A cellulase preparation and a method for its production

Abstract

This application provides a cellulase preparation and its preparation method, belonging to the field of enzyme preparation technology. The cellulase preparation comprises, by weight parts, component A: 500-600 parts modified cellulase, 60-90 parts pectinase, 80-120 parts inorganic salt, 40-60 parts protective agent, and 5-15 parts nano-silica; component B: 160-200 parts alcohol ether, 100-130 parts polyol, 60-90 parts anti-re-dyeing polymer, 1-3 parts preservative, and 550-650 parts deionized water. This compound system of the present invention enables the cellulase preparation to exhibit excellent low-temperature catalytic activity and good stability in various applications such as textile finishing and detergent addition.

Description

Technical Field

[0001] This application relates to the field of enzyme preparation technology, and in particular to a cellulase preparation and its preparation method. Background Technology

[0002] Cellulase is a general term for a class of enzymes that can hydrolyze the β-1,4 glycosidic bonds of cellulose. It mainly includes endoglucanase, exoglucanase and β-glucosidase, and is widely used in textiles, detergents, feed, papermaking and bioenergy.

[0003] In recent years, with the increasing demand for energy conservation and emission reduction, low-temperature processing technology has gradually become a trend in industrial development. In applications such as textile finishing (e.g., denim biopolishing), neutral detergents, and feed additives, cellulase is required to maintain high catalytic activity under low-temperature (10-25℃) and neutral (pH 6.0-7.5) conditions. However, most existing commercially available cellulases are mesophilic or thermophilic, with optimal temperatures typically between 40-60℃. Under low-temperature conditions, the enzyme molecular conformation tends to become rigid, significantly reducing catalytic efficiency; simultaneously, enzyme activity further decreases under neutral conditions, making it difficult to meet practical application requirements.

[0004] To address these issues, researchers have attempted to improve enzyme performance through chemical modification, immobilization, or the addition of stabilizers. Adding polyols, sugars, or salts as stabilizers to the enzyme solution is the most common method. However, these stabilizers bind to enzyme molecules only through weak interactions (such as hydrogen bonds and hydrophobic interactions), and are prone to dissociation during storage or use, leading to a decrease in protective efficacy. Furthermore, to obtain solid-state enzyme preparations, industrial processes typically employ freeze-drying, which is energy-intensive, time-consuming, and requires significant equipment investment, severely limiting the production efficiency and economic viability of enzyme preparations.

[0005] In recent years, some studies have attempted to combine cationic and anionic polymers for material modification. For example, Chinese patent application CN115715771A describes a bacterial cellulose / chitosan oligosaccharide / γ-polyglutamic acid composite membrane and its preparation method and application. This method uses a combination of dynamic and static fermentation and in-situ modification to prepare a covalently bonded double-network interpenetrating composite membrane by activating carboxyl groups. The resulting membrane material has excellent mechanical and swelling properties and can be used in the fields of drug carriers or sensors. However, this technology has the following limitations: (1) Its core is the construction of macroscopic membrane materials, which depends on the 3D network framework of bacterial cellulose. This is a completely different technical field from the microscopic modification of soluble enzyme proteins. Its application is limited to medical materials and does not involve the low-temperature activity and stability of enzyme preparations.

[0006] In summary, there is currently a lack of an enzyme preparation method that can simultaneously address the issues of maintaining enzyme protein activity at low temperatures, tolerance to high-temperature drying, and storage stability. Summary of the Invention

[0007] This application is made in view of the above-mentioned problems, and its purpose is to provide a cellulase preparation and a method for preparing the same.

[0008] Specifically, the first aspect of this application provides a cellulase preparation, in parts by mass, comprising: Component A: 500-600 parts modified cellulase, 60-90 parts pectinase, 80-120 parts inorganic salts, 40-60 parts protective agent, and 5-15 parts nano silica. Component B: 160-200 parts alcohol ether, 100-130 parts polyol, 60-90 parts anti-re-dyeing polymer, 1-3 parts preservative, 550-650 parts deionized water; The mass ratio of component A to component B is 1:1.2-2.0.

[0009] The inorganic salt is 70-90 parts sodium chloride and 10-30 parts calcium chloride; the protective agent is trehalose, the polyol is propylene glycol, the anti-re-dyeing polymer is polyvinylpyrrolidone, and the preservative is Kathon.

[0010] Furthermore, the method for preparing the modified cellulase includes the following steps: S1: Provide cellulase solution and adjust its pH to 6.0-7.5; S2: Introduce a cationic polypeptide polymer into the pH-adjusted enzyme solution and stir until homogeneous; S3: Introduce anionic polysaccharide polymers into the system, so that the anionic polysaccharide polymers and cationic polypeptide polymers form a complex through electrostatic interaction, forming a coating layer on the surface of cellulase; S4: Add polyvalent metal ions for cross-linking treatment to solidify the coating layer; concentrate and / or dry the resulting product to obtain an enzyme preparation.

[0011] Furthermore, the cationic polypeptide polymer is ε-polylysine with a number-average molecular weight of 2000-5000 Da; The anionic polysaccharide polymer is alginate with a weight-average molecular weight of 100-200 kDa.

[0012] Furthermore, the multivalent metal ion is Zn. 2+ Cu 2+ Fe 3+ or Co 2+ At least one of them.

[0013] Furthermore, the enzyme protein concentration of the cellulase solution is 18-22 g / L; After pH adjustment in step S1, the temperature is controlled at 5-20℃.

[0014] Furthermore, the amount of the cationic polypeptide polymer added is 5%-12% of the enzyme protein mass; And / or, the amount of the anionic polysaccharide polymer added is 10-25% of the enzyme protein mass.

[0015] Furthermore, the concentration of the polyvalent metal ions is 2-5 mM.

[0016] Furthermore, the cationic polypeptide polymer and the anionic polysaccharide polymer are added by dropwise addition at a rate of 0.1-1.0 L / (h·L enzyme solution) and a stirring speed of 80-160 rpm.

[0017] Furthermore, the crosslinking treatment also includes a curing step; the curing is carried out by standing at 10-20°C for 2-8 hours.

[0018] Furthermore, the drying is spray drying; the inlet air temperature of the spray dryer is 130-170℃, and the outlet air temperature is 60-80℃.

[0019] A second aspect of this application provides a method for preparing a cellulase preparation, comprising the following steps: Modified cellulase, pectinase, inorganic salts, protective agents and inorganic nanoparticles were mixed evenly in the liquid phase, and then freeze-dried, pulverized and sieved to obtain component A freeze-dried powder. Alcohol ether, polyol, anti-re-dyeing polymer and preservative are mixed in water and homogenized to obtain component B liquid carrier; Component A and Component B are packaged separately and then mixed together before use.

[0020] The present invention has the following beneficial effects: (1) The modified cellulase of the present invention forms a stable three-dimensional network structure by constructing a coating layer on the surface of the cellulase, which is formed by the electrostatic interaction of a cationic polypeptide polymer (ε-polylysine) and anionic polysaccharide polymer (alginate), and then cross-linking and curing it with multivalent metal ions. This coating layer can effectively maintain the active conformation of enzyme molecules under low temperature and neutral conditions, reduce the problem of increased enzyme molecule rigidity and decreased catalytic efficiency caused by low temperature, and significantly improve the catalytic activity of enzyme preparations. At the same time, the coating layer forms a physical protective barrier for enzyme molecules, enhances its tolerance to high temperature drying (such as spray drying, inlet air temperature 130-170℃), effectively reduces the high energy consumption and long cycle problem of traditional freeze drying process, and improves production efficiency and economy. In addition, the cured coating layer is tightly bound to the enzyme molecules and is not easily dissociated during storage, which can effectively resist the influence of external environmental factors (such as temperature fluctuations, humidity changes, etc.) on enzyme activity, and significantly improve the storage stability of enzyme preparations.

[0021] (2) The cellulase preparation of the present invention consists of lyophilized powder component A and liquid carrier component B. It is packaged independently and mixed before use. This design ensures the long-term stable storage of the enzyme active ingredients in component A and further optimizes the enzyme's action environment after mixing by the alcohol ethers, polyols and other components in component B. Among them, pectinase in component A can work synergistically with cellulase to improve the degradation efficiency of complex substrates; inorganic salts (sodium chloride and calcium chloride) help maintain the osmotic pressure balance and structural stability of enzyme molecules; trehalose, as a protective agent, can play a good protective role for enzyme molecules during freeze-drying and storage; nano silica can increase the dispersibility and adsorption of the enzyme preparation. Alcohol ethers and propylene glycol in component B, as solvents and co-solvents, can ensure that the lyophilized powder of component A dissolves quickly and uniformly during use; polyvinylpyrrolidone, as an anti-re-dyeing polymer, can effectively prevent the hydrolysis products from re-contaminating the substrate during the treatment process (such as biopolishing in the textile industry); Kathon, as a preservative, can inhibit the growth of microorganisms in the liquid carrier during storage and ensure the safety of the preparation. This compound system enables the cellulase preparation to exhibit excellent low-temperature catalytic activity and good stability in various application scenarios such as textile finishing and detergent addition. Detailed Implementation

[0022] To make the objectives, technical solutions, and advantages of this application clearer, the following description and illustration are provided in conjunction with embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of this application. All other embodiments obtained by those skilled in the art based on the embodiments provided in this application without inventive effort are within the scope of protection of this application.

[0023] Obviously, the following description is merely some examples or embodiments of this application. Those skilled in the art can apply this application to other similar scenarios without any inventive effort. Furthermore, it is understood that although the effort involved in such development may be complex and lengthy, for those skilled in the art related to the content disclosed in this application, any changes to design, manufacturing, or production based on the technical content disclosed in this application are merely conventional technical means and should not be construed as insufficient disclosure of the content of this application.

[0024] An embodiment of the first aspect of this application provides a cellulase preparation, comprising, by weight parts: Component A: 500-600 parts modified cellulase, 60-90 parts pectinase, 80-120 parts inorganic salts, 40-60 parts protective agent, and 5-15 parts nano silica. Component B: 160-200 parts alcohol ether, 100-130 parts polyol, 60-90 parts anti-re-dyeing polymer, 1-3 parts preservative, 550-650 parts deionized water; The mass ratio of component A to component B is 1:1.2-2.0.

[0025] The inorganic salt is 70-90 parts sodium chloride and 10-30 parts calcium chloride; the protective agent is trehalose, the polyol is propylene glycol, the anti-re-dyeing polymer is polyvinylpyrrolidone, and the preservative is Kathon.

[0026] In this embodiment, the method for preparing the modified cellulase includes the following steps: S1: Provide cellulase solution and adjust its pH to 6.0-7.5; S2: Introduce a cationic polypeptide polymer into the pH-adjusted enzyme solution and stir until homogeneous; S3: Introduce anionic polysaccharide polymers into the system, so that the anionic polysaccharide polymers and cationic polypeptide polymers form a complex through electrostatic interaction, forming a coating layer on the surface of cellulase; S4: Add polyvalent metal ions for cross-linking treatment to solidify the coating layer; concentrate and / or dry the resulting product to obtain an enzyme preparation.

[0027] This method introduces cationic polypeptide polymers and anionic polysaccharide polymers in steps, and utilizes the electrostatic interaction between the two under neutral pH conditions to self-assemble a uniform composite coating layer on the surface of cellulase molecules. Subsequently, the coating layer is solidified through the cross-linking effect of polyvalent metal ions.

[0028] In step S1, an industrial strain of *Trichoderma reesei* was used and cultured in a fermentation medium containing: 40 g / L corn straw enzymatic hydrolysate, 10 g lactose, 20 g corn steep liquor, 3 g (NH4)2SO4, 5 g KH2PO4, 1 g MgSO4·7H2O, 0.5 g CaCl2, 0.005 g FeSO4·7H2O, 0.0016 g MnSO4·H2O, 0.0014 g ZnSO4·7H2O, and 0.5 mL / L defoamer. The initial pH was 5.2. Fermentation was carried out at 25°C for 120 h, followed by filtration and concentration. The protein concentration in the concentrated enzyme solution was controlled at 18-22 g / L, the pH at 5.2, and the temperature at 15°C.

[0029] Slowly add 1M NaOH solution dropwise to the concentrated enzyme solution to adjust the pH from 5.2 to 6.1-6.3 while stirring at 80 rpm. At pH 6.1-6.3, the enzyme surface carries a net negative charge, and the zeta potential value should be -18 mV to -22 mV.

[0030] In this embodiment, the cationic polypeptide polymer is ε-polylysine with a number-average molecular weight of 2000-5000 Da. ε-polylysine is prepared as a 5% w / v aqueous solution and added at 5%-12% of the enzyme protein mass. It is slowly added dropwise at a rate of 3-5 mL / min, maintaining a temperature of 15°C and a stirring speed of 100 rpm during the addition process. After the addition is complete, stirring continues for 30 min to allow adsorption equilibrium to be reached. During this process, the positively charged ε-polylysine molecules bind to the negatively charged sites on the enzyme molecule surface through electrostatic attraction, initially forming a positively charged adsorption layer around the enzyme molecule. At this point, the Zeta potential value on the enzyme molecule surface changes from the original -18 mV to -22 mV to -2 mV to +2 mV, providing a charge basis for the subsequent binding of the anionic polysaccharide polymer.

[0031] In this embodiment, the anionic polysaccharide polymer is alginate with a weight-average molecular weight of 100-200 kDa, prepared as a 5% w / v aqueous solution. The amount of the anionic polysaccharide polymer added is 10-25% of the enzyme protein mass, slowly added dropwise at a rate of 5-8 mL / min, maintaining a temperature of 15°C and a stirring speed of 100 rpm. After the addition is complete, stirring is continued for 30 min. The Zeta potential is detected to decrease to -20 mV to -25 mV, indicating that sodium alginate has been successfully assembled. During this process, the negatively charged alginate molecules further assemble with the ε-polylysine already bound to the enzyme surface through strong electrostatic interactions, forming an ε-polylysine-alginate composite bilayer coating structure. This structure not only tightly encapsulates the enzyme molecule through electrostatic attraction, but the hydrogen bonds and hydrophobic interactions between its molecular chains further enhance the stability of the coating layer, providing a preliminary physical barrier for the enzyme molecule.

[0032] In this embodiment, the multivalent metal ion is Zn. 2+ Cu 2+ Fe 3+ or Co 2+ At least one of the following. Preferably Zn. 2+ .

[0033] A 1 mol / L ZnCl2 solution was used. The ZnCl2 solution was slowly added to the reaction system in step S3. The pH of the system was precisely adjusted to 6.5 ± 0.1 using dilute NaOH or HCl. Stirring was stopped, and the system was allowed to stand at 10-20°C for 2-8 hours to mature. The concentration of the polyvalent metal ions in the reaction system was 2-5 mM, preferably 3 mM. In this step, Zn... 2+ As a cross-linking agent, it can undergo coordination complexation with the carboxyl groups on the alginate molecular chain, forming a stable three-dimensional network structure within the composite coating layer. This cross-linking and curing effect significantly improves the mechanical strength and structural stability of the coating layer, enabling more effective fixation of the active conformation of enzyme molecules and enhancing its resistance to external environmental stresses. The static ripening process helps Zn... 2+ The reaction with alginate ensures the uniformity and integrity of the cross-linked network, thus laying a good foundation for subsequent concentration and drying steps.

[0034] In this embodiment, after maturation, a nanofiltration membrane with a molecular weight cutoff of 1000 Da was used to concentrate the protein to a concentration of 80-120 g / L at 25°C. Spray drying was then performed; the inlet air temperature was 130-170°C, the outlet air temperature was 60-80°C, and the atomizer speed was 18000-20000 rpm, ultimately yielding cellulase preparation powder.

[0035] A second aspect of this application provides a method for preparing a cellulase preparation, comprising the following steps: The modified cellulase, pectinase, sodium chloride, calcium chloride, and trehalose from Example 1 were mixed evenly in deionized water (the amount of water in component A was about 3-5 times the total mass of solids), and stirred at low speed for 100-200 rpm to obtain an enzyme solution, which was kept at 10-15℃. Nano-silica (particle size 20-50 nm) was slowly added to the enzyme solution and dispersed for 15-20 minutes using a high-shear emulsifier (speed 3000-5000 rpm) to form a uniform suspension. The suspension was pre-frozen at -40℃ for 4-6 hours. The freeze-dried block was then pulverized in a low-temperature pulverizer and passed through an 80-mesh sieve to obtain the freeze-dried powder of component A. Add the mixture of alcohol ether and propylene glycol to the polyvinylpyrrolidone solution, stir at 300-500 rpm, and homogenize 2-3 times under 20-30 MPa pressure; add Kathon, stir for 15 minutes, and let stand for 1-2 hours to obtain component B liquid carrier. Component A and Component B are packaged separately in a mass ratio of 1:1.5, and are mixed and compounded before use.

[0036] The cellulase preparation of this invention exhibits excellent catalytic activity under low temperature and neutral conditions. The main components of the modified cellulase include cellulase, ε-polylysine, alginate, and Zn. 2+ It contains polyvalent metal ions, among which ε-polylysine and alginate are naturally sourced biocompatible materials, ensuring the safety and biocompatibility of the enzyme preparation in textiles, detergents, feed, food, and other fields. It appears as a light yellow powder with good flowability, no irritating odor, and is easily soluble in water.

[0037] Example The following examples describe the disclosure of this invention in more detail. These examples are merely illustrative, as various modifications and variations will be apparent to those skilled in the art within the scope of this disclosure. Unless otherwise stated, all parts, percentages, and ratios reported in the following examples are based on weight. Unless otherwise stated, all reagents used in the examples are available commercially or synthesized using conventional methods and are ready for use without further processing. Unless otherwise stated, all instruments used in the examples are available commercially.

[0038] Preparation Example 1 The preparation method of modified cellulase includes the following steps: S1: Take 10 L of concentrated enzyme solution (protein concentration 18.2 g / L), place it in a reaction vessel, control the temperature at 15℃, stir at 80 rpm, and slowly adjust the pH to 6.1 with 1 M NaOH solution; take a sample and measure the Zeta potential, which is -19.8 mV; S2: Prepare a 5% w / v aqueous solution of ε-polylysine. Take 30 mL of the ε-polylysine aqueous solution (ε-polylysine addition = 1.5 g, protein mass ratio = 1:12) and add it dropwise to the protease solution in step S1 at a flow rate of 4 mL / min. After the addition is complete, continue stirring for 30 min. Take a sample and detect that the Zeta potential rises to -0.5 mV. S3: Add 60 mL of 5% (w / v) sodium alginate solution dropwise at 6 mL / min (sodium alginate addition = 3.0 g, protein ratio = 1:6). After the addition is complete, continue stirring for 30 min, and take a sample to detect that the zeta potential has dropped to -21.2 mV; S4: Add 30.5 mL of 1 M ZnCl2 solution, and adjust the pH to 6.5 with dilute NaOH. Stop stirring and let it stand at 15°C for 4 hours to mature. The polyvalent Zn in the reaction system... 2+ The concentration was 3 mM; after maturation, the protein was concentrated to a concentration of 100 g / L using a nanofiltration membrane with a molecular weight cutoff of 1000 Da at 25°C. Spray drying was then performed; the inlet air temperature was 150°C and the outlet air temperature was 70°C, ultimately yielding cellulase powder.

[0039] Preparation Example 2 This embodiment is basically the same as that of embodiment 1, except that in step S2: ε-polylysine is prepared into a 5% w / v aqueous solution, and 20 mL of ε-polylysine aqueous solution (ε-polylysine addition amount = 1.0 g, protein mass ratio = 1:20) is added dropwise to the protease solution in step S1 at a flow rate of 3 mL / min. After the addition is completed, stirring is continued for 30 min.

[0040] Preparation Example 3 This embodiment is basically the same as Example 1, except that in step S3: 90 mL of 5% (w / v) sodium alginate solution (sodium alginate addition = 4.5 g, protein ratio = 1:4) is added dropwise at a rate of 7 mL / min. After the addition is complete, stirring is continued for 30 min.

[0041] Preparation Example 4 This embodiment is basically the same as Example 1, except that in step S4: 10.1 mL of 1 M ZnCl2 solution is added, and the pH is adjusted to 6.5 with dilute NaOH. Stirring is stopped, and the mixture is allowed to stand at 15°C for 4 hours to mature. The polyvalent Zn in the reaction system... 2+ The concentration is 1 mM.

[0042] Preparation Example 5 This embodiment is basically the same as that of embodiment 1, except that in step S4, ZnCl2 is replaced with an equimolar amount of CuCl2 (30.5 mL 1M CuCl2).

[0043] Example 1 A method for preparing a cellulase preparation includes the following steps: The modified cellulase, pectinase, sodium chloride, calcium chloride, and trehalose from Preparation Example 1 were mixed evenly in deionized water (the amount of water in component A was approximately 4 times the total mass of solids), and stirred at low speed at 1500 rpm to obtain an enzyme solution, which was kept at 15°C. Nano-silica (particle size 20 nm) was slowly added to the enzyme solution and dispersed for 20 minutes using a high-shear emulsifier (speed 4000 rpm) to form a uniform suspension. The suspension was pre-frozen at -40°C for 4-6 hours, and the freeze-dried block was pulverized in a low-temperature pulverizer and passed through an 80-mesh sieve to obtain the freeze-dried powder of component A. The mixture of alcohol ether and propylene glycol was added to a polyvinylpyrrolidone solution and stirred at 400 rpm. The mixture was homogenized twice at 25 MPa pressure. Kathon was added, and the mixture was stirred for 15 minutes and allowed to stand for 1.5 hours to obtain component B liquid carrier. Component A and Component B are packaged separately in a mass ratio of 1:1.5, and then mixed and compounded before use.

[0044] Example 2 This embodiment is basically the same as Example 1, except that the modified cellulase from Preparation Example 2 is used.

[0045] Example 3 This embodiment is basically the same as Example 1, except that the modified cellulase from Preparation Example 3 is used.

[0046] Example 4 This embodiment is basically the same as Example 1, except that the modified cellulase from Preparation Example 4 is used.

[0047] Example 5 This embodiment is basically the same as Example 1, except that the modified cellulase of Preparation Example 5 is used.

[0048] Comparative Example 1 This comparative example is basically the same as Example 1, except that the method for preparing the modified cellulase is as follows: 10 L of concentrated enzyme solution is taken and concentrated to a protein concentration of 100 g / L at 25°C without any assembly treatment using a nanofiltration membrane with a molecular weight cutoff of 1000 Da; spray drying is then performed with an inlet air temperature of 150°C and an outlet air temperature of 70°C to finally obtain cellulase powder.

[0049] Comparative Example 2 This comparative example is basically the same as Example 1, except that the method for preparing the modified cellulase is as follows: take 10 L of concentrated enzyme solution, adjust the pH to 6.2, add 1.5 g of ε-polylysine, 3.0 g of sodium alginate and 30.5 mL of ZnCl2 at one time, stir rapidly for 30 min, adjust the pH to 6.5, then concentrate by nanofiltration and spray dry.

[0050] Comparative Example 3 This comparative example is basically the same as Example 1, except that the method for preparing the modified cellulase is as follows: take 10 L of concentrated enzyme solution, adjust the pH to 6.2, skip the ε-polylysine addition step in step S2, directly add 60 mL of 5% sodium alginate, then add 30.5 mL of ZnCl2, adjust the pH to 6.5, let it stand and mature for 4 h, and then proceed as in Example 1.

[0051] Comparative Example 4 This comparative example is basically the same as Example 1, except that the method for preparing the modified cellulase is: ZnCl2 is not added, while the rest is basically the same as the method for preparing Example 1.

[0052] Experimental Case 1 The modified cellulase samples from Examples 1-5 and Comparative Examples 1-4 were subjected to the following tests: Spray drying and yield calculation: The liquid to be dried (protein concentration approximately 18-20 g / L) was concentrated to a protein concentration of approximately 100 g / L via nanofiltration. Spray drying was then performed using a centrifugal spray drying tower (inlet air temperature 150℃, outlet air temperature 70℃, atomizer speed 18000 rpm). The powder collected from the bottom of the tower and the cyclone separator was weighed. Spray drying yield (%) = (Total protein content in the dried powder / Total protein content in the liquid before drying) × 100%.

[0053]

[0054] As shown in Table 1, the spray drying yields of the modified cellulase samples in Examples 1-5 of this application are all above 91%, significantly higher than Comparative Example 1 (65.2%) without coating and cross-linking treatment, Comparative Example 2 (82.1%) without stepwise assembly, Comparative Example 3 (78.5%) lacking cationic polypeptide polymers, and Comparative Example 4 (80.3%) without the addition of polyvalent metal ions for cross-linking. This indicates that the composite coating layer structure formed by the method of this application effectively improves the spray drying performance of the enzyme solution, reduces losses during the drying process, and increases the powder yield.

[0055] Experimental Case 2 Enzyme activity retention rate after spray drying: The cellulase preparations A and B components of Examples 1-5 and Comparative Examples 1-4 were mixed and the enzyme activity was measured. Retention rate (%) = (enzyme activity in reconstituted solution / enzyme activity in concentrated solution before spray drying) × 100%. The test results are shown in Table 1.

[0056] Storage stability: The cellulase preparations A and B components of Examples 1-5 and Comparative Examples 1-4 were mixed, sealed in aluminum foil bags, and placed in a 40°C constant temperature incubator for accelerated aging for 30 days (simulating storage at room temperature of 25°C for 6 months). The residual enzyme activity was then measured. Storage retention rate (%) = (enzyme activity after 30 days of storage at 40°C / initial enzyme activity) × 100%.

[0057] Thermal stability: The cellulase preparations A and B from Examples 1-5 and Comparative Examples 1-4 were mixed and treated in a 50°C water bath for 30 min, then immediately cooled in an ice bath, and the enzyme activity was measured. Thermal stability residual rate (%) = (enzyme activity after treatment / enzyme activity before treatment) × 100%. The test results are shown in Table 2.

[0058]

[0059] As shown in Table 2, regarding the enzyme activity retention rate after spray drying, Examples 1-5 all reached over 81%, with Examples 1 and 3 reaching 85.6% and 86.3%, respectively. In contrast, Comparative Example 1 only achieved 32.5%, Comparative Example 2 58.6%, Comparative Example 3 41.2%, and Comparative Example 4 68.5%. The enzyme activity retention rates of the Examples are significantly higher than those of the Comparative Examples, fully demonstrating that the coating layer formed by the stepwise introduction of cationic polypeptide polymers and anionic polysaccharide polymers and cross-linking with multivalent metal ions in this application can effectively protect the active conformation of cellulase during the harsh process of spray drying, significantly reducing enzyme activity loss. Comparative Example 2, due to the lack of stepwise assembly and the addition of all components at once, may have resulted in uneven or incomplete coating layer formation. Therefore, although its yield and enzyme activity retention rate were higher than Comparative Examples 1, 3, and 4, they were still lower than those of the Examples. Comparative Example 3 lacked the initial adsorption of ε-polylysine, making it difficult to effectively assemble and form a stable coating layer on the enzyme surface by directly adding sodium alginate. Comparative Example 4 did not undergo Zn treatment. 2+ Cross-linking and insufficient stability of the coating layer structure both lead to a decrease in enzyme protection efficacy.

[0060] The cellulase preparations of Examples 1-5 of this application all exhibited a storage retention rate exceeding 84% at 40°C, with Examples 1 and 3 reaching 90.3% and 90.2%, respectively, significantly superior to the comparative examples. This indicates that the cellulase preparations prepared according to this invention possess excellent storage stability, and the ε-polylysine-alginate composite bilayer coating structure, coated with Zn... 2+The three-dimensional network formed after cross-linking can effectively isolate the effects of external environmental factors (such as oxygen and humidity) on enzyme molecules and slow down the enzyme inactivation rate. In the 50°C thermal stability test, the retention rates of Examples 1-5 were all above 67%, with Example 3 reaching 74%, while the thermal stability retention rates of the comparative examples were generally lower, with Comparative Example 1 at only 18.5% and Comparative Example 3 at 15.2%. This further verifies the effective protective effect of the composite coating structure on enzyme molecules. Its stable three-dimensional network can resist enzyme protein denaturation caused by high temperature to a certain extent, thereby maintaining a high enzyme activity retention rate. Although Comparative Example 4, due to its alginate coating layer, has better storage stability and thermal stability than the uncoated Comparative Examples 1 and 3, it lacks Zn. 2+ The crosslinking of the components in Comparative Example 2 resulted in insufficient stability of the coating layer structure, and the protective effect was still inferior to that of the other examples. Furthermore, the uneven coating layer caused by the one-time addition of components in Comparative Example 2 also resulted in significantly lower stability compared to the examples that were assembled and crosslinked in stages.

[0061] Experimental Case 3 Protease resistance: Components A and B of the cellulase preparations from Examples 1-5 and Comparative Examples 1-4 were mixed, and trypsin was added to a final concentration of 0.1% (w / v). The mixture was treated in a 25°C water bath for 1 h, and the reaction was immediately terminated by adding benzyl sulfonyl fluoride to a final concentration of 1 mM. Enzyme activity was then measured. Protease resistance residual rate (%) = (enzyme activity after treatment / enzyme activity before treatment) × 100%. The test results are shown in Table 3.

[0062] Table 3 Test results of samples from Examples 1-5 and Comparative Examples 1-4

[0063] As shown in Table 3, the protease resistance retention rates of Examples 1-5 of this application all reached over 80%, with Examples 1 and 3 reaching 84.9% and 85.2% respectively, significantly higher than the comparative examples. Comparative Example 1, without any coating treatment, had a protease resistance retention rate of only 23.7%; Comparative Example 3, lacking ε-polylysine, had an even lower retention rate of 21.4%; Comparative Example 2, due to uneven coating caused by the simultaneous addition of all components, had a retention rate of 40.5%; Comparative Example 4, without the addition of Zn... 2+ Crosslinking was performed, and the residual rate was 56.7%. This indicates that the ε-polylysine-alginate composite bilayer coating structure of the present invention, after Zn crosslinking, has a high degree of crosslinking efficiency. 2+ After cross-linking, a physical barrier is formed, effectively preventing exogenous proteases such as trypsin from directly attacking and degrading cellulase molecules, thereby significantly improving the protease resistance of the enzyme preparation. This characteristic allows the enzyme preparation to better maintain its catalytic activity in practical applications, especially in environments where it may come into contact with proteases (such as detergents, feed, etc.).

[0064] Experimental Case 4 Low-temperature neutral polishing test of pure cotton knitted fabric: Pure cotton knitted fabric (32S single jersey, weight 160 g / m²) 2 The fabric (purchased from a textile factory) was cut into 20 cm × 20 cm samples, each weighing approximately 6.4 g. The fabric was pretreated in a 65°C water bath for 10 min (liquor ratio 1:20) to remove surface impurities, then thoroughly washed with water and squeezed dry for later use.

[0065] Add 8 times the amount of room temperature water (25°C) to the mixing tank, add component B, and stir for 30 seconds until evenly dispersed; slowly sprinkle the powder of component A into the above diluted solution, and stir gently for 2 minutes until completely dissolved to obtain the working solution mother liquor of Examples 1-5 and Comparative Examples 1-4.

[0066] The fabric was immersed in the above working solution stock solution at a temperature of 15°C for 60 minutes at a pH of 7.0. After treatment, the temperature was raised to 85°C and kept at that temperature for 10 minutes to completely inactivate the enzyme. The fabric was then removed, washed with 60°C hot water for 10 minutes, followed by washing with cold water for 10 minutes. It was then centrifuged to remove water and dried at 60°C to constant weight.

[0067] Fabrics were dried to constant weight before and after treatment, and weighed for calculation. Weight loss rate (%) = (W0 - W1) / W0 × 100%. Following GB / T 4802.1-2008, the pilling box method was used, and the fabrics were rated after 60 revolutions (grades 1-5, with grade 5 being the best). Test results are shown in Table 4.

[0068] Table 4. Test results of samples from Examples 1-5 and Comparative Examples 1-4

[0069] As shown in Table 4, the weight loss rate of the pure cotton knitted fabrics in Examples 1-5 of this application after low-temperature neutral polishing treatment was between 1.72% and 1.81%, and the anti-pilling performance reached level 3.7 or above. Among them, Example 3 showed the best performance with a weight loss rate of 1.81% and an anti-pilling performance of level 4.0. This indicates that the cellulase preparation prepared by this invention still has excellent catalytic activity and polishing effect under low-temperature (15℃) and neutral (pH 7.0) conditions, and can effectively hydrolyze the fuzz and loose fibers on the fabric surface, thereby improving the anti-pilling performance of the fabric. In contrast, Comparative Example 1, due to severe enzyme activity loss and poor stability, had a weight loss rate of only 0.62% and an anti-pilling property of only 2.5, indicating poor treatment effect; Comparative Example 2, due to uneven coating leading to insufficient enzyme activity and stability, had a weight loss rate of 1.02% and an anti-pilling property of 3.1; Comparative Example 3, lacking ε-polylysine, had poor enzyme protection, with a weight loss rate of 1.54% and an anti-pilling property of 3.4; Comparative Example 4 did not undergo Zn treatment. 2+The cross-linking and coating layer stability were insufficient, with a weight loss rate of 1.67% and a pilling resistance grade of 3.5. The weight loss rate and pilling resistance of each comparative example were significantly lower than those of the example, fully demonstrating that the modified cellulase preparation of this application can effectively function in practical applications, especially suitable for low-temperature neutral polishing processes sensitive to temperature and pH. It can reduce energy consumption while ensuring processing effects and has good prospects for industrial application.

[0070] It should be noted that this application is not limited to the above-described embodiments. The above embodiments are merely examples, and any embodiments with the same structure and effect as the technical concept within the scope of this application are included in the technical scope of this application. Furthermore, various modifications that can be conceived by those skilled in the art to the embodiments, and other ways of constructing by combining some of the constituent elements of the embodiments, without departing from the spirit of this application, are also included in the scope of this application.

Claims

1. A cellulase preparation, characterized in that, In parts by weight, including: Component A: 500-600 parts modified cellulase, 60-90 parts pectinase, 80-120 parts inorganic salts, 40-60 parts protective agent, and 5-15 parts nano silica. Component B: 160-200 parts alcohol ether, 100-130 parts polyol, 60-90 parts anti-re-dyeing polymer, 1-3 parts preservative, 550-650 parts deionized water; The mass ratio of component A to component B is 1:1.2-2.

0.

2. The cellulase preparation according to claim 1, characterized in that, The method for preparing the modified cellulase includes the following steps: S1: Provide cellulase solution and adjust its pH to 6.0-7.5; S2: Introduce a cationic polypeptide polymer into the pH-adjusted enzyme solution and stir until homogeneous; S3: Introduce anionic polysaccharide polymers into the system, so that the anionic polysaccharide polymers and cationic polypeptide polymers form a complex through electrostatic interaction, forming a coating layer on the surface of cellulase; S4: Add polyvalent metal ions for cross-linking treatment to solidify the coating layer; concentrate and / or dry the resulting product to obtain an enzyme preparation.

3. The cellulase preparation according to claim 2, characterized in that, The cationic polypeptide polymer is ε-polylysine, with a number-average molecular weight of 2000-5000 Da. The anionic polysaccharide polymer is alginate with a weight-average molecular weight of 100-200 kDa.

4. The cellulase preparation according to claim 2, characterized in that, The multivalent metal ion is Zn. 2+ Cu 2+ Fe 3+ or Co 2+ At least one of them.

5. The cellulase preparation according to claim 2, characterized in that, The enzyme protein concentration of the cellulase solution is 18-22 g / L; After pH adjustment in step S1, the temperature is controlled at 5-20℃.

6. The cellulase preparation according to claim 5, characterized in that, The amount of the cationic polypeptide polymer added is 5%-12% of the enzyme protein mass; And / or, the amount of the anionic polysaccharide polymer added is 10-25% of the enzyme protein mass.

7. The cellulase preparation according to claim 2, characterized in that, The concentration of the polyvalent metal ions is 2-5 mM.

8. The cellulase preparation according to claim 2, characterized in that, The cationic polypeptide polymer and the anionic polysaccharide polymer are added dropwise at a rate of 0.1-1.0 L / (h·L enzyme solution) and a stirring speed of 80-160 rpm.

9. The cellulase preparation according to claim 2, characterized in that, The crosslinking treatment is followed by a curing step; the curing is carried out at 10-20°C for 2-8 hours. And / or, the drying is spray drying; the inlet air temperature of the spray dryer is 130-170℃, and the outlet air temperature is 60-80℃.

10. A method for preparing a cellulase preparation, characterized in that, The method for preparing the cellulase preparation according to any one of claims 1-9 comprises the following steps: Modified cellulase, pectinase, inorganic salts, protective agents and inorganic nanoparticles were mixed evenly in the liquid phase, and then freeze-dried, pulverized and sieved to obtain component A freeze-dried powder. Alcohol ether, polyol, anti-re-dyeing polymer and preservative are mixed in water and homogenized to obtain component B liquid carrier; Component A and Component B are packaged separately and then mixed together before use.

Images