CN1245523C - Method for preparing leather using protease and method for treating wastes dervied from leather processing using same - Google Patents

Method for preparing leather using protease and method for treating wastes dervied from leather processing using same Download PDF

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CN1245523C
CN1245523C CN 01823478 CN01823478A CN1245523C CN 1245523 C CN1245523 C CN 1245523C CN 01823478 CN01823478 CN 01823478 CN 01823478 A CN01823478 A CN 01823478A CN 1245523 C CN1245523 C CN 1245523C
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hy
protease
step
leather
waste
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CN1529761A (en
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朴镐用
孙光熙
权容国
申东夏
闵升基
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昆虫生命工学株式会社
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    • CCHEMISTRY; METALLURGY
    • C14SKINS; HIDES; PELTS; LEATHER
    • C14CCHEMICAL TREATMENT OF HIDES, SKINS OR LEATHER, e.g. TANNING, IMPREGNATING, FINISHING; APPARATUS THEREFOR; COMPOSITIONS FOR TANNING
    • C14C1/00Chemical treatment prior to tanning
    • C14C1/08Deliming; Bating; Pickling; Degreasing
    • CCHEMISTRY; METALLURGY
    • C14SKINS; HIDES; PELTS; LEATHER
    • C14CCHEMICAL TREATMENT OF HIDES, SKINS OR LEATHER, e.g. TANNING, IMPREGNATING, FINISHING; APPARATUS THEREFOR; COMPOSITIONS FOR TANNING
    • C14C1/00Chemical treatment prior to tanning
    • C14C1/06Facilitating unhairing, e.g. by painting, by liming
    • C14C1/065Enzymatic unhairing

Abstract

本发明涉及使用蛋白酶制备皮革的方法和使用它处理来源于皮革加工的废物的方法,其在制备优异品质的皮革,通过减少化学品的量减少废物产量和以有利于环境的方式处理或再循环废物方面是有利的。 The present invention relates to methods for producing a leather protease and its use for treating wastes from leather processing, which is excellent in quality leather preparation, so as to facilitate recycling or processing environment by reducing the amount of chemicals to reduce waste production, and waste is advantageous. 将从Aranicola proteolyticus HY-3菌株生产的蛋白酶HY-3加至皮革加工中的浸水,浸灰,脱灰和软化步骤,从而制备皮革。 From Aranicola proteolyticus HY-3 strains produced protease HY-3 was added to the soaking, liming of leather processing, deliming and bating step, thereby preparing leather. 另外,将蛋白酶HY-3加至在浸水和浸灰步骤期间产生的废水和固体废物,和在浸灰,脱灰,软化和整理步骤期间产生的固体废物中,以便处理废物。 Further, protease was added to the HY-3 during soaking and liming step of generating the wastewater and solid waste, liming and in deliming, during softening and finishing step to produce a solid waste for disposal of waste.

Description

使用蛋白酶制备皮革的方法和使用它处理来自皮革加工的废物的方法 Use of a protease preparation methods and use of leather waste from its processing leather processing method

技术领域 FIELD

本发明涉及使用蛋白酶HY-3制备皮革的方法和使用它处理来自皮革加工的废物的方法。 The present invention relates to a process for preparing a protease leather HY-3 and its use method for processing waste from the processing of leather. 更具体地,本发明涉及制备皮革的方法,其中将从Aranicola proteolyticus HY-3菌株生产的蛋白酶HY-3加至浸水,浸灰,脱灰和软化步骤;和使用蛋白酶HY-3处理来源于浸水和浸灰步骤的废水和固体废物,和来自浸灰,脱灰,软化和整理步骤的固体废物的方法,其具有制备优秀品质的皮革和废物的有利于环境的处理或再循环的优点。 More particularly, the present invention relates to a process for the preparation of leather, wherein from Aranicola proteolyticus HY-3 strains produced protease HY-3 was added to the soaking, liming, deliming and bating step; and a protease derived from a soaking process HY-3 wastewater and solid waste and liming steps, and advantages of the process is conducive to the environment or recycled from liming, deliming method of solid waste, softening and finishing steps, and waste leather preparation having an excellent quality.

背景技术 Background technique

通常,作为需要昂贵的设备和技术的粗皮加工工业,皮革工业是决定皮革制品如鞋,袋,服装和皮带的质量的主要工业(chief industry)。 Conventionally, as techniques require expensive equipment and crude Pijia industry, leather industry is to determine the major industrial products such as leather shoes, bags quality, and clothing belt (chief industry). 在皮革工业中,当将各种动物的生皮或兽皮进行复杂的皮革制备过程时,生产终产品,即皮革。 In the leather industry, when various animal skins or hides for leather complicated production process, the production of the final product, i.e. leather. 将使用物理化学和生物方法的皮革制备方法分为需要水的湿法和干法。 The physical chemical and biological methods leather production method requires water into wet and dry.

湿法包含为了防止由细菌或霉菌导致生皮腐败的盐腌,为了去除生皮多余成分的浸水,为了产生用石灰处理的粗皮的浸灰,为了去除石灰的脱灰,为了生物处理多余成分的软化,为了降低pH值的浸酸,中和,复鞣,染色和乳液加油(或脂)的步骤。 Wet comprising To prevent bacterial or fungal cause salted rawhide corruption, in order to remove hides unwanted ingredients flooding, in order to produce rough skin treated with lime liming, in order to remove deashing lime to biological treatment to soften the undesired component, in order to reduce the pH of the pickling, neutralization, retanning, dyeing and fatliquoring (or fat) steps.

更具体地,在浸水步骤中,去除大量附着于生皮或兽皮的多余蛋白质,和盐类或污物。 More specifically, in the immersion step, the excess protein is removed, and a large number of salts or contaminants adhering to the hide or hide. 在浸灰步骤中,使用石灰去除毛发和非必需蛋白质。 In a liming step, the removal of hair and lime nonessential protein. 在脱灰步骤中,去除在先前步骤中使用的石灰。 In deliming step, the removal of lime used in the previous step. 在用于彻底的表面清洗的软化步骤中,去除多余蛋白质。 The softening step for cleaning the surface thoroughly in removing excess proteins. 一种保留毛发的方法,其是一种有利于环境的皮革制备方法,将蛋白水解酶用于浸灰和脱灰步骤。 One kind of hair retention method is a method of preparing leather conducive environment, proteolytic enzymes for the liming and deashing steps. 在皮革制备过程中浸灰步骤极大影响COD(化学需氧量)。 Immersed in the manufacturing process step greatly influenced gray leather COD (chemical oxygen demand).

尽管是主要的轻工业,皮革工业是有代表性的产生污染的工业,因为大量废水和固体废物导致水污染和土壤污染。 Although the major light industry, leather industry is polluting industry representative, because a lot of waste water and solid waste leads to water and soil pollution. 在湿法中使用它们之后将在各个步骤中使用的大部分化学品作为废水排放。 After they are used in a wet most of the chemicals used in each step as wastewater.

为了从原料皮制备皮革制品,使用了大量化学品。 For the preparation of leather from raw hides, using a large number of chemicals. 另外,使用大量水,活化剂,石灰,硫化物,盐类,酸类,铬,合成单宁,染料,乳液加油(或脂)试剂,粘合剂,补充剂,增亮剂和溶剂。 Further, a large amount of water, an activator, lime, sulfides, salts, acids, chromium, synthetic tannins, dyes, fatliquoring (or fat) agents, binders, extenders, brighteners and solvents. 然而,通过在浸灰步骤中过度使用去除原料皮中各种非结构蛋白质所需的硫化物和碱金属,废水污染变得严重。 However, the excessive use of liming step in removing the various raw hide nonstructural proteins required and an alkali metal sulfide, wastewater pollution becomes serious.

在从皮革制备中产生的废水中,包含大量有机和无机物质,如高浓度的含盐硫化物和有机物质,从而增加了污染水平。 In the waste water resulting from the preparation of leather, comprising a large number of organic and inorganic substances, such as high salt concentrations of sulfides and organic substances, thereby increasing the level of contamination. 另外,在皮革制备中,浸灰,脱灰,鞣革和染色步骤需要大量的水,因而将用过的水作为废水排放。 Further, in the manufacture of leather, liming, deliming, tanning and dyeing step requires a lot of water, so that the used water discharged as waste water. 排放的废水包含具有高污染负荷的兽皮碎屑,毛发,可溶性蛋白和中间降解产物,流入废水蓄水池。 The wastewater contains a high pollution load skins of debris, hair, soluble protein and intermediate degradation products, waste water flows into the reservoir. 该废水特征在于存在高毒性重金属一铬,并且BOD(生物需氧量)和COD非常高,其是因为大量的有机和无机物质,和浮游物。 The waste water characterized by the presence of highly toxic heavy metal a chromium and BOD (biological oxygen demand) and COD is very high, it is because a large number of organic and inorganic substances, and float.

为了纯化废水,需要各种设备和试剂,从而导致高三废水处理成本。 For purification of wastewater, requires a variety of equipment and reagents, resulting in the third year cost of wastewater treatment. 此外,从皮革制备过程中产生的生皮的各种固体废物,如去肉碎屑,毛皮碎屑,刮皮碎屑,削匀碎屑相当于50%或更大的原料皮重量。 Further, resulting from the preparation process of leather hides various solid waste, such as chips to meat, fur debris frying chips, shaved chips corresponding to 50% or more by weight of raw hides. 该固体废物的处理成本增加了生产成本。 The solid waste disposal costs increase the cost of production.

发明概述本发明人对皮革制备方法和废物处理方法进行彻底和深入的研究,其目的在于解决在现有技术中遇到的问题,结果发现使用蛋白水解酶制备皮革和处理废物,由此可以获得高质量皮革并且可以减少来自皮革加工的废物的量并可再循环废物,从而完成本发明。 SUMMARY The present invention is a method of preparing leather and waste treatment methods thorough and intensive studies with an object to solve the problems encountered in the prior art, and found that a proteolytic enzyme preparation and use of leather waste treatment, thereby obtaining the and a high-quality leather can reduce the amount of waste can be recycled from waste leather processing, thereby completing the present invention.

因此,本发明的一个目的是提供使用由Aranicola proteolyticus HY-3菌株生产的蛋白酶HY-3制备皮革的方法。 It is therefore an object of the present invention to provide a process for the preparation of leather by using Aranicola proteolyticus HY-3 strains produced protease HY-3.

本发明的另一个目的是提供使用由Aranicola proteolyticus HY-3菌株生产的蛋白酶HY-3处理来自皮革加工的废物的方法。 Another object of the present invention is to provide the use of a Aranicola proteolyticus HY-3 strains produced protease HY-3 treated waste from the method of leather processing.

在本发明的一个方面,提供使用由Aranicola proteolyticus HY-3菌株生产的蛋白酶HY-3制备皮革的方法。 In one aspect of the present invention, there is provided the use of a method of preparing leather Aranicola proteolyticus HY-3 strains produced protease HY-3.

在本发明的另一方面,提供使用由Aranicola proteolyticus HY-3菌株生产的蛋白酶HY-3处理来自皮革加工的废物的方法。 In another aspect of the present invention, there is provided the use Aranicola proteolyticus HY-3 strains produced protease HY-3 treated waste from the method of leather processing.

附图简述图1是显示能够使用本发明蛋白酶HY-3的皮革制备方法的结构示意图。 BRIEF DESCRIPTION FIG. 1 is a diagram showing the structure of the present invention HY-3 protease preparation can be used in the leather.

图2是显示在使用本发明的蛋白酶HY-3时从皮革洗脱的蛋白质的量的曲线图;●:对照■:上清液▲:浓缩的上清液发明详述本发明关于通过使用由Aranicola proteolyticus HY-3菌株生产的蛋白酶HY-3制备皮革的方法。 FIG 2 is a graph illustrating the present invention in an amount of protease HY-3 protein was eluted from leather when a display; ●: control ■: supernatant ▲: concentrated supernatant DETAILED DESCRIPTION The present invention relates to the use by the method leather Aranicola proteolyticus HY-3 strains produced protease HY-3 was prepared.

本发明的方法包含浸水,浸灰,脱灰和软化的步骤(参见图1)。 The method of the present invention comprises soaking, liming, deliming and bating step (see FIG. 1).

为了制备用于本发明皮革制备方法的蛋白酶HY-3,从培养Aranicolaproteolyticus HY-3菌株的培养基中分离微生物,从而使用剩余的包含酶的上清液,或者使用配制为制剂的蛋白酶HY-3与用于提高酶的稳定性的添加剂或者每步引入的物质的混合物。 To prepare HY-3 protease for leather production method of the present invention, isolated from the culture medium Aranicolaproteolyticus HY-3 strain of microorganism, thereby using the remaining supernatant containing enzymes, or using a protease formulation formulated as HY-3 and the mixture of substances for improving the stability of the enzyme or each additive introduction step.

优选以0.1-15重量%的量将本发明的蛋白酶HY-3加至浸水,浸灰,脱灰和软化步骤,并且其是来自微生物培养基的去除微生物的含酶液体,或者可以是与用于提高酶稳定性的添加剂或者每步引入物质混合的制剂。 Preferably in an amount of 0.1-15% by weight of the protease of the invention HY-3 was added to the soaking, liming, deliming and bating step, and removing the microorganism from which the enzyme-containing liquid medium, or may be used with or formulation additives to enhance the stability of the enzyme at each step of mixing the substance introduced.

本发明的蛋白酶HY-3在37℃具有最大活性,在20-40℃具有75%或更高的相对活性。 HY-3 protease of the present invention has a maximum activity at 37 ℃, having 75% or higher relative activity at 20-40 ℃. 此外,该蛋白酶在pH 8.0显示最大活性,在pH 7.0-9.5显示80%或更高的相对活性(韩国专利申请号2000-5479)。 Furthermore, the maximum activity displayed protease pH 8.0, 7.0-9.5 show 80% or higher relative activity (Korean Patent Application No. 2000-5479) at pH. 通常,浸水,浸灰和软化步骤是在25-35℃和pH 8-9下进行,因此使用蛋白酶HY-3可以稳定分解生皮中的蛋白质。 Typically, the soaking, liming and bating step is carried out at 25-35 deg.] C and pH 8-9, thus HY-3 protease can be stabilized protein decomposition rawhide. 此外,当将大量的盐类加至浸水,浸灰,脱灰和软化步骤时,大部分蛋白水解酶的活性降低,而本发明的蛋白酶HY-3保持它的活性,即使在10%的高盐浓度下,因此可以用于各个步骤。 Further, when a large amount of salt added to the soaking, liming, deliming and bating step, most of the activity of proteolytic enzyme is lowered, and HY-3 protease of the present invention retains its activity even at the high 10% salt concentration, can be used for each step.

通过使用蛋白酶HY-3,可以急剧减少常规使用的用于皮革制备的有机和无机化学品的量,从而可以进行有利于环境的皮革加工。 By using a protease HY-3, you can drastically reduce the amount of the inorganic and organic chemicals conventionally used for the preparation of leather, leather processing which can be beneficial to the environment.

因此,可以将该蛋白酶HY-3用于皮革加工,特别是可以用于贯穿浸水,浸灰,脱灰和软化步骤的表皮,毛发和可溶性蛋白质的去除。 Thus, the HY-3 protease for leather processing, in particular can be used throughout the soaking, liming, deliming and bating step of removing the skin, hair and soluble protein.

在浸水步骤中,当将进行揉盐处理的动物生皮或兽皮运送至皮革加工厂时,为了去除各种污物和多余成分应该将足量的水加至划槽或桶中,其后在长时间内吸收于生皮组织中的水使得生皮组织能够恢复活的动物的正常生皮柔度。 In the soaking step, when the salt kneading process will be an animal hide or hides to leather processing transport, in order to remove various contaminants and unwanted ingredients should be a sufficient amount of water was added to a bucket or paddle, thereafter a long time to absorb the water in the raw dermal tissue such that tissue can be restored rawhide living animal skin softness normal growth. 因此,可以分解附着在原料皮或兽皮的多余的可溶性蛋白质,污物和毛发,然后通过蛋白酶HY-3的处理去除。 Thus, the decomposition can be attached to the excess soluble protein, hair, dirt and raw skin or hide, and then removed by protease treatment of HY-3. 通过该方法,兽皮组织变得光滑,可以容易的进行接下来的浸灰步骤。 By this method, the hide tissue becomes smooth and can easily perform the next step liming.

在强烈影响从皮革加工产生的废水中COD的浸灰和脱灰步骤中,蛋白酶HY-3可以分解生发层细胞或毛根基底细胞(base cell),其应用不分解毛发的皮层而分解髓质的原理。 Strong impact of wastewater from leather processing liming and deashing steps the COD, the protease may be decomposed HY-3 cells or germinal layer Mao Genji bottom cell (base cell), the application does not decompose decomposed hair cortex medulla principle. 另外,去除毛根和毛发滤泡,这样毛腔肯定膨胀,于是消除毛发,因此石灰和其它化学品可以容易地渗透,这样可以减少化学品的处理量。 Further, removal of the hair root and hair follicle, and hair cell affirmative expansion, thus eliminating hair, lime and other chemicals and therefore can easily penetrate, so that the processing amount of chemicals can be reduced.

在软化步骤中,蛋白酶HY-3去除原料皮中多余的蛋白质并疏松皮肤组织,由此在软化步骤后供应的用于皮革加工的化学品可以容易地渗透于组织中并且提高了化学品和组织之间的粘合强度。 In the softening step, the HY-3 protease removing excess protein raw hide and loose skin tissue, thereby softening step after the supply for the leather processing chemicals can easily penetrate into the tissue and to improve the chemical and organizational between the adhesive strength. 因此,减少了该化学品所需的量,抑制了废水中由未结合化学品导致的COD和BOD的增大。 Thus, the amount required for the chemical, the wastewater is increased by the suppression of unbound COD and BOD caused by the chemicals. 通过使用蛋白酶HY-3可以生产柔软和柔顺的皮革并可去除生皮上的污染物。 By using a protease HY-3 can produce soft and supple leather and remove contaminants on the rawhide. 由此,在染色时,改善了颜色的染色均匀性,因此减少了染料的量和极大提高了皮革商品的质量。 Thus, in dyeing, dyed color uniformity is improved, thereby reducing the amount of dye and greatly improved the quality of the leather product.

在本发明的另一个实施方案中,提供使用蛋白酶HY-3处理来自皮革制备过程的废物的方法。 In another embodiment of the present invention, there is provided a method HY-3 protease treated waste from the manufacturing process of the leather.

用本发明的蛋白酶HY-3处理从浸水,浸灰,脱灰,软化和整理步骤产生的废物(参见图1)。 Waste with a protease HY-3 of the present invention, processes from soaking, liming, deliming, softening and finishing step (see FIG. 1).

废物可以以液态或固态的形式形成,并且还可以再循环。 Waste may be formed in the form of liquid or solid, and may also be recycled.

适用于本发明皮革制备的蛋白酶HY-3由微生物培养基,将微生物从培养基中分离之后剩余的含酶液体,或者与用于增加酶稳定性的添加剂或者每步引入物质混合的配制的制剂制成。 Suitable for the preparation of the present invention to a leather remaining after protease HY-3, a microorganism isolated from the microbial culture medium prepared from enzyme-containing liquid formulation, or with an additive for increasing the stability of the enzyme at each step or substance mixture is introduced production.

为了处理来自皮革制备过程的废物,将选自脂酶和淀粉酶中的一种与蛋白酶HY-3一起使用。 In order to dispose of waste from a leather manufacturing process, the selected lipase and amylase in a protease HY-3 used together.

为了处理来自皮革制备过程的废物,将生产蛋白酶HY-3的微生物培养基,将微生物从培养基中分离之后剩余的含酶液体,或者与用于增加酶稳定性的添加剂或者每步引入物质混合的配制制剂加至在浸水和浸灰步骤期间产生的废水和固体废物,在软化步骤期间产生的固体废物,和在整理步骤后的固体废物之中。 In order to dispose of waste from a leather manufacturing process, the HY-3 protease production medium of the microorganism, the microorganism remaining after the enzyme-containing culture medium is separated from the liquid, or an additive for increasing the stability of the enzyme at each step or substance mixture is introduced the formulation was added to the formulation of solid waste and waste water during soaking and liming step of generating, during a softening step in solid wastes, and solid waste at the finishing step.

从皮革制备中产生的废物相当于40-70重量%原料皮或兽皮的初始重量。 From waste leather preparation equivalent to 40-70 wt% of the initial weight of raw hides or skins. 大部分排放的固体废物被归类为工业废物并因此烧尽或简单地掩埋。 Most emissions from solid waste is classified as industrial waste and therefore simply burned or buried. 因此,从生产成本来看,这些固体废物的处理成本相当高。 Thus, the production cost point of view, the solid waste disposal costs relatively high.

可以通过蛋白酶HY-3处理的固体废物的实例有在浸水和浸灰步骤期间产生的来自原料皮的去肉碎屑,削匀碎屑和毛发;在软化步骤后的毛皮碎屑;和在最后染色后的整理步骤中产生的生皮碎屑。 Examples of the solid waste can by protease HY-3 debris from the treated meat material to the skin during the soaking and liming step of generating, shaving debris and hair; fur debris after the softening step; and a final finishing step after dyeing debris generated rawhide.

在去肉碎屑和削匀碎屑中,蛋白质和脂类未完全分离并以混合状态存在,其中脂类组分相当于总组分的30-50%。 In fleshing shaving debris and debris, proteins and lipids are not completely separated and is present in a mixed state, wherein the lipid component is equivalent to 30-50% of the total composition. 对于去肉碎屑和削匀碎屑,可以加入选自蛋白酶HY-3,脂酶和淀粉酶中的至少一种酶。 For fleshing shaving debris and chips, may be added selected from proteases HY-3, at least one enzyme lipase and amylase.

固体废物,如从在浸灰步骤之后的软化步骤产生的毛皮碎屑,包含大约40%的从皮革加工产生的总废物。 Solid waste, such as fur debris generated from the softening step after the liming step, comprising approximately 40% of the total waste generated from the processing of leather. 毛皮碎屑包含4.0%脂类,1.5%钙,5.5%灰分,50-55%水和35-40%蛋白质。 Fur debris containing 4.0% lipids, 1.5% calcium, 5.5% ash, 50-55% water and 35-40% protein. 本发明的蛋白酶HY-3,可用作金属蛋白酶,当金属离子存在时它的活性增大。 Proteases of the invention HY-3, can be used as metalloproteases, when the metal ions are present in its activity is increased. 在1mM的钙离子的存在下蛋白酶HY-3的活性增大约1.5倍,在其它金属离子的存在下增大1.2-1.4倍。 HY-3 protease in the presence of 1mM calcium ion activity increased about 1.5 times, 1.2 to 1.4-fold increase in the presence of other metal ions. 金属离子如钙离子在毛皮碎屑中大量存在,因此本发明的蛋白酶HY3有效分解毛皮碎屑中的蛋白质。 Metal ions such as calcium ions in the presence of a large amount of debris in the fur, the present invention is therefore a protease HY3 fur effective decomposition of protein debris.

为了从原料皮制备皮革商品,必须使用大量化学品。 To prepare the leather from raw hides trade, a large amount of chemicals must be used. 此外,使用水,活化剂,石灰,盐类,酸类,铬,合成单宁,染料,乳液加油(或脂)试剂,粘合剂,补充剂,增亮剂和溶剂。 In addition, the use of water, an activator, lime, salts, acids, chromium, synthetic tannins, dyes, fatliquoring (or fat) agents, binders, extenders, brighteners and solvents. 在浸灰步骤中过度使用去除生皮中各种非结构蛋白质所需的硫化物和碱金属导致严重的废水污染。 Removing the excessive use of the various rawhide nonstructural proteins required for the alkali metal sulfide and cause serious water pollution in the liming step. 从皮革加工设备排放的废水包括有机、无机和浮游物质,其特征在于废水中的BOD和COD非常高并且存在铬,一种具有高毒性的重金属。 Leather processing apparatus from the wastewater emissions include organic, inorganic, and floating material, characterized in that the wastewater BOD and COD is very high and the presence of chromium, having a highly toxic heavy metal. 通过使用蛋白酶HY-3,可以减少活化剂,石灰,硫化物,盐类,酸类和铬的量。 By using a protease HY-3, the amount of activator, lime, sulfides, salts, acids and chromium can be reduced.

同时,报道了使用蛋白水解酶的废物再循环(韩国专利号1994-0007333),然而,还未报道在废物中使用产生蛋白酶的微生物分解蛋白质。 At the same time, the recycling of waste reported (Korean Patent No. 1994-0007333) proteolytic enzymes, however, has not reported the use of a protease produced by microorganisms break down proteins in the waste. 本发明的微生物即使在少量碳源和氮源的存在下也能茁壮成长,由该微生物分泌的蛋白酶HY-3可以分解来自皮革加工的固体废物。 Microorganism of the invention even in the presence of small amounts of carbon and nitrogen sources can thrive, protease secreted by the microorganism may be decomposed HY-3 solid waste from the processing of leather. 使用蛋白酶HY-3,可以将废物中的蛋白质水解成肽来制备食品,化妆品和工业产品。 Protease HY-3, may waste proteolysis into peptides prepared foods, cosmetics and industrial products. 另外,可以将该蛋白酶用于在浸水和浸灰步骤期间产生的含盐废水,由此通过蛋白酶HY3的分解可以再循环废水中的蛋白质。 Further, the proteases may be used during the soaking and saline wastewater liming step of generating, whereby the waste water may be recycled by decomposition of proteins in the proteasome HY3.

此外,通过切割胶原蛋白分子中的多肽链可以将废皮革用作饲料和可食用明胶。 Further, the polypeptide chain cleavage collagen molecules may be used as feed and leather waste edible gelatin. 通过将蛋白酶HY-3用于固体皮革废物的处理,可以防止环境污染并且可以获得二级产品。 By HY-3 protease for processing solid wastes of leather, and to prevent environmental pollution can be obtained two products.

实施例借助下列实施例可以获得对本发明更好的理解,阐明所述实施例是为了举例说明而不可理解为是来限制本发明。 Example A better understanding of the following by means of embodiments of the invention may be obtained, the embodiments set forth for purposes of illustration and not to be understood as limiting the invention.

实施例1:生产蛋白酶HY-3为了生产蛋白酶HY-3,将用于培养微生物的标准培养基在高压下121℃灭菌20分钟,然后基于培养基总体积加入0.1-5体积%的量的产生蛋白酶HY-3的Aranicola proteolyticus HY-3菌株(KCTC 0268BP)并在25-30℃培养25-30小时。 Example 1: Production HY-3 protease for protease production HY-3, for standard microbiological culture medium was sterilized 121 ℃ 20 minutes at high pressure, then, based on the total volume of medium added in an amount of 0.1 to 5% by volume HY-3 protease generating the strain Aranicola proteolyticus HY-3 (KCTC 0268BP) and incubated at 25-30 ℃ 25-30 hours. 将培养基进行膜过滤,这样将上清液从生物量中分离。 The membrane filtration medium, so that the supernatant was separated from the biomass. 当需要时,通过10kDa的膜过滤将上清液浓缩3-10倍。 When required, by membrane filtration of 10kDa supernatant was concentrated 3-10 fold.

实施例2:分析洗脱自使用蛋白酶HY-3的浸灰和脱灰步骤的蛋白质为了研究在极大影响COD的浸灰和脱灰步骤中蛋白酶HY-3的作用,实施下列程序。 Example 2: Analysis of protease was eluted from the liming and HY-3 deashing step in order to study a protein protease HY-3 liming and in deliming step greatly affect the COD, the implementation of the following procedure.

具体地,将20g生皮切割成适当的大小,放入50ml试管中,然后向其中加入10ml水。 Specifically, the raw Peel 20g cut into an appropriate size, placed in 50ml test tube, and then 10ml of water was added thereto. 此后,加入0.5%亚硫酸氢钠和0.5%硫酸铵并且在室温下反应15分钟。 Thereafter, 0.5% sodium bisulphite and 0.5% ammonium sulfate and reacted at room temperature for 15 minutes. 反应后,将0.2%洗涤剂和0.5%脱脂剂加至得到的溶液中并在室温下反应30分钟。 After the reaction, 0.2% detergent and 0.5% degreasing agent added to the resultant solution and reacted at room temperature for 30 minutes. 将三支试管分为不含蛋白酶HY-3的对照,和含有如上述实施例1中制备的上清液和浓缩的上清液。 The divided into three test tubes containing no protease control HY-3, and containing supernatant and concentrated supernatant prepared as in Example 1 above. 使每支试管在室温静置,并获得样品。 Each tube was allowed to stand at room temperature to make and obtain a sample. 使用改进Bradford法(Bradford,M.,Anal.Biochem.,1976,72,248)的蛋白质分析试剂盒(Bio-rad,USA)来进行从样品洗脱的蛋白质的分析。 Using a modified Bradford method (Bradford, M., Anal.Biochem., 1976,72,248) protein assay kit (Bio-rad, USA) for analysis of protein eluted from the sample.

从实验结果,可以发现与对照相比,包含蛋白酶HY-3的试管含有更多的洗脱蛋白质,此外培养微生物的培养基的浓缩上清液比未浓缩上清液洗脱的蛋白质要多的多。 From the experimental results, it was found compared with the control, comprising a protease HY-3 tubes containing more protein was eluted, concentrated in addition microorganism culture medium supernatant eluted protein than non-concentrated supernatant to be more many.

实施例3:在软化步骤使用蛋白酶HY-3为了在皮革加工中提高蛋白酶HY-3的稳定性和性能,将蛋白酶配制为制剂并从而用在软化步骤中。 Example 3: HY-3 using a protease proteinase HY-3 in order to improve the stability and performance of leather processing, protease and thus formulated into preparations for use in the softening step, the softening step.

将冻干的本发明的蛋白酶HY-3以0.5-10重量%的量加至40-50%氯化铵(NH4Cl),40-50%硫酸铵((NH4)2SO4),0.005-0.01%氯化钙(CaCl3)和0.025-0.1%乳糖之中。 HY-3 protease of the present invention in an amount of lyophilized 0.5-10% by weight of ammonium chloride was added to 40-50% (NH4Cl), 40-50% ammonium sulfate ((NH4) 2SO4), 0.005-0.01% chlorine calcium (CaCl3) and 0.025-0.1% in lactose. 为了检验配制的蛋白酶HY-3的软化作用,将生皮进行脱灰步骤,然后进行使用不同酶制剂的软化步骤,其后对生皮的表面状态进行观察。 To test the effect of proteinase HY-3 formulated softening of the hides deashing step, followed by a softening step using different enzyme preparations, and thereafter the hides surface state was observed. 另外,将生皮进行鞣革和整理步骤,然后观察表面状态。 Further, the pelt tanning and finishing step, and then the surface state was observed. 作为在用于比较的软化步骤中使用的蛋白酶制剂,使用Amoron(ChungmuFermentation,Korea)和Oropon K(TFL,Germany)。 As the protease used in the softening step preparation for comparison using Amoron (ChungmuFermentation, Korea) and Oropon K (TFL, Germany). 实验步骤在下面表1中总结。 Experimental procedures are summarized in Table 1 below.

表1 Table 1

在软化步骤加入本发明的蛋白酶HY-3和对照(Amoron和Oropon K)。 Added softening step of the present invention and a control protease HY-3 (Amoron and Oropon K). 更具体地,在脱灰步骤,加入表1中所示化学品1小时,然后在软化步骤加入0.2重量%乳酸和0.1-15重量%蛋白酶。 More specifically, in the deliming step, addition of chemicals in Table 1 in FIG. 1 hour, followed by addition of 0.2 wt.% Lactic acid and 0.1-15% by weight protease softening step. 软化步骤进行80分钟,然后进行使用含有盐分的强酸化学品处理的浸酸步骤12小时或更久,含铬的鞣革步骤进行大约12小时,接着干燥生皮。 Softening step carried out for 80 minutes and then acid pickling step process using chemicals containing salt for 12 hours or longer, chromium tanning step is performed for about 12 hours, followed by drying hides. 此后,观察生皮。 Afterwards, the rawhide.

为了比较使用的酶制剂在每步的作用,将生皮进行脱灰,软化和浸酸步骤而仅在软化步骤使用不同的酶制剂。 In order to compare enzyme preparation used in each step of the action of the pelt deliming, pickling and softening steps and only using different enzyme preparations in the softening step. 观察软化后的生皮并分析生皮的湿铬鞣革状态。 Softened hides observed after analysis of raw hides and wet blue state.

在软化后,与对照相比,用配制的蛋白酶HY-3处理的生皮在perioplie真皮的粒面(grain)和表面方面更清洁和更光滑。 After softening, compared with a control, formulated hides treated HY-3 protease and surface cleaner and smoother aspects in perioplie dermis grain (grain). 至于湿铬鞣革的粒面,通过蛋白酶HY-3处理的生皮表面更软并且白度更高。 As grain wet blue, and green coat surface by higher HY-3 protease treated softer whiteness.

比较例1:比较通过在软化步骤加入脱灰剂制备的皮革的性能为了研究在不同脱灰剂的存在下蛋白酶HY-3的活性,除了在软化步骤加入每种脱灰剂,如3重量%氯化铵(NH4Cl),3重量%硫酸铵((NH4)2SO4),2重量%硫酸铵((NH4)2SO4)和1重量%氯化铵(NH4Cl),和上面表1同样的方法实行本实施例,然后往那里加入蛋白酶HY-3。 Comparative Example 1: Comparative properties of the leather softened by the step of preparing deliming agent is added in order to investigate the activity in the presence of various protease deliming agent of HY-3, except for adding the softening step each deliming agent, such as 3 wt% ammonium chloride (NH4Cl), 3 wt.% ammonium sulfate ((NH4) 2SO4), 2 wt% ammonium sulfate ((NH4) 2SO4) and 1 wt% ammonium chloride (NH4Cl), and the same method of practicing the above table 1 embodiment, where the protease is added to and HY-3. 对于每种脱灰剂的加入比较生皮的物理性质。 For each of the deliming agent is added to the physical properties of Comparative rawhide.

通常,加入脱灰剂以增大软化作用。 Typically, deliming agent is added to increase the softening effect. 在加入脱灰剂时,检查由该脱灰剂导致的对蛋白酶HY-3的影响。 When addition of deliming agents, examine the effects of proteinase HY-3 by the deliming agent caused.

1-1加入氯化铵在进行与表1相同步骤的同时,在软化步骤中加入3重量%氯化铵(NH4Cl)并搅拌60分钟。 1-1 while carrying out ammonium chloride was added and the same procedure as in Table 1, was added 3 wt% ammonium chloride (NH4Cl) in the softening step and stirred for 60 minutes. 此外,加入蛋白酶HY-3,Amoron和Oropon K中的每一个。 Furthermore, addition of the protease HY-3, each of Amoron and Oropon K. 将生皮切割成宽30cm×长20cm。 Peel raw cut into length of 30cm × width 20cm. 作为对照,在软化步骤未使用脱灰剂。 As a control, softening step deliming agent is not used.

比较获得的生皮它们的性能。 Hides obtained by comparing their performance. 结果在下面表2中给出。 The results are given in Table 2 below.

表2 Table 2

1-2加入硫酸铵在进行与表1相同步骤的同时,在软化步骤中加入3重量%硫酸铵((NH4)2SO4)并搅拌60分钟。 Ammonium sulfate was added 1-2 while carrying out the same procedure of Table 1, was added 3 wt.% Ammonium sulfate ((NH4) 2SO4) in the softening step and stirred for 60 minutes. 此外,使用蛋白酶HY-3,Amoron和OroponK。 In addition, protease HY-3, Amoron and OroponK. 将生皮切割成宽30cm×长20cm。 Peel raw cut into length of 30cm × width 20cm. 作为对照,在软化步骤未使用脱灰剂。 As a control, softening step deliming agent is not used.

比较获得的生皮它们的性能。 Hides obtained by comparing their performance. 结果在下面表3中给出。 The results are given in Table 3 below.

表3 table 3

1-3加入氯化铵和硫酸铵在进行与表1相同步骤的同时,在软化步骤中加入1重量%氯化铵(NH4Cl)和2重量%硫酸铵((NH4)2SO4)并搅拌60分钟。 Ammonium chloride, and ammonium sulfate was added 1-3 while carrying out the same procedure as in Table 1, the addition of 1 wt% ammonium chloride (NH4Cl) in the softening step and 2 wt% ammonium sulfate ((NH4) 2SO4) and stirred for 60 minutes . 此外,使用蛋白酶HY-3,Amoron和Oropon K。 In addition, protease HY-3, Amoron and Oropon K. 将生皮切割成宽30cm×长20cm。 Peel raw cut into length of 30cm × width 20cm. 作为对照,在软化步骤未使用脱灰剂。 As a control, softening step deliming agent is not used.

比较如此获得的生皮它们的性能。 Comparing the thus obtained hides their performance. 结果在下面表4中给出。 The results are given in Table 4 below.

表4 Table 4

如上所述,当在软化步骤中单独使用硫酸铵((NH4)2SO4)时,使用蛋白酶HY-3之后的生皮的表面类似于使用Amoron和Oropon K之后的生皮表面。 As described above, when used alone ammonium sulphate ((NH4) 2SO4) softening step, the surface of the green HY 3-skin after use similar proteases surface after Amoron rawhide and Oropon K. 当单独使用氯化铵(NH4Cl)和氯化铵(NH4Cl)与硫酸铵((NH4)2SO4)的混合物时,用蛋白酶HY-3制备的生皮的伸长率是优异的。 When using ammonium chloride (NH4Cl) and ammonium chloride (NH4Cl) and ammonium sulfate ((NH4) 2SO4) mixture alone, with a protease HY-3 Elongation prepared hides are excellent. 与Amoron和Oropon K相比,生皮具有更平滑的表面和更清洁的粒面。 Compared with Amoron and Oropon K, raw skin has a smoother surface and a grain cleaner. 在收缩率和柔软度方面,结果相似。 In the shrinkage and softness similar results.

当蛋白酶HY-3仅与硫酸铵一起使用时,发现中等效果,当单独加入氯化铵或氯化铵与硫酸铵的混合物时,发现协同效应。 When using only HY-3 protease with sulfate, found moderate effect, when added to the mixture with ammonium chloride or ammonium sulfate alone, a synergistic effect was found.

比较例2:测量实际使用的生皮的性能将具有1.2-1.4mm厚度的生皮进行浸灰步骤,分为两片,其中一片加入蛋白酶HY-3,另外一片加入进口软化剂。 Comparative Example 2: rawhide rawhide performance measurement actually used will have a thickness of 1.2-1.4mm were liming step, is divided into two, wherein a protease is added HY-3, an additional inlet was added softeners. 然后,进行软化。 Then, softening. 将生皮制成外皮(即进行鞣革过程以制备准备制备皮革商品(例如手提包和鞋)的生皮),其然后进行如下面表5中的方法。 The hides the skin is made (i.e. to prepare a process for tanning leather prepared ready goods (e.g. handbags and shoes) hides), which is then carried out as in the following Table 5 method. 观察生皮的表面状态并测量生皮的机械强度。 Observation of the surface state of the rawhide and rawhide mechanical strength was measured.

表5 table 5

当使用蛋白酶HY-3和进口Oropon K时,关于生皮的性能在下面表6中显示。 When using HY-3 protease and import Oropon K, performance with respect to the hides are shown in Table 6 below.

表6 Table 6

从表6的结果,可以发现蛋白酶HY-3在几乎所有性能上比进口的Oropon K更优异。 The results of Table 6, it was found proteinase HY-3 ratio in almost all imported Oropon K performance more excellent. 在拉伸强度方面,蛋白酶HY-3处理的皮革是2.5-2.7kg/mm2,而Oropon K处理的皮革是1.1-2.4kg/mm2。 Tensile strength, HY-3 protease treated leather is 2.5-2.7kg / mm2, and the leather is treated Oropon K 1.1-2.4kg / mm2. 在抗撕强度方面,前者是5.1-7.3kg/mm2,而后者是4.1-5.8kg/mm2。 In terms of the tear strength, the former is 5.1-7.3kg / mm2, while the latter is 4.1-5.8kg / mm2. 此外,在崩裂强度方面,前者是40kg/mm2或更大,而后者是32-40kg/mm2。 Further, in the bursting strength, the former is 40kg / mm2 or more, and the latter is 32-40kg / mm2. 至于延伸率和柔软度,Oropon K处理的皮革显示为59-82%和3.5-4.2%,而本发明的蛋白酶HY-3生产延伸率为60-77%并且柔软度为3.3-3.9%的皮革。 As elongation and softness, leather processing Oropon K is shown as 59-82% and 3.5-4.2%, while the protease of the invention HY-3 60-77%, and producing an elongation of 3.3-3.9% of the softness of the leather .

比较例3:通过蛋白酶HY-3的蛋白分解为了比较通过由Aranicola proteolyticus HY-3菌株(KCTC 0268BP)生产的蛋白酶HY-3和其它蛋白酶的蛋白质分解,使用本发明的蛋白酶HY3和进口的Oropon K。 Comparative Example 3: decomposition by a protease in order to compare protein HY3 decomposed by the Aranicola proteolyticus HY3 strain (KCTC 0268BP) produced HY3 protease proteins and other proteases, and the protease HY3 inlet invention Oropon K . 作为胰酶制剂,将Oropon K广泛用于增大粒面的拉伸强度和柔软度,并通过分解胶原蛋白而不损伤皮革的粒面来保持柔软的粒面。 As pancreatin, it is widely used to increase the Oropon K tensile strength and softness of the grain, by decomposing and without damaging the collagen to maintain grain leather soft grain.

用如下方法(Braun,V.& Schmitz,G.,Arch,Microbiol.1980,124:55-61)测量蛋白酶HY-3的活性。 HY-3 protease activity measurement:; the following manner (55-61 Schmitz, G., Arch, Microbiol.1980,124 Braun, V & amp.). 将0.24g偶氮酪蛋白(Sigma,USA)溶解在10ml 50mM的磷酸盐缓冲液,pH 7.5中来制备底物溶液。 The 0.24g azocasein (Sigma, USA) was dissolved in 10ml 50mM phosphate buffer, pH 7.5 to prepare a substrate solution. 将300μl的底物溶液与100μl培养基混合并在37℃反应30分钟。 300μl of substrate solution was mixed with 100μl of culture medium and reacted at 37 ℃ 30 minutes. 向反应物加入300μl 10%三氯乙酸盐并在室温下另外反应1小时。 Add 300μl 10% trichloroacetate was added to the reaction and additionally reacted at room temperature for 1 hour. 将得到的反应物在7,000rpm下离心并将沉淀从上清液中分离。 The resulting reaction was separated from the supernatant and the precipitate was centrifuged at 7,000rpm. 在300μl上清液中加入30μl10%氢氧化钠,然后在420nm测量吸光度。 30μl10% sodium hydroxide was added 300μl supernatant, and then absorbance was measured at 420nm. 将1单位本发明的酶定义为在37℃,消化偶氮-酪蛋白测试底物1分钟后释放足以将吸光度提高1.0的偶氮和酪蛋白的量的酶量。 1 unit of enzyme is defined in the present invention is 37 ℃, digested azo - casein test substrate released amount of enzyme sufficient to increase the absorbance amount of the azo casein and 1.0 after 1 minute.

将1单位蛋白酶HY-3和Oropon K各自加入酪蛋白,清蛋白,血红蛋白和角蛋白的蛋白质底物混合物(1mg/ml),和胶原蛋白与弹性蛋白的蛋白质底物混合物(5mg/ml),然后在37℃反应2小时。 The 1 unit of protease HY-3 and Oropon K are each added casein, albumin, a protein substrate a mixture of hemoglobin and keratin (1mg / ml), and substrate mixture of collagen and elastin proteins (5mg / ml), then reacted at 37 ℃ 2 hours. 此后,使用Bradford法测量样品中蛋白质的量(参见表7)。 Thereafter, using the Bradford method measuring the amount of protein in the sample (see Table 7). 然后将1单位酶定义为在37℃从底物的蛋白水解消化1分钟产生1μg蛋白质等价物(equivalent)所需的量。 Then, 1 unit of enzyme is defined as the proteolytic digestion of the substrate 1 minutes at 37 [deg.] C to produce the desired amount of 1μg protein equivalents (equivalent).

因此,与Oropon K相比,对于除了血红蛋白的大部分底物,本发明的蛋白酶HY-3显示更高的分解活性。 Therefore, compared with Oropon K, except for most of the hemoglobin substrate, the protease of the invention exhibits higher HY-3 decomposition activity. 通常,动物生皮包含胶原蛋白,弹性蛋白,角蛋白等结构蛋白,和清蛋白,球蛋白等非结构蛋白。 Typically, animal hides comprising structural protein collagen, elastin, keratin, albumin and globulin and other non-structural proteins. 蛋白酶HY-3可以分解在生皮中也存在的酪蛋白和清蛋白,特别是具有针对毛发主要成分角蛋白的极好分解活性,以致它因此可以在浸灰和脱灰步骤中发挥作用。 HY-3 protease may also decompose in the presence of rawhide casein and albumin, in particular having an excellent decomposition activity for hair keratin main component, so that it can play a role in liming and deashing steps.

通过用蛋白酶消化10分钟,可以将角蛋白和胶原蛋白分解40%或更多。 By digestion with proteases for 10 minutes, the keratin and collagen can be decomposed by 40% or more. 除了血红蛋白之外的蛋白质被蛋白酶HY-3高度分解,结果示于下表7。 In addition to the hemoglobin protein is a protease HY-3 highly decomposition results are shown in Table 7.

表7 Table 7

另外,与Oropon K相比,蛋白酶HY-3对胶原蛋白的活性高4倍多。 Further, as compared with Oropon K, proteinase HY-3 activity on collagen 4 times higher. 结果在下面的表8中显示。 The results are shown in Table 8 below. 结构蛋白,构成大多数动物生皮,由大约60%或更多的胶原蛋白组成,胶原蛋白另外在骨,肌肉和腱中存在。 Structural proteins, constituting the most animal hides from about 60% or more of collagen, collagen additionally present in the bone, muscles and tendons. 因此认为如果给定的蛋白酶可以有效分解胶原蛋白,它对于皮革加工具有极好的效果。 So that if a given protease may effectively breaks down collagen, which has an excellent effect for leather processing.

表8 Table 8

工业适用性如上所述,通过使用蛋白酶HY-3制备皮革的方法和本发明处理来自皮革加工的废物的方法,可以获得优秀品质的皮革并且还可以急剧减少在皮革加工中使用的化学品的量,由此以有利于环境的方式处理或者再循环废物。 Industrial Applicability As described above, the processing waste from the leather prepared by the leather processing method of the present invention and a protease 3 HY-process can be obtained and also excellent quality leather can be drastically reduce the amount of chemicals used in the processing of leather thus in a manner conducive to environmental waste treatment or recycling.

已经以举例说明的方式描述了本发明,应当理解使用的术语是意欲具有描述而不是限制的性质。 In the illustrated embodiment has been described with the present invention, it should be understood that the term is intended to have the nature of description rather than of limitation. 根据上述教导可对本发明进行许多修饰和改变。 Many modifications and variations may be made to the present invention of the above teachings. 因此,应当理解在后附权利要求的范围内,可以不同于具体描述来实施本发明。 Thus, it should be understood that within the scope of the appended claims, than as specifically described may be embodied in the present invention.

Claims (6)

1.一种制备皮革的方法,其包含浸水,浸灰,脱灰和软化步骤,其中将由Aranicola proteolyticus HY-3菌株生产的蛋白酶HY-3加至所述浸水步骤,浸灰步骤,脱灰步骤或软化步骤,其中所述Aranicola proteolyticus HY-3菌株的保藏编号为KCTC 0268BP。 A process for producing leather, which comprises soaking, liming, deliming and bating step, wherein the production by strain Aranicola proteolyticus HY-3 protease HY-3 was added to the soaking step, step liming, deliming step or softening step wherein the deposit number Aranicola proteolyticus HY-3 strain is KCTC 0268BP.
2.依照权利要求1的方法,其中所述蛋白酶HY-3是来自所述微生物培养基的去除微生物的含酶液体,或者配制的制剂。 2. The method according to claim 1, wherein said protease HY-3 is prepared from enzyme-containing liquid formulations of the microbe removal of the microbial culture, or.
3.依照权利要求1的方法,其中以每个所述步骤中所用溶液的量的0.1-15重量%的量使用所述蛋白酶HY-3。 3. The method according to claim 1, wherein said each step is in an amount of 0.1-15% by weight of the amount of the solution used with the HY-3 protease.
4.一种用于处理来源于皮革制备的废物的方法,其中将由Aranicolaproteolyticus HY-3菌株生产的蛋白酶HY-3加至来自所述浸水步骤和浸灰步骤的废水和固体废物,来自所述浸灰和脱灰步骤以及所述软化步骤的固体废物,或者来自所述整理步骤的固体废物中,其中所述Aranicolaproteolyticus HY-3菌株的保藏编号为KCTC 0268BP。 4. A process for the preparation of leather waste from the process, wherein by Aranicolaproteolyticus HY-3 strains produced protease HY-3 was added to the waste water and solid waste from the step of soaking and liming steps, from the dip gray and deashing step, and the step of softening the solid waste, or from solid waste in the finishing step, wherein the accession number Aranicolaproteolyticus HY-3 strain is KCTC 0268BP.
5.依照权利要求4的方法,其中所述蛋白酶HY-3是所述微生物培养基,来自所述培养基的去除微生物的含酶液体,或者配制的制剂。 The method according to claim 4, wherein the protease is HY-3 medium the microorganism, enzyme-containing liquid is removed from the culture medium of a microorganism, or prepared formulation.
6.权利要求4的方法,其中所述蛋白酶HY-3与选自脂酶,淀粉酶和其混合物中的一种组合加入。 The method of claim 4, wherein the protease is added to a composition HY-3 is selected from lipase, amylase and mixtures thereof with.
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