JPS59232092A - Immobilization of enzyme - Google Patents

Abstract

PURPOSE:To provide an immobilized enzyme resistant to the separation of the enzyme and having excellent thermal stability, by adsorbing an enzyme to a water-insoluble tannin, and including the product in a gelled carrier such as agar, carrageenen, etc. CONSTITUTION:An enzyme is adsorbed to a water-insoluble tannin prepared by adding one or more substances selected from insoluble polymers, activated carbon, chitin and gluten to tannin, and the adsorbed enzyme is stabilized by including in one or more gelled carrier selected from agar, carrageenan, alginic acid salt, gelatin and low-methylated peptide. The separation of the enzyme adsorbed to a water-insoluble tannin can be remarkably suppressed by the inclusion in the gelled carrier such as agar, carrageenan, etc. The enzyme is preferably aminopeptidase, lactic acid dehydrase, urease, glucose oxidase, glucoamylase, alcohol dehydrogenase, etc.

Description

【発明の詳細な説明】 本発明は包括法による酵素の固定化法に関する。[Detailed description of the invention] The present invention relates to a method for immobilizing enzymes using a comprehensive method.

酵素反応は医薬品、食品等の製造過程で工業的に広く用
いられているが、従来酵素を基質の水溶液に溶解させて
、溶液中で反応を゛行わせている。Enzyme reactions are widely used industrially in the manufacturing process of pharmaceuticals, foods, etc., but conventionally enzymes are dissolved in an aqueous solution of a substrate and the reaction is carried out in the solution.

しかしこのような方法では、反応条件を一定に維持しつ
＼、新鮮な酵素を補給したシ、又反応後に酵素を回収す
る操作等が著し“く複雑であるばかシでなく、反応生成
物の分離、精製も又著しく煩雑　１− な操作となる。このよう々欠点を解消する為に、近年酵
素を固定化して酵素反応を連続的に行なう試みがある。However, in this method, the reaction conditions are kept constant, fresh enzyme is supplied, and the operations for recovering the enzyme after the reaction are extremely complicated, and the reaction products are Separation and purification of the enzymes are also extremely complicated operations.In order to overcome these drawbacks, recent attempts have been made to immobilize enzymes and carry out enzyme reactions continuously.

酵素の固定化法としては、（１）担体結合法、（２）物
理的吸着法もしくはイオン結合法、（３）架橋法、（４
）包括法、（５）マイクロカプセル法等が知られている
。Enzyme immobilization methods include (1) carrier binding method, (2) physical adsorption method or ionic bonding method, (3) crosslinking method, (4)
) comprehensive method, (5) microcapsule method, etc. are known.

これらの固定化法のうち、（１）の担体結合法は特殊な
担体を必要とする上、反応条件の設定が難しく、一般に
高活性の固定化酵素を得るのが難、しい。Among these immobilization methods, the carrier binding method (1) requires a special carrier and is difficult to set reaction conditions, so that it is generally difficult to obtain a highly active immobilized enzyme.

高活性な固定化酵素が得られるが、担体と酵素との結合
力が弱い為、担体より酵素が脱離し易い欠点がある。（
３）の架橋法は、比較的苛酷な条件で反応きせる為、活
性度の低い固定化酵素しか得られない難点があシ、又（
４）の包括法は一般に簡単かつ低廉に製造出来る利点が
あるが、水溶性重合体を架更に（５）のマイクロカプセ
ル法は、固定化の際の調整が煩雑であることの他、有機
溶媒の使用により固定化酵素の活性が低下する等の欠点
がある。Although highly active immobilized enzymes can be obtained, the bonding force between the carrier and the enzyme is weak, so there is a drawback that the enzyme is more easily detached than the carrier. (
The cross-linking method (3) has the disadvantage that only immobilized enzyme with low activity can be obtained because the reaction is carried out under relatively harsh conditions.
The encapsulant method (4) generally has the advantage of being easy and inexpensive to produce, but the microcapsule method (5), in which a water-soluble polymer is cross-linked, requires complicated adjustments during immobilization and requires the use of organic solvents. There are drawbacks such as a decrease in the activity of the immobilized enzyme when used.

そこで本発明者等は上記諸欠点を考慮し、酵素の包括固
定化法に関し種々検討した結果、先ず水不溶性タンニン
は酵素蛋白質に対し特異的に強い吸着性を示すこと、そ
してこのような酵素を吸着した不溶性タンニンは、ｐＨ
１温度、イオン強度の変化によシ脱離し易い欠点がある
こと、更に検討した結果、該吸着物質を寒天、カラギー
ナン等のゲル化担体で包括することにより、酵素の脱離
が著しく抑制されること等の知見を得、本発明を完成し
た。Therefore, the present inventors took the above-mentioned drawbacks into account and conducted various studies on enzyme entrapment immobilization methods. First, the inventors found that water-insoluble tannins exhibit strong adsorption properties specifically for enzyme proteins, and that such enzymes The adsorbed insoluble tannins are
1) It has the disadvantage that it is easily desorbed due to changes in temperature and ionic strength.As a result of further investigation, we found that by enclosing the adsorbed substance in a gelling carrier such as agar or carrageenan, desorption of the enzyme can be significantly suppressed. Based on these findings, the present invention was completed.

即ち本発明は、タンニンに水溶性高分子物質、活性炭、
キチン及びグルテンより選ばれた少なくとも７種を加え
て得られる水不溶性タンニンに、酵素を吸着させた後、
これを寒天、カラギーナン、アルキン酸塩、ゼラチン及
び低メチル化ペクチンより選ばれた少なくとも７種のゲ
ル化担体で包括■ することを特徴とする酵素の固定化法である。That is, the present invention combines tannin with a water-soluble polymer substance, activated carbon,
After adsorbing enzymes to water-insoluble tannins obtained by adding at least seven types selected from chitin and gluten,
This enzyme immobilization method is characterized by enclosing the enzyme in at least seven kinds of gelling carriers selected from agar, carrageenan, alkinate, gelatin, and hypomethylated pectin.

以下、本発明を詳述する。The present invention will be explained in detail below.

先ず本発明に用いられるタンニンとしては、タンニン酸
の他、ピロガロールタンニン例えば没食子酸又は五倍子
タンニン、カテコール例えば茶、カカオ等からタンニン
物質（カテコール重合体）が用いられる。これらのタン
ニンはタンニン作用を有する限り精製されていないもの
でも良く、例えば市販の柿渋タンニン等も用いられる。First, as the tannins used in the present invention, in addition to tannic acid, pyrogallol tannins such as gallic acid or pentadol tannins, and catechols such as tannin substances (catechol polymers) from tea, cacao, etc. are used. These tannins may be unrefined as long as they have a tannin effect; for example, commercially available persimmon tannins may be used.

これらは単独でも２種以上のタンニン混合物としても用
いることが出来る。These tannins can be used alone or as a mixture of two or more tannins.

そして上記したタンニンに、水溶性高分子物質、活性炭
、キチン及びグルテンより選ばれた少なくとも７種のも
の（以下、これらをタンニン除去剤と言う）を加えて水
不溶、性のタンニンとする。Then, at least seven types selected from water-soluble polymer substances, activated carbon, chitin, and gluten (hereinafter referred to as tannin removers) are added to the above-mentioned tannins to obtain water-insoluble tannins.

なお水溶性高分子物質としては、例えばゼラチン、カゼ
イン、大豆分離蛋白、卵白、ポリビニルピロリドン、ア
セチルセルロース、メチルセルロース等であり、特に好
ましくは大豆分離蛋白、ゼラチン、メチルセルロース等
である。Examples of water-soluble polymeric substances include gelatin, casein, soybean protein isolate, egg white, polyvinylpyrrolidone, acetylcellulose, and methylcellulose, with soybean protein isolate, gelatin, and methylcellulose being particularly preferred.

そして水不溶性タンニンは、例えば次のようにして製造
することが出来る。Water-insoluble tannins can be produced, for example, as follows.

タンニン又はタンニン含有物を水中に溶解又は懸濁し、
好ましくはｐＨ７以下及び０−ＳＯ℃の温度に保持し、
これに上述のタンニン除去剤の水溶液又は水性懸濁液を
添加し、攪拌又は振盪等により約７０分以上作用させる
。Dissolving or suspending tannins or tannin-containing substances in water,
Preferably maintained at a pH of 7 or less and a temperature of 0-SO°C,
An aqueous solution or aqueous suspension of the tannin remover described above is added to this, and the mixture is allowed to act for about 70 minutes or more by stirring or shaking.

逆にタンニン除去剤の水溶液又は水性懸濁液に、タンニ
ン又はタンニン含有物の水溶液又は水性懸濁液を加えて
も良い。この操作によりタンニンは剤の比率は、通常／
：／ないしｔニア、好ましくはｌ：／ないし！：／、特
に３：ｌないしｔ；ｌである。Conversely, an aqueous solution or suspension of tannin or a tannin-containing substance may be added to an aqueous solution or suspension of the tannin remover. By this operation, the ratio of tannins and agents is usually /
:/ or t-nia, preferably l:/ or! :/, especially 3:l to t;l.

又本発明に用いられる酵素は、一般に固定化法に用いら
れるものであれば如何なるものでも良く、例えばアミノ
ペプチダーゼ、乳酸脱水素酵素、ウレアーゼ、グルコー
スオキシダーゼ、グルコアミラーゼ、アルコール脱水素
酵素、インベルターゼ、カルボキシペプチダーゼ等が好
ましい例として挙げられる。The enzyme used in the present invention may be any enzyme that is generally used in immobilization methods, such as aminopeptidase, lactate dehydrogenase, urease, glucose oxidase, glucoamylase, alcohol dehydrogenase, invertase, and carboxylic acid dehydrogenase. Preferred examples include peptidases and the like.

　５− これらの酵素含有溶液に、該酵素量に対し７〜２０倍量
（Ｗ／Ｗ　）の上述した水不溶性タンニンを攪拌しつ＼
加え、ｐＨＪ〜／２、温度θ〜７０℃で酵素を吸着させ
る。なお、この吸着操作はイオン強度／、０以下で行な
うのが望ましい。5- Add the above-mentioned water-insoluble tannin in an amount (W/W) 7 to 20 times the amount of the enzyme into these enzyme-containing solutions while stirring.
In addition, the enzyme is adsorbed at pHJ~/2 and temperature θ~70°C. Note that this adsorption operation is preferably performed at an ionic strength of /0 or less.

次に、水不溶性タンニンに酵素を吸着させたものを、寒
天、カラギーナン、アルギン酸塩、ゼラチン及び低メチ
ル化ペクチンより選ばれた少なくとも７種のゲル化担体
で包括し、目的とする固定化酵素を得る。Next, the enzyme adsorbed onto water-insoluble tannins is wrapped in at least seven types of gelling carriers selected from agar, carrageenan, alginate, gelatin, and hypomethylated pectin, and the desired immobilized enzyme is absorbed. obtain.

なお、寒天、カラギーナン又はゼラチンをゲル化担体と
して用いる場合、これらを水に所定量加熱溶解させた後
、冷却したものが用いられる。前記カラギーナンとして
は、カッパーカラギーナンが最も好ましい。Note that when agar, carrageenan, or gelatin is used as a gelling carrier, a predetermined amount of these is dissolved in water by heating, and then cooled. As the carrageenan, kappa carrageenan is most preferable.

又、アルギン酸Ｎａ、アルギン酸に１アルギン酸Ｎ　Ｈ
ｅ等のアルギン酸塩又は低メチル化ペクチンを担体とし
て用いる場合、これらを所定量の水で溶解したものをＣ
ａＣ１ｘ、Ａｌｘ（ＳＯｇ）３、Ｃａ　（Ｃ，２ＨＪＯ
Ｊ）、２等のゲル化剤中に押出し、固化したものが用い
ら　６− れる。Also, Na alginate, 1 alginate N H to alginic acid
When using alginates such as C or low methylated pectin as a carrier, dissolve them in a predetermined amount of water.
aC1x, Alx(SOg)3, Ca (C,2HJO
J), extruded into a gelling agent such as 2 and solidified is used.

本発明によれば、簡易な操作で安定性、殊に熱安定性の
優れた固定化酵素を効率良く得ることが出来、しかも該
酵素を種々の基質に繰り返し使用しても、該酵素の脱離
が著しく抑制される等、本発明は産業上極めて有意義で
ある。According to the present invention, it is possible to efficiently obtain an immobilized enzyme with excellent stability, especially thermostability, by a simple operation, and even when the enzyme is repeatedly used with various substrates, the enzyme can be desorbed. The present invention is extremely significant industrially, as separation is significantly suppressed.

以下、実施例を挙げて本発明を具体的に示す。Hereinafter, the present invention will be specifically illustrated by giving examples.

実施例１ タンニン酸〔和光紬薬（株）製〕／、＋０７ｎｇを５０
ｍｇの水に溶解したもの（１）Ｈ７，Ｏ）に、タンニン
除去剤としてメチルセルロース（本発明−１）、ゼラチ
ン（本発明−２）、ポリピロリドンに−６０（本発明−
３）、カゼイン（本発明−４）、活性炭（本発明−５）
を夫々ｚ　ｏ　ｏ　ｍｙ加えて混合し、水不溶性タンニ
ンを夫々得た。Example 1 Tannic acid [manufactured by Wako Tsumugi Co., Ltd.] /, +07 ng at 50
(1) H7,O) dissolved in mg of water, methyl cellulose (invention-1), gelatin (invention-2), polypyrrolidone-60 (invention-2) as tannin removers.
3), casein (present invention-4), activated carbon (present invention-5)
were added and mixed to obtain water-insoluble tannins.

次にアスペルギルス・オリゼーＦＥＲＭ　　Ｐ’−／／
≠りの皺培養物よυ硫安外画し、ＤＥＡＢ−１セルロー
スを用いて精製したロイシン・アミノペプチダーゼ標品
ｊＯ■をＯ２Ｏ３−Ｍ酢酸緩衝液（ｐＨ６，０）１００
ｍｌ！に溶解したものを、上記水不溶性タンニンに夫々
吸着させた。Next, Aspergillus oryzae FERM P'-//
Leucine aminopeptidase preparation jO, which was fractionated from the wrinkled culture and purified using DEAB-1 cellulose, was added to O2O3-M acetate buffer (pH 6,0) at 100%
ml! The water-insoluble tannins were adsorbed onto the water-insoluble tannins.

該酵素を吸着させた水不溶性タンニンをＯ，ＯＳＭのｐ
ＨＡ、０の酢酸緩衝液で２回洗滌し、次いでこれを２％
アルギン酸ソーダ１０ｍ１３に懸濁した後、注射器を用
いて５係ＣａＣ１，２溶液に滴下し球状に包括し、固定
化酵素を夫々得た（本発明１〜５）。The water-insoluble tannin adsorbed with the enzyme is O, the p of OSM.
Wash twice with HA, 0 acetate buffer, which was then combined with 2%
After suspending the enzyme in 10 ml of sodium alginate, it was dropped into a 5-functional CaC1,2 solution using a syringe and encapsulated in a spherical shape to obtain immobilized enzymes (Inventions 1 to 5).

なお第１表中、 対照−１：前述のロイシン・アミノペプチダーゼ（ＦＥ
ＲＭ　Ｐ−／／ｌ／Ｌり）の精製酵素標品。In Table 1, Control-1: the above-mentioned leucine aminopeptidase (FE
Purified enzyme preparation of RM P-//l/Lri).

対照−２：タンニン酸／ｊ０７ｎｇをよ０罰の水に溶解
したもの（ｐＥ（乙、（１））に、、ｔｏｏｍｇのポリ
ピロリドンに−６０を１０ｏ−の水に溶解したものを加
えて水不溶性タンニンを得、これに前述のロイシン・ア
ミノペプチダーゼ（ＦＥＲＭＰ−／／≠５？）の精製酵
素標品夕θ■を０．　Ｏｊ　Ｍの酢酸緩衝液に溶解した
ものを吸着させたもの。Control-2: To a solution of tannic acid/J07ng dissolved in 100% water (pE (B, (1)), add 0mg polypyrrolidone -60 dissolved in 100% water and add water. Insoluble tannin was obtained, and a solution of the purified enzyme preparation of the aforementioned leucine aminopeptidase (FERMP-//≠5?) dissolved in 0.0 M acetate buffer was adsorbed thereon.

対照−３：前述のロイシン・アミノペプチダーゼ（ＦＢ
ＲＭ　　Ｐ−／／４１り）の精製酵素標品夕Ｏｍｇを、
２％アルギン酸ソーダ１０ｍ１に懸濁した後、注射器を
用いてよ％ＣａＣＬｚ溶液に滴下し球状に包括した固定
化酵素。Control-3: Leucine aminopeptidase (FB
RM P-//41ri) purified enzyme preparation (Omg),
The immobilized enzyme was suspended in 10 ml of 2% sodium alginate and then dropped into a 2% CaCLz solution using a syringe to enclose it in a spherical shape.

第１表に於いて酵素の漏出試験は、本発明１〜５及び対
照−３はガーゼで包み、又対照−２は透析チューブに入
れた後、これらを２ｊ時間θ、Ｏ！Ｍ酢酸アンモニウム
緩衝液（ｐＨ７０）に透析し、固定化酵素の透析前の活
性値を基に残存活性値を測定した 々お対照−１は、固定化されていない為漏出率の算出は
出来なかった。In Table 1, in the enzyme leakage test, Inventions 1 to 5 and Control-3 were wrapped in gauze, and Control-2 was placed in a dialysis tube. Dialysis was performed against M ammonium acetate buffer (pH 70), and the residual activity was measured based on the activity value of the immobilized enzyme before dialysis. Control-1 was not immobilized, so the leakage rate could not be calculated. Ta.

又、残存酵素活性（４）は各試料を０．０　ｊ　Ｍ酢酸
緩衝液（ｐＨ６，Ｏ）中で、７０℃１．２０分放置した
後、酵素活性を測定した値である。Further, the residual enzyme activity (4) is the value obtained by measuring the enzyme activity after each sample was left at 70° C. for 1.20 minutes in a 0.0 j M acetate buffer (pH 6, O).

なお残存酵素の活性値（イ）は、各試料を５０〜７０℃
に３０分間放置後、ロイシン−Ｐ−ニトロアニリドを基
質として中台の方法〔中台忠信：置所り。The activity value (a) of the remaining enzyme is determined by heating each sample at 50 to 70°C.
After leaving it for 30 minutes, use leucine-P-nitroanilide as a substrate and use Nakadai's method [Tadanobu Nakadai: Co., Ltd.].

タタ（／り７７年）〕で測定した活性値を対照−１の標
品と比較した値（イ）である。The activity value measured by Tata (1977) was compared with the control-1 sample (a).

−９−−＾＾ 第　　　　　１　　　　　表 第１表から明らかな如く、本発明に係る固定化酵素は、
対照試料の何れに比しても、酵素の漏出率、残存酵素活
性（７０℃、２０分加熱処理）共に著しく優れたもので
あることが認められた。-9--^^ Table 1 As is clear from Table 1, the immobilized enzyme according to the present invention is
It was found that both the enzyme leakage rate and residual enzyme activity (heated at 70° C. for 20 minutes) were significantly superior to any of the control samples.

実施例２ タンニン酸３００■をｊＯｄの水に溶解したも　１０− のに、メチルセルロースフグを５Ｐθ℃の熱水２００ｙ
ｎｌに懸濁後冷却した液を、加え混合し、水不溶性タン
ニンを得た。Example 2 300 μm of tannic acid was dissolved in 10 μm of water, and methyl cellulose pufferfish was dissolved in 200 μm of hot water at 5 Pθ℃.
The suspension was suspended in water and then cooled, and then mixed to obtain water-insoluble tannins.

該水不溶性タンニンに、ナタ豆起源のウレアーゼ（タイ
プ■、シグマ社製）ｓｏｍグを夕℃で７時間攪拌するこ
とによシ吸着させた。Urease derived from rapeseed beans (type Ⅰ, manufactured by Sigma) was adsorbed onto the water-insoluble tannin by stirring at 7 hours in the evening at ℃.

次に該酵素を吸着させた水不溶性タンニンを遠心分離し
つ＼０．ＯｊＭトリス緩衝液（ｐＨ’７：Ｏ）で２回洗
滌し、次いでこれを１１０℃の２襲力ツパーカラギーナ
ン溶液２０ｍ１に懸濁した後、注射器を用いて冷０．３
ＭＫＣｌ溶液に滴下し球状に包括された固定化酵素を得
た。Next, the water-insoluble tannin with the enzyme adsorbed thereon is centrifuged and \0. Washed twice with OjM Tris buffer (pH'7:O), then suspended in 20 ml of 2-strength carrageenan solution at 110°C, and then injected using a syringe into a cold 0.3° C.
This was added dropwise to an MKCl solution to obtain a spherically encapsulated immobilized enzyme.

特許出願人　　キッコーマン株式会社 −１１− ５１０−Patent applicant: Kikkoman Corporation -11- 510-

Claims (1)

【特許請求の範囲】[Claims] タンニンに水溶性高分子物質、活性炭、キチン及びグル
テンよシ選ばれた少なくとも７種を加えて得られる水不
溶性タンニンに、酵素を吸着させた後、これを寒天、カ
ラギーナン、アルギン酸塩、ゼラチン及び低メチル化ペ
クチンよシ選ばれた少なくとも７種のゲル化担体で包括
することを特徴とする酵素の固定化法。Water-insoluble tannins obtained by adding at least seven selected water-soluble polymer substances, activated carbon, chitin, and gluten to tannins are adsorbed with enzymes, and then mixed with agar, carrageenan, alginate, gelatin, and A method for immobilizing an enzyme, which comprises enclosing it in at least seven kinds of gelling carriers selected from methylated pectin.