A lipase resistant to surface activity and metal ions, and a preparation method and application thereof

Abstract

The application relates to the technical field of lipases, and discloses a lipase resistant to surface activity and metal ions, a preparation method and application thereof. In the application, the lipase with the amino acid sequence shown in SEQ ID NO:1 has good alkali resistance and surfactant resistance, can be well adapted to an oil stain cleaning agent system, also has good metal ion resistance, the enzyme activity is less affected by the negative influence of metal ions in washing water, and the catalytic temperature is adapted to the oil stain cleaning process, so that when the lipase is used in the oil stain cleaning agent, efficient removal of oil stains (including polymerized heavy oil stains) can be realized; on the basis, the alkali resistance and surfactant resistance of the lipase can be further improved through specific chemical modification of the lipase, so that the stain removal performance and long-term storage stability of the oil stain cleaning agent are provided, the bactericidal capacity of the oil stain cleaning agent is improved, and the corrosiveness of the oil stain cleaning agent to common kitchen materials is reduced.

Description

Technical Field

[0001] This invention relates to the field of lipase technology, and in particular to a lipase resistant to surfactants and metal ions, its preparation method, and its applications. Background Technology

[0002] Kitchen grease is a stubborn pollutant that frequently occurs in homes and restaurants. It is mainly composed of animal and vegetable oils, food residues, oxidized and polymerized greases, dust, and gelatinous deposits. During high-temperature cooking and long-term sitting, grease undergoes oxidation, polymerization, and cross-linking reactions, eventually forming dense, highly adhesive, and aged heavy grease that firmly adheres to surfaces such as stovetops, range hoods, tiles, and stainless steel cookware, making it difficult to remove completely with conventional cleaning products.

[0003] Currently, mainstream kitchen degreasers on the market are mainly divided into two categories: chemically alkaline and bio-enzymatic. Chemically alkaline cleaners primarily rely on sodium hydroxide, sodium carbonate, organic solvents, and highly penetrating surfactants to remove grease. While they offer fast cleaning, they have the following drawbacks: the strong alkaline system can corrode stainless steel and aluminum alloy kitchenware and kitchen paint, accelerating oxidation and paint peeling with long-term use; the chemical solvents are highly irritating, and their volatile compounds can damage the respiratory tract; furthermore, they leave chemical residues that can contaminate tableware and food; and the chemical components in the wastewater have poor biodegradability, posing significant environmental safety hazards.

[0004] Compared to strong alkaline chemical detergents, bio-enzyme-based detergents have lower corrosiveness, irritation, and environmental safety risks. However, due to the poor compatibility of lipases with chemical cleaning systems, these detergents have weaker oil removal capabilities and poor stability. Specifically, oil cleaning systems typically require an alkaline environment to enhance cleaning efficiency and rely on surfactants to emulsify the oil. Existing lipases have weak alkali and surfactant resistance, easily undergoing rapid inactivation in oil cleaning systems. This leads to antagonism between the enzymatic and chemical cleaning processes, resulting in overall cleaning efficiency far lower than traditional purely chemical detergents. Furthermore, it shortens the product's shelf life, causing a rapid decline in the system's effective oil degradation capacity. Additionally, the calcium in the washing water... 2+ Mg 2+ Metal ions can also easily affect the conformation of lipases, thereby altering their catalytic activity. This is especially true for heavy kitchen grease, where the grease molecules are no longer fresh, free grease, but rather large molecular grease polymers formed through high-temperature oxidation and cross-linking polymerization. These polymers are more difficult to degrade, and existing bio-enzyme-based cleaners are insufficient to meet the application requirements for cleaning heavy kitchen grease.

[0005] Patent CN116732002A, based on the wild-type lipase derived from *Thermomyces lanuginosus*, introduced three site mutations: R84H, I86L, and N92A or N92V. This effectively improved the lipase's tolerance to two surfactants, fatty alcohol polyoxyethylene ether-9 (AEO-9) and sodium dodecyl sulfate (SDS), but did not improve the lipase's alkali resistance and metal ion resistance. Summary of the Invention

[0006] To address the technical problems of poor alkali resistance, surfactant resistance, and metal ion resistance in existing lipases, this invention provides a surfactant- and metal ion-resistant lipase, its preparation method, and its applications. The lipase of this invention exhibits good alkali resistance, surfactant resistance, and metal ion resistance, and possesses a catalytic temperature suitable for oil stain cleaning processes. When used in oil stain cleaners, it facilitates the efficient removal of oil stains (including polymerized heavy oil stains).

[0007] The specific technical solution of this invention is as follows: In a first aspect, the present invention provides a lipase resistant to surfactants and metal ions, the amino acid sequence of which is shown in SEQ ID NO:1.

[0008] Lipases with the aforementioned amino acid sequence exhibit good resistance to alkalis, surfactants, and metal ions, and possess catalytic temperatures suitable for oil stain cleaning processes. Experiments show that, without chemical modification, the lipase with the amino acid sequence shown in SEQ ID NO:1 has an optimal pH range of 7.5–9.5, an optimal catalytic temperature of 45°C, and a tolerance temperature range of 10–45°C. It also demonstrates good tolerance to various surfactants, including cocamidopropyl betaine (CAPB), alkyl glycosides (APG), and fatty alcohol polyoxyethylene ethers (AEO), and can tolerate Ca2+. 2+ and Mg 2+ .

[0009] The above-mentioned characteristics enable the lipase of the present invention to achieve good compatibility with the alkaline environment and surfactants in oil stain cleaners when used in oil stain cleaners. To a certain extent, it overcomes the problem of mutual antagonism between enzymatic decontamination and chemical decontamination processes. Furthermore, during the use of oil stain cleaners, the lipase is less affected by the negative impact of metal ions in the washing water and has higher enzyme activity in warm water environments, which is beneficial for achieving efficient removal of oil stains (including polymerized heavy oil stains).

[0010] Preferably, the lipase, based on the amino acid sequence shown in SEQ ID NO:1, includes the following chemical modifications: some surface amino groups are modified by polyethylene glycol segment grafting, and some surface amino groups are modified by acetylation.

[0011] For the lipase with a specific sequence in this invention, the above two chemical modifications can effectively improve its compatibility with oil-based cleaning agent systems while retaining the original oil hydrolysis activity of the lipase. Specifically: by grafting polyethylene glycol (PEG) segments onto the amino groups on the surface of the lipase, a PEG polymer layer can be formed, which can isolate the enzyme protein backbone from the damage caused by surfactants and organic solvents in the oil-based cleaning agent system; by acetylation modification of the amino groups on the surface of the lipase, the surface charge distribution of the enzyme can be optimized, improving the conformational stability of the lipase in a weakly alkaline system. Through the above methods, the chemically modified lipase, when used in oil-based cleaning agents, can enable the oil-based cleaning agents to have better detergency and long-term storage stability.

[0012] Furthermore, this invention has found that, compared to unmodified lipases, using chemically modified lipases can, to some extent, reduce the corrosiveness of oil stain cleaners to common kitchen materials (aluminum alloys and painted panels) and improve the antibacterial properties of oil stain cleaners.

[0013] Preferably, the modifier used for the polyethylene glycol segment grafting modification is polyethylene glycol-N-hydroxysuccinimide ester (i.e., a polyethylene glycol derivative with N-hydroxysuccinimide ester at one end of the molecular chain, abbreviated as PEG-NHS); the modifier used for the acetylation modification is acetic anhydride.

[0014] Preferably, the polyethylene glycol-N-hydroxysuccinimide ester is methoxy polyethylene glycol-N-hydroxysuccinimide ester (i.e., a polyethylene glycol derivative with methoxy and N-hydroxysuccinimide esters at both ends of the molecular chain, abbreviated as mPEG-NHS), wherein the number average molecular weight of the polyethylene glycol segments is 4000~6000 Da.

[0015] Preferably, the surfactant includes cocamidopropyl betaine, alkyl glycosides, and fatty alcohol polyoxyethylene ethers; the metal ion includes Ca. 2+ and Mg 2+ .

[0016] Secondly, the present invention provides a method for preparing the lipase, comprising the steps of: adding polyethylene glycol-N-hydroxysuccinimide ester to an enzyme solution containing unmodified lipase, performing a grafting reaction, terminating the reaction, adding acetic anhydride, performing an acetylation reaction, and separating and purifying the product; the amino acid sequence of the unmodified lipase is shown in SEQ ID NO:1.

[0017] Preferably, the mass ratio of the unmodified lipase, polyethylene glycol-N-hydroxysuccinimide ester, and acetic anhydride is 1:1~5:0.1~1.0; before adding polyethylene glycol-N-hydroxysuccinimide ester, the enzyme solution is adjusted to pH 8.3~8.7; the grafting reaction is carried out at a temperature of 20~30℃ for 1.5~2.5 h; after terminating the reaction, the pH is adjusted to 7.8~8.2 before adding acetic anhydride; during the addition of acetic anhydride, the pH is maintained at 7.8~8.2; the acetylation reaction is carried out at a temperature of 20~30℃ for 1~2 h.

[0018] Thirdly, the present invention provides the application of the lipase in an oil stain cleaner, the oil stain cleaner having a pH of 7.5 to 10.0 and containing the lipase and a surfactant.

[0019] Preferably, the oil stain cleaner is a heavy-duty kitchen oil stain cleaner; the oil stain cleaner comprises the following components in parts by weight: 0.2-0.8 parts lipase, 6-10 parts surfactant, 1-3 parts solubilizer, 2.5-6 parts enzyme stabilizer, 0.3-1.0 parts chelating agent, 0.4-1.2 parts pH buffer adjuster, 0-0.2 parts preservative, 0-0.4 parts fragrance, and 70-90 parts solvent.

[0020] In the above-mentioned oil stain cleaner formulation, the lipase of this invention solves the problem of antagonistic effects between chemical components and enzyme activity in the cleaner, as well as the inability of traditional enzyme preparations to degrade polymerized heavy oil stains. Based on this, the entire formulation forms a triple synergistic cleaning system of "enzymatic degradation - emulsification and stripping - penetration and softening," achieving highly efficient removal of heavy oil stains in the kitchen under a formulation system that is non-alkali, mild, low-irritant, and compatible with commonly used kitchen materials. Furthermore, the cleaning process does not require high-intensity scrubbing, reducing cleaning labor intensity. Specifically: 1) Penetration and softening stage: The co-solvent and surfactant produce a synergistic penetration effect, which can quickly penetrate into the interior of heavy oil stains, soften the dense oil stain layer formed by high temperature curing and oxidative polymerization, destroy the bonding force between the oil stains and the surface of the kitchenware, and achieve the initial loosening of the oil stains. 2) Emulsification and stripping stage: Surfactants can play an emulsifying, dispersing and solubilizing role, which can encapsulate and disperse the loosened free oil and fine oil in the aqueous phase, and prevent the oil from re-attaching. 3) Enzymatic degradation stage: The lipase of the present invention can maintain high catalytic activity and can specifically break down large molecular oil polymers and oxidized cross-linked oils that cannot be decomposed by conventional detergents, degrading them into small molecular fatty acids and glycerol, thereby achieving the complete decomposition of stubborn heavy oil stains and completing the cleaning and stain removal process from the root.

[0021] Furthermore, the lipase of the present invention has good compatibility with the alkaline environment and surfactants of the oil stain cleaner. At the same time, the enzyme stabilizer in the formulation can further maintain the catalytic activity of the enzyme, and the pH buffer adjuster can provide a better suitable environment for enzyme activity. Therefore, the above-mentioned oil stain cleaner formulation has good storage stability. After long-term storage, the lipase therein can still maintain high activity, which can meet the application requirements of industrial production, transportation and long-term storage.

[0022] Preferably, the surfactant includes amphoteric surfactants and / or nonionic surfactants, the amphoteric surfactants being alkyl glycosides and / or fatty alcohol polyoxyethylene ethers, and the nonionic surfactants including cocamidopropyl betaine; the enzyme stabilizer includes one or more of glycerol, sorbitol, glycine, and betaine.

[0023] Preferably, the surfactant comprises an amphoteric surfactant and a nonionic surfactant in a weight ratio of 12 to 15:1.

[0024] Preferably, the enzyme stabilizer comprises one or more of glycerol, sorbitol, glycine, and betaine in a weight ratio of 2~5:2~4:1~3:1.

[0025] Fourthly, the present invention provides an oil stain cleaner having a pH of 7.5 to 10.0 and containing a surfactant and the lipase.

[0026] Preferably, the oil stain cleaner is a heavy-duty kitchen oil stain cleaner; the oil stain cleaner comprises the following components in parts by weight: 0.2-0.8 parts lipase, 6-10 parts surfactant, 1-3 parts solubilizer, 2.5-6 parts enzyme stabilizer, 0.3-1.0 parts chelating agent, 0.4-1.2 parts pH buffer adjuster, 0-0.2 parts preservative, 0-0.4 parts fragrance, and 70-90 parts solvent.

[0027] Preferably, the surfactant includes amphoteric surfactants and / or nonionic surfactants, the amphoteric surfactants being alkyl glycosides and / or fatty alcohol polyoxyethylene ethers, and the nonionic surfactants including cocamidopropyl betaine; the enzyme stabilizer includes one or more of glycerol, sorbitol, glycine, and betaine.

[0028] Compared with the prior art, the present invention has the following advantages: (1) In this invention, the lipase with the amino acid sequence shown in SEQ ID NO:1 has good alkali resistance and surfactant resistance, and can be well adapted to the oil stain cleaning agent system. It also has good resistance to metal ions, and the enzyme activity is less affected by metal ions in the washing water. Moreover, the catalytic temperature is adapted to the oil stain cleaning process. Therefore, when used in oil stain cleaning agents, it is beneficial to achieve efficient removal of oil stains (including polymerized heavy oil stains).

[0029] (2) In this invention, by performing specific chemical modifications on the lipase with the amino acid sequence shown in SEQ ID NO:1 (part of the surface amino groups are modified by grafting polyethylene glycol segments and part of the surface amino groups are modified by acetylation), the lipase's resistance to alkali and surfactants can be further improved, thereby providing the cleaning performance and long-term storage stability of the oil stain cleaner, while also improving its antibacterial ability and reducing its corrosiveness to common kitchen materials.

[0030] (3) In the oil stain cleaner formula of the present invention, the lipase and chemical components have good compatibility and can form a triple synergistic cleaning system of "enzymatic degradation-emulsification stripping-penetration softening". It can achieve efficient removal of heavy oil stains in the kitchen under a formula system that is free of strong alkali, mild and low irritation and compatible with commonly used kitchen materials. Moreover, the cleaning process does not require high-intensity scrubbing. At the same time, the oil stain cleaner of the present invention also has good storage stability. Detailed Implementation

[0031] The present invention will be further described below with reference to specific embodiments.

[0032] In a first aspect, the present invention relates to a lipase resistant to surfactants and metal ions, the amino acid sequence of which is shown in SEQ ID NO:1.

[0033] In this invention, the amino acid sequence shown in SEQ ID NO:1 is specifically as follows: .

[0034] The present invention does not impose any particular restrictions on the synthesis method of lipase (without chemical modification) with an amino acid sequence as shown in SEQ ID NO:1, and conventional or common protein synthesis methods in the art can be used.

[0035] In some specific embodiments, the lipase, based on the amino acid sequence shown in SEQ ID NO:1, includes the following chemical modifications: some surface amino groups are modified by polyethylene glycol segment grafting, and some surface amino groups are modified by acetylation.

[0036] In some specific embodiments, the modifier used for the polyethylene glycol segment grafting modification is polyethylene glycol-N-hydroxysuccinimide ester (i.e., a polyethylene glycol derivative with N-hydroxysuccinimide ester at one end of the molecular chain, abbreviated as PEG-NHS). In this specific embodiment, optionally or preferably, the polyethylene glycol-N-hydroxysuccinimide ester is methoxy polyethylene glycol-N-hydroxysuccinimide ester (i.e., a polyethylene glycol derivative with methoxy and N-hydroxysuccinimide esters at both ends of the molecular chain, abbreviated as mPEG-NHS).

[0037] In some specific embodiments, the number-average molecular weight of the polyethylene glycol segments in the modifier used for the polyethylene glycol segment grafting modification is 4000~6000 Da.

[0038] In some specific embodiments, the modifying agent used for the acetylation modification is acetic anhydride.

[0039] In some specific embodiments, among the surfactants and metal ions, the surfactants include cocamidopropyl betaine, alkyl glycosides, and fatty alcohol polyoxyethylene ethers, and the metal ions include Ca... 2+ and Mg 2+ .

[0040] In a second aspect, the present invention relates to a method for preparing the lipase, comprising the steps of: adding polyethylene glycol-N-hydroxysuccinimide ester to an enzyme solution containing unmodified lipase, performing a grafting reaction, terminating the reaction, adding acetic anhydride, performing an acetylation reaction, and separating and purifying the product; the amino acid sequence of the unmodified lipase is shown in SEQ ID NO:1.

[0041] In some specific embodiments, the mass ratio of the unmodified lipase, polyethylene glycol-N-hydroxysuccinimide ester, and acetic anhydride is 1:1~5:0.1~1.0.

[0042] In some specific embodiments, the enzyme solution is adjusted to pH 8.3-8.7 before adding polyethylene glycol-N-hydroxysuccinimide ester; the grafting reaction is carried out at a temperature of 20-30°C for 1.5-2.5 h.

[0043] In some specific embodiments, the pH is adjusted to 7.8-8.2 before adding acetic anhydride after the reaction is terminated; the pH is maintained at 7.8-8.2 during the addition of acetic anhydride; the acetylation reaction is carried out at a temperature of 20-30°C for 1-2 hours.

[0044] Thirdly, the present invention relates to the application of the lipase in an oil stain cleaner having a pH of 7.5 to 10.0 and containing the lipase and a surfactant.

[0045] Fourthly, the present invention relates to an oil stain cleaner having a pH of 7.5 to 10.0 and containing a surfactant and the lipase.

[0046] Regarding the oil stain cleaners mentioned in the third and fourth aspects above: In some embodiments, the lipase is an unmodified lipase, and the pH of the oil stain cleaner is 7.5-9.5. In other embodiments, the lipase is a chemically modified lipase, and the pH of the oil stain cleaner is 8.5-10.0.

[0047] In some specific embodiments, the surfactant includes amphoteric surfactants and / or nonionic surfactants, the amphoteric surfactants being alkyl glycosides and / or fatty alcohol polyoxyethylene ethers, and the nonionic surfactants including cocamidopropyl betaine.

[0048] In some specific embodiments, the oil stain cleaner is a heavy-duty kitchen oil stain cleaner.

[0049] In some specific embodiments, the oil stain cleaner comprises the following components in parts by weight: 0.2-0.8 parts lipase, 6-10 parts surfactant, 1-3 parts solubilizer, 2.5-6 parts enzyme stabilizer, 0.3-1.0 parts chelating agent, 0.4-1.2 parts pH buffer adjuster, 0-0.2 parts preservative, 0-0.4 parts fragrance, and 70-90 parts solvent. In this specific embodiment, optional or preferred components include: The surfactants include zwitterionic surfactants and nonionic surfactants in a weight ratio of 12 to 15:1, zwitterionic surfactants alkyl glycosides and / or fatty alcohol polyoxyethylene ethers, and nonionic surfactants including cocamidopropyl betaine. The cosolvent includes acetone glycerol (ACM); ACM is a green cosolvent, a renewable plant-based raw material, which does not have a pungent odor. The plant-derived components have safe and environmentally friendly properties, can help break down the structure of solidified oil stains, and have excellent system compatibility and no adverse effects on enzyme activity. The enzyme stabilizer comprises one or more of glycerol, sorbitol, glycine, and betaine in a weight ratio of 2~5:2~4:1~3:1; The chelating agent includes tetrasodium diglutamate; The pH buffer regulator includes triethanolamine; triethanolamine not only regulates the pH value of the system, but also has weak emulsifying and rust-preventing functions, which can protect the materials of kitchenware. The preservatives include Kathon preservatives; Kathon preservatives have the characteristics of broad antibacterial spectrum, low addition amount, and will not have adverse effects on system stability and enzyme activity.

[0050] In some specific embodiments, the preparation steps of the oil stain cleaner include: taking a portion of the solvent, adding a chelating agent and a pH buffer adjuster in sequence, dissolving them, adding a surfactant in batches, mixing well, adding a co-solvent and mixing well, cooling to 25~30℃, adding an enzyme stabilizer and mixing well, then adding a lipase and mixing well, then adding a preservative and a fragrance, mixing well, then adding the remaining solvent, and then mixing, defoaming and filtering in sequence.

[0051] The present invention will now be described through specific embodiments and comparative examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, the raw materials and equipment used in the present invention are conventional materials and equipment in the art and can be obtained through conventional commercial channels; unless otherwise specified, the methods used in the present invention are conventional methods in the art.

[0052] Example 1: Performance comparison of different lipases (without chemical modification) This embodiment studies the enzyme activity of the lipase (hereinafter referred to as "LPS1") with the amino acid sequence shown in SEQ ID NO:1, and three other lipases derived from natural Aspergillus niger, Streptomyces and Thermophilus spp., with amino acid sequences shown in SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4, respectively (hereinafter referred to as "AspLP", "StrSP" and "TLL"). The studies include tests on alkali resistance, surfactant resistance and metal ion resistance.

[0053] The amino acid sequences of AspLP, StrSP, and TLL are as follows: (1) Amino acid sequence of AspLP (SEQ ID NO:2): MQSLSTLPLPLLGAAATAGPASAPSAQTRTSVAVQDYVAAIKQANVNGVLVLDGTNWGDLVCDGASQIGFVVTDRGKQFTYLSDAANRQGKANWILNLGGSWGLVAGLTSIGTADVNYTISVQHPHVKAVVLDHNAYGLNWEADVEVASQAVQAEVANAYKDA VVAALPKLRPDLYEALVRDVPKAYPTPLWGVGNPNFKDDNAIDPLFRLVDPVYANLKKQGKPNPFVLVGGYGNVLGGTQAAKRLANAGYVDAITGDLALVTTIGGAYRGAVPGSINDAVQRYVETLKDAVKELGAPVAVVASWGDGQDGAYWDLVANIGPKL.

[0054] (2) Amino acid sequence of StrSP (SEQ ID NO:3): MTPTPAAPLLAAVATLLGSAAPAQPAPADAPGQATVVAQDYAAAIKQADVNGVLVLDGTNWGDLVCDGASQIGFVVTDRGKQFTYLSDAAQRQGKANWILNLGGSWGLVAGLTSIGTADVNYTISVQHPHVKAVVLDHNAYGLNWEADVEVASQAVQAEVANAYKDAVVAALPKLRPDLYEALVRDVPKAYPTPLWGVGNPNFKDDNAIDPLFRLVDPVYANLKKQGKPNPFVLVGGYGNVLGGTQAAKRLANAGYVDAITGDLALVTTIGGAYRGAVPGSINDAVQRYVETLKDAVKELGAPVAVVASWGDGQDGAYWDLVANIGPKL.

[0055] (3) Amino acid sequence of TLL (SEQ ID NO:4): MQPLSVSLPLVAGALVQAGAAPAPSAKRTNVAVQDYVAAIKQANVNGVLVLDGTNWGDLVCDGASQIGFVVTDRGKQFTYLSDAAQRQGKANWILNLGGSWGLVAGLTSIGTADVNYTISVQHPHVKAVVLDHNAYGLNWEADVEVASQAVQAEVANAYKDAVVAALPKLRPDLYEALVRDVPKAYPTPLWGVGNPNFKDDNAIDPLFRLVDPVYANLKKQGKPNPFVLVGGYGNVLGGTQAAKRLANAGYVDAITGDLALVTTIGGAYRGAVPGSINDAVQRYVETLKDAVKELGAPVAVVASWGDGQDGAYWDLVANIGPKL.

[0056] The method for testing enzyme activity properties is as follows: (1) In all performance tests, the p-NPP colorimetric method was used to determine enzyme activity. Specifically, p-nitrophenol palmitate was used as the substrate. In the reaction system, lipase catalyzed the hydrolysis of the substrate to release p-nitrophenol. The enzyme activity was calculated by measuring the change in absorbance at 410 nm. Definition of enzyme activity unit: Under optimal reaction conditions, the amount of enzyme required to catalyze the hydrolysis of p-NPP to produce 1 μmol of p-nitrophenol per minute is defined as 1 enzyme activity unit (U). The "optimal pH range" mentioned in this invention refers to the pH range corresponding to when the enzyme activity of the lipase is not lower than 85% of its highest measured enzyme activity.

[0057] (2) Alkali resistance test: Each lipase was prepared into an enzyme solution with the same enzyme activity concentration and placed in a buffer system with pH values ​​of 8.5, 9.0, 9.5 and 10.0 respectively. After incubation at 25°C for 2 h, the remaining enzyme activity was measured. The enzyme activity retention rate was calculated with the initial enzyme activity of the untreated enzyme as 100%.

[0058] (3) Determination of surfactant resistance: Each lipase was prepared into an enzyme solution with the same enzyme activity concentration. Cocamidopropyl betaine (CAPB), alkyl glycoside 1214 (APG1214), and fatty alcohol polyoxyethylene ether 9 (AEO9) were added to each enzyme solution to a final concentration of 10%. After incubation at 25°C for 24 h, the remaining enzyme activity was measured. The enzyme activity of the enzyme solution without added surfactant was taken as 100%, and the enzyme activity retention rate was calculated.

[0059] (4) Determination of metal ion tolerance: Each lipase was prepared into an enzyme solution with the same enzyme activity concentration, and Ca2+ was added to each enzyme solution to a final concentration of 10 mmol / L. 2+ Mg 2+ After incubation at 25℃ for 2 hours, the remaining enzyme activity was measured. The enzyme activity of the enzyme solution without added metal ions was taken as 100%, and the enzyme activity retention rate was calculated.

[0060] Each of the four lipases was tested in triplicate using the methods described above, and the average value was recorded. The compatibility of each lipase in the alkaline cleaning system was then comprehensively evaluated. The results of the core performance indicators of the four lipases are shown in Table 1.

[0061] Table 1 Performance test results of different lipases

[0062] As shown in Table 1, the lipase with the amino acid sequence SEQ ID NO:1 exhibits the highest enzyme activity retention rate under weakly alkaline conditions, and is most effective against surfactants (including CAPB, APG1214, and AEO9) and metal ions (including Ca). 2+ and Mg 2 +Its tolerance is significantly better than the other three lipases, and its catalytic temperature range is suitable for room temperature applications in kitchen cleaning, making it the best in terms of overall performance.

[0063] Example 2: Chemical modification of lipase and comparison of its performance before and after chemical modification In this embodiment, the lipase (LPS1) with the amino acid sequence shown in SEQ ID NO:1 is chemically modified according to the following steps: S1. Preparation of enzyme solution: LPS1 was prepared into an enzyme solution with a protein concentration of 10 mg / mL using 0.05 mol / L Tris-HCl buffer at pH 8.0.

[0064] S2, PEG grafting modification: Take LPS1 enzyme solution and adjust the pH of the system to 8.5 with 0.1 mol / L sodium carbonate-sodium bicarbonate buffer; slowly add mPEG5000-NHS powder at a mass ratio of 1:5 (i.e., a polyethylene glycol derivative with methoxy and N-hydroxysuccinimide esters at both ends of the molecular chain, wherein the number average molecular weight of the polyethylene glycol segment is 5000 Da), stir at low speed until completely dissolved, and react at 25℃ for 2 h; after the reaction is completed, add glycine to terminate the reaction, and the final concentration of glycine is 0.1 mol / L.

[0065] S3, Acetylation Modification: Adjust the pH of the enzyme solution after step S2 to 8.0, and slowly add acetic anhydride dropwise at a mass ratio of enzyme protein to acetic anhydride of 1:0.8. During the dropwise addition, continuously maintain the pH of the system in the range of 7.8 to 8.2 with dilute alkali solution. After the dropwise addition is completed, continue the reaction at a constant temperature of 25°C for 1.5 h.

[0066] S4. Post-treatment concentration: Transfer the enzyme solution after step S3 into an ultrafiltration membrane with a molecular weight cutoff of 30 kDa, and wash it three times with Tris-HCl buffer at pH 8.5 to remove unreacted small molecule reagents and free PEG; finally concentrate it to an enzyme activity of not less than 20,000 U / g to obtain modified lipase (hereinafter referred to as "LPS2"), and store it at 4°C for later use.

[0067] Unmodified LPS1 and LPS2 modified in this example were taken and their core enzymatic properties were measured under the same conditions (the measurement method is the same as in Example 1). The results are shown in Table 2.

[0068] Table 2 Performance test results of lipase before and after modification

[0069] As shown in Table 2, after dual modification with PEG grafting and acetylation, the optimal pH range of lipase shifts towards alkalinity, and its alkali resistance, surfactant resistance and storage stability are significantly improved. At the same time, it retains high catalytic activity, which can meet the long-term storage and application requirements of weakly alkaline kitchen cleaning systems.

[0070] Example 3: Preparation of a kitchen heavy oil stain cleaner based on modified lipase In this embodiment, the lipase (LPS1) with the amino acid sequence shown in SEQ ID NO:1 and the modified lipase (LPS2) prepared in Example 2 were used in a kitchen heavy oil stain cleaner.

[0071] The formula for this heavy-duty kitchen degreaser is as follows: LPS1 or LPS2 0.2wt%, compound surfactant 6wt%, green cosolvent (acetone glyceryl acetate) 1wt%, compound enzyme stabilizer 2.5wt%, chelating agent (tetrasodium glutamate diacetate) 0.6wt%, pH buffer adjuster (triethanolamine) 1.0wt%, preservative (Kasone) 0.05wt%, plant-based soothing fragrance 0.3wt%, and deionized water as the balance. The compound surfactant is composed of cocamidopropyl betaine and APG1214 (alkyl chain C...). 12 ~C 14 The complex enzyme stabilizer is composed of alkyl glycosides with a degree of polymerization of 1.4 to 1.6 in a weight ratio of 12:1, and glycerol, sorbitol, glycine and betaine in a weight ratio of 4:3:2:1.

[0072] The preparation steps for a heavy-duty kitchen degreaser are as follows: S1. At room temperature, add 60% of the total weight of the formula of deionized water to the stirred reactor, turn on the stirrer, and control the speed at 300 r / min. S2. Add tetrasodium glutamate diacetate and triethanolamine in sequence, and stir continuously for 5 minutes until the materials are completely dissolved to obtain a uniform and transparent base solution. S3. Add the composite surfactant to the base solution in batches, stirring during the addition process. After the addition is complete, continue stirring for 10 minutes until the system is completely transparent, without layering or agglomerates. S4. Slowly add acetone glycerol while maintaining a constant stirring speed and continue stirring for 8 minutes to ensure the system is fully mixed. S5. Turn on the circulating cooling water to cool the system to 25°C, add the compound enzyme stabilizer, stir for 5 min until completely dissolved, then slowly and evenly sprinkle in LPS1 or LPS2, reduce the stirring speed to 200 r / min, and stir at low speed for 8 min until the enzyme preparation is completely dissolved and dispersed. S6. Finally, add Kathon preservative and plant-based soothing fragrance, stir for 3 minutes, add the remaining deionized water to 100% of the total weight, continue stirring for 10 minutes, then stop stirring, let stand to defoam for 30 minutes, and filter through a 200-mesh filter cloth to obtain the finished kitchen heavy oil stain cleaner.

[0073] Example 4: Comparison of the cleaning power of different kitchen heavy grease cleaners on aged heavy grease stains This embodiment refers to the washing method specified in Appendix A of GB / T 35833-2018 "Kitchen Oil Stain Cleaners" to test the removal efficacy of two kitchen heavy oil stain cleaners prepared in Example 3, a mainstream commercial strong alkaline kitchen heavy oil stain cleaner (formula composition: 3.5wt% NaOH, 6wt% fatty alcohol polyoxyethylene ether, the remainder being deionized water; the "mainstream commercial strong alkaline kitchen heavy oil stain cleaner" is the same in all embodiments) and a commercial ordinary lipase-type kitchen cleaner (lipase addition amount 0.3wt%, the surfactants used are AEO9 and APG0810; the "common commercial lipase-type kitchen cleaner" is the same in all embodiments) on simulated aged heavy oil stains in the kitchen. The specific test process is as follows: S1. Prepare a simulated kitchen aged oil stain according to the formula specified in Appendix A of GB / T 35833-2018: Weigh 64.0 g of refined soybean oil, 8.0 g of refined crude beef tallow, 8.0 g of refined crude lard, and 2.4 g of glyceryl monostearate into a plastic beaker and heat in a 70°C water bath until completely dissolved. After stirring and cooling to 30°C, add 8.0 g of ammonia-free caramel coloring and stir at a speed of not less than 1000 r / min for 30 min until emulsified evenly. Add 12.0 g of wheat flour and continue stirring for 10 min. After aging at room temperature for 24 h, store in a refrigerator and restore to 20°C~25°C before use.

[0074] S2. Preparation of test stains: S2.1. Wash the stainless steel test piece, dry it in an oven at 120℃±2℃ for 1 h, transfer it to a desiccator to cool for 30 min, weigh it, and record it as the blank mass of the test piece (m0), accurate to 0.0002 g.

[0075] S2.2 Apply the above-mentioned artificial dirt evenly to the specified area on one side of the test piece with a brush. The amount of dirt applied to each test piece should be controlled between 0.24 g and 0.26 g. After the application is completed, place the test piece on a clean ceramic plate and dry it in an oven at 200℃±2℃ for 10 min.

[0076] S2.3 After removing the test piece and cooling it for 20 minutes, transfer it to a UV constant temperature aging chamber and turn on the UV lamp for aging at 45℃±2℃. After irradiation for 1.5 hours, quickly change the position of the test piece diagonally and continue irradiation for another 1.5 hours. The total aging time is 3 hours.

[0077] S2.4. Take out the soiled tablets and place them in a petri dish. Place them in a desiccator to dry and age for 20 h to 22 h. Weigh the tablets before washing to obtain the mass (m1) of the soiled tablets, accurate to 0.001 g.

[0078] S3, Stain Removal: S3.1 Weigh 200 g of the cleaning agent sample to be tested and place it in a glass beaker. Preheat the beaker to 35℃±2℃ in a water bath. Prepare 6 sets of test systems in parallel for each sample.

[0079] S3.2. Place the prepared stained sample on the washing rack of the washing machine, keeping it vertical so that the stained surface is completely immersed in the sample solution. Let it stand and soak for 10 minutes, then wash it at a frequency of 40 times / min for 5 minutes.

[0080] S3.3. Remove the sample and transfer it to a beaker containing 210 mL of 35℃±2℃ clean water. Rinse for 30 seconds to complete the rinsing.

[0081] S3.4. Take out the test piece and place it in a tray. Place it in an oven at 120℃±2℃ and dry for 45 min. After cooling in a desiccator for 30 min, weigh it to obtain the mass (m2) of the dirty piece after washing, accurate to 0.001 g.

[0082] S4. Calculate the decontamination rate: The stain removal rate was calculated as (m1-m2) / (m1-m0)×100%. Each group of samples was tested in parallel three times, and the average value was taken. The results are shown in Table 3.

[0083] Table 3. Test results of the detergency performance of aged heavy oil stains

[0084] As shown in Table 3, the two kitchen heavy-duty oil stain cleaners of Example 3 of this invention have significantly higher cleaning power for aged heavy-duty oil stains than ordinary lipase-based kitchen cleaners on the market. Among them, the kitchen heavy-duty oil stain cleaner using LPS2 has a cleaning effect that reaches the level of mainstream strong alkaline kitchen heavy-duty oil stain cleaners on the market, and is better than the kitchen heavy-duty oil stain cleaner using LPS1. The above results prove that the lipase before and after modification in this invention has good compatibility with the weak alkaline kitchen cleaning system. When it is used in the kitchen heavy-duty oil stain cleaner, it can effectively cleave and polymerize aged heavy-duty oil stains in a mild and low-irritation formulation system without strong alkali, and its cleaning power is higher than that of ordinary lipase-based kitchen cleaners on the market. Moreover, the cleaning process does not require high-intensity scrubbing. Furthermore, after the lipase shown in SEQ ID NO:1 is modified by PEG grafting and acetylation as described in this invention, the compatibility between the lipase and the weak alkaline kitchen cleaning system can be further improved. The cleaning power of the kitchen heavy-duty oil stain cleaner formulated with it is comparable to that of traditional strong alkaline cleaners.

[0085] Example 5: Comparison of long-term storage stability of different heavy-duty kitchen degreasers This embodiment tests the long-term storage stability of two kitchen heavy oil stain cleaners prepared in Example 3 and a commercially available lipase-type kitchen cleaner against simulated aged heavy oil stains in the kitchen. The test method is as follows: The cleaner samples to be tested are sealed and packaged, and stored in a constant temperature and humidity environment of 25°C and 60% for 12 months. Samples are taken at 0, 3, 6 and 12 months of storage, and the lipase activity is determined by acid-base titration. The enzyme activity retention rate is calculated as enzyme activity after storage / initial enzyme activity × 100%. The results are shown in Table 4.

[0086] Table 4. Long-term storage stability test results

[0087] As shown in Table 4, the enzyme activity retention rates of the two heavy-duty kitchen degreasers in Example 3 of this invention during 12 months of storage at room temperature are higher than those of ordinary lipase-based kitchen cleaners on the market. Specifically, compared to LPS1, the heavy-duty kitchen degreaser using LPS2 exhibits a significantly higher enzyme activity retention rate during 12 months of storage at room temperature. These results demonstrate that the lipase shown in SEQ ID NO:1 has a certain tolerance to weakly alkaline kitchen cleaning systems containing surfactants. Furthermore, by performing the PEG grafting and acetylation dual modification of this lipase as described in this invention, its stability in the aforementioned kitchen cleaning systems can be further improved, thus more thoroughly solving the problem of lipase inactivation in weakly alkaline kitchen cleaning systems.

[0088] Example 6: Comparison of the corrosiveness of different heavy-duty kitchen cleaners to common kitchen materials This embodiment tested the corrosion of common kitchen materials by two types of heavy-duty kitchen degreasers prepared in Example 3, a mainstream strong alkaline heavy-duty kitchen degreaser, and a common lipase-based kitchen cleaner. The test method is as follows: three common kitchen materials, 304 stainless steel, aluminum alloy, and painted panels, were selected and completely immersed in the original solution of the cleaning agent to be tested. They were soaked at a constant temperature of 25°C for 72 hours. After being taken out, the surface appearance changes were observed, and the corrosion weight loss was tested. The results are shown in Table 5.

[0089] Table 5 Test results of corrosion resistance of common kitchen materials

[0090] As shown in Table 5, compared with strong alkaline kitchen heavy oil stain cleaners, the two kitchen heavy oil stain cleaners in Example 3 of this invention have significantly reduced corrosiveness to various commonly used kitchen materials and have better material compatibility. Therefore, they are safer when used for cleaning various surfaces such as stoves, range hoods, stainless steel kitchenware, and painted cabinets.

[0091] Example 7: Comparison of skin irritation of heavy-duty kitchen degreasers Referring to the acute skin irritation test method in the "Cosmetic Safety Technical Specifications" (2022 edition), six healthy adult rabbits were selected. 0.5 mL of the test substance was applied to the shaved skin area on the back of each rabbit, while physiological saline was applied to the other side as a blank control. The area was fixed with a dressing. After 4 hours, the test substance was removed, and skin erythema and edema were observed and scored at 24 hours, 48 ​​hours, and 72 hours. The results showed that the two kitchen heavy-duty oil stain cleaner samples in Example 3 showed no erythema or edema at any observation time point, with skin irritation scores of 0, indicating non-irritation. The mainstream commercially available strong alkaline kitchen heavy-duty oil stain cleaner samples showed mild erythema, with an average irritation score of 2.3, indicating mild irritation. This demonstrates that the kitchen heavy-duty oil stain cleaner of the present invention is mild and low-irritant, with higher safety in use.

[0092] Example 8: Comparison of the antibacterial performance of heavy-duty kitchen degreasers This embodiment refers to the evaluation method for instant antibacterial hard surface cleaners in Appendix C of T / CHCIA 032-2024 "General Technical Requirements for Antibacterial Detergents". The antibacterial performance of the two kitchen heavy-duty oil stain cleaners prepared in Example 3, mainstream commercially available strong alkaline kitchen heavy-duty oil stain cleaners, and ordinary commercially available lipase-based kitchen cleaners were tested. The specific test methods are as follows: (1) The test strains were Staphylococcus aureus (ATCC 6538) and Escherichia coli (ATCC 25922), both of which were commercially available standard preserved strains. The test medium was a 304 stainless steel sheet (50 mm × 50 mm) to simulate the hard surface material of a kitchen. The main reagents were: SCDLP neutralization medium, 0.03 mol / L phosphate buffer (PBS, pH 7.2), 342 mg / L standard hard water, and nutrient agar medium.

[0093] (2) Prepare a solution with a concentration of approximately 6.0 × 10⁻⁶ using standard methods. 5 A working bacterial suspension of CFU / mL was prepared; 0.4 mL of the bacterial suspension was dropped onto a sterile stainless steel sheet and allowed to air dry to prepare a contaminated carrier. 0.4 mL of the original cleaning agent sample was dropped onto the contaminated carrier, covered with a sterile film to ensure uniform contact, and incubated at 25±1℃ for 5 min. After rinsing with standard hard water to simulate the cleaning process, 10 mL of SCDLP neutralization medium was added to elute and recover residual viable bacteria. The eluent was serially diluted 10-fold, and appropriate dilutions were selected for pouring onto nutrient agar plates. After incubation at 36±1℃ for 48 h, colonies were counted. A positive control group was set up using sterile hard water instead of the sample, and the same procedure was followed.

[0094] (3) The sterilization rate was calculated by (average colony count of control group - average colony count of test group) / average colony count of control group × 100%. Each group was measured in parallel 3 times and the arithmetic mean was taken. The results are shown in Table 6.

[0095] Table 6. Results of sterilization performance test

[0096] As shown in Table 6, compared with strong alkaline kitchen heavy-duty oil stain cleaners, the two kitchen heavy-duty oil stain cleaners in Example 3 of this invention have better antibacterial effects. Among them, compared with LPS1, the kitchen heavy-duty oil stain cleaner using LPS2 has further improved antibacterial performance, reaching a level comparable to mainstream strong alkaline kitchen heavy-duty oil stain cleaners on the market. The above results prove that when the modified lipases of this invention are used in kitchen heavy-duty oil stain cleaners, efficient and broad-spectrum antibacterial effects can be achieved in a mild and low-irritation formulation system without strong alkali, effectively reducing the risk of microbial residues on hard surfaces in the kitchen, balancing cleaning ability and hygiene safety, and meeting the cleaning needs of households and restaurant kitchens; furthermore, after the lipase shown in SEQ ID NO:1 is modified by PEG grafting and acetylation as described in this invention, the antibacterial performance of the kitchen heavy-duty oil stain cleaner can be further improved.

[0097] Example 9: Acute oral toxicity test of a heavy-duty kitchen degreaser To evaluate the safety of the cleaning agents of this invention, this embodiment refers to GB 15193.3-2014 "National Food Safety Standard Acute Oral Toxicity Test" and uses the single maximum limit test method to conduct an acute oral toxicity evaluation on the two heavy-duty kitchen cleaners prepared in Example 3. The specific test method is as follows: (1) The experimental animals were SPF-grade ICR mice, half male and half female, with females being non-pregnant and having no history of reproduction; the weight range of the animals was 18.0~22.0g, and the weight difference between the same sex did not exceed ±20% of the mean; the animals were housed in a barrier environment with an ambient temperature of 21~23℃ and a relative humidity of 40%~60%, with free access to food and water, and were acclimatized for 5 days before the experiment. The cleaning agent samples to be tested were prepared into a homogeneous suspension using sterile water for injection as the solvent, and were prepared and used immediately.

[0098] (2) Animals were fasted for 4 hours before the experiment, but water was allowed. Mice were given a single dose of 5000 mg / kg body weight via gavage at a volume of 0.2 mL / 10 g body weight. After exposure, animals were fasted for another hour. The animals' physical appearance, behavior, poisoning symptoms, and mortality were observed and recorded daily. They were weighed once a week for 14 consecutive days. After the observation period, all surviving animals were euthanized and subjected to gross anatomical examination. The morphological changes of the major organs were recorded.

[0099] Experimental results showed that during the observation period, none of the tested mice exhibited symptoms of poisoning, and no animals died; the weight gain of both male and female mice was normal, with no abnormal fluctuations; gross anatomical examination at the end of the observation period revealed no significant pathological changes in major organs, including the heart, liver, spleen, lungs, and kidneys. The acute oral LD50 of the two tested samples in ICR mice was calculated to be... 50 >5000 mg / kg body weight. Based on the acute toxicity classification standard, the acute oral LD50 of the cleaning agent of this invention is... 50 >5000 mg / kg, which is practically non-toxic, indicates that the product is highly safe for oral contact. Even if a small amount of accidental contact occurs in a household setting, there will be no risk of acute toxicity, and it meets the safety requirements for kitchen cleaning products.

Claims

1. A lipase resistant to surfactants and metal ions, characterized in that, The amino acid sequence of the lipase is shown in SEQ ID NO:

1.

2. The lipase according to claim 1, characterized in that, The lipase, based on the amino acid sequence shown in SEQ ID NO:1, includes the following chemical modifications: some surface amino groups are modified by polyethylene glycol segment grafting, and some surface amino groups are modified by acetylation.

3. The lipase according to claim 1, characterized in that, The modifier used for the polyethylene glycol segment grafting modification is polyethylene glycol-N-hydroxysuccinimide ester; the modifier used for the acetylation modification is acetic anhydride.

4. The lipase according to claim 3, characterized in that, The polyethylene glycol-N-hydroxysuccinimide ester is a methoxy polyethylene glycol-N-hydroxysuccinimide ester, wherein the number average molecular weight of the polyethylene glycol segments is 4000~6000 Da.

5. A method for preparing the lipase according to claim 3 or 4, characterized in that, The steps include: Polyethylene glycol-N-hydroxysuccinimide ester was added to the enzyme solution containing unmodified lipase to carry out a grafting reaction. After terminating the reaction, acetic anhydride was added to carry out an acetylation reaction, and the product was separated and purified. The amino acid sequence of the unmodified lipase is shown in SEQ ID NO:

1.

6. The preparation method according to claim 5, characterized in that, The mass ratio of the unmodified lipase, polyethylene glycol-N-hydroxysuccinimide ester, and acetic anhydride is 1:1~5:0.1~1.0; before adding polyethylene glycol-N-hydroxysuccinimide ester, the enzyme solution is adjusted to pH 8.3~8.7; the grafting reaction is carried out at a temperature of 20~30℃ for 1.5~2.5 h; after terminating the reaction, the pH is adjusted to 7.8~8.2 before adding acetic anhydride; during the addition of acetic anhydride, the pH is maintained at 7.8~8.2; the acetylation reaction is carried out at a temperature of 20~30℃ for 1~2 h.

7. The application of the lipase according to any one of claims 1 to 4 in an oil stain cleaner, characterized in that, The oil stain cleaner has a pH of 7.5 to 10.0 and contains the lipase and surfactant.

8. The application according to claim 7, characterized in that, The oil stain cleaner is a heavy-duty kitchen oil stain cleaner; the oil stain cleaner comprises the following components in parts by weight: lipase 0.2-0.8 parts, surfactant 6-10 parts, solubilizer 1-3 parts, enzyme stabilizer 2.5-6 parts, chelating agent 0.3-1.0 parts, pH buffer adjuster 0.4-1.2 parts, preservative 0-0.2 parts, fragrance 0-0.4 parts, and solvent 70-90 parts.

9. The application according to claim 8, characterized in that, The surfactants include amphoteric surfactants and / or nonionic surfactants, the amphoteric surfactants being alkyl glycosides and / or fatty alcohol polyoxyethylene ethers, and the nonionic surfactants including cocamidopropyl betaine; the enzyme stabilizers include one or more of glycerol, sorbitol, glycine, and betaine.

10. An oil stain cleaner, characterized in that, The oil stain cleaner has a pH of 7.5 to 10.0 and contains a surfactant and a lipase according to any one of claims 1 to 4.

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