JP2002300875A - Method for producing alkaline earth metal-treated immobilized enzyme - Google Patents
Method for producing alkaline earth metal-treated immobilized enzymeInfo
- Publication number
- JP2002300875A JP2002300875A JP2001105696A JP2001105696A JP2002300875A JP 2002300875 A JP2002300875 A JP 2002300875A JP 2001105696 A JP2001105696 A JP 2001105696A JP 2001105696 A JP2001105696 A JP 2001105696A JP 2002300875 A JP2002300875 A JP 2002300875A
- Authority
- JP
- Japan
- Prior art keywords
- enzyme
- earth metal
- alkaline earth
- treated
- copolymer
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Landscapes
- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
Abstract
Description
【０００１】[0001]
【発明の属する技術分野】本発明は、担体に固定化され
た酵素を利用して、アミドやエステル等の合成、転移、
及び加水分解等の触媒反応を極性溶媒、特に水を含む極
性溶剤中で行って種々のペプチドや脂質等を有機合成す
る際に優れた性能を具備し、繰り返し使用する場合にも
安定した活性を発現する固定化酵素を得るための固定化
酵素の製造方法に関する。[0001] The present invention relates to the use of an enzyme immobilized on a carrier to synthesize and transfer amides and esters.
It has excellent performance when conducting a catalytic reaction such as hydrolysis in a polar solvent, particularly a polar solvent containing water, to synthesize various peptides and lipids, and has a stable activity even when used repeatedly. The present invention relates to a method for producing an immobilized enzyme for obtaining an expressed immobilized enzyme.
【０００２】[0002]
【従来の技術】近年、有機合成に酵素を利用する研究が
活発に行われている。それは酵素が常温、常圧で反応す
るため熱的に不安定な物質の合成が可能であるととも
に、反応が省エネルギーでクリーンであり、反応の特異
性に優れ、位置選択的、基質選択的反応や、不斉合成反
応が可能である等の多くの特徴を有するからである。特
に、極性溶媒中での酵素の反応は、水に不溶なアミドや
エステルの加水分解、非極性溶媒に不溶性の糖エステル
の合成、転移、加水分解等への応用が効率的にできる利
点がある。2. Description of the Related Art In recent years, researches utilizing enzymes for organic synthesis have been actively conducted. It is possible to synthesize a thermally unstable substance because the enzyme reacts at normal temperature and normal pressure, and the reaction is energy-saving and clean, and has excellent reaction specificity. This is because it has many features such as an asymmetric synthesis reaction. In particular, enzyme reactions in polar solvents have the advantage that they can be efficiently applied to the hydrolysis of water-insoluble amides and esters, the synthesis, transfer, and hydrolysis of sugar esters insoluble in non-polar solvents. .
【０００３】従来から、酵素を担体に固定化させるのに
担体をグルタルアルデヒド溶液で処理し、これに酵素を
固定化させることが極めて一般的に行われている。グル
タルアルデヒドは水中で酵素を固定させる時、約５℃か
ら室温程度の低温域で反応させることができ、比較的に
酵素の失活も少ない長所がある。しかし、巨大分子であ
る酵素と担体を架橋反応させるには、グルタルアルデヒ
ドの鎖長が短いので効率よく架橋反応させにくく、特に
水を含む極性溶媒中では酵素の離脱が多いという欠点が
あった。[0003] Conventionally, it is very common to immobilize an enzyme on a carrier by treating the carrier with a glutaraldehyde solution and immobilizing the enzyme on the solution. Glutaraldehyde can be reacted in a low temperature range of about 5 ° C. to about room temperature when immobilizing an enzyme in water, and has the advantage of relatively little enzyme deactivation. However, in order to carry out a cross-linking reaction between a macromolecule enzyme and a carrier, it is difficult to carry out a cross-linking reaction efficiently because the chain length of glutaraldehyde is short.
【０００４】生産と精製に多大な労力を要する酵素を工
業的に利用する上で、酵素の効率の高い固定化方法を開
発することは重要な課題であった。例えば、リパーゼの
固定化に関して、特開平１１−１６４６８７号公報に
は、再生粒状多孔質キトサン担体をビニルアルコールの
アルキルエーテルと無水マレイン酸との共重合体溶液で
処理した後、酵素水溶液を加えて固定化する固定化酵素
の製造方法が開示されている。この方法によれば、極性
溶剤、特に水を含む極性溶剤中で優れた活性を発現する
ことができる。しかしながら、この方法で得られた固定
化酵素は繰り返し使用する際に、無水マレイン酸がキト
サンのアミノ基や水と反応して生ずるカルボン酸が酵素
近傍を酸性雰囲気にするため、酵素の活性発現を損なう
結果となり、安定した酵素活性が得られない欠点があ
る。[0004] In order to industrially utilize enzymes that require a great deal of labor for production and purification, it has been an important task to develop a method for immobilizing enzymes with high efficiency. For example, regarding immobilization of lipase, JP-A-11-164687 discloses that after treating a regenerated granular porous chitosan carrier with a copolymer solution of an alkyl ether of vinyl alcohol and maleic anhydride, an enzyme aqueous solution is added. A method for producing an immobilized enzyme to be immobilized is disclosed. According to this method, excellent activity can be exhibited in a polar solvent, particularly a polar solvent containing water. However, when the immobilized enzyme obtained by this method is used repeatedly, maleic anhydride reacts with the amino group of chitosan or water, and the carboxylic acid generated by the reaction creates an acidic atmosphere near the enzyme. As a result, there is a disadvantage that stable enzyme activity cannot be obtained.
【０００５】[0005]
【発明が解決しようとする課題】本発明は、上記した従
来の欠点を克服し、極性溶媒、特に水を含む極性溶剤中
で優れた酵素活性を具備するとともに、繰り返し使用す
る場合にも安定した活性を発現する固定化酵素の製造方
法を提供することを目的とする。DISCLOSURE OF THE INVENTION The present invention overcomes the above-mentioned conventional disadvantages and has excellent enzyme activity in a polar solvent, particularly a polar solvent containing water, and is stable even when used repeatedly. An object of the present invention is to provide a method for producing an immobilized enzyme that exhibits activity.
【０００６】[0006]
【課題を解決するための手段】本発明者等は、上記課題
を解決するために鋭意検討した結果、再生粒状多孔質キ
トサン担体の処理に使用する無水マレイン酸が、キトサ
ンのアミノ基や水と反応して生ずるカルボン酸にアルカ
リ土金属を配位することにより、酵素の活性発現を損な
う原因となる酵素近傍の酸性雰囲気を緩和することを見
出し本発明に至った。Means for Solving the Problems The present inventors have conducted intensive studies in order to solve the above-mentioned problems, and as a result, the maleic anhydride used in the treatment of the regenerated granular porous chitosan carrier has been replaced with the amino group of chitosan and water. By coordinating an alkaline earth metal with a carboxylic acid generated by the reaction, the present inventors have found that the acidic atmosphere near the enzyme, which is a cause of impairing the expression of the activity of the enzyme, is alleviated.
【０００７】すなわち、本発明は再生粒状多孔質キトサ
ン担体をビニルアルコールのアルキルエーテルと無水マ
レイン酸との共重合体溶液で処理するに際し、再生粒状
多孔質キトサン担体１乾燥重量部に対して、ビニルアル
コールのアルキルエーテルと無水マレイン酸との共重合
体を０．０５〜０．６０乾燥重量部反応させ、次いで処
理後の再生粒状多孔質キトサン担体に酵素水溶液で酵素
を固定した後、得られた固定化酵素をアルカリ土金属を
含む水溶液に浸漬処理するアルカリ土金属処理固定化酵
素の製造方法であり、浸漬処理するアルカリ土金属を水
溶液中のアルカリ土金属の量が、湿潤状態の固定化酵素
１ｇ当たり０．０２〜０．２ｍｍｏｌであるアルカリ土
金属処理固定化酵素の製造方法である。That is, in the present invention, when the regenerated granular porous chitosan carrier is treated with a copolymer solution of an alkyl ether of vinyl alcohol and maleic anhydride, 1 part by weight of the regenerated granular porous chitosan carrier is dried with respect to 1 part by weight of vinyl. A copolymer of an alkyl ether of alcohol and maleic anhydride was allowed to react at 0.05 to 0.60 dry parts by weight, and then the enzyme was immobilized on the regenerated granular porous chitosan carrier after treatment with an aqueous enzyme solution, and the obtained product was obtained. A method for producing an alkaline-earth metal-treated immobilized enzyme in which the immobilized enzyme is immersed in an aqueous solution containing an alkaline-earth metal, wherein the amount of the alkaline-earth metal in the aqueous solution is a wet state. This is a method for producing an alkaline earth metal-treated immobilized enzyme in an amount of 0.02 to 0.2 mmol per 1 g.
【０００８】[0008]
【発明の実施の形態】本発明で用いる再生粒状多孔質キ
トサン担体は、キトサンを素材とした再生粒状多孔質体
であれば特に限定はされない。この再生粒状多孔質キト
サン担体は酵素や基質の拡散に優れ、強度が強く、カラ
ムに充填して使用するのに容易であるため固定化担体を
調整するのに好適である。具体的には例えば、特公平１
−１６４２０号公報で開示された方法により、再生粒状
多孔質キトサン担体を得ることができる。すなわち、平
均分子量が１０，０００〜２３０，０００の範囲である
低分子量キトサンを酸性溶液に溶解し、該溶解液を塩基
性溶液中に落下せしめ多孔質キトサンを凝固再生させる
ことによって得られる。再生粒状多孔質キトサン担体の
強度を向上させるために必要に応じてこのものを架橋し
てもよく、この時の架橋反応は特に制限されるものでは
ない。また、再生粒状多孔質キトサン担体を本発明に用
いる時は湿潤状態のものでも、水分を除いた乾燥状態の
ものでもよい。BEST MODE FOR CARRYING OUT THE INVENTION The regenerated granular porous chitosan carrier used in the present invention is not particularly limited as long as it is a regenerated granular porous material made of chitosan. This regenerated granular porous chitosan carrier is excellent in diffusing enzymes and substrates, has a high strength, and is easy to pack into a column for use, so that it is suitable for preparing an immobilized carrier. Specifically, for example,
According to the method disclosed in Japanese Patent Application No. -16420, a regenerated granular porous chitosan carrier can be obtained. That is, it is obtained by dissolving low molecular weight chitosan having an average molecular weight in the range of 10,000 to 230,000 in an acidic solution, dropping the dissolved solution into a basic solution, and coagulating and regenerating porous chitosan. The regenerated granular porous chitosan carrier may be crosslinked as needed in order to improve the strength, and the crosslinking reaction at this time is not particularly limited. When the regenerated granular porous chitosan carrier is used in the present invention, it may be in a wet state or in a dry state after removing moisture.
【０００９】本発明で用いるビニルアルコールのアルキ
ルエーテルと無水マレイン酸との共重合体（以下該重合
体という）としては、メチルビニルエーテル無水マレイ
ン酸共重合体、エチルビニルエーテル無水マレイン酸共
重合体溶液等が挙げられ、活性基である無水マレイン酸
の含有量と、更には該共重合体溶液を調整した時の溶液
粘度を適正にするため、その平均分子量は１０，０００
〜２，０００，０００が望ましく、好ましくは１００，
０００〜１，０００，０００の範囲のものが好適であ
る。The copolymer of an alkyl ether of vinyl alcohol and maleic anhydride (hereinafter referred to as the polymer) used in the present invention includes methyl vinyl ether maleic anhydride copolymer, ethyl vinyl ether maleic anhydride copolymer solution and the like. In order to make the content of the maleic anhydride as an active group and the solution viscosity of the copolymer solution adjusted properly, the average molecular weight is 10,000.
~ 2,000,000, preferably 100,
Those in the range of 000 to 1,000,000 are preferred.
【００１０】該共重合体を溶解する溶剤としては、該共
重合体が溶解する溶媒であれば制限されないが、該共重
合体は水やアルコールに溶解させると無水結合が開いて
脂肪酸やエステルができ、再生粒状多孔質キトサン担体
や酵素との結合に支障を生じるので、無水結合の開環に
あまり影響がない様に取扱う必要がある。また、該共重
合体は酸性水溶液中で加水分解が抑制されるので、酸性
水溶液に溶解させてもよい。有機溶剤としてはアセト
ン、メチルエチルケトン、テトラヒドロフラン、酢酸エ
チル、ジメチルアセトアミド、ジメチルホルムアミド、
メチルセルソルブ等が溶解度もよく、化学反応も起こら
ないので好ましい。The solvent for dissolving the copolymer is not limited as long as it is a solvent for dissolving the copolymer, but when the copolymer is dissolved in water or alcohol, an anhydride bond is opened to form a fatty acid or ester. It is possible to prevent the bond between the regenerated granular porous chitosan carrier and the enzyme. Further, since the copolymer is inhibited from being hydrolyzed in an acidic aqueous solution, it may be dissolved in an acidic aqueous solution. As an organic solvent, acetone, methyl ethyl ketone, tetrahydrofuran, ethyl acetate, dimethylacetamide, dimethylformamide,
Methylcellosolve and the like are preferable because they have good solubility and do not cause a chemical reaction.
【００１１】湿潤状態の再生粒状キトサン担体の１重量
部に対してビニルアルコールのアルキルエーテルと無水
マレイン酸との共重合体が０．２〜２０重量部、好まし
くは０．５〜１５重量部を上述の溶媒中に含有する様に
溶解させて反応溶液とする。該共重合体が０．２重量部
未満であると再生粒状多孔質キトサン担体との反応量が
少なく、その結果として酵素の固定化が不十分となる。
該共重合体が２０重量部を越えると再生粒状多孔質キト
サンと反応する該共重合体が増えるのにかかわらず再生
粒状多孔質キトサン担体との反応量が制限され、また該
共重合体溶液の粘度が高くなり取扱いの点からも好まし
くない。The copolymer of an alkyl ether of vinyl alcohol and maleic anhydride is used in an amount of 0.2 to 20 parts by weight, preferably 0.5 to 15 parts by weight, based on 1 part by weight of the wet regenerated granular chitosan carrier. It is dissolved so as to be contained in the above-mentioned solvent to obtain a reaction solution. When the amount of the copolymer is less than 0.2 parts by weight, the reaction amount with the regenerated granular porous chitosan carrier is small, and as a result, immobilization of the enzyme becomes insufficient.
When the amount of the copolymer exceeds 20 parts by weight, the amount of the copolymer reacting with the regenerated granular porous chitosan carrier is limited irrespective of an increase in the amount of the copolymer reacting with the regenerated granular porous chitosan. The viscosity increases, which is not preferable from the viewpoint of handling.
【００１２】該共重合体溶液中に再生粒状多孔質キトサ
ン担体を攪拌しながら反応させる際の反応温度は２５〜
９５℃で、該共重合体を有機溶剤に溶解した溶液を用い
る時は室温で溶解可能なので２５〜５０℃、水または弱
酸性の水に溶解した溶液を用いる時には該共重合体が５
０℃以上の温度で可溶であるので５０〜９０℃が好まし
い。そして、反応時間は０．５〜５時間で、特に水また
は弱酸性の水に溶解した溶液を用いる時は長時間反応さ
せると、該共重合体の無水結合が加水分解で開いて次工
程での酵素固定化に資する無水結合が消失するので、こ
の範囲以上の反応時間は避けなければならない。The reaction temperature when reacting the regenerated granular porous chitosan carrier in the copolymer solution with stirring is 25 to 25.
At a temperature of 95 ° C., when a solution of the copolymer dissolved in an organic solvent is used, the copolymer can be dissolved at room temperature.
50-90 ° C. is preferable since it is soluble at a temperature of 0 ° C. or higher. The reaction time is 0.5 to 5 hours, especially when a solution dissolved in water or weakly acidic water is used for a long time, when the anhydride bond of the copolymer is opened by hydrolysis and the next step is carried out. Since the anhydrous bonds contributing to enzyme immobilization disappear, reaction times longer than this range must be avoided.
【００１３】上記の処理により再生粒状多孔質キトサン
担体に該共重合体を結合させた後、用いられた溶液を除
去し、十分に水洗して未反応の該共重合体を除去し活性
化再生粒状多孔質キトサン担体を得る。After binding the copolymer to the regenerated granular porous chitosan carrier by the above-mentioned treatment, the used solution is removed and washed sufficiently with water to remove the unreacted copolymer and activate regeneration. A granular porous chitosan carrier is obtained.
【００１４】このような処理をしたときの該共重合体の
反応量は、該坦体１乾燥重量部に対して０．０５〜０．
６０乾燥重量部、好ましくは０．１０〜０．５０乾燥重
量部となる。０．０５乾燥重量部未満では、得られた固
定化酵素を極性溶剤または水を含む極性溶剤中で反応に
供する際に、酵素の脱離が起こり好ましくない。また、
０．６０乾燥重量部より多く反応させると、酵素の固定
化率低減や発現活性が低下したり、該共重合体が高分子
であるため反応が進まないといった問題を生ずる場合が
あり好ましくない。The amount of reaction of the copolymer upon such treatment is from 0.05 to 0.5% based on 1 part by weight of the carrier.
60 parts by dry weight, preferably 0.10 to 0.50 parts by dry weight. If the amount is less than 0.05 parts by dry weight, when the obtained immobilized enzyme is subjected to a reaction in a polar solvent or a polar solvent containing water, the enzyme is detached, which is not preferable. Also,
If the reaction is carried out in an amount of more than 0.60 parts by dry weight, problems such as a decrease in the immobilization rate of the enzyme and a decrease in the expression activity or a problem in that the reaction does not proceed because the copolymer is a polymer are not preferred.
【００１５】次いで得られた活性化再生粒状多孔質キト
サン担体に酵素水溶液を加えて振とうまたは攪拌しなが
ら活性化再生粒状多孔質キトサン担体に酵素を固定さ
せ、十分に水洗して未反応の酵素を除去して固定化酵素
を得る。Subsequently, an enzyme aqueous solution is added to the obtained activated regenerated granular porous chitosan carrier, and the enzyme is immobilized on the activated regenerated granular porous chitosan carrier while shaking or stirring. Is removed to obtain an immobilized enzyme.
【００１６】酵素の種類としては、リパーゼ、ホスホリ
パーゼ等の脂質関連酵素、パパイン、トリプシン、ペプ
シン、サーモライシン、トランスグルタミナーゼ、酸性
プロテアーゼ等の蛋白質、ペプチド関連酵素、アミラー
ゼ、グルコアミラーゼ、シクロデキストリングルコノト
ランスフェラーゼ等の糖質関連酵素等があげられる。ま
た、これら酵素水溶液濃度は酵素が溶解する濃度であれ
ばよく、特に制限はされず通常酵素は緩衝液に溶解させ
る。そして得られた活性化再生粒状多孔質キトサン担体
に酵素が固定された後の担体の処理は通常実施されてい
る処理方法による。Examples of the types of enzymes include lipid-related enzymes such as lipase and phospholipase, proteins such as papain, trypsin, pepsin, thermolysin, transglutaminase, and acidic protease, peptide-related enzymes, amylase, glucoamylase, and cyclodextrin gluconotransferase. Carbohydrate-related enzymes. The concentration of the aqueous enzyme solution may be any concentration at which the enzyme can be dissolved, and is not particularly limited. Usually, the enzyme is dissolved in a buffer solution. After the enzyme is immobilized on the obtained activated regenerated granular porous chitosan carrier, the carrier is treated by a commonly used treatment method.
【００１７】続いて、得られた固定化酵素はアルカリ土
金属を含む水溶液で浸漬処理されるが、この時のアルカ
リ土金属を含む水溶液に含まれるアルカリ土金属の量
は、湿潤状態の固定化酵素１ｇ当り０．０２〜０．２ｍ
ｍｏｌであることが好ましい。アルカリ土金属の量が少
ない場合は処理の効果が少なく、繰り返し使用する場合
の２回目の固定化酵素活性が１回目に比べて著しく低く
なり好ましくない。一方、アルカリ土金属の量が多すぎ
る場合は酵素活性が阻害され、１回目の使用時の活性値
が低くなり好ましくない。Next, the obtained immobilized enzyme is immersed in an aqueous solution containing an alkaline earth metal. 0.02-0.2m per 1g of enzyme
It is preferably mol. When the amount of the alkaline earth metal is small, the effect of the treatment is small, and the immobilized enzyme activity in the second use when used repeatedly is significantly lower than that in the first use, which is not preferable. On the other hand, when the amount of the alkaline earth metal is too large, the enzyme activity is inhibited, and the activity value at the first use is lowered, which is not preferable.
【００１８】用いるアルカリ土金属としては、ベリリウ
ム、マグネシウム、カルシウム、ストロンチウム、バリ
ウムの中で、固定化酵素中に存在するカルボン酸と強固
な配位結合を形成するマグネシウム、カルシウム、スト
ロンチウム、バリウムが有効で、特にカルシウムは強く
配位するので好ましい。またアルカリ土金属を含む水溶
液を調整するには、これら金属の化合物のなかで水溶性
のものであれば特に制限されないが、事実上は塩化マグ
ネシウム、塩化カルシウム、塩化ストロンチウム、塩化
バリウム等が好ましい。アルカリ土金属の化合物は水に
溶解してもよいが、酵素がｐＨの変化で失活するのを防
止するために、酵素に対して好適なｐＨの緩衝液に溶解
することが好ましい。Among the alkaline earth metals used, among beryllium, magnesium, calcium, strontium and barium, magnesium, calcium, strontium and barium which form a strong coordination bond with the carboxylic acid present in the immobilized enzyme are effective. In particular, calcium is preferred because it coordinates strongly. The aqueous solution containing an alkaline earth metal is not particularly limited as long as it is a water-soluble compound among these metal compounds, but magnesium chloride, calcium chloride, strontium chloride, barium chloride and the like are practically preferable. The alkaline earth metal compound may be dissolved in water, but is preferably dissolved in a buffer having a pH suitable for the enzyme in order to prevent the enzyme from being deactivated by a change in pH.
【００１９】アルカリ土金属を含む水溶液で固定化酵素
を浸漬処理する時の添加量と処理時間は、固定化酵素に
含まれるカルボキシル基の量にも影響されるが、通常は
湿潤状態の固定化酵素１ｇに対してアルカリ土金属を含
む水溶液が５〜５０倍容量程度の割合となるように添加
して、室温で０．５〜５時間処理すれば十分である。The amount of the immobilized enzyme and the treatment time when the immobilized enzyme is immersed in an aqueous solution containing an alkaline earth metal are affected by the amount of carboxyl groups contained in the immobilized enzyme. It is sufficient to add an aqueous solution containing an alkaline earth metal to 1 g of the enzyme so as to have a ratio of about 5 to 50 times the volume, and to perform treatment at room temperature for 0.5 to 5 hours.
【００２０】この処理により、固定化酵素に含まれてい
るカルボキシル基にカルシウム等のカルカリ土金属が配
位し、酵素近傍が酸性雰囲気になることを防ぐことがで
き、酵素活性が安定して発現できるようになる。[0020] By this treatment, calcarium earth metal such as calcium is coordinated to the carboxyl group contained in the immobilized enzyme, so that an acidic atmosphere near the enzyme can be prevented, and the enzyme activity is stably expressed. become able to.
【００２１】[0021]
【実施例】以下、本発明を実施例により具体的に説明す
るが、本発明はこの範囲に限定されるものではない。
尚、各測定値は以下に記載の試験方法により求めた。EXAMPLES Hereinafter, the present invention will be described specifically with reference to Examples, but the present invention is not limited to these ranges.
In addition, each measured value was determined by the test method described below.
【００２２】１．共重合体の反応量測定法 （１）再生粒状多孔質キトサン担体約２ｇの乾燥重量を
測定する。 （２）該担体をビニルアルコールのアルキルエーテルと
無水マレイン酸との共重合体溶液で活性化し、得られた
活性化再生粒状多孔質キトサン担体の乾燥重量を測定す
る。 （３）次式により担体１乾燥重量部に対する反応した該
共重合体の乾燥重量部の比率（共重合体の反応量）を求
める。1. (1) The dry weight of about 2 g of the regenerated granular porous chitosan carrier is measured. (2) The carrier is activated with a copolymer solution of an alkyl ether of vinyl alcohol and maleic anhydride, and the dry weight of the activated regenerated granular porous chitosan carrier obtained is measured. (3) The ratio of the dry weight part of the copolymer reacted to one dry weight part of the carrier (reaction amount of the copolymer) is determined by the following formula.
【数１】 (Equation 1)
【００２３】２．リパーゼの固定化率測定法 （１）オリーブ油（関東化学（株）製）約２０ｇを界面
活性剤（アデカトールＳＯ−１２０、旭電化工業（株）
製）２０ｇ、純水６０ｍｌと混ぜ、エマルジョン基質と
する。 （２）該基質２５ｍｌに純水１０ｍｌを加え３７℃で１
０分間予備加熱する。 （３）固定化操作に使用するリパーゼ水溶液０．１ｍｌ
を基質に加え３７℃で５分間反応させる。 （４）アセトンを５０％含むエタノールを８０ｍｌ加
え、攪拌し酵素反応を停止する。 （５）５０ｍＭのＮａＯＨで酵素反応により遊離した脂
肪酸を滴定し、酵素反応液の滴定量を求める。 （６）（３）の操作でリパーゼ水溶液のかわりに純水
０．１ｍｌを加え、（４） （５）の操作でブランクの滴定量を求める。 （７）固定化操作前のリパーゼ水溶液のエマルジョン脂
質分解発現活性値Ａ（ｕ／ｍｌ水溶液）を次式により求
める。2. Measuring method of lipase immobilization rate (1) About 20 g of olive oil (manufactured by Kanto Chemical Co., Ltd.) was added to a surfactant (Adecitol SO-120, Asahi Denka Kogyo Co., Ltd.)
20 g) and 60 ml of pure water to obtain an emulsion substrate. (2) Add 10 ml of pure water to 25 ml of the substrate, and add
Preheat for 0 minutes. (3) 0.1 ml of lipase aqueous solution used for the immobilization operation
Is added to the substrate and reacted at 37 ° C. for 5 minutes. (4) 80 ml of ethanol containing 50% of acetone is added and stirred to stop the enzyme reaction. (5) The fatty acid released by the enzyme reaction is titrated with 50 mM NaOH, and the titer of the enzyme reaction solution is determined. (6) Add 0.1 ml of pure water instead of the lipase aqueous solution in the operation of (3), and determine the titer of the blank in the operation of (4) and (5). (7) Emulsion lipolysis expression activity value A (u / ml aqueous solution) of the lipase aqueous solution before the immobilization operation is determined by the following equation.
【数２】 （８）固定化操作後の濾液のエマルジョン脂質分解発現
活性値Ｂ（ｕ／ｍｌ水溶液）を前述（７）と同様に測定
する。 （９）リパーゼの固定化率は（７）と（８）で求めたＡ
とＢにより次式で求める。(Equation 2) (8) Emulsion lipolysis expression activity value B (u / ml aqueous solution) of the filtrate after the immobilization operation is measured in the same manner as in (7) above. (9) The lipase immobilization rate was determined by A in (7) and (8).
And B by the following equation.
【数３】 (Equation 3)
【００２４】３．アルカリ土金属処理固定化リパーゼ中
の吸着アルカリ土金属量の測定方法 （１）所定量のアルカリ土金属塩化物をグッド緩衝液の
一種である１００ｍＭ−ＨＥＰＥＳ水溶液（ｐＨ７．
４）に溶解し、アルカリ土金属水溶液（原液）を作製す
る。 （２）湿潤固定化酵素１ｇをアルカリ土金属水溶液（原
液）２０ｍｌに浸漬し、２５℃で１時間振とう後、固定
化リパーゼを濾別しアルカリ土金属水溶液（残液）を得
る。 （３）得られたアルカリ土金属水溶液（原液）とアルカ
リ土金属水溶液（残液）それぞれのアルカリ土金属量Ｃ
ｍｏｌ、ＤｍｏｌをＩＣＰ（ＳＰＳ７０００、セイコー
電子工業（株）製）を用いて測定する。 （４）次式でアルカリ土金属処理固定化リパーゼ中の吸
着アルカリ土金属量を求める。3. Method for measuring the amount of adsorbed alkaline earth metal in immobilized lipase treated with alkaline earth metal (1) A predetermined amount of alkaline earth metal chloride is dissolved in 100 mM HEPES aqueous solution (pH 7.
4) to prepare an alkaline earth metal aqueous solution (stock solution). (2) 1 g of the wet immobilized enzyme is immersed in 20 ml of an alkaline earth metal aqueous solution (stock solution) and shaken at 25 ° C. for 1 hour. (3) Amount C of each of the obtained alkaline earth metal aqueous solution (stock solution) and alkaline earth metal aqueous solution (residual solution)
The mol and Dmol are measured using ICP (SPS7000, manufactured by Seiko Denshi Kogyo KK). (4) The amount of adsorbed alkaline earth metal in the lipase treated with alkaline earth metal is determined by the following equation.
【数４】 (Equation 4)
【００２５】４．水を含むアセトン溶液中でアルカリ土
金属処理固定化リパーゼの活性測定法 （１）３００ｍＭのモノラウリンと４０％の水を含有す
るアセトン溶液５ｍｌに１０ｍｇのアルカリ土金属処理
固定化リパーゼを添加し、３７℃で２０分間攪拌しなが
ら反応を行う。 （２）アルカリ土金属処理固定化リパーゼを濾別して反
応液を得る。 （３）この反応液１ｍｌと０．２規定の塩酸１ｍｌを混
合し、５０℃で１０分間振とうした後、２％界面活性剤
（トリトンＸ−１００、ローム・アンド・ハース製）２
０ｍｌを加え、塩酸混合液を得る。 （４）次の組成からなる試験液を調整する。 ５０ｍＭ−トリス塩酸緩衝液（ｐＨ７．５） ０．５％界面活性剤（トリトンＸ−１００） １ｍＭ−ＭｇＣｌ2 １ｍＭ−アデノシン３リン酸 １ｕ／ｍｌグリセロールキナーゼ ５ｕ／ｍｌグリセロフォスフェートオキシダーゼ ０．０３％４−アミノアンチピリン ０．０３％３，５−ジメトキシ−Ｎ−エチル−（２−ヒ
ドロキシ−３−スルフォプロピル）−アニリン、ナトリ
ウム塩 （５）（３）で得られた塩酸混合溶液を２０μｌ取り
（４）で示した試験液０．５ｍｌに添加し３７℃で１０
分間発色反応を行う。 （６）０．５％ドデシル硫酸ナトリウムを１ｍｌ加えて
吸光光度計を用いて６００ｎｍの吸光度を測定する。 （７）アルカリ土金属処理固定化リパーゼの活性を、次
式で得られる分解モノラウリン濃度より求める。4. Activity measurement method of lipase treated with alkaline earth metal in an acetone solution containing water (1) 10 mg of lipase treated with alkaline earth metal was added to 5 ml of acetone solution containing 300 mM monolaurin and 40% water. The reaction is carried out with stirring at 200C for 20 minutes. (2) The reaction solution is obtained by filtering the immobilized lipase treated with alkaline earth metal. (3) 1 ml of this reaction solution and 1 ml of 0.2 N hydrochloric acid were mixed and shaken at 50 ° C. for 10 minutes, and then 2% surfactant (Triton X-100, manufactured by Rohm and Haas) 2
0 ml is added to obtain a hydrochloric acid mixture. (4) Prepare a test solution having the following composition. 50mM- Tris-HCl buffer (pH 7.5) 0.5% surfactant (Triton X-100) 1mM-MgCl 2 1mM- adenosine triphosphate 1u / ml glycerol kinase 5u / ml glycerophosphate oxidase 0.03% 4-aminoantipyrine 0.03% 3,5-dimethoxy-N-ethyl- (2-hydroxy-3-sulfopropyl) -aniline, sodium salt (5) Take 20 μl of the hydrochloric acid mixed solution obtained in (3). Add to 0.5 ml of the test solution shown in (4) and add
Perform color reaction for 10 minutes. (6) Add 1 ml of 0.5% sodium dodecyl sulfate and measure the absorbance at 600 nm using an absorptiometer. (7) The activity of the immobilized lipase treated with alkaline earth metal is determined from the concentration of degraded monolaurin obtained by the following equation.
【数５】 （８）次いで（２）で濾別したアルカリ土金属処理固定
化リパーゼを、水を４０％含むアセトン溶液５ｍｌに入
れ、２５℃で１０分間振とうする。この濾別と振とうの
操作を合計３回繰り返しアルカリ土金属処理固定化リパ
ーゼを洗浄する。 （９）洗浄したアルカリ土金属固定化リパーゼを用い
（１）から（７）の操作で活性測定し、反応２回目の分
解モノラウリン濃度を求める。 （１０）１回目と２回目の分解モノラウリン濃度から次
式により２回目の残存活性値を求める。(Equation 5) (8) Next, the immobilized lipase treated with alkaline earth metal filtered in (2) is put into 5 ml of an acetone solution containing 40% of water, and shaken at 25 ° C. for 10 minutes. This filtration and shaking operation is repeated a total of three times to wash the lipase treated with alkaline earth metal. (9) Using the washed alkaline earth metal-immobilized lipase, the activity is measured by the operations of (1) to (7), and the concentration of degraded monolaurin in the second reaction is determined. (10) The residual activity value of the second time is determined from the concentrations of the decomposed monolaurin of the first time and the second time by the following equation.
【数６】 (Equation 6)
【００２６】５．ｐＨ緩衝液を含むアセトン溶液中での
アルカリ土金属処理固定化リパーゼの活性測定法 （１）３００ｎＭのモノラウリンと、濃度が１２５ｍＭ
の２−〔４−（ヒドロキシエチル）−１−ピペラジニ
ル〕エタンスルフォン酸緩衝液（ｐＨ７．０）を４０％
含有するアセトン溶液５ｍｌにアルカリ土金属処理固定
化リパーゼを１０ｍｇ添加し、３７℃で２０分間攪拌し
ながら反応を行う。 （２）水を含むアセトン溶液中でのアルカリ土金属処理
固定化リパーゼの活性測定法（上述４に記載）の（２）
から（１０）に記載した操作を行い、１回目の分解モノ
ラウリン濃度と２回目の分解モノラウリン濃度とから２
回目残存活性率を求める。[5] Activity measurement method of immobilized lipase treated with alkaline earth metal in acetone solution containing pH buffer (1) 300 nM monolaurin and 125 mM concentration
Of 2- [4- (hydroxyethyl) -1-piperazinyl] ethanesulfonic acid buffer (pH 7.0) at 40%
10 mg of the immobilized lipase treated with alkaline earth metal is added to 5 ml of the acetone solution contained, and the reaction is carried out at 37 ° C. with stirring for 20 minutes. (2) (2) of the method for measuring the activity of an immobilized lipase treated with an alkaline earth metal in an acetone solution containing water (described in 4).
The operations described in (10) to (10) are performed, and the concentration of monolaurin decomposed in the first time and the concentration of monolaurin decomposed in the second time are calculated as 2
The second-time residual activity rate is determined.
【００２７】〔実施例１〕脱アセチル化度８０％で平均
分子量６０，０００のキトサン１２０ｇを３．５％酢酸
水溶液１８８０ｇに溶解した。この水溶液を、７％水酸
化ナトリウム、２０％エタノール、７３％水よりなる塩
基性凝固浴中に滴下し、平均粒径０．１ｍｍの再生粒状
多孔質キトサンを凝固再生させた。次いで、中性になる
まで十分水洗して湿潤状態の粒状多孔質キトサン１００
０ｍｌを得た。Example 1 120 g of chitosan having a degree of deacetylation of 80% and an average molecular weight of 60,000 was dissolved in 1880 g of a 3.5% acetic acid aqueous solution. This aqueous solution was dropped into a basic coagulation bath composed of 7% sodium hydroxide, 20% ethanol, and 73% water to coagulate and regenerate regenerated granular porous chitosan having an average particle size of 0.1 mm. Next, the mixture is sufficiently washed with water until it becomes neutral, and the granular porous chitosan 100 in a wet state is obtained.
0 ml was obtained.
【００２８】この湿潤状態の再生粒状多孔質キトサン５
００ｍｌに水５００ｍｌとエチレングリコールジグリシ
ジルエーテル（エポキシ当量８７．１３）２．６２ｇを
加えて６０℃で１時間架橋反応を行い、反応終了後十分
に水洗し架橋粒状多孔質キトサンを得た。この湿潤状態
の架橋粒状多孔質キトサン２００ｍｌを秤量し、ジメチ
ルアセトアミドで処理し、これに含まれていた水をジメ
チルアセトアミドと置換、除去した。次いで、ジメチル
アセトアミド２００ｍｌに塩化ステアロイル１５．１ｇ
とトリエチルアミン５．１ｇを加えた溶液中で、２５℃
で１８時間攪拌しながら反応させた。反応終了後に、反
応に用いた溶液の残液を除去した後、ジメチルアセトア
ミドで洗浄し、次いで純水で処理して、ジメチルアセト
アミドを水と置換、除去し湿潤状態の再生粒状多孔質キ
トサン担体を得た。The regenerated granular porous chitosan 5 in the wet state
500 ml of water and 2.62 g of ethylene glycol diglycidyl ether (epoxy equivalent: 87.13) were added to 00 ml, and a crosslinking reaction was carried out at 60 ° C. for 1 hour. After the completion of the reaction, the mixture was sufficiently washed with water to obtain a crosslinked granular porous chitosan. 200 ml of the wet crosslinked granular porous chitosan was weighed, treated with dimethylacetamide, and the water contained therein was replaced with dimethylacetamide and removed. Then, 15.1 g of stearoyl chloride was added to 200 ml of dimethylacetamide.
In a solution containing 5.1 g of triethylamine and 25 ° C.
For 18 hours with stirring. After the completion of the reaction, after removing the residual liquid of the solution used for the reaction, the residue is washed with dimethylacetamide, and then treated with pure water to replace and remove dimethylacetamide with water to obtain a wet regenerated granular porous chitosan carrier. Obtained.
【００２９】湿潤状態の再生粒状多孔質キトサン担体２
０ｇをアセトンで処理し、含まれている水をアセトンと
置換、除去した。アセトン２００ｍｌにメチルビニルエ
ーテル無水マレイン酸共重合体である商品名ガントレッ
ト（ＧＡＮＴＲＥＺ）ＡＮ−１１９（ＣＨＥＭＩＣＡＬ
Ｓ ＣＯＲＰＯＲＡＴＩＯＮ製）の濃度が１．２重量部
になるように溶解させた。この溶液に湿潤状態の再生粒
状多孔質キトサン担体２０ｇを入れ、２５℃で１時間振
とうしながら反応させた。反応に用いた溶液の残液を除
去した後、アセトンで十分洗浄した後、純水で十分に水
洗処理し、アセトンを水と置換、除去し再生粒状多孔質
キトサン担体１乾燥重量に対してメチルビニルエーテル
無水マレイン酸共重合体反応量が０．１１乾燥重量部の
活性化粒状再生多孔質キトサン担体を得た。Regenerated granular porous chitosan carrier 2 in a wet state
0 g was treated with acetone, and the contained water was replaced with acetone and removed. Gantrez AN-119 (CHEMICAL) is a copolymer of methyl vinyl ether and maleic anhydride in 200 ml of acetone.
S CORPORATION) (1.2 parts by weight). 20 g of the regenerated granular porous chitosan carrier in a wet state was added to this solution, and the mixture was reacted at 25 ° C. for 1 hour with shaking. After removing the residual solution of the solution used for the reaction, the resultant was sufficiently washed with acetone, and then sufficiently washed with pure water, and the acetone was replaced with water. An activated granular regenerated porous chitosan carrier having a vinyl ether maleic anhydride copolymer reaction amount of 0.11 dry parts by weight was obtained.
【００３０】２０ｍＭリン酸緩衝液（ｐＨ７．５）５０
０ｍｌにリパーゼＴ−０１（旭化成工業（株）製）２０
０ｍｇを溶解させた後、得られた活性化粒状再生多孔質
キトサン担体を加え、２５℃で１８時間振とうしリパー
ゼを固定化させた。終了後、十分に水洗し、湿潤状態の
固定化リパーゼ６ｇを得た。この時リパーゼ固定化率は
９７％であった。20 mM phosphate buffer (pH 7.5) 50
Lipase T-01 (manufactured by Asahi Chemical Industry Co., Ltd.) 20 in 0 ml
After dissolving 0 mg, the obtained activated granular regenerated porous chitosan carrier was added, and the mixture was shaken at 25 ° C. for 18 hours to immobilize lipase. After the completion, the product was sufficiently washed with water to obtain 6 g of immobilized lipase in a wet state. At this time, the lipase immobilization rate was 97%.
【００３１】１００ｍＭ−ＨＥＰＳ緩衝液（ｐＨ７．
４）２０ｍｌに、表１のカルシウム量に相当する量の塩
化カルシウムを溶解させた。カルシウムを含む緩衝溶液
に湿潤状態の固定化リパーゼを夫々１ｇを加え、２５℃
で３時間振とうした後、十分に水洗し未反応の塩化カル
シウムを除去した。次いで、アセトンで処理し、水を置
換、除去した後、真空乾燥して乾燥状態のカルシウム処
理固定化リパーゼ試料Ｎｏ．２から試料Ｎｏ．７を得
た。この時のカルシウム処理固定化リパーゼ中の吸着カ
ルシウム量は表１に示した通りであった。A 100 mM HEPS buffer (pH 7.
4) An amount of calcium chloride corresponding to the amount of calcium in Table 1 was dissolved in 20 ml. 1 g of the immobilized lipase in a wet state was added to a buffer solution containing calcium at 25 ° C.
And shaken for 3 hours, and then sufficiently washed with water to remove unreacted calcium chloride. Next, the mixture was treated with acetone to replace and remove water, and then dried under vacuum to dry calcium-treated and immobilized lipase sample no. 2 to Sample No. 7 was obtained. At this time, the amount of adsorbed calcium in the calcium-treated immobilized lipase was as shown in Table 1.
【００３２】比較として上述と同様で得た湿潤状態の固
定化リパーゼ１ｇを、１００ｍＭ−ＨＥＰＳ緩衝液（ｐ
Ｈ７．４）２０ｍｌに入れ、２５℃で３時間振とう後、
水洗し、含まれる水をアセトン置換、除去した後、真空
乾燥してカルシウム処理しない乾燥状態の固定化リパー
ゼ試料Ｎｏ．１を得た。For comparison, 1 g of the immobilized lipase in a wet state obtained in the same manner as described above was added to a 100 mM HEPS buffer (p
H7.4) Put in 20 ml, shake at 25 ° C for 3 hours,
After washing with water and replacing the contained water with acetone and removing the same, the dried immobilized lipase sample no. 1 was obtained.
【００３３】得られた試料Ｎｏ．１から試料Ｎｏ．７夫
々について、水を含むアセトン溶液中での固定化リパー
ゼの活性測定を行い、その結果を表１に示した。The obtained sample No. 1 to sample no. For each of the seven samples, the activity of the immobilized lipase was measured in an acetone solution containing water, and the results are shown in Table 1.
【００３４】[0034]
【表１】 [Table 1]
【００３５】試料Ｎｏ．２は２回目残存活性率が低く、
試料Ｎｏ．７は２回目残存活性率が高いものの１回目分
解モノラウリン濃度が低く活性が劣っていた。一方、ア
ルカリ土金属を含む水溶液である塩化カルシウム緩衝液
で処理していない試料Ｎｏ．１は２回目残存活性率が低
く、繰り返し使用に好ましくない。Sample No. 2 has a low residual activity rate for the second time,
Sample No. Sample No. 7 had a high residual activity rate for the second time, but had a low concentration of degraded monolaurin at the first time and was inferior in activity. On the other hand, Sample No. which was not treated with a calcium chloride buffer solution which is an aqueous solution containing an alkaline earth metal. 1 has a low residual activity rate for the second time, and is not preferred for repeated use.
【００３６】〔実施例２〕実施例１と同様の操作で湿潤
状態の固定化リパーゼ６ｇを得た。この時リパーゼ固定
化率は９８％であった。Example 2 6 g of wet lipase were obtained in the same manner as in Example 1. At this time, the lipase immobilization rate was 98%.
【００３７】１００ｍＭ−ＨＥＰＳ緩衝役（ｐＨ７．
４）２０ｍｌに、表２のマグネシウム量に相当する量の
塩化マグネシウムを溶解させた。マグネシウムを含む緩
衝溶液に湿潤状態の固定化リパーゼを各々１ｇを加え、
２５℃で３時間振とうした後、十分に水洗し未反応の塩
化マグネシウムを除去した。次いで、アセトンで処理
し、水を置換、除去した後、真空乾燥して乾燥状態のマ
グネシウム処理固定化リパーゼ試料Ｎｏ．８から試料Ｎ
ｏ．１３を得た。この時のマグネシウム処理固定化リパ
ーゼ中の吸着マグネシウム量は表２に示した通りであっ
た。得られた試料Ｎｏ．８から試料Ｎｏ．１３夫々につ
いて、水を含むアセトン溶液中での固定化リパーゼの活
性測定を行い、その結果を表２に示した。100 mM HEPS buffer (pH 7.
4) An amount of magnesium chloride corresponding to the amount of magnesium in Table 2 was dissolved in 20 ml. 1 g of each of the immobilized lipase in a wet state was added to a buffer solution containing magnesium,
After shaking at 25 ° C. for 3 hours, it was sufficiently washed with water to remove unreacted magnesium chloride. Next, the mixture was treated with acetone to replace and remove water, and then dried in vacuum to dry and dry the magnesium-treated immobilized lipase sample No. 1; Sample N from 8
o. 13 was obtained. At this time, the amount of adsorbed magnesium in the magnesium-treated immobilized lipase was as shown in Table 2. The obtained sample No. 8 to Sample No. For each of the 13 samples, the activity of the immobilized lipase in an acetone solution containing water was measured, and the results are shown in Table 2.
【００３８】[0038]
【表２】 [Table 2]
【００３９】試料Ｎｏ．８は２回目残存活性率が低く、
試料Ｎｏ．１３は２回目残存活性率が高いものの１回目
分解モノラウリン濃度が低く活性が劣っていた。Sample No. 8 has a low residual activity rate for the second time,
Sample No. Sample No. 13 had a high residual activity rate for the second time, but had a low concentration of degraded monolaurin at the first time and was inferior in activity.
【００４０】〔実施例３〕実施例１と同様の操作で湿潤
状態の固定化リパーゼ６ｇを得た。この時リパーゼ固定
化率は９８％であった。Example 3 By the same operation as in Example 1, 6 g of immobilized lipase in a wet state was obtained. At this time, the lipase immobilization rate was 98%.
【００４１】１００ｍＭ−ＨＥＰＳ緩衝役（ｐＨ７．
４）２０ｍｌに、表３のストロンチウム量に相当する量
の塩化ストロンチウムを溶解させた。ストロンチウムを
含む緩衝溶液に湿潤状態の固定化リパーゼを夫々１ｇ加
え、２５℃で３時間振とうした後、十分に水洗し未反応
の塩化ストロンチウムを除去した。次いで、アセトンで
処理し、水を置換、除去した後、真空乾燥して乾燥状態
のストロンチウム処理固定化リパーゼ試料Ｎｏ．１４か
ら試料Ｎｏ．１９を得た。この時のストロンチウム処理
固定化リパーゼ中の吸着ストロンチウム量は表３に示し
た通りであった。得られた試料Ｎｏ．１４から試料Ｎ
ｏ．１９夫々について、水を含むアセトン溶液中での固
定化リパーゼの活性測定を行い、その結果を表３に示し
た。100 mM HEPS buffer (pH 7.
4) An amount of strontium chloride corresponding to the amount of strontium in Table 3 was dissolved in 20 ml. To the buffer solution containing strontium, 1 g of the immobilized lipase in a wet state was added, and each was shaken at 25 ° C. for 3 hours, and then sufficiently washed with water to remove unreacted strontium chloride. Next, the mixture was treated with acetone to replace and remove water, and then dried under vacuum to obtain a dried strontium-treated immobilized lipase sample no. 14 to sample no. 19 was obtained. At this time, the amount of adsorbed strontium in the strontium-treated immobilized lipase was as shown in Table 3. The obtained sample No. 14 to sample N
o. For each of the 19 samples, the activity of the immobilized lipase was measured in an acetone solution containing water, and the results are shown in Table 3.
【００４２】[0042]
【表３】 [Table 3]
【００４３】試料Ｎｏ．１４は２回目残存活性率が低
く、試料Ｎｏ．１９は２回目残存活性率が高いものの１
回目分解モノラウリン濃度が低く活性が劣っていた。Sample No. Sample No. 14 has a low second-stage residual activity rate, and 19 shows that although the second-stage residual activity ratio was high, 1
The concentration of monolaurin degraded the second time was low and the activity was inferior.
【００４４】〔実施例４〕実施例１と同様の操作で湿潤
状態の固定化リパーゼ６ｇを得た。この時リパーゼ固定
化率は９７％であった。Example 4 6 g of wet lipase were obtained in the same manner as in Example 1. At this time, the lipase immobilization rate was 97%.
【００４５】１００ｍＭ−ＨＥＰＳ緩衝役（ｐＨ７．
４）２０ｍｌに、表４のバリウム量に相当する量の塩化
バリウムを溶解させた。バリウムを含む緩衝溶液に湿潤
状態の固定化リパーゼを夫々１ｇを加え、２５℃で３時
間振とうした後、十分に水洗し未反応の塩化バリウムを
除去した。次いで、アセトンで処理し、水を置換、除去
した後、真空乾燥して乾燥状態のバリウム処理固定化リ
パーゼ試料Ｎｏ．２０から試料Ｎｏ．２５を得た。この
時のバリウム処理固定化リパーゼ中の吸着バリウム量は
表４に示した通りであった。得られた試料Ｎｏ．２０か
ら試料Ｎｏ．２５の夫々について、水を含むアセトン溶
液中での固定化リパーゼの活性測定を行い、その結果を
表４に示した。100 mM HEPS buffer (pH 7.
4) An amount of barium chloride corresponding to the amount of barium in Table 4 was dissolved in 20 ml. To the buffer solution containing barium was added 1 g of the immobilized lipase in a wet state, and the mixture was shaken at 25 ° C. for 3 hours, and then sufficiently washed with water to remove unreacted barium chloride. Next, the mixture was treated with acetone to replace and remove water, and then dried under vacuum to dry barium-treated immobilized lipase sample no. 20 to sample no. 25 was obtained. At this time, the amount of barium adsorbed in the barium-treated immobilized lipase was as shown in Table 4. The obtained sample No. 20 to sample no. For each of the 25 samples, the activity of the immobilized lipase in an acetone solution containing water was measured, and the results are shown in Table 4.
【００４６】[0046]
【表４】 [Table 4]
【００４７】試料Ｎｏ．２０は２回目残存活性率が低
く、試料Ｎｏ．２５は２回目残存活性率が高いものの１
回目分解モノラウリン濃度が低く活性が劣っていた。Sample No. Sample No. 20 has a low residual activity rate for the second time, and 25 indicates that although the second-stage residual activity rate was high, 1
The concentration of monolaurin degraded the second time was low and the activity was inferior.
【００４８】〔実施例５〕実施例１で得られた試料Ｎ
ｏ．１と試料Ｎｏ．４、実施例２で得られた試料Ｎｏ．
１０、実施例３で得られた試料Ｎｏ．１６、実施例４で
得られた試料Ｎｏ．２２の夫々について、ｐＨ緩衝液を
含むアセトン溶液中での固定化リパーゼの活性測定を行
い、その結果を表５に示した。Example 5 Sample N obtained in Example 1
o. 1 and Sample No. 4, sample No. 4 obtained in Example 2.
10, the sample No. obtained in Example 3. 16, the sample No. obtained in Example 4. For each of the 22 samples, the activity of the immobilized lipase was measured in an acetone solution containing a pH buffer, and the results are shown in Table 5.
【００４９】[0049]
【表５】 [Table 5]
【００５０】表５から明らかのようにアルカリ土金属処
理した固定化酵素は基質にｐＨ緩衝水溶液を含むアセト
ン中でも優れた活性を示した。As apparent from Table 5, the immobilized enzyme treated with the alkaline earth metal exhibited excellent activity even in acetone containing a pH buffer aqueous solution as a substrate.
【００５１】[0051]
【発明の効果】本発明は、再生粒状多孔質キトサン担体
をビニルアルコールのアルキルエーテルと無水マレイン
酸との共重合体で処理するに際し、該担体に共重合体を
反応させ、次いで酵素水溶液を加えて酵素を固定した後
に、更に得られた固定化酵素をアルカリ土金属を含む水
溶液に浸漬処理してアルカリ土金属処理固定化酵素とす
る方法である。本発明の方法で得た固定化酵素は、極性
溶媒、特に水を含む極性溶剤中で優れた酵素活性を具備
するとともに、繰り返し使用する場合にも安定した活性
を発現する効果があり、酵素の効率の高い固定化が達成
され、工業的有機合成に好ましく用いることが出来る。According to the present invention, when the regenerated granular porous chitosan carrier is treated with a copolymer of an alkyl ether of vinyl alcohol and maleic anhydride, the carrier is reacted with the copolymer, and then an aqueous enzyme solution is added. After immobilizing the enzyme, the obtained immobilized enzyme is further immersed in an aqueous solution containing an alkaline earth metal to obtain an alkaline earth metal-treated immobilized enzyme. The immobilized enzyme obtained by the method of the present invention has excellent enzymatic activity in a polar solvent, particularly a polar solvent containing water, and has an effect of exhibiting a stable activity even when used repeatedly. The immobilization with high efficiency is achieved and can be preferably used for industrial organic synthesis.
───────────────────────────────────────────────────── フロントページの続き Ｆターム(参考） 4B033 NA27 NB04 NB15 NB22 NB34 NB35 NB49 NB62 NB68 NC03 NC12 NC15 ND02 ──────────────────────────────────────────────────続 き Continued on the front page F term (reference) 4B033 NA27 NB04 NB15 NB22 NB34 NB35 NB49 NB62 NB68 NC03 NC12 NC15 ND02
Claims (2)
ルコールのアルキルエーテルと無水マレイン酸との共重
合体溶液で処理するに際し、該担体１乾燥重量部に対し
て、該共重合体を０．０５〜０．６０乾燥重量部反応さ
せ、次いで処理後の再生粒状多孔質キトサン担体に酵素
水溶液で酵素を固定した後、得られた固定化酵素をアル
カリ土金属を含む水溶液に浸漬処理することを特徴とす
るアルカリ土金属処理固定化酵素の製造方法。When a regenerated granular porous chitosan carrier is treated with a copolymer solution of an alkyl ether of vinyl alcohol and maleic anhydride, the copolymer is added in an amount of 0.05 to 1 part by weight of the carrier. 0.60 dry parts by weight, and then immobilizing the enzyme on the regenerated granular porous chitosan carrier after the treatment with an aqueous enzyme solution, and then immersing the obtained immobilized enzyme in an aqueous solution containing an alkaline earth metal. A method for producing an immobilized enzyme treated with an alkaline earth metal.
ルカリ土金属の量が湿潤状態の固定化酵素１ｇ当たり
０．０２〜０．２ｍｍｏｌであることを特徴とする請求
項１に記載のアルカリ土金属処理固定化酵素の製造方
法。2. The alkali according to claim 1, wherein the amount of the alkaline earth metal in the aqueous solution in which the immobilized enzyme is immersed is 0.02 to 0.2 mmol per 1 g of the wet immobilized enzyme. Method for producing immobilized enzyme treated with earth metal.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2001105696A JP2002300875A (en) | 2001-04-04 | 2001-04-04 | Method for producing alkaline earth metal-treated immobilized enzyme |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2001105696A JP2002300875A (en) | 2001-04-04 | 2001-04-04 | Method for producing alkaline earth metal-treated immobilized enzyme |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JP2002300875A true JP2002300875A (en) | 2002-10-15 |
Family
ID=18958336
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2001105696A Pending JP2002300875A (en) | 2001-04-04 | 2001-04-04 | Method for producing alkaline earth metal-treated immobilized enzyme |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP2002300875A (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1314801C (en) * | 2005-09-19 | 2007-05-09 | 江南大学 | Immobilized recombinant high temperature beta-galactosidase preparation method |
| CN1320124C (en) * | 2005-09-15 | 2007-06-06 | 武汉大学 | Preparation of low-molecular weight chitoglycan or chitooligose |
| JP2009515303A (en) * | 2005-11-02 | 2009-04-09 | セント・ルイス・ユニバーシティ | Direct electron transfer using enzymes in bioanodes, biocathodes, and biofuel cells |
| CN111467978A (en) * | 2020-03-03 | 2020-07-31 | 贵州省材料产业技术研究院(贵州省复合改性聚合物材料工程技术研究中心、国家复合改性聚合物材料工程技术研究中心) | Method for preparing chitosan crosslinked styrene maleic anhydride/polyether sulfone composite nanofiltration membrane |
-
2001
- 2001-04-04 JP JP2001105696A patent/JP2002300875A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1320124C (en) * | 2005-09-15 | 2007-06-06 | 武汉大学 | Preparation of low-molecular weight chitoglycan or chitooligose |
| CN1314801C (en) * | 2005-09-19 | 2007-05-09 | 江南大学 | Immobilized recombinant high temperature beta-galactosidase preparation method |
| JP2009515303A (en) * | 2005-11-02 | 2009-04-09 | セント・ルイス・ユニバーシティ | Direct electron transfer using enzymes in bioanodes, biocathodes, and biofuel cells |
| CN111467978A (en) * | 2020-03-03 | 2020-07-31 | 贵州省材料产业技术研究院(贵州省复合改性聚合物材料工程技术研究中心、国家复合改性聚合物材料工程技术研究中心) | Method for preparing chitosan crosslinked styrene maleic anhydride/polyether sulfone composite nanofiltration membrane |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Talekar et al. | Porous cross linked enzyme aggregates (p-CLEAs) of Saccharomyces cerevisiae invertase | |
| US4034139A (en) | Grafted mineral carriers for fixing enzymes | |
| HU179727B (en) | Process for producing water-insoluble enzyme composition | |
| JPH01285188A (en) | Lipase-immobilized polyacrylic acid-based material and utilization thereof | |
| JP2002300875A (en) | Method for producing alkaline earth metal-treated immobilized enzyme | |
| WO2011051145A1 (en) | Enzyme-functionalized supports | |
| JP3025947B2 (en) | Method for producing dry immobilized lipase carrier | |
| JPH0841106A (en) | Low-viscosity chitosan and its production | |
| JP2007517939A (en) | A simple method for recovering chitosan from its acidic solution | |
| JPH0956379A (en) | Regeneration of immobilized lipase | |
| JPH11164687A (en) | Production of immobilized enzyme | |
| JPH06197757A (en) | Method for separating beta-amylase | |
| JP4821168B2 (en) | Gas phase purification body, gas phase purification unit, and gas phase purification method | |
| JPS6015382B2 (en) | Adsorbent manufacturing method | |
| JP2824902B2 (en) | Method for producing enzyme-immobilizing carrier | |
| JP2021533776A (en) | Nucleic acid decontamination method | |
| George et al. | Flow rate dependent kinetics of urease immobilized onto diverse matrices | |
| JP2002233362A (en) | Method for stabilizing laccase activity and stabilized laccase composition | |
| IL43573A (en) | Enzymes fixed on a solid cellulose support | |
| US10626192B2 (en) | Process for reducing propylene chlorohydrin in hydroxypropylated products | |
| JP2613153B2 (en) | Method for producing carrier for immobilizing microorganisms | |
| JPH1042868A (en) | Activation of immobilized papain | |
| JP2556331B2 (en) | Method for removing acrylonitrile in water or aqueous dispersion | |
| JP2885353B2 (en) | Determination of chitin and chitosan in foods | |
| JPH04349883A (en) | Modified thermolysin-like enzyme and method for synthesizing alpha-l-aspartyl-l-phenylalanine methyl ester precursor using the same enzyme |