JP3359868B2 - Wash solution containing protease - Google Patents
Wash solution containing proteaseInfo
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- JP3359868B2 JP3359868B2 JP20276898A JP20276898A JP3359868B2 JP 3359868 B2 JP3359868 B2 JP 3359868B2 JP 20276898 A JP20276898 A JP 20276898A JP 20276898 A JP20276898 A JP 20276898A JP 3359868 B2 JP3359868 B2 JP 3359868B2
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- enzyme
- solution
- protease
- modified
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Description
【０００１】[0001]
【発明の属する技術分野】本発明は、蛋白分解酵素の安
定性が高く、酵素活性が維持された蛋白分解酵素含有洗
浄液に関するものである。BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a cleaning solution containing a protease, which has high stability of the protease and maintains the enzyme activity.
【０００２】[0002]
【従来の技術】蛋白分解酵素は、脂質分解酵素などと共
に医療、食品、化粧品、洗浄剤などに用いられ優れた効
果をあげている。しかしながら、蛋白分解酵素には、加
熱や界面活性剤の共存下などにより変成したり、自己消
化などを起こし失活しやすく、特に水溶液中では安定性
が著しく低下するという問題点がある。近年、洗濯用洗
剤や家庭用食器洗剤などにも配合されている蛋白分解酵
素は、顆粒状洗剤中で安定ではあるが、液体洗剤中では
不安定である。2. Description of the Related Art Proteolytic enzymes have been used in medicine, foods, cosmetics, detergents and the like together with lipolytic enzymes, and have produced excellent effects. However, proteolytic enzymes have the problem that they are denatured by heating or in the presence of a surfactant, or are easily deactivated due to self-digestion, and the stability is particularly remarkably reduced in an aqueous solution. In recent years, proteolytic enzymes, which are also incorporated in laundry detergents and household dishwashing detergents, are stable in granular detergents but unstable in liquid detergents.
【０００３】そこで、これらの問題を克服するために、
蛋白分解酵素を化学修飾することにより、安定性を増加
させる方法がいくつか報告されている。例えば、特開平
７−１５５１８２号公報には、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カル
シウムが含有されると安定性が増強することが記載され
ている。又、特開平６−２４０２９７号公報には、蛋白
分解酵素存在下でリパーゼあるいはアミラーゼをメチル
ビニールエーテルと無水マレイン酸共重合体で修飾する
とリパーゼ、アミラーゼの安定性が向上する方法が提案
されているが、単独酵素をメチルビニールエーテルと無
水マレイン酸共重合体で修飾してもその効果が低いこと
が記載されている。また、同公報には、各種酵素の存在
下、修飾された蛋白分解酵素の安定性についての記載が
なく、蛋白分解酵素の安定性については更に改良すべき
点がある。その上、リン酸緩衝液下では、メチルビニル
エ−テル−無水マレイン酸共重合体の溶解形態は、直線
状であるためメチルビニルエ−テル−無水マレイン酸共
重合体はランダムに蛋白分解酵素と反応する確率が高
い。そのため蛋白分解酵素の活性部位あるいはその近傍
で反応し、活性部位の作用を失わせたり、活性部位の近
傍に反応したメチルビニルエ−テル−無水マレイン酸共
重合体の立体障害のため基質との反応性の低下を招き易
い。更に溶解している修飾酵素が直線状の高分子である
ことから、分子間距離が糸まり状に溶解した高分子より
近かくなるため、分子−分子間の絡み合いによる高分子
の凝集が生じ易い。この様な修飾酵素の凝集は、修飾酵
素を製造する場合、澱や濁り、また酵素の安定性、活性
の低下につながるなどの大きな問題点がある。このため
蛋白分解酵素を修飾し、液状酵素の安定性および酵素活
性を長期に維持するには、従来の方法では充分でない。[0003] In order to overcome these problems,
Several methods have been reported to increase stability by chemically modifying proteolytic enzymes. For example, JP-A-7-155182 describes that stability is enhanced when calcium is contained in a modified protease in which a polysaccharide and a protease are bonded via a triazine ring. JP-A-6-240297 proposes a method for improving the stability of lipase or amylase by modifying lipase or amylase with methyl vinyl ether and maleic anhydride copolymer in the presence of a protease. However, it is described that even if a single enzyme is modified with methyl vinyl ether and maleic anhydride copolymer, the effect is low. In addition, the publication does not describe the stability of the modified protease in the presence of various enzymes, and the stability of the protease must be further improved. In addition, in a phosphate buffer, the dissolution form of the methylvinyl ether-maleic anhydride copolymer is linear, so that the methylvinyl ether-maleic anhydride copolymer is likely to react with the protease at random. Is high. Therefore, it reacts at or near the active site of the protease, losing the effect of the active site, or reacts with the substrate due to steric hindrance of the methyl vinyl ether-maleic anhydride copolymer reacted near the active site. Tends to decrease. Furthermore, since the dissolved modifying enzyme is a linear polymer, the intermolecular distance becomes closer than that of the polymer dissolved in a string form, so that aggregation of the polymer due to entanglement between molecules is likely to occur. . When the modified enzyme is produced, such aggregation of the modified enzyme has serious problems such as precipitation and turbidity, and leads to a decrease in the stability and activity of the enzyme. For this reason, the conventional methods are not sufficient for modifying the protease to maintain the stability and the enzyme activity of the liquid enzyme for a long period of time.
【０００４】更に、特開平１−１５３０８８号公報に
は、無水マレイン酸とポリエチレングリコールモノアリ
ルエーテルとの共重合体で修飾された酵素が高い安定性
を有し、バイオリアクター等に使用できることが記載さ
れており、また、特開平８−１４６３６３号公報には、
α−アリルポリオキシアルキレンと無水マレイン酸共重
合体で酵素を修飾しているが、この様な無水マレイン酸
共重合体で修飾した酵素でも、その安定性は、十分とは
言えず実用性が低い。この他、特開昭６３−１５９８２
２号公報や特開平１−１８０５１５号公報にも、蛋白分
解酵素を安定化させる方法として、水に混和性の多価ア
ルコールを含有する方法が提案されているが、このまま
では蛋白分解酵素の活性が低く、水で希釈すれば高まる
ものの、安定性が逆に低下するという欠点がある。又、
特開平６−１０２４７４号公報には、タンパク分解酵
素、脂質分解酵素、多糖類分解酵素をタブレット上に賦
形した洗浄剤が記載されているが、この洗浄剤はタブレ
ットの為、溶解させる容器、手間が必要となり、簡便に
使用する点で劣るという問題点がある。Furthermore, Japanese Patent Application Laid-Open No. 1-153088 discloses that an enzyme modified with a copolymer of maleic anhydride and polyethylene glycol monoallyl ether has high stability and can be used in bioreactors and the like. In addition, JP-A-8-146363 discloses that
Although the enzyme is modified with α-allylpolyoxyalkylene and maleic anhydride copolymer, the stability of such an enzyme modified with maleic anhydride copolymer is not sufficient, and practicality cannot be said. Low. In addition, JP-A-63-15982
No. 2 and JP-A-1-180515 also propose a method of stabilizing the protease by containing a water-miscible polyhydric alcohol. However, there is a drawback in that although the stability is lowered, the stability is lowered, though it is increased by dilution with water. or,
JP-A-6-102474 describes a detergent in which a proteolytic enzyme, a lipolytic enzyme, and a polysaccharide-degrading enzyme are formed on a tablet. However, since this detergent is a tablet, a container for dissolving the tablet, There is a problem that it requires time and effort and is inferior in terms of easy use.
【０００５】[0005]
【発明が解決しようとする課題】本発明は、上述の従来
技術における問題点を解決し、蛋白分解酵素の安定性が
高く、酵素活性が維持された蛋白分解酵素含有洗浄液を
提供することを課題とする。SUMMARY OF THE INVENTION An object of the present invention is to solve the above-mentioned problems in the prior art, and to provide a protease-containing cleaning solution in which the stability of the protease is high and the enzyme activity is maintained. And
【０００６】[0006]
【課題を解決するための手段】本発明の蛋白分解酵素含
有洗浄液は、蛋白分解酵素がポリエチレングリコール、
多糖類、蛋白質及びＣＨ2 ＝Ｃ＜基を有する化合物と無
水マレイン酸との共重合体から成る群より選ばれた水溶
性高分子によって化学修飾されている修飾酵素と、ポリ
グリセリン脂肪酸エステル及びポリオキシエチレンアル
キルフェニルエーテルから選ばれた少なくとも１種の非
イオン界面活性剤と、陰イオン界面活性剤であるミリス
トイルサルコシンナトリウム、ラウリル硫酸アンモニウ
ム及び、両性界面活性剤であるアルキルジメチルアミン
オキサイド、アルキルジメチルアミノ酢酸ベタインから
成る群より選ばれた少なくとも１種の界面活性剤と、ホ
ウ酸、メタホウ酸、四ホウ酸、ホウ砂、メタホウ酸塩及
び四ホウ酸塩から成る群より選ばれたホウ酸化合物とを
含有することを特徴とする。According to the present invention, there is provided a detergent solution containing a protease, wherein the protease comprises polyethylene glycol,
A modifying enzyme chemically modified by a water-soluble polymer selected from the group consisting of polysaccharides, proteins, and copolymers of a compound having a CH 2 ＣC <group and maleic anhydride; a polyglycerol fatty acid ester and a polyglycerol fatty acid ester; At least one nonionic surfactant selected from oxyethylene alkyl phenyl ethers; anionic surfactants such as sodium myristoyl sarcosine and ammonium lauryl sulfate; and amphoteric surfactants such as alkyldimethylamine oxide and alkyldimethylaminoacetic acid At least one surfactant selected from the group consisting of betaines and a boric acid compound selected from the group consisting of boric acid, metaboric acid, tetraboric acid, borax, metaborate and tetraborate It is characterized by containing.
【０００７】又、本発明の蛋白分解酵素含有洗浄液は、
上述の特徴を有するものにおいて、前記修飾酵素を、単
位容積当たりの酵素活性値が10,000〜200,000 U/mlであ
る修飾酵素溶液として０．１〜１０重量％の割合で含有
し、しかも当該修飾酵素溶液：前記各界面活性剤：ホウ
酸化合物の重量比率が１：０．０５〜１０：０．５〜１
０であることを特徴とするものである。[0007] The cleaning solution containing the protease of the present invention comprises:
In the above-mentioned feature, the modified enzyme is contained at a ratio of 0.1 to 10% by weight as a modified enzyme solution having an enzyme activity value per unit volume of 10,000 to 200,000 U / ml, and further comprises the modified enzyme The weight ratio of the solution: each of the surfactants and the boric acid compound is 1: 0.05 to 10: 0.5 to 1.
It is characterized by being 0.
【０００８】[0008]
【発明の実施の形態】本発明の蛋白分解酵素含有洗浄液
中に含まれる各成分について説明する。 (1) 修飾酵素 蛋白分解酵素が水溶性高分子によって化学修飾されたも
のであって、この蛋白分解酵素としては、微生物由来、
植物由来、動物由来から選ばれたものがいずれも使用で
き、特に微生物由来の中性又はアルカリ性のものが好適
である。代表的なものとしては、Bacillus属(B.subtili
s, amyloloquefaciens, cereus, licheniformis, pumil
is, natto, mesentericus, sphaericus)、Aspergillus
属 (A.sojae, oryzae, flavus, sulphureus, candidus,
terricola, melleus, nidulans,sydowi)、Streptmyces
属(S.fradiae, griseus, moderatus, rectus) 、Cepha
losporium sp., C.acremonium, Fusarium, Gliocladiu
m, Malbranchea pulchella, penicillium cyano-fulvu
m, P.notatum, Scopulariopsis, Tritirachium album,,
Achromobacter, Arthrobacter, E.coli, Pseudomonas a
eruginosa, P.maltophilia, Candida lipolytica, Toru
laなどから得られるものが挙げられ、市販の蛋白分解酵
素としては、例えば、ナガセ生化学社製のビオプラー
ゼ、ノボインダストリー社製サビナーゼやエスペラーゼ
等が使用できる。BEST MODE FOR CARRYING OUT THE INVENTION Each component contained in the cleaning solution containing a protease according to the present invention will be described. (1) Modification enzyme The protease is chemically modified with a water-soluble polymer, and the protease is derived from a microorganism,
Any of plant-derived and animal-derived ones can be used, and neutral or alkaline ones derived from microorganisms are particularly preferable. Representative examples include the genus Bacillus (B. subtili
s, amyloloquefaciens, cereus, licheniformis, pumil
is, natto, mesentericus, sphaericus), Aspergillus
Genus (A. sojae, oryzae, flavus, sulphureus, candidus,
terricola, melleus, nidulans, sydowi), Streptmyces
Genus (S.fradiae, griseus, moderatus, rectus), Cepha
losporium sp., C. acremonium, Fusarium, Gliocladiu
m, Malbranchea pulchella, penicillium cyano-fulvu
m, P. notatum, Scopulariopsis, Tritirachium album ,,
Achromobacter, Arthrobacter, E.coli, Pseudomonas a
eruginosa, P. maltophilia, Candida lipolytica, Toru
Examples of commercially available proteolytic enzymes include bioseparase manufactured by Nagase Biochemical Co., Ltd., Savinase and Esperase manufactured by Novo Industry Co., Ltd.
【０００９】本発明において、このような蛋白分解酵素
を修飾する水溶性高分子としては、ポリエチレングリコ
ール、多糖類、蛋白質、有機合成高分子重合体が挙げら
れ、蛋白分解酵素を修飾する多糖類としては、デキスト
ラン、デンプン、プルラン、アガロース、ヒドロキシプ
ロピルセルロース、メチオルセルロース、エチルセルロ
ース、カルボキシメチルセルロースなどが好ましく、蛋
白質としては、ゼラチン、カゼイン、ミルクカゼインな
どが好ましい。又、有機合成高分子重合体としては、Ｃ
Ｈ2 ＝Ｃ＜基を有する化合物と無水マレイン酸との共重
合体が好ましく、具体的には、メチルビニルエーテルな
どのアルキルビニルエーテル−無水マレイン酸共重合
体、スチレン−無水マレイン酸共重合体、イソブチレン
−無水マレイン酸共重合体などある。本発明の蛋白分解
酵素含有洗浄液中の単位容積当たりの酵素活性値は 100
〜10,000U/mlが好ましく、500 〜 5,000U/mlが特に好ま
しい。又、酵素活性値が10,000〜200,000 U/mlである修
飾酵素溶液として０．１〜１０重量％の割合で含有する
ことが好ましく、０．５〜５重量％の割合で含有するこ
とが特に好ましい。In the present invention, examples of the water-soluble polymer that modifies the protease include polyethylene glycol, polysaccharides, proteins, and organic synthetic high-molecular polymers. Are preferably dextran, starch, pullulan, agarose, hydroxypropylcellulose, methylcellulose, ethylcellulose, carboxymethylcellulose and the like, and as the protein, gelatin, casein, milk casein and the like are preferable. As the organic synthetic high molecular polymer, C
A copolymer of a compound having an H 2 ＣC <group and maleic anhydride is preferred. Specific examples include an alkyl vinyl ether such as methyl vinyl ether-maleic anhydride copolymer, a styrene-maleic anhydride copolymer, and isobutylene. -Maleic anhydride copolymers and the like. The enzyme activity value per unit volume in the detergent solution containing the protease of the present invention is 100.
1010,000 U / ml is preferred, and 500-5,000 U / ml is particularly preferred. Further, the modified enzyme solution having an enzyme activity value of 10,000 to 200,000 U / ml is preferably contained at a ratio of 0.1 to 10% by weight, particularly preferably at a ratio of 0.5 to 5% by weight. .
【００１０】(2) 界面活性剤 本発明の蛋白分解酵素含有洗浄液中には、ポリグリセリ
ン脂肪酸エステル及びポリオキシエチレンアルキルフェ
ニルエーテルから選ばれた非イオン界面活性剤の少なく
とも１種と、陰イオン界面活性剤であるミリストイルサ
ルコシンナトリウム、ラウリル硫酸アンモニウム及び、
両性界面活性剤であるアルキルジメチルアミンオキサイ
ド、アルキルジメチルアミノ酢酸ベタインから成る群よ
り選ばれた少なくとも１種の界面活性剤とが含有されて
おり、上記の群からそれぞれ複数の界面活性剤が選択さ
れて含有されても良い。これら界面活性剤の含有量（濃
度）については、非イオン界面活性剤、陰イオン界面活
性剤、両性界面活性剤とも、酵素洗浄液中に有効成分量
として、０．０１〜１０重量％、特に０．０５〜５重量
％程度の割合であることが好ましい。(2) Surfactant The proteolytic enzyme-containing washing solution of the present invention contains at least one nonionic surfactant selected from polyglycerin fatty acid ester and polyoxyethylene alkylphenyl ether, and an anionic surfactant. Activators myristoyl sarcosine sodium, ammonium lauryl sulfate and
It contains at least one surfactant selected from the group consisting of alkyl dimethyl amine oxide, which is an amphoteric surfactant, and betaine alkyl dimethyl amino acetate, and a plurality of surfactants are respectively selected from the above group. May be contained. Regarding the content (concentration) of these surfactants, the nonionic surfactant, anionic surfactant, and amphoteric surfactant are 0.01 to 10% by weight, particularly 0% by weight, as the active ingredient in the enzyme washing solution. The proportion is preferably about 0.05 to 5% by weight.
【００１１】(3) ホウ酸化合物 本発明の蛋白分解酵素含有洗浄液中に含有されるホウ酸
化合物としては、ホウ酸、メタホウ酸、四ホウ酸などの
ホウ酸と、ホウ砂、メタホウ酸塩、四ホウ酸塩などのホ
ウ酸塩が挙げられ、その濃度としては０．５〜１０％、
特に１．０〜５％が好ましい。(3) Boric acid compound The boric acid compound contained in the proteolytic enzyme-containing washing solution of the present invention includes boric acid such as boric acid, metaboric acid and tetraboric acid, borax, metaborate, and the like. Borates such as tetraborate and the like, the concentration of which is 0.5 to 10%,
Particularly, 1.0 to 5% is preferable.
【００１２】尚、本発明の洗浄液における修飾酵素溶液
（10,000〜200,000 U/ml）：界面活性剤：ホウ酸化合物
の割合は、修飾酵素の安定性の点から、１：０．０１〜
２０：０．１〜５０（重量比率）が好ましく、１：０．
０５〜１０：０．５〜１０が特に好ましい。The ratio of the modified enzyme solution (10,000 to 200,000 U / ml): surfactant: borate compound in the washing solution of the present invention is from 1: 0.01 to 1: 1 from the viewpoint of the stability of the modified enzyme.
20: 0.1-50 (weight ratio) is preferred, and 1: 0.
05 to 10: 0.5 to 10 are particularly preferred.
【００１３】本発明における上述の修飾酵素は、単独で
水溶液中に存在した場合には、実用に耐える十分な安定
性を示さないが、特定の非イオン界面活性剤と、特定の
陰イオン界面活性剤及び／又は両性界面活性剤から成る
群より選ばれた少なくとも一種類の界面活性剤と、ホウ
酸化合物とを併用することによって、実用に耐える十分
な酵素安定性を示し、これにより、洗浄効果（蛋白質除
去効果）が経時的に低下することなく、長期間に渡って
安定して蛋白分解酵素含有洗浄液を使用することが可能
となる。本発明の洗浄液は、蛋白質の付着が問題になる
種々の製品の洗浄に使用できるものであるが、特にコン
タクトレンズの洗浄に最適であり、安全性が高く、しか
も簡単な操作（一定時間、浸漬保存）にて洗浄を実施す
ることができるという利点を有している。The above-mentioned modified enzyme of the present invention does not show sufficient stability for practical use when used alone in an aqueous solution. However, the modified enzyme has a specific nonionic surfactant and a specific anionic surfactant. By using at least one surfactant selected from the group consisting of a surfactant and / or an amphoteric surfactant and a boric acid compound, sufficient enzymatic stability sufficient for practical use is exhibited. (Protein removing effect) can be stably used for a long period of time without using protein-containing enzyme-containing cleaning solution without decreasing over time. Although the cleaning solution of the present invention can be used for cleaning various products in which protein adhesion is a problem, it is particularly suitable for cleaning contact lenses, is highly safe, and has a simple operation (for a certain period of time, immersion). This has the advantage that the cleaning can be carried out during storage.
【００１４】次に、本発明の蛋白分解酵素含有洗浄液中
に含まれる修飾酵素を得るための、水溶性高分子を用い
た蛋白分解酵素の修飾方法について説明する。 (A) ポリエチレングリコールによる化学修飾（塩化シア
ヌル法） ポリエチレングリコールと塩化シアヌルを反応させ、得
られた反応生成物と蛋白分解酵素とをｐＨ調整後、冷却
しながら攪拌を行うと、蛋白分解酵素中のアミノ基がポ
リエチレングリコールで修飾された修飾酵素が得られ
る。 (B) 多糖類による化学修飾 多糖類を用いて蛋白分解酵素を修飾するには、従来より
知られている過ヨウ素酸酸化法、臭化シアン法、カルボ
ジイミド法、塩化シアヌル法、エピクロルヒドリン法、
ＳＰＤＰ（N-Succinimidyl 3-[2-pyridyldithio]propio
nate）試薬法、活性エステル法などが使用される。 (C) 蛋白質による化学修飾 蛋白質（カゼイン、ミルクカゼイン、ゼラチンなど）
を、蛋白分解酵素及びグルタルアルデヒドと混合し、ｐ
Ｈ調整した後、冷却しながら攪拌を行って架橋させる
と、蛋白分解酵素が蛋白質により化学修飾された修飾酵
素が得られる。 (D) 有機合成高分子重合体による化学修飾 ＣＨ2 ＝Ｃ＜基を有する化合物（例えばメチルビニルエ
ーテルなど）と無水マレイン酸との共重合体、蛋白分解
酵素、ホウ酸化合物に、蒸留水を加え、ｐＨ調整した
後、冷却しながら攪拌を行うと、蛋白分解酵素が有機合
成高分子重合体により化学修飾された修飾酵素が得られ
る。Next, a method for modifying a protease using a water-soluble polymer for obtaining the modification enzyme contained in the protease-containing washing solution of the present invention will be described. (A) Chemical modification with polyethylene glycol (cyanuric chloride method) After reacting polyethylene glycol with cyanuric chloride, adjusting the pH of the resulting reaction product and the protease, and stirring while cooling, the protease A modified enzyme in which the amino group is modified with polyethylene glycol is obtained. (B) Chemical modification with polysaccharide To modify a protease using a polysaccharide, a conventionally known periodate oxidation method, cyanogen bromide method, carbodiimide method, cyanuric chloride method, epichlorohydrin method,
SPDP (N-Succinimidyl 3- [2-pyridyldithio] propio
nate) A reagent method, an active ester method and the like are used. (C) Chemical modification by protein Protein (casein, milk casein, gelatin, etc.)
Is mixed with a protease and glutaraldehyde, and p
After the H adjustment, the mixture is stirred and cooled while being crosslinked to obtain a modified enzyme in which a protease is chemically modified with a protein. (D) Chemical modification with an organic synthetic high molecular polymer Distilled water is added to a copolymer of a compound having a CH 2 ＣC <group (for example, methyl vinyl ether) and maleic anhydride, a protease, and a boric acid compound. If the pH is adjusted and then stirred while cooling, a modified enzyme in which the proteolytic enzyme is chemically modified with the organic synthetic polymer is obtained.
【００１５】尚、本発明の洗浄液中には、前記の成分の
他に保存剤が含有されても良く、このような保存剤とし
ては、医療施設内における病原性微生物の感染防止等に
用いられるものがいずれも使用できる。このような保存
剤は、第四級アンモニウム塩系消毒剤である塩化ベンザ
ルコニウム、塩化ベンゼトニウム、チセルピリジウムク
ロライド、ジアルキルジメチルアンモニウムクロライド
や、グリシン系両性界面活性剤であるアルキルポリアミ
ノグリシン、ジアルキルアミノエチルグリシンや、ビグ
アナイド系消毒剤であるポリヘキサメチレンビグアニジ
ン塩酸塩、グルコン酸クロルヘキシジンまたはその塩か
らなる群から選択でき、これらは１種で使用されても２
種以上併用されてもよい。上記の化合物群において、特
に実用性のある消毒剤は、塩化ベンザルコニウム、塩化
ベンゼトニウム、ジアルキルアミノエチルグリシン、グ
ルコン酸クロルヘキシジンまたはその塩などである。保
存剤の使用量については、酵素、界面活性剤の濃度や緩
衝液の種類などによって変化するが、通常は、上述の消
毒液の１種以上を酵素洗浄液中に約０．００１〜０．５
重量％の割合となるように使用するのが好ましい。ま
た、必要に応じて、金属キレート剤、等張調整剤、多価
アルコール、ｐＨ調整剤、親水性高分子並びに他の酵素
剤、例えば脂肪分解酵素等の各種の成分を含有すること
も出来る。The cleaning solution of the present invention may contain a preservative in addition to the above-mentioned components. Such a preservative is used for preventing infection of pathogenic microorganisms in medical facilities. Any of them can be used. Such preservatives include quaternary ammonium salt-based disinfectants such as benzalkonium chloride, benzethonium chloride, tisserpyridium chloride, and dialkyldimethylammonium chloride, and glycine-based amphoteric surfactants such as alkylpolyaminoglycine and dialkylamino. It can be selected from the group consisting of ethyl glycine, polyhexamethylene biguanidine hydrochloride which is a biguanide disinfectant, chlorhexidine gluconate or a salt thereof.
More than one species may be used in combination. Among the above compounds, particularly useful disinfectants are benzalkonium chloride, benzethonium chloride, dialkylaminoethylglycine, chlorhexidine gluconate or salts thereof. The amount of the preservative used varies depending on the concentration of the enzyme and the surfactant, the type of the buffer, and the like.
It is preferred to use it in a proportion by weight. In addition, if necessary, various components such as a metal chelating agent, an isotonicity adjusting agent, a polyhydric alcohol, a pH adjusting agent, a hydrophilic polymer, and other enzyme agents such as lipolytic enzymes can be contained.
【００１６】次に、本発明の蛋白分解酵素含有洗浄液に
より、酵素洗浄液中の蛋白分解酵素を安定化する方法に
ついて説明する。この方法においては、前述の蛋白分解
酵素を前述の水溶性高分子を用いて化学修飾することに
よって得られた修飾酵素を準備し、この修飾酵素を、前
述の非イオン界面活性剤と、前述の陰イオン界面活性剤
及び両性界面活性剤から成る群より選ばれた少なくとも
１種の界面活性剤と共に、前述のホウ酸化合物を含有す
る水溶液に添加し、混合する。この際、修飾酵素は単離
された状態で添加されても、溶液中に分散された状態で
添加されても良く、ホウ酸化合物添加後の水溶液のｐＨ
値は、修飾酵素の安定化の点からｐＨ５〜１２の範囲で
あることが好ましく、ｐＨ６〜１０の範囲が特に好まし
い。このような方法では、界面活性剤とホウ酸化合物の
併用により、蛋白分解酵素の安定性が著しく向上し、酵
素活性が維持されるため、酵素洗浄液を長期間、安定し
て使用することが可能である。Next, a method for stabilizing the protease in the enzyme washing solution by using the protease-containing washing solution of the present invention will be described. In this method, a modified enzyme obtained by chemically modifying the above-mentioned protease with the above-mentioned water-soluble polymer is prepared, and the modified enzyme is combined with the above-mentioned nonionic surfactant and the above-mentioned nonionic surfactant. Along with at least one surfactant selected from the group consisting of an anionic surfactant and an amphoteric surfactant, the surfactant is added to the aqueous solution containing the boric acid compound and mixed. At this time, the modifying enzyme may be added in an isolated state or in a dispersed state in a solution, and the pH of the aqueous solution after the boric acid compound is added may be added.
The value is preferably in the range of pH 5 to 12 from the viewpoint of stabilizing the modifying enzyme, and particularly preferably in the range of pH 6 to 10. In such a method, the stability of the protease is significantly improved and the enzyme activity is maintained by the combined use of the surfactant and the boric acid compound, so that the enzyme washing solution can be used stably for a long time. It is.
【００１７】以下、本発明の蛋白分解酵素含有洗浄液に
ついての実施例を示すが、本発明はこれらに限定される
ものではない。尚、下記の調製例に従って得られた酵素
洗浄液中の蛋白分解酵素の安定化の評価については、蛋
白分解酵素活性を、カゼイン消化を用いる公知の方法
（例えば、Journal of General Phyalology, 30 (1947)
291参照）に準じて測定し、下記式を用いて蛋白分解酵
素活性残存率を算出することによって行った。蛋白分解
酵素活性残存率（％）＝（６０℃で１０時間加熱処理し
た後の蛋白分解酵素活性／溶液調製直後の蛋白分解酵素
活性）×１００Hereinafter, the present invention will be described in more detail with reference to Examples of the detergent solution containing a protease, but the present invention is not limited thereto. In addition, regarding the evaluation of the stabilization of the protease in the enzyme washing solution obtained according to the following preparation examples, the protease activity was determined by a known method using casein digestion (for example, Journal of General Phyalology, 30 (1947)).
291), and the residual rate of protease activity was calculated using the following equation. Protease activity residual rate (%) = (protease activity after heat treatment at 60 ° C. for 10 hours / protease activity immediately after solution preparation) × 100
【００１８】[0018]
【実施例】（実施例１） 分子量750 〜2,000 のモノメトキシポリエチレングリコ
ール５ｇと塩化シアヌル１ｇを反応させ得られた、2-O-
methoxypolyethylene glycol-4,6-dichloro-s-triazine
(activated PEG 1) を水溶液とし、この５％溶液５０ｇ
とBacillus属起源のアルカリ性プロテアーゼ（商品名：
ビオプラーゼ、ナガセ生化学社製）５０ｇを混合し、ホ
ウ砂でｐＨ９．２に調整した後、４℃で１時間反応さ
せ、蛋白分解酵素中のアミノ基をポリエチレングリコー
ルで修飾された修飾酵素を含む溶液（酵素活性値47,300
U/ml ）を得た。２％ホウ酸溶液１００ｍｌに、ラウリ
ルジメチルアミンオキサイド（３５％）０．５ｇ、モノ
ラウリン酸デカグリセル０．５ｇ、ミリストイルサルコ
シンナトリウム（９３％）０．５ｇを加え、更にポリエ
チレングリコールを修飾担体として得られた修飾蛋白分
解酵素を含有する上記修飾酵素溶液（酵素活性値47,300
U/ml ）を２．０ｇ加え、その後、ホウ砂を加えて混合
溶液のｐＨを８．０に調整し、約１時間程攪拌して本発
明の酵素洗浄液（酵素活性値946 U/ml）を得た。この酵
素洗浄液の蛋白分解酵素活性を測定した後、該洗浄液を
密封容器の中に入れ、６０℃の恒温器中で１０時間加熱
した。そして、加熱処理した後の酵素洗浄液の蛋白分解
酵素活性と洗浄効果の測定を行った。EXAMPLES (Example 1) 5-O-methoxy-polyethylene glycol having a molecular weight of 750 to 2,000 was reacted with 5 g of cyanuric chloride to obtain 2-O-.
methoxypolyethylene glycol-4,6-dichloro-s-triazine
(activated PEG 1) as an aqueous solution, 50 g of this 5% solution
And alkaline protease of Bacillus genus (trade name:
50 g of biopulase, manufactured by Nagase Seikagaku Co., Ltd.), adjusted to pH 9.2 with borax, reacted at 4 ° C. for 1 hour, and contained a modified enzyme in which the amino group in the protease was modified with polyethylene glycol. Solution (enzyme activity value 47,300
U / ml). To 100 ml of a 2% boric acid solution, 0.5 g of lauryl dimethylamine oxide (35%), 0.5 g of decaglycel monolaurate, and 0.5 g of sodium myristoyl sarcosine (93%) were added, and polyethylene glycol was obtained as a modified carrier. The above modified enzyme solution containing a modified protease (enzyme activity value 47,300
U / ml), and then add borax to adjust the pH of the mixed solution to 8.0 and stir for about 1 hour to wash the enzyme of the present invention (enzyme activity value 946 U / ml). I got After measuring the proteolytic enzyme activity of this enzyme washing solution, the washing solution was put in a sealed container and heated in a thermostat at 60 ° C. for 10 hours. Then, the proteolytic enzyme activity and the cleaning effect of the enzyme cleaning solution after the heat treatment were measured.
【００１９】（実施例２） １ｇのデキストランＴ４０を０．２Ｍ炭酸水素ナトリウ
ム溶液９０ｍｌ中に溶解し、４℃にてアセトニトリル
２．５ｇに溶解した臭化シアン１．５ｇを加えつつホウ
砂でｐＨを９．５に保持した。反応終了後ｐＨを８．５
に下げ、緩衝液に透析した。これに、実施例１と同じ蛋
白分解酵素５０ｇを加え、一夜放置後、エタノールアミ
ンを加えて活性基を除去し、透析後、カラムクロマトグ
ラフィーにより修飾蛋白分解酵素溶液（酵素活性値27,7
00 U/ml ）を得た。２％ホウ酸溶液１００ｍｌに、ラウ
リルジメチルアミンオキサイド（３５％）０．５ｇ、モ
ノラウリン酸デカグリセル０．５ｇ、ミリストイルサル
コシンナトリウム（９３％）０．５ｇを加え、更にデキ
ストランＴ４０を修飾担体として得られた上記修飾酵素
溶液（酵素活性値27,700 U/ml ）を３．０ｇ加え、その
後、ホウ砂を加えて混合溶液のｐＨを８．０に調整し、
約１時間程攪拌して本発明の酵素洗浄液（酵素活性値83
0 U/ml）を得た。実施例１と同様に、酵素洗浄液の加熱
処理前後の蛋白分解酵素活性と洗浄効果の測定を行っ
た。Example 2 1 g of dextran T40 was dissolved in 90 ml of a 0.2M sodium hydrogen carbonate solution, and pH was adjusted with borax at 4 ° C. while adding 1.5 g of cyanogen bromide dissolved in 2.5 g of acetonitrile. Was maintained at 9.5. After the completion of the reaction, the pH was adjusted to 8.5.
And dialyzed against buffer. To this, 50 g of the same protease as in Example 1 was added, and after standing overnight, ethanolamine was added to remove the active groups. After dialysis, the modified protease solution (enzyme activity value 27,7
00 U / ml). To 100 ml of a 2% boric acid solution were added 0.5 g of lauryl dimethylamine oxide (35%), 0.5 g of decaglycel monolaurate, and 0.5 g of sodium myristoyl sarcosine (93%), and dextran T40 was obtained as a modified carrier. 3.0 g of the modified enzyme solution (enzyme activity value 27,700 U / ml) was added, and then borax was added to adjust the pH of the mixed solution to 8.0,
After stirring for about 1 hour, the enzyme washing solution of the present invention (enzyme activity value 83
0 U / ml). As in Example 1, the proteolytic enzyme activity and the cleaning effect before and after the heat treatment of the enzyme cleaning solution were measured.
【００２０】（実施例３） ゼラチン１ｇを、実施例１と同じ蛋白分解酵素５０ｇ及
び２５％グルタルアルデヒド溶液０．２ｇと混合し、ホ
ウ砂でｐＨ８．２に調整し、４℃で１２時間反応させ架
橋させることにより修飾酵素溶液（酵素活性値34,700 U
/ml ）を得た。２％ホウ酸溶液１００ｍｌに、ラウリル
ジメチルアミンオキサイド（３５％）０．５ｇ、モノラ
ウリン酸デカグリセル０．５ｇ、ミリストイルサルコシ
ンナトリウム（９３％）０．５ｇを加え、更にゼラチン
を修飾担体として得られた上記修飾酵素溶液（酵素活性
値34,700 U/ml ）を２．５ｇ加え、その後、ホウ砂を加
えて混合溶液のｐＨを８．０に調整し、約１時間程攪拌
して本発明の酵素洗浄液（酵素活性値867 U/ml）を得
た。実施例１と同様に、酵素洗浄液の加熱処理前後の蛋
白分解酵素活性と洗浄効果の測定を行った。Example 3 1 g of gelatin was mixed with 50 g of the same protease as in Example 1 and 0.2 g of a 25% glutaraldehyde solution, adjusted to pH 8.2 with borax, and reacted at 4 ° C. for 12 hours. And then cross-link the modified enzyme solution (enzyme activity value 34,700 U
/ ml). To 100 ml of a 2% boric acid solution were added 0.5 g of lauryl dimethylamine oxide (35%), 0.5 g of decaglyceryl monolaurate, and 0.5 g of sodium myristoyl sarcosine (93%), and the obtained gelatin was used as a modified carrier. 2.5 g of the modified enzyme solution (enzyme activity value 34,700 U / ml) was added, and then the pH of the mixed solution was adjusted to 8.0 by adding borax, and the mixture was stirred for about 1 hour, and the enzyme cleaning solution of the present invention ( An enzyme activity value of 867 U / ml) was obtained. As in Example 1, the proteolytic enzyme activity and the cleaning effect before and after the heat treatment of the enzyme cleaning solution were measured.
【００２１】（実施例４） メチルビニルエーテルと無水マレイン酸との共重合体(G
ANTREZ AN:G.A.F.社製）１ｇ、実施例１と同じ蛋白分解
酵素５０ｇ、ホウ砂０．１ｇ、蒸留水５０ｍｌを混合
し、４℃にて２４時間攪拌することにより修飾酵素溶液
（酵素活性値58,900U/ml ）を得た。２％ホウ酸溶液１
００ｍｌに、ラウリルジメチルアミンオキサイド（３５
％）０．５ｇ、モノラウリン酸デカグリセル０．５ｇ、
ミリストイルサルコシンナトリウム（９３％）０．５ｇ
を加え、更にメチルビニルエーテル−無水マレイン酸共
重合体を修飾担体として得られた上記修飾酵素溶液（酵
素活性値58,900 U/ml ）を１．５ｇ加え、その後、ホウ
砂を加えて混合溶液のｐＨを８．０に調整し、約１時間
程攪拌して本発明の酵素洗浄液（酵素活性値883 U/ml）
を得た。実施例１と同様に、酵素洗浄液の加熱処理前後
の蛋白分解酵素活性と洗浄効果の測定を行った。Example 4 A copolymer of methyl vinyl ether and maleic anhydride (G
1 g of ANTREZ AN (manufactured by GAF), 50 g of the same protease as in Example 1, 0.1 g of borax and 50 ml of distilled water were mixed, and the mixture was stirred at 4 ° C. for 24 hours to obtain a modified enzyme solution (enzyme activity value 58,900). U / ml). 2% boric acid solution 1
In 00 ml, lauryl dimethylamine oxide (35
%) 0.5 g, decaglycerol monolaurate 0.5 g,
0.5g of myristoyl sarcosine sodium (93%)
, And 1.5 g of the above-mentioned modified enzyme solution (enzyme activity value 58,900 U / ml) obtained using a methyl vinyl ether-maleic anhydride copolymer as a modifying carrier, and then borax is added to adjust the pH of the mixed solution. Was adjusted to 8.0, and the mixture was stirred for about 1 hour, and the enzyme washing solution of the present invention (enzyme activity value: 883 U / ml)
I got As in Example 1, the proteolytic enzyme activity and the cleaning effect before and after the heat treatment of the enzyme cleaning solution were measured.
【００２２】（実施例５） ２％ホウ酸溶液１００ｍｌに、ラウリルジメチルアミノ
酢酸ベタイン（２６％）０．３ｇ、ポリオキシエチレン
ノニルフェニルエーテル（１００％）０．３ｇ、更に実
施例１と同様のポリエチレングリコールを修飾担体とし
て得られた上記修飾酵素を含む溶液（酵素活性値47,300
U/ml ）を２．０ｇ加え、その後、ホウ砂を加えて混合
溶液のｐＨを８．０に調整し、約１時間程攪拌して本発
明の酵素洗浄液を得た。実施例１と同様に、酵素洗浄液
の加熱処理前後の蛋白分解酵素活性と洗浄効果の測定を
行った。Example 5 In 100 ml of a 2% boric acid solution, 0.3 g of betaine lauryldimethylaminoacetate (26%), 0.3 g of polyoxyethylene nonylphenyl ether (100%), and the same as in Example 1 A solution containing the modified enzyme obtained using polyethylene glycol as a modified carrier (enzyme activity value 47,300
U / ml) was added, and borax was added to adjust the pH of the mixed solution to 8.0, followed by stirring for about 1 hour to obtain an enzyme washing solution of the present invention. As in Example 1, the proteolytic enzyme activity and the cleaning effect before and after the heat treatment of the enzyme cleaning solution were measured.
【００２３】（実施例６） ２％ホウ酸溶液１００ｍｌに、ヤシジメチルアミンオキ
サイド（３０％）０．４ｇ、ラウリル硫酸アンモニウム
（２５％）０．４ｇ、更に実施例１と同様のポリエチレ
ングリコールを修飾担体として得られた上記修飾酵素を
含む溶液（酵素活性値47,300 U/ml ）を２．０ｇ加え、
その後、ホウ砂を加えて混合溶液のｐＨを８．０に調整
し、約１時間程攪拌して本発明の酵素洗浄液を得た。実
施例１と同様に、酵素洗浄液の加熱処理前後の蛋白分解
酵素活性と洗浄効果の測定を行った。Example 6 In 100 ml of a 2% boric acid solution, 0.4 g of coconut dimethylamine oxide (30%), 0.4 g of ammonium lauryl sulfate (25%), and the same polyethylene glycol as in Example 1 were used as modified carriers. 2.0 g of a solution containing the above modified enzyme (enzyme activity value 47,300 U / ml) obtained as
Thereafter, borax was added to adjust the pH of the mixed solution to 8.0, and the mixture was stirred for about 1 hour to obtain an enzyme cleaning solution of the present invention. As in Example 1, the proteolytic enzyme activity and the cleaning effect before and after the heat treatment of the enzyme cleaning solution were measured.
【００２４】（比較例１） ０．９％塩化ナトリウム、０．５％リン酸水素二ナトリ
ウム溶液に適量の水酸化ナトリウムを加え、ｐＨを８．
０に調整し、緩衝液とした。この緩衝液１００ｍｌにラ
ウリルジメチルアミンオキサイド（３５％）０．５ｇ、
モノラウリン酸デカグリセル０．５ｇ、ミリストイルサ
ルコシンナトリウム（９３％）０．５ｇを加え、更に実
施例１と同様のポリエチレングリコールを修飾担体とし
て得られた修飾蛋白分解酵素を含む溶液（酵素活性値4
7,300 U/ml ）２．０ｇ加え、その後、水酸化ナトリウ
ムを加えて混合溶液のｐＨを８．０に調整し、約１時間
程攪拌し酵素洗浄液を得た。実施例１と同様に、酵素洗
浄液の加熱処理前後の蛋白分解酵素活性と洗浄効果の測
定を行った。Comparative Example 1 An appropriate amount of sodium hydroxide was added to a 0.9% sodium chloride, 0.5% disodium hydrogen phosphate solution to adjust the pH to 8.8.
It was adjusted to 0 and used as a buffer. 0.5 g of lauryl dimethylamine oxide (35%) was added to 100 ml of this buffer,
0.5 g of decaglycerol monolaurate and 0.5 g of myristoyl sarcosine sodium (93%) were added, and a solution containing a modified protease obtained using polyethylene glycol as the modification carrier as in Example 1 (enzyme activity value 4
7,300 U / ml) was added, and then sodium hydroxide was added to adjust the pH of the mixed solution to 8.0, followed by stirring for about 1 hour to obtain an enzyme washing solution. As in Example 1, the proteolytic enzyme activity and the cleaning effect before and after the heat treatment of the enzyme cleaning solution were measured.
【００２５】（比較例２） 比較例１と同様の緩衝液（ｐＨ８．０）１００ｍｌに、
ラウリルジメチルアミンオキサイド（３５％）０．５
ｇ、モノラウリン酸デカグリセル０．５ｇ、ミリストイ
ルサルコシンナトリウム（９３％）０．５ｇを加え、更
に実施例２と同様のデキストランＴ４０を修飾担体とし
て得られた修飾蛋白分解酵素を含む溶液（酵素活性値2
7,700 U/ml ）を３．０ｇ加え、その後、水酸化ナトリ
ウムを加えて混合溶液のｐＨを８．０に調整し、約１時
間程攪拌し酵素洗浄液を得た。実施例１と同様に、酵素
洗浄液の加熱処理前後の蛋白分解酵素活性と洗浄効果の
測定を行った。(Comparative Example 2) In 100 ml of the same buffer solution (pH 8.0) as in Comparative Example 1,
Lauryl dimethylamine oxide (35%) 0.5
g, 0.5 g of decaglycel monolaurate, and 0.5 g of myristoyl sarcosine sodium (93%), and a solution containing the modified protease obtained using dextran T40 as a modification carrier as in Example 2 (enzyme activity value 2).
7,700 U / ml) was added, and then the pH of the mixed solution was adjusted to 8.0 by adding sodium hydroxide, followed by stirring for about 1 hour to obtain an enzyme washing solution. As in Example 1, the proteolytic enzyme activity and the cleaning effect before and after the heat treatment of the enzyme cleaning solution were measured.
【００２６】（比較例３） 比較例１と同様の緩衝液（ｐＨ８．０）１００ｍｌに、
ラウリルジメチルアミンオキサイド（３５％）０．５
ｇ、モノラウリン酸デカグリセル０．５ｇ、ミリストイ
ルサルコシンナトリウム（９３％）０．５ｇを加え、更
に実施例３と同様のゼラチンを修飾担体として得られた
修飾蛋白分解酵素を含む溶液（酵素活性値34,700 U/ml
）を２．５ｇ加え、その後、水酸化ナトリウムを加え
て混合溶液のｐＨを８．０に調整し、約１時間程攪拌し
酵素洗浄液を得た。実施例１と同様に、酵素洗浄液の加
熱処理前後の蛋白分解酵素活性と洗浄効果の測定を行っ
た。(Comparative Example 3) In 100 ml of the same buffer solution (pH 8.0) as in Comparative Example 1,
Lauryl dimethylamine oxide (35%) 0.5
g, 0.5 g of decaglycel monolaurate and 0.5 g of myristoyl sarcosine sodium (93%), and a solution containing a modified proteolytic enzyme obtained using gelatin as a modification carrier (enzyme activity value 34,700 U / ml
) Was added, and then sodium hydroxide was added to adjust the pH of the mixed solution to 8.0, followed by stirring for about 1 hour to obtain an enzyme washing solution. As in Example 1, the proteolytic enzyme activity and the cleaning effect before and after the heat treatment of the enzyme cleaning solution were measured.
【００２７】（比較例４） 比較例１と同様の緩衝液（ｐＨ８．０）１００ｍｌに、
ラウリルジメチルアミンオキサイド（３５％）０．５
ｇ、モノラウリン酸デカグリセル０．５ｇ、ミリストイ
ルサルコシンナトリウム（９３％）０．５ｇを加え、更
に実施例４と同様のメチルビニルエーテル−無水マレイ
ン酸共重合体を修飾担体として得られた修飾蛋白分解酵
素を含む溶液（酵素活性値58,900 U/ml ）を１．５ｇ加
え、その後、水酸化ナトリウムを加えて混合溶液のｐＨ
を８．０に調整し、約１時間程攪拌し酵素洗浄液を得
た。実施例１と同様に、酵素洗浄液の加熱処理前後の蛋
白分解酵素活性と洗浄効果の測定を行った。(Comparative Example 4) In 100 ml of the same buffer solution (pH 8.0) as in Comparative Example 1,
Lauryl dimethylamine oxide (35%) 0.5
g, 0.5 g of decaglycel monolaurate and 0.5 g of myristoyl sarcosine sodium (93%), and the modified proteolytic enzyme obtained using the same methyl vinyl ether-maleic anhydride copolymer as in Example 4 as a modification carrier was used. 1.5 g of a solution (enzyme activity value 58,900 U / ml) containing sodium hydroxide was added, and then the pH of the mixed solution was added.
Was adjusted to 8.0 and stirred for about 1 hour to obtain an enzyme washing solution. As in Example 1, the proteolytic enzyme activity and the cleaning effect before and after the heat treatment of the enzyme cleaning solution were measured.
【００２８】（比較例５） 実施例１と同様に調製した緩衝液１００ｍｌに、ラウリ
ルジメチルアミンオキサイド（３５％）０．５ｇ、モノ
ラウリン酸デカグリセル０．５ｇ、ミリストイルサルコ
シンナトリウム（９３％）０．５ｇを加え、更に蛋白分
解酵素として未修飾の蛋白分解酵素（商品名：ビオプラ
ーゼ、ナガセ生化学社製）を０．３５ｇ加え、その後、
ホウ砂を加えて混合溶液のｐＨを８．０に調整し、約１
時間程攪拌し酵素洗浄液を得た。実施例１と同様に、酵
素洗浄液の加熱処理前後の蛋白分解酵素活性と洗浄効果
の測定を行った。(Comparative Example 5) 0.5 g of lauryl dimethylamine oxide (35%), 0.5 g of decaglyceryl monolaurate, 0.5 g of myristoyl sarcosine sodium (93%) were added to 100 ml of the buffer prepared in the same manner as in Example 1. , And 0.35 g of an unmodified proteolytic enzyme (trade name: Bioprase, manufactured by Nagase Biochemical Co., Ltd.) is added as a proteolytic enzyme.
The pH of the mixed solution was adjusted to 8.0 by adding borax, and
The mixture was stirred for about an hour to obtain an enzyme washing solution. As in Example 1, the proteolytic enzyme activity and the cleaning effect before and after the heat treatment of the enzyme cleaning solution were measured.
【００２９】（比較例６） 実施例１と同様に調製した緩衝液１００ｍｌに、実施例
１と同様の、ポリエチレングリコールを修飾担体として
得られた修飾蛋白分解酵素を含む溶液（酵素活性値47,3
00 U/ml ）を２．０ｇ加え、その後、ホウ砂を加えて混
合溶液のｐＨを８．０に調整し、約１時間程攪拌し酵素
洗浄液を得た。実施例１と同様に、酵素洗浄液の加熱処
理前後の蛋白分解酵素活性と洗浄効果の測定を行った。Comparative Example 6 A solution containing a modified proteolytic enzyme obtained using polyethylene glycol as a modified carrier (enzyme activity value: 47, 100 ml) in 100 ml of buffer prepared in the same manner as in Example 1 Three
(00 U / ml) was added, and borax was added to adjust the pH of the mixed solution to 8.0, followed by stirring for about 1 hour to obtain an enzyme washing solution. As in Example 1, the proteolytic enzyme activity and the cleaning effect before and after the heat treatment of the enzyme cleaning solution were measured.
【００３０】（比較例７） 実施例１と同様に調製した緩衝液１００ｍｌに、実施例
２と同様の、デキストランＴ４０を修飾担体として得ら
れた修飾蛋白分解酵素を含む溶液（酵素活性値27,700 U
/ml ）を３．０ｇ加え、その後、ホウ砂を加えて混合溶
液のｐＨを８．０に調整し、約１時間程攪拌し酵素洗浄
液を得た。実施例１と同様に、酵素洗浄液の加熱処理前
後の蛋白分解酵素活性と洗浄効果の測定を行った。(Comparative Example 7) A solution containing a modified proteolytic enzyme obtained using dextran T40 as a modified carrier (enzyme activity value: 27,700 U) in 100 ml of buffer prepared in the same manner as in Example 1
/ ml), and then borax was added to adjust the pH of the mixed solution to 8.0, followed by stirring for about 1 hour to obtain an enzyme washing solution. As in Example 1, the proteolytic enzyme activity and the cleaning effect before and after the heat treatment of the enzyme cleaning solution were measured.
【００３１】（比較例８） 実施例１と同様に調製した緩衝液１００ｍｌに、実施例
３と同様の、ゼラチンを修飾担体として得られた修飾蛋
白分解酵素を含む溶液（酵素活性値34,700 U/ml）を
２．５ｇ加え、その後、ホウ砂を加えて混合溶液のｐＨ
を８．０に調整し、約１時間程攪拌し酵素洗浄液を得
た。実施例１と同様に、酵素洗浄液の加熱処理前後の蛋
白分解酵素活性と洗浄効果の測定を行った。Comparative Example 8 A solution containing a modified proteolytic enzyme obtained using gelatin as a modified carrier (enzyme activity value 34,700 U / 100 ml) in 100 ml of buffer prepared in the same manner as in Example 1 2.5 g), and then add borax to adjust the pH of the mixed solution.
Was adjusted to 8.0 and stirred for about 1 hour to obtain an enzyme washing solution. As in Example 1, the proteolytic enzyme activity and the cleaning effect before and after the heat treatment of the enzyme cleaning solution were measured.
【００３２】（比較例９） 実施例１と同様に調製した緩衝液１００ｍｌに、実施例
４と同様の、メチルビニルエーテル−無水マレイン酸共
重合体を修飾担体として得られた修飾蛋白分解酵素を含
む溶液（酵素活性値58,900 U/ml ）を１．５ｇ加え、そ
の後、ホウ砂を加えて混合溶液のｐＨを８．０に調整
し、約１時間程攪拌し酵素洗浄液を得た。実施例１と同
様に、酵素洗浄液の加熱処理前後の蛋白分解酵素活性と
洗浄効果の測定を行った。(Comparative Example 9) A modified protease obtained by using a methylvinyl ether-maleic anhydride copolymer as a modified carrier in the same manner as in Example 4 was contained in 100 ml of a buffer solution prepared in the same manner as in Example 1. 1.5 g of a solution (enzyme activity value: 58,900 U / ml) was added, and then borax was added to adjust the pH of the mixed solution to 8.0, followed by stirring for about 1 hour to obtain an enzyme washing solution. As in Example 1, the proteolytic enzyme activity and the cleaning effect before and after the heat treatment of the enzyme cleaning solution were measured.
【００３３】上記実施例１〜６及び比較例１〜９の酵素
洗浄液中に含有されている成分を、以下の表１及び表２
にまとめた。The components contained in the enzyme washing liquids of Examples 1 to 6 and Comparative Examples 1 to 9 are shown in Tables 1 and 2 below.
Summarized in
【００３４】[0034]
【表１】 [Table 1]
【００３５】[0035]
【表２】 [Table 2]
【００３６】上記実施例１〜６及び比較例１〜９の酵素
洗浄液を用いて、それぞれ、溶液調製直後の蛋白分解酵
素活性と、６０℃で１０時間加熱処理した後の蛋白分解
酵素活性を測定した結果を、以下の表３に示す。尚、表
３には、前述の算出式を用いて算出された蛋白分解酵素
活性残存率（％）も示されている。Using the enzyme washing solutions of Examples 1 to 6 and Comparative Examples 1 to 9, the activity of the protease immediately after solution preparation and the activity of the protease after heat treatment at 60 ° C. for 10 hours were measured. The results obtained are shown in Table 3 below. Table 3 also shows the residual rate (%) of the protease activity calculated using the above formula.
【００３７】[0037]
【表３】 [Table 3]
【００３８】表３の実験結果から、本発明の蛋白分解酵
素含有洗浄液は、６０℃で１０時間加熱した後において
も、優れた酵素活性を有していることがわかる。From the experimental results shown in Table 3, it can be seen that the protease-containing cleaning solution of the present invention has excellent enzyme activity even after heating at 60 ° C. for 10 hours.
【００３９】次に、前記実施例１〜６及び比較例１〜９
の酵素洗浄液を用いて、洗浄力（汚れ除去率）の経時変
化について測定を行った。 （実験方法） 涙液とほぼ同様の下記組成からなる人工蛋白質溶液を調
製した。 アルブミン（牛血清） ０．３９％ γ−グロブリン（牛血清） ０．１６％ リゾチーム（卵白由来） ０．１２％ 残り 生理食塩水 上記蛋白質溶液中に、ジメチルシロキサン基及びフッ素
原子を含有している酸素透過性ハードコンタクトレンズ
を浸漬し、レンズを浸漬したまま６０℃に加熱して蛋白
質を変性させてレンズ表面に人工蛋白質汚れを付着させ
た。次いで、このようにして人工汚れを付着させたレン
ズを、蛋白質を特異的に染色するエリスロシンで染色
し、分光光度計（島津製作所製のＵＶ−１６０Ａ）で、
５４５ｎｍの吸光度を測定した。この時の吸光度をＡと
する。別に、人工蛋白質汚れを付着させたレンズを酵素
洗浄液中に６時間浸漬後、レンズを水洗いして再びエリ
スロシンで染色し、分光光度計で５４５ｎｍの吸光度を
測定した。この時の吸光度をＢとする。蛋白質汚れ除去
率は下記式にて算出される。 蛋白質汚れ除去率（％）＝（Ｂ／Ａ）×１００ 上記実験方法により、酵素洗浄液を調製した直後と、洗
浄液を６０℃にて１０時間加熱した後の、蛋白質汚れ除
去率をそれぞれ測定した。得られた実験結果を表４に示
す。Next, the above Examples 1 to 6 and Comparative Examples 1 to 9
Using the enzyme cleaning solution of No. 5, the change over time of the cleaning power (dirt removal rate) was measured. (Experimental method) An artificial protein solution having the following composition substantially similar to that of tears was prepared. Albumin (bovine serum) 0.39% γ-globulin (bovine serum) 0.16% Lysozyme (derived from egg white) 0.12% Remaining physiological saline The protein solution contains a dimethylsiloxane group and a fluorine atom The oxygen-permeable hard contact lens was immersed, and the protein was denatured by heating at 60 ° C. while the lens was immersed, so that artificial protein stain was attached to the lens surface. Next, the lens to which the artificial stain was attached in this manner was stained with erythrosine, which specifically stains a protein, and the spectrophotometer (UV-160A manufactured by Shimadzu Corporation) was used.
The absorbance at 545 nm was measured. The absorbance at this time is defined as A. Separately, the lens to which the artificial protein stain was adhered was immersed in an enzyme washing solution for 6 hours, then the lens was washed with water, stained again with erythrosine, and the absorbance at 545 nm was measured with a spectrophotometer. The absorbance at this time is B. The protein stain removal rate is calculated by the following equation. Protein stain removal rate (%) = (B / A) × 100 The protein stain removal rates were measured immediately after preparing the enzyme washing solution and after heating the washing solution at 60 ° C. for 10 hours, respectively, by the above-described experimental method. Table 4 shows the obtained experimental results.
【００４０】[0040]
【表４】 [Table 4]
【００４１】表４の実験結果から、本発明の蛋白分解酵
素含有洗浄液は、６０℃で１０時間加熱した後において
も、優れた蛋白質汚れ除去率を有していることがわか
る。From the experimental results shown in Table 4, it can be seen that the protease-containing cleaning solution of the present invention has an excellent protein stain removal rate even after heating at 60 ° C. for 10 hours.
【００４２】[0042]
【発明の効果】本発明の蛋白分解酵素含有洗浄液は、蛋
白分解酵素の安定性が高く、長期間に渡って酵素活性が
維持され、一定期間保存した後に使用した場合であって
も、充分な洗浄効果を有するものである。The proteolytic enzyme-containing washing solution of the present invention has high stability of proteolytic enzymes, maintains the enzymatic activity for a long period of time, and has a sufficient effect even when used after being stored for a certain period of time. It has a cleaning effect.
───────────────────────────────────────────────────── フロントページの続き (72)発明者 長勢 康志 京都府京都市中京区麸屋町通夷川上る笹 屋町475番地 株式会社サンコンタクト レンズ内 (72)発明者 神山 健児 京都府京都市中京区麸屋町通夷川上る笹 屋町475番地 株式会社サンコンタクト レンズ内 (72)発明者 土屋 明人 滋賀県甲賀郡甲西町中央５−116−１ (56)参考文献 特開 平９−51793（ＪＰ，Ａ) 特開 平６−240297（ＪＰ，Ａ) 特開 平８−146363（ＪＰ，Ａ) 特開 平９−241696（ＪＰ，Ａ) 特開 平８−327956（ＪＰ，Ａ) 特開 平９−87682（ＪＰ，Ａ) 特開 平７−265075（ＪＰ，Ａ) 特開 平７−155182（ＪＰ，Ａ) 特開 平６−189759（ＪＰ，Ａ) 特開 平６−62846（ＪＰ，Ａ) 特表 平９−507929（ＪＰ，Ａ) 特表2000−500513（ＪＰ，Ａ) 国際公開97／18288（ＷＯ，Ａ１) (58)調査した分野(Int.Cl.7，ＤＢ名) C11D 17/08 C11D 3/04 C11D 3/37 C11D 3/386 G02C 13/00 特許ファイル（ＰＡＴＯＬＩＳ) ＪＩＣＳＴファイル（ＪＯＩＳ)──────────────────────────────────────────────────続 き Continuing on the front page (72) Inventor Yasushi Nagase 475 Sasaya-machi, Furuya-cho, Nakagyo-ku, Kyoto-shi, Kyoto Sun Contact Lens Co., Ltd. (72) Inventor Kenji Kamiyama Nakagyo-ku, Kyoto-shi, Kyoto 475 Sasaya-machi, Fuya-cho, Toyogawa-kami Sun Contact Lens Co., Ltd. (72) Inventor Akito Tsuchiya 5-116-1, Kosai-cho, Koga-gun, Kiga-gun, Shiga Prefecture (56) References JP-A-9-51793 (JP) JP-A-6-240297 (JP, A) JP-A-8-146363 (JP, A) JP-A-9-241696 (JP, A) JP-A-8-327956 (JP, A) 9-87682 (JP, A) JP-A-7-265075 (JP, A) JP-A-7-155182 (JP, A) JP-A-6-189759 (JP, A) JP-A-6-62846 (JP, A) A) Special table Hei 9-507929 (JP, ) JP-T 2000-500513 (JP, A) WO 97/18288 (WO, A1) (58 ) investigated the field (Int.Cl. 7, DB name) C11D 17/08 C11D 3/04 C11D 3/37 C11D 3/386 G02C 13/00 Patent file (PATOLIS) JICST file (JOIS)
Claims (2)
ル、多糖類、蛋白質及びＣＨ2 ＝Ｃ＜基を有する化合物
と無水マレイン酸との共重合体から成る群より選ばれた
水溶性高分子によって化学修飾されている修飾酵素と、 ポリグリセリン脂肪酸エステル及びポリオキシエチレン
アルキルフェニルエーテルから選ばれた少なくとも１種
の非イオン界面活性剤と、 陰イオン界面活性剤であるミリストイルサルコシンナト
リウム、ラウリル硫酸アンモニウム及び、両性界面活性
剤であるアルキルジメチルアミンオキサイド、アルキル
ジメチルアミノ酢酸ベタインから成る群より選ばれた少
なくとも１種の界面活性剤と、 ホウ酸、メタホウ酸、四ホウ酸、ホウ砂、メタホウ酸塩
及び四ホウ酸塩から成る群より選ばれたホウ酸化合物と
を含有することを特徴とする蛋白分解酵素含有洗浄液。1. A method in which a protease is chemically modified with a water-soluble polymer selected from the group consisting of polyethylene glycol, polysaccharides, proteins and a copolymer of maleic anhydride and a compound having a CH 2 ＣC <group. Modifying enzyme, at least one nonionic surfactant selected from polyglycerin fatty acid ester and polyoxyethylene alkylphenyl ether, and anionic surfactants such as sodium myristoylsarcosine, ammonium lauryl sulfate, and amphoteric surfactant At least one surfactant selected from the group consisting of alkyldimethylamine oxide and betaine alkyldimethylaminoacetate, boric acid, metaboric acid, tetraboric acid, borax, metaborate and tetraborate And a boric acid compound selected from the group consisting of A cleaning solution containing a proteolytic enzyme.
活性値が10,000〜200,000 U/mlである修飾酵素溶液とし
て０．１〜１０重量％の割合で含有し、しかも当該修飾
酵素溶液：前記各界面活性剤：ホウ酸化合物の重量比率
が１：０．０５〜１０：０．５〜１０であることを特徴
とする請求項１記載の蛋白分解酵素含有洗浄液。2. The modified enzyme solution containing 0.1 to 10% by weight of the modified enzyme as a modified enzyme solution having an enzyme activity value per unit volume of 10,000 to 200,000 U / ml. The protease-containing cleaning solution according to claim 1, wherein the weight ratio of each surfactant: boric acid compound is 1: 0.05 to 10: 0.5 to 10.
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|---|---|---|---|
| JP20276898A JP3359868B2 (en) | 1998-07-01 | 1998-07-01 | Wash solution containing protease |
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|---|---|
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| JP (1) | JP3359868B2 (en) |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP4222771B2 (en) * | 2002-04-03 | 2009-02-12 | 株式会社サンコンタクトレンズ | Modified carrier for stabilizing proteolytic enzyme, proteolytic enzyme chemically modified by the modified carrier, and method for producing the proteolytic enzyme |
| NO20073834L (en) | 2006-07-21 | 2008-01-22 | Akzo Nobel Chemicals Int Bv | Sulfonated graft copolymers |
| CN102574961B (en) | 2009-07-31 | 2015-09-23 | 阿克佐诺贝尔股份有限公司 | Hybrid copolymer composition |
| JP5586321B2 (en) * | 2010-05-25 | 2014-09-10 | 株式会社メニコン | Cleaning / preserving solution for oxygen permeable hard contact lenses |
| US8679366B2 (en) | 2011-08-05 | 2014-03-25 | Ecolab Usa Inc. | Cleaning composition containing a polysaccharide graft polymer composition and methods of controlling hard water scale |
| US8841246B2 (en) | 2011-08-05 | 2014-09-23 | Ecolab Usa Inc. | Cleaning composition containing a polysaccharide hybrid polymer composition and methods of improving drainage |
| US8853144B2 (en) | 2011-08-05 | 2014-10-07 | Ecolab Usa Inc. | Cleaning composition containing a polysaccharide graft polymer composition and methods of improving drainage |
| US8636918B2 (en) | 2011-08-05 | 2014-01-28 | Ecolab Usa Inc. | Cleaning composition containing a polysaccharide hybrid polymer composition and methods of controlling hard water scale |
| BR112014008874A2 (en) | 2011-11-04 | 2017-04-25 | Akzo Nobel Chemicals Int Bv | dendrite hybrid copolymer composition |
| WO2013064648A1 (en) | 2011-11-04 | 2013-05-10 | Akzo Nobel Chemicals International B.V. | Graft dendrite copolymers, and methods for producing the same |
| US8945314B2 (en) | 2012-07-30 | 2015-02-03 | Ecolab Usa Inc. | Biodegradable stability binding agent for a solid detergent |
| US9365805B2 (en) | 2014-05-15 | 2016-06-14 | Ecolab Usa Inc. | Bio-based pot and pan pre-soak |
| JP6712019B1 (en) * | 2019-12-18 | 2020-06-17 | 株式会社アルボース | Cleaning agent for medical equipment |
Family Cites Families (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH0662846A (en) * | 1992-08-12 | 1994-03-08 | Eiken Chem Co Ltd | Stabilization of enzyme for analytical reagent and enzyme for solution-state analytical reagent |
| JPH06189759A (en) * | 1992-12-25 | 1994-07-12 | Eiken Chem Co Ltd | Peroxidase composition |
| JPH06240297A (en) * | 1993-02-16 | 1994-08-30 | Toray Ind Inc | Washing assistant containing immobilized enzyme |
| JP3081749B2 (en) * | 1993-12-06 | 2000-08-28 | 鐘紡株式会社 | Method for producing modified protease |
| JPH07265075A (en) * | 1994-03-30 | 1995-10-17 | Kanebo Ltd | Stable modified protease composition and stable enzyme-containing aqueous solution containing the same composition |
| JP3586903B2 (en) * | 1994-11-25 | 2004-11-10 | 日本油脂株式会社 | Cleaning preservative for contact lenses |
| JP3552838B2 (en) * | 1995-03-28 | 2004-08-11 | セイコーエプソン株式会社 | Contact lens solution and cleaning and disinfecting method using the same |
| US5723421A (en) * | 1995-06-07 | 1998-03-03 | Alcon Laboratories, Inc. | Stable liquid enzyme compositions and methods of use in contact lens cleaning and disinfecting systems |
| JPH0951793A (en) * | 1995-06-09 | 1997-02-25 | Nippon Oil & Fats Co Ltd | Enzyme monomer, polymer and stain-removing agent for contact lens |
| JP2745397B2 (en) * | 1995-09-20 | 1998-04-28 | 株式会社コスメサイエンス | Contact lens cleaning agent and cleaning and disinfecting method |
| US5718895A (en) * | 1995-11-16 | 1998-02-17 | Alcon Laboratories, Inc. | Enzymes with low isoelectric points for use in contact lens cleaning |
| JPH09241696A (en) * | 1996-03-06 | 1997-09-16 | Toray Ind Inc | Liquid composition and cleaning of contact lens using the same |
-
1998
- 1998-07-01 JP JP20276898A patent/JP3359868B2/en not_active Expired - Lifetime
Also Published As
| Publication number | Publication date |
|---|---|
| JP2000017299A (en) | 2000-01-18 |
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