Efficient energy-saving degreasing agent and preparation method thereof

Abstract

The invention relates to the technical field of degreasing agents, in particular to a high-efficiency energy-saving degreasing agent and a preparation method thereof, and the high-efficiency energy-saving degreasing agent comprises the following components in parts by weight: 6-9 parts of recombinant thermostable lipase; 4-7 parts of alkaline protease; 1.5 to 2.5 parts of nano silver particles wrapped by chitosan; 0.8 to 1.8 parts of beta-cyclodextrin modified carbon quantum dots; 12 to 18 parts of sodium alginate based intelligent hydrogel microspheres; 18 to 22 parts of lauryl glucoside; 12 to 18 parts of coconut oil-based betaine; 6 to 9 parts of propylene glycol; 4 to 7 parts of d-limonene; 10 to 14 parts of polyethylene glycol 400; 2 to 4 parts of dipotassium phosphate; according to the modified recombinant thermostable lipase, the low-temperature activity and the pH adaptability are remarkably improved while the high catalytic efficiency is maintained, and the antibacterial performance is enhanced by combining the nano-silver particles wrapped by the chitosan with the beta-cyclodextrin modified carbon quantum dots.

Description

Technical Field

[0001] The invention relates to the technical field of degreasing agents, and in particular to a high-efficiency and energy-saving degreasing agent and a preparation method thereof. Background Art

[0002] With the increasing awareness of environmental protection and the continuous upgrading of industrial cleaning needs, the research and development of efficient, energy-saving and environmentally friendly degreasers has become a top priority in the cleaning industry. Although traditional degreasers have significant cleaning effects, they often have problems such as high energy consumption, serious environmental pollution and limited applicable conditions. In recent years, the rapid development of bio-enzyme technology, nanomaterials and intelligent carrier systems has provided new possibilities for the innovation of degreasers.

[0003] In enzyme technology, significant progress has been made in the modification of lipase and protease by genetic engineering. For example, by combining directed evolution and rational design, researchers have successfully improved the thermal stability and low-temperature activity of Thermomyces lanuginosus lipase. However, these modified enzymes still face challenges such as insufficient pH adaptability and sensitivity to metal ions in practical applications, which limits their application in complex industrial environments.

[0004] Important progress has also been made in the application of nanomaterials in degreasing agents. Nanosilver has attracted much attention due to its excellent antibacterial properties, but it tends to agglomerate when used alone, affecting its long-term effectiveness. Carbon quantum dots, as emerging nanomaterials, exhibit good photocatalytic properties and biocompatibility, but their application research in degreasing agents is still in its infancy. How to effectively integrate these nanomaterials into degreasing formulas and work synergistically with other components remains an issue that needs to be addressed.

[0005] Smart responsive carriers have been widely used in the field of drug delivery, but their exploration in cleaning products is relatively limited. Although pH-responsive hydrogel microspheres have shown the potential to regulate the release of active ingredients on a laboratory scale, there are still many technical challenges in maintaining their stability and responsiveness in complex cleaning systems.

[0006] In addition, existing degreaser formulations often have difficulty maintaining high efficiency at low temperatures, wide pH ranges, and complex dirt conditions. Although they perform well at room temperature, their efficiency drops sharply below 10°C and is only effective in the pH range of 6-8. This highlights the urgent need to develop multifunctional degreasers that are efficient over the full temperature and pH range. Summary of the invention

[0007] In view of the above problems, the present invention aims to develop a new degreasing agent that can maintain high efficiency at low temperature and wide pH range, has antibacterial and self-cleaning functions, and is suitable for complex dirt. By integrating genetically engineered enzymes, functionalized nanomaterials and intelligent response carriers, the present invention strives to achieve synergistic enhancement of each component at the molecular level.

[0008] The invention discloses an efficient and energy-saving degreasing agent, which comprises the following components in parts by weight: 6-9 parts of recombinant thermostable lipase; 4-7 parts of alkaline protease; 1.5-2.5 parts of nano silver particles wrapped by chitosan; 0.8-1.8 parts of carbon quantum dots modified by beta-cyclodextrin; 12-18 parts of sodium alginate-based smart hydrogel microspheres; 18-22 parts of lauryl glucoside; 12-18 parts of coconut oil-based betaine; 6-9 parts of propylene glycol; 4-7 parts of d-limonene; 10-14 parts of polyethylene glycol 400; 2-4 parts of dipotassium hydrogen phosphate; and the balance of deionized water is up to 100 parts.

[0009] Specifically, the preparation method of the recombinant thermostable lipase comprises the following steps:

[0010] (1) Gene cloning: Genomic DNA was extracted from the Thermomyces lanuginosus strain, the lipase gene was amplified using specific primers, and the PCR product was cloned into the pET-22b(+) expression vector;

[0011] (2) Recombinant expression: The constructed plasmid was transformed into Escherichia coli BL21 (DE3) competent cells, cultured in LB medium and induced for expression with IPTG;

[0012] (3) Protein purification: collect the bacteria, disrupt them by ultrasonication, and purify the target protein using a Ni-NTA affinity chromatography column;

[0013] (4) Enzyme activity assay: Using olive oil as substrate, the enzyme activity was measured at 37°C to ensure that the activity was ≥100,000 U / g.

[0014] Specifically, the alkaline protease is derived from Bacillus subtilis, maintains ≥70% of the maximum activity within the range of 20-60° C., and has an optimum pH of 8.0-11.0.

[0015] Specifically, the preparation method of the chitosan-coated nanosilver particles comprises the following steps: (1) dissolving silver nitrate in deionized water, adding trisodium citrate and stirring; (2) slowly adding sodium borohydride solution and stirring at room temperature; (3) adding chitosan solution dissolved in 1% acetic acid and continuing to stir; (4) centrifuging and washing, and freeze-drying to obtain the product.

[0016] Specifically, the preparation method of the β-cyclodextrin modified carbon quantum dots comprises the following steps: (1) dissolving citric acid and ethylenediamine in water, and subjecting them to a hydrothermal reaction at 180°C for 4 hours; (2) cooling to room temperature, and subjecting them to dialysis at a molecular weight cutoff of 1000Da for 48 hours; (3) mixing the dialyzed solution with β-cyclodextrin, and subjecting them to a reaction at 50°C for 24 hours; and (4) freeze-drying to obtain the product.

[0017] Specifically, the preparation method of the sodium alginate-based smart hydrogel microspheres comprises the following steps: (1) dissolving sodium alginate in deionized water; (2) dripping the solution into a calcium chloride solution using a microfluidic device with a 100 μm nozzle and a pressure of 0.2 MPa; (3) cross-linking in the calcium chloride solution for 2 hours, filtering and washing with deionized water.

[0018] A method for preparing the highly efficient and energy-saving degreasing agent comprises the following steps: (1) preparing an enzyme solution: dissolving a recombinant thermostable lipase and an alkaline protease in a potassium hydrogen phosphate buffer solution and subjecting the solution to ultrasonic treatment; (2) preparing a nano-dispersion solution: dispersing chitosan-wrapped nano-silver particles and β-cyclodextrin-modified carbon quantum dots in deionized water and subjecting the solution to ultrasonic treatment; (3) preparing sodium alginate microspheres: dispersing sodium alginate-based smart hydrogel microspheres in deionized water and subjecting the solution to gentle stirring; (4) preparing a surfactant and solvent mixed solution: mixing lauryl glucoside, coconut oil-based betaine, propylene glycol, d-limonene and polyethylene glycol 400, heating and stirring; and (5) preparing a final product: mixing all the components prepared in steps (1) to (4), subjecting the mixture to high-speed shear emulsification, allowing the mixture to stand, and then subjecting the mixture to sterile filtration.

[0019] Specifically, in step (1), the conditions of the ultrasonic treatment are: frequency 40kHz, power 300-400W, and treatment time 5-10 minutes.

[0020] Specifically, in step (4), the heating and stirring conditions are: temperature 50±2°C, stirring speed 300-400rpm, and time 15-20 minutes.

[0021] Specifically, in step (5), the conditions for high-speed shear emulsification are: speed 7000±1000rpm, time 15-20 minutes, temperature 25±3°C; the conditions for standing are: temperature 25±2°C, time 24±2 hours; and the sterile filtration uses a 0.22μm polyethersulfone filter membrane.

[0022] Compared with the prior art, the present invention can achieve the following beneficial effects:

[0023] The present invention achieves performance breakthroughs through the following mechanisms: First, the recombinant thermostable lipase modified by site-directed mutagenesis technology significantly improves low-temperature activity and pH adaptability while maintaining high catalytic efficiency. Secondly, the innovative combination of chitosan-coated nanosilver particles and β-cyclodextrin-modified carbon quantum dots not only enhances the antibacterial properties, but also provides a new way to degrade organic pollutants through host-guest interactions and photocatalytic effects. Furthermore, the introduction of sodium alginate-based smart hydrogel microspheres achieves pH-responsive release of active ingredients, greatly broadening the applicable pH range of the degreasing agent. Finally, the carefully designed bio-based surfactant composite system ensures the effective removal of various polar dirt.

[0024] The synergistic effect of these innovative components produces a number of breakthrough effects: (1) It maintains high-efficiency degreasing capabilities over a wide temperature range of 5-80°C, with an activity retention rate of over 90% at low temperature (5°C); (2) It can work effectively in the pH range of 4-10, with the degreasing efficiency fluctuating by no more than 5%; (3) The inhibition rate of common bacteria exceeds 99.5%, and it has a sustained antibacterial effect for up to 72 hours; (4) The cleaned surface exhibits a self-cleaning effect, and the removal rate of newly contaminated dirt is increased by 30% within 48 hours; (5) The one-time removal rate of mixed grease-protein-inorganic dirt is increased by 40%.

[0025] In summary, the high-efficiency and energy-saving degreasing agent of the present invention achieves a comprehensive improvement in performance through multiple innovative mechanisms, which not only overcomes the limitations of the prior art, but also provides a new type of efficient, environmentally friendly and multifunctional solution for industrial cleaning, household cleaning, environmental governance and other fields, and is expected to promote the development of cleaning technology in a smarter and greener direction. DETAILED DESCRIPTION

[0026] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be clearly and completely described below in conjunction with the specific embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0027] Example 1

[0028] This embodiment provides an energy-efficient degreasing agent and a preparation method thereof. The degreasing agent comprises the following components: 6 parts by weight of recombinant thermostable lipase, 4 parts by weight of alkaline protease, 1.5 parts by weight of nanosilver particles wrapped in chitosan, 0.8 parts by weight of carbon quantum dots modified with β-cyclodextrin, 12 parts by weight of sodium alginate-based smart hydrogel microspheres, 18 parts by weight of lauryl glucoside, 12 parts by weight of coconut oil betaine, 6 parts by weight of propylene glycol, 4 parts by weight of d-limonene, 10 parts by weight of polyethylene glycol 400, 2 parts by weight of dipotassium hydrogen phosphate, and the balance of deionized water to 100 parts by weight.

[0029] The preparation method of the high-efficiency energy-saving degreasing agent comprises the following steps:

[0030] First, a recombinant thermostable lipase was prepared. Specifically, genomic DNA was extracted from the Thermomyces lanuginosus strain, and the lipase gene was amplified using the forward primer 5'-GGAATTCCATATGAAGCTACTTTCTT ATATACG-3' and the reverse primer 5'-CCGCTCGAGTTAATGATGATGATGATGATGCAG GAACACTGAGG-3'. The PCR reaction conditions were: 95°C for 5 minutes; 30 cycles (95°C for 30 seconds, 58°C for 30 seconds, 72°C for 1 minute); 72°C for 10 minutes. The PCR product was cloned into the pET-22b(+) expression vector and transformed into Escherichia coli BL21(DE3) for expression. In LB medium, culture at 37°C until OD600 reached 0.6, IPTG was added to a final concentration of 0.5mM, and expression was induced at 20°C for 16 hours. Afterwards, the target protein was purified by a Ni-NTA affinity chromatography column.

[0031] Secondly, alkaline protease was prepared. Alkaline protease was isolated from Bacillus subtilis and purified by ion exchange chromatography and gel filtration.

[0032] Then, prepare the nano silver particles wrapped by chitosan. Dissolve 0.5g silver nitrate in 100mL deionized water, add 1g trisodium citrate, and stir for 10 minutes. Slowly drop 10mL 1% sodium borohydride solution, stir at room temperature for 1 hour. Add 50mL 0.5% chitosan solution (dissolved in 1% acetic acid), continue stirring for 2 hours. Centrifuge and wash 3 times, freeze-dry to obtain the product.

[0033] Next, prepare β-cyclodextrin-modified carbon quantum dots. Dissolve 2 g of citric acid and 1 g of ethylenediamine in 30 mL of water and perform hydrothermal reaction at 180 ° C for 4 hours. Cool to room temperature and dialyze for 48 hours (molecular weight cutoff 1000 Da). Mix the dialyzed solution with 1 g of β-cyclodextrin and react at 50 ° C for 24 hours. Freeze-dry to obtain the product.

[0034] Again, sodium alginate-based smart hydrogel microspheres were prepared. 2 g of sodium alginate was dissolved in 100 mL of deionized water. The solution was dripped into a 0.1 M calcium chloride solution using a microfluidic device (100 μm nozzle, pressure 0.2 MPa). Crosslinked in the calcium chloride solution for 2 hours, filtered and washed 3 times with deionized water.

[0035] Finally, the components prepared above were mixed with other commercial components according to the specified weight parts. Specifically, the recombinant thermostable lipase and alkaline protease were dissolved in dipotassium hydrogen phosphate buffer and ultrasonically treated at 40kHz frequency and 300W power for 5 minutes. The chitosan-encapsulated nanosilver particles and β-cyclodextrin-modified carbon quantum dots were dispersed in deionized water and ultrasonically treated at 20kHz frequency for 10 minutes. The sodium alginate-based smart hydrogel microspheres were dispersed in deionized water and gently stirred at 100rpm for 30 minutes. Lauryl glucoside, coconut oil betaine, propylene glycol, d-limonene and polyethylene glycol 400 were mixed and stirred at 48°C and 300rpm for 15 minutes. Finally, all the components were mixed, sheared and emulsified at 6000rpm for 15 minutes, left to stand at 25°C for 22 hours, and then sterile filtered with a 0.22μm polyethersulfone filter membrane.

[0036] The energy-efficient degreasing agent in this embodiment has excellent low-temperature activity and wide pH adaptability. The recombinant thermostable lipase maintains ≥80% of its maximum activity in the range of 5-80°C, with an optimum pH of 7.5-9.0, and can effectively decompose various grease stains. Alkaline protease maintains ≥70% of its maximum activity in the range of 20-60°C, with an optimum pH of 8.0-11.0, and can effectively remove protein stains. Chitosan-coated nanosilver particles not only have antibacterial effects, but also enhance the degradation of organic pollutants through photocatalysis. β-cyclodextrin-modified carbon quantum dots selectively adsorb small molecular organic matter through host-guest interactions, further improving the cleaning effect. Sodium alginate-based smart hydrogel microspheres automatically adjust their release behavior according to the environmental pH, ensuring that good cleaning effects can be maintained under different pH conditions.

[0037] Example 2

[0038] This embodiment provides another high-efficiency and energy-saving degreasing agent and a preparation method thereof. The degreasing agent comprises the following components: 7.5 parts by weight of recombinant thermostable lipase, 5.5 parts by weight of alkaline protease, 2 parts by weight of nanosilver particles wrapped in chitosan, 1.3 parts by weight of carbon quantum dots modified with β-cyclodextrin, 15 parts by weight of sodium alginate-based smart hydrogel microspheres, 20 parts by weight of lauryl glucoside, 15 parts by weight of coconut oil betaine, 7.5 parts by weight of propylene glycol, 5.5 parts by weight of d-limonene, 12 parts by weight of polyethylene glycol 400, 3 parts by weight of dipotassium hydrogen phosphate, and the balance of deionized water to 100 parts by weight.

[0039] The preparation method of the high-efficiency energy-saving degreasing agent comprises the following steps:

[0040] First, recombinant thermostable lipase was prepared in the same manner as in Example 1, but during the protein expression stage, the final concentration of IPTG was adjusted to 0.7 mM and the induction expression time was extended to 18 hours.

[0041] Next, alkaline protease was prepared in the same manner as in Example 1, but a hydrophobic interaction chromatography step was added during the purification process to further improve the purity of the enzyme.

[0042] Then, prepare the nano silver particles wrapped by chitosan. Dissolve 0.75g silver nitrate in 120mL deionized water, add 1.2g trisodium citrate, and stir for 12 minutes. Slowly drop 12mL 1.2% sodium borohydride solution, stir at room temperature for 1.5 hours. Add 60mL 0.6% chitosan solution (dissolved in 1.2% acetic acid), continue stirring for 2.5 hours. Centrifuge and wash 4 times, freeze-dry to obtain the product.

[0043] Next, prepare β-cyclodextrin-modified carbon quantum dots. Dissolve 2.5 g of citric acid and 1.2 g of ethylenediamine in 35 mL of water and perform hydrothermal reaction at 185 ° C for 4.5 hours. Cool to room temperature and dialyze for 50 hours (molecular weight cutoff 1200 Da). Mix the dialyzed solution with 1.2 g of β-cyclodextrin and react at 55 ° C for 26 hours. Freeze-dry to obtain the product.

[0044] Again, sodium alginate-based smart hydrogel microspheres were prepared. 2.5 g of sodium alginate was dissolved in 110 mL of deionized water. The solution was dripped into a 0.12 M calcium chloride solution using a microfluidic device (110 μm nozzle, pressure 0.22 MPa). Crosslinked in the calcium chloride solution for 2.5 hours, filtered and washed 4 times with deionized water.

[0045] Finally, the components prepared above were mixed with other commercial components according to the specified weight parts. Specifically, the recombinant thermostable lipase and alkaline protease were dissolved in dipotassium hydrogen phosphate buffer and ultrasonically treated for 7 minutes at a frequency of 42kHz and a power of 350W. The chitosan-encapsulated nanosilver particles and β-cyclodextrin-modified carbon quantum dots were dispersed in deionized water and ultrasonically treated for 12 minutes at a frequency of 22kHz. The sodium alginate-based smart hydrogel microspheres were dispersed in deionized water and gently stirred at 125rpm for 35 minutes. Lauryl glucoside, coconut betaine, propylene glycol, d-limonene and polyethylene glycol 400 were mixed and stirred at 50℃ and 350rpm for 17 minutes. Finally, all the components were mixed, sheared and emulsified at 7000rpm for 17 minutes, left at 25℃ for 24 hours, and then sterile filtered with a 0.22μm polyethersulfone filter membrane.

[0046] The energy-efficient degreasing agent in this embodiment has stronger antibacterial properties and higher organic pollutant degradation efficiency than that in Example 1. This is mainly due to the increase in the content of chitosan-encapsulated nanosilver particles and the improvement in the performance of β-cyclodextrin-modified carbon quantum dots. At the same time, the increase in the content of sodium alginate-based smart hydrogel microspheres also improves the adaptability of the degreasing agent under different pH environments.

[0047] Example 3

[0048] This embodiment provides a third high-efficiency and energy-saving degreasing agent and a preparation method thereof. The degreasing agent comprises the following components: 9 parts by weight of recombinant thermostable lipase, 7 parts by weight of alkaline protease, 2.5 parts by weight of nanosilver particles wrapped in chitosan, 1.8 parts by weight of carbon quantum dots modified with β-cyclodextrin, 18 parts by weight of sodium alginate-based smart hydrogel microspheres, 22 parts by weight of lauryl glucoside, 18 parts by weight of coconut oil betaine, 9 parts by weight of propylene glycol, 7 parts by weight of d-limonene, 14 parts by weight of polyethylene glycol 400, 4 parts by weight of dipotassium hydrogen phosphate, and the balance of deionized water to 100 parts by weight.

[0049] The preparation method of the high-efficiency energy-saving degreasing agent comprises the following steps:

[0050] First, recombinant thermostable lipase was prepared. The method was the same as in Example 1, but in the gene cloning stage, a codon-optimized gene sequence was used to improve the protein expression level. In the protein expression stage, the final concentration of IPTG was adjusted to 1 mM, and the induction expression time was extended to 20 hours.

[0051] Secondly, alkaline protease was prepared in the same manner as in Example 1, but molecular imprinting technology was used in the purification process to prepare a specific adsorption column to obtain an enzyme preparation with higher purity and activity.

[0052] Then, prepare the nano silver particles wrapped by chitosan. Dissolve 1g of silver nitrate in 150mL of deionized water, add 1.5g of trisodium citrate, and stir for 15 minutes. Slowly drop 15mL of 1.5% sodium borohydride solution, and stir at room temperature for 2 hours. Add 75mL of 0.7% chitosan solution (dissolved in 1.5% acetic acid) and continue stirring for 3 hours. Centrifuge and wash 5 times, and freeze-dry to obtain the product.

[0053] Next, prepare β-cyclodextrin-modified carbon quantum dots. Dissolve 3 g of citric acid and 1.5 g of ethylenediamine in 40 mL of water and perform hydrothermal reaction at 190 ° C for 5 hours. Cool to room temperature and dialyze for 52 hours (molecular weight cutoff 1500 Da). Mix the dialyzed solution with 1.5 g of β-cyclodextrin and react at 60 ° C for 28 hours. Freeze-dry to obtain the product.

[0054] Again, sodium alginate-based smart hydrogel microspheres were prepared. 3 g of sodium alginate was dissolved in 120 mL of deionized water. The solution was dripped into a 0.15 M calcium chloride solution using a microfluidic device (120 μm nozzle, pressure 0.25 MPa). Crosslinked in the calcium chloride solution for 3 hours, filtered and washed 5 times with deionized water.

[0055] Finally, the components prepared above were mixed with other commercial components according to the specified weight parts. Specifically, the recombinant thermostable lipase and alkaline protease were dissolved in potassium hydrogen phosphate buffer and ultrasonically treated at 45kHz frequency and 400W power for 10 minutes. The chitosan-encapsulated nanosilver particles and β-cyclodextrin-modified carbon quantum dots were dispersed in deionized water and ultrasonically treated at 25kHz frequency for 15 minutes. The sodium alginate-based smart hydrogel microspheres were dispersed in deionized water and gently stirred at 150rpm for 40 minutes. Lauryl glucoside, coconut oil betaine, propylene glycol, d-limonene and polyethylene glycol 400 were mixed and stirred at 52℃ and 400rpm for 20 minutes. Finally, all the components were mixed, sheared and emulsified at 8000rpm for 20 minutes, left at 25℃ for 26 hours, and then sterile filtered with a 0.22μm polyethersulfone filter membrane.

[0056] The energy-efficient degreasing agent in this embodiment has higher enzyme activity and stronger pollutant degradation ability. The content of recombinant thermostable lipase is increased to 9 parts by weight, and the expression level and activity of lipase are significantly improved in combination with the codon-optimized gene sequence. The increase in the content of alkaline protease and the application of molecular imprinting technology further enhance the ability to remove protein dirt. The increase in the content of chitosan-coated nanosilver particles and β-cyclodextrin-modified carbon quantum dots improves the antibacterial properties of the degreasing agent and the degradation efficiency of organic pollutants. The increase in the content of sodium alginate-based smart hydrogel microspheres further optimizes the adaptability and sustained-release performance of the degreasing agent under different pH environments.

[0057] Example 4

[0058] This embodiment provides a fourth high-efficiency energy-saving degreasing agent and a preparation method thereof. The degreasing agent comprises the following components: 8 parts by weight of recombinant thermostable lipase, 6 parts by weight of alkaline protease, 2.2 parts by weight of nanosilver particles wrapped in chitosan, 1.5 parts by weight of carbon quantum dots modified with β-cyclodextrin, 16 parts by weight of sodium alginate-based smart hydrogel microspheres, 21 parts by weight of lauryl glucoside, 16 parts by weight of coconut oil betaine, 8 parts by weight of propylene glycol, 6 parts by weight of d-limonene, 13 parts by weight of polyethylene glycol 400, 3.5 parts by weight of dipotassium hydrogen phosphate, and the balance of deionized water to 100 parts by weight.

[0059] The preparation method of the high-efficiency energy-saving degreasing agent comprises the following steps:

[0060] First, a recombinant thermostable lipase was prepared. The method is based on Example 1, but in the gene cloning stage, site-directed mutations were introduced to enhance the thermostability of the enzyme. Specifically, PCR amplification was performed using the following primers: forward primer 5'-GGAATTCCATATGAAGCTACTTTCTTATATACG-3', reverse primer 5'-CC GCTCGAGTTAATGATGATGATGATGATGCAGGAACACTGAGG-3'. PCR reaction conditions were: 95°C for 5 minutes; 32 cycles (95°C for 30 seconds, 60°C for 30 seconds, 72°C for 1 minute); 72°C for 12 minutes. In the protein expression stage, the final concentration of IPTG was adjusted to 0.8 mM, and the induction expression time was 19 hours.

[0061] Secondly, alkaline protease was prepared. The method was based on Example 1, but a method combining affinity chromatography and ion exchange chromatography was used in the purification process to obtain an enzyme preparation with higher purity.

[0062] Then, prepare the nano silver particles wrapped by chitosan. Dissolve 0.85g silver nitrate in 130mL deionized water, add 1.3g trisodium citrate, and stir for 13 minutes. Slowly drop 13mL 1.3% sodium borohydride solution, stir at room temperature for 1.8 hours. Add 65mL 0.65% chitosan solution (dissolved in 1.3% acetic acid), continue stirring for 2.8 hours. Centrifuge and wash 4 times, freeze-dry to obtain the product.

[0063] Next, prepare β-cyclodextrin-modified carbon quantum dots. Dissolve 2.8 g of citric acid and 1.3 g of ethylenediamine in 38 mL of water and perform hydrothermal reaction at 188 ° C for 4.8 hours. Cool to room temperature and dialyze for 51 hours (molecular weight cutoff 1300 Da). Mix the dialyzed solution with 1.3 g of β-cyclodextrin and react at 58 ° C for 27 hours. Freeze-dry to obtain the product.

[0064] Again, sodium alginate-based smart hydrogel microspheres were prepared. 2.8 g of sodium alginate was dissolved in 115 mL of deionized water. The solution was dripped into a 0.13 M calcium chloride solution using a microfluidic device (115 μm nozzle, pressure 0.23 MPa). Crosslinked in the calcium chloride solution for 2.8 hours, filtered and washed 4 times with deionized water.

[0065] Finally, the components prepared above were mixed with other commercial components according to the specified weight parts. Specifically, the recombinant thermostable lipase and alkaline protease were dissolved in dipotassium hydrogen phosphate buffer and ultrasonically treated for 8 minutes at a frequency of 43kHz and a power of 375W. The chitosan-encapsulated nanosilver particles and β-cyclodextrin-modified carbon quantum dots were dispersed in deionized water and ultrasonically treated for 13 minutes at a frequency of 23kHz. The sodium alginate-based smart hydrogel microspheres were dispersed in deionized water and gently stirred at 135rpm for 38 minutes. Lauryl glucoside, coconut betaine, propylene glycol, d-limonene and polyethylene glycol 400 were mixed and stirred at 51°C and 375rpm for 18 minutes. Finally, all the components were mixed, sheared and emulsified at 7500rpm for 18 minutes, left to stand at 25°C for 25 hours, and then sterile filtered with a 0.22μm polyethersulfone filter membrane.

[0066] The high-efficiency and energy-saving degreasing agent in this embodiment is optimized in terms of the content of each component to achieve a better balance. The recombinant thermostable lipase has improved thermal stability through site-directed mutagenesis, so that the degreasing agent remains efficient over a wider temperature range. The improved purification method of alkaline protease improves the purity and specific activity of the enzyme. The content of chitosan-coated nanosilver particles and β-cyclodextrin-modified carbon quantum dots is moderate, which not only ensures antibacterial properties and organic pollutant degradation efficiency, but also takes cost factors into consideration. The content of sodium alginate-based smart hydrogel microspheres is optimized, so that the degreasing agent can maintain good performance under different pH environments, while having an appropriate sustained-release effect.

[0067] Comparative Example 1 (corresponding to Example 1)

[0068] This comparative example provides a degreasing agent formula without recombinant thermostable lipase. The degreasing agent includes 4 parts by weight of alkaline protease, 1.5 parts by weight of chitosan-encapsulated nanosilver particles, 0.8 parts by weight of carbon quantum dots modified by β-cyclodextrin, 12 parts by weight of sodium alginate-based smart hydrogel microspheres, 18 parts by weight of lauryl glucoside, 12 parts by weight of coconut oil betaine, 6 parts by weight of propylene glycol, 4 parts by weight of d-limonene, 10 parts by weight of polyethylene glycol 400, 2 parts by weight of dipotassium hydrogen phosphate, and the balance of deionized water to 100 parts by weight.

[0069] The preparation method of the degreasing agent is similar to that of Example 1, but the preparation step of the recombinant thermostable lipase is omitted. The preparation methods and parameters of other components are the same as those of Example 1.

[0070] By comparison with Example 1, it can be found that the lack of recombinant thermostable lipase leads to a significant decrease in the ability of the degreasing agent to remove grease dirt, especially in a low temperature environment (<20° C.). This proves the key role of the recombinant thermostable lipase in the present invention, especially its characteristic of maintaining high activity in a low temperature environment, which provides the present invention with a unique low-temperature degreasing ability.

[0071] Comparative Example 2 (corresponding to Example 2)

[0072] This comparative example provides a degreasing agent formula without chitosan-coated nanosilver particles. The degreasing agent includes 7.5 weight parts of recombinant thermostable lipase, 5.5 weight parts of alkaline protease, 1.3 weight parts of carbon quantum dots modified by β-cyclodextrin, 15 weight parts of sodium alginate-based smart hydrogel microspheres, 20 weight parts of lauryl glucoside, 15 weight parts of coconut oil betaine, 7.5 weight parts of propylene glycol, 5.5 weight parts of d-limonene, 12 weight parts of polyethylene glycol 400, 3 weight parts of dipotassium hydrogen phosphate, and the balance of deionized water to 100 weight parts.

[0073] The preparation method is similar to that of Example 2, but the preparation step of the chitosan-coated nanosilver particles is omitted. The preparation methods and parameters of other components are the same as those of Example 2.

[0074] Compared with Example 2, the antibacterial property of the degreasing agent in this comparative example is significantly reduced, and the degradation efficiency of some stubborn organic pollutants is also reduced. This proves the important role of chitosan-encapsulated nanosilver particles in providing antibacterial properties and enhancing photocatalytic degradation of organic pollutants, and reflects the synergistic effect of nanomaterials and other components in the present invention.

[0075] Comparative Example 3 (corresponding to Example 3)

[0076] This comparative example provides a degreasing agent formula without sodium alginate-based smart hydrogel microspheres. The degreasing agent includes 9 parts by weight of recombinant thermostable lipase, 7 parts by weight of alkaline protease, 2.5 parts by weight of chitosan-encapsulated nanosilver particles, 1.8 parts by weight of carbon quantum dots modified by β-cyclodextrin, 22 parts by weight of lauryl glucoside, 18 parts by weight of coconut oil betaine, 9 parts by weight of propylene glycol, 7 parts by weight of d-limonene, 14 parts by weight of polyethylene glycol 400, 4 parts by weight of dipotassium hydrogen phosphate, and the balance of deionized water to 100 parts by weight.

[0077] The preparation method is similar to that of Example 3, but the preparation step of sodium alginate-based smart hydrogel microspheres is omitted. The preparation methods and parameters of other components are the same as those of Example 3.

[0078] Compared with Example 3, the adaptability of the degreasing agent in this comparative example under different pH environments is significantly reduced, especially in extremely acidic or alkaline environments. This proves the key role of sodium alginate-based smart hydrogel microspheres in regulating the pH responsiveness and sustained release performance of the degreasing agent, highlighting the innovativeness of the smart carrier system in the present invention.

[0079] Comparative Example 4 (corresponding to Example 4)

[0080] This comparative example provides a degreasing agent formulation using a recombinant thermostable lipase without site-directed mutagenesis. The formulation components are the same as those in Example 4, but the preparation method of the recombinant thermostable lipase is different.

[0081] When preparing the recombinant thermostable lipase, the original gene sequence without codon optimization was used, and the PCR reaction conditions were: 95°C for 5 minutes; 30 cycles (95°C for 30 seconds, 58°C for 30 seconds, 72°C for 1 minute); 72°C for 10 minutes. The preparation methods and parameters of other components were the same as those in Example 4.

[0082] Compared with Example 4, the stability and activity of the degreasing agent in this comparative example at high temperature (>60°C) are significantly reduced. This proves the importance of improving the thermal stability of recombinant thermostable lipase by site-directed mutagenesis, reflects the innovation of the present invention in enzyme engineering, and provides a wider application temperature range for the degreasing agent.

[0083] Comparative Example 5 (corresponding to Example 1 and Example 3)

[0084] This comparative example provides a degreasing agent formula containing only a single surfactant. The degreasing agent includes 7.5 weight parts of recombinant thermostable lipase, 5.5 weight parts of alkaline protease, 2 weight parts of chitosan-wrapped nanosilver particles, 1.3 weight parts of β-cyclodextrin-modified carbon quantum dots, 15 weight parts of sodium alginate-based smart hydrogel microspheres, 35 weight parts of lauryl glucoside, 7.5 weight parts of propylene glycol, 5.5 weight parts of d-limonene, 12 weight parts of polyethylene glycol 400, 3 weight parts of dipotassium hydrogen phosphate, and the balance of deionized water to 100 weight parts.

[0085] The preparation method is similar to the mixing method of Example 1 and Example 3, but only lauryl glucoside is used as the surfactant. The preparation methods and parameters of other components are the same as those of the corresponding examples.

[0086] Compared with Example 1 and Example 3, the degreasing agent in this comparative example has a reduced ability to remove dirt of different polarities, especially when dealing with complex oil-water mixed dirt. This proves the importance of the composite micelle system formed by lauryl glucoside and coconut oil betaine in enhancing the cleaning ability and adaptability of the degreasing agent, and reflects the innovation of the present invention in the design of surfactant formula.

[0087] Comparative Example 6 (corresponding to Example 2 and Example 4)

[0088] This comparative example provides a degreasing agent formula without β-cyclodextrin modified carbon quantum dots. The degreasing agent includes 8 parts by weight of recombinant thermostable lipase, 6 parts by weight of alkaline protease, 2.2 parts by weight of chitosan-wrapped nanosilver particles, 16 parts by weight of sodium alginate-based smart hydrogel microspheres, 21 parts by weight of lauryl glucoside, 16 parts by weight of coconut oil betaine, 8 parts by weight of propylene glycol, 6 parts by weight of d-limonene, 13 parts by weight of polyethylene glycol 400, 3.5 parts by weight of dipotassium hydrogen phosphate, and the balance of deionized water to 100 parts by weight.

[0089] The preparation method is similar to that of Example 2 and Example 4, but the preparation step of the carbon quantum dots modified with β-cyclodextrin is omitted. The preparation methods and parameters of the other components are the same as those of the corresponding examples.

[0090] Compared with Example 2 and Example 4, the selective adsorption capacity of the degreasing agent for small molecular organic pollutants in this comparative example is significantly reduced, and the pollutant degradation efficiency under light conditions is also reduced. This proves the important role of β-cyclodextrin-modified carbon quantum dots in enhancing the removal ability of the degreasing agent for specific pollutants, and reflects the innovation of the present invention in the functional design of nanomaterials.

[0091] In order to comprehensively evaluate the performance of the energy-efficient degreasing agent of the present invention, we designed the following experiments:

[0092] 1. Degreasing efficiency test: First, we evaluated the degreasing efficiency of the degreasing agent at different temperatures. A 5cm×5cm stainless steel sheet was immersed in a standard oil mixture (an equal mixture of olive oil, motor oil, and animal fat), dried, and weighed. The oily sheet was then immersed in a 1% degreasing agent solution and treated at 5°C, 25°C, and 50°C for 15 minutes respectively. After treatment, it was rinsed with deionized water, dried, and weighed again. The degreasing efficiency was calculated by the percentage of mass loss.

[0093] 2. pH adaptability test: Secondly, we tested the cleaning ability of the degreaser under different pH environments. The same oily piece was immersed in 1% degreaser solution with pH values ​​of 4, 7 and 10, treated at 25°C for 15 minutes, and the degreasing efficiency was calculated as above.

[0094] 3. Antibacterial performance test: Then, we evaluated the antibacterial performance of the degreaser. Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) were used as test strains. 1 ml of bacterial suspension containing 106 CFU / ml was mixed with 9 ml of 1% degreaser solution and incubated at 37°C for 24 hours. The number of surviving bacteria was determined by plate counting method and the inhibition rate was calculated.

[0095] 4. Organic pollutant degradation test: Next, we tested the degreasing agent's ability to treat difficult-to-degrade organic pollutants. Methyl orange was selected as a model pollutant with an initial concentration of 10 mg / L. 50 ml of pollutant solution was mixed with 1 ml of degreasing agent, stirred under natural light, and samples were taken every 30 minutes to measure the absorbance and calculate the degradation rate.

[0096] 5. Low temperature activity test: Finally, we specifically tested the activity of the degreasing agent in a low temperature environment. The lipase activity was measured at 5°C using olive oil as a substrate and compared with the activity at 25°C.

[0097] The experimental results are shown in Tables 1 to 5:

[0098] Table 1: Degreasing efficiency at different temperatures (%)

[0099] sample 5℃ 25℃ 50℃ Example 1 182.5 91.3 94.8 Example 2 85.7 93.6 96.2 Example 3 88.9 95.8 97.9 Example 4 87.2 94.5 97.1 Comparative Example 1 45.3 68.7 79.2 Comparative Example 2 83.1 92.8 95.5 Comparative Example 3 86.4 94.1 96.7 Comparative Example 4 79.8 91.9 93.6 Comparative Example 5 81.2 90.5 93.9 Comparative Example 6 84.6 93.2 95.8

[0100] Table 2: Degreasing efficiency at different pH (%)

[0101] sample pH4 pH7 pH10 Example 1 85.2 91.3 88.7 Example 2 87.9 93.6 90.8 Example 3 90.5 95.8 93.2 Example 4 89.1 94.5 91.9 Comparative Example 1 62.3 68.7 65.1 Comparative Example 2 86.5 92.8 89.4 Comparative Example 3 78.3 94.1 82.6 Comparative Example 4 85.7 91.9 89.3 Comparative Example 5 83.9 90.5 87.2 Comparative Example 6 87.1 93.2 90.3

[0102] Table 3: Antibacterial performance (inhibition rate %)

[0103] sample E. coli S. aureus Example 1 99.2 98.7 Example 2 99.5 99.1 Example 3 99.8 99.5 Example 4 99.6 99.3 Comparative Example 1 98.9 98.3 Comparative Example 2 92.4 91.8 Comparative Example 3 99.7 99.4 Comparative Example 4 99.4 99 Comparative Example 5 99.3 98.9 Comparative Example 6 99.4 99.1

[0104] Table 4: Degradation rate of organic pollutants (after 2 hours)

[0105] sample Degradation rate (%) Example 1 78.5 Example 2 82.3 Example 3 86.7 Example 4 84.9 Comparative Example 1 75.2 Comparative Example 2 79.6 Comparative Example 3 84.1 Comparative Example 4 82.7 Comparative Example 5 77.8 Comparative Example 6 71.4

[0106] Table 5: Low temperature activity retention rate (5°C relative to 25°C)

[0107]

[0108]

[0109] Based on the above experimental results, we can draw the following conclusions:

[0110] 1. Degreasing efficiency: All examples of the present invention showed excellent degreasing efficiency under various temperature conditions, especially under low temperature (5°C). Example 3 performed best, with a degreasing efficiency of 88.9% at 5°C, much higher than that of Comparative Example 1 (45.3%). This proves the key role of the recombinant thermostable lipase under low temperature conditions.

[0111] 2. pH adaptability: Examples 1-4 maintained efficient degreasing capabilities in a wide pH range (pH 4-10), with Example 3 achieving degreasing efficiencies of 90.5% and 93.2% at pH 4 and pH 10, respectively. In contrast, the efficiency of Comparative Example 3 (without sodium alginate-based smart hydrogel microspheres) was significantly reduced under extreme pH conditions, demonstrating the importance of smart hydrogel microspheres in regulating pH responsiveness.

[0112] 3. Antibacterial properties: All examples showed excellent antibacterial effects, with the antibacterial rates against Escherichia coli and Staphylococcus aureus exceeding 98.5%. In particular, Example 3 had an antibacterial rate of 99.8% and 99.5%. In contrast, the antibacterial effect of Comparative Example 2 (without chitosan-coated nanosilver particles) was significantly poor, highlighting the key role of nanosilver in providing antibacterial properties.

[0113] 4. Degradation of organic pollutants: Examples 1-4 performed well in degrading organic pollutants, with Example 3 achieving a degradation rate of up to 86.7%. Comparative Example 6 (without β-cyclodextrin-modified carbon quantum dots) had a significantly lower degradation rate (71.4%), demonstrating the importance of carbon quantum dots in enhancing the degradation of organic pollutants.

[0114] 5. Low temperature activity: The examples of the present invention maintained high activity under low temperature conditions, and the low temperature activity retention rate of Example 3 reached 92.1%. In contrast, the low temperature activity retention rate of Comparative Example 4 (using an enzyme without site-directed mutagenesis) was only 82.4%, highlighting the importance of improving enzyme thermal stability through site-directed mutagenesis.

[0115] Unexpected technical effects:

[0116] 1. Ultra-wide pH adaptability: The degreasing agent of the present invention maintains efficient cleaning ability in the pH range of 4-10, which greatly expands its application range. This wide pH adaptability is derived from the synergistic effect of sodium alginate-based smart hydrogel microspheres, recombinant thermostable lipase and alkaline protease. The smart hydrogel microspheres automatically adjust the release behavior according to the environmental pH, and the combination of the two enzymes ensures that the enzymes maintain high activity at different pH levels.

[0117] 2. Low-temperature and efficient cleaning: Under low-temperature conditions of 5°C, the degreasing agent of the present invention still maintains a degreasing efficiency of more than 80%, which is very rare in the industry. This low-temperature and efficient cleaning ability is mainly due to the synergistic effect of the recombinant thermostable lipase that has undergone site-directed mutagenesis and other components (such as nanomaterials) at low temperatures.

[0118] 3. Long-lasting antibacterial effect: The experimental results show that the degreasing agent of the present invention not only has immediate and efficient antibacterial ability, but also can continue to play an antibacterial role on the surface after cleaning. This long-lasting antibacterial effect may be due to the formation of a microscopic antibacterial film by the nanosilver particles wrapped in chitosan during the cleaning process.

[0119] 4. Self-cleaning effect: In the organic pollutant degradation experiment, we unexpectedly found that the cleaned surface showed a certain self-cleaning ability. This may be because the carbon quantum dots and nanosilver particles modified by β-cyclodextrin formed a photocatalytic active layer on the surface, which can continuously degrade trace pollutants under light conditions.

[0120] 5. Synergistic effect: The components show unexpected synergistic effects. For example, nanomaterials not only enhance the antibacterial and pollutant degradation capabilities of the degreasing agent, but also improve the stability and activity of the enzyme. This multiple synergistic effect enables the degreasing agent of the present invention to reach a high level in various performance indicators.

[0121] In summary, the high-efficiency and energy-saving degreasing agent of the present invention achieves many unexpected technical effects such as ultra-wide pH adaptability, low-temperature high-efficiency cleaning, long-term antibacterial, and self-cleaning effects through the synergistic effect of multiple innovative components. These characteristics make the present invention have broad application prospects in many fields such as industrial cleaning, household cleaning, and environmental management.

[0122] The above description is only an embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent substitution, improvement, etc. made within the spirit and principle of the present invention should be included in the scope of the claims of the present invention.

Claims

1. A highly efficient and energy-saving degreasing agent, characterized in that: The composition comprises the following components in parts by weight: 6-9 parts of recombinant thermostable lipase; 4-7 parts of alkaline protease; 1.5-2.5 parts of chitosan-encapsulated nanosilver particles; 0.8-1.8 parts of β-cyclodextrin-modified carbon quantum dots; 12-18 parts of sodium alginate-based smart hydrogel microspheres; 18-22 parts of lauryl glucoside; 12-18 parts of coconut oil betaine; 6-9 parts of propylene glycol; 4-7 parts of d-limonene; 10-14 parts of polyethylene glycol 400; 2-4 parts of dipotassium hydrogen phosphate; the balance is up to 100 parts of deionized water.

2. The high-efficiency and energy-saving degreasing agent according to claim 1, characterized in that: The preparation method of the recombinant thermostable lipase comprises the following steps: (1) Gene cloning: Genomic DNA was extracted from the Thermomyces lanuginosus strain, the lipase gene was amplified using primers, and the PCR product was cloned into the pET-22b(+) expression vector; (2) Recombinant expression: The constructed plasmid was transformed into Escherichia coli BL21 (DE3) competent cells, cultured in LB medium and induced for expression with IPTG; (3) Protein purification: collect the bacteria, disrupt them by ultrasonication, and purify the target protein using a Ni-NTA affinity chromatography column; (4) Enzyme activity assay: Using olive oil as substrate, the enzyme activity was measured at 37°C to ensure that the activity was ≥100,000 U / g.

3. The high-efficiency and energy-saving degreasing agent according to claim 1, characterized in that: The alkaline protease is derived from Bacillus subtilis, maintains a maximum activity of ≥70% within the range of 20-60° C., and has an optimum pH of 8.0-11.

0.

4. The high-efficiency and energy-saving degreasing agent according to claim 1, characterized in that: The preparation method of chitosan-coated nano-silver particles comprises the following steps: (1) dissolving silver nitrate in deionized water, adding trisodium citrate and stirring; (2) slowly dropping sodium borohydride solution and stirring at room temperature; (3) adding chitosan solution and dissolving it in 1% acetic acid and continuing to stir; (4) centrifuging and washing, and freeze-drying to obtain the product.

5. The high-efficiency and energy-saving degreasing agent according to claim 1, characterized in that: The preparation method of the β-cyclodextrin modified carbon quantum dots comprises the following steps: (1) dissolving citric acid and ethylenediamine in water, and subjecting them to a hydrothermal reaction at 180° C. for 4 hours; (2) cooling to room temperature, and subjecting them to dialysis at a molecular weight cutoff of 1000 Da for 48 hours; (3) mixing the dialyzed solution with β-cyclodextrin, and subjecting them to a reaction at 50° C. for 24 hours; and (4) freeze-drying to obtain the product.

6. The high-efficiency and energy-saving degreasing agent according to claim 1, characterized in that: The preparation method of the sodium alginate-based smart hydrogel microspheres comprises the following steps: (1) dissolving sodium alginate in deionized water; (2) dripping the solution into a calcium chloride solution using a microfluidic device with a 100 μm nozzle and a pressure of 0.2 MPa; and (3) cross-linking in the calcium chloride solution for 2 hours, filtering and washing with deionized water.

7. A method for preparing the high-efficiency energy-saving degreasing agent according to any one of claims 1 to 6, characterized in that: The method comprises the following steps: (1) preparing an enzyme solution: dissolving recombinant thermostable lipase and alkaline protease in a potassium hydrogen phosphate buffer solution and subjecting the solution to ultrasonic treatment; (2) preparing a nano-dispersion solution: dispersing chitosan-wrapped nano-silver particles and β-cyclodextrin-modified carbon quantum dots in deionized water and subjecting the solution to ultrasonic treatment; (3) preparing sodium alginate microspheres: dispersing sodium alginate-based smart hydrogel microspheres in deionized water and subjecting the solution to gentle stirring; (4) preparing a surfactant and solvent mixed solution: mixing lauryl glucoside, coconut oil-based betaine, propylene glycol, d-limonene and polyethylene glycol 400, heating and stirring; and (5) preparing a final product: mixing all the components prepared in steps (1) to (4), subjecting the mixture to high-speed shear emulsification, allowing the mixture to stand, and then subjecting the mixture to sterile filtration.

8. The preparation method according to claim 7, characterized in that: In step (1), the conditions of the ultrasonic treatment are: frequency 40kHz, power 300-400W, and treatment time 5-10 minutes.

9. The preparation method according to claim 7, characterized in that: In step (4), the heating and stirring conditions are: temperature 50±2°C, stirring speed 300-400rpm, and time 15-20 minutes.

10. The preparation method according to claim 7, characterized in that: In step (5), the conditions for high-speed shear emulsification are: speed 7000±1000rpm, time 15-20 minutes, temperature 25±3°C; the conditions for standing are: temperature 25±2°C, time 24±2 hours; and the sterile filtration uses a 0.22μm polyethersulfone filter membrane.

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