JP5699261B2 - Keratinase and production method thereof - Google Patents
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Description
本発明は、ケラチナーゼおよびその製造法に関する。詳細には、本発明は、芋、豆、穀物類にAspergillus属の微生物を接種して固体培養を行い、得られた培養物を比較的高温の水に浸漬し、抽出を行うことを特徴とするケラチナーゼの製造方法、および該方法により得られるケラチナーゼに関する。 The present invention relates to keratinase and a method for producing the same. Specifically, the present invention is characterized by inoculating a microorganism of the genus Aspergillus on straw, beans, and grains, performing solid culture, and immersing the obtained culture in relatively high temperature water to perform extraction. The present invention relates to a method for producing keratinase, and keratinase obtained by the method.
従来、ケラチナーゼを選抜・取得するための方法としては、ケラチナーゼ活性を有する菌株を養鶏場等の土壌からスクリーニングし、得られた菌体そのものを利用する、あるいはそれらの菌体が有するケラチナーゼをコードする遺伝子をクローニングし、組換え発現させるというものであった（非特許文献１および２）。しかしスクリーニングにより得られた菌株には病原性を有する場合もあり、安全性に問題がある。したがって、安全性の高い菌株を用いてケラチナーゼを製造すること、ならびに安全性の高い菌株由来のケラチナーゼに対する必要性が生じている。 Conventionally, as a method for selecting and obtaining keratinase, a strain having keratinase activity is screened from soil such as a poultry farm, and the obtained microbial cells themselves are used or the keratinases possessed by these microbial cells are encoded. The gene was cloned and recombinantly expressed (Non-patent Documents 1 and 2). However, the strains obtained by screening may have pathogenicity and have a safety problem. Therefore, there is a need to produce keratinase using a highly safe strain, as well as a keratinase derived from a highly safe strain.
さらに、新たな性質を有するケラチナーゼを探索する必要性もある。例えば、近年よく用いられている洗剤は弱酸性のものが多く、かかる条件下でも十分に作用するケラチナーゼを洗剤に配合する必要性がある。 There is also a need to search for keratinases with new properties. For example, many detergents frequently used in recent years are weakly acidic, and it is necessary to add keratinase that works well under such conditions to the detergent.
本発明は、安全な微生物菌株から、新たな性質を有するケラチナーゼを効率よく製造する方法を開発すること、ならびにそのような方法により得られるケラチナーゼを提供することを課題とした。 An object of the present invention is to develop a method for efficiently producing keratinase having new properties from a safe microbial strain, and to provide a keratinase obtained by such a method.
本発明者らは、上記現状および課題に鑑みて鋭意研究を重ね、芋、豆、穀物類にAspergillus属の微生物を接種して固体培養を行い、得られた培養物を比較的高温の水に浸漬し、抽出を行うことにより得られる抽出液がケラチナーゼを含有すること、得られたケラチナーゼが酸性領域において高い活性を有する新規なタイプのものであること等を見出し、本発明を完成させるに至った。 The present inventors have conducted extensive research in view of the above-mentioned present situation and problems, inoculated microorganisms belonging to the genus Aspergillus to straw, beans and grains, and conducted solid culture, and the obtained culture was made into relatively high-temperature water. It has been found that the extract obtained by soaking and extracting contains keratinase, and that the obtained keratinase is a novel type having high activity in the acidic region, leading to the completion of the present invention. It was.
すなわち、本発明は以下のものを提供する：
（１）芋、豆、穀物類にAspergillus属の微生物を接種して固体培養を行い、得られた培養物を２５℃〜６０℃の水に浸漬し、ケラチナーゼを抽出することを特徴とするケラチナーゼの製造方法。
（２）穀物類が米または麦である（１）記載の方法。
（３）Aspergillus属が、酒造、焼酎、味噌、醤油製造用のものである（１）または（２）記載の方法。
（４）培養物を５０〜６０℃の水に浸漬する（１）〜（３）のいずれかに記載の方法。
（５）（１）〜（４）のいずれかに記載の方法により得られるケラチナーゼ。
（６）至適ｐＨがｐＨ約３．５〜ｐＨ約５である（５）記載のケラチナーゼ。
（７）下記の特徴：
ケラチンに作用し、エラスチン、コラーゲンに作用しない
至適ｐＨがｐＨ約３．５〜ｐＨ約５である
ｐＨ４〜ｐＨ６で２５℃、１６時間置いた後に失活が認められない
約５５℃までの温度にてｐＨ４．５で１時間置いた後に失活が認められない
を有するAspergillus属由来のケラチナーゼ。
（８）（５）、（６）または（７）記載のケラチナーゼを含有する洗剤、化粧品または入浴剤。
That is, the present invention provides the following:
(1) Keratinase characterized by inoculating a microorganism of the genus Aspergillus on straw, beans, and grains, performing solid culture, immersing the obtained culture in water at 25 ° C to 60 ° C, and extracting keratinase Manufacturing method.
(2) The method according to (1), wherein the cereal is rice or wheat.
(3) The method according to (1) or (2), wherein the genus Aspergillus is for producing sake, shochu, miso, or soy sauce.
(4) The method according to any one of (1) to (3), wherein the culture is immersed in water at 50 to 60 ° C.
(5) Keratinase obtained by the method according to any one of (1) to (4).
(6) The keratinase according to (5), wherein the optimum pH is about 3.5 to about
(7) The following features:
Acts on keratin and does not act on elastin or collagen. Optimal pH is about pH 3.5 to
(8) A detergent, cosmetic or bath containing the keratinase according to (5), (6) or (7).
本発明によれば、効率的なケラチンの製造方法、ならびに酸性領域および高温において高い活性を有する新規ケラチナーゼが提供される。しかも本発明において使用する菌株は麹菌などの食品醸造用の菌株であるため安全性が高く、得られるケラチナーゼの毒性に関しても心配はない。また本発明により得られるケラチナーゼは、廃棄物中の毛髪などのケラチン含有タンパク質の分解や皮膚、爪及び毛髪のケア用化粧品・入浴剤などに適用可能である。 ADVANTAGE OF THE INVENTION According to this invention, the manufacturing method of an efficient keratin and the novel keratinase which has high activity in an acidic region and high temperature are provided. Moreover, since the strain used in the present invention is a food brewing strain such as koji mold, it is highly safe and there is no concern about the toxicity of the keratinase obtained. The keratinase obtained by the present invention can be applied to decomposition of keratin-containing proteins such as hair in wastes, cosmetics for taking care of skin, nails and hair, bathing agents and the like.
本発明は、１の態様において、芋、豆、穀物類にAspergillus属の微生物を接種して固体培養を行い、得られた培養物を約２５℃〜約６０℃の水に浸漬し、ケラチナーゼの抽出を行うことを特徴とするケラチナーゼの製造方法に関するものである。 In one embodiment, the present invention, in one embodiment, inoculates persimmons, beans, and cereals with microorganisms of the genus Aspergillus, performs solid culture, immerses the obtained culture in water at about 25 ° C. to about 60 ° C., and The present invention relates to a method for producing keratinase characterized by performing extraction.
本発明のケラチナーゼの製造方法の第１の工程は、芋、豆、穀物類にAspergillus属の微生物を接種して固体培養を行う工程である。Aspergillus属の微生物を接種する芋、豆、穀物類はAspergillus属の微生物が増殖しケラチナーゼを産生しうるものであればいずれの種類であってもよく、限定されない。芋の例としてはバレイショ、サツマイモ、キャッサバなどが挙げられる。豆の例としてはダイズ、アズキ、ナタマメなどが挙げられる。穀物類の例としては米、麦、アワ、キビ、ヒエ、コウリャンなどが例示される。Aspergillus属の微生物を接種して固体培養を行うのに好ましい穀物類は米および麦であり、なかでも米が好ましい。芋、豆、穀物類へのAspergillus属の微生物の接種方法はいずれの方法であってもよいが、好ましくは胞子を形成したAspergillus属の微生物の菌体またはAspergillus属の微生物の胞子を適量接種する。接種後、よく混合することが好ましい。典型的には種麹を芋、豆、穀物類に適量接種する。芋、豆、穀物類は加熱処理をしていなくてもよく、蒸煮などの加熱処理をしたものであってもよい。接種量は、使用する芋、豆、穀物類の種類、量および加熱処理の有無、接種されるAspergillus属の微生物の種類、固体培養条件などに応じて当業者が適宜決定することができる。 The first step of the method for producing keratinase of the present invention is a step of inoculating a microorganism of the genus Aspergillus on straw, beans and cereals and performing solid culture. There are no limitations on the candy, beans, and cereals inoculated with microorganisms of the genus Aspergillus, as long as the microorganisms of the genus Aspergillus can grow and produce keratinase. Examples of salmon include potato, sweet potato and cassava. Examples of beans include soybeans, azuki bean and jujube. Examples of cereals include rice, wheat, millet, millet, barnyard millet and cucumber. The preferred cereals for inoculating microorganisms belonging to the genus Aspergillus and performing solid culture are rice and wheat, with rice being particularly preferred. Any method may be used for inoculating strawberry, beans, and cereals with Aspergillus microorganisms, but preferably an appropriate amount of Aspergillus microorganisms or spores of Aspergillus microorganisms that have formed spores are inoculated. . It is preferable to mix well after inoculation. Typically, seed potatoes are inoculated in appropriate amounts in straw, beans, and grains. Rice cakes, beans, and grains need not be heat-treated, and may be heat-treated such as steaming. The inoculation amount can be appropriately determined by those skilled in the art according to the type and amount of straw, beans, and grains used, the presence or absence of heat treatment, the type of microorganism of the genus Aspergillus to be inoculated, solid culture conditions, and the like.
固体培養は、Aspergillus属の微生物の発芽および増殖、ならびにケラチナーゼ産生に適した条件下で行うことが好ましい。温度、培養時間、水分量、通気量などの固体培養の諸条件もまた、使用する芋、豆、穀物類の種類、量および加熱処理の有無、接種されるAspergillus属の微生物の種類などに応じて当業者が適宜決定することができる。固体培養中は培養物の温度を適温に保ち、通気を図って菌の増殖および酵素の産生を促進するために、いわゆる「切返し」「盛り」「手入れ」等の作業を適宜行うことが好ましい。菌株や培養条件により異なるが、十分な菌糸の生育が見られた時点ないし十分な胞子の着生が見られた時点で、次の浸漬工程に移す。 The solid culture is preferably performed under conditions suitable for germination and growth of microorganisms of the genus Aspergillus and keratinase production. The conditions of solid culture, such as temperature, incubation time, moisture content, aeration rate, etc., also depend on the type, amount, and presence of heat treatment, the type of Aspergillus microorganisms to be inoculated, etc. Thus, those skilled in the art can appropriately determine. During solid culture, it is preferable to appropriately perform operations such as so-called “turn-over”, “pick-up”, and “care” in order to keep the temperature of the culture at an appropriate temperature and promote aeration to promote the growth of bacteria and the production of enzymes. Although depending on the strain and culture conditions, when sufficient hypha growth is observed or when sufficient spore growth is observed, the next dipping step is performed.
上で説明した固体培養は、公知の製麹方法に従って行うこともできる。麹は、糸状菌を米、麦等の穀物類、大豆等の豆類、あるいは芋類などの食品素材に接種して発育させたものをいう。本発明において用いる芋、豆、穀物類は１種類であってもよく、２種類以上を混合して用いてもよい。本発明に使用されるAspergillus属の微生物の菌株は、ケラチナーゼを産生しうる菌株であればいずいれの菌株であってもよいが、Aspergillus属の微生物が好ましい。なかでも酒造、焼酎、味噌、醤油製造用の麹菌が安全性の点から特に好ましい。好ましい麹菌の具体例としては、Aspergillus awamori、Aspergillus saitoi、Aspergillus saitoi var. kagoshimaensis、Aspergillus usami、Aspergillus sojae、Aspergillus oryzae、Aspergillusvar. kawachiiなどが挙げられる。本発明に用いるAspergillus属の菌株は１種類であってもよく、２種類以上であってもよい。また、本発明に使用するAspergillus属の菌株として、種麹を用いてもよい。種麹は市販されており、適宜選択して使用することができる。本発明に使用できる好ましい種麹としては、株式会社菱六から市販されている「菱六もやし 糖化マイティ」、「菱六もやし 改良長白菌」、「菱六もやし SR-108」、「菱六もやし 長白菌」、「菱六もやし A-27」、「菱六もやし 白夜」、「菱六もやし 月下氷吟」、「菱六もやし 焼酎用白麹菌No.198」、「菱六もやし 焼酎用くろ」、「菱六もやし 醤油用旭菌」、「菱六もやし 特撰自動製麹用」または「菱六もやし 特殊吟醸用」などが例示される。 The solid culture described above can also be performed according to a known iron making method. Koji refers to foods such as rice, wheat and other grains, beans such as soybeans, or food materials such as koji that are grown. One kind of straw, beans and grains used in the present invention may be used, or two or more kinds may be mixed and used. The strain of the microorganism belonging to the genus Aspergillus used in the present invention may be any strain as long as it can produce keratinase, but a microorganism belonging to the genus Aspergillus is preferred. Of these, sake brewing, shochu, miso, and koji mold for producing soy sauce are particularly preferable from the viewpoint of safety. Specific examples of preferable koji molds include Aspergillus awamori, Aspergillus saitoi, Aspergillus saitoi var. Kagoshimaensis, Aspergillus usami, Aspergillus sojae, Aspergillus oryzae, Aspergillus var. Kawachii and the like. The strain of the genus Aspergillus used in the present invention may be one type or two or more types. Moreover, you may use a seed cocoon as a Aspergillus genus strain used for this invention. Seeds are commercially available and can be selected and used as appropriate. Preferred seeds that can be used in the present invention include “Rishoku Sprout Saccharification Mighty”, “Ryokuroku Sprout Improved Chogaku”, “Ryokuroku Sprout SR-108”, “Ryokuroku Sprout” commercially available from Ryokuroku Co. "Nagahaku fungus", "Ryokuroku bean sprouts A-27", "Ryokuroku bean sprouts", "Ryokuroku bean sprout Tsukishita Hyogin", "Ryokuroku bean sprout white fungus No. 198", "Ryokuroku bean sprouts" ”,“ Rokuroku Moyashi Asahi fungus for soy sauce ”,“ Ryoroku Moyashi Tokushu automatic brewing ”or“ Ryokuroku Moyashi special brewing sake ”.
本発明のケラチナーゼの製造方法の第２の工程は、上記工程で得られた培養物を約２５℃〜約６０℃の水に浸漬し、ケラチナーゼを抽出する工程である。浸漬は培養物と水が十分に混ざるようにすればよい。浸漬の態様は特に限定はないが、培養物を水に浸してもよく、懸濁してもよい。水の量は、浸漬中にケラチナーゼの抽出が十分に行われるような量とする。浸漬中に適宜撹拌してケラチナーゼの抽出を促進してもよい。浸漬中に菌が増殖してもよく、増殖しなくてもよい。浸漬に用いる水の温度は約２５℃〜約６０℃、好ましくはより高温の約４５℃〜約６０℃、さらに好ましくは約５０℃〜約６０℃であり、例えば、約５０℃〜約５５℃、約５３℃〜約５７℃、あるいは約５５℃などの水に浸漬することができる。浸漬温度が低すぎるとケラチナーゼ以外の酵素の失活が抑制され、得られるケラチナーゼの純度が低下する。さらに酵素の抽出効率も低下する。浸漬温度が高すぎるとケラチナーゼの失活も進み、ケラチナーゼの収量が低下する。浸漬時間は、ケラチナーゼが十分に抽出される時間であればよい。通常は約２４時間〜約９６時間、好ましくは約３６時間〜約９６時間、例えば約３６時間〜約８４時間、約４０時間〜約４８時間、約４８時間〜約７２時間などである。浸漬時間が短すぎるとケラチナーゼの抽出が不十分となり、その収量が低下する。浸漬時間が長すぎるとケラチナーゼの失活が進み、さらには雑菌が繁殖してケラチナーゼが分解され、雑菌酵素の混入によりケラチナーゼの純度が低下してしまう。浸漬の水温および浸漬時間は上記範囲でなくてもよく、当業者が諸条件に応じて適宜決定することができる。 The second step of the method for producing keratinase of the present invention is a step of extracting keratinase by immersing the culture obtained in the above step in water at about 25 ° C. to about 60 ° C. The immersion may be performed so that the culture and water are sufficiently mixed. Although the aspect of immersion is not particularly limited, the culture may be immersed in water or suspended. The amount of water is such that keratinase is sufficiently extracted during immersion. The extraction of keratinase may be promoted by stirring as appropriate during the immersion. Bacteria may or may not grow during the immersion. The temperature of the water used for immersion is about 25 ° C. to about 60 ° C., preferably about 45 ° C. to about 60 ° C., more preferably about 50 ° C. to about 60 ° C., for example, about 50 ° C. to about 55 ° C. , About 53 ° C to about 57 ° C, or about 55 ° C. If the soaking temperature is too low, inactivation of enzymes other than keratinase is suppressed, and the purity of the resulting keratinase decreases. Furthermore, the extraction efficiency of the enzyme also decreases. If the soaking temperature is too high, the deactivation of keratinase proceeds and the yield of keratinase decreases. The immersion time may be a time for sufficiently extracting keratinase. It is usually about 24 hours to about 96 hours, preferably about 36 hours to about 96 hours, such as about 36 hours to about 84 hours, about 40 hours to about 48 hours, about 48 hours to about 72 hours, and the like. If the soaking time is too short, the extraction of keratinase becomes insufficient and the yield decreases. If the soaking time is too long, the deactivation of keratinase proceeds, and further, germs propagate and keratinase is decomposed, and the purity of keratinase decreases due to contamination with the germicidal enzyme. The immersion water temperature and the immersion time do not have to be in the above ranges, and those skilled in the art can appropriately determine them according to various conditions.
浸漬・抽出が終了したら抽出液をケラチナーゼ酵素液として得る。抽出液を固液分離して上清をケラチナーゼ酵素液としてもよい。したがって、本発明の製造方法における抽出は、上清を得る操作も包含しうる。上清は、公知の固液分離方法、例えば、遠心分離、ろ過、布で絞る等の方法を用いて得ることができる。得られたケラチナーゼ酵素液をそのままケラチナーゼとして使用してもよいが、酵素液を硫安等で濃縮してもよく、凍結乾燥等の処理を行ってから用いてもよい。さらに、酵素液を公知の手段、例えばイオン交換クロマトグラフィー、ゲルろ過クロマトグラフィー、アフィニィークロマトグラフィー、疎水クロマトグラフィー等の各種クロマトグラフィー、ＨＰＬＣ、電気泳動などの手段にて精製して、ケラチナーゼの純度を高めてから用いてもよい。 When immersion and extraction are completed, an extract is obtained as a keratinase enzyme solution. The extract may be subjected to solid-liquid separation, and the supernatant may be used as a keratinase enzyme solution. Therefore, extraction in the production method of the present invention can also include an operation for obtaining a supernatant. The supernatant can be obtained using a known solid-liquid separation method such as centrifugation, filtration, or squeezing with a cloth. The obtained keratinase enzyme solution may be used as it is as keratinase, but the enzyme solution may be concentrated with ammonium sulfate or the like, or may be used after treatment such as freeze-drying. Furthermore, the purity of the keratinase is purified by purifying the enzyme solution by a known means such as ion exchange chromatography, gel filtration chromatography, affinity chromatography, various chromatography such as hydrophobic chromatography, HPLC, electrophoresis, etc. You may use it after raising.
本発明のケラチナーの製造方法において、例えば５０℃以上の生体においては高温とされる温度で、例えば４８時間以上の比較的長時間の浸漬・抽出を行うことが特徴である。これらの条件下で安定して存在する酵素は少ないので、比較的簡単な操作、かつ低コストで、かつ遺伝子組換え技術等を用いることなく純度の高いケラチナーゼを得ることができる。また、かかる比較的高温での工程はバイオリアクターにおける酵素生産において冷却エネルギーを削減できる。さらに、本発明の製造方法は、既存の清酒や味噌、醤油などに代表される発酵食品を製造する糖化の工程を流用あるいは部分的に改変するだけで実施しうるという利点もある。 The keratinar production method of the present invention is characterized in that immersion and extraction are performed for a relatively long time, for example, 48 hours or more, for example, at a high temperature in a living body of 50 ° C. or higher. Since few enzymes exist stably under these conditions, a highly pure keratinase can be obtained at a relatively simple operation, at low cost, and without using a gene recombination technique. Further, such a relatively high temperature process can reduce cooling energy in enzyme production in a bioreactor. Furthermore, the production method of the present invention has an advantage that it can be carried out by diverting or partially modifying the saccharification process for producing a fermented food represented by existing sake, miso and soy sauce.
そのうえ、酒造、焼酎、味噌、醤油製造用の麹菌を用いてケラチナーゼを製造する場合には、得られるケラチナーゼ製品の安全性が保証されているので、ヒトやペットが日常使用する洗剤、爪または毛髪のケア用化粧品、入浴剤などへの適用において有利である。 In addition, when keratinase is produced using koji molds for sake brewing, shochu, miso, and soy sauce production, the safety of the resulting keratinase product is guaranteed. This is advantageous in application to cosmetics for care and bathing agents.
本発明により得られるAspergillus属由来のケラチナーゼは、酸性・高温条件下、例えばｐＨ約３．５〜ｐＨ約５、約５５℃において高いケラチン分解活性を有し、安定である等の従来のケラチナーゼには見られない特徴を有する。すなわち、本発明により得られるケラチナーゼは、
ケラチンに作用し、エラスチン、コラーゲンに作用しない
ケラチン分解活性の至適ｐＨがｐＨ約３．５〜ｐＨ約５である
ｐＨ４〜ｐＨ６で２５℃、１６時間置いた後にケラチン分解活性の失活が認められない
約５５℃までの温度にてｐＨ４．５で６０分間置いた後にケラチン分解活性の失活が認められない
という特徴を有する。
The keratinase derived from the genus Aspergillus obtained by the present invention has high keratin degradation activity under acidic and high temperature conditions, for example, pH of about 3.5 to about 5 and about 55 ° C., and is stable. Has features not seen. That is, the keratinase obtained by the present invention is
Acts on keratin and does not act on elastin or collagen. The optimum pH for keratin degradation activity is about pH 3.5 to about
上述のごとく、従来のケラチナーゼは中性からアルカリ性領域で活性を有するものが多いが、本発明のケラチナーゼは酸性領域において安定で高い活性を有する。最近の洗剤や化粧品は弱酸性のものが多く、本発明のケラチナーゼはこれらの洗剤や化粧品に好適に用いられる。本発明のケラチナーゼは、廃棄物中の毛髪などのケラチン含有タンパク質の分解、例えば、廃棄物処理剤や水質改善剤、下水処理剤の有効成分として用いられるほか、皮膚、爪および毛髪のケア用化粧品、入浴剤、洗剤などに添加して用いることができる。 As described above, many conventional keratinases have an activity in the neutral to alkaline region, but the keratinase of the present invention has a stable and high activity in the acidic region. Recent detergents and cosmetics are often weakly acidic, and the keratinase of the present invention is suitably used for these detergents and cosmetics. The keratinase of the present invention is used as an active ingredient for decomposition of keratin-containing proteins such as hair in wastes, for example, waste treatment agents, water quality improvers, and sewage treatment agents, as well as skin, nail and hair care cosmetics. It can be used by adding to bathing agents, detergents and the like.
以下に実施例を示して本発明をさらに詳細かつ具体的に説明するが、実施例はあくまでも例示説明であり、本発明を限定するものではない。 The present invention will be described in more detail and specifically below with reference to examples, but the examples are merely illustrative and do not limit the present invention.
実施例１：ケラチナーゼの製造
洗米、浸漬、蒸煮が終了した原料米を４０℃程度に冷却し、種麹「菱六もやし 糖化マイティ」を所定重量比（０．３％ｗ／ｗ）だけ接種し、両者が均一となるまで混合した。その後、混合物を製麹装置内に投入して、初期温度を３２〜３４℃程度に加温した状態で固体培養（製麹）を行った。製麹装置内の培養物の温度は、製麹が進行するに従って時間的に変化していくので、「切返し」「盛り」「手入れ」等の作業により、３６〜４０℃程度を保持し、種麹接種後４０〜４８時間で製麹を終了した。
Example 1: Production of keratinase The raw rice after washing, soaking and cooking is cooled to about 40 ° C. and inoculated with a seed weight “Ryokuro Moyashi Saccharification Mighty” in a predetermined weight ratio (0.3% w / w). The two were mixed until uniform. Thereafter, the mixture was put into a koji making apparatus, and solid culture (koji making) was performed with the initial temperature heated to about 32 to 34 ° C. Since the temperature of the culture in the iron making apparatus changes with time as the iron making progresses, the temperature of 36 to 40 ° C. is maintained by operations such as “turning over”, “pickup”, “care”, and so on. The iron making was completed 40 to 48 hours after vaginal inoculation.
得られた固体培養物（麹）と水を２：３（ｗ／ｗ）の比率で混合し、５５℃にて７２時間浸漬し、酵素を抽出した。得られた抽出物を０．４５μｍのフィルターにてろ過し、ろ液に７０％の飽和硫酸アンモニウムを加えて十分に沈殿を生じさせた後、１０，０００ｇにて１時間遠心し、上清画分を回収した。この上清画分を２Ｍ 硫酸アンモニウムにて平衡化した疎水カラム（Phenyl sepharose fast flow）にかけタンパク質を吸着させ、２Ｍ 硫酸アンモニウムで非結合のタンパク質を洗い流した後、１．３Ｍ硫酸アンモニウムで溶出させ回収した。この回収画分について透析を行い酵素溶液とした。 The obtained solid culture (strawberry) and water were mixed at a ratio of 2: 3 (w / w) and immersed at 55 ° C. for 72 hours to extract the enzyme. The obtained extract was filtered through a 0.45 μm filter, 70% saturated ammonium sulfate was added to the filtrate to cause sufficient precipitation, and then centrifuged at 10,000 g for 1 hour to obtain a supernatant fraction. Was recovered. The supernatant fraction was applied to a hydrophobic column (Phenyl sepharose fast flow) equilibrated with 2M ammonium sulfate, the protein was adsorbed, unbound protein was washed away with 2M ammonium sulfate, and then eluted and collected with 1.3M ammonium sulfate. The collected fraction was dialyzed to obtain an enzyme solution.
実施例２：ケラチナーゼの性質
実施例で得られた酵素溶液にケラチナーゼ活性を以下のようにして測定した。ケラチン分解の活性測定に用いた基質を下記のごとく調製した。トリ羽毛２．０ｇに対してジメチルスルホキシド１００ｍｌを加え１００℃にて２時間処理し、ろ過を行いケラチンを含むタンパク溶出液を回収した。回収したタンパク溶液に２倍量のアセトンを加え−２０℃で１６時間静置しタンパク質を沈殿させた。１６時間後、沈殿溶液を６，０００ｇで遠心し、沈殿を蒸留水で洗い、再度蒸留水を加えたものを基質とした。
Example 2: Properties of keratinase Keratinase activity was measured in the enzyme solutions obtained in the examples as follows. The substrate used for measuring the activity of keratin degradation was prepared as follows. To 2.0 g of chicken feathers, 100 ml of dimethyl sulfoxide was added and treated at 100 ° C. for 2 hours, followed by filtration to recover a protein eluate containing keratin. Two times the amount of acetone was added to the recovered protein solution and allowed to stand at −20 ° C. for 16 hours to precipitate the protein. After 16 hours, the precipitation solution was centrifuged at 6,000 g, the precipitate was washed with distilled water, and distilled water was added again as a substrate.
ケラチン分解活性測定方法について説明する。０〜５０ｍＭ 酢酸‐ナトリウムバッファー（ｐＨ４．５）中に、８ｍｇ／ｍｌ濃度になるように基質を加え基質溶液とした。基質溶液に１／１０ ｖ／ｖの酵素溶液を加え５５℃にて一時間反応させた。氷冷することで反応を停止させた。酵素を加えずに基質溶液のみのものも５５℃一時間反応させ、氷冷したのちに酵素を１／１０ ｖ／ｖ加えゼロタイム（反応開始時）のサンプルとした。反応停止させた液を１２，０００ｇ、４℃で１０分間遠心し、上清を回収した。回収した上清に２倍量のニンヒドリン溶液を加え１００℃で５分発色反応を行い、５７０ｎｍの吸光度を測定し、１時間反応液とゼロタイム反応液の吸光度を比較することで活性の有無を判定した。図１に１０倍に希釈した培養液を用いて酵素活性の時間経過を追ったデータを示す。グラフから明らかなように時間経過とともに基質と酵素溶液を含む反応液においてはアミノ酸量が増加していることが認められている一方で、酵素溶液のみの反応液ではアミノ酸量が増加していないことから酵素の自己消化は認められず、アミノ酸の増加が基質であるケラチンが分解されたためであることが確認された。比活性は５７０ｎｍ／２８０ｎｍで計算すると、培養抽出液の段階で０．００４６、疎水カラム溶出後のサンプルで１．２４であった。 A method for measuring keratin degradation activity will be described. Substrate was added to a concentration of 8 mg / ml in 0-50 mM acetate-sodium buffer (pH 4.5) to give a substrate solution. 1/10 v / v enzyme solution was added to the substrate solution and reacted at 55 ° C. for 1 hour. The reaction was stopped by cooling with ice. The substrate solution alone without the enzyme was reacted at 55 ° C. for 1 hour, and after ice cooling, the enzyme was added to 1/10 v / v to obtain a zero time sample (at the start of the reaction). The solution whose reaction was stopped was centrifuged at 12,000 g and 4 ° C. for 10 minutes, and the supernatant was collected. Add twice the amount of ninhydrin solution to the collected supernatant, perform color reaction at 100 ° C. for 5 minutes, measure the absorbance at 570 nm, and compare the absorbance of the reaction solution for 1 hour with that of the zero-time reaction solution. Judged. FIG. 1 shows data following the time course of enzyme activity using a culture solution diluted 10-fold. As is apparent from the graph, the amount of amino acids in the reaction solution containing the substrate and the enzyme solution was found to increase over time, while the amount of amino acids in the reaction solution containing only the enzyme solution was not increased. Thus, autolysis of the enzyme was not observed, and it was confirmed that the increase in amino acid was due to degradation of keratin as a substrate. The specific activity calculated at 570 nm / 280 nm was 0.0046 at the stage of the culture extract, and 1.24 for the sample after elution from the hydrophobic column.
次に、疎水カラム溶出後のサンプルについて、ケラチナーゼの諸性質を調べた。活性測定は上記ごとくケラチンを基質として行った。
（ａ）至適ｐＨ
それぞれのｐＨで基質と酵素を５５℃で１時間反応させ活性測定を行った。それぞれのバッファーは、５０ｍＭ ギ酸−ナトリウムバッファー（ｐＨ２．５〜３．５）、５０ｍＭ 酢酸−ナトリウムバッファー（ｐＨ３．５〜６）、５０ｍＭ リン酸−ナトリウムバッファー（ｐＨ６〜８）を用いた。結果を図２に示す。本発明のケラチナーゼは酸性側で活性が高く、ｐＨ３．５〜ｐＨ５が至適ｐＨと考えられた。本発明のケラチナーゼはｐＨ４．５において最も活性が高かった。
Next, various properties of keratinase were examined for the sample after elution with a hydrophobic column. As described above, the activity was measured using keratin as a substrate.
(A) Optimum pH
Activity was measured by reacting the substrate and enzyme at 55 ° C. for 1 hour at each pH. As each buffer, 50 mM formic acid-sodium buffer (pH 2.5 to 3.5), 50 mM acetic acid-sodium buffer (pH 3.5 to 6), and 50 mM phosphoric acid-sodium buffer (
（ｂ）ｐＨ安定性
酵素溶液をそれぞれのｐＨ下で１６時間インキュベートした後、５０ｍＭ 酢酸−ナトリウムバッファー（ｐＨ４．５）中で，基質と酵素を５５℃で１時間反応させて活性測定を行った。それぞれのバッファーは、１０ｍＭ ギ酸−ナトリウムバッファー（ｐＨ３）、１０ ｍＭ酢酸−ナトリウムバッファー（ｐＨ４〜６）、１０ｍＭ リン酸−ナトリウムバッファー（ｐＨ６〜８）、１０ｍＭ Ｔｒｉｓ−ＨＣｌバッファー（ｐＨ９）を用いた。結果を図３に示す。本発明のケラチナーゼは酸性側で安定で、ｐＨ４〜６で最も安定であり、これらのｐＨに２５℃、１６時間置いた後であっても失活は認められなかった。
(B) pH stability After the enzyme solution was incubated for 16 hours at each pH, the activity was measured by reacting the substrate with the enzyme at 55 ° C. for 1 hour in 50 mM acetic acid-sodium buffer (pH 4.5). . As each buffer, 10 mM formate-sodium buffer (pH 3), 10 mM acetate-sodium buffer (pH 4-6), 10 mM phosphate-sodium buffer (pH 6-8), 10 mM Tris-HCl buffer (pH 9) were used. The results are shown in FIG. The keratinase of the present invention is stable on the acidic side, most stable at
（ｃ）温度安定性
基質以外の反応液をそれぞれの温度にて事前に１時間インキュベートし（５０ｍＭ酢酸−ナトリウムバッファー中）、一度氷中で冷却したあと、５０ｍＭ 酢酸−ナトリウムバッファー（ｐＨ４．５）中で基質と酵素を５５℃で、１時間反応させた。結果を図４に示す。本発明のケラチナーゼは５５℃までの温度（１時間保持）では失活が認められなかった。
(C) Temperature stability The reaction solution other than the substrate was incubated for 1 hour in advance at each temperature (in 50 mM acetate-sodium buffer), once cooled in ice, and then 50 mM acetate-sodium buffer (pH 4.5). The substrate and the enzyme were reacted at 55 ° C. for 1 hour. The results are shown in FIG. The keratinase of the present invention was not inactivated at temperatures up to 55 ° C. (held for 1 hour).
（ｄ）基質特異性
５０ｍＭ酢酸−ナトリウムバッファー（ｐＨ４．５）中それぞれの基質濃度が８ｍｇ／ｍｌとなるように調製し、酵素溶液を加え５５℃にて１時間反応させて活性を測定した。結果を表１に示す。本発明のケラチナーゼはケラチンを最も強く分解した。ゼラチンおよびカゼインも分解されたが、ケラチンほどではなかった。コラーゲンおよびエラスチンは本発明のケラチナーゼによっては分解されなかった。
分解活性の強さ ＋＋＞＋
−は分解が認められないことを示す。
(D) Substrate specificity Each substrate concentration was adjusted to 8 mg / ml in 50 mM acetic acid-sodium buffer (pH 4.5), and the enzyme solution was added and reacted at 55 ° C. for 1 hour to measure the activity. The results are shown in Table 1. The keratinase of the present invention most strongly degraded keratin. Gelatin and casein were also degraded, but not as much as keratin. Collagen and elastin were not degraded by the keratinase of the present invention.
Degradation activity strength ++> +
-Indicates that no decomposition is observed.
（ｅ）阻害剤
５０ｍＭ酢酸−ナトリウムバッファー（ｐＨ４．５）中の阻害剤（表２参照）および基質に酵素溶液を加え、５５℃にて１時間反応させて活性を測定した。本発明のケラチナーゼはＳＤＳ、ＰＭＳＦおよびペプスタチンＡによって阻害されることがわかった。
本発明は、洗剤、爪または毛髪のケア用化粧品、入浴剤などの製造、ならびに下水処理や水質改善等に利用可能である。 INDUSTRIAL APPLICABILITY The present invention can be used for the manufacture of detergents, nail or hair care cosmetics, bath preparations, sewage treatment and water quality improvement.
Claims (7)
ケラチンに作用し、エラスチン、コラーゲンに作用しない
至適ｐＨがｐＨ３．５〜ｐＨ５である
ｐＨ４〜ｐＨ６で２５℃、１６時間置いた後に失活が認められない
５５℃までの温度にてｐＨ４．５で１時間置いた後に失活が認められない
を有するアスペルギルス属由来のケラチナーゼ。 The following features:
Acts on keratin and does not act on elastin or collagen. Optimal pH is pH 3.5 to pH 5. No deactivation after 25 hours at pH 4 to pH 6 at pH 5 to pH 55 at a temperature up to 55 ° C. Aspergillus- derived keratinase having no inactivation after 1 hour.
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