JP2824902B2 - Method for producing enzyme-immobilizing carrier - Google Patents
Method for producing enzyme-immobilizing carrierInfo
- Publication number
- JP2824902B2 JP2824902B2 JP27704695A JP27704695A JP2824902B2 JP 2824902 B2 JP2824902 B2 JP 2824902B2 JP 27704695 A JP27704695 A JP 27704695A JP 27704695 A JP27704695 A JP 27704695A JP 2824902 B2 JP2824902 B2 JP 2824902B2
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- Prior art keywords
- enzyme
- immobilized
- carrageenan
- carrier
- aqueous solution
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- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
Description
【０００１】[0001]
【発明の属する技術分野】本発明は、天然高分子である
キトサンとカラギーナンを複合化した、バイオリアクタ
ーによる食品素材の製造に好適な酵素固定化用担体の製
造方法に関するものである。TECHNICAL FIELD The present invention relates to a method for producing a carrier for immobilizing enzymes suitable for producing a food material by a bioreactor, in which a natural polymer chitosan and carrageenan are complexed.
【０００２】[0002]
【従来の技術】近年、固定化酵素を用いたバイオリアク
ターによる食品素材の製造が、その高い生産性と高価な
酵素の再使用可能性によるコスト意識から、食品産業を
中心に研究・実用化が計られている。酵素の固定化用担
体として、合成高分子イオン交換体、多糖類系イオン交
換体及びシリカゲル、ガラス等の無機系の担体等が使用
されている。本出願人は特公平１−１６４２０号で開示
した粒状多孔質キトサン担体以外に、これをベースとし
て特公昭６３−５４２８５号及び特開平６−２３７６９
号等で各種の官能基を導入した固定化用担体を開示し
た。しかし、上述の特公平１−１６４２０号以外の固定
化用担体は、イオン交換基として塩基性を有するもので
あったり、担体に疎水性が導入され酵素が疎水吸着され
るものであった。固定化用担体の塩基性基は等電点の低
い酸性蛋白の吸着には有効に作用するが、塩基性蛋白を
吸着固定させることは不可能で、又、強い疎水吸着は往
々にして酵素との適合性が悪く失活の原因ともなる欠点
がある。2. Description of the Related Art In recent years, the production and production of food materials by bioreactors using immobilized enzymes have been studied and commercialized mainly in the food industry, due to their high productivity and cost consciousness due to the reusability of expensive enzymes. Is measured. As carriers for immobilizing enzymes, synthetic polymer ion exchangers, polysaccharide ion exchangers, and inorganic carriers such as silica gel and glass are used. The applicant of the present invention has disclosed, in addition to the granular porous chitosan carrier disclosed in Japanese Examined Patent Publication No. 1-16420, based on this, Japanese Patent Publication No. 63-54285 and Japanese Unexamined Patent Publication No.
And the like, disclosed a carrier for immobilization into which various functional groups were introduced. However, the carriers for immobilization other than the above-mentioned Japanese Patent Publication No. 1-16420 were those having basicity as an ion exchange group, or those having hydrophobicity introduced into the carrier and the enzyme being hydrophobically adsorbed. The basic group of the immobilization carrier effectively acts on the adsorption of acidic proteins having a low isoelectric point, but cannot immobilize and immobilize basic proteins, and strong hydrophobic adsorption often occurs with enzymes. Has a drawback that it is incompatible and causes deactivation.
【０００３】又、酵素を、キトサンをベースとした固定
化用担体に強固に固定化させる目的で使用されるグルタ
ルアルデヒドは、酵素の失活を招く上に、酵素を固定化
用担体から取り除いて固定化用担体として再使用に供す
ることが不可能であり、更に食品素材生産に供する場合
は、できればグルタルアルデヒドを使用することは好ま
しくない。[0003] Glutaraldehyde used for the purpose of firmly immobilizing an enzyme on a chitosan-based immobilization carrier not only inactivates the enzyme but also removes the enzyme from the immobilization carrier. It is impossible to re-use as a carrier for immobilization, and when it is further used for production of food materials, it is not preferable to use glutaraldehyde if possible.
【０００４】又、特開昭５７−１３２８８３号に、カラ
ギーナン水溶液に酵素活性物質と多カチオン性高分子化
合物を添加混合し、カラギーナンをゲル化させ、そのゲ
ル格子内に酵素活性物質を包括させることが開示されて
いるが、この固定化酵素活性物質は強度も弱く、酵素が
包括されているために活性が低下したときにその活性を
再活性することが出来ず再利用出来ない欠点がある。Japanese Patent Application Laid-Open No. 57-132883 discloses that carrageenan is gelled by adding an enzyme active substance and a polycationic polymer compound to an aqueous solution of carrageenan, and the enzyme active substance is included in the gel lattice. However, this immobilized enzyme active substance has a weakness, and has a disadvantage that when the activity is reduced due to the inclusion of the enzyme, the activity cannot be reactivated and cannot be reused.
【０００５】[0005]
【発明が解決しようとする課題】本発明の酵素固定化用
担体は、天然高分子であるキトサンにカラギーナンで処
理し、多官能試薬でカラギーナンをキトサンと複合した
もので安全性に優れ、カラギーナンが有する硫酸基の酸
性によって塩基性酵素の吸着に優れ、更に硫酸基が強酸
性陽イオン交換基であることから、塩基性酵素を強固に
吸着するので酵素を固定化する目的でグルタルアルデヒ
ド等の多官能性試薬を使用しなくても、塩基性酵素を用
いたときに強固に吸着するので、固定化酵素の繰り返し
反応が可能な上、固定化酵素として使用後に不要になっ
た失活酵素を容易に離脱・再生することが出来る。The carrier for immobilizing enzymes of the present invention is obtained by treating a natural polymer, chitosan, with carrageenan.
And complexed carrageenan with chitosan with a multifunctional reagent
It has excellent safety and basic enzyme adsorption due to the acidity of the sulfate group of carrageenan.Since the sulfate group is a strongly acidic cation exchange group, it strongly adsorbs the basic enzyme and immobilizes the enzyme. use without using the polyfunctional reagents such as glutaraldehyde purposes, the basic enzyme of
Since it is strongly adsorbed when immobilized, the immobilized enzyme can be repeatedly reacted, and the inactivated enzyme that has become unnecessary after use as the immobilized enzyme can be easily detached and regenerated.
【０００６】[0006]
【課題を解決するための手段】本発明は、低分子量キト
サンの酸性水溶液を塩基性水溶液中に滴下凝固再生せし
めて得た再生粒状多孔質キトサンを、カラギーナンの水
溶液中に分散し、次いで多官能性試薬を反応させる酵素
固定化用担体の製造方法である。According to the present invention, a regenerated granular porous chitosan obtained by subjecting an acidic aqueous solution of low molecular weight chitosan to coagulation and regeneration in a basic aqueous solution is dispersed in an aqueous solution of carrageenan, and then dispersed in a carrageenan aqueous solution. This is a method for producing a carrier for immobilizing an enzyme, which is reacted with a sex reagent.
【０００７】[0007]
【発明の実施の形態】本発明で用いられる再生粒状多孔
質キトサンは、特公平１−１６４２０号で開示された方
法によって得られる。即ち、平均分子量が１０，０００
〜２３０，０００の低分子量キトサンのみを蟻酸，酢
酸，ジクロル酢酸，乳酸等の有機酸の単独又は混合、又
は塩酸，硝酸等の無機酸の酸性水溶液に溶解し、該キト
サン酸性水溶液を、水酸化ナトリウム，水酸化カリウ
ム，炭酸ナトリウム，炭酸カリウム，アンモニア，エチ
レンジアミン等のアルカリ性物質を水、又はメタノー
ル，エタノール等のアルコール類、又は水とアルコール
類の混合液に加えた塩基性溶液中に、一定量づつ滴下せ
しめて所望の平均粒径の粒状多孔質キトサンを凝固再生
析出させることにより得られる。尚、本発明に用いられ
る再生粒状多孔質キトサンの平均粒径は特に限定される
ものではない。DETAILED DESCRIPTION OF THE INVENTION The regenerated granular porous chitosan used in the present invention can be obtained by the method disclosed in Japanese Patent Publication No. 1-16420. That is, the average molecular weight is 10,000
Only 230,000 low molecular weight chitosan is dissolved alone or in a mixture of organic acids such as formic acid, acetic acid, dichloroacetic acid and lactic acid, or dissolved in an acidic aqueous solution of an inorganic acid such as hydrochloric acid and nitric acid. A certain amount of a basic solution in which an alkaline substance such as sodium, potassium hydroxide, sodium carbonate, potassium carbonate, ammonia, ethylenediamine or the like is added to water, an alcohol such as methanol or ethanol, or a mixture of water and alcohol. It is obtained by dropping one by one, and coagulating, regenerating and depositing granular porous chitosan having a desired average particle size. The average particle size of the regenerated granular porous chitosan used in the present invention is not particularly limited.
【０００８】本発明で用いられるカラギーナンとは、ユ
ーキューマ，コンドラス，イルディヤ，ギガルティーナ
等の原藻の紅藻類より抽出される天然多糖類の１種で、
カッパ，イオタ，ラムダの３種のフラクションがあり、
詳しくは１、３結合したガラクトース−４−サルフェイ
トと１、４結合した３、６−アンヒドロ−Ｄ−ガラクト
ースから成るカッパカラギーナン，１、３結合したガラ
クトース−４−サルフェイトと１、４結合した３、６−
アンヒドロ−Ｄ−ガラクトース−２−サルフェイトから
成るイオタカラギーナン，１、３結合したガラクトース
−２−サルフェイトと１、４結合したＤ−ガラクトース
−２、６−サルフェイトから成るラムダカラギーナン
で、これらの単独又は混合のいずれも用いることが出来
る。[0008] Carrageenan used in the present invention is one of natural polysaccharides extracted from red algae of protoal algae such as Eucuma, Kondras, Irdiya, Gigartina, etc.
There are three types of kappa, iota, lambda,
Specifically, kappa carrageenan consisting of 1,3-linked galactose-4-sulfate and 1,4-linked 3,6-anhydro-D-galactose, 1,3-linked galactose-4-sulfate 1,4-linked 3,6-
Iota carrageenan consisting of anhydro-D-galactose-2-sulfate, lambda carrageenan consisting of 1,3 linked galactose-2-sulfate and 1,4 linked D-galactose-2,6-sulfate. Either alone or a mixture can be used.
【０００９】粒状多孔質キトサンを分散するカラギーナ
ン水溶液としては、濃度が０．１〜１５％のものが用い
られる。０．１％以下では効果が低く、１５％以上の濃
度にすると低分子量のカラギーナンを用いても粘度が高
くなり過ぎ取り扱い難い欠点があるので、カラギーナン
水溶液の濃度は好ましくは１〜１０％である。As the aqueous solution of carrageenan in which the granular porous chitosan is dispersed, one having a concentration of 0.1 to 15% is used. When the concentration is 0.1% or less, the effect is low, and when the concentration is 15% or more, even if a low molecular weight carrageenan is used, the viscosity becomes too high and it is difficult to handle. Therefore, the concentration of the carrageenan aqueous solution is preferably 1 to 10%. .
【００１０】粒状多孔質キトサンを分散させる方法とし
ては、再生粒状多孔質キトサン１重量部をカラギーナン
水溶液１〜１０重量部に入れて５〜９０℃，好ましくは
２５〜７０℃で１〜１００時間，好ましくは２〜４８時
間攪拌し分散する。この操作でカラギーナンは再生粒状
多孔質キトサンとポリイオンコンプレックスを形成し吸
着される。この様にしてカラギーナンと複合化した粒状
多孔質キトサンを得る。As a method for dispersing the granular porous chitosan, 1 part by weight of the regenerated granular porous chitosan is added to 1 to 10 parts by weight of an aqueous solution of carrageenan at 5 to 90 ° C., preferably at 25 to 70 ° C. for 1 to 100 hours. Preferably, it is stirred and dispersed for 2 to 48 hours. In this operation, carrageenan is adsorbed by forming a polyion complex with the regenerated granular porous chitosan. Thus, granular porous chitosan complexed with carrageenan is obtained.
【００１１】カラギーナンの硫酸基とキトサンのアミノ
基間で形成されたポリイオンコンプレックスは比較的強
固な結合と考えられるが、酵素を固定化して酵素固定化
用担体として長時間使用する間、及び使用後の固定化酵
素を再生させる操作で粒状多孔質キトサンからカラギー
ナンが脱落するので、次いで多官能性試薬により粒状多
孔質キトサンとカラギーナンを共有結合させる処理をす
る必要がある。[0011] The polyion complex formed between the sulfate group of carrageenan and the amino group of chitosan is considered to be a relatively strong bond, but the enzyme is immobilized and used for a long time as a carrier for enzyme immobilization, and after use. Since carrageenan falls off from the granular porous chitosan by the operation of regenerating the immobilized enzyme, it is necessary to perform a treatment for covalently bonding the granular porous chitosan and carrageenan with a polyfunctional reagent.
【００１２】この際の多官能性試薬としては、エピクロ
ロヒドリンやエチレングリコール，ポリエチレングリコ
ール又はポリプロピレングリコールのジグリシジルエー
テルが挙げられる。好ましくはエピクロロヒドリン，エ
チレングリコールジグリシジルエーテル，プロピレング
リコールジグリシジルエーテルである。Examples of the polyfunctional reagent in this case include epichlorohydrin and diglycidyl ether of ethylene glycol, polyethylene glycol or polypropylene glycol. Preferred are epichlorohydrin, ethylene glycol diglycidyl ether, and propylene glycol diglycidyl ether.
【００１３】この反応は濃度１〜３０％の多官能性試薬
水溶液を使用し、カラギーナンと複合化された粒状多孔
質キトサン１重量部に対し多官能性試薬水溶液１〜１０
重量部を加え、２５〜９０℃、好ましくは５０〜８０℃
で１〜４８時間，好ましくは６〜４８時間反応させる。
この時の水溶液は、アルカリ性域とすることが好まし
い。反応終了後充分に水洗し、本発明の酵素固定化用担
体を得る。In this reaction, an aqueous solution of a polyfunctional reagent having a concentration of 1 to 30% is used, and 1 part by weight of a granular porous chitosan complexed with carrageenan is used in an amount of 1 to 10%.
25 to 90 ° C, preferably 50 to 80 ° C, by weight
For 1 to 48 hours, preferably 6 to 48 hours.
The aqueous solution at this time is preferably in an alkaline region. After the completion of the reaction, the substrate is sufficiently washed with water to obtain the carrier for immobilizing the enzyme of the present invention.
【００１４】[0014]
【実施例】以下に本発明実施例を詳述するが、本発明は
この範囲に限定されるものではない。尚、酵素の吸着
率，固定化パパイン及び固定化トリプシンの活性は、次
の測定方法により求めた。EXAMPLES Examples of the present invention will be described below in detail, but the present invention is not limited to this range. The enzyme adsorption rate and the activities of immobilized papain and immobilized trypsin were determined by the following measurement methods.
【００１５】〈１〉酵素の吸着率測定方法 (1) 吸着前の酵素液を、水で希釈し２８０nmにおける吸
光度を水を対照として分光光度計（ベックマン（株）
製、商品名；ＤＵ−６４０）で光路長１cmの石英セルで
測定（ａ）する。測定に際しては吸光度が０．５以下に
なる如く希釈する。 (2) 吸着後の上清の吸光度を(1) と同様に測定（ｂ）す
る。その希釈率は(1) と同様とする。 (3) 次式により酵素の吸着率を求める。 酵素の吸着率（％）＝｛（ａ−ｂ）×１００｝<1> Method for Measuring Enzyme Adsorption Rate (1) The enzyme solution before adsorption is diluted with water, and the absorbance at 280 nm is measured with water as a spectrophotometer (Beckman KK)
(DU) (trade name, manufactured by DU-640) in a quartz cell having an optical path length of 1 cm. At the time of measurement, dilution is performed so that the absorbance becomes 0.5 or less. (2) Measure the absorbance of the supernatant after adsorption in the same manner as in (1) (b). The dilution ratio is the same as in (1). (3) Obtain the adsorption rate of the enzyme by the following equation. Enzyme adsorption rate (%) = {(ab) × 100}
【００１６】〈２〉固定化パパインの活性測定方法 (1) １００ｇのカゼインを、０．０５Ｍ・トリス塩酸緩
衝液（ｐＨ８．０）１，０００mlに、湯浴中で加温して
溶解させ、基質溶液とする。 (2) 固定化用担体にパパインを固定した固定化パパイン
１mlに、基質溶液５０mlを添加し、３７℃１０分間攪拌
する。 (3) 反応終了後、上清を１ml採取し、５％トリクロロ酢
酸水溶液３mlを添加する。 (4) ３，５００rpm で遠心分離を１５分行い、上清の２
８０nmにおける吸光度（ｃ）を〈１〉で用いた分光光度
計で光路長１cmの石英セルで測定する。測定に際して
は、測定値が０．５を越えない様に希釈する（希釈倍
率）。 (5) ブランクとして、固定化パパインの代わりに未酵素
固定の固定化用担体を用いて上述と同様の操作により２
８０nmにおける吸光度（ｄ）を測定する。このときの希
釈倍率は(4) と同様とする。 (6) 固定化パパインの活性は、次式により算出する。 固定化パパインの活性（Ｕ／ml・担体）＝０．４×（ｃ
−ｄ）×希釈倍率<2> Method for measuring the activity of immobilized papain (1) 100 g of casein was dissolved in 1,000 ml of 0.05 M Tris-HCl buffer (pH 8.0) by heating in a water bath. Use as substrate solution. (2) 50 ml of a substrate solution is added to 1 ml of immobilized papain in which papain is immobilized on a carrier for immobilization, and the mixture is stirred at 37 ° C. for 10 minutes. (3) After completion of the reaction, 1 ml of the supernatant is collected, and 3 ml of a 5% aqueous solution of trichloroacetic acid is added. (4) Centrifuge at 3,500 rpm for 15 minutes,
The absorbance (c) at 80 nm is measured with a spectrophotometer using <1> in a quartz cell having an optical path length of 1 cm. At the time of measurement, dilution is performed so that the measured value does not exceed 0.5 (dilution ratio). (5) As a blank, a non-enzyme-immobilized carrier for immobilization was used in place of immobilized papain, followed by the same operation as described above.
The absorbance (d) at 80 nm is measured. The dilution ratio at this time is the same as in (4). (6) The activity of immobilized papain is calculated by the following equation. Activity of immobilized papain (U / ml · carrier) = 0.4 × (c
-D) x dilution ratio
【００１７】〈３〉固定化トリプシンの活性測定法 (1) ５０ｇのカゼインを、０．１Ｍ・りん酸緩衝液（ｐ
Ｈ７．６）５００mlに湯浴中で加温して溶解させ基質溶
液とする。 (2) 固定化用担体にトリプシンを固定化させた固定化ト
リプシン１mlに、基質溶液５０mlを添加し、３５℃１０
分間攪拌する。 (3) 反応終了後、反応液を１ml採取し、５％トリクロロ
酢酸水溶液３mlを添加する。 (4) ３，５００rpm で遠心分離を１５分行い、上清の２
８０nmにおける吸光度（ｅ）を〈１〉で用いた分光光度
計で光路長１cmの石英セルで測定する。測定に際して
は、測定値が０．５を越えない様に希釈する（希釈倍
率）。 (5) ブランクとして、固定化トリプシンの代わりに未酵
素固定の固定化用担体を用いて上述と同様の操作により
２８０nmにおける吸光度（ｆ）を測定する。このときの
希釈は(4) と同様とする。 (6) 固定化トリプシンの活性は、次式により算出する。 固定化トリプシンの活性（Ｕ／ml・担体）＝０．４×
（ｅ−ｆ）×希釈倍率<3> Method for measuring the activity of immobilized trypsin (1) 50 g of casein was added to a 0.1 M phosphate buffer (p
H7.6) Heat and dissolve in 500 ml of hot water bath to obtain a substrate solution. (2) 50 ml of a substrate solution was added to 1 ml of immobilized trypsin in which trypsin was immobilized on a carrier for immobilization, and the solution was added at 35 ° C
Stir for a minute. (3) After completion of the reaction, 1 ml of the reaction solution is collected, and 3 ml of a 5% aqueous solution of trichloroacetic acid is added. (4) Centrifuge at 3,500 rpm for 15 minutes,
The absorbance (e) at 80 nm is measured with a spectrophotometer using <1> in a quartz cell having an optical path length of 1 cm. At the time of measurement, dilution is performed so that the measured value does not exceed 0.5 (dilution ratio). (5) As a blank, the absorbance (f) at 280 nm is measured in the same manner as described above, using a non-enzyme-fixed immobilizing carrier instead of immobilized trypsin. The dilution at this time is the same as in (4). (6) The activity of immobilized trypsin is calculated by the following equation. Activity of immobilized trypsin (U / ml · carrier) = 0.4 ×
(Ef) x dilution ratio
【００１８】〔実施例１〕脱アセチル化度７９％、平均
分子量４６，０００のキトサン２１０ｇを３．５％酢酸
水溶液２，７９０ｇに溶解した。該キトサン酸性水溶液
を、７％水酸化ナトリウム，２０％エタノール，７３％
水よりなる凝固溶液中に落下し、キトサンを粒状多孔質
に凝固再生後、中性になるまで充分水洗し、平均粒径１
mmの再生粒状多孔質キトサン３，０００ml（湿潤）を得
た。Example 1 210 g of chitosan having a deacetylation degree of 79% and an average molecular weight of 46,000 was dissolved in 2,790 g of a 3.5% acetic acid aqueous solution. The chitosan acidic aqueous solution was diluted with 7% sodium hydroxide, 20% ethanol, 73%
After falling into a coagulation solution composed of water and coagulating and regenerating the chitosan into a granular porous material, it was thoroughly washed with water until it became neutral.
3,000 ml (wet) of regenerated granular porous chitosan having a thickness of mm were obtained.
【００１９】次に再生粒状多孔質キトサン各５０mlを、
ハーキュリーズ・ジャパン（株）製、商品名ジェノヴィ
スコ型（ＧＥＮＯＶＩＳＣＯ Ｔｙｐｅ）ＣＳＷ−２の
カッパカラギーナンを用いて、表１に示す濃度のカッパ
カラギーナン水溶液１００mlに加え、２４時間攪拌した
後、カッパカラギーナン水溶液をろ過して除き、５．６
％エピクロロヒドリン５０ml、５Ｎ−ＫＯＨ１２mlをそ
れぞれ加え７０℃で２時間攪拌し反応させた。反応終了
後、充分水洗し、酵素固定化用担体（担体Ａ〜Ｆ）を得
た。Next, 50 ml each of the regenerated granular porous chitosan is
Using Hercules Japan Co., Ltd. product name GENOVISCO Type CSW-2 kappa carrageenan, add it to 100 ml of kappa carrageenan aqueous solution of the concentration shown in Table 1, stir for 24 hours, and then add the kappa carrageenan aqueous solution. 5.6.
50 ml of 5% epichlorohydrin and 12 ml of 5N-KOH were added thereto, and the mixture was stirred and reacted at 70 ° C. for 2 hours. After completion of the reaction, the resultant was sufficiently washed with water to obtain enzyme-immobilizing carriers (carriers A to F).
【００２０】[0020]
【表１】 [Table 1]
【００２１】担体Ａ〜Ｆをそれぞれ２０ml秤量し、１％
パパイン水溶液各１００mlを加え、２５℃にて３時間攪
拌した後、水洗して固定化パパインＡ* 〜Ｆ* 夫々２０
mlを得た。固定化パパインを夫々１ml取り、パパインの
吸着率及び固定化パパインの活性を測定し、表２に示し
た。20 ml of each of the carriers A to F was weighed and 1%
After adding 100 ml of each papain aqueous solution and stirring at 25 ° C. for 3 hours, the mixture was washed with water and immobilized papain A * to F * for 20 minutes each.
ml was obtained. 1 ml of the immobilized papain was taken, and the adsorption rate of the papain and the activity of the immobilized papain were measured.
【００２２】[0022]
【表２】 [Table 2]
【００２３】表２から明らかなように、カッパカラギー
ナン濃度が０．１〜１０％、好ましくは、１〜１０％の
とき優れた吸着率と活性を示した。さらに上述で得られ
た固定化パパインＤ* を１ml採取し、固定化パパインの
活性測定前に０．０５Ｍ−ＥＤＴＡと０．１Ｍ−システ
インを含む混合水溶液５mlに入れ、１０℃で１２時間賦
活化処理した後、充分水洗して、固定化パパインの活性
を測定した（第１回）。この操作を繰り返し行い、５回
迄固定化パパインの活性を測定し、表３に示した。As apparent from Table 2, when the concentration of kappa carrageenan is 0.1 to 10%, preferably 1 to 10%, excellent adsorption rate and activity were exhibited. Further, 1 ml of the immobilized papain D * obtained above was collected, put into 5 ml of a mixed aqueous solution containing 0.05 M-EDTA and 0.1 M-cysteine before measuring the activity of the immobilized papain, and activated at 10 ° C. for 12 hours. After the treatment, the plate was thoroughly washed with water and the activity of immobilized papain was measured (first time). This operation was repeated, and the activity of the immobilized papain was measured up to five times.
【００２４】[0024]
【表３】 この結果より、固定化パパインを賦活化処理する事によ
り、活性値が著しく高くなり、繰り返し使用にも適する
高い活性を持続することが明らかである。[Table 3] From this result, it is clear that the activation value of the immobilized papain significantly increases the activity value and maintains a high activity suitable for repeated use.
【００２５】〔実施例２〕実施例１と同様にして得られ
た再生粒状多孔質キトサン各５０mlを採取し、夫々２％
カッパカラギーナン水溶液１００mlに加え、表４に示す
処理時間で攪拌した後、カッパカラギーナン水溶液をろ
過して除き、５．６％エピクロロヒドリン５０ml、５Ｎ
−ＫＯＨ１２mlを夫々に加え７０℃で２時間攪拌し反応
させた。反応終了後、充分水洗し、酵素固定化用担体
（担体Ｇ〜Ｌ）を得た。Example 2 50 ml each of the regenerated granular porous chitosan obtained in the same manner as in Example 1 was collected, and 2% each.
After adding to 100 ml of kappa carrageenan aqueous solution and stirring for the treatment time shown in Table 4, the aqueous solution of kappa carrageenan was removed by filtration, and 50 ml of 5.6% epichlorohydrin and 5N
-12 ml of -KOH was added to each and stirred at 70 ° C for 2 hours to react. After the completion of the reaction, the resultant was sufficiently washed with water to obtain enzyme-immobilizing carriers (carriers G to L).
【００２６】[0026]
【表４】 [Table 4]
【００２７】担体Ｇ〜Ｌを夫々２０ml秤量し、１％パパ
イン水溶液各１００mlを加え、２５℃にて３時間攪拌し
た後、水洗して固定化パパインＧ* 〜Ｌ* 夫々２０mlを
得た。固定化パパインを夫々１ml取り、パパインの吸着
率及び固定化パパインの活性を測定し、表５に示した。20 ml of each of the carriers GL was weighed, 100 ml of a 1% aqueous solution of papain was added, and the mixture was stirred at 25 ° C. for 3 hours and washed with water to obtain 20 ml of each of immobilized papain G * to L * . 1 ml of the immobilized papain was taken, and the adsorption rate of papain and the activity of the immobilized papain were measured.
【００２８】[0028]
【表５】 表５から明らかなように、２％のカッパカラギーナン水
溶液の処理時間が１〜４８時間、好ましくは、６〜４８
時間のとき、優れた吸着率と活性を示した。[Table 5] As is clear from Table 5, the treatment time of the 2% aqueous solution of kappa carrageenan is 1 to 48 hours, preferably 6 to 48 hours.
At time, it showed excellent adsorption rate and activity.
【００２９】〔実施例３〕実施例１と同様にして得られ
た酵素固定化用担体Ｄより５mlを採取し、１％トリプシ
ン水溶液２５mlに入れ、３時間攪拌し吸着後、充分水洗
して固定化トリプシンを５ml得た。この１mlを取り、既
述の固定化トリプシンの活性測定方法により活性を測定
した（第１回）。この固定化トリプシンを充分水洗した
後、再び同様の方法で活性測定をした（第２回）。これ
を５回迄繰り返し活性を測定しその結果を表６に示した
（Ａ）。更に５回目の活性測定終了後、この固定化トリ
プシンに０．５Ｎ−ＮａＯＨを１０ml加え、６０℃で３
時間処理した後、充分に水洗してトリプシンを除き、１
％トリプシン水溶液の５mlに入れ、３時間攪拌し再吸着
後、水洗し活性を測定した（第１回）。この固定化トリ
プシンを充分水洗した後、再び同様の方法で活性測定を
した（第２回）。これを５回迄繰り返し活性を測定しそ
の結果を表６に示した（Ｂ）。Example 3 5 ml of the enzyme-immobilizing carrier D obtained in the same manner as in Example 1 was collected, put into 25 ml of a 1% aqueous solution of trypsin, stirred for 3 hours, adsorbed, and sufficiently washed with water to fix. 5 ml of trypsin was obtained. An aliquot of 1 ml was taken and the activity was measured by the above-described method for measuring the activity of immobilized trypsin (first time). After sufficiently washing the immobilized trypsin with water, the activity was measured again in the same manner (second time). This was repeated up to five times, and the activity was measured. The results are shown in Table 6 (A). After the fifth activity measurement, 10 ml of 0.5N NaOH was added to the immobilized trypsin,
After treatment for a long time, wash thoroughly with water to remove trypsin.
5% aqueous trypsin solution, stirred for 3 hours, re-adsorbed, washed with water and measured for activity (1st time). After sufficiently washing the immobilized trypsin with water, the activity was measured again in the same manner (second time). This was repeated up to 5 times, and the activity was measured. The results are shown in Table 6 (B).
【００３０】[0030]
【表６】 表６から明らかなように、本法で得られた酵素固定化用
担体は、繰り返し使用、再吸着後の再繰り返し使用に於
いても、活性の持続性が高く、優れた性能を有する酵素
固定化用担体が得られた。[Table 6] As is clear from Table 6, the enzyme immobilization carrier obtained by this method has high activity persistence and excellent performance even in repeated use and re-use after re-adsorption. The resulting carrier was obtained.
【００３１】〔実施例４〕実施例１と同様にして得られ
た再生粒状多孔質キトサン各５０mlを、和光純薬工業
（株）製ラムダカラギーナンを用いて表７に示す濃度の
ラムダカラギーナン水溶液１００mlに加え、２４時間攪
拌した後、ラムダカラギーナン水溶液をろ過して除き、
５．６％エピクロロヒドリン５０ml、５Ｎ−ＫＯＨ１２
mlをそれぞれ加え、７０℃で２時間攪拌し反応させた。
反応終了後、充分水洗し、酵素固定化用担体（担体Ｍ〜
Ｒ）を得た。Example 4 50 ml of the regenerated granular porous chitosan obtained in the same manner as in Example 1 was applied to 100 ml of an aqueous solution of lambda carrageenan having a concentration shown in Table 7 using lambda carrageenan manufactured by Wako Pure Chemical Industries, Ltd. After stirring for 24 hours, the aqueous solution of lambda carrageenan was removed by filtration.
50 ml of 5.6% epichlorohydrin, 5N-KOH12
ml was added thereto, and the mixture was stirred and reacted at 70 ° C. for 2 hours.
After the completion of the reaction, the plate is thoroughly washed with water, and the enzyme immobilization carrier (carrier M to
R) was obtained.
【００３２】[0032]
【表７】 [Table 7]
【００３３】担体Ｍ〜Ｒをそれぞれ２０ml秤量し、１％
パパイン水溶液各１００mlを加え、２５℃にて３時間攪
拌した後、充分水洗し、固定化パパインＭ* 〜Ｒ* 夫々
２０mlを得た。固定化パパインを夫々１ml取り、パパイ
ンの吸着率及び固定化パパインの活性を測定し、表８に
示した。20 ml of each of the carriers M to R was weighed, and 1%
After adding 100 ml of each papain aqueous solution and stirring at 25 ° C. for 3 hours, the mixture was sufficiently washed with water to obtain 20 ml of each of immobilized papain M * to R * . 1 ml of the immobilized papain was taken, and the adsorption rate of papain and the activity of the immobilized papain were measured.
【００３４】[0034]
【表８】 表８から明らかなように、ラムダカラギーナン濃度が
０．１〜１０％好ましくは、１〜１０％のとき優れた吸
着率と活性を示した。[Table 8] As is clear from Table 8, when the concentration of lambda carrageenan is 0.1 to 10%, preferably 1 to 10%, excellent adsorption rate and activity were exhibited.
【００３５】〔比較例〕 実施例１と同様にして得られた再生粒状多孔質キトサン
１００ｍｌが含む水を、ジメチルホルムアミドで充分に
置換除去した後、ジメチルホルムアミド１００ｍｌに、
ヘキサメチレンジイソシアナート２．８ｇを溶解した溶
解液に加え、２５℃で１時間反応させた。反応残液を除
去した後、ジメチルホルムアミドで洗浄し、次いで充分
水洗して酵素固定化用担体（担体Ｓ）を得た。同様に、
実施例１と同様にして得られた再生粒状多孔質キトサン
１００ｍｌが含む水をジメチルホルムアミドで充分に置
換除去した後、ジメチルホルムアミド１００ｍｌにジフ
ェニルメタンジイソシアナート４．２ｇを溶解した溶解
液に加え２５℃で１時間反応させた。反応残液を除去し
た後、ジメチルホルムアミドで洗浄し、次いで充分水洗
して酵素固定化用担体（担体Ｔ）を得た。上記の担体
Ｓ，Ｔ及び実施例１で得られた担体Ｄの各１０ｍｌを１
％パパイン水溶液５０ｍｌに加え、２５℃で２時間攪拌
した。吸着後の吸着液を除き、水洗して固定化パパイン
Ｄ＊，Ｓ＊，Ｔ＊を得た。これらの固定化パパイン各１
ｍｌの吸着率、活性を測定した結果を表９に示した。[Comparative Example] Water contained in 100 ml of regenerated granular porous chitosan obtained in the same manner as in Example 1 was sufficiently displaced and removed with dimethylformamide, and then added to 100 ml of dimethylformamide.
Hexamethylene diisocyanate (2.8 g) was added to the solution, and reacted at 25 ° C. for 1 hour. After removal of the reaction residue was washed with dimethyl Chi le formamide, then obtain an enzyme-immobilized carrier (carrier S) and sufficiently washed with water. Similarly,
After the water contained in 100 ml of the regenerated granular porous chitosan obtained in the same manner as in Example 1 was sufficiently displaced and removed with dimethylformamide, the solution was added to a solution obtained by dissolving 4.2 g of diphenylmethane diisocyanate in 100 ml of dimethylformamide and added at 25 ° C. For 1 hour. After removing the reaction residue, the residue was washed with dimethylformamide and then sufficiently washed with water to obtain a carrier for enzyme immobilization (carrier T). 10 ml of each of the carriers S and T and the carrier D obtained in Example 1 was
% Papain aqueous solution, and stirred at 25 ° C. for 2 hours. The adsorbed solution after the adsorption was removed and washed with water to obtain immobilized papain D * , S * , T * . Each of these immobilized papain
Table 9 shows the results of the measurement of the adsorption rate and the activity in ml.
【００３６】[0036]
【表９】 [Table 9]
【００３７】表９から明らかなように、本発明の方法で
得られた酵素固定化用担体Ｄ* を用いれば、優れた吸着
率、活性を有する固定化パパインが得られるが、本発明
以外の方法で得た酵素固定化用担体を用いた場合には、
所望する活性が得られなかった。As is clear from Table 9, when the enzyme-immobilized carrier D * obtained by the method of the present invention is used, immobilized papain having an excellent adsorption rate and activity can be obtained. When using the enzyme immobilization carrier obtained by the method,
The desired activity was not obtained.
【００３８】[0038]
【発明の効果】低分子量キトサンの酸性水溶液を塩基性
溶液中に滴下凝固再生せしめて得た再生粒状多孔質キト
サンを、カラギーナンの水溶液中に分散し、次いで多官
能性試薬と反応させ、カラギーナンを再生粒状多孔質キ
トサンに共有結合させる方法で得られた本発明の酵素固
定化用担体は、天然高分子であるキトサンとカラギーナ
ンが多官能試薬で複合化したことにより、天然物を母体
とすることから安全性に極めて優れ、また、カラギーナ
ンの硫酸基に由来するカチオン性により塩基性酵素の吸
着率も高く、固定化酵素の活性にも優れ、繰り返し使用
しても、優れた性能を発揮する効果がある。The regenerated granular porous chitosan obtained by dropping and coagulating an acidic aqueous solution of low molecular weight chitosan into a basic solution is dispersed in an aqueous solution of carrageenan, and then reacted with a polyfunctional reagent to form carrageenan. The carrier for enzyme immobilization of the present invention obtained by the method of covalently binding to regenerated granular porous chitosan comprises chitosan, a natural polymer, and carrageena.
Is highly safe because it is made of natural products, and the adsorption rate of basic enzymes is high due to the cationic nature derived from the sulfate group of carrageenan. It has an excellent activity and exhibits excellent performance even when used repeatedly.
Claims (1)
溶液中に滴下凝固再生せしめて得た再生粒状多孔質キト
サンを、カラギーナンの水溶液に分散し、次いで多官能
性試薬を反応させることを特徴とする酵素固定化用担体
の製造方法。A regenerated granular porous chitosan obtained by coagulating and regenerating an acidic aqueous solution of low molecular weight chitosan in a basic solution is dispersed in an aqueous solution of carrageenan, and then reacted with a polyfunctional reagent. A method for producing a carrier for immobilizing an enzyme.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP27704695A JP2824902B2 (en) | 1995-09-29 | 1995-09-29 | Method for producing enzyme-immobilizing carrier |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP27704695A JP2824902B2 (en) | 1995-09-29 | 1995-09-29 | Method for producing enzyme-immobilizing carrier |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH0994090A JPH0994090A (en) | 1997-04-08 |
| JP2824902B2 true JP2824902B2 (en) | 1998-11-18 |
Family
ID=17578037
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP27704695A Expired - Fee Related JP2824902B2 (en) | 1995-09-29 | 1995-09-29 | Method for producing enzyme-immobilizing carrier |
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| Country | Link |
|---|---|
| JP (1) | JP2824902B2 (en) |
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| CN107647112B (en) * | 2017-09-18 | 2021-07-02 | 江苏瑞牧生物科技有限公司 | Feed enzyme preparation carrier and preparation method thereof |
| CN114940776A (en) * | 2022-06-28 | 2022-08-26 | 中山大学 | Porous chitosan microsphere and method for fixing alkaline protease by using same |
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| Publication number | Publication date |
|---|---|
| JPH0994090A (en) | 1997-04-08 |
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