Application of modifying enzymes in treating animal skins and leather

Abstract

The present invention belongs to the technical field of leather processing and manufacturing, and discloses an application of a modified enzyme in treating animal skins and leather. The application of the modified enzyme in treating animal skins, wherein the modified enzyme is a modified protease, and the absolute value of the difference between its isoelectric point and the pH value of the animal skin treatment process is not greater than 0.5. The present invention can improve the transfer rate of the protease in the skin, shorten the action time of the protease on the grain layer of the animal skin, and improve the uniformity of the action of the protease on the skin. The present invention can solve the technical problems of easy surface damage or loose surface when using protease to treat animal skins, and improve the quality of leather products.

Description

Technical Field

[0001] The present invention belongs to the technical field of leather processing and manufacturing, and specifically relates to the application of modifying enzymes in treating animal skins and leather. Background Art

[0002] Replacing traditional leather-making chemicals with proteases is an important direction for the leather industry to achieve clean and efficient production. In recent years, leather workers have used protease preparations in processes such as soaking, dehairing, liming, and bating to remove non-collagen proteins in the skin, such as albumin, globulin, elastin, proteoglycan, keratin, etc., in order to achieve rapid soaking, efficient dehairing, and moderate dispersion of collagen fibers, etc., creating suitable conditions for the penetration and combination of materials in subsequent tanning and finishing sections.

[0003] In order to obtain high-quality leather, it is necessary to minimize the damage to skin collagen fibers and ensure the basic integrity of the structure of skin collagen fibers during the process of treating animal skins with proteases. However, the specificity of protease catalysis belongs to relative specificity (i.e., bond specificity), rather than absolute specificity (i.e., substrate specificity). Although this relative specificity still endows proteases with the ability to selectively hydrolyze a certain peptide bond, it cannot enable proteases to highly selectively hydrolyze only a certain protein. For non-collagen proteins and collagen, although their molecular structures are quite different, they contain some of the same types of peptide bonds. Therefore, almost every protease has the ability to hydrolyze both types of proteins simultaneously, but the degree of hydrolysis will vary due to differences in affinity, catalytic efficiency, etc. For example, trypsin, the core component of the enzyme preparation for leather bating, is recognized as one of the proteases that act most gently on skin collagen fibers. However, while it hydrolyzes and removes non-collagen proteins such as albumin, globulin, and elastin, it may still damage the collagen fibers on the surface of the leather due to improper design or control of the leather-making process (enzyme dosage, temperature, time, etc.), resulting in product quality problems such as surface damage and loose surface. In addition, protease molecules with a relative molecular mass of 20 - 50 kDa penetrate slowly in the porous medium of leather and will accumulate more in the surface layer of the leather, with uneven spatial distribution. Coupled with the high catalytic efficiency of proteases, the collagen fibers on the surface of the leather are undoubtedly exposed to the enzyme for a longer time than those in the middle layer, with uneven time distribution, which makes the leather enzyme treatment technology prone to damage the surface morphology of the leather (surface damage) and reduce the surface strength (loose surface).

[0004] Chinese Patent CN109880939B, "An Auxiliary Agent for Enhancing the Mass Transfer of Protease in the Skin and Its Application", discloses a method of using a protein with an isoelectric point < 5.5 as an auxiliary agent together with an existing protease preparation in the processes of soaking, dehairing, or softening to reasonably regulate the electrostatic interaction between the protease and the animal skin, thereby increasing the mass transfer rate of the enzyme molecules in the skin, shortening the residence time and action time of the protease in the granular layer, improving the uniformity of the hydrolysis of collagen fibers in each layer of the skin by the protease, and reducing the degree of hydrolysis / damage of the skin collagen fibers. This method solves to a certain extent the technical problem that the existing protease preparations for leather making are prone to damage the skin collagen fibers during the treatment of animal skins and seriously reduce the quality of leather. However, by adding this kind of auxiliary agent to regulate the electrostatic interaction between the protease and the animal skin to improve the mass transfer rate of the enzyme, due to the large volume of the bath liquid, a large amount of the auxiliary agent is required. In addition, because the protease and the skin collagen fibers have some similar protein common characteristics, the auxiliary agent will simultaneously change the charged states of both the protease and the leather (i.e., introducing positive or negative charges into both), resulting in a limited range of adjustment of the enzyme-leather electrostatic force and being difficult to precisely control. These problems are obviously not conducive to the efficient improvement of the mass transfer rate of the enzyme by this technology.

[0005] In view of this, the present invention is specifically proposed. Summary of the Invention

[0006] The object of the present invention is to overcome the deficiencies existing in the prior art and provide an application of a modified enzyme in treating animal skins and leather.

[0007] To achieve the above object, the technical solution provided by the present invention is as follows:

[0008] The application of a modified enzyme in treating animal skins, wherein the modified enzyme is a modified protease, and the absolute value of the difference between its isoelectric point and the pH of the animal skin treatment process is not greater than 0.5.

[0009] Preferably, the absolute value of the difference between the isoelectric point of the modified enzyme and the pH of the animal skin treatment process is not greater than 0.3.

[0010] Preferably, the animal skin treatment process includes at least one of leather soaking, dehairing, liming, softening, and acid softening.

[0011] Preferably, the modified enzyme is obtained by modifying a protease with a small molecule compound, the adjustable range of the isoelectric point of the modified enzyme is 1.0 - 13.0, and the small molecule compound can react with the side chain groups of the protease to introduce charged groups or block the dissociable groups of the protease molecule.

[0012] Preferably, the small molecule compound includes aldehydes, acid anhydrides, cyanates, esters, amines, alkanolamines, and haloamides.

[0013] Preferably, the protease includes any one or more of microbial protease, animal protease and plant protease.

[0014] Preferably, the animal skin includes cowhide, sheepskin, pigskin, horsehide, deerhide, ostrich skin, fish skin.

[0015] The present invention also discloses leather obtained by applying any of the above-mentioned modifying enzymes in the treatment of animal skin.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] 1. The method for treating animal skin with a modifying enzyme provided by the present invention precisely changes the charge properties of the enzyme molecules for leather making through the use of small molecule modification technology. The obtained modified protease still has high catalytic activity and little change in molecular size, which can meet the application requirements of the leather industry.

[0018] 2. The present invention uses the obtained modified protease for leather soaking, hair removal, liming, softening, acid softening, etc. By controlling the absolute value of the difference between the isoelectric point of the modified enzyme molecule and the pH of the leather making process to be no more than 0.5, the electrostatic force between the protease and the hide can be significantly reduced, the penetration rate of the protease into the hide can be increased, and the time difference of the action of the protease on the inner and outer layers of the animal skin can be shortened, solving the technical problems of easy surface damage and loose surface in the existing leather making enzyme treatment technology.

[0019] 3. The method for treating animal skin with a modifying enzyme provided by the present invention uses a modified protease with a pH difference of no more than ±0.5 from the leather making process pH to treat animal skin, which can more precisely and effectively regulate the electrostatic interaction between the protease and the animal skin compared with the method of adding external auxiliaries or adjusting the pH.

[0020] 4. The method for treating animal skin with a modifying enzyme provided by the present invention can make the distribution of the protease in the inner and outer layers of the hide more uniform in different enzyme treatment processes, improve the dispersion uniformity of the leather collagen fibers, and is beneficial to obtaining high-quality leather. Detailed Embodiments

[0021] To make the objectives, technical solutions and advantages of the present invention clearer and more understandable, the present invention will be further described in detail below in conjunction with specific embodiments. It should be understood that these descriptions are only exemplary and are not intended to limit the scope of the present invention. In addition, in the following description, the descriptions of well-known structures and technologies are omitted to avoid unnecessarily confusing the concepts of the present invention. For those not specified in the embodiments, they are carried out under conventional conditions or conditions recommended by the manufacturer. For reagents or instruments not specified by the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0022] The electrostatic interaction between enzyme molecules and skin collagen fibers is a key factor affecting the mass transfer behavior of proteases during leather-making processes, and its influence even exceeds process parameters such as enzyme molecule size and enzyme treatment temperature. Although, in existing leather-making processes, the electrostatic interaction between enzymes and leather can also be controlled by adjusting the bath solution pH, which can accelerate the penetration of enzyme molecules into the leather to a certain extent, this is not convenient for the subsequent leather-making processes (the modern leather-making process system is relatively mature, and the pH of each process is basically fixed. Adjusting the process pH requires a large amount of chemicals and increases the operation time on the one hand, and may change the state of the leather and the penetration and binding effects of other chemicals on the other hand). In addition, adding auxiliaries to regulate the electrostatic interaction between enzymes and leather has disadvantages such as a large amount of auxiliaries required and a limited range of adjustment of the enzyme-leather electrostatic force.

[0023] The prior art discloses that when the protease and the leather surface carry the same charge, the electrostatic repulsion between the two can promote the penetration of the protease in the leather. However, the applicant has found that too strong electrostatic repulsion will instead hinder the penetration of the protease in the leather.

[0024] Based on this, an embodiment of the present invention provides an application of a modified enzyme in treating animal skins, wherein the modified enzyme is a modified protease, and the absolute value of the difference between its isoelectric point and the pH of the animal skin treatment process is not greater than 0.5.

[0025] The key point of the embodiment of the present invention is to control the isoelectric point of the modified protease within a range where the difference from the pH of the animal skin treatment process is not greater than ±0.5. The animal skin treatment process described in the embodiment of the present invention includes at least one of soaking, dehairing, liming, bating, and acid bating. For example, the pH of the soaking, dehairing, initial liming, and bating processes is generally 8.0 - 9.0, so a modified enzyme with an isoelectric point of 7.5 - 9.5 is used to treat the leather for soaking, dehairing, liming, and bating; the pH of the acid bating process is generally about 3.5, so a modified enzyme with an isoelectric point of 3.0 - 4.0 is used to treat the wet blue leather for acid bating. The applicant has found in the process of implementing the present invention that the closer the isoelectric point of the protease is to the pH of the leather-making process, the smaller the mass transfer resistance of the enzyme molecules in the leather. Especially when the absolute value of the difference between the isoelectric point of the protease and the pH of the leather-making process is not greater than 0.3, the mass transfer resistance of the enzyme molecules in the leather is the smallest, the penetration is the fastest, the distribution is more uniform, and the leather obtained has the best performance.

[0026] During the existing leather-making process, the isoelectric point of neutral protease is generally around 6.1, that of alkaline protease is generally around 8.9, that of trypsin is generally around 9.2, that of papain is generally around 9.5, that of acidic protease is generally around 2.4, and that of pepsin is generally around 1.9. Moreover, in the existing leather-making process, the difference between the isoelectric point of the protease used due to the different requirements of skin protein hydrolysis and the pH value of the leather-making process is generally 0.8 - 2.0, and the rapid penetration of the protease into the leather cannot be achieved. Especially in the acidic softening treatment stage of leather, the acidic protease used is difficult to achieve rapid penetration because its isoelectric point (generally less than 2.5) is significantly lower than the pH value of 3.5 - 4.0 in the treatment process. Therefore, in this application, the protease is chemically modified by using a small molecule compound, which can react with the side chain groups of the protease to introduce charged groups or block the dissociable groups of the protease molecule, thereby regulating the isoelectric point of the protease, making the absolute value of the difference between the isoelectric point of the modified enzyme and the pH value of the leather-making process not greater than 0.5, and having little impact on the relative molecular mass of the protease. The specific modification method of the protease in the present invention is a commonly used enzyme modification method in the prior art, and the isoelectric point of the modified enzyme can be adjusted within the range of 1.0 - 13.0 by controlling the type, dosage of the reaction reagent and the reaction conditions.

[0027] In some preferred embodiments, the small molecule compound includes aldehydes, acid anhydrides, cyanates, esters, amines, alkanolamines, haloamides and other compounds.

[0028] In some preferred embodiments, the protease includes any one or more of microbial protease, animal protease and plant protease.

[0029] The raw hides, soaked hides, limed hides, delimed hides, wet blue leathers are all prepared from commonly used animal skins in the leather-making field, such as cowhide, sheepskin, pigskin, horsehide, deerhide, ostrich skin, fish skin, etc.

[0030] There is no special limitation on the dosage of the modified enzyme, and those skilled in the art can select the actual dosage according to the treatment effect to be achieved.

[0031] The present invention will be further described in detail below with reference to the embodiments. The following embodiments are only used to further illustrate the present invention and should not be construed as limiting the protection scope of the present invention. Those skilled in the art can make some non-essential improvements and adjustments to the present invention according to the above-mentioned inventive content.

[0032] Example 1

[0033] In this embodiment, multiple groups of samples were set up, differing only in the isoelectric point of the modified enzyme. By measuring the penetration rate of the modified enzyme in the treated animal skin, the influence of the difference between the isoelectric point of the modified enzyme and the pH of the animal skin treatment process on the permeability of the modified enzyme was illustrated.

[0034] The following material dosages are all calculated as mass percentages (wt%) of the raw skin. The preparation method for each sample is as follows:

[0035] Put the sheepskin into a rotating drum, add 200% water, adjust the temperature to 20 °C, add 0.3% sodium carbonate, 0.4% degreasing agent, and 0.05% modified enzyme (protease activity is 2000 U / g). After rotating for 30 min, adjust it to intermittent rotation (rotate for 10 min every hour), with a total time of 8 h to obtain soaked skin. Among them, the pH during the soaking process is 8.7.

[0036] Among them, the preparation method of the modified enzyme is: Mix papain with carboxybenzaldehyde, react at 8 °C for 3 h, and purify and dry.

[0037] Table 1 Comparison table of the penetration rates of modified enzymes with different isoelectric points in sheepskin (soaked for 8 h)

[0038] 。

[0039] Note: a. In the embodiments of the present invention, the penetration rate of the modified enzyme = the penetration depth of the modified enzyme in the skin / the thickness of the skin × 100%.

[0040] Example 2

[0041] The following material dosages are all calculated as mass percentages (%) of the raw skin.

[0042] Put the cowhide into a rotating drum, add 300% water, adjust the temperature to 30 °C, add 0.3% sodium carbonate, 0.2% degreasing agent, and 0.3% modified enzyme (protease activity is 4000 U / g, isoelectric point is 8.3). After rotating for 30 min, adjust it to intermittent rotation (rotate for 10 min every hour), with a total time of 12 h to obtain soaked skin. Among them, the pH during the soaking process is 8.5.

[0043] Among them, the preparation method of the modified enzyme is: Mix 1 part of Bacillus subtilis neutral protease with 30 parts of ethylenediamine, react at 2 °C for 2.5 h, and purify and dry to obtain the modified enzyme.

[0044] In this example, the penetration rate of the modified enzyme in the cowhide is 97.5%.

[0045] Example 3

[0046] The following material dosages are all calculated as mass percentages (wt%) of the raw skin.

[0047] Place the pigskin in a rotating drum, add 400% water, adjust the temperature to 25°C, add 0.2% sodium carbonate, 0.5% degreasing agent, and 0.5% modified enzyme (protease activity of 5000 U / g, isoelectric point of 8.8), rotate for 30 minutes, then adjust to intermittent rotation (10 minutes per hour) for a total of 18 hours to obtain soaked skin. The pH during the soaking process is 8.5.

[0048] The preparation method of the modified enzyme is as follows: 1 part of trypsin is mixed with 10 parts of glycine methyl ester, reacted at 4°C for 6 hours, purified and dried to obtain the modified enzyme.

[0049] In this example, the penetration rate of the modified enzyme in pig skin was 98.3%.

[0050] The following material amounts are all calculated based on the mass percentage (wt%) of the original hide.

[0051] Place horse hides in a rotating drum, add 300% water, adjust the temperature to 22°C, add 0.3% sodium carbonate, 0.3% degreasing agent, and 1% modified enzyme (protease activity of 2000 U / g, isoelectric point of 8.8), rotate for 30 minutes, then adjust to intermittent rotation (10 minutes per hour) for a total of 12 hours to obtain soaked hides. The pH during the soaking process is 8.6.

[0052] The preparation method of the modified enzyme is as follows: 1 part of papain is mixed with 15 parts of disodium 4,4'-diisothiocyanato-2,2'-styrenesulfonate, reacted at 4°C for 6 hours, purified and dried to obtain the modified enzyme.

[0053] In this example, the penetration rate of the modified enzyme into horse hide was 98.6%.

[0054] Example 5

[0055] The following material amounts are all calculated based on the mass percentage (wt%) of the soaked leather.

[0056] Place the soaked cowhide in a drum, add 300% water, adjust the temperature to 30°C, add 3.0% modified enzyme (protease activity 20,000 U / g, isoelectric point 8.3), rotate for 30 minutes, then adjust to intermittent rotation (20 minutes per hour) for a total of 2.5 hours to obtain the dehaired bare hide. The pH during the dehairing process is 8.1.

[0057] The preparation method of the modified enzyme is as follows: 1 part of Bacillus licheniformis alkaline protease is mixed with 15 parts of succinic anhydride, reacted at 4°C for 2 hours, purified and dried to obtain the modified enzyme.

[0058] Comparative Example 1

[0059] The following material amounts are all calculated based on the mass percentage (wt%) of the soaked leather.

[0060] Put the soaked cowhide into a rotary drum, add 300% water, adjust the temperature to 30 °C, add 3.0% of the modified enzyme (protease activity is 20000 U / g, isoelectric point is 8.7), rotate for 30 min, then adjust to intermittent rotation (rotate for 20 min every hour), with a total time of 2.5 h to obtain the dehaired raw hide. Among them, the pH during the dehairing process is 8.1.

[0061] Among them, the preparation method of the modified enzyme is as follows: Mix 1 part of Bacillus licheniformis alkaline protease with 5 parts of succinic anhydride, react at 4 °C for 2 h, and purify and dry to obtain the modified enzyme.

[0062] Comparative Example 2

[0063] The following material dosages are all calculated based on the mass percentage (wt%) of the soaked hide.

[0064] Put the soaked cowhide into a rotary drum, add 300% water, adjust the temperature to 30 °C, add 3.0% of Bacillus licheniformis alkaline protease (protease activity is 20000 U / g, isoelectric point is 8.9), rotate for 30 min, then adjust to intermittent rotation (rotate for 20 min every hour), with a total time of 2.5 h to obtain the dehaired raw hide. Among them, the pH during the dehairing process is 8.1.

[0065] Comparative Example 3

[0066] The following material dosages are all calculated based on the mass percentage (wt%) of the soaked hide.

[0067] Put the soaked cowhide into a rotary drum, add 300% water, adjust the temperature to 30 °C, add 3.0% of Bacillus licheniformis alkaline protease (protease activity is 20000 U / g, isoelectric point is 8.9) and 1.0% of casein (isoelectric point 4.7), rotate for 30 min, then adjust to intermittent rotation (rotate for 20 min every hour), with a total time of 2.5 h to obtain the dehaired raw hide. Among them, the pH during the dehairing process is 8.1.

[0068] Perform corresponding dehairing effect tests on Example 5, Comparative Example 1, Comparative Example 2 and Comparative Example 3. The test results are shown in Table 2. It can be seen that the dehairing rate of the protease in Example 5 is higher than that of Comparative Example 1, Comparative Example 2 and Comparative Example 3, and the hydroxyproline concentration in the waste liquid is lower than that of Comparative Example 1, Comparative Example 2 and Comparative Example 3. This shows that the method for treating animal skins with the modified enzyme provided by the present invention can improve the dehairing effect of animal skins in the dehairing process, and at the same time can reduce the damage of protease to skin collagen fibers.

[0069] Table 2 Comparison table of dehairing effects (dehairing for 2.5 h)

[0070] 。

[0071] Note: a. In the embodiments of the present invention, the hair removal rate = the area of the hairless part of the grain surface of the dehaired raw hide / the total area of the grain surface of the dehaired raw hide × 100%;

[0072] b. The higher the concentration of hydroxyproline (a characteristic amino acid of collagen) in the hair removal waste liquid, the greater the degree of damage to the skin collagen fibers.

[0073] Example 6

[0074] The following material dosages are all based on the mass percentage (wt%) of the soaked hide.

[0075] Load the soaked deer hide into a rotating drum, add 200% water, adjust the temperature to 28 °C, add 0.15% modified enzyme (protease activity is 10000 U / g, isoelectric point is 8.1), rotate for 45 min, add 1.0% lime, rotate for 45 min, add 1.5% sodium sulfide, rotate for 60 min, adjust to intermittent rotation (rotate for 15 min per hour), and the total time is 600 min to obtain the limed raw hide. Among them, the pH at the initial stage of liming is 8.3.

[0076] Among them, the preparation method of the modified enzyme is: mix 1 part of Bacillus licheniformis alkaline protease with 25 parts of succinic anhydride, react at 5 °C for 1 h, and purify and dry to obtain the modified enzyme.

[0077] In this example, the penetration rate of the modified enzyme in the soaked deer hide is 98.6%.

[0078] Example 7

[0079] The following material dosages are all based on the mass percentage (wt%) of the limed raw hide.

[0080] Load the limed ostrich hide into a rotating drum, add 150% water, adjust the temperature to 33 °C, add 1.5% ammonia-free dehairing agent and 0.1% degreasing agent, rotate for 60 min for dehairing, then add 0.5% modified enzyme (protease activity is 5000 U / g, isoelectric point is 8.4), rotate for 30 min, stop for 30 min, adjust to intermittent rotation (rotate for 15 min per hour), and the total time is 180 min to obtain the softened raw hide. Among them, the pH during the softening process is 8.3.

[0081] Among them, the preparation method of the modified enzyme is: mix 1 part of Bacillus subtilis neutral protease with 30 parts of 3-dimethylaminopropylamine, react at 4 °C for 2 h, and purify and dry to obtain the modified enzyme.

[0082] In this example, the penetration rate of the modified enzyme in the limed ostrich hide is 100%.

[0083] Example 8

[0084] The dosages of the following materials are all based on the mass percentage (wt%) of the limed fish skin.

[0085] Load the limed fish skin into a rotating drum, add 150% water, adjust the temperature to 30 °C, add 3.0% ammonium sulfate and 0.1% degreasing agent, rotate for 60 min for deliming, then change the liquid, add 200% water, adjust the temperature to 32 °C, add 0.3% modifying enzyme (protease activity is 10,000 U / g, isoelectric point is 8.2), rotate for 30 min, stop for 30 min, to obtain softened fish skin. Among them, the pH during the softening process is 8.3.

[0086] Among them, the preparation method of the modifying enzyme is: mix 1 part of Bacillus licheniformis alkaline protease with 25 parts of citric acid, react at 8 °C for 3 h, purify and dry to obtain the modifying enzyme.

[0087] In this example, the penetration rate of the modifying enzyme in the limed fish skin is 100%.

[0088] Example 9

[0089] The dosages of the following materials are all based on the mass percentage (wt%) of the limed bare skin.

[0090] Load the limed cowhide into a rotating drum, add 150% water, adjust the temperature to 32 °C, add 2.5% ammonium sulfate and 0.2% degreasing agent, rotate for 120 min for deliming, then change the liquid, add 100% water, adjust the temperature to 32 °C, add 0.2% modifying enzyme (protease activity is 4000 U / g, isoelectric point is 8.5), rotate for 30 min, stop for 15 min, rotate for 15 min, to obtain softened bare skin. Among them, the pH during the softening process is 8.4.

[0091] Among them, the preparation method of the modifying enzyme is: mix 1 part of trypsin with 20 parts of maleic anhydride, react at 4 °C for 2 h, purify and dry to obtain the modifying enzyme.

[0092] Comparative Example 4

[0093] The dosages of the following materials are all based on the mass percentage (wt%) of the limed bare skin.

[0094] Load the limed cowhide into a rotating drum, add 150% water, adjust the temperature to 32 °C, add 2.5% ammonium sulfate and 0.2% degreasing agent, rotate for 120 min for deliming, then change the liquid, add 100% water, adjust the temperature to 32 °C, add 0.2% modifying enzyme (protease activity is 4000 U / g, isoelectric point is 9.0), rotate for 30 min, stop for 15 min, rotate for 15 min, to obtain softened bare skin. Among them, the pH during the softening process is 8.4.

[0095] Among them, the preparation method of the modified enzyme is as follows: 1 part of trypsin is mixed with 5 parts of maleic anhydride, reacted at 4°C for 2 h, purified and dried to obtain the modified enzyme.

[0096] Comparative Example 5

[0097] The following material dosages are all based on the mass percentage (wt%) of the limed hide.

[0098] Put the limed cowhide into a drum, add 150% water, adjust the temperature to 32°C, add 2.5% ammonium sulfate and 0.2% degreasing agent, rotate for 120 min for deliming, then change the liquid, add 100% water, adjust the temperature to 32°C, add 0.2% protease (trypsin, protease activity is 4000 U / g, isoelectric point is 9.2), rotate for 30 min, stop for 15 min, and rotate for 15 min to obtain the softened hide. Among them, the pH during the softening process is 8.4.

[0099] Comparative Example 6

[0100] The following material dosages are all based on the mass percentage (wt%) of the limed hide.

[0101] Put the limed cowhide into a drum, add 150% water, adjust the temperature to 32°C, add 2.5% ammonium sulfate and 0.2% degreasing agent, rotate for 120 min for deliming, then change the liquid, add 100% water, adjust the temperature to 32°C, add 0.2% protease (trypsin, protease activity is 4000 U / g, isoelectric point is 9.2) and 2.0% bovine serum albumin (isoelectric point 4.6), rotate for 30 min, stop for 15 min, and rotate for 15 min to obtain the softened hide. Among them, the pH during the softening process is 8.4.

[0102] Perform corresponding softening effect tests on Example 9, Comparative Example 4, Comparative Example 5, and Comparative Example 6; in addition, subject the softened hides obtained from Example 9, Comparative Example 4, Comparative Example 5, and Comparative Example 6 to the same conventional pickling, tanning, dyeing and finishing treatments to obtain leather, and conduct physical property tests on the leather. The test results are shown in Table 3. It can be seen that the penetration rate of the protease in Example 9 is higher than that in Comparative Example 4, Comparative Example 5, and Comparative Example 6, and the hydroxyproline concentration in the softening waste liquid is lower than that in Comparative Example 4, Comparative Example 5, and Comparative Example 6. This shows that the method provided by the present invention can significantly improve the penetration rate of the protease in the hide and reduce the damage to the skin collagen fibers. The physical properties (tensile strength and tear strength) of the leather in Example 9 are better than those in Comparative Example 4, Comparative Example 5, and Comparative Example 6. This shows that the method provided by the present invention can improve the leather quality. In summary, the method provided by the present invention can solve the technical problems of easily damaging collagen fibers and reducing leather quality in the softening technology by effectively increasing the penetration rate of the protease in the hide, shortening the residence time of the protease in the grain layer, and increasing the uniformity of the hydrolysis effect of the protease on collagen fibers in each layer.

[0103] Table 3 Comparison Table of Softening Effects (Softening for 60 min)

[0104] 。

[0105] Example 10

[0106] The following material dosages are all calculated based on the mass percentage (wt%) of the shaved blue wet leather.

[0107] Put the shaved blue wet leather into a rotating drum, add 200% water, adjust the temperature to 35°C, add 0.3% formic acid and 0.3% degreasing agent, and rotate for 60 min for rewetting; then change the liquid, add 200% water, adjust the temperature to 40°C, add 0.5% modified enzyme (protease activity is 20000 U / g, isoelectric point is 3.1), rotate for 30 min, stop for 30 min, and adjust to intermittent rotation (rotate for 15 min per hour) until the softening is completed. The total softening time is 480 min to obtain softened tanned leather. Among them, the pH value during the acidic softening process is 3.3.

[0108] Among them, the preparation method of the modified enzyme is: mix 1 part of pepsin with 25 parts of ethanolamine, react at 8°C for 4 h, and purify and dry to obtain the modified enzyme.

[0109] In this example, the penetration rate of the modified enzyme in the blue wet leather is 100%.

[0110] Example 11

[0111] In this example, multiple groups of samples are set up, and the difference is only the isoelectric point of the modified enzyme. By measuring the penetration rate of the modified enzyme in the treated animal skin, the influence of the difference between the isoelectric point of the modified enzyme and the pH value of the animal skin treatment process on the permeability of the modified enzyme is illustrated.

[0112] The following material dosages are all calculated based on the mass percentage (wt%) of the shaved blue wet leather.

[0113] Put the shaved blue wet leather into a rotating drum, add 150% water, adjust the temperature to 40°C, add 0.1% formic acid and 0.1% degreasing agent, and rotate for 30 min for rewetting; add 2% modified enzyme (protease activity is 5000 U / g), rotate for 30 min and stop for 30 min, and adjust to intermittent rotation (rotate for 20 min per hour) until the softening is completed. The total softening time is 6 h to obtain softened tanned leather. Among them, the pH value during the acidic softening process is 3.6.

[0114] Among them, the preparation method of the modified enzyme is: mix Aspergillus usamii acidic protease with 2,2-dichloroacetamide, react at 2°C for 3 h, and purify and dry to obtain the modified enzyme.

[0115] Table 4 Comparison Table of Penetration Rates of Modified Enzymes with Different Isoelectric Points in Wet Blue Leather (Acid Softening for 6 h)

[0116]

[0117] 。

[0118] Example 12

[0119] The following material dosages are all based on the mass percentage (wt%) of the shaved wet blue leather.

[0120] Put the shaved wet blue leather into a rotating drum, add 150% water, adjust the temperature to 42 °C, add 0.2% formic acid, rotate for 45 min for rewetting; add 3% modified enzyme (protease activity is 4000 U / g, isoelectric point is 3.7), rotate for 30 min and stop for 30 min, adjust to intermittent rotation (rotate for 15 min per hour) until the softening is completed. The total softening time is 240 min to obtain softened tanned leather. Among them, the pH during the acid softening process is 3.5.

[0121] Among them, the preparation method of the modified enzyme is: mix 1 part of papain with 60 parts of citric acid, react at 4 °C for 4 h, purify and dry to obtain the modified enzyme.

[0122] Comparative Example 7

[0123] The following material dosages are all based on the mass percentage (wt%) of the shaved wet blue leather.

[0124] Put the shaved wet blue leather into a rotating drum, add 150% water, adjust the temperature to 42 °C, add 0.2% formic acid, rotate for 45 min for rewetting; add 3% modified enzyme (protease activity is 4000 U / g, isoelectric point is 4.2), rotate for 30 min and stop for 30 min, adjust to intermittent rotation (rotate for 15 min per hour) until the softening is completed. The total softening time is 240 min to obtain softened tanned leather. Among them, the pH during the acid softening process is 3.5.

[0125] Among them, the preparation method of the modified enzyme is: mix 1 part of papain with 50 parts of citric acid, react at 4 °C for 4 h, purify and dry to obtain the modified enzyme.

[0126] Comparative Example 8

[0127] The following material dosages are all based on the mass percentage (wt%) of the shaved wet blue leather.

[0128] Put the shaved wet blue leather into a rotating drum, add 150% water, adjust the temperature to 42 °C, add 0.2% formic acid, and rotate for 45 min for rewetting; add 3% modifying enzyme (protease activity is 4000 U / g, isoelectric point is 6.5), rotate for 30 min and stop for 30 min, adjust to intermittent rotation (rotate for 15 min per hour) until the softening is completed. The total softening time is 240 min to obtain softened tanned leather. Among them, the pH value during the acid softening process is 3.5.

[0129] Among them, the preparation method of the modifying enzyme is as follows: Mix 1 part of papain with 35 parts of citric acid, react at 4 °C for 4 h, and purify and dry to obtain the modifying enzyme.

[0130] Comparative Example 9

[0131] The following material dosages are all based on the mass percentage (wt%) of the shaved wet blue leather.

[0132] Put the shaved wet blue leather into a rotating drum, add 150% water, adjust the temperature to 42 °C, add 0.2% formic acid, and rotate for 45 min for rewetting; add 3% protease (papain, protease activity is 4000 U / g, isoelectric point is 9.5), rotate for 30 min and stop for 30 min, adjust to intermittent rotation (rotate for 15 min per hour) until the softening is completed. The total softening time is 240 min to obtain softened tanned leather. Among them, the pH value during the acid softening process is 3.5.

[0133] Perform corresponding acid softening effect tests on Example 12, Comparative Example 7, Comparative Example 8, and Comparative Example 9; in addition, perform the same dyeing and finishing treatments on the softened tanned leathers obtained in Example 12, Comparative Example 7, Comparative Example 8, and Comparative Example 9 to obtain finished leathers, and conduct physical property tests on the finished leathers. The test results are shown in Table 5. It can be seen that the penetration rate of protease in Example 12 is higher than that in Comparative Example 7, Comparative Example 8, and Comparative Example 9, and the hydroxyproline concentration in the softened waste liquid is lower than that in Comparative Example 7, Comparative Example 8, and Comparative Example 9. This shows that the method provided by the present invention can significantly improve the penetration rate of protease in the nude skin during the acid softening process and reduce the damage of skin collagen fibers. The physical properties (tensile strength and tear strength) of the finished leather in Example 12 are better than those in Comparative Example 7, Comparative Example 8, and Comparative Example 9. This shows that the method provided by the present invention can improve the quality of the finished leather. In summary, the method provided by the present invention can effectively improve the penetration rate of protease in the wet blue leather, shorten the residence time of protease in the grain layer, and improve the softening effect of the wet blue leather.

[0134] Table 5 Comparison table of acid softening effects (softening for 240 min)

[0135] 。

[0136] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. Use of a modifying enzyme in treating animal skins, characterized in that, The modified enzyme is a modified protease, and the absolute value of the difference between its isoelectric point and the pH value of the animal skin treatment process is not greater than 0.5; The modified enzyme is obtained by modifying a protease with a small molecule compound. The adjustable range of the isoelectric point of the modified enzyme is 1.0 to 13.0, and the small molecule compound can react with the side chain groups of the protease to introduce charged groups or block the dissociable groups of the protease molecule.

2. The application according to claim 1, characterized in that The absolute value of the difference between the isoelectric point of the modified enzyme and the pH value of the animal skin treatment process is not greater than 0.

3.

3. The application according to claim 1, characterized in that The animal skin treatment process includes at least one of soaking, dehairing, liming, softening, and acid softening.

4. The application according to claim 1, characterized in that, The small molecule compound includes aldehydes, acid anhydrides, cyanates, esters, amines, alkanolamines, and haloamides.

5. The application according to claim 1, wherein The protease includes any one or more of microbial proteases, animal proteases, and plant proteases.

6. The application according to claim 1, characterized in that The animal skin includes cowhide, sheepskin, pigskin, horsehide, deerhide, ostrich skin, and fish skin.

7. Leather obtained by applying the modified enzyme according to any one of claims 1-6 to the treatment of animal skin.

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