JPH0448437B2 - - Google Patents
Info
- Publication number
- JPH0448437B2 JPH0448437B2 JP62248215A JP24821587A JPH0448437B2 JP H0448437 B2 JPH0448437 B2 JP H0448437B2 JP 62248215 A JP62248215 A JP 62248215A JP 24821587 A JP24821587 A JP 24821587A JP H0448437 B2 JPH0448437 B2 JP H0448437B2
- Authority
- JP
- Japan
- Prior art keywords
- enzyme
- immobilized
- membrane
- silk fibroin
- solution
- 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.)
- Expired - Lifetime
Links
- 239000012528 membrane Substances 0.000 claims description 66
- 108010022355 Fibroins Proteins 0.000 claims description 44
- 108090000790 Enzymes Proteins 0.000 claims description 43
- 102000004190 Enzymes Human genes 0.000 claims description 43
- 229940088598 enzyme Drugs 0.000 claims description 43
- 238000011282 treatment Methods 0.000 claims description 16
- 239000000243 solution Substances 0.000 claims description 12
- 238000004519 manufacturing process Methods 0.000 claims description 10
- 108010015776 Glucose oxidase Proteins 0.000 claims description 9
- 239000004366 Glucose oxidase Substances 0.000 claims description 9
- 229940116332 glucose oxidase Drugs 0.000 claims description 9
- 235000019420 glucose oxidase Nutrition 0.000 claims description 9
- 239000011259 mixed solution Substances 0.000 claims description 2
- 239000000203 mixture Substances 0.000 claims 1
- 230000006641 stabilisation Effects 0.000 claims 1
- 238000011105 stabilization Methods 0.000 claims 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 24
- 239000008103 glucose Substances 0.000 description 24
- 230000000694 effects Effects 0.000 description 23
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 21
- 241000255789 Bombyx mori Species 0.000 description 18
- 238000010586 diagram Methods 0.000 description 12
- 238000006243 chemical reaction Methods 0.000 description 9
- 238000005259 measurement Methods 0.000 description 9
- 230000035699 permeability Effects 0.000 description 6
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 5
- 238000000034 method Methods 0.000 description 5
- 239000001301 oxygen Substances 0.000 description 5
- 229910052760 oxygen Inorganic materials 0.000 description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 5
- 238000005481 NMR spectroscopy Methods 0.000 description 4
- 239000007864 aqueous solution Substances 0.000 description 3
- 230000008901 benefit Effects 0.000 description 3
- 238000004737 colorimetric analysis Methods 0.000 description 3
- 238000010828 elution Methods 0.000 description 3
- 238000003028 enzyme activity measurement method Methods 0.000 description 3
- 230000007774 longterm Effects 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 2
- 108010093096 Immobilized Enzymes Proteins 0.000 description 2
- 239000004809 Teflon Substances 0.000 description 2
- 229920006362 Teflon® Polymers 0.000 description 2
- 230000003247 decreasing effect Effects 0.000 description 2
- 238000002329 infrared spectrum Methods 0.000 description 2
- AMXOYNBUYSYVKV-UHFFFAOYSA-M lithium bromide Chemical compound [Li+].[Br-] AMXOYNBUYSYVKV-UHFFFAOYSA-M 0.000 description 2
- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Chemical compound [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 230000004044 response Effects 0.000 description 2
- 238000001228 spectrum Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- 230000007704 transition Effects 0.000 description 2
- 241000228245 Aspergillus niger Species 0.000 description 1
- 241000255794 Bombyx mandarina Species 0.000 description 1
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- NIXOWILDQLNWCW-UHFFFAOYSA-N acrylic acid group Chemical group C(C=C)(=O)O NIXOWILDQLNWCW-UHFFFAOYSA-N 0.000 description 1
- 150000001408 amides Chemical class 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 238000009792 diffusion process Methods 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- 150000002303 glucose derivatives Chemical class 0.000 description 1
- 125000002791 glucosyl group Chemical group C1([C@H](O)[C@@H](O)[C@H](O)[C@H](O1)CO)* 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 230000003100 immobilizing effect Effects 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 238000000691 measurement method Methods 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 238000000655 nuclear magnetic resonance spectrum Methods 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 229910052697 platinum Inorganic materials 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 238000002791 soaking Methods 0.000 description 1
- 239000000344 soap Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 238000012916 structural analysis Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09J—ADHESIVES; NON-MECHANICAL ASPECTS OF ADHESIVE PROCESSES IN GENERAL; ADHESIVE PROCESSES NOT PROVIDED FOR ELSEWHERE; USE OF MATERIALS AS ADHESIVES
- C09J4/00—Adhesives based on organic non-macromolecular compounds having at least one polymerisable carbon-to-carbon unsaturated bond ; adhesives, based on monomers of macromolecular compounds of groups C09J183/00 - C09J183/16
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
Description
［産業上の利用分野］
本発明は、酵素固定化膜及びその製造方法、特
に絹フイブロインの製造特性を生かした酵素固定
化膜及び製造方法に関するものである。
［従来の技術］
従来、包括法で酵素の固定化を行う場合、固定
化担体には、不溶化のためにグルタルアルデヒド
等の架橋処理が必要である場合が多く、熱、強
酸、強アルカリ、有機溶媒等による二次的な酵素
失活が免れず、１ケ月も経過すると膜中の酵素活
性はほとんどなくなるという欠点があつた。
［発明が解決しようとする課題］
本発明の目的は、絹フイブロインをその構造特
性を生かして酵素の包括固定化材料とすることに
より、酵素含有膜の活性が高く、酵素の溶出の極
めて少ない、長期間酵素活性を有する酵素固定化
膜及びその製造方法を提供することにある。
［課題を解決するための手段及び作用］
この問題点を解決するために、本発明の酵素固
定化膜は、延伸処理により固定化絹フイブロイン
膜の構造安定化がなされている。
又、本発明の酵素固定化膜の製造方法は、絹フ
イブロイン溶液に所定量の酵素溶液を混合し、該
混合溶液を板上に塗布して乾燥させた酵素固定化
絹フイブロイン膜を所定温度及び温度雰囲気内で
延伸して、所定の伸度で構造安定化させる。
［実施例］
以下添付図面を参照しながら、本発明の一実施
例を説明する。
＜絹フイブロイン溶液の作成方法＞
家蚕繭を、0.5％のマルセル石鹸で溶比200倍に
て、100℃で30分の精錬を２回施した後、蒸溜水
で洗浄して精錬繭を得た。
この精錬繭を、9MのLiBr水溶液に40℃で溶
解・透析後、約4w／ｗ％の家蚕再生絹フイブロ
ン溶液を得た。
＜酵素固定化膜の作製方法＞
家蚕再生絹フイブロイン溶液に、0.2重量％の
グルコースオキシターゼ（以下GOD）を混合し
た。これをアクリル板上にキヤストして４℃で乾
燥させた後、GOD固定化家蚕再生絹フイブロイ
ン膜を得た（膜厚27μｍ）。
＜不溶化処理＞
メタノール処理例
メタノール処理は、80％水溶液にそれぞれ30
秒（以下ME30）、３分（以下ME3）、24時間
（以下ME24）浸漬後水洗いした。
延伸処理例
延伸処理は、以下のように行つた。膜を延伸
器に取り付け、20℃、相対湿度90％雰囲気のシ
ールドボツクスに30分格納した後、引張速度
0.2mm／秒で延伸し、所定の伸度1.25、1.5、
2.0、3.0倍（以下DR1.25等で表す）で10分間、
相対湿度40％雰囲気下で10分間それぞれ構造安
定化させた。
＜酵素固定化膜の保存＞
作成された酵素固定化膜は、酵素活性測定まで
は４℃の乾燥状態で保存され、酵素活性測定の前
に各酵素固定化膜を0.1Mリン酸緩衝液（PH7.0）
中に浸漬した。
＜酵素活性測定法＞
GOD固定化家蚕再生絹フイブロイン膜の酵素
活性は、比色法および溶存酵素電極で定量した。
分光光度計はＵ−3200（株式会社日立製作所製）、
酵素電極はBO型（株式会社石川製作所製）を使
用した。尚、比較例として遊離GODでも測定し
た。
溶出蛋白質の定量は、ラウリー（Lowry）法
で行つた。
膜透過性は、0.1Mグルコース水溶液を用いて、
透過量を示差屈折計（日本分析工業株式会社製）
で測定した。
＜構造解析測定法＞
13CNMR測定は、FX−90Q（JEOL製）を用
い、22.49MHz、25℃で行つた。
IR測定（赤外線分析）は、IR−435（株式会社
島津製作所製）を用いて行つた。
＜IR測定結果＞
各種延伸処理されたGOD固定化家蚕再生絹フ
イブロイン膜のIRスペクトルを第１図に示した。
延伸処理により、アミドＶバンドの700cm-1のピ
ーク強度が増加し、一部、β化が起きていること
が確認できる。製膜直後では、絹フイブロイン膜
のランダムコイル領域が多く、このままでは水浸
により再度溶解してしまうが、延伸だけで絹フイ
ブロイン分子鎖間の結晶性を増加させることによ
り、不溶性の膜を作製できた。ここでいうβ化と
は、分子鎖間で水素結合がなされて緻密な構造を
有し、固定化された酵素が外に溶出せず、構造安
定化していることを言う。また、ランダムコイル
領域とは、比較的分子鎖間の相互作用が弱く、容
易に水に溶けることを意味する。
実際、GODを含まない延伸家蚕再生絹フイブ
ロイン膜をリン酸緩衝液（PH7.0）に浸漬してお
いても、溶出蛋白質家蚕絹フイブロインは10日間
で、いずれも0.02±0.01w／ｗ％以下であつた。
＜ 13CNMR測定結果＞
第２図に、不溶化処理されたGOD固定化家蚕
再生絹フイブロイン膜の 13CNMR（核磁気共鳴）
スペクトルを示した。メタノール処理膜Ｂでは、
高分解能スペクトルを与える易動成分がかなり残
存しているが、延伸膜Ｃではその成分が減少す
る。これは、メタノール処理の場合、表面付近の
β構造領域の内部の易動領域との間で著しい不均
一構造を有するが、延伸処理の場合には、膜全体
にわたりβ化が進み易いことを示している。
＜比色法による酵素活性測定結果＞
表１に、GOD固定化家蚕再生絹フイブロイン
膜の酵素反応に伴なう過酸化水素生成速度を、比
色法で定量した結果を示した。
[Industrial Application Field] The present invention relates to an enzyme-immobilized membrane and a manufacturing method thereof, and particularly to an enzyme-immobilized membrane and a manufacturing method that take advantage of the manufacturing characteristics of silk fibroin. [Prior art] Conventionally, when immobilizing enzymes using the entrapment method, the immobilization carrier often needs to be cross-linked with glutaraldehyde or the like to make it insolubilized, and There was a drawback that secondary enzyme inactivation due to solvents and the like was inevitable, and that the enzyme activity in the membrane was almost gone after one month had passed. [Problems to be Solved by the Invention] The purpose of the present invention is to utilize silk fibroin as an entrapping immobilization material for enzymes by taking advantage of its structural characteristics. An object of the present invention is to provide an enzyme-immobilized membrane having long-term enzyme activity and a method for producing the same. [Means and effects for solving the problem] In order to solve this problem, in the enzyme-immobilized membrane of the present invention, the structure of the immobilized silk fibroin membrane is stabilized by stretching treatment. Further, the method for producing an enzyme-immobilized membrane of the present invention includes mixing a predetermined amount of an enzyme solution with a silk fibroin solution, applying the mixed solution onto a plate, and drying the enzyme-immobilized silk fibroin membrane at a predetermined temperature and temperature. The structure is stabilized by stretching in a temperature atmosphere at a predetermined degree of elongation. [Example] An example of the present invention will be described below with reference to the accompanying drawings. <Method for preparing silk fibroin solution> Domestic silkworm cocoons were refined twice with 0.5% Marcel soap at a dissolution ratio of 200 times at 100°C for 30 minutes, and then washed with distilled water to obtain refined cocoons. . The refined cocoons were dissolved in a 9M LiBr aqueous solution at 40°C and dialyzed to obtain a solution of approximately 4 w/w% domestic silkworm regenerated silk fibrous. <Method for producing enzyme-immobilized membrane> 0.2% by weight of glucose oxidase (hereinafter referred to as GOD) was mixed into a solution of domestic silkworm regenerated silk fibroin. This was cast on an acrylic plate and dried at 4°C to obtain a GOD-immobilized domestic silkworm regenerated silk fibroin film (film thickness: 27 μm). <Insolubilization treatment> Example of methanol treatment For methanol treatment, add 30% each to an 80% aqueous solution.
After soaking for seconds (hereinafter referred to as ME30), 3 minutes (hereinafter referred to as ME3), and 24 hours (hereinafter referred to as ME24), they were washed with water. Example of stretching treatment The stretching treatment was performed as follows. After attaching the membrane to a stretcher and storing it in a shielded box at 20℃ and 90% relative humidity for 30 minutes, the tensile speed was increased.
Stretched at 0.2 mm/sec to the specified elongation of 1.25, 1.5,
2.0, 3.0 times (hereinafter expressed as DR1.25, etc.) for 10 minutes,
Each structure was stabilized for 10 minutes in an atmosphere of 40% relative humidity. <Storage of enzyme-immobilized membranes> The prepared enzyme-immobilized membranes are stored in a dry state at 4°C until enzyme activity measurement. PH7.0)
immersed in it. <Enzyme activity measurement method> The enzyme activity of the GOD-immobilized domestic silkworm regenerated silk fibroin membrane was quantified using a colorimetric method and a dissolved enzyme electrode.
The spectrophotometer is U-3200 (manufactured by Hitachi, Ltd.),
The enzyme electrode used was a BO type (manufactured by Ishikawa Seisakusho Co., Ltd.). As a comparative example, free GOD was also measured. Quantification of eluted protein was performed by the Lowry method. Membrane permeability was determined using a 0.1M glucose aqueous solution.
Measure the amount of transmission using a differential refractometer (manufactured by Japan Analytical Industry Co., Ltd.)
It was measured with <Structural analysis measurement method> 13 CNMR measurement was performed at 22.49MHz and 25°C using FX-90Q (manufactured by JEOL). IR measurement (infrared analysis) was performed using IR-435 (manufactured by Shimadzu Corporation). <IR Measurement Results> Figure 1 shows the IR spectra of GOD-immobilized domestic silkworm regenerated silk fibroin films subjected to various stretching treatments.
As a result of the stretching treatment, the peak intensity at 700 cm -1 of the amide V band increases, and it can be confirmed that some beta conversion has occurred. Immediately after film formation, there are many random coil regions in the silk fibroin film, which will dissolve again when immersed in water, but an insoluble film can be created by increasing the crystallinity between silk fibroin molecular chains just by stretching. Ta. Here, β-ization means that the enzyme has a dense structure due to hydrogen bonding between molecular chains, and that the immobilized enzyme does not elute to the outside and has a stable structure. Furthermore, the random coil region means that the interaction between molecular chains is relatively weak and it is easily dissolved in water. In fact, even if the stretched silkworm regenerated silk fibroin membrane containing no GOD is immersed in phosphate buffer (PH7.0), the eluted protein domestic silkworm silk fibroin remains less than 0.02±0.01w/w% in 10 days. It was hot. < 13 CNMR measurement results> Figure 2 shows the 13 CNMR (nuclear magnetic resonance) of the insolubilized GOD-immobilized domestic silkworm regenerated silk fibroin membrane.
The spectrum was shown. In methanol treatment membrane B,
Although a considerable amount of mobile components that provide a high-resolution spectrum remain, these components are reduced in the stretched film C. This indicates that in the case of methanol treatment, there is a significant non-uniform structure between the β-structure region near the surface and the mobile region inside, but in the case of stretching treatment, β-ization tends to progress throughout the film. ing. <Results of enzymatic activity measurement by colorimetric method> Table 1 shows the results of quantifying the rate of hydrogen peroxide production accompanying the enzymatic reaction of the GOD-immobilized regenerated silk fibroin membrane using a colorimetric method.
【表】
メタノール処理膜と延伸膜を比較すると、単位
表面積当たりの反応速度には、著しい違いは認め
られないが、単位酵素重量当たりの反応速度に換
算すると、延伸膜はいずれもメタノール処理膜に
比べて反応速度が大きくなる傾向がある。
また、延伸倍率の酵素活性への効果は、単位表
面積当たりの反応速度では、延伸倍率が大きくな
るに従つて徐々に低下したが、単位酵素重量当た
りの反応速度に換算すると延伸倍率の違いは有意
差として認められなかつた。
＜グルコース透過性の測定結果＞
固定化酵素の反応速度は、多くの場合担体内で
の基質の拡散速度に左右されることがある。絹フ
イブロイン膜のグルコース透過性を測定したとこ
ろ、第３図に示すように透過係数は延伸に伴ない
著しく低下し、延伸処理された家蚕再生絹フイブ
ロイン膜の酵素反応は、基質であるグルコースの
透過性と相関がある。
＜溶存酸素電極による酵素活性測定結果＞
第４図ａ，ｂは、GOD固定化家蚕再生絹フイ
ブロイン膜の酵素活性を溶存酸素電極で定量した
結果を示す図、第５図は第４図ａ，ｂから求めら
れたミハエリス（Michaelis）定数Knと最大反応
速度Vnである。ここで、［Ｓ］は基質濃度を示
す。この結果から次のことが分つた。
延伸したGOD固定化家蚕再生絹フイブロイン
膜の酵素活性は、メタノール処理膜に比べて全体
に高く、伸度が高い程より高い。
次に、第６図には、PH変化に対する酵素活性の
安定性を示す図である。GOD固定化家蚕再生絹
フイブロイン膜は、遊離GODに比べてPH変化に
対する活性が安定しており、80％以上の相対活性
を示すPHレンジは、PH５〜８と広域にわたつてい
る。
＜グルコースセンサへの適用例＞
酸素電極を本実施例のGOD固定化家蚕再生絹
フイブロイン膜で覆い、第９図ａ，ｂにその模式
図を示すグルコースセンサを作成した。ここで、
９１は公知の酸素電極、９２はGOD固定化家蚕
再生絹フイブロイン膜、９３は電圧測定器、９４
はグルコース溶液９６を撹拌する撹拌器、９５は
恒温温水を流してグルコース溶液９６を一定温度
に保つ容器、９６はグルコース溶液である。又第
９図ｂはグルコースセンサの先端部を拡大した図
であつて、９１ａは白金電極、９１ｂは内部液
室、９７はガス透過性を有するテフロン膜であ
り、このテフロン膜９７の上にGOD固定化家蚕
再生絹フイブロイン膜９２が被覆される。このグ
ルコースセンサの特性を電圧測定器９３でボルト
メトリツクに検出した。
第７図、第１０図はグルコース濃度に対するグ
ルコースセンサの出力電圧を示す図、第８図はPH
変化に対するグルコースセンサの出力の安定性を
示す図である。この結果、本実施例のGOD固定
化家蚕再生絹フイブロイン膜がグルコースセンサ
の酵素膜として十分使用できることを示し、PHに
対する安定性もPH５〜８で非常に安定しているこ
とが分つた。
更に以下に示した性能を発揮した。
延伸した絹フイブロイン膜を酵素固定化担体と
するグルコースセンサーの性能
保存期間 ４カ月
PH依存性 PH５〜８で安定（90％出力）
酵素溶出率 0.01％／10日
応答時間 8.5秒（90％応答）
測定範囲 １〜500mg／１
繰り返し測定誤差 0.9％以下（30回）
但し、
GOD：Aspergillus niger
固定化法：延伸による包括法
膜厚：23μｍ
有効膜面積：0.126cm2
以上、絹フイブロインをその構造特性を生かし
て酵素の包括固定材料とすることにより、酵素含
有膜の活性が高く酵素の溶出の極めて少ない、長
期間酵素活性を有する本実施例の酵素固定化膜を
酵素センサに適用できることが認められた。
更に詳細には、本実施例の酵素固定化膜を用い
た酵素センサは、酵素含有家蚕再生絹フイブロイ
ン膜の構造転移を何ら化学処理することなく、延
仲処理のみで発現させたため、
(1) 酵素含有膜の活性収率が高い。
(2) PHを変化させたときの安定性が優れる。
(3) 膜中の酵素活性が４ケ月間変化しない。等の
特徴を示す。
尚、本実施例では、酵素としてグルコースを代
表させたが、他の酵素にも適用できることは自明
である。又、本発明の技術思想は家蚕に限らず、
野蚕の再生絹フイブロイン膜をはじめとした他の
酵素固定化膜、更に微生物膜や動・植物細胞膜等
にも適用できる。
［発明の効果］
本発明により、絹フイブロインをその構造特性
を生かして酵素の包括固定化材料とすることによ
り、酵素含有膜の活性が高く、酵素の溶出の極め
て少ない、長期間酵素活性を有する酵素固定化膜
及びその製造方法を提供できる。
そして、本発明の酵素固定化膜を適用した酵素
センサは、酵素含有家蚕再生絹フイブロイン膜の
構造転移を何ら化学処理することなく、延伸処理
のみで発現させたため、
(1) 酵素含有膜の活性収率が高い。
(2) PHを変化させたときの安定性が優れる。
(3) 膜中の酵素活性が４ケ月間変化しない。
等の特長を示す。[Table] Comparing methanol-treated membranes and stretched membranes, there is no significant difference in reaction rate per unit surface area, but when converted to reaction rate per unit enzyme weight, stretched membranes are significantly different from methanol-treated membranes. The reaction rate tends to be faster. In addition, the effect of stretching ratio on enzyme activity was that the reaction rate per unit surface area gradually decreased as the stretching ratio increased, but the difference in stretching ratio was significant when converted to the reaction rate per unit enzyme weight. It was not recognized as a difference. <Results of Glucose Permeability Measurement> The reaction rate of an immobilized enzyme often depends on the diffusion rate of the substrate within the carrier. When the glucose permeability of the silk fibroin membrane was measured, as shown in Figure 3, the permeability coefficient decreased significantly as it was stretched. There is a correlation with gender. <Results of enzyme activity measurement using a dissolved oxygen electrode> Figures 4a and 4b show the results of quantifying the enzyme activity of the GOD-immobilized domestic silkworm regenerated silk fibroin membrane using a dissolved oxygen electrode. These are the Michaelis constant K n and the maximum reaction rate V n determined from b. Here, [S] indicates the substrate concentration. From this result, we found the following. The enzyme activity of the stretched GOD-immobilized regenerated domestic silk fibroin membrane is generally higher than that of the methanol-treated membrane, and the higher the degree of stretching, the higher the enzyme activity. Next, FIG. 6 is a diagram showing the stability of enzyme activity against PH changes. GOD-immobilized domestic silkworm regenerated silk fibroin membrane has more stable activity against pH changes than free GOD, and the pH range in which it exhibits relative activity of 80% or more is over a wide range of pH 5 to 8. <Example of Application to Glucose Sensor> An oxygen electrode was covered with the GOD-immobilized domestic silkworm regenerated silk fibroin membrane of this example to create a glucose sensor, the schematic diagram of which is shown in FIGS. 9a and 9b. here,
91 is a known oxygen electrode, 92 is a GOD-immobilized domestic silkworm regenerated silk fibroin membrane, 93 is a voltage measuring device, 94
95 is a stirrer for stirring the glucose solution 96; 95 is a container for keeping the glucose solution 96 at a constant temperature by flowing constant-temperature water; and 96 is the glucose solution. FIG. 9b is an enlarged view of the tip of the glucose sensor, in which 91a is a platinum electrode, 91b is an internal liquid chamber, 97 is a Teflon membrane having gas permeability, and GOD is placed on top of this Teflon membrane 97. An immobilized domestic silkworm regenerated silk fibroin film 92 is coated. The characteristics of this glucose sensor were detected voltmetrically using a voltage measuring device 93. Figures 7 and 10 are diagrams showing the output voltage of the glucose sensor relative to the glucose concentration, and Figure 8 is the PH
FIG. 3 is a diagram showing the stability of the output of a glucose sensor with respect to changes. The results showed that the GOD-immobilized regenerated domestic silk fibroin membrane of this example could be sufficiently used as an enzyme membrane for a glucose sensor, and it was also found to be extremely stable at pH levels of 5 to 8. Furthermore, it demonstrated the performance shown below. Performance of glucose sensor using stretched silk fibroin membrane as enzyme immobilization carrier Storage period 4 months PH dependence Stable at PH 5 to 8 (90% output) Enzyme elution rate 0.01%/10 days Response time 8.5 seconds (90% response) Measurement range: 1 to 500 mg/1 Repeated measurement error: 0.9% or less ( 30 times) However, GOD: Aspergillus niger Immobilization method: Inclusive method by stretching Film thickness: 23 μm Effective film area: 0.126 cm It was confirmed that the enzyme-immobilized membrane of this example, which has high enzyme-containing membrane activity, very little enzyme elution, and long-term enzyme activity, can be applied to enzyme sensors by making the most of the enzyme-encompassing immobilization material. Ta. More specifically, in the enzyme sensor using the enzyme-immobilized membrane of this example, the structural transition of the enzyme-containing regenerated silk fibroin membrane of domestic silkworms was caused by Nobunaka treatment alone without any chemical treatment; (1) The activity yield of the enzyme-containing membrane is high. (2) Excellent stability when changing pH. (3) Enzyme activity in the membrane remains unchanged for 4 months. It shows the characteristics such as. In this example, glucose is used as a representative enzyme, but it is obvious that the present invention can be applied to other enzymes as well. In addition, the technical idea of the present invention is not limited to domestic silkworms,
It can also be applied to other enzyme-immobilized membranes, including regenerated silk fibroin membranes from wild silkworms, as well as microbial membranes and animal/plant cell membranes. [Effects of the Invention] According to the present invention, silk fibroin is used as a material for enzyme entrapping immobilization by taking advantage of its structural characteristics, so that the enzyme-containing membrane has high activity, extremely little enzyme elution, and long-term enzyme activity. An enzyme-immobilized membrane and a method for producing the same can be provided. In addition, the enzyme sensor to which the enzyme-immobilized membrane of the present invention is applied has the ability to express the structural transition of the enzyme-containing regenerated domestic silk fibroin membrane only by stretching treatment without any chemical treatment. (1) The activity of the enzyme-containing membrane High yield. (2) Excellent stability when changing pH. (3) Enzyme activity in the membrane remains unchanged for 4 months. The following features are shown.
第１図はグルコースオキシターゼ固定化家蚕再
生絹フイブロイン膜の赤外線スペクトル図、第２
図はグルコースオキシターゼ固定化家蚕再生絹フ
イブロイン膜の 13C核磁気共鳴スペクトル図、第
３図はグルコースオキシターゼ固定化家蚕再生絹
フイブロイン膜の延伸率とグルコース透過率を示
す図、第４図ａ，ｂはグルコースオキシターゼ固
定化家蚕再生絹フイブロイン膜の溶存酸素電極に
よる酵素活性の測定結果を示す図、第５図はグル
コースオキシターゼ固定化家蚕再生絹フイブロイ
ン膜のミハエリス（Michaelis）定数Knと最大反
応速度Vnを示す図、第６図はグルコースオキシ
ターゼ固定化家蚕再生絹フイブロイン膜のPH変化
に対する酵素活性の安定性を示す図、第７図、第
１０図はグルコースオキシターゼ固定化家蚕再生
絹フイブロイン膜のグルコース濃度に対するグル
コースセンサの出力電圧を示す図、第８図はグル
コースオキシターゼ固定化家蚕再生絹フイブロイ
ン膜のPH変化に対するグルコースセンサの出力の
安定性を示す図、第９図ａ，ｂは酸素電極を本実
施例のGOD固定化家蚕再生絹フイブロイン膜で
覆つたグルコースセンサの模式図である。
図中、９１……酵素電極、９２……GOD固定
化家蚕再生絹フイブロイン膜である。
Figure 1 is an infrared spectrum diagram of the regenerated domestic silk fibroin membrane immobilized with glucose oxidase, Figure 2
The figure is a 13 C nuclear magnetic resonance spectrum diagram of the regenerated domestic silk fibroin membrane immobilized with glucose oxidase, Figure 3 is a diagram showing the elongation rate and glucose permeability of the regenerated domestic silk fibroin membrane immobilized with glucose oxidase, and Figure 4 a, b. Figure 5 shows the measurement results of the enzyme activity of the regenerated domestic silk fibroin membrane immobilized with glucose oxidase using a dissolved oxygen electrode. Figure 5 shows the Michaelis constant K n and the maximum reaction rate V of the regenerated domestic silk fibroin membrane immobilized with glucose oxidase. Figure 6 is a diagram showing the stability of enzyme activity with respect to PH changes in the regenerated domestic silk fibroin membrane immobilized with glucose oxidase, Figures 7 and 10 are the glucose in the regenerated domestic silk fibroin membrane immobilized with glucose oxidase. Figure 8 is a diagram showing the output voltage of the glucose sensor versus concentration, Figure 8 is a diagram showing the stability of the output of the glucose sensor with respect to pH changes of the glucose oxidase-immobilized regenerated domestic silk fibroin membrane, and Figures 9a and b are diagrams showing the output voltage of the glucose sensor with respect to the pH change of the glucose oxidase-immobilized silk fibroin membrane. FIG. 2 is a schematic diagram of a glucose sensor covered with a GOD-immobilized domestic silkworm regenerated silk fibroin membrane according to an example. In the figure, 91... Enzyme electrode, 92... GOD-immobilized domestic silkworm regenerated silk fibroin membrane.
Claims (1)
造安定化がなされていることを特徴とする酵素固
定化膜。 ２ 絹フイブロイン溶液に所定量の酵素溶液を混
合し、 該混合溶液を板上に塗布して乾燥させた酵素固
定化絹フイブロイン膜を所定温度及び湿度雰囲気
内で延伸して、所定の伸度で構造安定化させるこ
とを特徴とする酵素固定化膜の製造方法。 ３ 酵素溶液として、グルコースオキシターゼ溶
液を混合することを特徴とする特許請求の範囲第
２項記載の酵素固定化膜の製造方法。 ４ 酵素固定化絹フイブロイン膜を延伸器に取り
付けて、温度20℃、相対湿度90％雰囲気下に30分
置き、引張速度0.2mm／秒で延伸し、所定の伸度
で10分間、相対湿度40％で10分間置いて構造安定
化させることを特徴とする特許請求の範囲第２項
記載の酵素固定化膜の製造方法。[Scope of Claims] 1. An enzyme-immobilized membrane characterized in that the structure of the immobilized silk fibroin membrane is stabilized by stretching treatment. 2 Mix a predetermined amount of an enzyme solution with a silk fibroin solution, apply the mixed solution onto a plate, dry the enzyme-immobilized silk fibroin membrane, and stretch it at a predetermined temperature and humidity atmosphere to a predetermined degree of elongation. A method for producing an enzyme-immobilized membrane characterized by structural stabilization. 3. The method for producing an enzyme-immobilized membrane according to claim 2, characterized in that a glucose oxidase solution is mixed as the enzyme solution. 4. Attach the enzyme-immobilized silk fibroin membrane to a stretching machine and place it in an atmosphere at a temperature of 20°C and a relative humidity of 90% for 30 minutes, stretch it at a tensile speed of 0.2 mm/sec, and stretch it at the specified elongation rate for 10 minutes at a relative humidity of 40%. % for 10 minutes to stabilize the structure.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP62248215A JPS6460382A (en) | 1987-05-08 | 1987-10-02 | Immobilized enzyme membrane, its production and enzyme sensor using said immobilized enzyme membrane |
| US07/176,687 US4999295A (en) | 1987-05-08 | 1988-04-01 | Biocatalyst entrapped in a silk fibroin membrane |
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP11068187 | 1987-05-08 | ||
| JP62248215A JPS6460382A (en) | 1987-05-08 | 1987-10-02 | Immobilized enzyme membrane, its production and enzyme sensor using said immobilized enzyme membrane |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS6460382A JPS6460382A (en) | 1989-03-07 |
| JPH0448437B2 true JPH0448437B2 (en) | 1992-08-06 |
Family
ID=26450253
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP62248215A Granted JPS6460382A (en) | 1987-05-08 | 1987-10-02 | Immobilized enzyme membrane, its production and enzyme sensor using said immobilized enzyme membrane |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPS6460382A (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5328847A (en) * | 1990-02-20 | 1994-07-12 | Case George D | Thin membrane sensor with biochemical switch |
-
1987
- 1987-10-02 JP JP62248215A patent/JPS6460382A/en active Granted
Also Published As
| Publication number | Publication date |
|---|---|
| JPS6460382A (en) | 1989-03-07 |
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