JPH0739377A - Method for producing immobilized body of physiologically active substance - Google Patents
Method for producing immobilized body of physiologically active substanceInfo
- Publication number
- JPH0739377A JPH0739377A JP20835593A JP20835593A JPH0739377A JP H0739377 A JPH0739377 A JP H0739377A JP 20835593 A JP20835593 A JP 20835593A JP 20835593 A JP20835593 A JP 20835593A JP H0739377 A JPH0739377 A JP H0739377A
- Authority
- JP
- Japan
- Prior art keywords
- physiologically active
- active substance
- immobilized
- acid
- film
- 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
- 239000013543 active substance Substances 0.000 title claims abstract description 29
- 238000004519 manufacturing process Methods 0.000 title claims description 6
- TWNQGVIAIRXVLR-UHFFFAOYSA-N oxo(oxoalumanyloxy)alumane Chemical compound O=[Al]O[Al]=O TWNQGVIAIRXVLR-UHFFFAOYSA-N 0.000 claims abstract description 30
- XAGFODPZIPBFFR-UHFFFAOYSA-N aluminium Chemical compound [Al] XAGFODPZIPBFFR-UHFFFAOYSA-N 0.000 claims description 32
- 229910052782 aluminium Inorganic materials 0.000 claims description 32
- 238000010438 heat treatment Methods 0.000 claims description 5
- 230000000694 effects Effects 0.000 abstract description 26
- 230000007423 decrease Effects 0.000 abstract description 7
- 230000003100 immobilizing effect Effects 0.000 abstract description 2
- 239000010408 film Substances 0.000 description 38
- 238000000034 method Methods 0.000 description 21
- 239000010409 thin film Substances 0.000 description 18
- 102000004190 Enzymes Human genes 0.000 description 16
- 108090000790 Enzymes Proteins 0.000 description 16
- 229940088598 enzyme Drugs 0.000 description 16
- 239000000243 solution Substances 0.000 description 16
- 239000007974 sodium acetate buffer Substances 0.000 description 12
- BHZOKUMUHVTPBX-UHFFFAOYSA-M sodium acetic acid acetate Chemical compound [Na+].CC(O)=O.CC([O-])=O BHZOKUMUHVTPBX-UHFFFAOYSA-M 0.000 description 12
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 10
- 239000000126 substance Substances 0.000 description 10
- MUBZPKHOEPUJKR-UHFFFAOYSA-N Oxalic acid Chemical compound OC(=O)C(O)=O MUBZPKHOEPUJKR-UHFFFAOYSA-N 0.000 description 9
- 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 8
- 239000002253 acid Substances 0.000 description 8
- 239000007864 aqueous solution Substances 0.000 description 8
- 239000008103 glucose Substances 0.000 description 8
- 239000000956 alloy Substances 0.000 description 7
- 229910045601 alloy Inorganic materials 0.000 description 7
- 238000006243 chemical reaction Methods 0.000 description 7
- 102000005367 Carboxypeptidases Human genes 0.000 description 6
- 108010006303 Carboxypeptidases Proteins 0.000 description 6
- OFOBLEOULBTSOW-UHFFFAOYSA-N Malonic acid Chemical compound OC(=O)CC(O)=O OFOBLEOULBTSOW-UHFFFAOYSA-N 0.000 description 6
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 6
- 239000007983 Tris buffer Substances 0.000 description 6
- 238000007743 anodising Methods 0.000 description 6
- 239000000872 buffer Substances 0.000 description 6
- 230000002255 enzymatic effect Effects 0.000 description 6
- CPQCSJYYDADLCZ-UHFFFAOYSA-N n-methylhydroxylamine Chemical compound CNO CPQCSJYYDADLCZ-UHFFFAOYSA-N 0.000 description 6
- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Chemical compound [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 description 6
- 238000003756 stirring Methods 0.000 description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 6
- PNEYBMLMFCGWSK-UHFFFAOYSA-N aluminium oxide Inorganic materials [O-2].[O-2].[O-2].[Al+3].[Al+3] PNEYBMLMFCGWSK-UHFFFAOYSA-N 0.000 description 5
- 238000001179 sorption measurement Methods 0.000 description 5
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 4
- 238000002835 absorbance Methods 0.000 description 4
- 239000011148 porous material Substances 0.000 description 4
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 3
- 241000235545 Rhizopus niveus Species 0.000 description 3
- 239000003513 alkali Substances 0.000 description 3
- 230000004888 barrier function Effects 0.000 description 3
- 238000005342 ion exchange Methods 0.000 description 3
- 235000006408 oxalic acid Nutrition 0.000 description 3
- 229910052697 platinum Inorganic materials 0.000 description 3
- XLUMOZQZGPJGTL-ROUUACIJSA-N (4s)-5-[[(1s)-1-carboxy-2-(4-hydroxyphenyl)ethyl]amino]-5-oxo-4-(phenylmethoxycarbonylamino)pentanoic acid Chemical compound N([C@@H](CCC(=O)O)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(O)=O)C(=O)OCC1=CC=CC=C1 XLUMOZQZGPJGTL-ROUUACIJSA-N 0.000 description 2
- OWEGMIWEEQEYGQ-UHFFFAOYSA-N 100676-05-9 Natural products OC1C(O)C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(OC2C(OC(O)C(O)C2O)CO)O1 OWEGMIWEEQEYGQ-UHFFFAOYSA-N 0.000 description 2
- WXHLLJAMBQLULT-UHFFFAOYSA-N 2-[[6-[4-(2-hydroxyethyl)piperazin-1-yl]-2-methylpyrimidin-4-yl]amino]-n-(2-methyl-6-sulfanylphenyl)-1,3-thiazole-5-carboxamide;hydrate Chemical compound O.C=1C(N2CCN(CCO)CC2)=NC(C)=NC=1NC(S1)=NC=C1C(=O)NC1=C(C)C=CC=C1S WXHLLJAMBQLULT-UHFFFAOYSA-N 0.000 description 2
- SDGNNLQZAPXALR-UHFFFAOYSA-N 3-sulfophthalic acid Chemical compound OC(=O)C1=CC=CC(S(O)(=O)=O)=C1C(O)=O SDGNNLQZAPXALR-UHFFFAOYSA-N 0.000 description 2
- 239000004382 Amylase Substances 0.000 description 2
- 102000013142 Amylases Human genes 0.000 description 2
- 108010065511 Amylases Proteins 0.000 description 2
- XKRFYHLGVUSROY-UHFFFAOYSA-N Argon Chemical compound [Ar] XKRFYHLGVUSROY-UHFFFAOYSA-N 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 108010015776 Glucose oxidase Proteins 0.000 description 2
- 239000004366 Glucose oxidase Substances 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- ZDLDXNCMJBOYJV-YFKPBYRVSA-N L-arginine, methyl ester Chemical compound COC(=O)[C@@H](N)CCCN=C(N)N ZDLDXNCMJBOYJV-YFKPBYRVSA-N 0.000 description 2
- GUBGYTABKSRVRQ-PICCSMPSSA-N Maltose Natural products O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-PICCSMPSSA-N 0.000 description 2
- GRYLNZFGIOXLOG-UHFFFAOYSA-N Nitric acid Chemical compound O[N+]([O-])=O GRYLNZFGIOXLOG-UHFFFAOYSA-N 0.000 description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 2
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 description 2
- 241000209140 Triticum Species 0.000 description 2
- 235000021307 Triticum Nutrition 0.000 description 2
- 102000004142 Trypsin Human genes 0.000 description 2
- 108090000631 Trypsin Proteins 0.000 description 2
- MCMNRKCIXSYSNV-UHFFFAOYSA-N Zirconium dioxide Chemical compound O=[Zr]=O MCMNRKCIXSYSNV-UHFFFAOYSA-N 0.000 description 2
- 150000007513 acids Chemical class 0.000 description 2
- 150000007933 aliphatic carboxylic acids Chemical class 0.000 description 2
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 2
- 235000019418 amylase Nutrition 0.000 description 2
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 2
- 108010061696 benzyloxycarbonylglutamyltyrosine Proteins 0.000 description 2
- 238000004132 cross linking Methods 0.000 description 2
- 239000008151 electrolyte solution Substances 0.000 description 2
- 239000011888 foil Substances 0.000 description 2
- 229940116332 glucose oxidase Drugs 0.000 description 2
- 235000019420 glucose oxidase Nutrition 0.000 description 2
- 238000007654 immersion Methods 0.000 description 2
- 229910052500 inorganic mineral Inorganic materials 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- VZCYOOQTPOCHFL-UPHRSURJSA-N maleic acid Chemical compound OC(=O)\C=C/C(O)=O VZCYOOQTPOCHFL-UPHRSURJSA-N 0.000 description 2
- 239000011976 maleic acid Substances 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 239000011707 mineral Substances 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 230000007935 neutral effect Effects 0.000 description 2
- 229910017604 nitric acid Inorganic materials 0.000 description 2
- 239000001301 oxygen Substances 0.000 description 2
- 229910052760 oxygen Inorganic materials 0.000 description 2
- 239000006174 pH buffer Substances 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- 150000003457 sulfones Chemical class 0.000 description 2
- VZCYOOQTPOCHFL-UHFFFAOYSA-N trans-butenedioic acid Natural products OC(=O)C=CC(O)=O VZCYOOQTPOCHFL-UHFFFAOYSA-N 0.000 description 2
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 2
- 239000012588 trypsin Substances 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- YCELPWGPXSJYMB-OWOJBTEDSA-N (e)-2-sulfobut-2-enedioic acid Chemical compound OC(=O)\C=C(/C(O)=O)S(O)(=O)=O YCELPWGPXSJYMB-OWOJBTEDSA-N 0.000 description 1
- JAHNSTQSQJOJLO-UHFFFAOYSA-N 2-(3-fluorophenyl)-1h-imidazole Chemical compound FC1=CC=CC(C=2NC=CN=2)=C1 JAHNSTQSQJOJLO-UHFFFAOYSA-N 0.000 description 1
- BFMVMMMQVFULDO-UHFFFAOYSA-N 2-(sulfomethylidene)butanedioic acid Chemical compound OC(=O)CC(C(O)=O)=CS(O)(=O)=O BFMVMMMQVFULDO-UHFFFAOYSA-N 0.000 description 1
- ULUAUXLGCMPNKK-UHFFFAOYSA-N Sulfobutanedioic acid Chemical compound OC(=O)CC(C(O)=O)S(O)(=O)=O ULUAUXLGCMPNKK-UHFFFAOYSA-N 0.000 description 1
- 238000002441 X-ray diffraction Methods 0.000 description 1
- 150000001450 anions Chemical class 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 229910052786 argon Inorganic materials 0.000 description 1
- 239000002585 base Substances 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 239000000919 ceramic Substances 0.000 description 1
- KRVSOGSZCMJSLX-UHFFFAOYSA-L chromic acid Substances O[Cr](O)(=O)=O KRVSOGSZCMJSLX-UHFFFAOYSA-L 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 238000005868 electrolysis reaction Methods 0.000 description 1
- 239000003792 electrolyte Substances 0.000 description 1
- 238000005538 encapsulation Methods 0.000 description 1
- 239000002532 enzyme inhibitor Substances 0.000 description 1
- 229960002089 ferrous chloride Drugs 0.000 description 1
- AWJWCTOOIBYHON-UHFFFAOYSA-N furo[3,4-b]pyrazine-5,7-dione Chemical compound C1=CN=C2C(=O)OC(=O)C2=N1 AWJWCTOOIBYHON-UHFFFAOYSA-N 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 230000036571 hydration Effects 0.000 description 1
- 238000006703 hydration reaction Methods 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 239000011261 inert gas Substances 0.000 description 1
- NMCUIPGRVMDVDB-UHFFFAOYSA-L iron dichloride Chemical compound Cl[Fe]Cl NMCUIPGRVMDVDB-UHFFFAOYSA-L 0.000 description 1
- 238000002844 melting Methods 0.000 description 1
- 230000008018 melting Effects 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- LVHBHZANLOWSRM-UHFFFAOYSA-N methylenebutanedioic acid Natural products OC(=O)CC(=C)C(O)=O LVHBHZANLOWSRM-UHFFFAOYSA-N 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 230000001590 oxidative effect Effects 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 239000011347 resin Substances 0.000 description 1
- 229920005989 resin Polymers 0.000 description 1
- 239000012266 salt solution Substances 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 239000000377 silicon dioxide Substances 0.000 description 1
Landscapes
- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
- Peptides Or Proteins (AREA)
Abstract
(57)【要約】
【構成】 多孔質陽極酸化アルミニウム膜を３００℃を
越える温度で加熱処理後、これを生理活性物質の固定化
担体として生理活性物質を固定する。
【効果】 生理活性物質の活性が高く、かつ、経時的な
活性低下の少ない生理活性物質の固定化体を得ることが
できる。(57) [Summary] [Structure] The porous anodic aluminum oxide film is heat-treated at a temperature exceeding 300 ° C., and then the physiologically active substance is immobilized using this as a carrier for immobilizing the physiologically active substance. [Effect] It is possible to obtain an immobilized body of a physiologically active substance, which has a high activity of the physiologically active substance and whose activity does not decrease over time.
Description
【０００１】[0001]
【産業上の利用分野】本発明は、耐薬品性に優れた多孔
質陽極酸化アルミニウム膜を使用した生理活性物質固定
化体の製造方法に関する。BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for producing a physiologically active substance-immobilized body using a porous anodic aluminum oxide film having excellent chemical resistance.
【０００２】[0002]
【従来の技術とその課題】酵素、酵素阻害剤、抗原、抗
体、ホルモンなどの生理活性物質をイオン交換能を有す
る樹脂や、シリカ、アルミナなどの無機物に固定化した
ものがバイオリアクタ−、酵素センサ−などに応用され
ている。2. Description of the Related Art Bioreactors and enzymes in which physiologically active substances such as enzymes, enzyme inhibitors, antigens, antibodies and hormones are immobilized on a resin having ion exchange ability or an inorganic substance such as silica or alumina It is applied to sensors.
【０００３】ここで、各種材料に生理活性物質を固定化
する方法としては、（１）架橋法、（２）包括法、
（３）担体結合法などがある。しかし、架橋法や包括法
は、比較的激しい条件下で酵素を固定化するため、固定
化した生理活性物質の活性度が低下する問題がある。ま
た、担体結合法も、共有結合法、イオン結合法、物理的
吸着法に分けられ、用いる基材の特性に合わせて選択さ
れるが、特にイオン結合法と物理吸着法は、比較的温和
な条件下で生理活性物質を固定化できるので、活性の高
い固定化体が得られるものの、結合力が弱いため、経時
により生理活性物質が脱離して活性が低下する問題があ
る。Here, as a method for immobilizing a physiologically active substance on various materials, (1) crosslinking method, (2) encapsulation method,
(3) There is a carrier binding method and the like. However, the cross-linking method and the entrapping method have a problem that the activity of the immobilized physiologically active substance is lowered because the enzyme is immobilized under relatively violent conditions. The carrier binding method is also divided into a covalent binding method, an ionic binding method, and a physical adsorption method, and is selected according to the characteristics of the base material used. In particular, the ionic binding method and the physical adsorption method are relatively mild. Since a physiologically active substance can be immobilized under the conditions, an immobilized substance with high activity can be obtained, but since the binding force is weak, there is a problem that the physiologically active substance is released over time and the activity decreases.
【０００４】そこで、本発明者等は、陽極酸化アルミニ
ウム膜の吸着活性とイオン交換能の特性に着目し、多孔
質陽極酸化アルミニウム膜に生理活性物質を固定してな
る生理活性物質の固定化体について先に提案した（特公
平３−７３２７８号など）。多孔質陽極酸化アルミニウ
ム膜は吸着活性が高く、しかも、イオン交換能を有して
いるため、生理活性物質は多孔質陽極酸化膜の孔に吸着
し、かつ、イオン結合し、それゆえ非常に安定であり、
また、固定化は温和に行えるため、活性が高く経時安定
性に優れている。Therefore, the present inventors have focused on the characteristics of the adsorption activity and ion exchange capacity of the anodic aluminum oxide film, and have immobilized the physiologically active substance on the porous anodic aluminum oxide film. Was previously proposed (Japanese Patent Publication No. 3-73278, etc.). Since the porous anodized aluminum film has a high adsorption activity and also has an ion exchange capacity, the physiologically active substance is adsorbed to the pores of the porous anodized film and is ionically bonded, and therefore is very stable. And
Further, since the immobilization can be carried out mildly, the activity is high and the stability over time is excellent.
【０００５】本発明は、これら先の提案に続くものであ
る。一般に、生理活性物質の固定化体を用いる水溶液
は、生理活性物質の安定なｐＨ領域で用いられ、そのｐ
Ｈは４〜９程度の領域である。これらの領域で生理活性
物質を固定化した多孔質陽極酸化アルミニウム膜を用い
ると、ｐＨ緩衝剤の種類などによっては、ｐＨ４．５以
下あるいは８．５以上の場合において、経時により多孔
質陽極酸化アルミニウム膜の溶解が発生し、固定された
生理活性物質の脱離が発生し、活性の低下が発生するこ
とがある。また、ｐＨ７付近の中性溶液中で、多孔質陽
極酸化アルミニウム膜が水分を吸着し、孔を塞ぐいわゆ
る封孔現象が発生し、生理活性物質の脱離はないもの
の、反応物質との接触が低下し、反応速度を低下させる
こともある。The present invention follows these earlier proposals. In general, an aqueous solution using an immobilized body of a physiologically active substance is used in a stable pH range of the physiologically active substance, and its p
H is an area of about 4 to 9. When a porous anodized aluminum film having a physiologically active substance immobilized in these regions is used, depending on the type of pH buffer and the like, when the pH is 4.5 or less or 8.5 or more, the porous anodized aluminum film is used with time. The dissolution of the membrane may occur, the immobilized physiologically active substance may be released, and the activity may be reduced. In a neutral solution of around pH 7, the porous anodic aluminum oxide film adsorbs water, causing a so-called sealing phenomenon that blocks the pores, and the physiologically active substance is not detached, but contact with the reaction substance is not caused. It may decrease the reaction rate.
【０００６】一般に、硫酸、燐酸等の鉱酸、シュウ酸、
マロン酸、マレイン酸などの脂肪族カルボン酸、スルホ
サルチル酸、スルホフタル酸に少量の硫酸を添加した混
酸中でアルミニウム及びその合金を陽極酸化することに
より、多孔質の酸化皮膜は形成されている。これらの多
孔質酸化皮膜は非晶質であり、水分と陽極酸化に用いる
酸アニオンとを少量吸着している。この多孔質酸化皮膜
は、強酸、強アルカリ水溶液によって溶解することがあ
る。そのため、用いるｐＨ緩衝剤との組合せ如何によっ
て、ｐＨ４．５以下あるいはｐＨ８．５以上で、徐々に
ではあるが溶解することがある。ここで、生理活性物質
の種類によっては、これらのｐＨ領域で安定なものもあ
る。利用価値をより高める上でも耐薬品性の向上が必要
と考えられるところである。尚、ｐＨ７付近の中性水溶
液中で、水を吸着し、この吸着水によって陽極酸化皮膜
の体積が増加し、その結果、孔の径が狭まり、反応物質
との接触性が低下し、反応速度の低下が発生する原因に
ついては、物理的に吸着しているＨ2Ｏが影響し、この
Ｈ2Ｏにより酸アルカリを皮膜中に吸収し、多孔質陽極
酸化アルミニウム膜が溶解し、また、非晶質のため酸化
アルミニウムの結合が低く、吸着されたＨ2Ｏにより酸
化アルミニウムの結合が部分的に切れ、体積が増加する
ものと考えられる。Generally, mineral acids such as sulfuric acid and phosphoric acid, oxalic acid,
A porous oxide film is formed by anodizing aluminum and its alloy in a mixed acid obtained by adding a small amount of sulfuric acid to an aliphatic carboxylic acid such as malonic acid and maleic acid, sulfosalicylic acid, and sulfophthalic acid. These porous oxide films are amorphous and adsorb a small amount of water and acid anions used for anodic oxidation. This porous oxide film may be dissolved by a strong acid or strong alkaline aqueous solution. Therefore, depending on the combination with the pH buffer used, it may dissolve gradually but at a pH of 4.5 or lower or a pH of 8.5 or higher. Here, some physiologically active substances are stable in these pH regions. It is considered necessary to improve chemical resistance in order to further increase the utility value. It should be noted that water is adsorbed in a neutral aqueous solution near pH 7, and the volume of the anodized film is increased by this adsorbed water, resulting in a reduction in the diameter of the pores and a decrease in the contact property with the reaction substance. the cause of decrease in occurs, H 2 O is affected that are physically adsorbed, the H 2 O by absorbing the alkali in the coating, dissolving porous anodic aluminum oxide films, also, non It is considered that the bond of aluminum oxide is low due to the crystallinity, and the bond of aluminum oxide is partially broken by the adsorbed H 2 O to increase the volume.
【０００７】[0007]
【課題を解決するための手段】本発明は、多孔質陽極酸
化アルミニウム膜に含まれている水分とその皮膜の非晶
質に着目し、熱処理により、物理的に吸着しているＨ2
Ｏの除去と酸化アルミニウムの化学結合の強化をなし、
もって、耐薬品性を向上し、経時安定性に優れた生理活
性物質の固定化体の製造方法を提供するものであり、そ
の要旨は、多孔質陽極酸化アルミニウム膜を３００℃を
越える温度で加熱処理後、該多孔質陽極酸化アルミニウ
ム膜に生理活性物質を固定してなることを特徴とする生
理活性物質の固定化体の製造方法である。The present invention focuses on the moisture contained in the porous anodized aluminum film and the amorphous state of the film, and H 2 is physically adsorbed by heat treatment.
It removes O and strengthens the chemical bond of aluminum oxide,
Accordingly, the present invention provides a method for producing an immobilized body of a physiologically active substance having improved chemical resistance and excellent stability over time, the gist of which is to heat a porous anodized aluminum film at a temperature exceeding 300 ° C. A method for producing an immobilized body of a physiologically active substance, which comprises fixing a physiologically active substance on the porous anodized aluminum film after the treatment.
【０００８】固定化担体として使用する多孔質陽極酸化
アルミニウム膜は、公知方法、例えば、硫酸、リン酸、
クロム酸などの鉱酸、シュウ酸、マロン酸、マレイン
酸、イタコン酸などの脂肪属カルボン酸、スルホサルチ
ル酸、スルホフタル酸、スルホマレイン酸、スルホイタ
コン酸、スルホコハク酸等のスルホン酸に少量の硫酸を
添加した混酸の電解液中においてアルミニウムまたはそ
の合金を陽極酸化するとにより形成できるが、多孔質陽
極酸化アルミニウム膜をアルミニウムまたはその合金表
面より得るためには、特公昭４７−４４４４１８号公
報、特公昭４７−４５８５４号公報、特公平２−２５４
１９２号公報等に記載されているようにアルミニウム箔
を陽極酸化させることによりアルミニウム箔全てを酸化
させる方法、特公昭５３−３１８８３号公報に記載され
ているようにアルミニウムまたはその合金を陽極酸化
し、次にこの電解液中で陰極として電気分解を行い、ア
ルミニウムと酸化アルミニウムの間に水素ガスを発生さ
せ、多孔質酸化アルミニウム膜を剥離する方法、ＥＰＡ
１７８８３１に記載されているようにアルミニウムまた
はその合金を陽極酸化し、その後電圧を徐々に降下さ
せ、バリヤ−層を薄くしてから、この電解液中で陰極と
して電気分解を行い、アルミニウムと酸化アルミニウム
の間に水素ガスを発生させ、多孔質酸化アルミニウム膜
を剥離する方法、アルミニウムまたはその合金を陽極酸
化後、選択的にアルミニウムまたはその合金を溶質させ
る方法がある。得られた多孔質陽極酸化アルミニウム膜
の厚さは、１〜２００μｍ程度の厚さであればよいもの
である。また、これらの方法で得られた多孔質陽極酸化
アルミニウム膜は、片側に緻密な酸化皮膜（バリヤ−
層）が残っている場合もあり、目的に応じて、酸、アル
カリに浸漬して除去してもよい。The porous anodic aluminum oxide film used as the immobilization carrier can be prepared by a known method, for example, sulfuric acid, phosphoric acid,
Mineral acids such as chromic acid, aliphatic carboxylic acids such as oxalic acid, malonic acid, maleic acid, and itaconic acid, sulfosalicylic acid, sulfophthalic acid, sulfomaleic acid, sulfoitaconic acid, sulfosuccinic acid, and a small amount of sulfuric acid. It can be formed by anodizing aluminum or its alloy in an electrolytic solution of an added mixed acid, but in order to obtain a porous anodized aluminum film from the surface of aluminum or its alloy, Japanese Patent Publication Nos. 47-44418 and 47-47418 are used. -45854, Japanese Patent Publication No. 2-254
A method of oxidizing all aluminum foil by anodizing the aluminum foil as described in Japanese Patent Publication No. 192, etc., anodizing aluminum or its alloy as described in Japanese Patent Publication No. 53-31883. Next, a method of electrolyzing as a cathode in this electrolytic solution to generate hydrogen gas between aluminum and aluminum oxide and peeling the porous aluminum oxide film, EPA
Aluminum or its alloys are anodized as described in 178831, after which the voltage is gradually reduced to thin the barrier layer and then electrolyzed as a cathode in this electrolyte to form aluminum and aluminum oxide. There is a method in which hydrogen gas is generated during the process to peel off the porous aluminum oxide film, and a method in which aluminum or its alloy is selectively solute after anodizing aluminum or its alloy. The thickness of the obtained porous anodized aluminum film may be about 1 to 200 μm. The porous anodized aluminum film obtained by these methods has a dense oxide film (barrier) on one side.
The layer) may remain, and it may be removed by immersing it in an acid or alkali depending on the purpose.
【０００９】熱処理は３００℃を越える温度で行うが、
１３００℃といった高温でもよい。真空中、大気中、酸
素中、窒素やアルゴン等の不活性ガス雰囲気中で行う。
尚、熱処理によって多孔質陽極酸化アルミニウム膜が変
形することもあるので、アルミナ、ジルコニア等のセラ
ミクス板でサンドイッチして処理を行ってもよい。The heat treatment is performed at a temperature exceeding 300 ° C.
A high temperature such as 1300 ° C may be used. It is performed in vacuum, in the air, in oxygen, or in an atmosphere of an inert gas such as nitrogen or argon.
Since the porous anodized aluminum oxide film may be deformed by the heat treatment, the treatment may be carried out by sandwiching it with a ceramic plate of alumina, zirconia or the like.
【００１０】こうして得た多孔質陽極酸化アルミニウム
膜は、物理的強度が高く、また、耐酸、耐アルカリ性も
向上し、経時的な水和化による膜の体積増加に基づく孔
の狭まりもないことから、経時安定性が高く、広範囲の
条件下で使用できる。The porous anodized aluminum film thus obtained has a high physical strength, has improved acid resistance and alkali resistance, and has no pore narrowing due to the increase in the volume of the film due to hydration over time. It has high stability over time and can be used under a wide range of conditions.
【００１１】また、生理活性物質を固定化担体である多
孔質陽極酸化アルミニウム膜に物理的に吸着、固定化さ
せるには、固定化させる生理活性物質をその安定なｐＨ
域にある塩水溶液や緩衝液に溶解させ、この溶液に固定
化担体を浸漬、撹拌させればよい。吸着させる際の温度
は、生理活性物質の安定温度域であればよく、また、撹
拌時間は、生理活性物質の陽極酸化皮膜への吸着状態を
溶液中の活性量や蛋白量を測定しながら調節すればよ
い。通常は、温度５℃〜３０℃、２〜２４時間の撹拌で
固定化は完了する。In order to physically adsorb and immobilize the physiologically active substance on the porous anodic aluminum oxide film which is the immobilization carrier, the physiologically active substance to be immobilized should have a stable pH.
It may be dissolved in an aqueous salt solution or a buffer solution in the region, and the immobilized carrier may be dipped and stirred in this solution. The temperature for adsorbing may be in the stable temperature range of the physiologically active substance, and the stirring time can be adjusted by adjusting the adsorption state of the physiologically active substance on the anodized film while measuring the active amount and protein amount in the solution. do it. Usually, the immobilization is completed by stirring at a temperature of 5 ° C to 30 ° C for 2 to 24 hours.
【００１２】[0012]
＜実施例１＞２０ｍｍ×２０ｍｍ、厚さ０．５ｍｍのア
ルミニウムの板（９９．８％）を１０％水酸化ナトリウ
ムに１０秒浸漬し（５０℃）、その後３０％硝酸に浸漬
した（前処理）。その後１．５ｍｏｌ／Ｌの硫酸水溶液
中で、白金をカソ−ドとし、１５℃、２０Ｖの定電圧に
て１時間陽極酸化することによりアルミニウム表面に透
明な多孔質の酸化皮膜を形成し、その後電圧を２Ｖずつ
低下させ、バリヤ−層を薄くした。その後、カソ−ドを
酸化皮膜を形成したアルミニウム、アノ−ドを白金と
し、電気分解をすることによりアルミニウムと酸化皮膜
の間に水素を発生させ、厚さ５０μｍで透明な多孔質陽
極酸化アルミニウム膜を得た。Example 1 An aluminum plate (99.8%) having a size of 20 mm × 20 mm and a thickness of 0.5 mm was dipped in 10% sodium hydroxide for 10 seconds (50 ° C.), and then dipped in 30% nitric acid (pretreatment. ). Then, in a 1.5 mol / L sulfuric acid aqueous solution, platinum was used as a cathode and anodized at a constant voltage of 20 V at 15 ° C. for 1 hour to form a transparent porous oxide film on the aluminum surface. The voltage was reduced by 2 V to thin the barrier layer. After that, the cathode is made of aluminum having an oxide film, the anode is made of platinum, and hydrogen is generated between the aluminum and the oxide film by electrolysis to give a transparent porous anodized aluminum film having a thickness of 50 μm. Got
【００１３】この多孔質陽極酸化アルミニウム膜をアル
ミナ基盤にサンドイッチし、電気炉で５０℃／時の速度
で昇温し、６００℃で１時間熱処理を行った。得られた
ものは、ｐＨ３およびｐＨ９の水溶液に４時間浸漬して
も重量変化はなく、耐薬品性の高いことが確認された。This porous anodized aluminum film was sandwiched on an alumina substrate, heated in an electric furnace at a rate of 50 ° C./hour, and heat-treated at 600 ° C. for 1 hour. It was confirmed that the obtained product did not change in weight even when immersed in an aqueous solution of pH 3 and pH 9 for 4 hours, and had high chemical resistance.
【００１４】これを純水にて十分水洗後、小麦カルボキ
シペプチターゼＷ（ぺんてる（株）製、発売元：生化学
工業（株））２ｍｇを１０ｍｌの５０ｍＭ酢酸−酢酸ナ
トリウム緩衝液（ｐＨ３．０）に溶解させた液に２０℃
にて３時間浸漬撹拌し、その後、５０ｍｌの５０ｍＭ酢
酸−酢酸ナトリウム緩衝液（ｐＨ３．０）で２回洗浄
し、カルボキシペプチターゼＷ固定化酸化アルミニウム
薄膜を得た。After thoroughly washing this with pure water, 2 mg of wheat carboxypeptidase W (manufactured by Pentel Co., Ltd., distributor: Seikagaku Corporation) was added to 10 ml of 50 mM acetic acid-sodium acetate buffer (pH 3.0). ) In a liquid dissolved in
After 3 hours of immersion and stirring, the mixture was washed twice with 50 ml of 50 mM acetic acid-sodium acetate buffer (pH 3.0) to obtain a carboxypeptidase W-immobilized aluminum oxide thin film.
【００１５】１ｍＭになるようにベンジルオキシカルボ
ニル−グルタミル−チロシンを５０ｍＭ酢酸−酢酸ナト
リウム緩衝液（ｐＨ３．０）に溶解し、この溶液１０ｍ
ｌにカルボキシペプチターゼＷ固定化酸化アルミニウム
薄膜を浸漬し、３０℃で１時間反応させ、５７０nmの吸
光度測定にて酵素活性を求めた。１日１回５日間連続操
作して酵素活性を求め、また経時的な残存酵素活性
（％）を求めた結果を表１に示す。Benzyloxycarbonyl-glutamyl-tyrosine was dissolved in 50 mM acetic acid-sodium acetate buffer solution (pH 3.0) so as to be 1 mM, and the solution was 10 m.
The aluminum oxide thin film on which carboxypeptidase W was immobilized was immersed in 1 and reacted at 30 ° C. for 1 hour to measure the enzyme activity by measuring the absorbance at 570 nm. Table 1 shows the results of determining the enzyme activity by continuously operating once a day for 5 days and determining the residual enzyme activity (%) over time.
【００１６】[0016]
【表１】 [Table 1]
【００１７】また、トリプシン（Ｓｉｇｍａ社製）１ｍ
ｇを１０ｍｌのトリス（ヒドロキシアミノメタン）緩衝
液（ｐＨ９．０）に溶解させ、この溶液に上記の多孔質
酸化アルミニウム薄膜を４℃にて１２時間浸漬撹拌し、
次いでこの薄膜を１００ｍｌの０．０４Ｍトリス（ヒド
ロキシアミノメタン）緩衝液（ｐＨ９．０）で２回洗浄
し、トリプシン固定化薄膜を得た。ＴＡＭＥ（ｐ−トル
エンスルフォンアルギニンメチルエステル）を０．０４
Ｍトリス（ヒドロキシアミノメタン）緩衝液（ｐＨ９．
０）に溶解し、１ｍＭのＴＡＭＥ溶液（ｐＨ９．０）を
調製し、この溶液２０ｍｌにトリプシン固定化薄膜を浸
漬し、３０℃で１０分間反応させ、酵素活性を２４７ｎ
ｍの吸光度の増加にて測定した。１日１回５日間連続操
作して酵素活性を求め、また経時的な残存酵素活性
（％）を求めた結果を表２に示す。Also, trypsin (manufactured by Sigma) 1 m
g was dissolved in 10 ml of tris (hydroxyaminomethane) buffer (pH 9.0), and the above porous aluminum oxide thin film was immersed and stirred in this solution at 4 ° C. for 12 hours,
Next, this thin film was washed twice with 100 ml of 0.04 M tris (hydroxyaminomethane) buffer (pH 9.0) to obtain a trypsin-immobilized thin film. TAME (p-toluene sulfone arginine methyl ester) 0.04
M Tris (hydroxyaminomethane) buffer (pH 9.
0) to prepare a 1 mM TAME solution (pH 9.0), the trypsin-immobilized thin film is immersed in 20 ml of this solution, and the reaction is carried out at 30 ° C. for 10 minutes to obtain an enzyme activity of 247 n.
It was measured by the increase in absorbance at m. Table 2 shows the results of the enzymatic activity determined by continuous operation once a day for 5 days and the residual enzymatic activity (%) over time.
【００１８】[0018]
【表２】 [Table 2]
【００１９】また、リゾプスニベウス（Rhizopusniveu
s）のグルコースアミラーゼ（生化学工業（株）製）１
ｍｇを５０ｍＭ酢酸−酢酸ナトリウム緩衝液（ｐＨ４．
５）２０ｍｌに溶解させ、この溶液に上記多孔質酸化ア
ルミニウム薄膜を４℃にて４時間浸漬撹拌し、その後５
０ｍｌの５０ｍＭ酢酸−酢酸ナトリウム緩衝液（ｐＨ
４．５）で２回洗浄し、グルコースアミラーゼ固定化酸
化アルミニウム薄膜を得た。１％となるようにマルトー
ス（Grade３Ｈ：生化学工業（株）製）を５０ｍＭ酢酸
−酢酸ナトリウム緩衝液（ｐＨ４．５）に溶解し、この
溶液２０ｍｌにグルコースアミラーゼ固定化酸化アルミ
ニウム薄膜を浸漬し、３０℃で３０分間反応させた。生
成するグルコースをグルコースオキシターゼペルオキシ
ターゼ−Ｏ−ジアニシジン法により定量することにより
酵素活性を測定した。１日１回５日間連続操作して酵素
活性を求め、また経時的な残存酵素活性（％）を求めた
結果を表３に示す。In addition, Rhizopus niveu
s) glucose amylase (manufactured by Seikagaku Corporation) 1
mg to 50 mM acetic acid-sodium acetate buffer (pH 4.
5) Dissolve in 20 ml, dip and stir the porous aluminum oxide thin film in this solution at 4 ° C. for 4 hours, and then
0 ml of 50 mM acetic acid-sodium acetate buffer (pH
It was washed twice with 4.5) to obtain a glucose amylase-immobilized aluminum oxide thin film. Maltose (Grade 3H: manufactured by Seikagaku Corporation) was dissolved in 50 mM acetic acid-sodium acetate buffer (pH 4.5) so that the concentration became 1%, and 20 ml of this solution was dipped with a glucose amylase-immobilized aluminum oxide thin film, The reaction was carried out at 30 ° C for 30 minutes. The enzyme activity was measured by quantifying the produced glucose by the glucose oxidase peroxidase-O-dianisidine method. Table 3 shows the results of the enzymatic activity determined by continuous operation once a day for 5 days and the residual enzymatic activity (%) over time.
【００２０】[0020]
【表３】 [Table 3]
【００２１】＜実施例２＞２０ｍｍ×２０ｍｍ、厚さ
０．５ｍｍのアルミニウムの板（９９．８％）を１０％
水酸化ナトリウムに１０秒浸漬し（５０℃）、その後３
０％硝酸に浸漬した（前処理）。その後０．３ｍｏｌ／
Ｌのシュウ酸水溶液で、白金をカソ−ドとし、１５℃、
６０Ｖの定電圧で１時間陽極酸化することによりアルミ
ニウム表面に薄い黄色に発色した多孔質の酸化皮膜を形
成し、その後塩化第一鉄と塩酸を含む１０℃の水溶液に
浸漬することにより選択的にアルミニウムを溶解させ、
４５μｍの厚さの薄い黄色に発色した多孔質陽極酸化ア
ルミニウム膜を得た。Example 2 An aluminum plate (99.8%) having a size of 20 mm × 20 mm and a thickness of 0.5 mm is 10%.
Soak in sodium hydroxide for 10 seconds (50 ° C), then 3
It was immersed in 0% nitric acid (pretreatment). Then 0.3 mol /
L with an aqueous solution of oxalic acid, using platinum as a cathode, at 15 ° C,
By anodizing at a constant voltage of 60 V for 1 hour, a porous oxide film that develops a pale yellow color is formed on the aluminum surface, and then it is selectively immersed in an aqueous solution containing ferrous chloride and hydrochloric acid at 10 ° C. Melting aluminum,
A porous anodized aluminum film having a light yellow color and a thickness of 45 μm was obtained.
【００２２】この多孔質陽極酸化アルミニウム膜をアル
ミナ基盤にサンドイッチし、電気炉で酸素を１０ｍｌ／
秒で流しながら、５０℃／時の速度で昇温し、８００
℃、１時間熱処理を行った。得られたものは透明で、皮
膜はＸ線回折で分析するとγ−アルミナであることが確
認された。また、ｐＨ３およびｐＨ９の水溶液に４時間
浸漬しても重量変化はなく、耐薬品性の高いことも確認
された。This porous anodic aluminum oxide film was sandwiched on an alumina substrate, and oxygen was added in an electric furnace at 10 ml /
While flowing for 2 seconds, raise the temperature at a rate of 50 ° C / hour to 800
Heat treatment was performed at 1 ° C. for 1 hour. The obtained product was transparent, and the film was confirmed to be γ-alumina by X-ray diffraction analysis. It was also confirmed that there was no weight change even when immersed in an aqueous solution of pH 3 and pH 9 for 4 hours, and high chemical resistance.
【００２３】これを純水にて十分水洗後、小麦カルボキ
シペプチターゼＷ（ぺんてる（株）製、発売元：生化学
工業（株））２ｍｇを１０ｍｌの５０ｍＭ酢酸−酢酸ナ
トリウム緩衝液（ｐＨ３．０）に溶解させた液に２０℃
にて３時間浸漬撹拌し、その後、５０ｍｌの５０ｍＭ酢
酸−酢酸ナトリウム緩衝液（ｐＨ３．０）で２回洗浄
し、カルボキシペプチターゼＷ固定化酸化アルミニウム
薄膜を得た。After thoroughly washing this with pure water, 2 mg of wheat carboxypeptidase W (manufactured by Pentel Co., Ltd., distributor: Seikagaku Corporation) was added to 10 ml of 50 mM acetic acid-sodium acetate buffer (pH 3.0). ) In a liquid dissolved in
After 3 hours of immersion and stirring, the mixture was washed twice with 50 ml of 50 mM acetic acid-sodium acetate buffer (pH 3.0) to obtain a carboxypeptidase W-immobilized aluminum oxide thin film.
【００２４】１ｍＭになるようにベンジルオキシカルボ
ニル−グルタミル−チロシンを５０ｍＭ酢酸−酢酸ナト
リウム緩衝液（ｐＨ３．０）に溶解し、この溶液１０ｍ
ｌにカルボキシペプチターゼＷ固定化酸化アルミニウム
薄膜を浸漬し、３０℃で１時間反応させ、５７０nmの吸
光度測定にて酵素活性を求めた。１日１回５日間連続操
作して酵素活性を求め、また経時的な残存酵素活性
（％）を求めた結果を表４に示す。Benzyloxycarbonyl-glutamyl-tyrosine was dissolved in 50 mM acetic acid-sodium acetate buffer (pH 3.0) so that the concentration was 1 mM, and the solution was 10 m.
The aluminum oxide thin film on which carboxypeptidase W was immobilized was immersed in 1 and reacted at 30 ° C. for 1 hour to measure the enzyme activity by measuring the absorbance at 570 nm. Table 4 shows the results of determining the enzyme activity by continuously operating once a day for 5 days and determining the residual enzyme activity (%) over time.
【００２５】[0025]
【表４】 [Table 4]
【００２６】また、トリプシン（Ｓｉｇｍａ社製）１ｍ
ｇを１０ｍｌのトリス（ヒドロキシアミノメタン）緩衝
液（ｐＨ９．０）に溶解させ、この溶液に上記の多孔質
酸化アルミニウム薄膜を４℃にて１２時間浸漬撹拌し、
次いでこの薄膜を１００ｍｌの０．０４Ｍトリス（ヒド
ロキシアミノメタン）緩衝液（ｐＨ９．０）で２回洗浄
し、トリプシン固定化薄膜を得た。ＴＡＭＥ（ｐ−トル
エンスルフォンアルギニンメチルエステル）を０．０４
Ｍトリス（ヒドロキシアミノメタン）緩衝液（ｐＨ９．
０）に溶解し、１ｍＭのＴＡＭＥ溶液（ｐＨ９．０）を
調製し、この溶液２０ｍｌにトリプシン固定化薄膜を浸
漬し、３０℃で１０分間反応させ、酵素活性を２４７ｎ
ｍの吸光度の増加にて測定した。１日１回５日間連続操
作して酵素活性を求め、また経時的な残存酵素活性
（％）を求めた結果を表５に示す。Also, trypsin (manufactured by Sigma) 1 m
g was dissolved in 10 ml of tris (hydroxyaminomethane) buffer (pH 9.0), and the above porous aluminum oxide thin film was immersed and stirred in this solution at 4 ° C. for 12 hours,
Next, this thin film was washed twice with 100 ml of 0.04 M tris (hydroxyaminomethane) buffer (pH 9.0) to obtain a trypsin-immobilized thin film. TAME (p-toluene sulfone arginine methyl ester) 0.04
M Tris (hydroxyaminomethane) buffer (pH 9.
0) to prepare a 1 mM TAME solution (pH 9.0), the trypsin-immobilized thin film is immersed in 20 ml of this solution, and the reaction is carried out at 30 ° C. for 10 minutes to obtain an enzyme activity of 247 n.
It was measured by the increase in absorbance at m. Table 5 shows the results of the enzymatic activity obtained by continuously operating once a day for 5 days and the residual enzymatic activity (%) over time.
【００２７】[0027]
【表５】 [Table 5]
【００２８】また、リゾプスニベウス（Rhizopusniveu
s）のグルコースアミラーゼ（生化学工業（株）製）１
ｍｇを５０ｍＭ酢酸−酢酸ナトリウム緩衝液（ｐＨ４．
５）２０ｍｌに溶解させ、この溶液に上記多孔質酸化ア
ルミニウム薄膜を４℃にて４時間浸漬撹拌し、その後５
０ｍｌの５０ｍＭ酢酸−酢酸ナトリウム緩衝液（ｐＨ
４．５）で２回洗浄し、グルコースアミラーゼ固定化酸
化アルミニウム薄膜を得た。１％となるようにマルトー
ス（Grade３Ｈ：生化学工業（株）製）を５０ｍＭ酢酸
−酢酸ナトリウム緩衝液（ｐＨ４．５）に溶解し、この
溶液２０ｍｌにグルコースアミラーゼ固定化酸化アルミ
ニウム薄膜を浸漬し、３０℃で３０分間反応させた。生
成するグルコースをグルコースオキシターゼペルオキシ
ターゼ−Ｏ−ジアニシジン法により定量することにより
酵素活性を測定した。１日１回５日間連続操作して酵素
活性を求め、また経時的な残存酵素活性（％）を求めた
結果を表６に示す。Further, Rhizopus niveu (Rhizopus niveu)
s) glucose amylase (manufactured by Seikagaku Corporation) 1
mg to 50 mM acetic acid-sodium acetate buffer (pH 4.
5) Dissolve in 20 ml, dip and stir the porous aluminum oxide thin film in this solution at 4 ° C. for 4 hours, and then
0 ml of 50 mM acetic acid-sodium acetate buffer (pH
It was washed twice with 4.5) to obtain a glucose amylase-immobilized aluminum oxide thin film. Maltose (Grade 3H: manufactured by Seikagaku Corporation) was dissolved in 50 mM acetic acid-sodium acetate buffer (pH 4.5) so that the concentration became 1%, and 20 ml of this solution was dipped with a glucose amylase-immobilized aluminum oxide thin film, The reaction was carried out at 30 ° C for 30 minutes. The enzyme activity was measured by quantifying the produced glucose by the glucose oxidase peroxidase-O-dianisidine method. Table 6 shows the results of determining the enzyme activity by continuously operating once a day for 5 days and determining the residual enzyme activity (%) over time.
【００２９】[0029]
【表６】 [Table 6]
【００３０】[0030]
【発明の効果】上記各表は、各例のものが５日後でも良
好な残存酵素活性を有し、経時的な活性低下が防止され
ているという結果を示すが、このように、本発明の製造
方法により得られた生理活性物質の固定化体は、生理活
性物質の活性が高く、かつ、経時的な活性低下の少ない
ものとなる。INDUSTRIAL APPLICABILITY The above-mentioned tables show the results that each of the examples had a good residual enzyme activity even after 5 days, and the decrease in activity over time was prevented. The immobilized body of the physiologically active substance obtained by the production method has a high activity of the physiologically active substance and less decrease in activity over time.
Claims (1)
℃を越える温度で加熱処理後、該多孔質陽極酸化アルミ
ニウム膜に生理活性物質を固定してなることを特徴とす
る生理活性物質の固定化体の製造方法。1. A porous anodic aluminum oxide film 300
A method for producing an immobilized body of a physiologically active substance, which comprises subjecting the porous anodized aluminum film to a physiologically active substance after heat treatment at a temperature exceeding ° C.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP20835593A JP3154205B2 (en) | 1993-07-30 | 1993-07-30 | Method for producing immobilized body of physiologically active substance |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP20835593A JP3154205B2 (en) | 1993-07-30 | 1993-07-30 | Method for producing immobilized body of physiologically active substance |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH0739377A true JPH0739377A (en) | 1995-02-10 |
| JP3154205B2 JP3154205B2 (en) | 2001-04-09 |
Family
ID=16554923
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP20835593A Expired - Fee Related JP3154205B2 (en) | 1993-07-30 | 1993-07-30 | Method for producing immobilized body of physiologically active substance |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP3154205B2 (en) |
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1993
- 1993-07-30 JP JP20835593A patent/JP3154205B2/en not_active Expired - Fee Related
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| Publication number | Publication date |
|---|---|
| JP3154205B2 (en) | 2001-04-09 |
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