Modified zeolite, immobilized protease and preparation method and application thereof

Abstract

The invention belongs to the technical field of biochemistry, and particularly relates to modified zeolite, immobilized protease and a preparation method and application thereof. Wherein the structural formula of the modified zeolite is shown in figure 1. The immobilized protease prepared based on the modified zeolite has excellent stability, especially immobilized neutral protease, and the enzyme activity is only reduced by about 6% after the immobilized neutral protease is stored for 6 months under normal storage conditions; in addition, after shaking on a shaking table for 2 hours, the enzyme activity is not obviously changed, and the enzyme activity of the lower layer is only reduced by about 2%. And the softness, the part difference, the surface cleanliness and the fluff condition of the leather prepared after being treated by the immobilized protease prepared based on the modified zeolite are superior to those of the conventional enzyme.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biochemistry, and particularly relates to modified zeolite, immobilized protease, and preparation method and application thereof. BACKGROUND

[0002] Enzymes, as efficient biological catalysts, can realize protein hydrolysis and polypeptide distribution regulation under mild conditions. The high efficiency and clean production characteristics make them become the key technology in the industrial field such as leather processing. However, enzyme catalysis is highly sensitive to reaction conditions (such as temperature, pH, medium dispersibility, etc.), and only 0.01% to 1% of trace enzymes are needed in large-scale application. In order to realize the uniform action of enzymes, two strategies are usually adopted in industry: one is to dilute the enzyme by using a porous inert carrier to slow down the dispersion, but the carrier and the enzyme need to be highly compatible and depend on a large amount of aqueous medium; the other is to enhance the anti-interference ability of the enzyme to adapt to the complex environment, but the increase of enzyme dosage will lead to the increase of cost. Therefore, the development of immobilized protease technology with stability (including storage stability, shear resistance) and catalytic efficiency has become a core challenge for industrial enzyme application.

[0003] Although the existing immobilized protease technology has been developed for many years, there are still significant deficiencies in stability (especially long-term storage stability and anti-external interference stability): 1) embedding method mixes and embeds the carrier precursor with the enzyme, which can shield the environmental interference by using the carrier, but the precursor may directly damage the enzyme molecular conformation, resulting in the decrease of enzyme activity in the early stage of storage; and the carrier pore size limits the substrate size (only suitable for small molecule substrates), and the carrier aging in long-term use will further destroy the enzyme activity space; 2) adsorption method fixes the enzyme by weak forces such as hydrophobic interaction and electrostatic adsorption, which has mild reaction conditions, but the binding force between the enzyme and the carrier is weak, and the enzyme is easy to fall off under the action of shear force (such as shaking table oscillation), the enzyme activity recovery rate is low after repeated use, and the anti-mechanical interference stability is very poor; 3) covalent binding method connects the enzyme and the carrier by covalent bond, which can improve the structural stability of the enzyme, but the chemical modification will destroy the active groups of the enzyme, resulting in serious loss of initial enzyme activity after immobilization, and the covalent bond may be broken due to hydrolysis or oxidation in long-term storage, so the stability is difficult to guarantee; 4) inorganic material method (such as silicon-based material, metal oxide) has low cost and high mechanical strength, but the pore size of nano-silicon oxide carrier is insufficient, which cannot effectively embed macromolecular enzymes; COFs / HOFs framework material is easy to collapse in long-term storage due to poor stability of hydrogen bond or covalent bond, resulting in rapid loss of enzyme activity; 5) zeolite-like method (such as ZIF-8, MOFs) prepares porous carrier by using organic template, which has a higher enzyme loading capacity (15.5 μmol / g), but the embedding process may shield the active groups of the enzyme; and the framework material will slowly shrink or swell under thermodynamic equilibrium, gradually compressing the active space required by the enzyme, and finally leading to enzyme inactivation.

[0004] In summary, the common defects of the existing immobilized protease technology are that the stability (including storage stability and shear resistance) and catalytic activity are difficult to be considered together. For example, the enzyme may fall off due to weak binding force between the carrier and the enzyme (such as adsorption method), the enzyme activity group may be damaged due to the structure of the carrier (such as covalent bonding method, zeolite-like method), or the enzyme may be inactivated due to the aging of the carrier (such as embedding method, inorganic material method). Therefore, it is of great significance to develop an immobilized protease technology which can effectively protect the enzyme conformation through the carrier and maintain high enzyme activity under long-term storage and mechanical interference, so as to promote the large-scale application of industrial enzyme catalysis. SUMMARY

[0005] Based on this, the present application prepares a modified zeolite by de-aluminizing 4A zeolite, and the stability (including storage stability and shear resistance) of the immobilized protease prepared based on the modified zeolite is excellent.

[0006] In order to achieve the above-mentioned purpose, the present application can adopt the following technical solutions: The present application provides a preparation method of the modified zeolite.

[0007] Preferably, the preparation method comprises: (1) mixing 4A zeolite with acid to obtain a suspension slurry; (2) filtering the suspension slurry to obtain a precipitate; (3) centrifuging the precipitate to obtain the modified zeolite.

[0008] Preferably, the preparation method satisfies one or more of the following conditions: (i) in step (1), the mixing comprises stirring and dissolving for 20 min (ii) in step (1), the acid is a mixed acid comprising hydrochloric acid, sulfuric acid and citric acid; (iii) in step (2), the particle size of the solid particles in the suspension slurry is <10 μm; (iv) in step (2), the mesh number of the filter is 10 μm; (v) in step (3), the centrifugation comprises centrifuging for 5 min to 10 min at a centrifugal force of 10 kg to 15 kg.

[0009] More preferably, in condition (i) of the preparation method, the mass ratio of hydrochloric acid, sulfuric acid and citric acid is (4-5):(2-3):3.

[0010] The present application provides a modified zeolite prepared by the preparation method of the modified zeolite.

[0011] The present application further provides an immobilized protease comprising the modified zeolite and a protease.

[0012] Preferably, in the immobilized protease, the protease is selected from one or more of acid protease, neutral protease or alkaline protease.

[0013] In another aspect of the present application, a preparation method of the immobilized protease is provided, which comprises: mixing modified zeolite and protease, and drying the mixture to obtain the immobilized protease; and performing rolling stirring during the mixing and / or drying.

[0014] Preferably, in the preparation method, the added amount of the protease is 10% to 20% of the mass of the modified zeolite.

[0015] In another aspect of the present application, the immobilized protease is applied in leather softening.

[0016] The present application has the following advantages: (1) The immobilized protease prepared based on the modified zeolite provided by the present application has excellent stability, especially the immobilized neutral protease, which only has about 6% reduction in enzyme activity after 6 months of storage under normal storage conditions; in addition, after 2 hours of shaking on a shaking table, the enzyme activity does not change significantly, only the enzyme activity in the lower layer is reduced by about 2%.

[0017] (2) The leather prepared after treatment with the immobilized protease prepared based on the modified zeolite provided by the present application is superior to conventional enzymes in softness, site difference, surface cleanliness and nap condition; the immobilized protease in the present application has normal softness, better uniformity; small site difference, firm and good belly; bright and clean without root hair; and uniform nap.

[0018] (3) The modified zeolite provided by the present application is easy to be separated from waste water, and does not produce pollution indexes such as COD, BOD and NH3-N, which provides convenience for waste liquid treatment. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 It is a schematic diagram of the structure of the modified zeolite; Figure 2 It is a schematic diagram of the reaction mechanism of the modified zeolite prepared by the present application; Figure 3 It is a schematic diagram of the formation of the modified zeolite and the immobilized enzyme of the present application; Figure 4 It is a SEM diagram of the artificial 4A zeolite (400 mesh, 40-50 μm, industrial grade, purchased from Hebei Huipeng New Material Technology Co., Ltd.), modified zeolite and immobilized protease (neutral protease) selected in the examples; wherein A is artificial 4A zeolite × 10000, B is modified zeolite × 10000, and C is immobilized protease × 10000; Figure 5Release ratio of activity versus time for immobilized protease (acid protease) prepared in Example 1; Figure 6 Release ratio of activity versus time for immobilized protease (neutral protease) prepared in Example 1; Figure 7 Release ratio of activity versus time for immobilized protease (alkaline protease) prepared in Example 1. DETAILED DESCRIPTION

[0020] The examples are provided to better illustrate the present application but are not intended to limit the present application thereto. Therefore, the skilled in the art, based on the disclosure of the present application, can make some non-essential improvements and modifications to the embodiments, which are still within the scope of the present application.

[0021] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the disclosure. Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms "comprises" and / or "comprising," when used in this specification, specify the presence of stated features, integers, steps, operations, elements, and / or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or groups thereof. As used herein, "and / or" can be interpreted as "and," "or," or both, depending on the context.

[0022] In a first aspect, the embodiments of the present application provide a preparation method of modified zeolite, the preparation method comprising: subjecting 4A zeolite to dealumination treatment to obtain modified zeolite.

[0023] It should be noted that the present application can obtain modified zeolite with better performance than 4A zeolite that has not been treated, for example, the natural micropore number and binding stability of the unmodified 4A zeolite are far inferior to those of the modified modified zeolite. In addition, 4A zeolite is a zeolite material based on silicate, which has ion exchange, adsorption, catalytic, acid resistance (aqua regia), heat resistance (> 100℃), radiation resistance and other properties; the most common is to use the surface activity and porosity of zeolite as an adsorbent material to be applied to chemical adsorption, heavy metal ion filtration and other fields such as petroleum chemical industry, environmental protection, biological engineering, food industry, pharmaceutical chemical industry, and become an important raw material for "green chemistry".

[0024] In some specific examples, the above preparation method comprises: (1) mixing 4A zeolite with acid to obtain a suspension slurry; (2) filtering the suspension slurry to obtain a precipitate; (3) centrifuging the precipitate to obtain modified zeolite.

[0025] It should be noted that the "mixing", "filtering" and "centrifuging" in the above preparation method are all well-known operations in the art and have no specific meaning. In addition, the modified zeolite obtained in step (3) is a modified zeolite in the form of a slurry containing water, i.e., in a state containing a certain amount of water.

[0026] In some specific examples, the above preparation method satisfies one or more of the following conditions: (i) In step (1), the mixing comprises stirring for 20 min. (ii) In step (1), the acid is a mixed acid comprising hydrochloric acid, sulfuric acid and citric acid. (iii) In step (2), the particle size of the solid particles in the suspension slurry is <10 μm. (iv) In step (2), the mesh size of the filtering is 10 μm. (v) In step (3), the centrifuging comprises centrifuging for 5 min to 10 min at a centrifugal force of 10 kg to 15 kg.

[0027] It should be noted that in condition (i) of the above preparation method, the mixing can be agitated at a speed of 100 r / min to 150 r / min (e.g., 110 r / min, 120 r / min, 130 r / min or 140 r / min, etc.) at 10°C to 50°C (e.g., 20°C, 30°C or 40°C, etc.) to increase the dissolution rate, and the stirring time can be 10 min. In addition, in condition (ii), the acid can be one acid or a mixed acid of multiple acids, and is preferably a mixed acid. The mixed acid preferably comprises hydrochloric acid, sulfuric acid and citric acid. The mass ratio of hydrochloric acid, sulfuric acid and citric acid can be (4-5):(2-3):3, e.g., 5:2:3 or 4:3:3, etc. In addition, in condition (iii), the particle size of the solid particles in the suspension slurry can be 2 μm to 10 μm, e.g., 4 μm, 6 μm or 9 μm, etc. Furthermore, in condition (iv), the mesh size of the filtering is 1000 mesh to 2000 mesh, e.g., 1300 mesh, 1500 mesh or 1700 mesh, etc. In addition, in condition (v), the number of centrifuging can be preferably two or more times. The centrifuging can comprise: centrifuging the precipitate in a centrifuge at a centrifugal force of 10 kg to 20 kg for 5 min to 10 min, washing once with 50 kg of distilled water, and centrifuging once again at a centrifugal force of 10 kg to 20 kg for 5 min to 10 min to remove the aluminum ions eluted, to obtain the modified zeolite.

[0028] It should be further noted that the above preparation method can satisfy one of the above conditions, preferably simultaneously satisfy two of the above conditions, and more preferably simultaneously satisfy all of the above conditions.

[0029] In a second aspect, the embodiments of the present application provide a modified zeolite prepared by the method for preparing the modified zeolite.

[0030] It should be noted that the structural formula of the modified zeolite in the present application is as shown in Figure 1 In addition, after the modified zeolite in the present application is applied in production, the modified zeolite precipitates into waste water and is easily separated into liquid and solid, without the generation of soluble substances and suspended substances, thereby providing convenience for waste liquid treatment. In addition, the modified zeolite in the present application can adsorb power supply elements of chemicals by means of acid elution of Al ions and formation of high-potential hydrogen element holes after water washing; and the H elements on the surface of the exchanged zeolite can participate in cation exchange of enzymes, as shown in Figure 2

[0031] In a third aspect, the embodiments of the present application provide an immobilized protease comprising the modified zeolite and the protease.

[0032] It should be noted that the modified zeolite after dealumination in the present application reduces the crystal volume, increases the surface area and internal voids, and creates a high-density and high-potential electrophilic function on the surface, thereby increasing the chemical and physical combination ability of the protein surface and free side chains. In use, the modified zeolite satisfies the slow-release function and has a good homogenization effect on the protease. In addition, when the immobilized protease in the present application is used in an aqueous solution, the slow-release and activation can be achieved according to the pH, temperature and mechanical action, thereby achieving functionalization.

[0033] In addition, it should be understood that high activity and low dosage make it difficult for the protease to act uniformly in hydrolysis. The present application has practical significance in the preparation of the protease by using the adsorption activity of the modified zeolite, reducing the particle size by means of acidic resolution, and increasing the surface area and active pores. The enzyme immobilized by the modified zeolite in the present application has good storage and transportation stability, and improves the moisture and heat resistance. In addition, during the catalysis of the immobilized protease in the present application, the enzyme is released from the carrier by slow release, which not only solves the dispersion and uniform action, but also ensures the most effective activity of the enzyme.

[0034] In some specific examples, the immobilized protease comprises one or more of an acid protease, a neutral protease or an alkaline protease.

[0035] It should be noted that the modified zeolite in the present application can be loaded with various enzymes to prepare different immobilized proteases, and the enzyme can be selected from one or more of an acid protease, a neutral protease or an alkaline protease; preferably, the neutral protease. The present application tests the activity release process of different immobilized proteases at different times by means of the enzyme activity release test method, and the results show that, except that the activity release of the acid enzyme and the alkaline enzyme is 9.2% and 7.6% lower, respectively, the activity release of the neutral protease is only 4.7% lower.​

[0036] In a fourth aspect, the present application provides a preparation method of the immobilized protease, which comprises: mixing the modified zeolite and the protease, and drying the mixture to obtain the immobilized protease; and rolling stirring during the mixing and / or drying.

[0037] It should be noted that the reaction principle of the modified zeolite and the protease in the present application is as shown in FIG. 1. Figure 3 In addition, the preparation method can comprise: pouring the modified zeolite into a horizontal stirrer, adding the protease, and then rolling stirring at 23-25°C for 20-30 min, and then continuing rolling stirring under blowing of dry air at 35-40°C for 30-60 min until drying, and then packaging the product to obtain the immobilized protease. In addition, the horizontal roller stirrer can work at a speed of 5-8 r / min, at 35-40°C, for 20-30 min, and then dry hot air dehumidification can achieve the purpose of "low-temperature dehumidification stirring", and the amount of the added enzyme is 0.1-0.2 g / g of the maximum adsorption capacity of the modified zeolite.

[0038] In some specific examples, in the preparation method, the amount of the added protease is 10-20% of the mass of the modified zeolite.

[0039] It should be noted that the amount of the added protease can be 10-20% of the mass of the modified zeolite, for example, 13%, 15% or 17%, etc.

[0040] In a fifth aspect, the present application provides an application of the immobilized protease in leather softening.

[0041] It should be noted that the softness, site difference, surface cleanliness and nap condition of the leather prepared after treatment by the immobilized protease in the present application are all better than those of conventional enzymes, the softness of the immobilized protease in the present application is normal and uniform, the site difference is small, the belly is compact and good, the surface is bright and clean, and the nap is uniform.

[0042] In order to better understand the present application, the content of the present application will be further illustrated below in combination with specific examples, but the content of the present application is not limited to the following examples.

[0043] Preparation Example 1 Example 1 In the open mixer, 100 kg of artificial 4A zeolite (400 mesh, industrial grade, purchased from Hebei Hui Peng New Material Technology Co., Ltd.) with a particle size of 40-50 μm was added, then 100 kg of mixed acid (with mass fraction of 50% hydrochloric acid, 20% sulfuric acid and 30% citric acid) was added, and then 100 kg of deionized water was added. The average particle size of the zeolite was <8 μm after stirring at 50°C and 120 r / min to form a suspension slurry. The liquid was removed by pressure filtration with 2000 mesh filter cloth, and the precipitated slurry was poured into a centrifuge to centrifuge at 10 kg for 5 min. After washing once with 50 kg of distilled water, the aluminum ions were removed by centrifuging once at 10 kg for 5 min. The modified zeolite was obtained.

[0044] The modified zeolite prepared in Example 1 was poured into a horizontal drum-type mixer, and 15% of acid protease (powdered acid protease from Spain Cromogenia) was added based on the weight of the modified zeolite. The mixture was stirred at 25°C for 20 min, and then dry air at 35°C was blown in for continuous stirring for 30 min until dryness. The immobilized protease (acid protease) was packaged as a finished product.

[0045] In addition, the modified zeolite prepared according to the preparation method of the immobilized protease (acid protease) in Example 1 was used to prepare immobilized protease (neutral protease) and immobilized protease (alkaline protease).

[0046] Example 2 In the open mixer, 100 kg of artificial 4A zeolite (400 mesh, industrial grade, purchased from Hebei Hui Peng New Material Technology Co., Ltd.) with a particle size of 40-50 μm was added, then 100 kg of mixed acid (with mass fraction of 50% hydrochloric acid, 20% sulfuric acid and 30% citric acid) was added, and then 100 kg of deionized water was added. The average particle size of the zeolite was <8 μm after stirring at 50°C and 120 r / min to form a suspension slurry. The liquid was removed by pressure filtration with 2000 mesh filter cloth, and the precipitated slurry was poured into a centrifuge to centrifuge at 10 kg for 5 min. After washing once with 50 kg of distilled water, the aluminum ions were removed by centrifuging once at 10 kg for 5 min. The modified zeolite was obtained.

[0047] The modified zeolite prepared in Example 2 was poured into a horizontal drum-type mixer, and 15% of acid protease (powdered acid protease from Spain Cromogenia) was added based on the weight of the modified zeolite. The mixture was stirred at 25°C for 20 min, and then dry air at 35°C was blown in for continuous stirring for 30 min until dryness. The immobilized protease (acid protease) was packaged as a finished product.

[0048] In addition, the modified zeolite prepared in Example 2 was used to prepare immobilized protease (acid protease) and immobilized protease (alkaline protease) according to the above preparation method of immobilized protease (acid protease).

[0049] Example 3 In an open beater, 100 kg of artificial 4A zeolite with a particle size of 40-50 μm (400 mesh, industrial grade, purchased from Hebei Huipeng New Material Technology Co., Ltd.) was added, followed by 100 kg of mixed acid (with mass fraction of 40% hydrochloric acid, 10% sulfuric acid and 50% citric acid), and the zeolite was stirred at a speed of 120 r / min at 50°C to obtain a suspension slurry with an average particle size of <5 μm. The liquid was removed by pressure filtration with 2000 mesh filter cloth, and the precipitated slurry was poured into a centrifuge to centrifuge at a centrifugal force of 15 kg for 10 min. The precipitated slurry was washed once with 50 kg of distilled water, and then centrifuged at a centrifugal force of 15 kg for 10 min to remove the aluminum ions eluted, thereby obtaining the modified zeolite.

[0050] The modified zeolite prepared in Example 3 was poured into a horizontal beater, 15% alkaline protease (powdered alkaline protease from Spain Cromogenica) was added based on the weight of the modified zeolite, and the mixture was rolled and stirred at 25°C for 20 min. Then, dry air at 35°C was blown in to continue rolling and stirring for 30 min until dryness, and the immobilized protease (alkaline protease) was packaged as a finished product.

[0051] In addition, the modified zeolite prepared in Example 3 was used to prepare immobilized protease (acid protease) and immobilized protease (neutral protease) according to the above preparation method of immobilized protease (acid protease).

[0052] Comparative Example 1 Comparative Example 1 used artificial 4A zeolite with a particle size of 40-50 μm as a comparison.

[0053] The artificial 4A zeolite in Comparative Example 1 was poured into a horizontal beater, 15% acid protease was added based on the weight of the modified zeolite, and the mixture was rolled and stirred at 25°C for 20 min. Then, dry air at 35°C was blown in to continue rolling and stirring for 30 min until dryness, and the immobilized protease (acid protease) was packaged as a finished product.

[0054] In addition, the artificial 4A zeolite in Comparative Example 1 was used to prepare immobilized protease (neutral protease) and immobilized protease (alkaline protease) according to the above preparation method of immobilized protease (acid protease).

[0055] Comparative Example 2 Comparative Example 2 follows the existing reported methods for immobilizing proteases (Feng Yuxiao. Preparation and Application of Immobilized Enzymes Based on Zeolite-like Imidazole Ester Framework Materials [D]. 202, Tianjin University Doctoral Dissertation; P Zucca, E Sanjust. Inorganic materials as supports for covalent enzyme immobilization: methods and mechanisms [J]. Molecules, 2014, Immobilized protease was prepared in 19(9):14139-14194.

[0056] The artificial 4A zeolite, modified zeolite, and immobilized protease (neutral protease) prepared in Example 1 were observed using scanning electron microscopy (SEM), and the results are as follows: Figure 4 As shown, the results revealed that the size and saturation of zeolite particles increased after enzyme immobilization, indicating a large amount of adsorption on the surface.

[0057] Performance testing (a) Immobilized protease enzyme activity release test When immobilized protease enters an aqueous solution, enzyme molecules with weak or shallow surface binding are initially released into the solution; only a portion of the enzyme's activity is released. The majority of enzyme molecules are released based on time, mechanical action, pH, and temperature, allowing for control over the substrate hydrolysis process. This has several advantages: first, it provides the necessary osmotic environment, preventing excessive interaction with the substrate surface; second, slow release prolongs the enzyme's half-life; and third, the high density of zeolite combined with a small enzyme dosage disperses the enzyme's action, preventing rapid localized adsorption and differential hydrolysis. The following tests demonstrate the method for testing the release activity of immobilized protease: (1) Determination of acidic enzyme activity release: Add 100 mL of a solution with pH=4.0±0.1 adjusted with formic acid to a three-necked flask, add 1% enzyme (based on water volume) at 30℃, stir at 10 r / min, and take the solution after 5 min to determine the activity; then take another three-necked flask, add 100 mL of a solution with pH=4.0±0.1, add 1% enzyme (based on water volume) at 30℃, stir at 10 r / min, and take the solution after stirring for different times to determine the activity (SB / T10317-1999). 2) Neutral enzyme activity release determination: add 100 mL solution with pH = 7.5 ± 0.1 adjusted by sodium bicarbonate into a three-neck flask, add 1% enzyme (based on water) at 30°C, stirring speed 10 r / min, take liquid sample for activity determination after 5 min. Take another three-neck flask, add 100 mL solution with pH = 4.0 ± 0.1, add 1% enzyme (based on water) at 30°C, stirring speed 10 r / min, take liquid sample for activity determination after stirring for different time (SB / T 10317-1999); 3) Alkaline enzyme activity release determination: add 100 mL solution with pH = 9.5 ± 0.1 adjusted by sodium bicarbonate into a three-neck flask, add 1% enzyme (based on water) at 30°C, stirring speed 10 r / min, take liquid sample for activity determination after 5 min. Take another three-neck flask, add 100 mL solution with pH = 4.0 ± 0.1, add 1% enzyme (based on water) at 30°C, stirring speed 10 r / min, take liquid sample for activity determination after stirring for different time (SB / T 10317-1999).

[0058] The immobilized protease (acidic protease), immobilized protease (neutral protease) and immobilized protease (alkaline protease) prepared in Example 1 were respectively tested for activity release process of different immobilized proteases at different time according to the above-mentioned enzyme activity release test method (SB / T 10317-1999) of immobilized protease. The results are shown in Table 2. Figures 5 to 7 The results show that the activity release of neutral protease is only 4.7% lower, while the activity release of acidic and alkaline enzymes is 9.2% and 7.6% lower, respectively.

[0059] (B) Stability determination of neutral immobilized protease activity The enzyme activity of the immobilized protease (neutral protease) prepared in Example 1 was tested immediately after preparation (before leaving the factory), after being stored at room temperature for a period of time and after being shaken on a shaker for 2 h (the results were the average of 3 tests) to verify the stability of the immobilized protease. The method of shaking on a shaker for 2 h includes: placing 500 mL of the above-prepared immobilized protease (neutral protease) in a beaker, shaking at 50 r / min on a shaker for 2 h, and taking samples from the middle and bottom of the beaker for enzyme activity determination (according to the reference standard SB / T 10317-1999).

[0060] The test results are shown in Table 1.

[0061] Table 1 Stability determination of immobilized protease (neutral protease) prepared in Example 1 As shown in Table 1 above, the enzyme activity was reduced by only about 6% after 6 months of storage under normal storage conditions. In addition, the enzyme activity did not change significantly after shaking for 2 hours on a shaker, and only the enzyme activity of the lower layer was reduced by about 2% (a small amount of stratification occurred due to the difference in particle density of the immobilized enzyme), and the release increased after mechanical shaking. The above data show that the stability of the immobilized protease (neutral protease) prepared in Example 1 is excellent.

[0062] In addition, the immobilized protease (neutral protease) prepared in Example 2 was tested for stability according to the above method, and the results are shown in Table 2 below.

[0063] Table 2 Stability test results of the immobilized protease (neutral protease) prepared in Example 2 In addition, the immobilized protease (neutral protease) prepared in Example 3 was tested for stability according to the above method, and the results are shown in Table 3 below.

[0064] Table 3 Stability test results of the immobilized protease (neutral protease) prepared in Example 3 In addition, the immobilized protease (neutral protease) prepared in Comparative Example 1 was tested for stability according to the above method, and the results are shown in Table 4 below.

[0065] Table 4 Stability test results of the immobilized protease (neutral protease) prepared in Comparative Example 1 In addition, the immobilized protease (neutral protease) prepared in Comparative Example 2 was tested for stability according to the above method, and the results are shown in Table 5 below.

[0066] Table 5 Stability test results of the immobilized protease (neutral protease) prepared in Comparative Example 2 As shown above, the enzyme activity of the immobilized protease prepared in Example 1 was significantly higher than that of Comparative Examples 1 and 2, and the stability was also better than that of Comparative Examples 1 and 2.

[0067] (Three) Test of the immobilized protease (neutral protease) prepared in Example 1 for softening of leather In two 4.2m overloading drums, 8 tons of unhairing hide (according to the weight of the alkali hide) were respectively added, 16 kg of protease with activity of 700 U and 40 kg of immobilized protease (protease) prepared in Example 1 with the same total effective activity (U / L) were respectively added, at 32℃, according to the softening requirements, they were rotated for 30 min (protease) and 35 min (immobilized protease) respectively, then they were washed with water, immersed in acid, and tanned to obtain blue wet leather. The condition of the blue wet leather was checked, and the results are shown in Table 6.

[0068] Table 6 Comparison of softening of conventional trypsin and immobilized protease (protease) From the above Table 6, it can be seen that under the same enzyme activity, the treatment effect of immobilized protease (protease) (immobilized protease dosage 0.6%) is significantly better than that of conventional protease. It shows that the modified zeolite plays a role of filling from zeolite and surface cleaning.

[0069] Finally, it should be pointed out that the above examples are only used to illustrate the technical solutions of the present application and are not limiting. Although the present application has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solutions of the present application can be modified or replaced equivalently without departing from the purpose and scope of the technical solutions of the present application, and they should be covered in the scope of the claims of the present application.

Claims

1. A method for preparing a modified zeolite, characterized by, The preparation method comprises: modifying 4A zeolite by dealuminization.

2. The production method according to claim 1, characterized by, The preparation method comprises: (1) mixing 4A zeolite with acid to obtain a suspension slurry; (2) filtering the suspension slurry to obtain a precipitate; (3) centrifuging the precipitate to obtain modified zeolite.

3. The production method according to claim 2, characterized by, The preparation method satisfies one or more of the following conditions: (i) in step (1), the mixing comprises stirring and dissolving for 20 min; (ii) in step (1), the acid is a mixed acid comprising hydrochloric acid, sulfuric acid and citric acid; (iii) in step (2), the particle size of the solid particles in the suspension slurry is <10 μm; (iv) in step (2), the mesh size of the filter is 10 μm; (v) in step (3), the centrifugation comprises centrifuging for 5 min to 10 min at a centrifugal force of 10 kg to 15 kg.

4. The production method according to claim 3, characterized by, In condition (i), the mass ratio of hydrochloric acid, sulfuric acid and citric acid is (4-5):(2-3):

3.

5. Modified zeolite, characterized in that, The modified zeolite is prepared by the preparation method of any one of claims 1 to 4.

6. An immobilized protease characterized in that, The modified zeolite and the protease.

7. The immobilized protease according to claim 6, characterized in that, The protease is selected from one or more of acid protease, neutral protease or alkaline protease.

8. A method for the production of the immobilized protease according to claim 6 or 7, characterized in that, The preparation method comprises: mixing the modified zeolite and the protease, and drying after the mixing to obtain immobilized protease; and rolling stirring is performed during the mixing and / or drying.

9. The production method according to claim 8, characterized by, The addition amount of the protease is 10% to 20% of the mass of the modified zeolite.

10. Use of the immobilized protease of claim 6 or 7 in leather softening.

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