JPH10191969A - Enzyme recovery - Google Patents
Enzyme recoveryInfo
- Publication number
- JPH10191969A JPH10191969A JP9005262A JP526297A JPH10191969A JP H10191969 A JPH10191969 A JP H10191969A JP 9005262 A JP9005262 A JP 9005262A JP 526297 A JP526297 A JP 526297A JP H10191969 A JPH10191969 A JP H10191969A
- Authority
- JP
- Japan
- Prior art keywords
- enzyme
- aqueous solution
- isoelectric point
- polymer
- precipitate
- 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
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- Enzymes And Modification Thereof (AREA)
- Peptides Or Proteins (AREA)
Abstract
Description
【０００１】[0001]
【発明の属する技術分野】本発明は、不純物を含有する
酵素水溶液から酵素を失活させることなく、該酵素を効
率的に分離、回収する方法に関する。BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for efficiently separating and recovering an enzyme from an aqueous solution of the enzyme containing impurities without deactivating the enzyme.
【０００２】[0002]
【従来の技術】不純物を含有する酵素水溶液から酵素を
分離する方法として、従来、硫酸ナトリウム、硫酸アン
モニウム、塩化ナトリウム等の無機塩類による塩析法、
水溶性ポリマーを用いた沈澱分画法、エタノール、アセ
トン、イソプロピルアルコール等の有機溶媒による溶剤
沈澱法等が提案されている。2. Description of the Related Art As a method for separating an enzyme from an enzyme aqueous solution containing impurities, a salting-out method using an inorganic salt such as sodium sulfate, ammonium sulfate, sodium chloride, and the like has been conventionally used.
A precipitation fractionation method using a water-soluble polymer, a solvent precipitation method using an organic solvent such as ethanol, acetone, isopropyl alcohol and the like have been proposed.
【０００３】しかし、塩析法及び水溶性ポリマーを用い
た沈澱分画法では塩類及びポリマーを大量に使用するた
め、環境に対して問題があり、溶剤沈澱法ではコスト、
安全性の点からこれらの使用に限界があることが知られ
ている。[0003] However, the salting-out method and the precipitation fractionation method using a water-soluble polymer use a large amount of salts and polymers, and thus pose environmental problems.
It is known that their use is limited in terms of safety.
【０００４】また、酵素を含有する水溶液をその酵素の
等電点以上のpHとし、高分子アニオン電解質と高分子カ
チオン電解質とを添加し、酵素を不溶化させ分離する方
法が報告されている（特開昭６３−３０４９８３号公
報）。[0004] Further, a method has been reported in which an aqueous solution containing an enzyme is adjusted to a pH above the isoelectric point of the enzyme, a polymer anion electrolyte and a polymer cation electrolyte are added, and the enzyme is insolubilized and separated (Japanese Patent Application Laid-Open (kokai) No. 2000-282). JP-A-63-30983).
【０００５】しかしながら、この方法は酵素を含有する
水溶液のpHをその酵素の等電点以上にしなければならな
いため、洗剤に使用されるアルカリ酵素のうち等電点の
高い酵素は、等電点を超えるpH、すなわち強アルカリ下
においては失活するおそれがあり、好ましくない。However, in this method, the pH of the aqueous solution containing the enzyme must be higher than the isoelectric point of the enzyme. Therefore, among the alkaline enzymes used in detergents, the enzyme having a high isoelectric point has a high isoelectric point. If the pH is higher than that, that is, under a strong alkali, there is a possibility of deactivation, which is not preferable.
【０００６】[0006]
【発明が解決しようとする課題】従って、本発明の目的
は、酵素を失活させることなく、不純物を含有する酵素
水溶液から酵素を効率的に分離、回収する方法を提供す
ることにある。SUMMARY OF THE INVENTION Accordingly, an object of the present invention is to provide a method for efficiently separating and recovering an enzyme from an aqueous enzyme solution containing impurities without deactivating the enzyme.
【０００７】[0007]
【課題を解決するための手段】斯かる実情に鑑み本発明
者は鋭意研究を行った結果、酵素を含有する水溶液に、
その酵素の等電点未満のpHで、アニオン性高分子とカチ
オン性高分子とを添加すれば、酵素とこれら高分子とで
沈澱を生じ、これを分離すれば、効率よく酵素を回収す
ることができることを見出し本発明を完成した。Means for Solving the Problems In view of such circumstances, the present inventors have conducted intensive studies and have found that an aqueous solution containing an enzyme is
If the anionic polymer and the cationic polymer are added at a pH below the isoelectric point of the enzyme, precipitation occurs between the enzyme and the polymer, and if this is separated, the enzyme can be efficiently recovered. It has been found that the present invention has been completed and the present invention has been completed.
【０００８】すなわち本発明は、酵素を含有する水溶液
に、その酵素の等電点未満のpHにおいて、アニオン性高
分子とカチオン性高分子とを添加し、生じた沈澱を分離
することを特徴とする酵素の回収方法を提供するもので
ある。That is, the present invention is characterized in that an anionic polymer and a cationic polymer are added to an aqueous solution containing an enzyme at a pH lower than the isoelectric point of the enzyme, and the resulting precipitate is separated. The present invention provides a method for recovering an enzyme.
【０００９】[0009]
【発明の実施の形態】本発明が適用される酵素は、特に
限定されないが、酵素を含有する水溶液をその酵素の等
電点未満のpHにする関係上、洗浄剤等に配合されるアル
カリ酵素、特に等電点が５以上の酵素、更に等電点が７
以上の酵素が好ましい。このような酵素としては、アル
カリプロテアーゼ、アルカリエステラーゼ、アルカリカ
ルボヒドラーゼ、リアーゼ等が挙げられる。このうち、
プロテアーゼの具体例としては、ペプシン、トリプシ
ン、キモトリプシン、コラゲナーゼ、ケラチナーゼ、エ
ラスターゼ、ズブチリシン、パパイン、アミノペプチダ
ーゼ、カルボキシペプチダーゼ等を挙げることができ
る。エステラーゼの具体例としては、ガストリックリパ
ーゼ、パンクレアチックリパーゼ、植物リパーゼ類、ホ
スホリパーゼ類、コリンエステラーゼ類、ホスファター
ゼ類等が挙げられる。カルボヒドラーゼとしては、セル
ラーゼ、マルターゼ、サッカラーゼ、アミラーゼ、ペク
チナーゼ、α−及びβ−グリコシターゼ等が挙げられ
る。リアーゼとしては、カルボキシ脱離酵素、アルデヒ
ド脱離酵素、オキソ酸脱離酵素、ヒドロリアーゼ、多糖
に作用する脱離酵素、アンモニアリアーゼ等が挙げられ
る。BEST MODE FOR CARRYING OUT THE INVENTION The enzyme to which the present invention is applied is not particularly limited. However, since an aqueous solution containing the enzyme is adjusted to a pH lower than the isoelectric point of the enzyme, an alkaline enzyme to be added to a detergent or the like. Enzymes having an isoelectric point of 5 or more, especially an isoelectric point of 7 or more
The above enzymes are preferred. Examples of such enzymes include alkaline protease, alkaline esterase, alkaline carbohydrase, lyase and the like. this house,
Specific examples of proteases include pepsin, trypsin, chymotrypsin, collagenase, keratinase, elastase, subtilisin, papain, aminopeptidase, carboxypeptidase and the like. Specific examples of esterases include gastric lipase, pancreatic lipase, plant lipases, phospholipases, cholinesterases, phosphatases, and the like. Examples of carbohydrase include cellulase, maltase, saccharase, amylase, pectinase, α- and β-glycosidase. Examples of lyases include carboxy lyase, aldehyde lyase, oxoacid lyase, hydrolyase, lyase that acts on polysaccharides, and ammonia lyase.
【００１０】本発明で用いられるアニオン性高分子とし
ては界面活性剤高分子が好ましく、具体的には、ナフタ
レンスルホン酸ホルマリン縮合物、カルボン酸型高分子
活性剤等の合成高分子化合物、並びにタンニン酸、リグ
ニンスルホン酸等の天然高分子化合物を例示できる。こ
れらの高分子化合物は２種以上を併用してもよい。ま
た、本発明で用いられるカチオン性高分子はカチオン凝
集剤、特にポリアクリル酸エステル系、ポリアミン系、
ジシアンジアミド系等が好ましいものとして例示でき
る。As the anionic polymer used in the present invention, a surfactant polymer is preferable. Specifically, a synthetic polymer compound such as a naphthalenesulfonic acid formalin condensate, a carboxylic acid type polymer activator, and tannin Examples include natural high molecular compounds such as acids and ligninsulfonic acid. Two or more of these polymer compounds may be used in combination. Further, the cationic polymer used in the present invention is a cationic flocculant, particularly a polyacrylate ester type, a polyamine type,
Dicyandiamide-based compounds can be exemplified as preferred.
【００１１】アニオン性高分子及びカチオン性高分子
は、対象とする酵素を含有する水溶液の酵素に対してそ
れぞれ５０〜２００重量％添加することが好ましい。こ
の量が２００％を超えると、沈澱物中の酵素の濃度が低
くなり、回収に手間取るため好ましくない。The anionic polymer and the cationic polymer are preferably added in an amount of 50 to 200% by weight based on the amount of the enzyme in the aqueous solution containing the target enzyme. If this amount exceeds 200%, the concentration of the enzyme in the precipitate becomes low, and it is not preferable because it takes time to recover.
【００１２】上記の如く、２種の高分子を酵素水溶液を
添加すれば、酵素はその等電点未満のpHではカチオン性
の高分子となっているため、アニオン性高分子が酵素に
結合し、次いでカチオン性高分子が結合し沈澱を生ず
る。ここでpHの調整は等電点未満であれば特に必要な
い。このようにして得られた沈澱は遠心分離や濾過等に
より簡単に分離することができる。As described above, when two kinds of polymers are added to an aqueous enzyme solution, the enzyme becomes a cationic polymer at a pH lower than its isoelectric point, so that the anionic polymer binds to the enzyme. The cationic polymer then binds and precipitates. Here, the adjustment of pH is not particularly required as long as it is lower than the isoelectric point. The precipitate thus obtained can be easily separated by centrifugation or filtration.
【００１３】[0013]
【発明の効果】本発明によれば、酵素を含有する水溶液
から、酵素を失活させることなく、効率的に分離するこ
とができる。According to the present invention, an enzyme can be efficiently separated from an aqueous solution containing the enzyme without deactivating the enzyme.
【００１４】[0014]
【実施例】以下、実施例を挙げて本発明を更に詳細に説
明するが、本発明はこれらに限定されるものではない。EXAMPLES The present invention will be described in more detail with reference to the following Examples, but it should not be construed that the invention is limited thereto.
【００１５】実施例１ （１）酵素水溶液として、プロテアーゼ活性を有する粗
酵素（Bacillus sp. KSM-K16(FERM P-3367)由来、酵素
等電点１０．６）を含む水溶液（酵素蛋白質濃度：１．
５重量％）を用いた。この水溶液に、水溶液中の酵素蛋
白質量に対し、重量比で０．５〜２倍となるようにアニ
オン性高分子としてナフタレンスルホン酸ホルマリン縮
合物（商品名：デモールSC−30（花王（株）））（表１
中Ａと略す）及びカチオン性高分子としてジシアンジア
ミド系凝集剤（商品名：ダイヤフロックK405（ダイヤフ
ロック社））（表１中Ｂと略す）を添加し、pH８にてサ
ンプルを調製した。 （２）液中に沈澱物が形成されたので、これを遠心分離
により沈澱物と上清にわけ、それぞれの酵素活性を測定
して活性収率を計算により求めた。この結果を表１に示
す。Example 1 (1) As an aqueous enzyme solution, an aqueous solution containing a crude enzyme having protease activity (derived from Bacillus sp. KSM-K16 (FERM P-3367), isoelectric point of enzyme 10.6) (concentration of enzyme protein: 1.
5% by weight). In this aqueous solution, a naphthalenesulfonic acid formalin condensate (trade name: Demol SC-30 (Kao Corp.) is used as an anionic polymer so that the weight ratio is 0.5 to 2 times the mass of the enzyme protein in the aqueous solution. )) (Table 1)
Medium A) and a dicyandiamide-based flocculant (trade name: Diafloc K405 (Diafloc)) (abbreviated as B in Table 1) as a cationic polymer were added to prepare a sample at pH 8. (2) Since a precipitate was formed in the solution, the precipitate was separated into a precipitate and a supernatant by centrifugation, and the respective enzyme activities were measured to determine the activity yield. Table 1 shows the results.
【００１６】[0016]
【表１】 サンプル（添加剤、酵素蛋白質量：添加剤量（重量比）） 活性収率（％） コントロール ０ サンプル１（Ａ，１：１．８） ６８ サンプル２（Ａ，１：１．８、Ｂ，１：０．２５） ７２ サンプル３（Ａ，１：１．８、Ｂ，１：０．５） ８０ サンプル４（Ａ，１：１．８、Ｂ，１：１） ９０ サンプル５（Ａ，１：１．８、Ｂ，１：２） ９５Table 1 Samples (additive, enzyme protein mass: additive amount (weight ratio)) Activity yield (%) Control 0 Sample 1 (A, 1: 1.8) 68 Sample 2 (A, 1: 1. 8, B, 1: 0.25) 72 Sample 3 (A, 1: 1.8, B, 1: 0.5) 80 Sample 4 (A, 1: 1.8, B, 1: 1) 90 samples 5 (A, 1: 1.8, B, 1: 2) 95
【００１７】実施例２ （１）酵素水溶液として、プロテアーゼ活性を有する粗
酵素（Bacillus sp. KSM-K16(FERM P-3367)由来、酵素
等電点１０．６）を含む水溶液（酵素蛋白質濃度：１．
５重量％）を用いた。この水溶液に、水溶液中の酵素蛋
白質量に対し、重量比で０．５〜２倍となるようにアニ
オン性高分子としてカルボン酸型高分子活性剤（表２中
Ａと略す）（商品名：デモールEP（花王（株）））及び
カチオン性高分子としてジシアンジアミド系凝集剤（商
品名：ダイヤフロックK405（ダイヤフロック社））（表
２中Ｂと略す）を添加し、pH６にてサンプルを調製し
た。 （２）液中に沈澱物が形成されたので、これを遠心分離
により沈澱物と上清にわけ、それぞれの酵素活性を測定
して活性収率を計算により求めた。この結果を表２に示
す。Example 2 (1) As an enzyme aqueous solution, an aqueous solution containing a crude enzyme having protease activity (derived from Bacillus sp. KSM-K16 (FERM P-3367), isoelectric point of enzyme 10.6) (enzyme protein concentration: 1.
5% by weight). In this aqueous solution, a carboxylic acid type polymer activator (abbreviated as A in Table 2) as an anionic polymer (trade name: Demol EP (Kao Corp.)) and a dicyandiamide-based flocculant (trade name: Diafloc K405 (Diafloc)) (abbreviated as B in Table 2) as a cationic polymer were added to prepare a sample at pH 6. did. (2) Since a precipitate was formed in the solution, the precipitate was separated into a precipitate and a supernatant by centrifugation, and the respective enzyme activities were measured to determine the activity yield. Table 2 shows the results.
【００１８】[0018]
【表２】 サンプル（添加剤、酵素蛋白質量：添加剤量（重量比）） 活性収率（％） コントロール ０ サンプル１（Ａ，１：０．５） １０ サンプル２（Ａ，１：０．５、Ｂ，１：０．５） ４５ サンプル３（Ａ，１：０．５、Ｂ，１：２） ８０Table 2 Sample (additive, enzyme protein mass: additive amount (weight ratio)) Activity yield (%) Control 0 Sample 1 (A, 1: 0.5) 10 Sample 2 (A, 1: 0. 5, B, 1: 0.5) 45 Sample 3 (A, 1: 0.5, B, 1: 2) 80
【００１９】実施例３ （１）酵素水溶液として、プロテアーゼ活性を有する粗
酵素（Bacillus sp. KSM-K16(FERM P-3367)由来、酵素
等電点１０．６）を含む水溶液（酵素蛋白質濃度：１．
５重量％）を用いた。この水溶液に、水溶液中の酵素蛋
白質量に対し、重量比で０．５〜２倍となるようにアニ
オン性高分子としてナフタレンスルホン酸ホルマリン縮
合物（商品名：デモールSC−30（花王（株）））（表３
中Ａと略す）及びカチオン性高分子としてポリアクリル
酸エステル系凝集剤（商品名：クリフロックLC541（栗
田工業（株）））（表３中Ｂと略す）を添加し、pH８に
てサンプルを調製した。 （２）液中に沈澱物が形成されたので、これを遠心分離
により沈澱物と上清にわけ、それぞれの酵素活性を測定
して活性収率を計算により求めた。この結果を表３に示
す。Example 3 (1) As an aqueous enzyme solution, an aqueous solution containing a crude enzyme having protease activity (derived from Bacillus sp. KSM-K16 (FERM P-3367), isoelectric point of enzyme 10.6) (enzyme protein concentration: 1.
5% by weight). In this aqueous solution, a naphthalenesulfonic acid formalin condensate (trade name: Demol SC-30 (Kao Corp.) is used as an anionic polymer so that the weight ratio is 0.5 to 2 times the mass of the enzyme protein in the aqueous solution. )) (Table 3)
Medium A) and a polyacrylate coagulant (trade name: Cliffloc LC541 (Kurita Kogyo Co., Ltd.)) (abbreviated as B in Table 3) as a cationic polymer, and the sample was prepared at pH 8. Prepared. (2) Since a precipitate was formed in the solution, the precipitate was separated into a precipitate and a supernatant by centrifugation, and the respective enzyme activities were measured to determine the activity yield. Table 3 shows the results.
【００２０】[0020]
【表３】 サンプル（添加剤、酵素蛋白質量：添加剤量（重量比）） 活性収率（％） コントロール ０ サンプル１（Ａ，１：１．８） ６５ サンプル２（Ａ，１：１．８、Ｂ，１：０．５） ７０ サンプル３（Ａ，１：１．８、Ｂ，１：１） ８５Table 3 Samples (additive, enzyme protein mass: additive amount (weight ratio)) Activity yield (%) Control 0 Sample 1 (A, 1: 1.8) 65 Sample 2 (A, 1: 1. 8, B, 1: 0.5) 70 Sample 3 (A, 1: 1.8, B, 1: 1) 85
Claims (3)
電点未満のpHにおいて、アニオン性高分子とカチオン性
高分子とを添加し、生じた沈澱を分離することを特徴と
する酵素の回収方法。1. An enzyme comprising adding an anionic polymer and a cationic polymer to an aqueous solution containing the enzyme at a pH lower than the isoelectric point of the enzyme, and separating the resulting precipitate. Collection method.
上である請求項１記載の回収方法。2. The method according to claim 1, wherein the isoelectric point of the enzyme contained in the aqueous solution is 5 or more.
を酵素に対してそれぞれ５０〜２００重量％添加するこ
とを特徴とする請求項１又は２記載の酵素の回収方法。3. The method according to claim 1, wherein the anionic polymer and the cationic polymer are added in an amount of 50 to 200% by weight based on the enzyme.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP9005262A JPH10191969A (en) | 1997-01-16 | 1997-01-16 | Enzyme recovery |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP9005262A JPH10191969A (en) | 1997-01-16 | 1997-01-16 | Enzyme recovery |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH10191969A true JPH10191969A (en) | 1998-07-28 |
Family
ID=11606324
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP9005262A Pending JPH10191969A (en) | 1997-01-16 | 1997-01-16 | Enzyme recovery |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH10191969A (en) |
-
1997
- 1997-01-16 JP JP9005262A patent/JPH10191969A/en active Pending
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