3
- 239000004471 Glycine Substances 0.000 abstract description 3
- HZAXFHJVJLSVMW-UHFFFAOYSA-N 2-Aminoethan-1-ol Chemical compound NCCO HZAXFHJVJLSVMW-UHFFFAOYSA-N 0.000 abstract description 2
- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 abstract description 2
- 239000004472 Lysine Substances 0.000 abstract description 2
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 abstract description 2
- 230000003213 activating effect Effects 0.000 abstract description 2
- 239000012670 alkaline solution Substances 0.000 abstract description 2
- 150000001875 compounds Chemical class 0.000 abstract description 2
- 230000007935 neutral effect Effects 0.000 abstract description 2
- 239000007767 bonding agent Substances 0.000 abstract 1
- 102000004190 Enzymes Human genes 0.000 description 23
- 108090000790 Enzymes Proteins 0.000 description 23
- 229940088598 enzyme Drugs 0.000 description 23
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 19
- UHOVQNZJYSORNB-UHFFFAOYSA-N Benzene Chemical compound C1=CC=CC=C1 UHOVQNZJYSORNB-UHFFFAOYSA-N 0.000 description 9
- 238000006243 chemical reaction Methods 0.000 description 9
- 239000000243 solution Substances 0.000 description 9
- 230000014759 maintenance of location Effects 0.000 description 8
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 description 7
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 6
- 239000011230 binding agent Substances 0.000 description 6
- 239000008280 blood Substances 0.000 description 6
- 210000004369 blood Anatomy 0.000 description 6
- 238000000034 method Methods 0.000 description 6
- 239000002244 precipitate Substances 0.000 description 6
- 239000000047 product Substances 0.000 description 6
- 239000003208 petroleum Substances 0.000 description 5
- WGCNASOHLSPBMP-UHFFFAOYSA-N hydroxyacetaldehyde Natural products OCC=O WGCNASOHLSPBMP-UHFFFAOYSA-N 0.000 description 4
- -1 Oxyethylene-oxypropylene Chemical group 0.000 description 3
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 description 3
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 3
- 239000013078 crystal Substances 0.000 description 3
- 238000005259 measurement Methods 0.000 description 3
- 239000008363 phosphate buffer Substances 0.000 description 3
- 238000000108 ultra-filtration Methods 0.000 description 3
- JYEUMXHLPRZUAT-UHFFFAOYSA-N 1,2,3-triazine Chemical compound C1=CN=NN=C1 JYEUMXHLPRZUAT-UHFFFAOYSA-N 0.000 description 2
- 241000283690 Bos taurus Species 0.000 description 2
- 102000018832 Cytochromes Human genes 0.000 description 2
- 108010052832 Cytochromes Proteins 0.000 description 2
- 102000004316 Oxidoreductases Human genes 0.000 description 2
- 108090000854 Oxidoreductases Proteins 0.000 description 2
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 2
- 238000002835 absorbance Methods 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- PFKFTWBEEFSNDU-UHFFFAOYSA-N carbonyldiimidazole Chemical compound C1=CN=CN1C(=O)N1C=CN=C1 PFKFTWBEEFSNDU-UHFFFAOYSA-N 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- MGNCLNQXLYJVJD-UHFFFAOYSA-N cyanuric chloride Chemical compound ClC1=NC(Cl)=NC(Cl)=N1 MGNCLNQXLYJVJD-UHFFFAOYSA-N 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 239000000706 filtrate Substances 0.000 description 2
- NUJOXMJBOLGQSY-UHFFFAOYSA-N manganese dioxide Chemical compound O=[Mn]=O NUJOXMJBOLGQSY-UHFFFAOYSA-N 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 238000006011 modification reaction Methods 0.000 description 2
- 239000002504 physiological saline solution Substances 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 238000010992 reflux Methods 0.000 description 2
- 239000012085 test solution Substances 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- 102000015790 Asparaginase Human genes 0.000 description 1
- 108010024976 Asparaginase Proteins 0.000 description 1
- 101100381481 Caenorhabditis elegans baz-2 gene Proteins 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- NQTADLQHYWFPDB-UHFFFAOYSA-N N-Hydroxysuccinimide Chemical compound ON1C(=O)CCC1=O NQTADLQHYWFPDB-UHFFFAOYSA-N 0.000 description 1
- 241000283977 Oryctolagus Species 0.000 description 1
- RVGRUAULSDPKGF-UHFFFAOYSA-N Poloxamer Chemical compound C1CO1.CC1CO1 RVGRUAULSDPKGF-UHFFFAOYSA-N 0.000 description 1
- 239000004698 Polyethylene Substances 0.000 description 1
- 101100372762 Rattus norvegicus Flt1 gene Proteins 0.000 description 1
- 108010092464 Urate Oxidase Proteins 0.000 description 1
- 239000002671 adjuvant Substances 0.000 description 1
- 230000003110 anti-inflammatory effect Effects 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 206010003246 arthritis Diseases 0.000 description 1
- 229960003272 asparaginase Drugs 0.000 description 1
- DCXYFEDJOCDNAF-UHFFFAOYSA-M asparaginate Chemical compound [O-]C(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-M 0.000 description 1
- 229920001400 block copolymer Polymers 0.000 description 1
- 210000004204 blood vessel Anatomy 0.000 description 1
- 229910021538 borax Inorganic materials 0.000 description 1
- 150000001718 carbodiimides Chemical class 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 230000009849 deactivation Effects 0.000 description 1
- 125000004185 ester group Chemical group 0.000 description 1
- 150000002148 esters Chemical class 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 125000000956 methoxy group Chemical group [H]C([H])([H])O* 0.000 description 1
- 239000012046 mixed solvent Substances 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 238000002715 modification method Methods 0.000 description 1
- 239000008055 phosphate buffer solution Substances 0.000 description 1
- 229920001993 poloxamer 188 Polymers 0.000 description 1
- 229920000573 polyethylene Polymers 0.000 description 1
- 229920005604 random copolymer Polymers 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 239000004328 sodium tetraborate Substances 0.000 description 1
- 235000010339 sodium tetraborate Nutrition 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- 125000001424 substituent group Chemical group 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 230000002123 temporal effect Effects 0.000 description 1
- 239000002023 wood Substances 0.000 description 1
Landscapes
- Enzymes And Modification Thereof (AREA)
Abstract
Description
【発明の詳細な説明】
〔産業上の利用分野〕
この発明は、化粧品、医薬品等に利用することのできる
修飾スーパーオキシドジスムターゼに関するものである
。DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] This invention relates to a modified superoxide dismutase that can be used in cosmetics, pharmaceuticals, and the like.
スーパーオキシドジスムターゼは、動物、植物。 Superoxide dismutase is found in animals and plants.
微生物等の生体内に広く分布する酵素である。It is an enzyme that is widely distributed in living organisms such as microorganisms.
最近、上記スーパーオキシドジスムターゼがリュウマチ
や関節炎に対して抗炎症効果を示すとの報告がなされ、
医療面への応用が期待されている。Recently, it has been reported that superoxide dismutase has anti-inflammatory effects on rheumatism and arthritis.
It is expected to have medical applications.
しかしながら、上記人由来以外のスーパーオキシドジス
ムターゼは、生体内で抗原抗体反応を生起させる抗原性
を有するとともに血中半減期が短いため、そのままでは
医薬等に利用することが困難である。これは、スーパー
オキシドジスムターゼを微生物から産生ずる場合に特に
重大な問題とな゛る。However, superoxide dismutase derived from non-human sources has antigenicity that causes an antigen-antibody reaction in vivo and has a short half-life in the blood, so it is difficult to use it as it is in medicine. This is a particularly serious problem when superoxide dismutase is produced from microorganisms.
酵素活性を保持しつつその抗原性を抑制する一例として
、ウリカーゼおよびアスパラギナーゼを、塩化シアヌル
を結合剤としてポリエチレングリコールで修飾する方法
が提案されている（特公昭６１−４２５５８号）、また
、ウシ血液から得たスーパーオキシドジスムターゼにつ
いて、ポリエチレングリコールで修飾すると血中半減期
が延長、改善されることが報告されている（ベロネーズ
他ジャーナル オブ ファーマシイ アンド ファーマ
コロジイ（Ｖｅｒｏｎｅｓｅ Ｆ、Ｍ、ｅｔ ａｌ、
Ｊｏｕｒｎａｌ ｏｆＰｈａｒｍａｃｙ ａｎｄ Ｐｈ
ａｒｍａｃｏｌｏｇｙ ） ３５． ７５７〜７５８
（１９８３）参照）。As an example of suppressing antigenicity while retaining enzyme activity, a method has been proposed in which uricase and asparaginase are modified with polyethylene glycol using cyanuric chloride as a binder (Japanese Patent Publication No. 61-42558). It has been reported that the half-life in the blood of superoxide dismutase obtained from 100% polyethylene glycol is extended and improved by modifying it with polyethylene glycol (Veronese et al., Journal of Pharmacy and Pharmacology).
Journal of Pharmacy and Ph.
armacology) 35. 757-758
(1983)).
しかしながら、これらの方法をバチルス属菌産生のマン
ガン型スーパーオキシドジスムターゼに適用した報告は
見られない。また、スーパーオキシドジスムターゼは、
ウシ血液からは大量生産しに＜＜、バチルス属菌からの
方が量産しやすいという利点を有する。したがって、バ
チルス属菌産生のマンガン型スーパーオキシドジスムタ
ーゼの抗原性を抑制する方法の開発が望まれている。However, there have been no reports on the application of these methods to manganese-type superoxide dismutase produced by Bacillus bacteria. In addition, superoxide dismutase
Bovine blood has the advantage of being easier to mass produce, whereas Bacillus bacteria has the advantage of being easier to mass produce. Therefore, it is desired to develop a method for suppressing the antigenicity of manganese-type superoxide dismutase produced by Bacillus bacteria.
この発明は、このような事情に鑑みなされたもので、抗
原性と血中での代謝性が改善された修飾スーパーオキシ
ドジスムターゼの提供をその目的とする。The present invention was made in view of these circumstances, and its purpose is to provide a modified superoxide dismutase with improved antigenicity and improved metabolism in blood.
上記の目的を達成するため、この発明の修飾スーパーオ
キシドジスムターゼは、バチルス属菌によって産生され
るマンガン型スーパーオキシドジスムターゼにポリアル
キレングリコールを結合させたという構成をとる。In order to achieve the above object, the modified superoxide dismutase of the present invention has a structure in which a polyalkylene glycol is bonded to a manganese-type superoxide dismutase produced by a bacterium belonging to the genus Bacillus.
つぎに、この発明の詳細な説明する。Next, this invention will be explained in detail.
この発明が対象とするスーパーオキシドジスムターゼは
、バチルス属の菌によって産生されるマンガン型スーパ
ーオキシドジスムターゼであり、特にバチルス・ステア
ロサーモフィルス菌からのものが好ましい、このスーパ
ーオキシドジスムターゼは、ウシ血液等から産生される
スーパーオキシドジスムターゼよりも大量生産しやす（
、工業゛的利用に適したものである。The superoxide dismutase targeted by this invention is a manganese-type superoxide dismutase produced by a bacterium belonging to the genus Bacillus, and in particular, one from Bacillus stearothermophilus is preferable. It is easier to produce in large quantities than superoxide dismutase produced from
, suitable for industrial use.
この発明に用いるポリアルキレングリコールとしては、
ポリアルキレングリコール、ポリプロピレングリコール
、エチレンオキサイド−プロピレンオキサイド共重合体
等があげられ、単独で使用しても併用してもよい。なお
、これらのポリアルキレングリコールには、側鎖として
メトキシ基等の置換基が導入されているものも含まれる
。これらはいずれも親木疎水両親媒性を示す。上記ポリ
アルキレングリコールの分子量は特に限定されるもので
はないが、抗原性抑制の観点から１０００以上であるこ
とが好適である。The polyalkylene glycol used in this invention is
Examples include polyalkylene glycol, polypropylene glycol, ethylene oxide-propylene oxide copolymer, etc., and they may be used alone or in combination. Note that these polyalkylene glycols include those in which a substituent such as a methoxy group is introduced as a side chain. All of these exhibit parent wood hydrophobic and amphipathic properties. The molecular weight of the polyalkylene glycol is not particularly limited, but from the viewpoint of suppressing antigenicity, it is preferably 1000 or more.
この発明の修飾スーパーオキシドジスムターゼは、上記
スーパーオキシドジスムターゼと上記ポリアルキレング
リコールとを用い、例えばっぎのようにして製造するこ
とができる。すなわち、まず上記ポリアルキレングリコ
ールを適当な結合剤との結合によって活性化する。そし
て、上記活性化ポリアルキレングリコールの活性部位と
スーパーオキシドジスムターゼのアミノ基とを結合させ
ることにより、スーパーオキシドジスムターゼの修飾を
行う。活性化ポリアルキレングリコールの過剰な活性基
は、上記修飾反応後、リジン、グリシン、アミノエタノ
ール等のアミノ基を有する化合物と反応させて後処理を
行う、また、残存している未反応の結合剤等は、限外濾
過等によって分離除去する。このようにして目的とする
修飾スーパーオキシドジスムターゼを製造することがで
きる。The modified superoxide dismutase of the present invention can be produced using the above-mentioned superoxide dismutase and the above-mentioned polyalkylene glycol, for example, as described below. That is, first, the polyalkylene glycol is activated by binding with a suitable binder. Then, superoxide dismutase is modified by bonding the active site of the activated polyalkylene glycol with the amino group of superoxide dismutase. After the above modification reaction, the excess active groups of the activated polyalkylene glycol are post-treated by reacting them with a compound having an amino group such as lysine, glycine, aminoethanol, etc., and any remaining unreacted binding agent is removed. etc. are separated and removed by ultrafiltration or the like. In this way, the desired modified superoxide dismutase can be produced.
上記製法において、ポリアルキレングリコールを活性化
する結合剤および結合方法は特に限定されるものではな
く、酵素を失活させるような反応条件によるものでなけ
ればどのようなものであってもよい。このような結合剤
としては、例えばｐ−ニトロフェニルクロロホルマート
、２，４．６−トリクロロ−Ｓ−トリアジン、１，１′
−カルボニルジイミダゾール等があげられる。修飾の方
法としては、例えば、ポリアルキレングリコールの末端
水酸基を二酸化マンガン等によりカルボキシル基に変換
したのちこのカルボキシル基と酵素のアミノ基とをカル
ボジイミド等で結合させる方法や、Ｎ−ヒドロキシコハ
ク酸イミド等でカルボキシル基を活性エステルに変えた
のちこのエステル基と酵素のアミノ基とを結合させる方
法等があげられる。In the above production method, the binding agent and binding method for activating the polyalkylene glycol are not particularly limited, and any binding agent may be used as long as the reaction conditions do not deactivate the enzyme. Such binders include, for example, p-nitrophenylchloroformate, 2,4,6-trichloro-S-triazine, 1,1'
-carbonyldiimidazole and the like. Modification methods include, for example, converting the terminal hydroxyl group of polyalkylene glycol into a carboxyl group using manganese dioxide or the like, and then bonding this carboxyl group to the amino group of the enzyme using carbodiimide or the like, or using N-hydroxysuccinimide or the like. Examples include a method in which the carboxyl group is converted into an active ester, and then this ester group is bonded to the amino group of the enzyme.
なお、上記スーパーオキシドジスムターゼとポリアルキ
レングリコールの修飾反応は、酵素の失活を防ぐために
、中性もしくは弱アルカリ溶液中において室温で行うこ
とが好適である。The modification reaction between superoxide dismutase and polyalkylene glycol is preferably carried out in a neutral or weakly alkaline solution at room temperature in order to prevent deactivation of the enzyme.
このようにして得られる修飾スーパーオキシドジスムタ
ーゼは、高い活性を維持しており、しかも、抗原性が殆
どもしくは完全に抑制されている。The modified superoxide dismutase thus obtained maintains high activity and, moreover, has almost or completely suppressed antigenicity.
また、修飾していないスーパーオキシドジスムターゼに
比べて熱安定性が向上する場合も見られる。In addition, there are cases where the thermostability is improved compared to unmodified superoxide dismutase.
し゛たがって、この修飾スーパーオキシドジスムターゼ
は、医薬品、化粧品等に有効に利用することができる。Therefore, this modified superoxide dismutase can be effectively used in pharmaceuticals, cosmetics, etc.
なお、この発明において、スーパーオキシドジスムター
ゼの活性、抗原性、熱安定性の測定は、つぎのようにし
て行った。In this invention, the activity, antigenicity, and thermostability of superoxide dismutase were measured as follows.
く活性の測定〉
キサンチン−キサンチンオキシダーゼによるスーパーオ
キシド生成系にチトクロームＣと酵素または修飾酵素を
共存させチトクロームＣの還元速度を低下させる量を指
標として酵素の活性を測定した。そして、キサンチンー
キサンチンオキシターゼ系で生成させたスーパーオキシ
ドに対しスーパーオキシドジスムターゼ（未修飾）を用
いた場合の測定値を１としてこれに対する百分率で示し
た。Measurement of activity> Cytochrome C and an enzyme or a modifying enzyme were coexisted in a superoxide production system using xanthine-xanthine oxidase, and the activity of the enzyme was measured using the amount that reduces the reduction rate of cytochrome C as an index. The measured value when superoxide dismutase (unmodified) was used for superoxide generated by the xanthine-xanthine oxidase system was set as 1, and the value was expressed as a percentage of this value.
く抗原性の測定〉
まず、つぎのようにして抗血清を調製した。すなわち、
２重量％（以下「％」と略す）のスーパーオキシドジス
ムターゼ生理食塩水１シを（フロイント コンプリート
アジュバント（Ｆｒｅｕｎｄ’ ｓＣｏｍｐｌｅｔｅ
ａｄｊｕｖａｎｔ ） ） ｔｔｌと混和し、この
混和液を、家兎の四肢の足動に各０．２５ｖｎｌずつ皮
下投与した。そして、最初の投与から２週間目に再び同
様の皮下投与を行い、その１週間後にスーパーオキ・シ
トジスムターゼ生理食塩水０．５ｄを静脈内・に注射し
た。さらにその５日後、耳の血管から血液を採取して８
０００ｒｐｍで１０分間遠心分離を行って上清割分の血
清を得た。これをスーパーオキシドジスムターゼに対す
る抗血清とした。Measurement of antigenicity> First, antiserum was prepared as follows. That is,
One volume of 2% by weight (hereinafter abbreviated as "%") superoxide dismutase physiological saline was added (Freund's Complete Adjuvant).
The mixture was subcutaneously administered to each of the four limbs of domestic rabbits at a dose of 0.25 vnl. Two weeks after the first administration, the same subcutaneous administration was performed again, and one week later, 0.5 d of superoxycytodismutase physiological saline was injected intravenously. Furthermore, 5 days later, blood was collected from the ear blood vessel and 8
Centrifugation was performed at 000 rpm for 10 minutes to obtain a portion of the supernatant serum. This was used as an antiserum against superoxide dismutase.
つぎに、所定濃度の修飾酵素溶液（０〜１■プロテイン
／ｆｌｔ１） ０．４ ＭＬｌに上記抗血清０．４−を
加えて３０℃で２時間インキュベーションを行った。Next, 0.4 ml of the above antiserum was added to 0.4 MLl of a modified enzyme solution (0 to 1 protein/flt1) at a predetermined concentration and incubated at 30°C for 2 hours.
そして、生成した沈澱を遠心分離により分取し、０．０
７５Ｍ−リン酸緩衝液（ｐＨ７，８）１櫂りで３回洗浄
したのち０．ｌＮ−ＮａＯＨ３Ｊを加えてこれを溶解し
、２８５ｎｍにおける吸光度を測定した。Then, the generated precipitate was separated by centrifugation, and 0.0
After washing three times with one paddle of 75M phosphate buffer (pH 7,8), 0. This was dissolved by adding 1N-NaOH3J, and the absorbance at 285 nm was measured.
く熱安定性の測定〉
０．０７５Ｍ−リン酸緩衝液（ｐＨ７，８）に修飾酵素
を溶解して０．１■プロテイン／Ｊとして検液を得た。Measurement of thermostability> The modified enzyme was dissolved in 0.075M phosphate buffer (pH 7, 8) to obtain a test solution at a concentration of 0.1 protein/J.
上記検液１０ｒｎ１を０．０７５Ｍ−リン酸緩衝液５．
５ ｍＬに加え、７０℃で所定時間インキュベーション
を行ったのち、前記のようにして溶液中の酵素活性を測
定した。Add the above test solution 10rn1 to 0.075M phosphate buffer solution 5.
After adding 5 mL of the solution and incubating at 70°C for a predetermined time, the enzyme activity in the solution was measured as described above.
つぎに、この発明を実施例にもとづいて説明する。Next, the present invention will be explained based on examples.
〔実施例１〕
ポリエチレングリコール（平均分子量１９００）５．０
ｇ、ｐ−ニトロフェニルクロロホルマート０゜６ｇを無
水アセトニトリル３０ｍＬに溶解したのちこれにトリエ
チルアミン０．３ｇを加えた。この溶液を室温２５℃で
一昼夜攪拌したのちジエチルエーテル２００ｆＲ１−を
加え４℃で一昼夜静置して結晶を析出させた。この結晶
を濾別したのちジエチルエーテル−アセトニトリル混合
溶媒を用いて再結晶を行い、ジエチルエーテルでよく洗
浄してから乾燥し、活性化ポリエチレングリコールの白
色結晶４．５ｇを得た。[Example 1] Polyethylene glycol (average molecular weight 1900) 5.0
After dissolving 0.6 g of p-nitrophenyl chloroformate in 30 mL of anhydrous acetonitrile, 0.3 g of triethylamine was added thereto. This solution was stirred at room temperature of 25° C. for a day and night, and then 200 fR1− of diethyl ether was added and allowed to stand at 4° C. for a day and night to precipitate crystals. After the crystals were filtered, they were recrystallized using a mixed solvent of diethyl ether and acetonitrile, thoroughly washed with diethyl ether, and then dried to obtain 4.5 g of white crystals of activated polyethylene glycol.
一方、バチルス・ステアロサーモフィラス菌から産生さ
れたマンガン型スーパーオキシドジスムターゼ（ユニチ
カ社製、８２００Ｕ／■）５０■を０．０７５Ｍ−リン
酸緩衝液（ｐＨ７，８）２０−に溶解し、上記活性化ポ
リエチレングリコール１００■を室温２５℃で一昼夜攪
拌した。得られた反応液にグリシン０．１ｇを加え５時
間処理反応を行“つたのち溶液を限外濾過により精製、
濃縮し、凍゛結乾燥して修飾スーパーオキシドジスムタ
ーゼを得た。On the other hand, 50 μm of manganese-type superoxide dismutase (manufactured by Unitika, 8200 U/■) produced from Bacillus stearothermophilus was dissolved in 20 μm of 0.075 M phosphate buffer (pH 7,8). 100 μl of the above activated polyethylene glycol was stirred at room temperature of 25° C. all day and night. 0.1 g of glycine was added to the resulting reaction solution, and the reaction was carried out for 5 hours. After that, the solution was purified by ultrafiltration.
It was concentrated and lyophilized to obtain modified superoxide dismutase.
この修飾酵素の活性保持率は７８％であった。The activity retention rate of this modified enzyme was 78%.
〔実施例２〕
ポリエチレングリコールに代えてモノメトキシポリエチ
レングリコール（平均分子量５０００）を用いた。それ
以外は実施例１と同様にして修飾スーパーオキシドジス
ムターゼを得た。[Example 2] Monomethoxypolyethylene glycol (average molecular weight 5000) was used in place of polyethylene glycol. Modified superoxide dismutase was obtained in the same manner as in Example 1 except for the above.
この修飾酵素の活性保持率は７６％であった。The activity retention rate of this modified enzyme was 76%.
〔実施例３〕
ポリエチレングリコールに代えてオキシエチレン−オキ
シプロピレンランダム共重合体（平均分子量３７５０、
三洋化成社製、ニューボール５０ＨＢ５１００）を用い
た。それ以外は実施例１と同様にして修飾スーパーオキ
シドジスムターゼを得た。゛
この修飾酵素の活性保持率は９４％であった。[Example 3] Oxyethylene-oxypropylene random copolymer (average molecular weight 3750,
New Ball 50HB5100 (manufactured by Sanyo Chemical Co., Ltd.) was used. Modified superoxide dismutase was obtained in the same manner as in Example 1 except for the above. ``The activity retention rate of this modified enzyme was 94%.
〔実施例４〕
ポリエチレングリコールに代えてオキシエチレン−オキ
シプロピレンブロック共重合体（平均分子量７５００〜
９０００、旭電化工業社製、プルロニックＦ−６８）を
用いた。それ以外は実施例１と同様にして修飾スーパー
オキシドジスムターゼを得た。[Example 4] Oxyethylene-oxypropylene block copolymer (average molecular weight 7,500~
9000, manufactured by Asahi Denka Kogyo Co., Ltd., Pluronic F-68) was used. Modified superoxide dismutase was obtained in the same manner as in Example 1 except for the above.
この修飾酵素の活性保持率は８０％であった。The activity retention rate of this modified enzyme was 80%.
〔実施例５〕
モノメトキシポリエチレングリコール（平均分子量５０
００）１０．０ｇと２． ４． ６−）リクロローＳ−
トリアジン１．１ｇとを無水ベンゼン８０鑓に溶解し、
これに無水炭酸ナトリウム２．０ｇを加えた。この溶液
を室温２５℃で４８時間攪拌して反応させたのち反応残
留物を濾別した。そして、濾液に石油エーテルを徐々に
滴下して白色沈澱物を得た。この沈澱を再び無水ベンゼ
ン７０Ｗｔ１に溶解し、石油エーテルを徐々に滴下して
再沈澱を行い、石油エーテルでよく洗浄したのち乾燥し
て２−０−メトキシポリエチレングリコール−４，６−
ジクロロ−８−トリアジンを得た。[Example 5] Monomethoxypolyethylene glycol (average molecular weight 50
00) 10.0g and 2. 4. 6-) Rekuro S-
1.1 g of triazine was dissolved in 80 g of anhydrous benzene,
To this was added 2.0 g of anhydrous sodium carbonate. This solution was reacted by stirring at room temperature of 25° C. for 48 hours, and then the reaction residue was filtered off. Then, petroleum ether was gradually added dropwise to the filtrate to obtain a white precipitate. This precipitate was dissolved again in 70 Wt1 of anhydrous benzene, and petroleum ether was gradually added dropwise to re-precipitate. After thoroughly washing with petroleum ether, it was dried to obtain 2-0-methoxypolyethylene glycol-4,6-
Dichloro-8-triazine was obtained.
一方、バチルス・ステアロサーモフィラス菌から産生さ
れたマンガン型スーパーオキシドジスムターゼ（ユニチ
カ社製、８２００Ｕ／■）２５■を０．１Ｍホウ砂水溶
液（ｐＨ９，５）に溶解し、上記活性化ポリエチレング
リコール４００ｏｗを加えて室温２５℃で１時間攪拌し
反応させた。この反応液を限外濾過により精製、濃縮し
、凍結乾燥して修飾スーパーオキシドジスムターゼを得
た。On the other hand, 25 μm of manganese-type superoxide dismutase (manufactured by Unitika, 8200 U/■) produced from Bacillus stearothermophilus was dissolved in a 0.1 M borax aqueous solution (pH 9.5), and the activated polyethylene 400 ow of glycol was added and stirred at room temperature of 25° C. for 1 hour to react. This reaction solution was purified by ultrafiltration, concentrated, and lyophilized to obtain modified superoxide dismutase.
この修飾酵素の活性保持率は７９％であった。The activity retention rate of this modified enzyme was 79%.
〔実施例６〕
ポリエチレングリコール（平均分子ｉｔ５０００）５．
０ｇを無水ベンゼン５０ に溶解し、無水炭酸ナトリウ
ム２．５ｇを加えて８０℃で３０分間加熱還流した。そ
して、２，４．６−）ジクロロ−８−トリアジン１８０
■を加えて一昼夜８０℃で還流上反応を行ったのち反応
残留物を濾別した。そして、濾液に石油エーテルを徐々
に滴下して沈澱を生じさせ、この沈澱物を石油エーテル
でよく洗浄したのち乾燥して２，４−ビス（０−メトキ
シポリエチレングリコール）−６−クロロ−３−）リア
ジンを得た。[Example 6] Polyethylene glycol (average molecule it5000)5.
0 g was dissolved in 50 g of anhydrous benzene, 2.5 g of anhydrous sodium carbonate was added, and the mixture was heated under reflux at 80° C. for 30 minutes. and 2,4,6-)dichloro-8-triazine 180
(2) was added and the reaction was carried out under reflux at 80°C for a day and night, and then the reaction residue was filtered off. Then, petroleum ether was gradually added dropwise to the filtrate to form a precipitate, and this precipitate was thoroughly washed with petroleum ether and then dried. ) obtained riazine.
以下の操作は上記実施例５と同様であるが、２−０−メ
トキシポリエチレングリコール−４，６−ジクロロ−８
−トリアジンに代えて上記２，４−ヒス（０−メトキシ
ポリエチレングリコール）−６−クロロ−８−トリアジ
ンを用いた。このようにして修飾スーパーオキシドジス
ムターゼを得た。The following operations are the same as in Example 5 above, except that 2-0-methoxypolyethylene glycol-4,6-dichloro-8
- The above 2,4-his(0-methoxypolyethylene glycol)-6-chloro-8-triazine was used in place of triazine. In this way, a modified superoxide dismutase was obtained.
この修飾酵素の活性保持率は８１％であった。The activity retention rate of this modified enzyme was 81%.
上記のようにして得られた６種類の実施例品について、
前記の手順に従って抗原性と熱安定性を測定した。その
結果を第１図および第２図に示す・なお、それぞれの図
において、１は実施例１品の特性曲線、２は実施例２品
の特性曲線、３は実施例３品の特性曲線、４は実施例４
品の特性曲線、５は実施例５品の特性曲線、６は実施例
６品の特性曲線、７は未修飾スーパーオキシドジスムタ
ーゼの特性曲線を示している。Regarding the six types of example products obtained as described above,
Antigenicity and thermostability were determined according to the procedure described above. The results are shown in Figures 1 and 2. In each figure, 1 is the characteristic curve of Example 1 product, 2 is the characteristic curve of Example 2 product, 3 is the characteristic curve of Example 3 product, 4 is Example 4
5 shows the characteristic curve of the product of Example 5, 6 shows the characteristic curve of the product of Example 6, and 7 shows the characteristic curve of unmodified superoxide dismutase.
第１図は、修飾酵素添加量と添加によって生成する抗原
抗体反応による免疫複合体量（２８５ｎｍの吸光度）と
の関係を示すグラフである。FIG. 1 is a graph showing the relationship between the amount of modified enzyme added and the amount of immune complexes (absorbance at 285 nm) generated by the antigen-antibody reaction generated by the addition.
このグラフによると、修飾スーパーオキシドジスムター
ゼの抗原性は、修飾の仕方によって多少異なるものの、
未修飾のスーパーオキシドジスムターゼに比べて著しく
低下、あるいは消失していることがわかる。According to this graph, although the antigenicity of modified superoxide dismutase varies somewhat depending on the manner of modification,
It can be seen that the amount of superoxide dismutase is significantly reduced or eliminated compared to unmodified superoxide dismutase.
第２図は、修飾スーパーオキシドジスムターゼ溶液の熱
処理に伴う酵素活性保持率の経時的変化を示すグラフで
ある。FIG. 2 is a graph showing changes over time in enzyme activity retention due to heat treatment of a modified superoxide dismutase solution.
このグラフによると、修飾スーパーオキシドジスムター
ゼの熱安定性は、未修飾のスーパーオキシトジスムター
ゼに比べて向上する場合が多いことがわかる。This graph shows that the thermostability of modified superoxide dismutase is often improved compared to unmodified superoxide dismutase.
以上のように、この発明の修飾スーパーオキシドジスム
ターゼは、バチルス属菌から産生されるマンガン型スー
パーオキシドジスムターゼを原料とするため、ウシ血液
等から産生されたスーパーオキシドジスムターゼと異な
り大量生産が可能で、安全性および安定性に優れており
、化粧品、医薬品等に広く応用することができるもので
ある。As described above, the modified superoxide dismutase of the present invention uses manganese-type superoxide dismutase produced from Bacillus bacteria as a raw material, and therefore, unlike superoxide dismutase produced from bovine blood, etc., it can be mass-produced. It has excellent safety and stability and can be widely applied to cosmetics, pharmaceuticals, etc.
第１図は修飾酵素添加量と免疫複合体量の関係を示す特
性曲線図、第２図は修飾酵素の酵素活性保持率の経時的
変化を示す特性曲線図である。FIG. 1 is a characteristic curve diagram showing the relationship between the amount of modified enzyme added and the amount of immune complex, and FIG. 2 is a characteristic curve diagram showing the temporal change in the enzyme activity retention rate of the modified enzyme.
Claims (2)
パーオキシドジスムターゼにポリアルキレングリコール
を結合させたことを特徴とする修飾スーパーオキシドジ
スムターゼ。(1) A modified superoxide dismutase characterized in that a polyalkylene glycol is bonded to a manganese-type superoxide dismutase produced by Bacillus bacteria.
コール、ポリプロピレングリコールおよびエチレンオキ
サイド−プロピレンオキサイド共重合体からなる群から
選ばれた少なくとも一つのポリアルキレングリコールで
ある特許請求の範囲第１項記載の修飾スーパーオキシド
ジスムターゼ。(2) The modified superoxide dismutase according to claim 1, wherein the polyalkylene glycol is at least one polyalkylene glycol selected from the group consisting of polyethylene glycol, polypropylene glycol, and ethylene oxide-propylene oxide copolymer. .
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP8069887A JPS63245671A (en) | 1987-03-31 | 1987-03-31 | Modified superoxide dismutase |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP8069887A JPS63245671A (en) | 1987-03-31 | 1987-03-31 | Modified superoxide dismutase |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPS63245671A true JPS63245671A (en) | 1988-10-12 |
Family
ID=13725547
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP8069887A Pending JPS63245671A (en) | 1987-03-31 | 1987-03-31 | Modified superoxide dismutase |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPS63245671A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU657695B2 (en) * | 1990-08-03 | 1995-03-23 | Microbiological Research Authority | Pharmaceutical compositions containing superoxide dismutase from bacillus stearothermophilus and bacillus caldotenax |
| US5772996A (en) * | 1990-08-03 | 1998-06-30 | Public Health Laboratory Service Board | Pharmaceutical compositions containing superoxide dismutase from Bacillus Stearothermophilus and Bacillus Caldotenax |
| CN100360666C (en) * | 2005-11-29 | 2008-01-09 | 沈阳农业大学 | Corn SOD molecular modifying agent and modifying process thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS5911180A (en) * | 1982-07-07 | 1984-01-20 | Eisai Co Ltd | Modified elastase and its preparation |
| JPS59204130A (en) * | 1983-04-30 | 1984-11-19 | Nippon Chemiphar Co Ltd | Oral administration drug containing novel plasminogen activator derivative |
| JPS60156395A (en) * | 1984-01-17 | 1985-08-16 | Mihama Hisaharu | Preparation of ester by modification lipase |
-
1987
- 1987-03-31 JP JP8069887A patent/JPS63245671A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS5911180A (en) * | 1982-07-07 | 1984-01-20 | Eisai Co Ltd | Modified elastase and its preparation |
| JPS59204130A (en) * | 1983-04-30 | 1984-11-19 | Nippon Chemiphar Co Ltd | Oral administration drug containing novel plasminogen activator derivative |
| JPS60156395A (en) * | 1984-01-17 | 1985-08-16 | Mihama Hisaharu | Preparation of ester by modification lipase |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU657695B2 (en) * | 1990-08-03 | 1995-03-23 | Microbiological Research Authority | Pharmaceutical compositions containing superoxide dismutase from bacillus stearothermophilus and bacillus caldotenax |
| US5772996A (en) * | 1990-08-03 | 1998-06-30 | Public Health Laboratory Service Board | Pharmaceutical compositions containing superoxide dismutase from Bacillus Stearothermophilus and Bacillus Caldotenax |
| CN100360666C (en) * | 2005-11-29 | 2008-01-09 | 沈阳农业大学 | Corn SOD molecular modifying agent and modifying process thereof |
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