JPH07265075A - Stable modified protease composition and stable enzyme-containing aqueous solution containing the same composition - Google Patents
Stable modified protease composition and stable enzyme-containing aqueous solution containing the same compositionInfo
- Publication number
- JPH07265075A JPH07265075A JP6085893A JP8589394A JPH07265075A JP H07265075 A JPH07265075 A JP H07265075A JP 6085893 A JP6085893 A JP 6085893A JP 8589394 A JP8589394 A JP 8589394A JP H07265075 A JPH07265075 A JP H07265075A
- Authority
- JP
- Japan
- Prior art keywords
- protease
- modified protease
- modified
- stable
- composition
- 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
- Cosmetics (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Detergent Compositions (AREA)
- Enzymes And Modification Thereof (AREA)
- Medicinal Preparation (AREA)
Abstract
Description
【0001】[0001]
【産業上の利用分野】本発明は、高度に安定な、化粧
料、洗浄剤、医薬品用途に好適に利用される修飾プロテ
アーゼ組成物に関する。TECHNICAL FIELD The present invention relates to a highly stable modified protease composition suitable for use in cosmetics, detergents and pharmaceuticals.
【0002】[0002]
【従来の技術】酵素は、洗剤、繊維製品の精練、油脂や
デンプン等の分解、食品加工、医薬品、臨床検査、バイ
オセンサー、化粧品、更に有用物質の転換・製造など各
種の産業分野に広く用いられている。こうした利用を計
る上での一つの問題点は、酵素の安定性が一般的に低
く、その要求に対し満足でない場合が多いことである。
即ち、熱を加えられたり、極端に高いpH条件や逆に低
いpH条件下、界面活性剤や有機溶媒等の混合物の共存
下、更に長期保存によって殆どの酵素は容易に変性して
失活する。特にプロテアーゼの場合、水分率の高い媒体
や水溶液等の剤形中では変性の他に自己消化分解が起こ
り、室温で保存する間に速やかに失活するため安定な商
品を供給することが難しいという問題がある。また利用
される酵素は人体にとって異種起源のものであるため、
医薬品、化粧品、洗剤等に応用する場合、その抗原性や
皮膚感作性、刺激性が問題となる。BACKGROUND OF THE INVENTION Enzymes are widely used in various industrial fields such as detergents, scouring of textile products, decomposition of oils and fats, starch, food processing, pharmaceuticals, clinical tests, biosensors, cosmetics, and conversion / production of useful substances. Has been. One problem in measuring such utilization is that the stability of the enzyme is generally low, and the requirement is often not satisfied.
That is, most of the enzymes are easily denatured and inactivated by being heated, under extremely high pH conditions or, conversely, under low pH conditions, in the presence of a mixture of a surfactant, an organic solvent and the like, and further stored for a long time. . In particular, in the case of protease, it is difficult to supply stable products because in addition to denaturation, it undergoes autolytic digestion in dosage forms such as high-moisture content media and aqueous solutions, and is rapidly inactivated during storage at room temperature. There's a problem. Also, the enzymes used are of different origin to the human body,
When it is applied to medicines, cosmetics, detergents, etc., its antigenicity, skin sensitization and irritation become problems.
【0003】こうした問題に対処する方法として、酵素
の化学修飾が試みられている。例えば、治療用酵素とし
て用いられるウリカーゼ、アスパラギナーゼ、ストレプ
トキナーゼ等をポリエチレングリコールで修飾し、血中
でのクリアランスや抗原性を改善する方法(特公昭61
−42558号公報,特開昭57−118789号公
報)、スーパーオキシドジスムターゼを多糖類,ポリエ
チレングリコールで修飾し、抗原性抑制や熱安定性向上
を計る方法(特開昭58−16685号公報)、あるい
はキモトリプシンに分子内架橋を与えるような修飾を施
し、安定化を計る方法(Biochimica et Biophysica Act
a 522,277 〜283 (1978),ibd 485,1 〜12(1977) )等
が開示されている。Chemical modification of enzymes has been attempted as a method for coping with these problems. For example, a method for improving clearance and antigenicity in blood by modifying uricase, asparaginase, streptokinase, etc., which are used as therapeutic enzymes, with polyethylene glycol (Japanese Patent Publication No. Sho 61).
No. 42558, JP-A-57-118789), a method of modifying superoxide dismutase with a polysaccharide or polyethylene glycol to suppress antigenicity and improve heat stability (JP-A-58-16685), Alternatively, chymotrypsin is modified to give intramolecular cross-linking, and stabilization is performed (Biochimica et Biophysica Act).
a 522,277 to 283 (1978), ibd 485,1 to 12 (1977)) and the like are disclosed.
【0004】特にプロテーゼに関しては、基質となるも
のがタンパク質という高分子であり修飾により一般に活
性が大幅に損なわれることに加え、皮膚感作性の抑制と
共に高度の安定化を付与し実用化を図った例は知られて
いなかったことから、本発明者らは皮膚感作性抑制と安
定化の双方の目的を同時に達成する方法を検討した結
果、トリアジン環を介して多糖類で修飾したプロテアー
ゼ及びその製造法(特開平2−219572号公報,特
開平4−27388号公報,特開平4−88982号公
報等)を提案し、適切な条件でプロテアーゼに化学修飾
を施すと活性、安定性、安全性及び水溶性等の物性面で
優れた修飾酵素が得られることを報告している。[0004] In particular, regarding prostheses, the substrate is a polymer called a protein, and the modification generally impairs the activity to a large extent. In addition, it suppresses the skin sensitization and imparts a high degree of stabilization for practical use. Since no example was known, the present inventors investigated a method for simultaneously achieving both the purpose of suppressing skin sensitization and stabilizing, and as a result, a protease modified with a polysaccharide through a triazine ring and Proposal of its production method (Japanese Patent Laid-Open No. 2-219572, Japanese Patent Laid-Open No. 4-27388, Japanese Patent Laid-Open No. 4-88982, etc.) and chemical modification of a protease under appropriate conditions result in activity, stability, and safety. It has been reported that a modified enzyme excellent in physical properties such as solubility and water solubility can be obtained.
【0005】また、酵素の安定化に金属イオン、特にプ
ロテアーゼの場合カルシウムが有効であることは広く知
られている。こうした効果は酵素の種類によっても異な
るが、安定化を目的として酵素表面のアミノ酸残基を変
換し金属イオンに対する親和性を高める方法も研究され
ている(Biochemistry 27,8311〜8317(1988)など)。It is widely known that metal ions, particularly calcium in the case of protease, are effective for stabilizing the enzyme. Although such effects differ depending on the type of enzyme, methods for converting amino acid residues on the surface of the enzyme to increase its affinity for metal ions have also been studied for the purpose of stabilization (Biochemistry 27,8311-8317 (1988), etc.). .
【0006】こうした手法により金属イオンに対する親
和性を高めることは有効ではあるが、プロテアーゼの場
合は自己消化現象を示すため、これだけでは高度な安定
性を確保することが難しい。また、カルシウム(Ca)
等の金属イオンを高濃度に添加することは、場合によっ
ては製品としての価値を損なうことがある。即ち、アル
カリ性条件下や、混合物との不溶性物質形成により沈澱
や濁りを与えたり、界面活性剤を含む洗剤等にあっては
製品機能を減少せしめることがあり、できるだけ微量の
金属イオンの添加により安定化されることが望ましい。[0006] Although it is effective to increase the affinity for metal ions by such a method, it is difficult to secure a high degree of stability by using protease alone because it exhibits an autodigestion phenomenon. Also, calcium (Ca)
Addition of a metal ion such as, etc. to a high concentration may impair its value as a product in some cases. That is, it may cause precipitation or turbidity under alkaline conditions or due to the formation of an insoluble substance with the mixture, or may reduce the product function in the case of detergents containing surfactants, etc. Is desirable.
【0007】[0007]
【発明が解決しようとする課題】本発明者らは上述の修
飾プロテアーゼ(特開平2−219572号公報,特開
平4−27388号公報,特開平4−88982号公報
等)を各種成分を含有する実際の製品に配合利用するに
当り、より高度に安定な系を調製すべく鋭意検討の結
果、本発明に到達したものであって、微量のカルシウム
の添加により製品の目的とするところや性状に影響を与
えることなく、広い条件範囲での熱安定性及び保存安定
性に優れた酵素組成物が得られることを見いだしたもの
である。DISCLOSURE OF THE INVENTION The inventors of the present invention contain various components of the above-mentioned modified protease (Japanese Patent Application Laid-Open Nos. 2-219572, 4-27388, 4-88982, etc.). As a result of diligent studies to prepare a more highly stable system for use in actual products, the present invention has been achieved. It was found that an enzyme composition excellent in heat stability and storage stability in a wide range of conditions can be obtained without affecting.
【0008】[0008]
【課題を解決するための手段】本発明は、トリアジン環
を介して多糖類とプロテアーゼが結合した修飾プロテア
ーゼにおいて、カルシウムを含有することを特徴とする
安定な修飾プロテアーゼ組成物であって、前述の課題を
解決するものである。The present invention provides a modified protease composition in which a polysaccharide and a protease are bound to each other through a triazine ring, and a stable modified protease composition characterized by containing calcium. It solves the problem.
【0009】本発明の一つの態様は、該修飾プロテアー
ゼ組成物を含有する安定な酵素含有水溶液である。One aspect of the present invention is a stable enzyme-containing aqueous solution containing the modified protease composition.
【0010】本発明の修飾プロテアーゼは、基本的には
本発明者らの提案した上述の特許に述べた方法に従って
製造できる。例えば多糖類水溶液に塩化シアヌルのアセ
トン溶液を添加混合することにより活性化多糖類を合成
する。この際、修飾率を高め安定性等の品質の確保を計
る目的から、NaOH水溶液等によりpHを8〜9に維
持する。活性化反応終了後、直ちに塩酸等によりpHを
3程度とし活性化体を比較的安定な状態として、大量の
アセトン溶液中に添加し析出させ固体として分離し洗浄
・乾燥して活性化多糖類を得る。或は、活性化体を一旦
分離せず直接酵素との反応に進める場合は、酸性化した
活性化体溶液を限外濾過や透析により精製し未結合の塩
化シアヌル分解物やアセトン溶媒等を除く。該活性化体
溶液を透析により精製する場合は、ホローファイバー型
の透析チューブを用いることが好ましい。尚、多糖類と
しては特に限定はないが、溶液粘度が低く反応操作が容
易であり修飾反応を均一に進められる点でデキストラ
ン、プルランが好ましい。また、分子量は10,000
以上であることが好ましい。The modified protease of the present invention can be produced basically according to the method described in the above-mentioned patent proposed by the present inventors. For example, the activated polysaccharide is synthesized by adding and mixing an acetone solution of cyanuric chloride to an aqueous polysaccharide solution. At this time, the pH is maintained at 8 to 9 with an aqueous NaOH solution or the like for the purpose of increasing the modification rate and ensuring quality such as stability. Immediately after completion of the activation reaction, adjust the pH to about 3 with hydrochloric acid or the like to make the activator relatively stable, add it to a large amount of acetone solution, precipitate it, separate it as a solid, wash and dry it to remove the activated polysaccharide. obtain. Alternatively, if the activator is not separated once and proceeded directly to the reaction with the enzyme, the acidified activator solution is purified by ultrafiltration or dialysis to remove unbound cyanuric chloride decomposition products, acetone solvent, etc. . When the activated solution is purified by dialysis, it is preferable to use a hollow fiber type dialysis tube. The polysaccharide is not particularly limited, but dextran and pullulan are preferable in that the solution viscosity is low, the reaction operation is easy, and the modification reaction can proceed uniformly. The molecular weight is 10,000.
The above is preferable.
【0011】該活性化多糖類によるプロテアーゼの修飾
は、pH7〜9程度の緩衝液中で両者を混合反応させる
ことにより行うことができる。プロテアーゼとしては、
特に限定しないが、バチルス属由来の微生物プロテアー
ゼが好適に用いられる。緩衝液としてはほう酸塩緩衝
液、炭酸塩緩衝液等を好適に用いることができる。ま
た、この際、活性化多糖類の反応性塩素量が0.4〜
1.2mmol/gであると共に、この反応性塩素量が
プロテアーゼの反応性アミノ基に対してモル比で2倍以
上であり、更に活性化多糖類をプロテアーゼに対して重
量比で3倍以上にすることで安全かつ安定な水溶性修飾
プロテアーゼを再現性よく得ることができる。こうして
得られた修飾プロテアーゼは、未だトリアジン環に活性
残基を幾分有しているため、粉末状態とした際に不溶化
を起こすことがあり、通常アルカリ条件下、60〜65
℃程度でこの失活処理を行い、トリアジン環由来の塩素
量を修飾酵素に対し500ppm以下とする。この際、
グリシン、アラニン、β−アミノプロピオン酸、リジ
ン、セリン等のアミノ酸やタウリン、モノエタノールア
ミン等を添加しておくと効率よく進めることができる。
また通常、これらアミノ化合物は多糖類−酵素結合体に
対し5〜25重量%添加される。The modification of the protease with the activated polysaccharide can be carried out by mixing and reacting the two in a buffer solution having a pH of about 7-9. As a protease,
Although not particularly limited, a microbial protease derived from Bacillus is preferably used. As the buffer solution, a borate buffer solution, a carbonate buffer solution or the like can be preferably used. At this time, the reactive chlorine content of the activated polysaccharide is 0.4 to
In addition to 1.2 mmol / g, this reactive chlorine amount is more than twice the molar ratio of the reactive amino groups of the protease, and the activated polysaccharide is more than three times the weight ratio of the protease. By doing so, a safe and stable water-soluble modified protease can be obtained with good reproducibility. The modified protease thus obtained still has some active residues on the triazine ring, so it may cause insolubilization when it is made into a powder state.
This deactivation treatment is carried out at about 0 ° C. to adjust the amount of chlorine derived from the triazine ring to 500 ppm or less based on the modifying enzyme. On this occasion,
It is possible to efficiently proceed by adding amino acids such as glycine, alanine, β-aminopropionic acid, lysine and serine, taurine, monoethanolamine and the like.
Further, usually, these amino compounds are added in an amount of 5 to 25% by weight based on the polysaccharide-enzyme conjugate.
【0012】本発明者らの提案になる上述の修飾プロテ
アーゼは、自己消化性も完全に抑制され、水溶性であ
り、それ自体でも水系媒体中で非常に安定であるが、利
用される剤形中には通常様々の成分が含有されているた
め安定性が若干損なわれる場合もある。しかし、Ca,
Mg,Ba,Sr等の多価金属を添加すると安定性を高
めることができる。特にCaが著しい安定化効果を示
し、修飾プロテアーゼ自体についても含水粉末状態で高
い安定性を示す。この理由の一つとして、トリアジン環
残基の水酸基やトリアジン環残基に結合した活性基処理
剤によるキレート能の増大が考えられ、活性基処理剤の
種類によってかなりの違いが見られるが、修飾プロテア
ーゼ調製に際しグリシン、β−アミノプロピオン酸、タ
ウリンのいずれか、あるいはこれらの混合物を活性基処
理剤として用いたものが好適である。多価金属の含有量
としては、安定化効果の点から修飾プロテアーゼ中に
0.01重量%以上含有することが好ましく、修飾プロ
テアーゼに配位結合またはイオン結合等で結合したもの
でなければならない。但し、上記条件が満たされれば、
他に固体状の金属塩粉末等を含有していてもよい。一般
にプロテアーゼを剤形に添加して使用する場合、Caイ
オン濃度が大きくなる程安定性も高くなるが、アルカリ
性条件下や、Caイオンが剤形中の成分と結合したりす
る場合は濁り・沈澱を生じる欠点がある。これに対し、
本修飾プロテアーゼの場合、含有金属イオン量が0.0
1〜1重量%であれば通常の使用に於てこうしたトラブ
ルが発生せず、高度に安定かつ清澄な製品を得ることが
できる。製品として外観上の制約がなく、性能面でも濁
り・沈澱等が発生しても特に問題がない場合は、含有金
属イオン量を1重量%以上としてもよい。[0012] The above-mentioned modified protease proposed by the present inventors has a completely suppressed autodigestibility, is water-soluble, and is itself very stable in an aqueous medium. Since various components are usually contained therein, the stability may be slightly impaired. However, Ca,
Stability can be improved by adding a polyvalent metal such as Mg, Ba, or Sr. In particular, Ca shows a remarkable stabilizing effect, and the modified protease itself also shows high stability in a water-containing powder state. One of the reasons for this is considered to be the increase in chelating ability due to the active group treating agent bonded to the hydroxyl group of the triazine ring residue or the triazine ring residue, and there are considerable differences depending on the type of active group treating agent. It is preferable to use any one of glycine, β-aminopropionic acid, taurine, or a mixture thereof as an active group treating agent in the preparation of protease. The content of the polyvalent metal is preferably 0.01% by weight or more in the modified protease from the viewpoint of the stabilizing effect, and it must be bound to the modified protease by a coordinate bond or an ionic bond. However, if the above conditions are met,
In addition, solid metal salt powder and the like may be contained. Generally, when a protease is used by adding it to the dosage form, the stability increases as the Ca ion concentration increases, but under alkaline conditions or when Ca ions bind to the components in the dosage form, turbidity / precipitation occurs. There is a drawback that causes. In contrast,
In the case of this modified protease, the content of metal ions is 0.0
If it is 1 to 1% by weight, such trouble does not occur in normal use, and a highly stable and clear product can be obtained. When there is no restriction on the appearance of the product and there is no particular problem in terms of performance such as turbidity or precipitation, the content of metal ions may be 1% by weight or more.
【0013】Caによる修飾プロテアーゼの安定化は、
構造的にはプロテアーゼに対するCaイオンの配位によ
ると考えられるが、カルシウムを添加しこうした状態と
するためには、修飾プロテアーゼ量に対し0.01重量
%以上のCaイオンを含有する水溶液に修飾プロテアー
ゼを溶解した後、溶媒を除去すればよい。この際、溶液
pHはその効果及び酵素失活を防ぐ観点から6.0〜1
0.0とすることが好ましく、更に好ましくは7.0〜
9.5である。また、pH調整に於て緩衝塩を添加する
こともできるが、Caイオンと結合して濁り・不溶性沈
澱を生じないものを選ぶ必要がある。溶媒除去に先立
ち、必要に応じて透析・限外濾過等により一旦精製を加
え、緩衝塩等を除くこともできるが、本発明の修飾プロ
テアーゼはキレート能が高いので、Caイオン濃度を若
干高めに設定しておくとこうした操作を加えても修飾プ
ロテアーゼ中の含有カルシウム量を通常0.01重量%
以上とすることができる。Stabilization of modified proteases with Ca
Although it is structurally considered to be due to the coordination of Ca ions to the protease, in order to add calcium to bring about such a state, the modified protease should be added to an aqueous solution containing 0.01% by weight or more of Ca ion relative to the amount of modified protease. After dissolving, the solvent may be removed. At this time, the solution pH is 6.0 to 1 from the viewpoint of its effect and enzyme deactivation.
It is preferably 0.0, and more preferably 7.0.
It is 9.5. A buffer salt may be added for pH adjustment, but it is necessary to select one that does not cause turbidity or insoluble precipitation by binding with Ca ions. Prior to removal of the solvent, if necessary, purification can be performed once by dialysis, ultrafiltration, etc. to remove the buffer salts and the like, but since the modified protease of the present invention has a high chelating ability, the Ca ion concentration should be slightly increased. Even if such an operation is added by setting it, the content of calcium in the modified protease is usually 0.01% by weight.
The above can be done.
【0014】本発明のカルシウムを含有した修飾プロテ
アーゼ組成物は、水系剤形製品に添加して安定に用いる
ことができるが、目的に応じて該剤形中に水以外の溶媒
を混合してもよい。剤形系中のCaイオン濃度は、他に
添加されていなければ修飾プロテアーゼ量(濃度)によ
って変化する。例えばCaイオンを0.2%含有する修
飾プロテアーゼを10mg/mlの濃度で溶解した場
合、系中のCaイオン濃度は0.5mmolであるにも
かかわらず、Caイオン非添加系に較べ非常に安定性に
優れる効果を示すと共に、リン酸緩衝液中のようにCa
と不溶性塩を生ずる系に於いても濁りを与えないことか
ら広い用途に使用できるものである。The calcium-containing modified protease composition of the present invention can be stably used by adding it to an aqueous dosage form product, but a solvent other than water may be mixed in the dosage form depending on the purpose. Good. The Ca ion concentration in the dosage form system varies depending on the amount (concentration) of the modified protease unless otherwise added. For example, when a modified protease containing 0.2% of Ca ions was dissolved at a concentration of 10 mg / ml, the Ca ion concentration in the system was 0.5 mmol, but it was much more stable than the Ca ion-free system. In addition to exhibiting excellent effect, it has the same Ca content as in phosphate buffer.
Since it does not give turbidity even in a system which produces an insoluble salt, it can be used for a wide range of purposes.
【0015】[0015]
【発明の効果】本発明の、カルシウムを含有し、トリア
ジン環を介して多糖類とプロテアーゼが結合された修飾
プロテアーゼ組成物は、それ自体湿潤状態でも高度に安
定であるのみならず、水系媒体中に溶解して用いる場合
に於いても、系中Caイオン濃度が低い場合でも著しく
高い安定性を示し、製品としての性状面でも濁り・沈澱
を生じることがないことから、化粧品、薬品、洗浄剤そ
の他の工業用途に於いて極めて有用に用いられるもので
ある。INDUSTRIAL APPLICABILITY The modified protease composition of the present invention in which a polysaccharide and a protease are bound to each other through a triazine ring is not only highly stable even in a wet state, but also in an aqueous medium. Even when it is dissolved in water and used, it shows extremely high stability even when the Ca ion concentration in the system is low, and it does not cause turbidity or precipitation in terms of product properties. It is extremely useful in other industrial applications.
【0016】[0016]
【実施例】以下、実施例を挙げて本発明の効果を詳細に
説明する。EXAMPLES The effects of the present invention will be described in detail below with reference to examples.
【0017】実施例1 4%デキストラン水溶液(平均分子量4×104 )5l
に、室温下、pHを7.5〜9.5に保ちながら、塩化
シアヌルを5%含有するアセトン溶液1.5lを添加し
た後、pH3に調整した。この溶液をアセトン浴に加
え、析出した活性化デキストランの沈澱を濾別採取し
た。該活性化デキストラン90gを溶解した0.1Mほ
う酸緩衝液(pH9.0)2lに、バチルス・レンタス
菌(従来バチルス・リケニフォルミス菌に帰属されてい
た)由来のプロテアーゼ粉末(ノボ・ノルディスク社
製,エスペラーゼTM)10gを加え25℃で20時間反
応させた後、グリシン20gを添加し更に60℃で35
時間加熱処理を施し、デキストランによるプロテアーゼ
修飾を行った。反応終了後の水溶液をポリスルホン製の
ダイアライザー((株)クラレ製,PS−1.6UW,
膜面積1.6m2 )により透析精製した。処理条件とし
て、透析液(イオン交換水を使用)流量1l/min,
反応液の通液流量85ml/min,排出流量63ml
/min,出口側圧力100mmHgで実施した。処理
溶液を高速液体クロマトグラフィー(東ソー株式会社
製,G−3000SW)で分析したところ反応溶液中の
不純物である塩化シアヌル分解物及びそのグリシン誘導
体は全く検出されなかった。減圧下溶媒溜去することに
より、水溶液からデキストラン修飾プロテアーゼを粉末
状として得た。蛍光X線分析により求められた該修飾プ
ロテアーゼ中のCa含量は450ppmであったが、こ
のCaは原料酵素に由来するものであった。尚、原料酵
素中のCa含有量は12300ppmであった。50m
Mほう酸ナトリウム緩衝液(pH8.5)100ml
に、上記で得た修飾プロテアーゼ5gを溶解し、更に塩
化カルシウム(2水塩)を25mg加えて溶解させた
後、クプロファン透析チューブに入れ、イオン交換水1
0lに対して3回透析した。水溶媒を減圧溜去すること
により、修飾プロテアーゼを粉末状に回収した。蛍光X
線分析により求められた該修飾プロテアーゼ中のCa含
量は1350ppmであった(DMP+Caと略記)。
また、50mMほう酸ナトリウム緩衝液(pH8.5)
100mlに上記修飾プロテアーゼ5gを溶解してクプ
ロファン透析チューブに入れ、pH8.5の5mM−E
DTA・2Na塩(エチレンヂアミン4酢酸・2Na
塩)溶液10lに対して3回透析後、イオン交換水10
l中で透析し、続いて水溶媒を減圧溜去することによ
り、Caを除去した修飾プロテアーゼ試料を粉末状で得
た。この修飾プロテアーゼ(DMP−Caと略記)のC
a含量は80ppmであった。上記で得たDMP+C
a、DPM−Ca及び未修飾エスペラーゼについて、湿
熱及び溶液条件下で安定性を評価した。条件、及び結果
を表1に示す。尚、本実験は無菌状態で実施した。いず
れの条件に於いても、未修飾酵素の較べ修飾体は著しく
高い安定性を示すが、Ca含有量によっても有意な差の
あることが明確である。Example 1 5 l of 4% dextran aqueous solution (average molecular weight 4 × 10 4 ).
At room temperature, 1.5 l of an acetone solution containing 5% of cyanuric chloride was added while keeping the pH at 7.5 to 9.5, and then the pH was adjusted to 3. This solution was added to an acetone bath, and the precipitated activated dextran was collected by filtration. 2 l of 0.1 M borate buffer (pH 9.0) in which 90 g of the activated dextran was dissolved was added to a protease powder derived from Bacillus lentus (previously belonging to Bacillus licheniformis) (Novo Nordisk, 10g of Esperase ™ ) was added and reacted at 25 ° C for 20 hours, then 20g of glycine was added, and 35 ° C at 60 ° C.
Heat treatment was performed for a period of time, and the protease was modified with dextran. After the reaction, the aqueous solution of polysulfone was used as a dialyzer (PS-1.6UW, manufactured by Kuraray Co., Ltd.).
It was purified by dialysis with a membrane area of 1.6 m 2 ). As processing conditions, dialysate (using ion exchange water) flow rate 1 l / min,
Flow rate of reaction solution: 85 ml / min, discharge flow rate: 63 ml
/ Min, the outlet side pressure was 100 mmHg. When the treated solution was analyzed by high performance liquid chromatography (G-3000SW, manufactured by Tosoh Corporation), the decomposition product of cyanuric chloride and its glycine derivative which were impurities in the reaction solution were not detected at all. The solvent was distilled off under reduced pressure to obtain a dextran-modified protease as a powder from the aqueous solution. The Ca content in the modified protease determined by fluorescent X-ray analysis was 450 ppm, but this Ca was derived from the starting enzyme. The Ca content in the raw material enzyme was 12300 ppm. 50m
100 ml of M sodium borate buffer (pH 8.5)
5 g of the modified protease obtained above is dissolved in 25 ml of calcium chloride (dihydrate), and then dissolved in a cuprophane dialysis tube.
It was dialyzed 3 times against 0 l. The modified protease was recovered in powder form by distilling off the water solvent under reduced pressure. Fluorescent X
The Ca content in the modified protease determined by line analysis was 1350 ppm (abbreviated as DMP + Ca).
In addition, 50 mM sodium borate buffer (pH 8.5)
5 g of the above modified protease was dissolved in 100 ml and placed in a cuprophane dialysis tube, and 5 mM-E of pH 8.5 was added.
DTA / 2Na salt (ethylenediamine tetraacetic acid / 2Na
(Salt) solution was dialyzed 3 times against 10 l of ion-exchanged water
A modified protease sample with Ca removed was obtained in powder form by dialysis in 1 and subsequent evaporation of the aqueous solvent under reduced pressure. C of this modified protease (abbreviated as DMP-Ca)
The a content was 80 ppm. DMP + C obtained above
a, DPM-Ca and unmodified Esperase were evaluated for stability under moist heat and solution conditions. The conditions and the results are shown in Table 1. The experiment was carried out in a sterile condition. Under all of the conditions, the modified form of the unmodified enzyme shows remarkably high stability, but it is clear that there is a significant difference depending on the Ca content.
【0018】[0018]
【表1】 実験条件:(A) 粉末状,40℃,75%RH,3カ
月. (B) 0.1Mリン酸緩衝液(pH7.0),70
℃,1時間,修飾酵素濃度2mg/ml,(未修飾酵素
の濃度は0.2mg/ml).[Table 1] Experimental condition: (A) Powder, 40 ° C., 75% RH, 3 months. (B) 0.1 M phosphate buffer (pH 7.0), 70
C, 1 hour, modified enzyme concentration 2 mg / ml, (concentration of unmodified enzyme is 0.2 mg / ml).
【0019】実施例2 実施例1で調製したCa除去修飾プロテアーゼ試料(D
MP−Ca,Ca含有量80ppm)を、0.1Mほう
酸緩衝液(pH9.2)に1mg/mlの濃度で溶解し
た。これに塩化カルシウムを添加し各種Ca濃度に調整
した後、75℃、1時間の熱処理を施し、残存活性を評
価した。結果を表2に示す。エチレンジアミン4酢酸
(EDTA)塩添加によりCaイオンを除去(封鎖)し
た系に較べ、特に0.01重量%以上のCa添加系では
著しい安定化効果が認められる。Example 2 Ca removal modified protease sample prepared in Example 1 (D
MP-Ca, Ca content 80 ppm) was dissolved in 0.1 M borate buffer (pH 9.2) at a concentration of 1 mg / ml. After adding calcium chloride to this and adjusting to various Ca concentrations, it heat-processed at 75 degreeC and 1 hour, and evaluated residual activity. The results are shown in Table 2. Compared with a system in which Ca ions are removed (blocked) by adding ethylenediaminetetraacetic acid (EDTA) salt, a remarkable stabilizing effect is recognized particularly in a system containing 0.01% by weight or more of Ca.
【0020】[0020]
【表2】 *1 :修飾プロテアーゼに対するCa含有比率。 *2 :EDTA・2Na塩を添加(5mM濃度)。[Table 2] * 1: Ca content ratio to modified protease. * 2: EDTA ・ 2Na salt was added (5 mM concentration).
【0021】実施例3 実施例1と同様のスキームでデキストランとプロテアー
ゼとを結合せしめた後、グリシンの替わりに表3に示し
た各種アミノ化合物を加えて後処理を行い、更に実施例
1に準じて透析精製、粉末化、Ca添加処理を行って各
Ca含有修飾プロテアーゼ試料を得た。但し、用いたア
ミノ化合物量はいずれも0.25molとした。各Ca
含有修飾プロテアーゼ試料のCa含有量を蛍光X線分析
により定量した結果を併せて表3に示した。各試料10
0mgを水25mlに溶解し(濃度0.4重量%)、各
々Ca量が修飾プロテアーゼに対し1800ppmとな
るように塩化カルシウム溶液を加えて調整した後、水を
加えて50mlとした。更に0.2Mりん酸緩衝液(p
H7.2)50mlを加えて、各修飾酵素の0.1重量
%溶液を調製した。いずれも濁り等の発生は認められな
かった。この緩衝液系で、70℃,1時間の加熱処理を
行い、残存活性を評価した結果を表3に示す。但し、比
較に示した未修飾酵素エスペラーゼは0.01重量%溶
液で安定性評価を行った。いずれも修飾を施すことによ
り安定性が著しく向上していることが明かであるが、ア
ミノ化合物としてグリシン,タウリン,β−アミノプロ
ピオン酸を用いた場合は、特に良好な結果が得られるこ
とが解る。Example 3 After dextran and protease were bound in the same scheme as in Example 1, various amino compounds shown in Table 3 were added in place of glycine to perform post-treatment, and further, in accordance with Example 1. Then, dialysis purification, pulverization, and Ca addition treatment were performed to obtain each Ca-containing modified protease sample. However, the amount of amino compound used was 0.25 mol in all cases. Each Ca
Table 3 also shows the result of quantifying the Ca content of the modified protease sample containing by the fluorescent X-ray analysis. Each sample 10
0 mg was dissolved in 25 ml of water (concentration 0.4% by weight), calcium chloride solution was added to adjust the Ca content to 1800 ppm based on the modified protease, and water was added to make 50 ml. Furthermore, 0.2M phosphate buffer (p
H7.2) 50 ml was added to prepare a 0.1 wt% solution of each modifying enzyme. Neither turbidity nor the like was observed. Table 3 shows the results of evaluating the residual activity by subjecting this buffer system to heat treatment at 70 ° C for 1 hour. However, the stability of the unmodified enzyme Esperase shown in the comparison was evaluated in a 0.01 wt% solution. It is clear that the modification significantly improves the stability, but it is found that particularly good results are obtained when glycine, taurine, or β-aminopropionic acid is used as the amino compound. .
【0022】[0022]
【表3】 [Table 3]
【0023】実施例4 実施例1で調製したCa除去修飾プロテアーゼ試料(D
MP−Ca,Ca含有量80ppm)100mgを、
0.1Mほう酸緩衝液(pH8.5)に10mlに溶解
し、更に各種濃度で塩化カルシウム(2水塩)を加えて
溶解させた後、クプロファン透析チューブに入れ、イオ
ン交換水1lに対して3回透析した。水溶媒を減圧溜去
することにより、修飾プロテアーゼを粉末状に回収し
た。蛍光X線分析により求められた該修飾プロテアーゼ
中のCa含量、及び0.1Mりん酸緩衝液(pH7.
2)に2重量%で溶解させた場合の溶状を評価した。結
果を表4に示す。本発明の修飾酵素組成物はりん酸緩衝
液中でも清澄な水溶液を与えるが、Caを1重量%以上
含むものは濁りを生じた。Example 4 Ca removal modified protease sample prepared in Example 1 (D
MP-Ca, Ca content 80ppm) 100mg,
It is dissolved in 0.1 M borate buffer (pH 8.5) in 10 ml, and calcium chloride (dihydrate) is added at various concentrations to dissolve it, and then placed in a cuprophane dialysis tube to prepare 3 parts per 1 l of ion-exchanged water. He dialyzed twice. The modified protease was recovered in powder form by distilling off the water solvent under reduced pressure. Ca content in the modified protease determined by X-ray fluorescence analysis and 0.1 M phosphate buffer (pH 7.
The dissolution state when 2% by weight was dissolved in 2) was evaluated. The results are shown in Table 4. The modified enzyme composition of the present invention gave a clear aqueous solution even in a phosphate buffer, but those containing 1 wt% or more of Ca produced turbidity.
【0024】[0024]
【表4】 [Table 4]
───────────────────────────────────────────────────── フロントページの続き (51)Int.Cl.6 識別記号 庁内整理番号 FI 技術表示箇所 A61K 38/46 47/02 J C11D 3/386 // C12N 9/54 (C12N 9/54 C12R 1:07) ─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. 6 Identification code Internal reference number FI Technical display location A61K 38/46 47/02 J C11D 3/386 // C12N 9/54 (C12N 9/54 C12R 1 : 07)
Claims (4)
ーゼが結合した修飾プロテアーゼにおいて、カルシウム
を含有することを特徴とする安定な修飾プロテアーゼ組
成物。1. A modified protease composition in which a polysaccharide and a protease are bound to each other via a triazine ring and which contains calcium and is stable.
ーゼが結合した修飾プロテアーゼが、多糖類に塩化シア
ヌルを反応させて活性化多糖類を得、次いで該活性化多
糖類とプロテアーゼとを反応させて得られる修飾プロテ
アーゼである請求項1記載の修飾プロテアーゼ組成物。2. A modified protease in which a polysaccharide and a protease are bound to each other through a triazine ring is reacted with cyanuric chloride to obtain an activated polysaccharide, and then the activated polysaccharide is reacted with the protease. The modified protease composition according to claim 1, which is the obtained modified protease.
ロテアーゼとの反応後、グリシン、β−アミノプロピオ
ン酸、タウリンの中から選ばれる少なくとも1種を含有
する水溶液中で処理し、トリアジン環残基に由来する塩
素量を修飾プロテアーゼに対し500ppm以下とした
ものである請求項2記載の修飾プロテアーゼ組成物。3. The modified protease is treated with an aqueous solution containing at least one selected from glycine, β-aminopropionic acid, and taurine after the reaction between the activated polysaccharide and the protease, to give a triazine ring residue. The modified protease composition according to claim 2, wherein the amount of chlorine derived from the modified protease is 500 ppm or less based on the modified protease.
を含有する安定な酵素含有水溶液。4. A stable enzyme-containing aqueous solution containing the modified protease composition according to claim 1.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP6085893A JPH07265075A (en) | 1994-03-30 | 1994-03-30 | Stable modified protease composition and stable enzyme-containing aqueous solution containing the same composition |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP6085893A JPH07265075A (en) | 1994-03-30 | 1994-03-30 | Stable modified protease composition and stable enzyme-containing aqueous solution containing the same composition |
Publications (1)
Publication Number | Publication Date |
---|---|
JPH07265075A true JPH07265075A (en) | 1995-10-17 |
Family
ID=13871571
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP6085893A Pending JPH07265075A (en) | 1994-03-30 | 1994-03-30 | Stable modified protease composition and stable enzyme-containing aqueous solution containing the same composition |
Country Status (1)
Country | Link |
---|---|
JP (1) | JPH07265075A (en) |
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2000017299A (en) * | 1998-07-01 | 2000-01-18 | San Contact Lens:Kk | Proteolytic enzyme-containing cleaning fluid and method for stabilizing proteolytic enzyme in enzymatic cleaning fluid |
WO2002033035A1 (en) * | 2000-10-20 | 2002-04-25 | Innu-Science Canada Inc. | Hard surface cleaning composition |
JP2009000128A (en) * | 2001-01-31 | 2009-01-08 | Asahi Kasei Pharma Kk | Composition for assaying glycated protein |
CN105831388A (en) * | 2015-12-15 | 2016-08-10 | 浙江海洋学院 | Preparation method of taurine-rich protein powder |
-
1994
- 1994-03-30 JP JP6085893A patent/JPH07265075A/en active Pending
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2000017299A (en) * | 1998-07-01 | 2000-01-18 | San Contact Lens:Kk | Proteolytic enzyme-containing cleaning fluid and method for stabilizing proteolytic enzyme in enzymatic cleaning fluid |
WO2002033035A1 (en) * | 2000-10-20 | 2002-04-25 | Innu-Science Canada Inc. | Hard surface cleaning composition |
JP2009000128A (en) * | 2001-01-31 | 2009-01-08 | Asahi Kasei Pharma Kk | Composition for assaying glycated protein |
CN105831388A (en) * | 2015-12-15 | 2016-08-10 | 浙江海洋学院 | Preparation method of taurine-rich protein powder |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Villalonga et al. | Preparation and functional properties of trypsin modified by carboxymethylcellulose | |
JPS6289630A (en) | Hemoglobin-polyoxyalkylene conjugate and freeze-dried pharmaceutical thereof | |
JPH02219571A (en) | Modified protease and production thereof | |
JP3139563B2 (en) | Method for producing rice bran protein-derived peptide | |
JPH03147784A (en) | Production of modified superoxide dismutase | |
JPH07265075A (en) | Stable modified protease composition and stable enzyme-containing aqueous solution containing the same composition | |
JPS6027680B2 (en) | Method for producing keratin hydrolyzate | |
JPS5931520B2 (en) | Method for producing silk fibroin peptide aqueous solution | |
JPH034791A (en) | Stabilization of enzyme | |
JP2915575B2 (en) | Liquid enzyme detergent composition | |
JP2560363B2 (en) | Method for purifying chitin or chitosan | |
JP2919912B2 (en) | Modified protease and method for producing the same | |
JPH1052267A (en) | Activation of prothrombin and production of thrombin based on activation thereof | |
JPH034790A (en) | Stabilization of enzyme | |
CA1181282A (en) | Process for preparing modified protein compositions | |
JP3081749B2 (en) | Method for producing modified protease | |
JP3126578B2 (en) | Purification method of modified enzyme | |
JPH0829744A (en) | Cleaning composition for contact lens, storing solution composition for contact lens, cleaning method of contact lens and disinfecting method of contact lens | |
JPH01269499A (en) | Production of casein peptide | |
JP2902664B2 (en) | Water-soluble modified protease and method for producing the same | |
RU2795425C1 (en) | Method for obtaining hybrid preparation of papain and carboxymethylcellulose in the form of a dense solution | |
JPH10298199A (en) | New peptide and angiotensin i converting enzyme inhibitor containing the peptide | |
JPH0488982A (en) | Production of modified protease | |
JP2501543B2 (en) | Freeze-dried hemoglobin-polyoxyalkylene conjugate | |
JP2002233362A (en) | Method for stabilizing laccase activity and stabilized laccase composition |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
S531 | Written request for registration of change of domicile |
Free format text: JAPANESE INTERMEDIATE CODE: R313531 |
|
R350 | Written notification of registration of transfer |
Free format text: JAPANESE INTERMEDIATE CODE: R350 |
|
R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
RVTR | Cancellation of determination of trial for invalidation |