JP4114046B2 - Enzymatic hair removal treatment and enzymatic hair removal method - Google Patents

Description

【００３１】
【表２】

【００３２】
これらの表に示される結果から、本酵素は市販酵素である製品Aと比較して、ケラチン分解活性が2倍高く、コラーゲン分解活性およびエラスチン分解活性は同等もしくは低く、ケラチンに対する特異性の高いことがわかる。従って、本酵素は市販酵素に比して、コラーゲンおよびエラスチンへの作用が少なく、獣毛処理に際して真皮への影響が小さいことがわかる。
【００３３】
本発明酵素脱毛処理剤は、通常、上記特定酵素をpH調整剤と共に含む脱毛用処理液形態に調製される。ここで用いられるpH調整剤としては、得られる処理液のpHを皮革の脱毛処理に適した約9-11とするものであれば特に限定されない。一般には、水酸化カルシウムを用いるのが好ましい。処理液には、更にpH緩衝作用を持たせるためホウ酸などを加えることができる。なお、該処理液には、従来必須とされている脱毛促進剤としての硫化水素ナトリウム乃至チオグリコール酸などの還元剤を添加する必要はないが、これらの添加配合を禁止するものではない。
【００３４】
本発明酵素脱毛用処理液における酵素、pH調整剤などの配合量(濃度)は、得られる処理液を適用する原皮の種類、脱毛処理の方法、条件などに応じて適宜決定でき、特に限定されるものではない。通常酵素は、原皮重量1gに対して約30-180APU程度、好ましくは約60-150APU程度の範囲から選ばれる。pH調整剤としての水酸化カルシウムは、約0.2-2%、好ましくは約0.4-1%の範囲から選ぶことができる。所定pHの調整に用いられるホウ酸は、約0.3-3%、好ましくは約0.6-1.5%の範囲から選ぶことができる。
【００３５】
本発明酵素脱毛用処理液には、更に必要に応じて、通常この種の脱毛用処理液に添加配合されることの知られている界面活性剤などを適宜添加配合することができる。該界面活性剤としては、例えば、薬品浸透促進効果のある界面活性剤や脱脂効果のある界面活性剤など、具体的には「スプラランUF」(Zschimmer & Schwarz社製)などを挙げることができる。また該界面活性剤には、防腐効果をも奏し得る界面活性、具体的には「シスモランBH」(Bayer社製)なども含まれる。これらの界面活性剤などの添加配合量は、通常それらが用いられる量と特に異ならない。一般にはそれぞれ0.1-1%程度とされるのが普通である。
【００３６】
上記の如くして調製される本発明酵素脱毛用処理液は、従来知られているドラムまたはパドルを用いた皮革鞣製の脱毛工程に利用して、これと原皮とを一般にはゆるやかに接触させることによって、本発明所期の脱毛処理を実施することができる。本発明はこのような本発明酵素脱毛処理剤を利用した獣毛の酵素脱毛処理方法を提供する。
【００３７】
例えば、ドラムを用いた本発明酵素脱毛処理方法は、常法に従い予め水漬け処理した原皮に、本発明酵素脱毛処理剤を浴比1:2-5となる割合で使用して、温度20-30℃、pH9-11に調整しながら、10-24時間程度を要して実施される。
【００３８】
上記で脱毛された処理革は、引続き、革を膨潤させるために消石灰液を用いた石灰漬け処理後、常法に従う鞣製処理の工程に供することができる。このように、本発明方法では、酵素脱毛処理後に石灰漬け(消石灰液のみ)を行うことによって、上記消石灰液を繰返し使用できる利点がある。
【００３９】
また、本発明酵素脱毛処理によって得られる毛は、前述したように毛根から脱毛されており、従来技術にみられるような硫化物を使用したことに基づく分解、溶解、変質などは殆どなく、従って、これは例えば濾過などの操作により容易に脱毛処理液中より回収することができる。かくして回収された毛は、硫化物に曝されていないため、フェルト材料、ブラシ材料などの繊維材料として一般工業材料分野で有用であり、また化粧品原料、動物飼料などとしても期待できる。更に、本発明酵素脱毛処理によれば、フレッシング屑および床皮も硫化物に曝されていないので、これらもコラーゲン原料として化粧品分野でより有利に利用できる。
【００４０】
本発明方法の適用できる皮革としては、牛を代表例として挙げることができ、その他、豚、ウマ、ヒツジ、ヤギなどであることもできる。
【００４１】
本発明脱毛処理方法は、従来のこの種脱毛処理方法に比して、以下の如き優れた実用的価値を有している。
(a)硫化物を使用する必要がなく、従って、該硫化物による排水汚濁負荷を軽減できる、
(b)脱毛に要する水酸化カルシウムの使用量も低減でき、更に石灰漬けで利用する消石灰(水酸化カルシウム)は、これを繰り返し利用できるため、排水中への水酸化カルシウムの流出量を大幅に低減できる、
(c)脱毛した毛は実質的に処理液中に溶解せず、その大半を回収できるため、溶解毛およびこれに由来する蛋白質の排水中への混入を大幅に減少させ得る、
(d)得られる皮革は、ソフトであり、毛根跡が残らず、表面が美しく、明度の高い染色などを行い得る、
(e)鞣製における酵解工程をも省略して、歩留りの向上をはかり得る、
【００４２】
【実施例】
以下、本発明を更に詳しく説明するため、本発明酵素脱毛処理剤に利用するアルカリプロテアーゼの製造例を参考例として挙げ、次いで本発明酵素脱毛処理方法の実施例を挙げる。尚、各例における％は原皮重量に対する重量％を示し、使用酵素における力価および活性測定法は特記しない限り前述したそれらに従うものである。
【００４３】
【参考例１】
粗酵素標品の調製
ノカルディオプシス エスピー TOA-1株(FERM P-18676)の前培養液を、スキムミルク0.5％、酵母エキス0.1％および別殺菌して添加した炭酸ナトリウム1.0％を含む培地（pH10.5）4000 mLを入れた小型ジャーファーメンタ―に植菌し、30℃、通気量1v/v/分、200回転/分で3日間培養した。培養終了後、培養液を8,000回転/分で10分間遠心分離して菌体を除去した。
【００４４】
上記により、45APU/mLの粗酵素液約3,800 mLを得た。この粗酵素液に硫安粉末を80％飽和になるまで加え、一昼夜5℃で暗所に静置後、生じた沈殿を8,000回転/分で遠心分離して回収し、凍結乾燥した。上記により、15,100APU/gの粗酵素標品を8.9g得た。
【００４５】
このもののケラチン分解活性は、72,500AKU/gであり、コラーゲン分解活性は、77,900ACU/gであり、エラスチン分解活性は、106,000AEU/gであった。これらのことから、本酵素のケラチン分解活性に対するコラーゲン分解活性比は1.08、エラスチン分解活性比は1.46と算出される。
【００４６】
【実施例１】
牛皮の脱毛処理
原皮として、北米産ステヤー皮27kg、国内産生皮27kg、キップ皮12kgおよびカーフ皮4.5kgを用いた。
【００４７】
まず、各原皮を各々ドラム中で、浴比1：3、25℃で1時間水に浸した後、流水で10分間水洗した。次に、浴比1：3、脱脂目的の界面活性剤として「スプラランUF」(Zschimmer & Schwarz社)0.2％および炭酸ナトリウム0.2％を含む処理水中に上記で水洗した皮を25℃で一晩放置して水漬け処理を行った。処理後のpHは8付近であった。更に、浴比1：3、25℃で10分間、水中で攪拌しながら十分に水洗した後、以下に示す酵素脱毛処理を行った。
【００４８】
即ち、消石灰0.5％、ホウ酸0.8％および「スプラランUF」0.1％を含む処理液(pH10.5)を用いて、上記で得られた処理皮を、浴比1：3、27℃、1時間処理した後、原皮重量1gに対して90APUのアルカリプロテアーゼ(参考例1で調製したもの)を上記処理液に加え、更に27℃で、1時間に1分間の緩速攪拌を16時間行い、脱毛処理を完結した。処理後のpHは9付近であった。
【００４９】
また、処理液中に存在するマリモ状形態の脱毛を、フィルターで回収した。
【００５０】
次いで、上記で得られた脱毛処理皮を、石灰漬け(浴比1：3、消石灰3％、25℃、20時間)後、分割、脱灰、浸酸、クロム鞣、シェービング、中和、再鞣、染色、加脂、馬掛け、乾燥、味取り、ミリングおよびネット張りの各工程に付して、皮革製品サンプルを得た。
【００５１】
以上の結果、いずれの原皮を用いた場合も、毛は毛切れすることなく毛根より完全に全て脱毛されており、容易に回収できた。元の毛の量は、重量当たり、北米産ステヤー皮で6％、国内産生皮で7％、キップ皮で10％およびカーフ皮で10％であり、毛の回収量は各々95％以上であった。さらに、垢も完全に除去されており、浴中のpHは経時的に低下して行くため、銀面への過剰な反応も抑制され、革表面は美しかった。
【００５２】
本酵素処理を採用して得られた皮革サンプル(本発明品)の外観を、従来のヘアーバーン法に従って脱毛処理(浴比1：3、硫化水素ナトリウム1.5％、硫化ナトリウム1.5％および消石灰3％を含む処理液使用、25℃、一晩浸漬）し、以降、同様にして得た皮革サンプル(比較品)と比較検討した。
【００５３】
その結果、いずれの原皮を用いた場合も、本発明品は、比較品に比して、革がより柔らかく面積も大きくなっており、染色ムラもなく鮮明に染色されていた。また、本発明品では、比較品に認められる耐熱性、引裂き強度などの低下は、認められなかった。
【００５４】
また、上記本発明品と比較品とのそれぞれの製造における脱毛処理後の処理液(排水)の水質分析を以下の通り実施した。即ち、SSは、JIS環境庁告示第59号付表8の方法に従って分析した。T-Nは、JIS K0102-45.2の方法に従って測定した。CODは、JIS K0102-17の方法に従って測定した。BODはJIS K0102-21.注(14)および32.1の方法に従って測定した。
【００５５】
得られた結果を、下記表3に示す。
【００５６】
【表３】

【００５７】
表3に示される結果より明らかなとおり、本発明方法によれば、従来のヘアバーン法を採用する場合に比して、排水中のSSは99％削減でき、全窒素(T-N)は67％削減でき、CODは55％削減でき、またBODは26%削減できる。
【００５８】
更に、従来のヘアバーン法では、硫化物の使用を必須とするために、その処理に、例えば一次濾過、塩酸による中和、凝集剤による凝集沈殿などの操作や、硫酸マンガンなどの酸化触媒による中和処理などが必要であり、しかもこれらの処理操作の間に、硫化水素が発生する危険があり、作業者の安全性にも問題がある。これに対して、本発明酵素脱毛処理方法では、硫化物を使用しないので、上記硫化物の処理は不要であり、この処理に伴われる労力、経費などをも軽減できる利点があり、作業者の安全性の面でも非常に優れた方法である。
【００５９】
【実施例２】
豚皮
豚皮原皮(5kg×5枚、計25kg)を流水で30分間水洗した後、「トリロン」(松本油脂製薬界面活性剤)0.4％および炭酸ナトリウム0.4％を含む水中に、一晩放置(浴比1：2、25℃)して水漬け処理を行った。次に、「トリロン」3％を含む水中に、浴比1：2、30℃、3時間浸して脱脂処理を十分に行った後、流水で十分に水洗し、その後、以下に示す酵素脱毛処理を行った。
【００６０】
即ち、消石灰0.5％、ホウ酸0.8％および「スプラランUF」0.3％を含む処理液(pH10.5)を用いて、上記で得られた処理皮を、浴比1：3、27℃、1時間処理した後、原皮重量1gに対して120APUのアルカリプロテアーゼ(参考例1で調製したもの)を加え、27℃で、1時間に1分間の緩速攪拌を一夜続けて、脱毛処理を完結した。処理後のpHは9.3付近であった。
【００６１】
以上の結果、毛は毛切れすることなく毛根より抜け、大部分が脱毛され、容易に回収できた。また、残毛も軽く引っ張るだけで簡単に脱毛できた。得られた脱毛処理皮は、石灰漬け後、牛皮と同工程で処理して皮革製品とした。
【００６２】
【配列表】

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to an enzyme hair removal treatment agent for animal skin, more specifically, an enzyme hair removal treatment agent used in a hair removal step made of leather, and an animal hair removal treatment method using the same.
[0002]
[Prior art]
The process of smoking leather such as cowhide can be roughly divided into three processes: a preparation process, a tanning process, and a finishing process. The preparation process varies slightly depending on the type of product leather, the type of tanning process that follows this preparation process, etc., but in general, the raw leather is soaked, lined, depigmented (lime pickled), divided, exuded, re-lime It consists of each step of pickling, decalcification and fermentation.
[0003]
At present, the hair removal process of animal skin in the above-mentioned leather smelting is carried out in a drum or paddle in an integrated manner using a high concentration of calcium hydroxide and sulfide as a hair removal treatment agent from the viewpoint of shortening the work time and process. (Hair-burn method, hair-burn method). In other words, the skin that has been salted and dried for storage is generally softened by water absorption and returned to a state similar to fresh skin, and then immersed in a treatment bath of lime milk to which sodium sulfide or sodium hydrogen sulfide is added as a hair loss promoter. Then, the epidermal tissue is decomposed and removed, and the hair is decomposed and dissolved in the treatment bath.
[0004]
However, since this method decomposes and dissolves hair, there is a disadvantage that hair cannot be collected. In addition, the treatment bath once used contains dissolved hair and a large amount of keratin degradation products derived therefrom, and the treatment bath cannot be easily reused and must be discarded. In addition, the treatment liquid (wastewater) to be discarded contains high concentrations of calcium hydroxide and sulfide together with the dissolved hair and keratin degradation products, and has a very high pollution load (BOD, COD, SS, etc.) It causes environmental pollution. Therefore, this waste water requires waste water treatment including treatment of sulfides that are dangerous such as generation of hydrogen sulfide, and this waste water treatment requires excessive facilities and costs. As described above, the conventional hairburn method has a fatal defect that requires wastewater treatment with a high pollution load.
[0005]
In recent years, as a method that can reduce the pollution load of waste water, which is a drawback of the above hair burn method, a hair save method that incorporates a treatment for protecting the hair shaft from decomposition by a hair removal treatment agent has been proposed based on the hair burn method. Has been. Typical examples include the Blair method (Blair method, Leather, 1988 (Feb): 23-26 (1988)) and the SIROLIME method (Cranston, RW, Davis, MH, Scroggie, JG, JSLTC, 70 , 50-55 (1986)].
[0006]
However, even with these proposed methods, the use of sulfides as hair loss promoters is essential, and the effect of reducing the waste water pollution load is still not satisfactory. Moreover, the SIROLIME method has a drawback that hydrogen sulfide gas and chlorine gas are generated from the sodium hydrogen sulfide and hypochlorite used, and these treatments are newly required. These generated gases also have a problem of deteriorating the working environment.
[0007]
As another hair saving method, a disulfide bond-breaking substance such as thioglycolic acid (Patent No. 1432630) or thiourea dioxide (US Pat. No. 5,508,195) can be used as a reducing agent in place of sulfide as a hair loss promoter. There has also been proposed an enzyme hair-save method in which a raw skin is pretreated by using it and then hair is removed using an enzyme (protease).
[0008]
However, there has been little research on proteases used in enzyme hair-save methods. Generally known proteases generally have low keratin degrading activity, and when they are used, hair removal treatment cannot be performed sufficiently after pretreatment using the above reducing agent. Moreover, ordinary proteases have a considerably higher degradation activity for dermal components such as collagen and elastin than keratin degradation activity that works effectively for hair degradation. In addition, there is a problem in that a leather product with good quality cannot be obtained due to the remarkable adverse effects on the silver surface due to decomposition of the dermis, such as a decrease in leather texture, deterioration in leather quality, and decrease in leather strength.
[0009]
The present inventors previously established a practical animal hair removal method for animal hair using a specific enzyme (Japanese Patent Laid-Open No. 2001-164300). Although this method was able to perform hair removal treatment without adversely affecting the silver surface based on the use of an enzyme with high keratin degradation activity, it was accompanied by the combined use of sulfides even though the concentration was low. There was still room for improvement in terms of the effect of reducing the wastewater pollution load caused by this sulfide.
[0010]
[Problems to be solved by the invention]
The object of the present invention is to use no hair removal accelerator such as sulfide nor any reducing agent such as thioglycolic acid or thiourea dioxide, and therefore can significantly reduce the pollution load of the hair removal effluent, and has good quality. An object of the present invention is to provide an animal skin depilation technique with good workability, in which a simple leather product can be provided and hair can be collected and used.
[0011]
[Means for Solving the Problems]
As a result of continuous researches to achieve the above object, the present inventors have newly found an alkaline protease effective for hair removal treatment of animal hair, and improved this to meet the above object using this enzyme. We succeeded in establishing enzyme hair removal treatment technology. The present invention has been completed based on this finding.
[0012]
The gist of the present invention is the following items 1-7.
Item 1. Enzymatic hair removal of animal skin comprising as an active ingredient an alkaline protease having an keratin degradation activity of 70 AKU or more, a collagen degradation activity ratio to keratin degradation activity of 2 or less, and an elastinolysis activity ratio to keratin degradation activity of 4 or less Processing agent.
Item 2. The enzyme hair removal treatment according to Item 1, wherein the alkaline protease is derived from actinomycetes.
Item 3. The enzyme hair removal treatment according to Item 1, wherein the alkaline protease is produced by Nocardiopsis sp. TOA-1 strain (FERM P-18676).
Item 4. The enzyme hair removal treatment according to Item 1, which is used in a hair removal step made of leather.
Item 5. An enzyme hair removal treatment method for animal skin, which comprises bringing the enzyme hair removal treatment agent according to Item 1 into contact with a raw skin.
Item 6. Using an alkaline protease with an enzyme titer of 15-150 APU / g per gram of raw skin weight, contact ratio is 1: 2-5, temperature 20-30 ° C, pH 9-11 and time 10-24 hours Item 6. The method for enzymatic hair loss treatment according to Item 5, wherein
Item 7. A method for recovering hair in leather smoking, wherein the enzyme hair removal treatment agent according to Item 1 is contacted with a raw leather, and then the hair loss is recovered.
[0013]
The method of the present invention using the enzyme hair removal treatment of the present invention can reduce the amount of calcium hydroxide used to about 1/10 or less, compared with the conventional hairburn method. Further, it is not necessary to use a hair removal accelerator or a reducing agent such as sulfide. Therefore, in the method of the present invention, the pollution load (BOD, COD, sludge amount (SS), etc.) of the hair removal and drainage can be greatly reduced. The enzyme hair removal treatment method of the present invention also has an advantage that animal hair can be recovered without dissolving it. This means that the amount of hair protein degradation product in hair removal and drainage can be greatly reduced. For example, according to the method of the present invention shown in the Examples, COD in the hair removal and drainage can be reduced by about 50%, BOD can be reduced by about 26%, and SS can be reduced to 99% or less as compared with the hairburn method. In addition, the total nitrogen can be reduced by about 70%.
[0014]
The method of the present invention is also very advantageous in terms of hair removal and drainage treatment. For example, there is an advantage that the amount of sludge used in activated sludge treatment can be greatly reduced. The operation operation of the drainage treatment can be made simpler, the cost required for the drainage treatment can be reduced, and the danger associated with the drainage treatment can be greatly reduced. In particular, this drainage treatment does not require special equipment, and can be sufficiently performed using an existing apparatus. Furthermore, the method of the present invention does not require special mechanical means for hair removal.
[0015]
Furthermore, in the enzyme hair removal treatment method of the present invention, animal hair is removed from the hair roots, and the hair roots that cause darkening do not remain in the treated leather. Further, the treated leather surface (silver surface) is smooth and beautiful, and has a soft finish without any adverse effects such as a decrease in texture and a decrease in strength associated with the use of an enzyme. Furthermore, there is an advantage that no sulfide is contained in the freshening waste and the floor skin used as a raw material for collagen as a cosmetic. Taking these into account, the hair removal treatment method of the present invention is practically very excellent from the viewpoint of obtaining a leather product and recovered hair with good quality as well as reducing the wastewater treatment load.
[0016]
DETAILED DESCRIPTION OF THE INVENTION
Hereinafter, the enzyme hair removal treatment agent of the present invention and the hair removal treatment method using the hair removal treatment agent will be described in detail.
[0017]
The enzyme depilatory treatment agent of the present invention has an keratin degradation activity of 70 AKU or more as an active ingredient thereof, an alkaline protease having a collagen degradation activity ratio to keratin degradation activity of 2 or less and an elastin degradation activity ratio to keratin degradation activity of 4 or less. Must be included. Particularly preferred alkaline proteases include those having a keratin degradation activity of 120 AKU or more.
[0018]
The keratin degradation activity (AKU) is measured by the following measurement method. That is, 1 mL of enzyme solution prepared to 25 APU / mL of casein degradation activity was mixed with 3 mL of 100 mmol / L borax-sodium carbonate buffer (pH 10.5) containing 0.04 g of finely pulverized Keratin Azure made by Sigma. It is determined by measuring the absorbance of the dye released with decomposition at 595 nm after slow stirring at 35 ° C. for 60 minutes. Here, 1AKU is defined as the amount of enzyme that increases the absorbance at 595 nm by 0.001 per hour under the above reaction conditions.
[0019]
The casein degrading activity (titer; APU / mL) of the enzyme solution is measured by the following measurement method. That is, 1 mL of 100 mmol / L borax-sodium carbonate buffer (pH 10.5) containing 1% hamal-sten dairy casein was mixed with 1 ml of enzyme solution, reacted at 35 ° C. for 10 minutes, and then 7.2% trichloroacetic acid solution. Add 2 mL to stop the reaction, leave it at 35 ° C. for 20 minutes, then filter with filter paper (ADVANTEC, No.6, manufactured by TOYO) and measure the proteolysate in the filtrate by the Folin method. It is done. In this measurement method, the amount of enzyme that releases 1 μg of tyrosine per minute is defined as 1 APU.
[0020]
Collagen degrading activity (ACU / mL) and elastin degrading activity (AEU / mL) indicated by the relative ratio to the keratin degrading activity are determined by the following measuring methods, respectively. That is, 1 mL of an enzyme solution prepared to 1 mL of 100 mmol / L borax-sodium carbonate buffer solution (pH 10.5) containing 2% of collagen Type I (manufactured by SIGMA) or elastin (manufactured by SIGMA) to 50 APU / mL of casein decomposition activity And the mixture is allowed to react with stirring at 35 ° C. for 2 hours. Then, 2 mL of 7.2% trichloroacetic acid solution is added to stop the reaction, and the mixture is left at 35 ° C. for 20 minutes. Next, it is filtered through a filter paper (ADVANTEC, No.6, manufactured by TOYO), and the proteolysate in the filtrate is measured by the Folin method, and the amount of increase in absorbance at 660 nm (based on the measured value of 660 nm at 0 hours of reaction). Calculate the amount of increase for that). Here, 1ACU and 1AEU are defined as the amount of enzyme that increases the absorbance at 660 nm by 0.001 per hour under the above reaction conditions.
[0021]
Collagen-degrading activity and elastin-degrading activity indicated by the relative ratios based on the keratin-degrading activity indicate the specificity or selectivity of the enzyme for keratin. That is, the smaller the value of the relative ratio of collagen degrading activity and elastin degrading activity, the smaller the action of the enzyme on collagen and elastin, thus indicating that it is keratin specific.
[0022]
An enzyme having such characteristics, that is, an enzyme that satisfies a specific ratio of keratin degradation activity, collagen degradation activity relative to the keratin degradation activity, and relative ratio of elastin degradation activity, exhibits an excellent hair removal treatment effect of the present invention. obtain. The reason is considered as follows.
[0023]
The raw skin to be treated in the enzyme hair removal treatment method of the present invention is composed of two layers, the epidermis and the dermis (consisting of a nipple layer and a reticular layer), and animal hair is formed with the epidermis depressed in the dermis. The hair follicle has a structure extending obliquely from the bottom to the surface of the epidermis. Further, the animal hair is distinguished from the hair shaft by the exposed part on the skin, the hair root surrounded by the hair follicle, and the swollen part of the bottom of the hair root from the hair bulb. According to the enzyme hair removal treatment method of the present invention, based on the fact that the specific enzyme to be used has a high keratin degradation activity as described above, it acts on animal hair having keratin as a main constituent protein, particularly on the bottom of the hair root, This is considered to decompose and remove hair. On the other hand, the dermis is an important part that becomes leather after smoking, and the outer papillary layer has an entangled structure of collagen fibers and elastin fibers. According to the enzyme hair removal treatment method of the present invention, the enzyme used acts on the cell structure constituting the epidermis, decomposes and disappears (peels), and reaches the dermis (papillary layer) immediately inside the epidermis. However, based on having low collagen degradation activity and elastin degradation activity compared to keratin degradation activity, the papillary layer is not substantially degraded, and hence the texture of the silver surface obtained after treatment is reduced, It is considered that there is no deterioration of the leather quality or a decrease in the strength of the leather.
[0024]
Examples of the alkaline protease having the above-described properties that are preferably used in the enzyme hair removal treatment method of the present invention include those derived from actinomycetes. A representative example thereof is one produced by the present inventors of the alkaliphilic Nocardiopsis sp. TOA-1 strain (Japanese Patent Application No. 2002-161009). Hereinafter, the alkaline protease produced by this TOA-1 strain is referred to as “the present enzyme”.
[0025]
The TOA-1 strain will become Nocardiopsis sp. TOA-1 on January 16, 2002 at the National Institute of Advanced Industrial Science and Technology, National Institute of Advanced Industrial Science and Technology, 1-3-1 East Tsukuba, Ibaraki, Japan. Deposited as indicated, the deposit number is FERM P-18676.
[0026]
Cultivation of the above-mentioned bacteria and collection of the desired alkaline protease can be performed according to conventional methods. For example, since the above bacterium is an alkalophilic actinomycete, the culture is performed in an alkaline region obtained by adding a suitable alkali to a normal medium. The nutrient sources such as carbon source, nitrogen source, and other inorganic salts used in the medium may be those commonly used for culturing this type of enzyme-producing bacterium. For example, glucose, soluble starch, cellulose and the like can be exemplified as the carbon source. Examples of the nitrogen source include inorganic substances such as nitrates and ammonium salts, urea, peptone, dry yeast, yeast extract, skim milk, soy flour, corn steep liquor, casein, meat extract, amino acids, and the like. Examples of other inorganic salts include magnesium salts, potassium salts, sodium salts, and phosphates. These nutrient sources may be used alone or in combination of two or more. Their combination is also arbitrary. Examples of the alkali added to the medium include aqueous solutions of carbonates such as sodium carbonate and sodium bicarbonate having a concentration of about 0.5-2%, aqueous sodium hydroxide, and aqueous ammonia. The pH of the medium is usually preferably about 8-11. Culturing can be carried out at a temperature of about 20-40 ° C., preferably about 30-35 ° C., for 2-7 days with aerobic stirring or shaking. The desired enzyme is secreted and accumulated mainly in the culture medium.
[0027]
The enzyme can be easily collected and purified from the culture solution according to a conventional method using the physicochemical properties of the enzyme. For example, microbial cells can be removed by filtration, centrifugation, etc. to obtain a crude enzyme solution. The crude enzyme solution can be further purified by conventional methods, for example, salting out, organic solvent precipitation, ultrafiltration, gel filtration chromatography, ion exchange chromatography, hydrophobic chromatography and the like. One particularly preferred purification method is to first salt out by adding 80% saturated ammonium sulfate to the culture filtrate, dissolve the resulting precipitate in a buffer solution, then CM-Toyopearl 650M (manufactured by Tosoh Corporation), An example is a method of performing ion exchange chromatography using DEAE-Toyopearl I 650M (manufactured by the same company). By this method, a purified enzyme that is uniform by SDS electrophoresis can be obtained.
[0028]
The enzyme thus obtained has the following properties.
(1) Action and substrate specificity
Acts on proteins and peptides and breaks peptide bonds by an endo-type mechanism to produce low molecular weight oligopeptides and amino acids. It also exhibits strong activity against insoluble proteins such as keratin.
(2) Optimum pH and stable pH
Buffers include HCl / KCl (pH 1.0-1.5), glycine / NaCl / HCl (pH 2.0-3.0), acetic acid (pH 4.0-5.0), phosphoric acid (pH 6.0-7.0), Tris-HCl (pH 7 .0-9.0), glycine / NaCl / NaOH (pH 9.0-12.0) and KCl / NaOH (pH 12.0-13.0), the optimum pH of the enzyme determined according to the activity measurement method described above is At 30 ° C., 11.0-11.5 when casein is used as a substrate, and 12.0 or more when keratin is used as a substrate.
[0029]
Similarly, the stable pH range of the enzyme determined by measuring the residual protease activity after holding the enzyme in a buffer solution of each pH at 30 ° C. for 24 hours may be in a wide range of 1.5-12.0. confirmed.
(3) Optimal and stable temperature
The optimum temperature of this enzyme is 70-75 ° C when casein is used as a substrate, and 65-70 ° C when keratin is used as a substrate. In addition, the enzyme was added to 100 mmol / L Tris-HCl buffer (pH 7.0) and kept at a temperature range of 40-80 ° C. for 10 minutes, and then the residual protease activity was measured. It turns out to be stable. In addition, regarding the temperature stability of this enzyme, the effect of calcium addition (10 mM) is not recognized.
(4) Molecular weight
The molecular weight of the enzyme was measured by SDS electrophoresis. As a result, the molecular weight was about 20,000. The molecular weight calculated from the amino acid sequence described in SEQ ID NO: 2 described later is 19,150.
(5) Isoelectric point
The isoelectric point of this enzyme was measured by isoelectric focusing. As a result, the isoelectric point was 10.0 or more.
(6) Inhibition
General enzyme inhibitors PMSF (phenylmethanesulfonyl fluoride), EDTA (ethylenediaminetetraacetic acid) and SSI (Streptomyces subtilisin inhibitor) are each adjusted to a predetermined concentration of 50 mmol / L Tris-HCl buffer (pH 9). 0.0), and after the addition of this enzyme, the mixture was treated at 30 ° C. for 30 minutes, and then a certain amount was taken from the treated solution and its residual activity was measured. As a result, this enzyme was inhibited by PMSF and SSI, and not inhibited by EDTA. This revealed that this enzyme is a serine protease.
(7) Amino terminal sequence
The sequence from the amino terminus of the enzyme to the 25th position was determined using a gas phase protein sequencer (manufactured by Shimadzu Corporation, PPSQ-21). As a result, the 1-25th sequence of SEQ ID NO: 2 was confirmed.
(8) Base sequence and amino acid sequence
Use the gene and amino acid sequences of this enzyme according to conventional methods (see, for example, J. Sambrook, EF Fritsch, T. Maniatis: Molecular Cloning. A Laboratory Manual, 2nd. Ed. Cold Spring Harbor Laboratory Press, 1989, etc.) The determination was made according to the protocol of the instrument, reagent kit, etc. First, the purified enzyme was treated with urea and then decomposed with lysyl endopeptidase (Wako Pure Chemical Industries), and the amino acid sequence of the obtained fragment was determined with a gas phase protein sequencer. From the amino acid sequence information thus obtained, two appropriate oligonucleotide primers were synthesized by the phosphoramidite method, and using these primers, gene amplification was performed according to PCR (Biometra, T-Gradient Thermoblock 050-801). went. As a result, a specific amplified fragment was observed around 0.5 kbp. Using this fragment as a probe, a full-length gene encoding this enzyme was screened from the genomic library of this strain TOA-1. The base sequence of the obtained clone was determined by the dideoxy method (F. Sanger et. Al., Proc. Natl. Acad. Sci., 74 , 5463-5467, 1977) based on a DNA sequencer (LICOR-4000, manufactured by LICOR). The base sequence (564 bp) is shown in SEQ ID NO: 1. The amino acid sequence (188 amino acids) determined based on the base sequence is shown in SEQ ID NO: 2.
(9) Keratin specificity
This enzyme is characterized by having particularly excellent keratin specificity. For example, Table 1 shows the results of comparing the keratin degradation activity, collagen degradation activity, and elastin degradation activity of this enzyme and a commercially available representative alkaline protease (referred to as product A). In addition, Table 2 shows the results of determining the relative activity ratio of collagen degradation activity and elastin degradation activity based on keratin degradation activity.
[0030]
[Table 1]

[0031]
[Table 2]

[0032]
From the results shown in these tables, this enzyme has a keratin-degrading activity that is twice as high as that of product A, which is a commercially available enzyme, and has the same or lower collagen-degrading activity and elastin-degrading activity, and high specificity for keratin I understand. Therefore, it can be seen that this enzyme has less action on collagen and elastin than commercially available enzymes, and has little effect on the dermis during animal hair treatment.
[0033]
The enzyme hair removal treatment of the present invention is usually prepared in the form of a treatment solution for hair removal containing the specific enzyme together with a pH adjusting agent. The pH adjusting agent used here is not particularly limited as long as the pH of the resulting treatment solution is about 9-11 suitable for leather hair removal treatment. In general, calcium hydroxide is preferably used. To the treatment liquid, boric acid or the like can be added in order to further have a pH buffering action. Although it is not necessary to add a reducing agent such as sodium hydrogen sulfide or thioglycolic acid as a hair removal accelerator, which has been conventionally required, to the treatment liquid, addition of these additives is not prohibited.
[0034]
The blending amount (concentration) of the enzyme, pH adjusting agent, etc. in the enzyme hair removal treatment solution of the present invention can be appropriately determined according to the type of raw skin to which the treatment solution obtained is applied, the method of hair removal treatment, conditions, etc., and is particularly limited. It is not something. Usually, the enzyme is selected from the range of about 30-180 APU, preferably about 60-150 APU, per 1 g of raw skin weight. Calcium hydroxide as a pH adjusting agent can be selected from a range of about 0.2-2%, preferably about 0.4-1%. The boric acid used for adjusting the predetermined pH can be selected from the range of about 0.3-3%, preferably about 0.6-1.5%.
[0035]
If necessary, the enzyme hair removal treatment liquid of the present invention can be appropriately mixed with a surfactant or the like that is generally known to be added to this kind of hair removal treatment liquid. Examples of the surfactant include a surfactant having a chemical penetration promoting effect and a surfactant having a degreasing effect. Specifically, “Spralan UF” (manufactured by Zschimmer & Schwarz) can be used. The surfactant also includes a surface activity that can also exhibit an antiseptic effect, specifically, “cismoran BH” (manufactured by Bayer). The amount of these surfactants added is not particularly different from the amount in which they are usually used. Generally, it is usually about 0.1-1%.
[0036]
The enzyme hair removal treatment solution of the present invention prepared as described above is used in a conventionally known leather-made hair removal process using a drum or paddle, and this is generally brought into gentle contact with the raw leather. By this, the intended hair removal treatment of the present invention can be carried out. The present invention provides an enzymatic hair removal treatment method for animal hair using the enzyme hair removal treatment of the present invention.
[0037]
For example, the enzyme hair removal treatment method of the present invention using a drum uses the enzyme hair removal treatment agent of the present invention in a ratio of a bath ratio of 1: 2-5 to a raw skin previously soaked in accordance with a conventional method, at a temperature of 20- It takes about 10-24 hours while adjusting to 30 ° C and pH 9-11.
[0038]
The treated leather that has been depilated as described above can be subsequently subjected to a smoking process according to a conventional method after lime pickling using slaked lime solution to swell the leather. Thus, in the method of the present invention, there is an advantage that the slaked lime solution can be used repeatedly by performing lime pickling (only slaked lime solution) after the enzyme hair removal treatment.
[0039]
Further, the hair obtained by the enzyme hair removal treatment of the present invention has been removed from the hair root as described above, and there is almost no degradation, dissolution, alteration, etc. based on the use of sulfides as found in the prior art. This can be easily recovered from the hair removal treatment liquid by an operation such as filtration. Since the hair thus recovered is not exposed to sulfides, it is useful in the field of general industrial materials as a fiber material such as felt material and brush material, and can also be expected as a raw material for cosmetics, animal feed and the like. Furthermore, according to the enzyme hair removal treatment of the present invention, the fleshing waste and the floor skin are not exposed to sulfides, and these can be used more advantageously as a collagen raw material in the cosmetic field.
[0040]
Examples of leather to which the method of the present invention can be applied include cattle, and other examples include pigs, horses, sheep and goats.
[0041]
The hair removal treatment method of the present invention has the following excellent practical value as compared with this conventional hair removal treatment method.
(a) It is not necessary to use a sulfide, and therefore, it is possible to reduce drainage pollution load due to the sulfide.
(b) The amount of calcium hydroxide required for hair removal can be reduced, and slaked lime (calcium hydroxide) used for lime pickling can be used repeatedly, greatly increasing the amount of calcium hydroxide flowing into the wastewater. Can be reduced,
(c) The hair that has been depilated does not substantially dissolve in the treatment liquid, and most of the hair can be recovered, so that mixing of the dissolved hair and protein derived therefrom into the wastewater can be greatly reduced.
(d) The obtained leather is soft, has no hair root traces, has a beautiful surface, and can be dyed with high brightness, etc.
(e) The fermentation process in smoking can be omitted, and the yield can be improved.
[0042]
【Example】
Hereinafter, in order to describe the present invention in more detail, a production example of an alkaline protease used for the enzyme hair removal treatment of the present invention is given as a reference example, and then an example of the method of enzyme hair removal treatment of the present invention is given. In addition,% in each example shows the weight% with respect to the weight of a raw skin, and unless otherwise indicated, the titer and activity measuring method in an enzyme used are based on those mentioned above.
[0043]
[Reference Example 1]
Preparation of crude enzyme preparation
Nocardiopsis SP TOA-1 strain (FERM P-18676) pre-culture solution, 4000 ml of medium (pH 10.5) containing 0.5% skim milk, 0.1% yeast extract and 1.0% sodium carbonate added separately sterilized The small jar fermenter was inoculated and cultured at 30 ° C., aeration rate 1 v / v / min, 200 rpm / min for 3 days. After completion of the culture, the culture was centrifuged at 8,000 rpm for 10 minutes to remove the cells.
[0044]
As a result, approximately 3,800 mL of a 45 APU / mL crude enzyme solution was obtained. To this crude enzyme solution, ammonium sulfate powder was added until it was 80% saturated. After standing overnight in the dark at 5 ° C., the resulting precipitate was collected by centrifugation at 8,000 rpm, and lyophilized. As a result, 8.9 g of a crude enzyme preparation of 15,100 APU / g was obtained.
[0045]
This product had a keratin degradation activity of 72,500 AKU / g, a collagen degradation activity of 77,900 ACU / g, and an elastin degradation activity of 106,000 AEU / g. From these facts, the ratio of collagen degradation activity to the keratin degradation activity of this enzyme is calculated as 1.08, and the ratio of elastin degradation activity is calculated as 1.46.
[0046]
[Example 1]
Hair removal treatment of cowhide
North American steer skin 27 kg, domestically produced skin 27 kg, Kip skin 12 kg and calf skin 4.5 kg were used as the raw skin.
[0047]
First, each raw hide was immersed in water at a bath ratio of 1: 3 and 25 ° C. for 1 hour in each drum, and then washed with running water for 10 minutes. Next, the skin washed with the above in treated water containing 0.2% of "Spraran UF" (Zschimmer & Schwarz) as a surfactant for degreasing and 0.2% of sodium carbonate as a degreasing surfactant and left at 25 ° C overnight Then, it was soaked. The pH after treatment was around 8. Furthermore, after thoroughly washing with water at a bath ratio of 1: 3 and 25 ° C. for 10 minutes while stirring in water, the following enzymatic hair removal treatment was performed.
[0048]
That is, using a treatment solution (pH 10.5) containing 0.5% slaked lime, 0.8% boric acid and 0.1% "Supuraran UF", the treatment skin obtained above is bath ratio 1: 3, 27 ° C, 1 hour After the treatment, 90 APU alkaline protease (prepared in Reference Example 1) per 1 g of raw skin weight was added to the above treatment solution, and further gently stirred at 27 ° C. for 1 minute per hour for 16 hours. The process was completed. The pH after treatment was around 9.
[0049]
Further, the marimo-like hair loss present in the treatment liquid was collected with a filter.
[0050]
Next, the hair removal treated skin obtained above is soaked in lime (bath ratio 1: 3, slaked lime 3%, 25 ° C, 20 hours), then divided, decalcified, soaked, chromed, shaved, neutralized, re-treated. A leather product sample was obtained through the steps of wrinkling, dyeing, greasing, riding, drying, taste-removing, milling and netting.
[0051]
As a result of the above, when any of the raw hides was used, the hair was completely removed from the hair root without being broken and could be easily recovered. The amount of original hair was 6% by weight for North American steer skin, 7% for domestic skin, 10% for Kip skin and 10% for calf skin, and the amount of hair recovered was over 95% each. It was. Furthermore, the dirt was completely removed, and the pH in the bath gradually decreased with time, so that excessive reaction to the silver surface was suppressed and the leather surface was beautiful.
[0052]
The appearance of the leather sample obtained by adopting this enzyme treatment (product of the present invention) is subjected to hair removal treatment according to the conventional hairburn method (bath ratio 1: 3, sodium hydrogen sulfide 1.5%, sodium sulfide 1.5% and slaked lime 3% Using a treatment solution containing 25 ° C. overnight, followed by comparison with a leather sample (comparative product) obtained in the same manner.
[0053]
As a result, in any of the raw leathers, the product of the present invention was softer and larger in area than the comparative product, and was clearly dyed without uneven dyeing. Further, in the product of the present invention, no decrease in heat resistance, tear strength, etc. observed in the comparative product was observed.
[0054]
In addition, the water quality analysis of the treatment liquid (drainage) after the hair removal treatment in the production of the product of the present invention and the comparative product was performed as follows. In other words, SS was analyzed according to the method of Appendix 8 of JIS Environment Agency Notification No. 59. TN was measured according to the method of JIS K0102-45.2. COD was measured according to the method of JIS K0102-17. BOD was measured according to the method of JIS K0102-21. Note (14) and 32.1.
[0055]
The obtained results are shown in Table 3 below.
[0056]
[Table 3]

[0057]
As is apparent from the results shown in Table 3, according to the method of the present invention, SS in wastewater can be reduced by 99% and total nitrogen (TN) can be reduced by 67% compared to the case where the conventional hairburn method is adopted. COD can be reduced by 55% and BOD can be reduced by 26%.
[0058]
Further, since the use of sulfides is essential in the conventional hairburn method, for example, primary filtration, neutralization with hydrochloric acid, coagulation precipitation with an aggregating agent, and the use of an oxidation catalyst such as manganese sulfate. There is a risk of hydrogen sulfide being generated during these processing operations, and there is also a problem in worker safety. On the other hand, in the enzyme hair removal treatment method of the present invention, since no sulfide is used, the treatment of the sulfide is unnecessary, and there is an advantage that labor, cost, etc. associated with this treatment can be reduced. It is a very good method in terms of safety.
[0059]
[Example 2]
Pig skin
After washing the skin of pork skin (5kg x 5 sheets, total 25kg) for 30 minutes with running water, leave it overnight in water containing 0.4% "Trilon" (Matsumoto Yushi Pharmaceutical Surfactant) and 0.4% sodium carbonate (bath ratio) (1: 2, 25 ° C.) and soaked in water. Next, after degreasing treatment by immersing in water containing 3% “Trilone” for 3 hours at a bath ratio of 1: 2, 30 ° C., thoroughly wash with running water, and then perform the following enzymatic hair removal treatment Went.
[0060]
That is, using a treatment solution (pH 10.5) containing 0.5% slaked lime, 0.8% boric acid and 0.3% "Supuraran UF", the treatment skin obtained above is bath ratio 1: 3, 27 ° C, 1 hour After the treatment, 120 APU alkaline protease (prepared in Reference Example 1) was added to 1 g of the raw skin weight, and the hair removal treatment was completed by continuing agitation at 27 ° C. for 1 minute for 1 hour overnight. The pH after treatment was around 9.3.
[0061]
As a result, the hair was removed from the hair root without being broken, and most of the hair was removed and could be easily recovered. The remaining hair could be easily removed by simply pulling it. The obtained hair removal treated skin was lime pickled and then processed in the same process as cowhide to obtain a leather product.
[0062]
[Sequence Listing]

Claims (7)

An enzyme hair removal treatment for animal skin comprising as an active ingredient an alkaline protease having an keratin degradation activity of 70 AKU or more, a collagen degradation activity ratio to keratin degradation activity of 2 or less, and an elastinolysis activity ratio to keratin degradation activity of 4 or less. 2. The enzyme hair removal treatment according to claim 1, wherein the alkaline protease is derived from actinomycetes. 2. The enzyme hair removal treatment according to claim 1, wherein the alkaline protease is produced by Nocardiopsis sp. No. TOA-1 strain (FERM P-18676). 2. The enzyme hair removal treatment according to claim 1, which is used in a hair removal step made of leather. 2. An animal hair removal treatment method for animal skin comprising contacting the enzyme hair removal treatment agent according to claim 1 with a raw skin. The contact is carried out using an alkaline protease with an enzyme titer of 15-150 APU per gram of raw skin weight, at a bath ratio of 1: 2-5, temperature 20-30 ° C., pH 9-11 and time 10-24 hours. Item 6. The enzymatic hair loss treatment method according to Item 5. A method for recovering hair in leather smoking, comprising contacting the hair removal treatment agent according to claim 1 with a raw leather, and then recovering the hair loss.