Bio-enzyme preparation for catalytically degrading grease as well as preparation method and application of bio-enzyme preparation

Abstract

The invention discloses a bio-enzyme preparation for catalytic degradation of grease as well as a preparation method and application thereof, and belongs to the technical field of bio-enzymes. The gel carrier is formed by cross-linking a chitosan-lauric acid-dopamine conjugate and Fe < 3 + > ions through covalent coupling; the bonding adaptive compound enzyme is a mixed enzyme of which the surface is grafted with a catechol group through an N-hydroxysuccinimide-dopamine ester modifier; the technical bottleneck that efficient catalysis, firm fixation, convenient recovery and long-acting stability of an industrial cleaning enzyme preparation are difficult to consider at the same time is solved, and the prepared biological enzyme preparation has the high-activity recovery rate, excellent recycling stability, intelligent separation and recovery characteristics and efficient broad-spectrum cleaning capacity; and the method has remarkable industrial application advantages and market competitiveness.

Description

Technical Field

[0001] This invention belongs to the field of bioenzyme technology, and relates to a bioenzyme preparation for catalytic degradation of oils and fats, its preparation method, and its application. Background Technology

[0002] In the field of industrial cleaning, particularly for cleaning large amounts of grease residue in food processing equipment and pipelines, bio-enzyme preparations have attracted widespread attention due to their advantages such as high catalytic efficiency, mild reaction conditions, and environmental friendliness. However, in practical applications, traditional free enzymes are prone to inactivation under high temperatures, extreme pH conditions, or the presence of surfactants, and their free state cannot be recovered and reused from complex cleaning waste liquids, resulting in high costs.

[0003] In existing technologies, immobilized enzyme technology is an effective method to improve enzyme stability and achieve enzyme reuse. To facilitate recycling, various types of solid carriers have been disclosed, such as magnetic nanomaterials or porous microspheres. While magnetic carriers can be easily recycled using an external magnetic field, their core magnetic materials (such as Fe3O4) not only lack chemical stability in strongly alkaline or oxidizing cleaning environments but are also easily corroded and degraded, potentially contaminating the system. Furthermore, in complex industrial wastewater, nanoparticles are easily lost due to aggregation or embedding in contaminants, and their separation process faces challenges in large-scale applications. Porous microspheres, especially chitosan-based microspheres, are widely used in immobilized enzyme research due to their natural raw materials, ease of modification, and good biocompatibility. Patents CN115058052A and CN114940776A, for example, introduce nano-silica into chitosan gel and optimize the molding process to prepare porous chitosan microspheres. Their aim is to increase the specific surface area and improve the pore structure, thereby enhancing the loading rate and stability of alkaline proteases. While this technology improves immobilization efficiency to some extent, its inherent porous nature becomes a new obstacle when dealing with food industry cleaning wastewater rich in oils, protein colloids, and solid particles. Specifically, these micropores are easily clogged by contaminants, causing a sharp decline in mass transfer efficiency and preventing the full utilization of enzyme active sites. At the same time, these microspheres with a density similar to water are difficult to completely separate from suspended impurities in the wastewater through sedimentation or filtration, making the recovery process cumbersome and prone to carrier loss.

[0004] In contrast, hydrogel-based carriers provide enzymes with a hydrophilic three-dimensional network that more closely resembles their natural environment, theoretically offering superior enzyme stability and compatibility. These carriers have been widely used in fields where separation and recovery requirements are relatively less stringent and the system purity is crucial, such as controlled drug release, biosensor construction, and biocatalytic reactors. However, when applications shift to industrial equipment cleaning with complex compositions and frequent solid-liquid separation, the inherent limitations of traditional hydrogels become apparent. First, excessive swelling leads to poor mechanical strength, making them prone to breakage in fluids. Second, the lack of a clear physical boundary between their hydrated state and the waste liquid makes rapid and thorough separation impossible in mixtures containing emulsified oils and solid residues. This recovery challenge has resulted in a long-standing lack of efficient and reliable practical technologies for high-performance gel-immobilized enzymes in industrial cleaning, especially in demanding scenarios involving complex compositions and frequent solid-liquid separation, creating a technological gap in practical applications.

[0005] In summary, existing technologies have consistently failed to address a core contradiction: how to enable a carrier to simultaneously possess the excellent enzyme-stabilizing environment of hydrogels and the convenient recyclability of solid particles. Summary of the Invention

[0006] The purpose of this invention is to provide a bio-enzyme preparation for catalytic degradation of oils and fats, its preparation method, and its application. By constructing a synergistic system of a smart gel carrier with pH-responsive characteristics and a bonded compatible complex enzyme, this invention addresses the core problem in existing technologies where immobilized enzyme carriers struggle to achieve both efficient and stable catalysis and convenient and thorough recovery in complex cleaning wastewater systems. This improves the catalytic efficiency, operational stability, and recyclability of the enzyme preparation in harsh industrial cleaning environments.

[0007] The technical solution of this invention is a bio-enzyme preparation for catalytic degradation of oils and fats. The key feature is that it comprises: a bonded, compatible complex enzyme immobilized on a pH-responsive gel carrier; the gel carrier is composed of a chitosan-lauric acid-dopamine conjugate covalently coupled with Fe... 3+ The above-mentioned bond-matched complex enzyme is a mixed enzyme whose surface is grafted with catechol groups through an N-hydroxysuccinimide-dopamine ester modifier.

[0008] Specifically, the above-mentioned mixed enzyme contains lipase, protease and coenzyme in a mass ratio of (3-5):1:(0-0.3).

[0009] Furthermore, the aforementioned coenzyme is selected from at least one of amylase, pectinase, and cellulase.

[0010] Specifically, the aforementioned gel carrier shrinks and hardens at pH 6.5–7.0 and swells at pH 7.5–9.5.

[0011] A method for preparing the above-mentioned biological enzyme preparation, the key feature of which is that it includes the following steps: S1. Preparation of lauric acid-dopamine derivatives; S2. Synthesize chitosan-lauric acid-dopamine conjugate; S3. Preparation of bond-matched complex enzymes; S4. Immobilization and gel formation: The above-mentioned conjugate, the above-mentioned bond-matched complex enzyme, cross-linking agent, and coupling agent are mixed, and then covalently coupled with Fe... 3+ Ionic cross-linking reaction forms an enzyme preparation with a gel as a carrier.

[0012] Furthermore, in step S1, the process of lauric acid-dopamine derivative includes: adding a laurate solution activated with EDC·HCl and NHS dropwise to a phosphate buffer solution containing dopamine hydrochloride and sodium bicarbonate at 0℃~4℃ and in an inert atmosphere, stirring the reaction at room temperature in the dark for 6h~14h, and acidifying to pH 1~3 after the reaction is completed to precipitate the product.

[0013] Furthermore, in step S3, the process of preparing the bonded matching complex enzyme includes: reacting the above mixed enzyme with the above modifying agent at 0℃~4℃ and pH7.8~8.2 for 1.5h~2.5h.

[0014] Furthermore, in step S4, the conditions for the covalent coupling are: reaction at 0℃~4℃ for 1h~3h in the presence of EDC·HCl and NHS; the above Fe 3+ The ionic crosslinking reaction conditions are: adding FeCl3·6H2O aqueous solution, so that Fe... 3+ The final concentration is 1.6 mmol / L to 2.5 mmol / L, and the gel is formed by standing at room temperature for 5 min to 15 min. During the gel formation process, the stirring speed is controlled at 600 r / min to 800 r / min so that the particle size D50 of the formed gel particles is distributed in the range of 180 μm to 250 μm.

[0015] A cleaning agent, crucially, contains the aforementioned biological enzyme preparation.

[0016] The key to using the above-mentioned cleaning agent is that it includes: diluting the cleaning agent with water at a volume ratio of 1:(50-200) to prepare a cleaning working solution, and adjusting the pH of the cleaning working solution to 7.5-9.5 for cleaning; after cleaning, adjusting the pH of the cleaning working solution to 6.5-7.0 to cause the biological enzyme preparation to shrink and harden, so as to facilitate recycling.

[0017] Compared with the prior art, the present invention has the following advantages: This invention provides a bio-enzyme preparation for catalytic degradation of lipids and its intelligent immobilization method. By constructing a synergistic technology system integrating an intelligent responsive carrier, a synergistic anchoring mechanism, and a composite enzyme formulation, it achieves a systematic breakthrough in immobilization efficiency, operational stability, ease of application, and broad-spectrum cleaning effectiveness. Specific technical effects are reflected in: First, this invention establishes a system based on "Fe 3+ The intelligent responsive gel carrier, centered on a "catechin coordination network," fundamentally solves the long-standing technical problem in the industry of low mechanical strength and easy loss during recycling of immobilized enzymes. Using chitosan as a backbone, the carrier achieves a combination of hydrophobic microenvironment construction and metal coordination site introduction through covalent grafting of lauric acid-dopamine conjugates. This design endows the carrier with dual intelligent properties: firstly, excellent pH responsiveness, allowing it to shrink and harden during storage for easy separation, and fully swell during operation to facilitate mass transfer; secondly, the formation of a high-strength dynamic cross-linked network. This fundamental innovation enables enzyme preparations to maintain extremely high structural integrity and activity recovery rates even under harsh reusable conditions, laying the material foundation for industrial-grade recyclable applications.

[0018] Secondly, this invention innovatively proposes a strategy for catecholization modification of the enzyme surface, achieving synergistic, directional, and multi-point anchoring of enzyme molecules and the carrier network, greatly improving the immobilization robustness. Traditional immobilization methods rely on random coupling, which easily leads to the embedding of enzyme active sites or single binding sites. This invention, through specific modification of the enzyme, introduces catechol groups on its surface, enabling it not only to undergo conventional covalent coupling with the carrier, but also to bind to Fe in the carrier network through the catechol groups. 3+ Additional coordination bonds are formed. This dual anchoring mechanism of "covalent + coordination" significantly enhances the binding force and uniformity between the enzyme and the carrier, effectively inhibiting enzyme detachment caused by physical shearing or chemical stress during use, thus achieving operational stability far superior to traditional methods.

[0019] Third, this invention achieves integrated optimization across the entire chain, from carrier design and immobilization process to application formulation, realizing the optimal balance between performance, stability, and practicality. At the carrier level, the precise selection of lauric acid chain length creates the most suitable hydrophobic microenvironment for lipase and optimizes its pH response behavior. At the application level, the accompanying specialized cleaning agent formulation, through a precise pH buffer system, gel suspension stabilization technology, and enzyme-friendly surfactant blend, provides an ideal storage and working environment for the immobilized enzyme particles, ensuring long-term stability of their activity throughout the shelf life and full release during use. Furthermore, the composite enzyme formulation design targeting complex dirt components demonstrates excellent synergistic degradation capabilities, significantly broadening the product's application range.

[0020] In summary, the innovative design of this invention successfully solves the technical bottleneck of industrial cleaning enzyme preparations, which struggle to simultaneously achieve high-efficiency catalysis, robust fixation, convenient recovery, and long-term stability. The prepared bio-enzyme preparation possesses high activity recovery rate, excellent cyclic stability, intelligent separation and recovery characteristics, and efficient broad-spectrum cleaning capabilities, exhibiting significant advantages for industrial application and market competitiveness. Attached Figure Description

[0021] Figure 1 This is a graph showing the swelling rate-pH response of enzyme preparation sample 1 of the present invention. Detailed Implementation

[0022] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0023] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0024] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0025] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0026] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0027] Unless otherwise specified in the examples, conventional conditions may be followed. Reagents or instruments whose manufacturers are not specified or otherwise described are all commercially available products. Room temperature in this invention refers to the temperature of a normal laboratory or factory within the range of 22℃ to 26℃. In the embodiments or comparative examples of this invention, the room temperature is controlled at 25℃ ± 1℃.

[0028] The lipases, proteases, amylases, pectinases, and cellulases used in this invention are all commercially available, conventional, water-soluble, food-grade enzyme products, and are all in powder form. To ensure consistency and comparability of conditions between examples, unless otherwise specified, the enzyme products with the following activity requirements are used in all the following examples or comparative examples of this invention: lipase activity of 100,000 U / g, protease activity of 50,000 U / g, amylase activity of 100,000 U / g, and pectinase and cellulase activities of 5,000 U / g.

[0029] It should be noted that the above-mentioned activity values ​​are exemplary selections of the present invention. In fact, as long as the activity of each enzyme used is not lower than the minimum value required by the present invention (lipase ≥ 100,000 U / g, protease ≥ 50,000 U / g, amylase ≥ 100,000 U / g, pectinase / cellulase ≥ 5,000 U / g), the purpose of the present invention can be achieved. Those skilled in the art will understand that, under the premise that the compound enzyme ratio and immobilization method remain unchanged, using enzyme products with higher activity can theoretically obtain better cleaning effects, which also falls within the protection scope of the present invention.

[0030] The N-hydroxysuccinimide-dopamine ester modifier used in this invention is a customized raw material. Starting materials, succinimide dioctanoate and dopamine hydrochloride, are added to anhydrous dimethyl sulfoxide (DMSO) solvent at a mass ratio of 2:1. Then, 1.5 times the amount of dopamine hydrochloride, N,N-diisopropylethylamine (DIPEA) is added. The reaction system is stirred at room temperature and in the dark for 6 hours. After the reaction, the reaction solution is added dropwise to pre-cooled anhydrous diethyl ether to precipitate. The precipitate is obtained by filtration, washing with cold water, and vacuum drying. The crude product is purified by silica gel column chromatography (eluent: dichloromethane / methanol, volume ratio gradually changing from 15:1 to 8:1). The main fraction is collected, the solvent is removed by rotary evaporation, and then vacuum dried to obtain a white solid, which is N-hydroxysuccinimide-dopamine ester (C). 20 H 26 N2O7 (molecular weight 406.4 g / mol) was found to have a chemical purity of 99.3% (purity ≥ 99.0%) by high performance liquid chromatography.

[0031] Its synthetic route is as follows:

[0032] The chitosan solution used in this invention uses commercially available chitosan products with a degree of deacetylation of 90%. The preparation process of the chitosan solution is as follows: commercially available chitosan powder is dispersed in a 2-morpholine ethanesulfonic acid buffer solution containing glacial acetic acid; wherein the mass and volume ratio of chitosan to the 2-morpholine ethanesulfonic acid buffer solution is 1g:100mL; the concentration of the 2-morpholine ethanesulfonic acid buffer solution is 0.1mol / L, and the volume ratio of the 2-morpholine ethanesulfonic acid buffer solution to glacial acetic acid is 100:1; the mixture is stirred continuously at 55°C for 3 hours until completely dissolved, resulting in a clear chitosan solution with a concentration of 10mg / mL, which is then stored at 4°C for later use. Example 1

[0033] This embodiment provides a bio-enzyme preparation for catalytic degradation of lipids. The bio-enzyme preparation is a bonded, well-matched complex enzyme immobilized on a pH-responsive gel carrier. The gel carrier is composed of a chitosan-lauric acid-dopamine conjugate covalently coupled with Fe... 3+ Ionic cross-linking forms; the bonded suitable type complex enzyme is a mixed enzyme sample 1 whose surface is grafted with catechol groups through an N-hydroxysuccinimide-dopamine ester modifier. This mixed enzyme sample 1 contains lipase and protease in a mass ratio of 4:1, without the addition of auxiliary enzymes.

[0034] The specific preparation process is as follows: S1. Preparation of lauric acid-dopamine derivative (Lau-Dopa): S11, Activation of Lauric Acid: Lauric acid is dissolved in anhydrous dimethyl sulfoxide in a dry reactor; Under nitrogen protection and at room temperature, N-hydroxysuccinimide (NHS) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl) were added while stirring. Under light-protected conditions, the reaction was stirred for another 3 to 5 hours to obtain an activated ester solution. The molar and volume ratio of lauric acid and anhydrous dimethyl sulfoxide used is 1 mol: 10 L; the molar ratio of lauric acid, NHS and EDC·HCl is 1:1.05:1.10.

[0035] S12, Lau-Dopa coupling and decomposition: Dopamine hydrochloride and sodium bicarbonate were dissolved in a phosphate buffer solution to prepare a dopamine solution; wherein the molar ratio of dopamine hydrochloride to phosphate buffer solution was 1 mol: 10 L; the molar ratio of dopamine hydrochloride to sodium bicarbonate was 1: 2.0; the concentration of the phosphate buffer solution was 0.1 mol / L, and the pH was 8.0. The activated ester solution was added dropwise to the dopamine solution at a volume ratio of 1:1.0 under stirring in an ice bath at 2°C. After the addition was complete, the mixture was stirred continuously at room temperature, protected from light and under inert protection and reacted for 10 hours. After the reaction was completed, the pH of the reaction solution was acidified to 2.2, and a precipitate was formed. After filtration, washing with cold water, and freeze-drying, a white solid product, namely Lau-Dopa, was obtained.

[0036] S2. Synthesis of chitosan-lauric acid-dopamine conjugate: Add 50 mL of a 10 mg / mL chitosan solution to the reactor; Lau-Dopa was dissolved in dimethyl sulfoxide to prepare a Lau-Dopa solution with a concentration of 0.2 mol / L, and then mixed with the chitosan solution at a volume ratio of 1:10. Stir and mix for 60 minutes at room temperature and in the dark. Add EDC·HCl to adjust the pH of the reaction system to 5.4; wherein the molar ratio of Lau-Dopa to EDC·HCl in the system is 1:1.05; Continue stirring the reaction at 37°C in the dark for 24 hours; After the reaction was completed, the reaction solution was filtered and dialyzed with 0.1 mol / L sodium bicarbonate solution and water. The retained solid was freeze-dried to obtain a light brown chitosan-lauric acid-dopamine conjugate, abbreviated as chitosan-Lau-Dopa conjugate, with a weight-average molecular weight of 215 kDa.

[0037] S3. Preparation of bond-matched complex enzymes: S31. Material Weighing and Dissolving: Weigh the above mixed enzyme powder and dissolve it in a 0.1 mol / L, pH 8.0 borate buffer solution to prepare a mixed enzyme solution with a concentration of 50 g / L.

[0038] S32, Catecholization modification on the enzyme surface: The modifier N-hydroxysuccinimide-dopamine ester was rapidly dissolved in anhydrous dimethyl sulfoxide to prepare a stock solution of modifier with a concentration of 0.1 mol / L. Under light-protected conditions, stirring, and at 2°C, the modifier stock droplet was added to the mixed enzyme solution, and the reaction was continuously stirred for 2.0 h; the volume ratio of the modifier stock droplet to the mixed enzyme solution was 1:12.

[0039] S33. Termination of the reaction and crude purification: After the reaction was completed, the sample was repeatedly centrifuged and ultrafiltered 4 times at 2℃ and 8000×g centrifugation force. Each time, the sample was replenished to its original volume with ice-cold phosphate buffer at pH 7.0 with 0.05mol / L. The bond-matched complex enzyme was purified by gel filtration chromatography and stored at 2℃ for later use. This was designated as complex enzyme sample 1.

[0040] S4. Immobilized enzymes and gel forming: Chitosan-Lau-Dopa conjugate, complex enzyme sample 1, cross-linking agent, and coupling agent were mixed and then covalently coupled with Fe. 3+ Ionic cross-linking reaction forms an enzyme preparation with a gel as a carrier, specifically as follows: Chitosan-Lau-Dopa conjugate was dissolved in a phosphate buffer solution with a concentration of 0.1 mol / L and a pH of 7.0 to prepare a working solution of chitosan-Lau-Dopa conjugate with a concentration of 20 mg / mL, which is denoted as chitosan working solution. A 0.1 mol / L FeCl3·6H2O aqueous solution was prepared as a stock solution for the crosslinking agent; EDC·HCl and NHS were added sequentially to the chitosan working solution, and the mixture was stirred and activated at room temperature for 15 min to obtain the activated chitosan working solution. The molar-volume ratio of EDC·HCl to chitosan working solution was 10 mmol: 1 L, and the molar-volume ratio of NHS to chitosan working solution was 10 mmol: 1 L. Add compound enzyme sample 1 to the activated chitosan working solution and stir at 2°C for 2 hours; wherein the mass and volume ratio of compound enzyme sample 1 to chitosan working solution is 22g:1L. Add sodium ascorbate and continue stirring at room temperature for 30 minutes; wherein the mass ratio of sodium ascorbate to complex enzyme sample 1 is 1:325; Under vigorous stirring at 700 r / min, the crosslinking agent stock solution was added dropwise at a rate of 1.0 mL / min; wherein the volume ratio of the crosslinking agent stock solution to the chitosan working solution was 1:50; in this embodiment, Fe... 3+ The final concentration was 1.96 mmol / L; After the addition is complete, let it stand for 10 minutes to prepare an enzyme preparation with the gel as a carrier, which is recorded as enzyme preparation sample 1. Example 2

[0041] This embodiment provides a bio-enzyme preparation for catalytic degradation of lipids. The bio-enzyme preparation is a bonded, well-matched complex enzyme immobilized on a pH-responsive gel carrier. The gel carrier is composed of a chitosan-lauric acid-dopamine conjugate covalently coupled with Fe... 3+ Ionic cross-linking forms a bonded, well-matched complex enzyme, which is a mixed enzyme sample 1 with catechol groups grafted onto its surface by an N-hydroxysuccinimide-dopamine ester modifier. This mixed enzyme sample 1 is the same as in Example 1.

[0042] The specific preparation process of this embodiment is the same as that of Embodiment 1, except that in step S4, the composite enzyme sample 1 is added to the activated chitosan working solution, and after stirring and reacting, sodium ascorbate is not added. Instead, the crosslinking agent stock solution is added dropwise at the same rate to carry out subsequent operations, thereby preparing an enzyme preparation with gel as the carrier, which is denoted as enzyme preparation sample 2. Example 3

[0043] This embodiment provides a bio-enzyme preparation for catalytic degradation of lipids. The bio-enzyme preparation is a bonded, well-matched complex enzyme immobilized on a pH-responsive gel carrier. The gel carrier is composed of a chitosan-lauric acid-dopamine conjugate covalently coupled with Fe... 3+ Ionic cross-linking forms a bonded, well-matched complex enzyme, which is a mixed enzyme sample 1 with catechol groups grafted onto its surface by an N-hydroxysuccinimide-dopamine ester modifier. This mixed enzyme sample 1 is identical to that in Example 1.

[0044] The specific preparation process is as follows: S1. Preparation of Lau-Dopa: S11, Activation of Lauric Acid: Lauric acid is dissolved in anhydrous dimethyl sulfoxide in a dry reactor; NHS and EDC·HCl were added while stirring under nitrogen protection and at room temperature; Under light-protected conditions, the reaction was stirred for another 5 hours to obtain an activated ester solution; The molar and volume ratio of lauric acid and anhydrous dimethyl sulfoxide used is 1 mol: 10 L; the molar ratio of lauric acid, NHS and EDC·HCl is 1:1.0:1.05.

[0045] S12, Lau-Dopa coupling and decomposition: Dopamine hydrochloride and sodium bicarbonate were dissolved in a phosphate buffer solution to prepare a dopamine solution; wherein the molar and volume ratio of dopamine hydrochloride to phosphate buffer solution was 1 mol: 10 L; the molar ratio of dopamine hydrochloride to sodium bicarbonate was 1:1.7; the concentration of the phosphate buffer solution was 0.1 mol / L, and the pH was 8.0. The activated ester solution was added dropwise to the dopamine solution at a volume ratio of 1:0.9 under stirring in an ice bath at 0°C. After the addition was complete, the mixture was stirred continuously at room temperature, protected from light and under inert protection and reacted for 14 hours. After the reaction was completed, the pH of the reaction solution was acidified to 1.1, and a precipitate was formed. After filtration, washing with cold water, and freeze-drying, a white solid product, namely Lau-Dopa (C), was obtained. 20 H 33 NO3, molecular weight 335.48 g / mol).

[0046] S2. Synthesis of chitosan-Lau-Dopa conjugate: Add 50 mL of a 10 mg / mL chitosan solution to the reactor; Lau-Dopa was dissolved in dimethyl sulfoxide to prepare a Lau-Dopa solution with a concentration of 0.2 mol / L, and then mixed with the chitosan solution at a volume ratio of 1:8. Stir and mix for 80 minutes at room temperature and in the dark. Add EDC·HCl to adjust the pH of the reaction system to 5.0; wherein the molar ratio of Lau-Dopa to EDC·HCl in the system is 1:1.0; The reaction was continued for 30 hours at 35.0℃ in the dark. After the reaction was completed, the reaction solution was filtered and dialyzed with 0.1 mol / L sodium bicarbonate solution and water. The retained solid was freeze-dried to obtain a light brown chitosan-Lau-Dopa conjugate, which was tested to have a weight-average molecular weight of 208 kDa.

[0047] S3. Preparation of bond-matched complex enzymes: S31. Material Weighing and Dissolving: Weigh out the mixed enzyme sample 1 and dissolve it in a 0.1 mol / L, pH 7.8 borate buffer solution to prepare a mixed enzyme solution with a concentration of 40 g / L.

[0048] S32, Catecholization modification on the enzyme surface: The modifier N-hydroxysuccinimide-dopamine ester was rapidly dissolved in anhydrous dimethyl sulfoxide to prepare a stock solution of modifier with a concentration of 0.1 mol / L. Under light-protected, stirred conditions and at 0°C, the modifier stock droplet was added to the mixed enzyme solution, and the reaction was stirred continuously for 2.5 h; the volume ratio of the modifier stock droplet to the mixed enzyme solution was 1:15.

[0049] S33. Termination of the reaction and crude purification: After the reaction was completed, the sample was repeatedly centrifuged and ultrafiltered three times at 0℃ and 10000×g centrifugation force. Each time, the sample was replenished to its original volume with ice-cold phosphate buffer solution of 0.05mol / L and pH7.0. The bond-matched complex enzyme was purified by gel filtration chromatography and stored at 0℃ for later use. This was designated as complex enzyme sample 2.

[0050] S4. Immobilized enzymes and gel forming: Chitosan-Lau-Dopa conjugate, complex enzyme sample 2, cross-linking agent, and coupling agent were mixed and then covalently coupled with Fe. 3+ Ionic cross-linking reaction forms an enzyme preparation with a gel as a carrier, specifically as follows: Chitosan-Lau-Dopa conjugate was dissolved in a phosphate buffer solution with a concentration of 0.1 mol / L and a pH of 7.0 to prepare a working solution of chitosan-Lau-Dopa conjugate with a concentration of 20 mg / mL, which is denoted as chitosan working solution. A 0.1 mol / L FeCl3·6H2O aqueous solution was prepared as a stock solution for the crosslinking agent; EDC·HCl and NHS were added sequentially to the chitosan working solution, and the mixture was stirred and activated at room temperature for 10 min to obtain the activated chitosan working solution. The molar-volume ratio of EDC·HCl to chitosan working solution was 8 mmol:1 L, and the molar-volume ratio of NHS to chitosan working solution was 8 mmol:1 L. Add compound enzyme sample 2 to the activated chitosan working solution and stir at 0℃ for 3 hours; wherein the mass and volume ratio of compound enzyme sample 2 to chitosan working solution is 16g:1L. Add sodium ascorbate and continue stirring at room temperature for 30 minutes; wherein the mass ratio of sodium ascorbate to complex enzyme sample 2 is 1:300; Under vigorous stirring at 600 r / min, the crosslinking agent stock solution was added dropwise at a rate of 0.5 mL / min; wherein the volume ratio of the crosslinking agent stock solution to the chitosan working solution was 1:60; in this embodiment, Fe... 3+ The final concentration was 1.67 mmol / L; After the addition was complete, the mixture was allowed to stand for 15 minutes to prepare an enzyme preparation using a gel as a carrier, which was then designated as enzyme preparation sample 3. Example 4

[0051] This embodiment provides a bio-enzyme preparation for catalytic degradation of oils and fats. The bio-enzyme preparation is a bonded-matching complex enzyme immobilized on a pH-responsive gel carrier. The gel carrier is formed by covalently coupling a chitosan-lauric acid-dopamine conjugate with Fe3+ ions. The bonded-matching complex enzyme is a mixed enzyme sample 1 with catechol groups grafted onto its surface by an N-hydroxysuccinimide-dopamine ester modifier. This mixed enzyme sample 1 is the same as in Example 1.

[0052] The specific preparation process is as follows: S1. Preparation of Lau-Dopa: S11, Activation of Lauric Acid: Lauric acid is dissolved in anhydrous dimethyl sulfoxide in a dry reactor; NHS and EDC·HCl were added while stirring under nitrogen protection and at room temperature; Under light-protected conditions, the reaction was stirred for another 3 hours to obtain an activated ester solution; The molar and volume ratio of lauric acid and anhydrous dimethyl sulfoxide used is 1 mol: 10 L; the molar ratio of lauric acid, NHS and EDC·HCl is 1:1.1:1.15.

[0053] S12, Lau-Dopa coupling and decomposition: Dopamine hydrochloride and sodium bicarbonate were dissolved in a phosphate buffer solution to prepare a dopamine solution; wherein the molar and volume ratio of dopamine hydrochloride to phosphate buffer solution was 1 mol: 10 L; the molar ratio of dopamine hydrochloride to sodium bicarbonate was 1: 2.3; the concentration of the phosphate buffer solution was 0.1 mol / L, and the pH was 8.0; The activated ester solution was added dropwise to the dopamine solution at a volume ratio of 1:1.1 under stirring in an ice bath at 4°C. After the addition was complete, the mixture was stirred continuously at room temperature, protected from light and under inert conditions and reacted for 6 hours. After the reaction was completed, the pH of the reaction solution was acidified to 3.0, and a precipitate was formed. After filtration, washing with cold water, and freeze-drying, a white solid product, namely Lau-Dopa, was obtained.

[0054] S2. Synthesis of chitosan-Lau-Dopa conjugate: Add 50 mL of a 10 mg / mL chitosan solution to the reactor; Lau-Dopa was dissolved in dimethyl sulfoxide to prepare a Lau-Dopa solution with a concentration of 0.2 mol / L, and then mixed with the chitosan solution at a volume ratio of 1:12. Stir and mix for 80 minutes at room temperature and in the dark. Add EDC·HCl to adjust the pH of the reaction system to 6.0; wherein the molar ratio of Lau-Dopa to EDC·HCl in the system is 1:1.1; Continue stirring the reaction at 40°C in the dark for 18 hours; After the reaction was completed, the reaction solution was filtered and dialyzed with 0.1 mol / L sodium bicarbonate solution and water. The retained solid was freeze-dried to obtain a light brown chitosan-Lau-Dopa conjugate, which was tested to have a weight-average molecular weight of 221 kDa.

[0055] S3. Preparation of bond-matched complex enzymes: S31. Material Weighing and Dissolving: Weigh out the mixed enzyme sample 1 and dissolve it in a 0.1 mol / L, pH 8.2 borate buffer solution to prepare a mixed enzyme solution with a concentration of 60 g / L.

[0056] S32, Catecholization modification on the enzyme surface: The modifier N-hydroxysuccinimide-dopamine ester was rapidly dissolved in anhydrous dimethyl sulfoxide to prepare a stock solution of modifier with a concentration of 0.1 mol / L. Under light-protected conditions, stirring, and at 4°C, the modifier stock droplet was added to the mixed enzyme solution, and the reaction was continuously stirred for 1.5 h; the volume ratio of the modifier stock droplet to the mixed enzyme solution was 1:8.

[0057] S33. Termination of the reaction and crude purification: After the reaction was completed, the sample was repeatedly centrifuged and ultrafiltered 5 times at 4℃ and 6000×g centrifugation force. Each time, the sample was replenished to its original volume with 0.05mol / L, pH7.0 ice-cold phosphate buffer. The bond-matched complex enzyme was purified by gel filtration chromatography and stored at 4°C for later use. This was designated as complex enzyme sample 3.

[0058] S4. Immobilized enzymes and gel forming: Chitosan-Lau-Dopa conjugate, complex enzyme sample 3, cross-linking agent, and coupling agent were mixed and then covalently coupled with Fe. 3+ Ionic cross-linking reaction forms an enzyme preparation with a gel as a carrier, specifically as follows: Chitosan-Lau-Dopa conjugate was dissolved in a phosphate buffer solution with a concentration of 0.1 mol / L and a pH of 7.0 to prepare a working solution of chitosan-Lau-Dopa conjugate with a concentration of 20 mg / mL, which is denoted as chitosan working solution. A 0.1 mol / L FeCl3·6H2O aqueous solution was prepared as a stock solution for the crosslinking agent; EDC·HCl and NHS were added sequentially to the chitosan working solution and stirred at room temperature for 20 min to obtain the activated chitosan working solution. The molar-volume ratio of EDC·HCl to chitosan working solution was 12 mmol:1 L, and the molar-volume ratio of NHS to chitosan working solution was 12 mmol:1 L. Add compound enzyme sample 3 to the activated chitosan working solution and stir at 4°C for 1 hour; wherein the mass and volume ratio of compound enzyme sample 3 to chitosan working solution is 28 g: 1 L. Add sodium ascorbate and continue stirring at room temperature for 30 minutes; wherein the mass ratio of sodium ascorbate to complex enzyme sample 3 is 1:350; Under vigorous stirring at 800 r / min, the crosslinking agent stock solution was added dropwise at a rate of 1.5 mL / min; wherein the volume ratio of the crosslinking agent stock solution to the chitosan working solution was 1:40; in this embodiment, Fe... 3+ The final concentration was 2.50 mmol / L; After the addition is complete, let it stand for 5 minutes to prepare the enzyme preparation with the gel as the carrier, and record it as enzyme preparation sample 4. Example 5

[0059] This embodiment provides a bio-enzyme preparation for catalytic degradation of lipids. The bio-enzyme preparation is a bonded, well-matched complex enzyme immobilized on a pH-responsive gel carrier. The gel carrier is composed of a chitosan-lauric acid-dopamine conjugate covalently coupled with Fe... 3+ Ionic cross-linking forms; the bonded suitable complex enzyme is a mixed enzyme sample 2 whose surface is grafted with catechol groups through an N-hydroxysuccinimide-dopamine ester modifier. This mixed enzyme sample 2 contains lipase, protease and amylase in a mass ratio of 3:1:0.1.

[0060] The specific preparation process of this embodiment is the same as that of Embodiment 1, except that in step S3, mixed enzyme sample 1 is replaced with mixed enzyme sample 2 of equal mass. The subsequent operations remain unchanged, and a composite enzyme sample 4 is prepared and used in subsequent steps to prepare an enzyme preparation with a gel as a carrier, which is denoted as enzyme preparation sample 5. Example 6

[0061] This embodiment provides a bio-enzyme preparation for catalytic degradation of lipids. The bio-enzyme preparation is a bonded, well-matched complex enzyme immobilized on a pH-responsive gel carrier. The gel carrier is composed of a chitosan-lauric acid-dopamine conjugate covalently coupled with Fe... 3+ Ionic cross-linking forms; the bond-matched complex enzyme is a mixed enzyme sample 3 whose surface is grafted with catechol groups through an N-hydroxysuccinimide-dopamine ester modifier. This mixed enzyme sample 2 contains lipase, protease and coenzyme in a mass ratio of 5:1:0.3. The coenzyme includes equal masses of amylase, pectinase and cellulase.

[0062] The specific preparation process of this embodiment is the same as that of Embodiment 1, except that in step S3, mixed enzyme sample 1 is replaced with mixed enzyme sample 3 of equal mass. The subsequent operations remain unchanged, and a composite enzyme sample 5 is prepared and used in subsequent steps to prepare an enzyme preparation with a gel as a carrier, which is denoted as enzyme preparation sample 6. Comparative Example 1

[0063] This comparative example provides a bio-enzyme preparation for catalytic degradation of lipids. The specific implementation method is the same as in Example 1, except that the dopamine structural unit of the gel carrier is omitted to study Fe 3+ The effect of the catechol coordination network on gel stability and enzyme immobilization ability is denoted as enzyme preparation control 1.

[0064] The preparation process of enzyme preparation control 1 in this comparative example is as follows: S1. Preparation of Lau-Dopa: This step is omitted. In this comparative example, commercially available lauric acid monoethanolamide is used directly.

[0065] S2. Synthesis of chitosan-lauric acid conjugate: Same as step S2 in Example 1, except that an equal amount of lauric acid monoethanolamide is used instead of Lau-Dopa used in Example 1 to prepare a light brown chitosan-lauric acid conjugate, referred to as chitosan-Lau conjugate, with a weight-average molecular weight of 183 kDa.

[0066] S3. Preparation of bond-matched complex enzyme: Same as step S3 in Example 1.

[0067] S4. Immobilized enzyme and gel forming: Same as step S4 in Example 1, except that an equal amount of chitosan-Lau conjugate is used instead of the chitosan-Lau-Dopa conjugate used in Example 1 to prepare enzyme preparation control 1. Comparative Example 2

[0068] This comparative example provides a bio-enzyme preparation for catalytic degradation of lipids. The specific implementation method is the same as in Example 1, except that the catecholization modification step of the mixed enzyme is omitted to study the effect of the catechol groups on the enzyme surface on the interaction between the enzyme and the carrier Fe. 3+ The necessity of network collaborative anchoring is denoted as enzyme preparation control 2.

[0069] The preparation process of enzyme preparation control 2 in this comparative example is as follows: S1. Preparation of Lau-Dopa: Same as step S1 in Example 1.

[0070] S2. Synthesis of chitosan-lauric acid-dopamine conjugate: Same as step S2 in Example 1.

[0071] S3. Preparation of ordinary mixed enzyme solution: Perform only step S31 of Example 1, weigh dry powder of lipase and protease at a mass ratio of 4:1, dissolve in 0.1mol / L, pH 8.0 borate buffer solution to prepare a mixed enzyme solution with a concentration of 50g / L, which can be used directly as the enzyme solution to be fixed. Steps S32 (catecholization modification of enzyme surface) and S33 (purification) of Example 1 are omitted.

[0072] S4. Immobilized enzyme and gel forming: Same as step S4 in Example 1, except that the ordinary mixed enzyme solution prepared in step S3 of this comparative example is used to replace the bond-matched complex enzyme, and the total mass of the added mixed enzyme and the mass-volume ratio of the chitosan working solution are maintained at 22g:1L, to prepare enzyme preparation control 2. Comparative Example 3

[0073] This comparative example provides a bio-enzyme preparation for catalytic degradation of grease. The specific implementation method is the same as in Example 1, except that the bond-matched complex enzyme contains only lipase and no protease, in order to study the synergistic effect of the protease component in the complex enzyme on the degradation of protein-containing grease stains. It is designated as enzyme preparation control 3.

[0074] The preparation process of enzyme preparation control 3 in this comparative example is as follows: S1. Preparation of lauric acid-dopamine derivative (Lau-Dopa): Same as step S1 in Example 1.

[0075] S2. Synthesis of chitosan-lauric acid-dopamine conjugate: Same as step S2 in Example 1.

[0076] S3. Preparation of bond-compatible lipases: S31. Weighing and dissolving materials: Dissolve the weighed lipase powder in a 0.1 mol / L, pH 8.0 borate buffer solution to prepare a lipase solution with a concentration of 50 g / L.

[0077] S32. Catecholization modification of enzyme surface: Same as step S32 in Example 1, but the modification target is only the above-mentioned lipase solution.

[0078] S33. Termination of reaction and crude purification: Same as step S33 in Example 1, the obtained product is denoted as lipase sample 1.

[0079] S4. Immobilized enzyme and gel forming: Same as step S4 in Example 1, except that an equal mass of lipase sample 1 is used to replace the bond-matched complex enzyme to prepare enzyme preparation control 3. Comparative Example 4

[0080] This comparative example provides a bio-enzyme preparation for catalytic degradation of oils and fats. The specific implementation method is the same as in Example 1, except that the hydrophobic chain of the carrier is replaced with caprylic acid instead of lauric acid, in order to study the effect of the length of the hydrophobic chain on the pH response behavior of the carrier and the enzyme stabilization microenvironment. This is referred to as enzyme preparation control 4.

[0081] The preparation process of enzyme preparation control 4 in this comparative example is as follows: S1. Preparation of caprylic acid-dopamine derivative (Cap-Dopa): S11. Activation of octanoic acid: In a dry reactor, octanoic acid was dissolved in anhydrous dimethyl sulfoxide; under nitrogen protection and at room temperature, NHS and EDC·HCl were added while stirring; the reaction was continued for 4 hours under light-protected conditions to obtain an activated ester solution. The molar and volume ratio of octanoic acid to anhydrous dimethyl sulfoxide was 1 mol:10 L; the molar ratio of octanoic acid, NHS, and EDC·HCl was 1:1.05:1.10.

[0082] S12, Coupling and Precipitation of Cap-Dopa: Same as step S12 in Example 1, except that an equal amount of octanoic acid is used instead of lauric acid in Example 1, resulting in a white solid product denoted as Cap-Dopa (C 16 H 25 NO3, molecular weight 279.37 g / mol.

[0083] S2. Synthesis of chitosan-octanoic acid-dopamine conjugate: Same as step S2 in Example 1, except that an equal amount of Cap-Dopa is used instead of Lau-Dopa used in Example 1 to prepare chitosan-octanoic acid-dopamine conjugate, referred to as chitosan-Cap-Dopa conjugate, and its weight-average molecular weight is 196 kDa.

[0084] S3. Preparation of bond-matched complex enzyme: Same as step S3 in Example 1.

[0085] S4. Immobilized enzyme and gel forming: Same as step S4 in Example 1, except that chitosan-Cap-Dopa conjugate is used instead of chitosan-Lau-Dopa conjugate to prepare enzyme preparation control 4. Comparative Example 5

[0086] This comparative example provides a bio-enzyme preparation for catalytic degradation of oils and fats. The specific implementation method is the same as in Example 1, except that the gel crosslinking ions are changed from Fe... 3+ Replace with Ca 2+ To study the effect of different metal ion coordination bond strengths on the mechanical stability and service life of gel networks, this was designated as enzyme preparation control 5.

[0087] The preparation process of enzyme preparation control 5 in this comparative example is as follows: S1. Preparation of Lau-Dopa: Same as step S1 in Example 1.

[0088] S2. Synthesis of chitosan-lauric acid-dopamine conjugate: Same as step S2 in Example 1.

[0089] S3. Preparation of bond-matched complex enzyme: Same as step S3 in Example 1.

[0090] S4. Immobilized enzyme and gel forming: Same as step S4 in Example 1, except that: a 0.2 mol / L CaCl2 aqueous solution is prepared as the crosslinking agent stock solution; in the dropwise crosslinking step, the CaCl2 solution is used instead of the FeCl3 solution, and the volume ratio of the crosslinking agent stock solution to the chitosan working solution is adjusted to 1:25, thus preparing enzyme preparation control 5. Analysis and Testing

[0091] To verify the technical effects of the present invention and to compare it with the comparative scheme, the prepared enzyme preparation samples 1-6 and enzyme preparation control products 1-5 were systematically analyzed and tested.

[0092] I. Immobilization efficiency and catalytic activity analysis Immobilized enzyme activity assay: Take the sample of each enzyme preparation to be tested, add it to the 1% olive oil emulsion reaction system at pH 8.5 and 50℃, and use the sodium hydroxide titration method to determine the free fatty acids released per unit time, calculate its total enzyme activity, and express it as enzyme activity units per gram of gel (U / g gel).

[0093] Activity recovery rate calculation: Activity recovery rate (%) = (total enzyme activity of immobilized enzyme / total enzyme activity of free enzyme added before immobilization) × 100%, wherein the total enzyme activity of the added free enzyme is calculated based on the mass ratio of the mixed enzymes in each example and comparative example and the enzyme activity of each component (i.e., lipase activity is 100,000 U / g, protease activity is 50,000 U / g, amylase activity is 100,000 U / g, and pectinase and cellulase activity is 5,000 U / g).

[0094] Operational stability test: The sample was reacted under standard washing conditions (pH 8.5, 50℃) for 1 hour. After the reaction, the pH of the system was adjusted to 6.8 to cause the gel carrier to shrink and settle, and the sample was separated and recovered. The next cycle was started in a new batch of substrate. This process was repeated 10 times. The residual enzyme activity of the sample after the 10th cycle was measured, and the enzyme activity retention rate (%) after 10 cycles was calculated.

[0095] The results are shown in Table 1.

[0096] Table 1: Comparison of Immobilization Efficiency and Operational Stability of Various Enzyme Preparations

[0097] As shown in Table 1, the immobilized enzyme preparations prepared using the technical solution of this invention all exhibited high enzyme activity recovery rates and excellent operational stability. This indicates that the chitosan-lauric acid-dopamine smart gel carrier constructed in this invention, combined with catecholization modification of the enzyme surface, can achieve efficient and robust immobilization of the composite enzyme and provide it with a stable microenvironment.

[0098] Example 1 demonstrated the best performance across all three key indicators, particularly its cyclic stability of 85.1%, verifying the superiority of the process including sodium ascorbate. In contrast, Example 2 exhibited similar initial immobilization efficiency to Example 1, but its cyclic stability was lower, indicating that sodium ascorbate in this invention can act as a protective agent, maintaining Fe... 3+- The important role of the catechol coordination network in long-term stability. Examples 5 and 6, after using different complex enzyme formulations, showed performance indicators that were basically consistent with those of Example 1, indicating that the immobilization platform of this invention has good compatibility and robustness to different complex enzyme formulations, further demonstrating the universality of this carrier-immobilization method.

[0099] However, all comparative examples exhibited performance deficiencies. Reference 1 showed the lowest performance across all aspects, indicating that the lack of dopamine resulted in the loss of strong Fe. 3+ - The catechol coordination network leads to a fragile carrier structure and poor enzyme immobilization, causing the core cross-linking mechanism of this invention to fail. The cycling stability of control 2 is significantly lower than that of the embodiments of this invention, which proves that enzymes without catechol modification cannot form a synergistic and robust multi-point anchoring with the carrier network, and it is difficult to maintain stability in repeated use by relying solely on a single covalent link. Control 3 only selected a single lipase, which did not significantly affect the immobilization efficiency and stability. All indicators of controls 4 and 5, especially cycling stability, showed a systematic decline. This respectively confirms that the appropriate hydrophobic chain length provided by lauric acid is the key to constructing a stable enzyme microenvironment, and Fe 3+ Strong coordination ability (compared to) Both are essential conditions for forming a durable gel network that can withstand repeated use; both are part of the precision design of this invention and neither can be omitted.

[0100] II. Analysis of Physical Properties and Operational Stability To evaluate the key performance aspects of the gel carrier of the bio-enzyme of this invention in industrial cleaning applications, such as separation, recovery, and durability, its physical properties and stability for repeated use were systematically tested.

[0101] Particle size and distribution: The sample to be tested was placed in phosphate buffer at pH 6.8. After shrinkage and stabilization, the volume average particle size (D50) and the polydispersity index (PDI) of the particle size distribution were determined using a laser particle size analyzer.

[0102] Swelling properties: Weigh and record the gel in pH 6.8 buffer (i.e., fully shrunken state). W c ) and pH 8.5 buffer (i.e., fully swollen working state, W s Calculate the swelling ratio based on the wet weight of the sample. S The formula is as follows: S =( W s - W c ) / W c ×100%.

[0103] Operational stability test: The test method is the same as in "I. Immobilization efficiency and catalytic activity analysis". The gel mass recovery rate after the 10th cycle is tested to evaluate the durability of the physical structure.

[0104] The results are shown in Table 2.

[0105] Table 2: Comparison of physical properties and operational stability of various enzyme preparation samples

[0106] Remark : Reference standard 1 lacks a stable cross-linking network, and its structure disintegrates during the swelling process, so its effective equilibrium swelling rate cannot be determined; its D50 and PDI are the initial shrinkage state values, but its particle size distribution is extremely wide.

[0107] As can be seen from the results in Table 2, the physical properties and operational stability of the samples in each embodiment of the present invention are in good consistency with their specific preparation process parameters, and are all significantly better than those of the comparative examples.

[0108] All samples in the embodiments of this invention exhibit excellent physical properties. Sample 4, in particular, demonstrates superior physical properties due to the use of a higher stirring rate and higher Fe content during the gelation stage. 3+ The crosslinking concentration resulted in a network with higher crosslinking density and a more compact structure, thus exhibiting the smallest D50, the most uniform distribution, a relatively low swelling ratio, and the highest gel recovery rate after cycling. Sample 3, conversely, had the largest D50 due to its lower stirring rate and lower crosslinking agent concentration, slightly poorer distribution uniformity, and a slightly higher swelling ratio. However, due to the addition of sodium ascorbate for protection, its gel recovery rate remained at a relatively high level of 95.0%. Samples 1, 2, 5, and 6 had similar process conditions, resulting in similar physical properties. Notably, Sample 2, lacking sodium ascorbate, had a slightly weaker resistance to oxidative damage during long-term cycling, hence its gel recovery rate was slightly lower than other samples with added protective agents. The above experimental data demonstrate the precise control of the process parameters of this invention over the physical properties of the carrier, and also prove that within the parameter range provided by this invention, immobilized enzyme carriers with excellent separation characteristics, significant pH responsiveness, and superior cycling durability can be prepared.

[0109] Reference standard 1, however, was completely devoid of Fe. 3+- The catechol coordination network leads to loss of gel mechanical strength, structural disintegration during swelling-shrinkage cycles, and ineffective recovery. Reference 2, due to the enzyme-carrier connection via a single covalent bond and lack of co-anchoring, exhibits unstable binding interfaces under physical stress, resulting in a significantly lower gel recovery rate than the sample of this invention. Reference 3's physical properties are essentially consistent with Sample 1, which uses the same carrier and immobilization process, further confirming the versatility of this immobilization platform. Reference 4, due to shortened hydrophobic chains, exhibits weakened hydrophobic association, leading to abnormal swelling behavior and decreased structural stability, demonstrating that lauric acid chain length is crucial for constructing intelligent carriers with ideal swelling-shrinkage reversibility and mechanical strength. Reference 5, due to insufficient metal-ligand bond strength, has a loose gel network, exhibiting an abnormally high swelling rate and the lowest post-cycle recovery rate, indicating that Fe... 3+ Strong coordination bonds are the decisive factor in maintaining the structural integrity of the carrier during repeated use.

[0110] III. Application Effect Test Enzyme preparation sample 1 and enzyme preparation sample 6, which are designed to handle more complex contaminants, were selected as representatives of this invention. Based on the special cleaning agent formulation of this invention, cleaning agents (denoted as cleaning agent samples 1 and 2) were constructed and their catalytic properties and application performance were evaluated in depth.

[0111] Cleaning agent control 1 was constructed by combining enzyme preparation sample 6 with a common cleaning agent formulation; cleaning agent control 2-5 was constructed by combining enzyme preparation control 2-5 with the special cleaning agent formulation of the present invention.

[0112] The components of each cleaning agent sample and reference standard are shown in Table 3. By mass parts, the special cleaning agent formulation contains 10 parts of biological enzyme preparation, 10 parts of alkyl glycoside (APG-1214), 5 parts of fatty alcohol polyoxyethylene ether (AEO-7), 4 parts of glycerol, 2 parts of sodium dihydrogen phosphate / disodium hydrogen phosphate buffer, 1.5 parts of trehalose, 0.2 parts of xanthan gum, and 0.2 parts of potassium sorbate, to be made up to 100 parts with deionized water; the general cleaning agent formulation contains 10 parts of biological enzyme preparation, 8 parts of linear alkylbenzene sulfonate (LAS), 4 parts of fatty alcohol polyoxyethylene ether (AEO-9), 3 parts of glycerol, 2 parts of sodium dihydrogen phosphate / disodium hydrogen phosphate buffer, and 0.2 parts of sodium benzoate, to be made up to 100 parts with deionized water.

[0113] Table 3: Component Description of Cleaning Agent Samples and Reference Standards

[0114] The cleaning agent samples or controls in Table 3 were diluted with deionized water at a ratio of 1:100. Standard stainless steel sheets (5cm × 5cm, 304 stainless steel) treated with different test substrates (substrate A or B) were then soaked in the solution for 8 hours at 30℃ and pH 8.5. The solution was then stirred at 20 r / min to simulate water flow for 30 minutes. The stainless steel sheets were then removed, residual dirt was scraped off, the mass of the residual dirt was measured, and the dirt removal rate was calculated. The results are shown in Table 4.

[0115] Meanwhile, to explore the dilution ratio of the cleaning agent, cleaning agent sample 1 was diluted with deionized water at ratios of 1:50, 1:150, and 1:200. Substrate A was then treated in the same manner, and cleaning tests 8, 9, and 10 were conducted again to calculate the dirt removal rate. The results are shown in Table 4.

[0116] Substrate A is a high-fat, high-protein dirt, made by mixing lard and bovine serum albumin in a mass ratio of 3:1.

[0117] Substrate B is a complex composite fouling substance, which is made by mixing lard, bovine serum albumin, soluble starch, sodium carboxymethyl cellulose and pectin in a mass ratio of 5:2:2:1:0.5.

[0118] The method for substrate treatment of stainless steel sheets is to take a fixed mass of substrate A / B, apply it evenly to both sides of the stainless steel sheet, and simulate the high-temperature weathering process (60℃, 30min) in a blower dryer.

[0119] Table 4: Cleaning Test Results

[0120] Based on Tables 3 and 4, the special cleaning agent formulation of the present invention, in synergy with the enzyme preparation sample of the present invention, exhibits excellent cleaning performance and is significantly superior to ordinary formulations and defective enzyme preparations.

[0121] First, the comparison between the dedicated formulation and the ordinary formulation showed that, despite using the same enzyme sample, the cleaning agent sample 2 with the dedicated formulation achieved a removal rate of 82.7% for complex substrate B, while the removal rate of the cleaning agent control 1 (ordinary formulation) with the ordinary formulation was only 60.5%. This difference stems from the ordinary formulation's lack of xanthan gum and the use of unsuitable surfactants, which may lead to insufficient contact between the immobilized enzyme and the substrate, and an imperfect protection system that could affect enzyme stability.

[0122] Under specialized formulations, the cleaning efficacy of different enzyme preparations is closely related to their structural design. Cleaning agent sample 1 showed a significantly higher removal rate of dirt A than cleaning agent control 3 (control 3, lipase only), confirming the indispensability of proteases in decomposing protein components and synergistically breaking down the lipoprotein complex structure. Cleaning agent sample 2 exhibited a superior removal rate of substrate B compared to cleaning agent sample 1 for substrate A, demonstrating the strong synergistic effect of coenzymes such as amylase and cellulase in dealing with complex dirt, achieving broad-spectrum and efficient cleaning. The lower removal rate of cleaning agent control 2 is consistent with its poor cyclic stability conclusions in Tables 1 and 2, indicating that the catecholization modification of the enzyme is crucial for achieving firm fixation and preventing detachment during use. The removal rates of cleaning agent controls 4 and 5 were both significantly lower than those of the present invention sample, respectively demonstrating the importance of the lauric acid hydrophobic chain in constructing a suitable enzyme microenvironment and the Fe... 3+ - The strong coordination bond of catechins plays a decisive role in maintaining the stability of the carrier structure and ensuring the long-term effectiveness of the enzyme.

[0123] Furthermore, the study on the effect of dilution ratio showed that for cleaning agent sample 1, within the dilution range of 1:50 to 1:200, the dirt removal rate gradually decreased with increasing dilution ratio (i.e., decreasing enzyme concentration), exhibiting a clear dose-dependent effect. The highest removal rate was achieved at a 1:50 dilution, but a 1:100 dilution already achieved excellent cleaning results and was more economical. This indicates that the cleaning agent of the present invention can be flexibly diluted according to the severity of dirt accumulation in practical applications, possessing good operational flexibility.

[0124] IV. Determination of swelling ratio-pH curve Using enzyme preparation sample 1 as the test sample, its swelling rate was measured at different pH values. The specific procedure is as follows: The sufficient amount of gel sample prepared according to Example 1 was fully shrunk in pH 6.8 buffer solution to obtain the initial state; Accurately weigh multiple equal portions of the shrunken gel (wet weight, W c Each of the above-mentioned buffer solutions of different pH values ​​was placed in a capped container containing 30 mL of the buffer solution. Place all containers in a constant temperature shaker and shake at low speed at room temperature for a sufficient time (12 hours) away from light to ensure that the gel reaches swelling equilibrium at each pH. Once the time is reached, quickly scoop out the gel particles with a sieve, gently blot dry the surface droplets with filter paper, weigh immediately, and record as the equilibrium swelling wet weight at that pH. W s ); For each pH point, the swelling ratio was calculated. A scatter plot was created with pH value on the x-axis and swelling ratio S on the y-axis, and the points were connected to form a smooth curve. The results are shown in [Figure number missing]. Figure 1 .

[0125] Figure 1 The results showed that the swelling effect of the gel carrier used in the enzyme preparation of this invention exhibited a significant pH dependence. When the pH value was in the range of 6.5–7.0, the swelling rate remained at a low level, indicating that the gel was in a shrinkage and hardening state. When the pH value rose to around 7.5, the swelling rate increased sharply and stabilized at a high level of over 400%, forming a clear high-level plateau, indicating that the gel had entered a fully swollen working state. Although there were slight fluctuations in the swelling rate within the normal range of pH 8.5–9.5, its value was always significantly higher than the shrinkage plateau, proving that the gel could maintain a stable swollen state within a wide pH range of 7.5–9.5.

[0126] To enable those skilled in the art and end-users to more clearly understand how to fully utilize the intelligent characteristics and recycling advantages of the bio-enzyme cleaning agent of this invention, and to properly handle the cleaned system, a brief operating instruction is provided below. This instruction not only demonstrates the ease of use of the product but also reflects the design concept of this invention to solve the problem of enzyme residue and recycling from the source. The specific operating steps are as follows: 1. Preparation and cleaning of working solution: Dilute the cleaning agent of the present invention with water at the recommended ratio (e.g., 1:50 to 1:200) to obtain the working solution; adjust the pH of the working solution to about 8.5, at which point the gel carrier is fully swollen and the immobilized enzyme is in a highly active working state; immerse the object to be cleaned in the working solution and clean it after soaking for 8 to 12 hours.

[0127] 2. Carrier Separation and Recovery: After cleaning, add a small amount of acidic substance (such as food-grade citric acid, dilute acetic acid solution, etc.) to the system, or discharge the cleaning waste liquid into a recovery device with built-in pH adjustment function to adjust the pH value of the system to 6.5-7.0 (preferably 6.8). At this time, the smart gel carrier immediately shrinks and hardens, its density increases, and it quickly settles to the bottom of the container. Subsequently, the carrier particles can be quickly and thoroughly separated from other suspended dirt in the aqueous phase by simple static decantation, filtration, or low-speed centrifugation. If there is too much solid residue in the cleaned equipment, the cleaning liquid can be discharged first for coarse filtration to remove solid impurities before adjusting the pH.

[0128] 3. Carrier Reuse or Disposal: The collected bio-enzyme particles can be directly used for the next batch of cleaning after rinsing with clean water, maintaining good catalytic performance and physical stability (see Tables 1 and 2). To optimize enzyme activity and carrier stability during long-term cycling, it is recommended to add the special cleaning agent of this invention during reuse. The buffer system, enzyme protectant (glycerol, trehalose), and stabilizer in this special cleaning agent effectively repair minor carrier network losses during cycling and maintain the enzyme activity microenvironment, thereby significantly extending the overall lifespan of the immobilized enzyme preparation. If final disposal is required, its main components are biodegradable chitosan and natural product derivatives, making it environmentally friendly.

[0129] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A bio-enzyme preparation for catalytically degrading oils and fats, characterized in that, Contains: a bonded, compatible complex enzyme immobilized on a pH-responsive gel carrier; the gel carrier is composed of a chitosan-lauric acid-dopamine conjugate covalently coupled with Fe... 3+ Ionic cross-linking forms the complex enzyme; the bonded appropriate complex enzyme is a mixed enzyme whose surface is grafted with catechol groups through an N-hydroxysuccinimide-dopamine ester modifier.

2. The biological enzyme preparation according to claim 1, characterized in that, The mixed enzyme comprises lipase, protease and coenzyme in a mass ratio of (3-5):1:(0-0.3).

3. The biological enzyme preparation according to claim 2, characterized in that, The coenzyme is selected from at least one of amylase, pectinase and cellulase.

4. The biological enzyme preparation according to claim 1, characterized in that, The gel carrier shrinks and hardens at pH 6.5–7.0 and swells at pH 7.5–9.

5.

5. A method for preparing a biological enzyme preparation as described in any one of claims 1-4, characterized in that, Includes the following steps: S1. Preparation of lauric acid-dopamine derivatives; S2. Synthesize chitosan-lauric acid-dopamine conjugate; S3. Preparation of bond-matched complex enzymes; S4. Immobilization and gel formation: The conjugate, the bonded matching complex enzyme, the cross-linking agent, and the coupling agent are mixed, and then covalently coupled with Fe... 3+ Ionic cross-linking reaction forms an enzyme preparation with a gel as a carrier.

6. The preparation method according to claim 5, characterized in that, In step S1, the process of lauric acid-dopamine derivative includes: adding a laurate solution activated with EDC·HCl and NHS dropwise to a phosphate buffer solution containing dopamine hydrochloride and sodium bicarbonate at 0℃~4℃ and under an inert atmosphere, stirring the mixture at room temperature in the dark for 6h~14h, and acidifying the solution to pH 1~3 after the reaction is completed to precipitate the product.

7. The preparation method according to claim 5, characterized in that, In step S3, the process of preparing the bonded matching complex enzyme includes: reacting the mixed enzyme with the modifier at 0℃~4℃ and pH7.8~8.2 for 1.5h~2.5h.

8. The preparation method according to claim 5, characterized in that, In step S4, the covalent coupling conditions are: reaction at 0℃~4℃ for 1h~3h in the presence of EDC·HCl and NHS; the Fe 3+ The ionic crosslinking reaction conditions are: adding FeCl3·6H2O aqueous solution, so that Fe... 3+ The final concentration is 1.6 mmol / L to 2.5 mmol / L, and the gel is formed by standing at room temperature for 5 min to 15 min. During the gel formation process, the stirring speed is controlled at 600 r / min to 800 r / min so that the particle size D50 of the formed gel particles is distributed in the range of 180 μm to 250 μm.

9. A cleaning agent, characterized in that, The preparation comprises a biological enzyme as described in any one of claims 1-8.

10. The method of using the cleaning agent according to claim 9, characterized in that, include: The cleaning agent is diluted with water at a volume ratio of 1:(50-200) to prepare a cleaning working solution, and the pH of the cleaning working solution is adjusted to 7.5-9.5 for cleaning. After cleaning, the pH of the cleaning working solution is adjusted to 6.5-7.0 to cause the biological enzyme preparation to shrink and harden, so as to facilitate recovery.

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