Method for preparing lysophospholipid through catalytic hydrolysis of immobilized phospholipase LUat-ADS-17

Abstract

The invention belongs to the technical field of immobilized enzyme biological catalysis, and constructs efficient and stable LU (at) ADS-17 immobilized phospholipase and a method for preparing lysophosphatide by using the LU (at) ADS-17 immobilized phospholipase in catalytic hydrolysis of soybean phospholipids. Phospholipase LU is immobilized on macroporous resin ADS-17 through physical adsorption and chemical adsorption, and the prepared LUatADS-17 is good in stability, has high catalytic activity, can promote a hydrolysis reaction to reach balance within a short time, and can effectively prevent acyl migration. And finally, the reinforced cross-linked immobilized phospholipase is further obtained through the cross-linking agent, so that the problems that the traditional adsorption immobilized phospholipase is easy to fall off and the operation stability is insufficient are solved, and the reusability of the immobilized phospholipase is remarkably improved. The method is helpful for preparing lysophospholipid from soybean phospholipid, can reduce the production cost and improve the yield, and has a good application prospect.

Description

Technical Field

[0001] The present invention relates to the technical field of immobilized enzyme biocatalysis, and in particular to a method for immobilizing LU enzyme by using macroporous resin ADS-17 and applying the immobilized phospholipase to catalytic hydrolysis to prepare lysophospholipid. Background Art

[0002] Phospholipase Lecitase® Ultra (LU) is a phospholipase produced by genetically modified Aspergillus oryzae that specifically hydrolyzes phospholipids to produce α-lysophospholipids and free fatty acids. Despite its widespread industrial application, LU is currently only available in its free form, which has limitations such as poor stability, difficulty in recovery and reuse, and increased production costs. Common methods for modifying phospholipids include physical, chemical, and enzymatic methods. Enzymatic phospholipid modification offers advantages such as mild reaction, high efficiency, strong specificity, no disruption of the natural phospholipid structure, few byproducts, and high safety. Currently, the most commonly used enzymes for phospholipid modification are highly specific phospholipases, including phospholipase A1 and phospholipase A2. Phospholipases catalyze the hydrolysis of phospholipids and, in the presence of certain acyl group acceptors and donors, catalyze esterification and transesterification reactions, thereby altering or modifying the structure of phospholipids to produce phospholipids with diverse structures and applications. Although free phospholipases A1 and A2 are biocatalysts with high catalytic activity and high specificity, they have the following disadvantages: (1) they are less stable and prone to aggregation, which affects catalytic efficiency; (2) they have poor tolerance to high temperatures, strong acids, strong bases, and organic solvents, and their stereochemical structure is easily changed; (3) they are difficult to recycle and reuse, and are prone to contaminating products, resulting in increased production costs and being unfavorable for industrial applications.

[0003] To solve these problems, immobilized enzyme technology is often used to fix free enzymes to various solid materials to form immobilized enzymes. The methods for immobilizing enzymes mainly include physical methods (adsorption and embedding) and chemical methods (cross-linking). Enzyme immobilization technology is used to improve the stability and reusability of enzymes. By combining the enzyme with a solid support, the immobilized enzyme is not only easy to separate, but also maintains activity in multiple cycles, making it suitable for continuous and automated production.

[0004] Chinese patent publication number CN108486179B discloses a method for the continuous catalytic production of phosphatidic acid using immobilized phospholipase D. The method uses a macroporous adsorption resin selected from LX-1000, XAD761, SP207, or ESR1 to adsorb the phospholipase D. The resin provides a porous network structure, and the immobilization process is simple to operate under mild conditions. However, there is no interaction between the support and the enzyme, and the enzyme is easily detached during application.

[0005] Chinese patent publication number CN110760504B discloses a co-crosslinking immobilization method for phospholipase A1. The method uses oil-soluble oxazine diacrylate as a crosslinking agent. The reactants in the aqueous phase are phospholipase A1 containing an amino group and a supramolecular complex formed by an aminated epoxy resin and β-cyclodextrin. The Michael addition reaction between the double bond and the amino group is used to cause a co-crosslinking polymerization reaction at a relatively low temperature to prepare immobilized phospholipase A1 with different loading amounts. This method improves the catalytic activity and storage stability of the immobilized enzyme, but does not involve repeated use of the phospholipase after immobilization to maintain a high conversion rate.

[0006] Therefore, finding an ideal support for enzyme immobilization that can form a protective microenvironment around the enzyme to improve its tolerance and thermodynamic stability, while also preventing it from falling off after immobilization and allowing for easy separation and recovery from the product for reuse remains a major technical challenge for immobilizing phospholipase. Summary of the Invention

[0007] To address the above-mentioned issues, the present invention aims to propose a method for preparing an immobilized phospholipase, thereby resolving one or more technical problems existing in the prior art and at least providing a beneficial alternative or creating conditions. The present invention aims to provide a method for immobilizing phospholipase LU using the macroporous resin ADS-17. To address the issues of poor stability and difficulty in recycling of free phospholipase (LU) in industrial applications, the present invention immobilizes the macroporous resin and phospholipase through adsorption to construct a highly efficient and stable LU@ADS-17 immobilized enzyme system. The LU@ADS-17 is further strengthened through cross-linking, aiming to address bottlenecks such as easy shedding, insufficient operational stability, low activity, and low reusability of traditional adsorbed immobilized enzymes. This method provides theoretical support and technical solutions for the development of industrial-grade biocatalysts. Finally, the system is used to catalyze the hydrolysis of soybean lecithin to produce lysophospholipids, and the catalytic process and performance are systematically optimized to improve catalytic activity and reusability.

[0008] The purpose of the present invention is achieved through the following technical solutions: A method for preparing an immobilized enzyme using a macroporous resin ADS-17 and a phospholipase LU, and applying the immobilized enzyme to catalytic hydrolysis to prepare lysophospholipids. The method for preparing lysophospholipids using immobilized phospholipase LU@ADS-17 for catalytic hydrolysis is to use the macroporous resin ADS-17 and the LU enzyme to prepare an immobilized phospholipase, and apply it to catalytic hydrolysis to generate lysophospholipids. The method comprises the following steps: soaking the functionalized macroporous resin ADS-17 in an LU enzyme solution, shaking on a shaker, and immobilizing the LU enzyme on the macroporous resin ADS-17 by physical adsorption and chemical adsorption to prepare immobilized phospholipase LU@ADS-17. Then, soybean lecithin is mixed with deionized water for homogeneity, and then the immobilized phospholipase is added. The product is obtained after stirring the reaction. Specifically, the following steps are included: S01, activation of macroporous resin ADS-17: Place macroporous resin ADS-17 in a beaker, add 95% ethanol, seal and avoid light and soak for 24 hours. Subsequently, use a circulating water vacuum pump to filter and wash with ultrapure water several times until there is no ethanol smell. Transfer the resin to a new beaker, add 5% hydrochloric acid solution, seal and avoid light and soak for 4 hours. After filtering the hydrochloric acid solution, wash repeatedly with ultrapure water until the pH of the filtrate is adjusted to 7.0. Then transfer the resin to a beaker, add 5% sodium hydroxide solution, and continue soaking for 4 hours. After filtration, wash with ultrapure water several times until the pH returns to 7.0. Finally, add pH 7.0 phosphate buffer solution, soak for 2 hours, filter, and wash with phosphate buffer solution until the pH of the filtrate remains at 7.0.

[0009] S02, functionalization of macroporous resin ADS-17: the surface of the macroporous resin is subjected to a self-polymerization reaction of dopamine and then grafted with cellulose acetate to obtain a functionalized macroporous resin ADS-17; The activated macroporous resin was added to an ethanol-water solution (volume ratio of 1:1), mixed thoroughly, and then dopamine hydrochloride was added and stirred. Then, 20 mmol / L tris (hydroxymethyl)aminomethane) buffer was added dropwise, the pH was adjusted to 8.5, and the mixture was stirred for 60-90 minutes. Cellulose acetate was added, followed by ammonium persulfate, and the mixture was stirred at 50°C for 30-40 minutes. The mixture was then filtered and washed to obtain a polydopamine-surface-modified macroporous resin grafted with cellulose acetate, namely, the functionalized macroporous resin ADS-17. The mass ratio of dopamine hydrochloride to macroporous resin is 1:5-8; the mass ratio of cellulose acetate to dopamine hydrochloride is 1:5, and the amount of ammonium persulfate is 0.1-0.5% of the mass of dopamine hydrochloride; In an alkaline environment, dopamine undergoes self-polymerization on the surface of a macroporous resin to form polydopamine particles. These particles possess multiple reactive groups that act as crosslinkers, forming non-covalent interactions with cellulose acetate (π-π stacking and hydrogen bonding). The dynamic interactions between the polydopamine particles and the macromolecular chains create dynamic bonds that exhibit multiple synergistic and reversible effects. The large surface area and porous structure of the macroporous resin provide abundant attachment sites for dopamine, allowing for self-polymerization. Polydopamine attached to the macroporous resin possesses numerous functional groups, such as amino and phenolic hydroxyl groups, which can bind to phospholipase molecules through covalent bonding, physical adsorption, or chemical adsorption, thereby immobilizing the phospholipase. Cellulose acetate grafted onto the polydopamine improves the hydrophilicity and surface polarity of the macroporous resin, making it more conducive to subsequent phospholipase immobilization, substrate adsorption, and catalysis. S03, LU enzyme immobilization: LU enzyme was immobilized on the functionalized macroporous resin ADS-17 by physical adsorption and chemical adsorption to prepare immobilized phospholipase LU@ADS-17; Mix the LU enzyme solution with an appropriate amount of phosphate buffer (pH 4-8) to prepare an enzyme phosphate buffer solution. Place the macroporous resin in a conical flask and add the enzyme phosphate buffer solution. After shaking and adsorbing on a 25°C constant temperature shaker for 4 hours, the LU enzyme is immobilized on the macroporous resin ADS-17. Filter using a circulating water vacuum pump to obtain immobilized phospholipase LU@ADS-17. Dry the solution in a 30°C vacuum oven for 6 hours, collect it, seal it, and store it in a refrigerator at 4°C.

[0010] S04, catalytic hydrolysis: after homogenizing soybean lecithin with water, add immobilized phospholipase, and then stir in a water bath at 30-70°C for 0.5-48 hours to obtain the product. The product mainly contains lysophosphatidylethanolamine PE, lysophosphatidylinositol PI and lysophosphatidylcholine PC; The amount of soybean lecithin and water used is closely related to the lysophospholipid to be prepared. Lysophospholipid is prepared by using a soybean lecithin and water concentration of 0.5-12 wt %. Glycolysophospholipid is prepared by changing the substrate concentration and the reaction temperature.

[0011] In the preparation process of the immobilized phospholipase, the preferred enzyme concentration is 20-60 mg / mL, and the more preferred enzyme concentration is 40 mg / mL; In the preparation process of the immobilized phospholipase, 50 ml of enzyme phosphate buffer is added to every 0.5 g of macroporous resin.

[0012] The reaction temperature is preferably 30-70°C, more preferably 50°C.

[0013] The reaction time is preferably 0.5 to 48 hours, more preferably 0.5 hour.

[0014] The stirring speed is preferably 200 to 400 rpm, more preferably 300 rpm.

[0015] We found that by preparing immobilized phospholipase, the stability of LU was improved to a certain extent, but there was only a weak force between the enzyme and the carrier fixed by the adsorption method, so the enzyme was easy to lose and the stability was general, which hindered the reuse of the material in the application. Cross-linking is a common immobilization method. The enzyme molecules are fixed on the surface of the carrier or form a cross-linked network structure through chemical cross-linking agents, which can improve the stability, reusability and catalytic efficiency of the enzyme. The cross-linking method still faces some challenges in practical applications: excessive cross-linking may lead to an increase in the steric hindrance effect of the enzyme active center, while insufficient cross-linking makes it difficult to maintain the structural stability of the enzyme cluster. The effect of enzyme immobilization by cross-linking is affected by many factors, including the type and concentration of the cross-linking agent, reaction time and temperature. Therefore, in order to obtain efficient and stable immobilized enzymes and to further improve the stability of immobilized LU, polyethylene glycol diglycidyl ether was creatively introduced as a cross-linking agent to fix LU by cross-linking. The specific steps are as follows: The LU enzyme solution was mixed with an appropriate amount of phosphate buffer solution (pH = 6, 25mM) to prepare an enzyme phosphate buffer solution. After the adsorption reaction was completed, a cross-linking agent was immediately added. After shaking on a constant temperature shaker to fully cross-link, the solution was filtered using a circulating water vacuum pump and washed three times with a pH 6.0 phosphate buffer solution to obtain a cross-linked immobilized phospholipase. The solution was placed in a vacuum drying oven at 30°C for 6 hours, collected, sealed, and stored in a refrigerator at 4°C for later use. The cross-linking agent is polyethylene glycol diglycidyl ether, the cross-linking temperature is 40°C, the amount-to-volume ratio of the cross-linking agent is 0.005-0.02, and the cross-linking time is 8-10 hours; The immobilized enzyme LU@ADS-17, obtained through a dual crosslinking and macroporous resin adsorption process, exhibited significant recycling advantages: after five cycles of reuse, the PE and PC conversion rates dropped to 78.87% and 75.17% of the initial conversion rates, respectively, representing 48% to 49.18% increases in activity compared to the uncrosslinked system (LU@ADS-17) over the same period. After 10 cycles of reuse, the PE and phosphatidylcholine PC conversion rates remained at 57.73% and 51.75%, respectively.

[0016] Through multiple reversible interactions and irreversible covalent bond-mediated interactions, a uniform, moderately cross-linked hierarchical porous network is constructed. The synergistic effect of polydopamine, the highly entangled dual network structure, and the bridging cross-linking effect of cellulose acetate collectively enhance the loading rate of the macroporous resin and the stability of the immobilized enzyme. More importantly, the ethylene glycol diglycidyl ether molecule allows the immobilized lipase to be removed from the surface of the functionalized macroporous resin particles to a certain extent, effectively reducing steric hindrance and providing sufficient space to stretch the three-dimensional enzyme structure. This allows for high loading capacity while also offering advantages such as high activity recovery and reusability.

[0017] This may be because when the amount of cross-linking agent is excessive, the cross-links between enzymes become too tight, resulting in a significant steric hindrance during the enzymatic reaction. Prior to cross-linking and immobilizing the enzyme, the anionic long-chain cellulose acetate and diepoxide cross-linking agent are first adsorbed onto the surface of the polydopamine macroporous resin through electrostatic interactions. Phospholipase is then immobilized through various methods, including physical adsorption, covalent cross-linking, and chemical cross-linking. The immobilized phospholipase obtained in this manner exhibits high activity and stability during the catalytic process. Not only does it exhibit better thermal stability and pH tolerance than free phospholipase, but it also significantly improves storage stability and reusability.

[0018] Compared with the prior art, the beneficial effects of this invention are: 1. The present invention uses macroporous resin ADS-17 and phospholipase LU as raw materials to prepare immobilized phospholipase. Both raw materials are abundant and have good selection specificity. By self-polymerizing the macroporous resin with dopamine and then grafting the surface treatment of cellulose acetate, the phospholipase is immobilized by physical adsorption and chemical adsorption, constructing an efficient and stable LU@ADS-17 immobilized enzyme system. LU@ADS-17 is further strengthened by cross-linking, aiming to solve the bottleneck problems of traditional adsorbed immobilized enzymes such as easy shedding, insufficient operational stability, low activity and low reuse rate, providing theoretical support and technical solutions for the development of industrial-grade biocatalysts. Finally, it is used to catalyze the hydrolysis of soybean lecithin to prepare hemolyzed lecithin, and its catalytic process and performance are systematically optimized to improve catalytic activity and reusability.

[0019] 2. In the LU@ADS-17 immobilized enzyme system, this invention innovatively introduces polyethylene glycol diglycidyl ether as a crosslinking agent to immobilize LU through crosslinking. The crosslinking temperature is 40°C, the crosslinker volume ratio is 0.005-0.02, and the crosslinking time is 8-10 hours. The enzymatic activity of the crosslinked immobilized enzyme reaches 844.3 U / g. After five cycles, the PE and PC conversion rates of the crosslinked immobilized LU remain at 78.87% and 75.17%, respectively, representing 48-49.18% improvements compared to the uncrosslinked system (53.29% and 50.39%). Furthermore, the reusability of the crosslinked immobilized LU is significantly improved, with PE and PC still maintaining their initial conversion rates of 57.73% and 51.75%, respectively, after 10 cycles.

[0020] 3. Traditional cross-linking methods often require enzyme molecules to precipitate and aggregate before cross-linking, which not only causes enzyme waste, but also blocks mass transfer channels, making it impossible to fully utilize the catalytic efficiency of the enzyme. The present invention provides a co-cross-linking method for the immobilization of phospholipase, which utilizes the amino groups on the phospholipase molecules to react with the functional groups on the macroporous resin. At the same time, cellulose acetate and diepoxy cross-linking agents containing long anionic chains are introduced to remove the immobilized lipase from the surface of the functionalized macroporous resin particles to a certain extent, thereby effectively reducing steric hindrance and providing sufficient space to stretch the three-dimensional enzyme structure, so that it has high activity recovery rate and reusability on the basis of high loading capacity. This can provide space for catalytic reaction, reduce mass transfer resistance, and increase hydrophilicity and improve enzyme activity. Using this co-cross-linking method, the enzyme loading capacity and catalytic activity are high, the stability is good, the immobilized enzyme is granular, and the catalytic reaction is easy to operate.

[0021] 4. The prepared immobilized phospholipase has good catalytic activity and reusability when applied to the preparation of lysophospholipids. Soybean lecithin is used as raw material for preparing lysophospholipids. The raw material source is abundant and inexpensive. By converting soybean lecithin into lysophospholipids, the conversion rate of phospholipids can be effectively improved by changing the reaction conditions, and the content of lysophospholipids in the reaction product is high. The present invention has the effects of increasing the lysophospholipid rate and reducing production costs, improving the catalyst reuse rate in the continuous production process, and reducing industrial production costs. The application of this technology not only provides a solution for improving the utilization value of phospholipid resources, but also promotes the development of green biomanufacturing technology. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 , morphology and structure diagrams of ADS-17, functionalized ADS-17, and LU@ADS-17; Figure 2 , compared with the SEM-EDS images of the changes in elemental composition observed after the ADS-17 matrix LU was fixed on the macroporous resin; Figure 3 , pH stability change diagram of free enzyme and immobilized phospholipase prepared in Example 1; Figure 4 , thermal stability changes of free enzyme and immobilized phospholipase prepared in Example 1 in phosphate buffer (25 mM, pH 7.0); Figure 5 , the thermal stability change diagram of the free enzyme and the immobilized phospholipase prepared in Example 1 in air; Figure 6 , a graph showing the thermal stability change of the immobilized phospholipase prepared in Example 1 in a n-hexane system; Figure 7 , a graph showing the storage thermal stability change of the immobilized phospholipase prepared in Example 1; Figure 8 , histogram of structural stability of immobilized phospholipase enhanced by different cross-linking agents and changes in enzyme activity; Figure 9 , the reusability activity change diagram of LU@ADS-17 catalytic hydrolysis to prepare lysophospholipid in Example 1; Figure 10 , the reusability activity change diagram of the cross-linked immobilized LU@ADS-17 catalytic hydrolysis to prepare lysophospholipids in Example 6; DETAILED DESCRIPTION Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiments are only used to illustrate the present invention and are not used in limiting the scope of the present invention.In addition, should be understood that after reading the content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms fall equally within the scope limited by the appended claims of the application. The phospholipid conversion rate, phospholipid and lysophospholipid contents described in the examples were calculated using the area normalization method.

[0023] Example 1 A method for preparing lysophospholipids by catalytic hydrolysis using immobilized phospholipase LU@ADS-17, first preparing a functionalized macroporous resin ADS-17, specifically: placing 100g of macroporous resin ADS-17 in a beaker, adding 300ml of 95% ethanol, sealing and dark-proofing the beaker and soaking for 24 hours; then, using a circulating water vacuum pump to filter, and washing with ultrapure water multiple times until there is no ethanol smell; transferring the resin to a new beaker, adding 300ml of 5% hydrochloric acid solution, sealing and dark-proofing the beaker and soaking for 4 hours; after filtering the hydrochloric acid solution, repeatedly washing with ultrapure water until the pH of the filtrate is adjusted to 7.0; then transferring the resin to a beaker, adding 300ml of 5% sodium hydroxide solution, and continuing to soak for 4 hours; after filtering, washing with ultrapure water multiple times until the pH returns to 7.0; finally, adding 400ml of phosphate buffer solution with a pH of 7.0, soaking for 2 hours, filtering, and washing with phosphate buffer solution until the pH of the filtrate remains at 7.0, to obtain an activated macroporous resin; 100 g of activated macroporous resin was added to 500 mL of ethanol aqueous solution (volume ratio of 1:1), mixed evenly, and then 20 g of dopamine hydrochloride was added and stirred; then, 100 mL of 20 mmol / L tris(hydroxymethyl)aminomethane buffer was added dropwise, the pH was adjusted to 8.5 and stirred for 90 minutes, 4 g of cellulose acetate was added, and then 0.05 g of ammonium persulfate was added dropwise and stirred at 50°C for 40 minutes, filtered and washed to obtain a polydopamine surface-modified macroporous resin grafted with cellulose acetate, namely, the functionalized macroporous resin ADS-17.

[0024] Furthermore, the LU enzyme was immobilized as follows: a 40 mg / mL phospholipase solution was selected and mixed with a phosphate buffer solution (pH 4-8) to prepare an enzyme phosphate buffer solution. 50 ml of enzyme phosphate buffer was added for every 0.5 g of functionalized macroporous resin. 5 g of functionalized macroporous resin was placed in a conical flask, and 500 ml of enzyme buffer solution was added. After shaking and adsorbing on a 25°C constant temperature shaker for 4 hours, the LU enzyme was immobilized on the macroporous resin ADS-17 by physical adsorption. The solution was then filtered using a circulating water vacuum pump to obtain immobilized phospholipase LU@ADS-17. The solution was then dried in a vacuum oven at 30°C for 6 hours, collected, sealed, and stored in a refrigerator at 4°C.

[0025] Finally, catalytic hydrolysis was performed as follows: 0.9 g of soybean lecithin and 29.1 g of deionized water were placed in a single-necked round-bottom flask and homogenized for 10 minutes. 0.05 g of immobilized phospholipase was then added. The mixture was stirred in a water bath at 50°C and 300 rpm for 2 hours, and the reaction product was then removed. The composition of the product was analyzed using HPLC-ELSD. The conversion rates of phospholipids PE, PI, and PC in the product obtained in this example were 84.77%, 29.83%, and 83.64%, respectively.

[0026] Example 2: 3.6 g of soybean lecithin and 26.4 g of deionized water were placed in a single-necked round-bottom flask and homogenized for 10 minutes. 0.1 g of the immobilized phospholipase prepared in Example 1 was added thereto. The mixture was stirred in a water bath at 50° C. for 2 hours, and the reaction product was then taken out.

[0027] The composition of the product was analyzed by HPLC-ELSD. The conversion rates of phospholipids PE, PI, and PC in the product obtained in this example were 74.54%, 87.12%, and 57.93%, respectively.

[0028] Example 3: 0.3 g soybean lecithin and 29.7 g deionized water were placed in a single-necked round-bottom flask and homogenized for 10 minutes before adding 0.1 g immobilized phospholipase prepared in Example 1, 40 μg Ca 2+ After stirring in a water bath at 50°C for 0.5 hours, the reaction product was taken out.

[0029] The composition of the product was analyzed by HPLC-ELSD. The conversion rates of phospholipids PE, PI, and PC in the product obtained in this example were 63.45%, 71.80%, and 87.18%, respectively.

[0030] Example 4: 0.45 g soybean lecithin and 29.55 g deionized water were placed in a single-necked round-bottom flask and homogenized for 10 minutes before adding 0.05 g immobilized phospholipase prepared in Example 1 and 60 μg Ca 2+ After stirring and reacting in a water bath at 40°C for 0.5 hours, the reaction product was taken out.

[0031] The composition of the product was analyzed by HPLC-ELSD. The conversion rates of phospholipids PE, PI and PC in the product obtained in this example were 86.28%, 72.72% and 79.01% respectively. The results showed that the addition of a small amount of Ca 2+ Can improve conversion rate.

[0032] Example 5: 0.45 g soybean lecithin and 29.55 g deionized water were placed in a single-necked round-bottom flask and homogenized for 10 minutes before adding 0.05 g immobilized phospholipase prepared in Example 1 and 60 μg Ca 2+ After stirring in a water bath at 40°C for 0.5 hours, remove the reaction product. Filter to remove the enzyme and place in a refrigerator at 4°C for later use. Add the collected immobilized phospholipase to the reaction system under the same reaction conditions. After the reaction is complete, repeat the above steps.

[0033] The composition of the product was analyzed by HPLC-ELSD. The conversion rates of the phospholipids PE, PI, and PC obtained in this example still maintained relative activities of 53.30%, 97.88%, and 50.39% after five repetitions.

[0034] Example 6: A method for preparing lysophospholipids by catalytic hydrolysis using immobilized phospholipase LU@ADS-17, first preparing a functionalized macroporous resin ADS-17, specifically: placing 100g of macroporous resin ADS-17 in a beaker, adding 300ml of 95% ethanol, sealing and dark-proofing and soaking for 24 hours; then, using a circulating water vacuum pump to filter, and washing with ultrapure water multiple times until there is no ethanol smell; transferring the resin to a new beaker, adding 300ml of 5% hydrochloric acid solution, sealing and dark-proofing and soaking for 4 hours; after filtering the hydrochloric acid solution, repeatedly washing with ultrapure water until the pH of the filtrate is adjusted to 7.0; then transferring the resin to a beaker, adding 300ml of 5% sodium hydroxide solution, and continuing to soak for 4 hours; after filtering, washing with ultrapure water multiple times until the pH returns to 7.0; finally, adding 400ml of phosphate buffer solution with a pH of 7.0, soaking for 2 hours, filtering, and washing with phosphate buffer solution until the pH of the filtrate remains at 7.0, to obtain an activated macroporous resin; 100g of activated macroporous resin was added to 500mL of ethanol-water solution (1:1 volume ratio). After mixing thoroughly, 12.5g of dopamine hydrochloride was added and stirred. Then, 100mL of 20mmol / L Tris(hydroxymethyl)aminomethane buffer was added dropwise, the pH was adjusted to 8.5, and the mixture was stirred for 60 minutes. 2.5g of cellulose acetate was added, followed by 0.02g of ammonium persulfate. The mixture was stirred at 50°C for 30 minutes, filtered, and washed to obtain a polydopamine-surface-modified macroporous resin grafted with cellulose acetate, namely, the functionalized macroporous resin ADS-17. Furthermore, the LU enzyme was immobilized as follows: a 40 mg / mL phospholipase solution was prepared, mixed with phosphate buffer (pH 4-8) to prepare an enzyme phosphate buffer solution. 50 ml of enzyme phosphate buffer was added for every 0.5 g of functionalized macroporous resin. 5 g of functionalized macroporous resin was placed in a conical flask, and 500 ml of enzyme buffer solution was added. After adsorption on a 25°C constant temperature shaker for 4 hours, 1% (v / v) polyethylene glycol diglycidyl ether was immediately added. Cross-linking was carried out on a 40°C constant temperature shaker for 8 hours. After completion of the reaction, the system was filtered and washed with PBS (pH 6.0) to obtain the immobilized enzyme. The system was dried in a 30°C vacuum oven for 6 hours, filtered using a circulating water vacuum pump, and washed three times with phosphate buffer (pH 6.0) to obtain the cross-linked immobilized phospholipase. The system was dried in a 30°C vacuum oven for 6 hours, collected, sealed, and stored in a 4°C refrigerator until further use.

[0035] Finally, catalytic hydrolysis was carried out as follows: 0.45 g soybean lecithin and 29.55 g deionized water were placed in a single-necked round-bottom flask, homogenized for 10 minutes, and then 0.05 g cross-linked immobilized phospholipase and 60 μg Ca 2+ After stirring in a water bath at 40°C for 0.5 hours, remove the reaction product. Filter to remove the enzyme and place in a refrigerator at 4°C for later use. Add the collected immobilized phospholipase to the reaction system under the same reaction conditions. After the reaction is complete, repeat the above steps.

[0036] HPLC-ELSD analysis of the product composition revealed that the conversion rates of PE and PC in this example decreased to 78.87% and 75.17% of the initial conversion rates after five repetitions, respectively, representing increases of 48% and 49.18% compared to the uncrosslinked system. After 10 repetitions, the conversion rates of PE and PC remained at 57.73% and 51.75%, respectively.

[0037] Comparative Example 1, the macroporous resin is DA201, and the rest is the same as in Example 1; Comparative Example 2, the macroporous resin is DM301, and the rest is the same as in Example 1; Comparative Example 3, the macroporous resin is NKA-9, and the rest is the same as in Example 1; Comparative Example 4, the macroporous resin is D3520, and the rest is the same as in Example 1; In Comparative Example 5, the macroporous resin was ADS-17, but was not functionalized with dopamine and cellulose acetate, and the other conditions were the same as in Example 1; Comparative Example 6, the crosslinking agent is glutaraldehyde (GA), and the rest is the same as Example 6; Comparative Example 7, the crosslinking agent is ethylene glycol diglycidyl ether (EGDE), and the other conditions are the same as those in Example 6; Test data and result analysis 1. Scanning Electron Microscope-Energy Dispersive Spectrometer (SEM-EDS) The morphology and particle size distribution of the samples were observed using a JSM-7610FPlus scanning electron microscope equipped with EDS (Ultim Max 40, Oxford 189 Instruments, UK), and the elemental semi-quantitative analysis of ADS-17 and LU@ADS-17 was performed.

[0038] Transmission electron microscopy (TEM) and scanning electron microscopy-energy dispersive spectroscopy (SEM-EDS) were used to characterize the morphology and structure of ADS-17 and the immobilized product LU@ADS-17. Figure 1 、 Figure 2 shown. Figure 1 The SEM image of Figure a shows that the surface of ADS-17 is smooth and clean. Figure 1Figure b shows the morphology of functionalized ADS-17, which shows a microporous, rough surface of polydopamine grafted with cellulose acetate. Figure 1 Figures c and d show that LU@ADS-17 is slightly rough and has some obvious surface irregularities. ADS-17 and LU@ADS-17 were further analyzed by SEM-EDS. Figure 2 The changes in elemental composition were observed after LU was fixed on the macroporous resin, compared with the ADS-17 matrix ( Figure 2 a in), LU@ADS-17 ( Figure 2 In figure b), a characteristic sulfur signal (atomic content 0.13%) appears at 2.15 keV, while the carbon-oxygen ratio (C / O) decreases from 8.72 to 5.57. This change in elemental distribution confirms the successful and stable immobilization of the phospholipase LU molecules on the resin surface. Combined morphological and elemental analysis results indicate that the LU enzyme molecules were successfully loaded onto the ADS-17 support via surface grafting, and the immobilization process did not disrupt the support's spherical skeletal structure. This surface-modified immobilization method not only maintains the support's macroscopic mechanical properties but also provides a suitable microenvironment for enzymatic reactions through micro- and nanostructural modification, laying the structural foundation for subsequent catalytic performance studies.

[0039] LU@ADS-17 was characterized using SEM-EDS and TGA. The characteristic sulfur peaks and changes in the C / O ratio confirmed the successful immobilization of LU on the support surface, with no significant structural damage to ADS-17 during the adsorption process. The immobilized enzyme exhibited significantly improved enzymatic properties: 95% activity was retained at pH 6.0-8.0, 70.5% activity was retained after 4 hours in a 70°C dry heat environment, stability in n-hexane was 3.2 times greater than that of the free enzyme, and 89.36% of its initial activity was retained after 28 days of storage, meeting the long-term storage requirements of industrial enzyme preparations.

[0040] 2. Enzyme activity test results after immobilization Determination of LU enzyme hydrolysis activity Phospholipase activity (LU) was determined by hydrolysis of tributyrin. 1.0 mL of tributyrin was added to 50 mL of phosphate buffer (25 mM, pH 7.0), followed by the addition of 0.05 g of the immobilized phospholipase prepared in the Examples and Comparative Examples. The reaction was stirred at 500 rpm / min at 40°C for 15 minutes, and then 10 mL of anhydrous ethanol was added to terminate the reaction. Phenolphthalein was then added dropwise, and the mixture was titrated with 0.1 mol / L NaOH solution to the endpoint. The volume of NaOH consumed was recorded. A blank experiment was performed without the addition of phospholipase. All experiments were performed in triplicate. One unit (U) of phospholipase activity was defined as the amount of lipase required to hydrolyze tributyrin to produce 1.0 μmol of butyric acid per minute under the assay conditions. The calculation formula is as follows:

[0041] in, V is the volume of NaOH solution consumed after adding lipase (mL); V 0 is the volume of NaOH solution consumed by the blank control (mL); N is the concentration of NaOH solution (mol / L); T is the reaction time (min); M is the amount of phospholipase used (L or g) Immobilization effect evaluation parameters The immobilization effect of immobilized phospholipase can be evaluated by the immobilization efficiency (IE), which is calculated as follows:

[0042] Table 1 Effects of different resins on the immobilization of LU

[0043] As shown in the data of Table 1, when using macroporous resin as a carrier, as the pore size increases, the adsorption amount of protein and the activity of the enzyme will increase accordingly, which is mainly attributed to the fact that the larger pore structure is conducive to the diffusion of enzyme molecules and the full exposure of active sites. The functionalized macroporous resin ADS-17 immobilizes enzyme molecules by hydrogen bonds, van der Waals forces or chemical bonding, and its physicochemical properties play a key role in immobilization ability and catalytic efficiency. As can be seen from Table 1, among the five macroporous adsorption resins under the same immobilization conditions, ADS-17 in Example 1 and Example 6 and NKA-9 in Comparative Example 3 show excellent immobilization performance for phospholipase LU, and the enzyme activities after immobilization reach 1048.82U / g, 844.3U / g and 1005.7U / g, respectively, and the immobilization rates IE reach 74.8%, 76.1% and 68.7, respectively. However, in actual operation, it was found that the skeleton structure of NKA-9 resin was easily deformed during use, resulting in poor enzyme activity stability and affecting the test results. In comparison, ADS-17 is a hydrogen-bonding selective adsorption resin, and its three-dimensional network structure forms a stable bond with the enzyme molecules through multiple hydrogen bonds. It also has excellent stability under conditions such as heat, acid, alkali, and organic solvents. Based on the above analysis, the immobilized enzyme obtained by using ADS-17 functionalized as a carrier to immobilize phospholipase LU in Examples 1 and 6 has the highest activity and the best stability. It can be seen that after cross-linking with a cross-linking agent, the enzyme activity is slightly reduced, because cross-linking further restricts the flexibility of the enzyme.

[0044] 3. Stability of immobilized phospholipase pH stability In order to improve the application range of the enzyme and expand the reaction conditions, the relative activity of the enzyme after being placed in phosphate buffer solutions of different pH values (pH 4.0-8.0, 25 mM) for 0.5 h at room temperature was investigated and compared with the free enzyme. The difference in catalytic reaction stability between the free state and the immobilized phospholipase LU prepared in Example 1 in a wide pH range was systematically evaluated. Figure 3 . Using the hydrolysis reaction of tributyrin as a model, experimental data showed that the enzyme activities of both reached their maximum at pH 6.0, but showed significant differences when deviating from the optimal pH. In the acidic range (pH 4.0-5.0), the free enzyme showed better structure retention ability than the immobilized enzyme, maintaining 35.38% relative activity at pH 5.0, while the immobilized enzyme maintained 25.82%. This may be because immobilization limits the flexibility of the enzyme, reducing its ability to bind to the substrate and catalyze the reaction, which is more obvious under acidic conditions. It is worth noting that under alkaline conditions (pH 6.0-8.0), the activity of the free enzyme showed a continuous downward trend, reaching 71.83% at pH 8.0, while the relative activity of LU@ADS-17 was stable in the range of 90-100%. This phenomenon confirms that the immobilization process significantly improves the enzyme's tolerance to alkaline environments, likely due to the buffering effect of the carrier interface microenvironment on the medium pH. Furthermore, immobilization may limit protein mobility and reduce its exposure to extreme pH, thereby reducing protease denaturation and maintaining enzyme activity. This improvement in alkaline pH stability lays a foundation for the application of immobilized LU enzymes in complex systems.

[0045] Thermal stability Good thermal stability means that the support material provides a suitable microenvironment for the lipase, reducing the thermal denaturation of the free enzyme and effectively improving its efficiency. To test the thermal stability of the immobilized LU@ADS-17 prepared in Example 1, its relative activity was examined after 1, 2, 3, and 4 hours in three media (pH 7.0 phosphate buffer, air, and n-hexane) at 70°C, and compared with that of the free enzyme.

[0046] The thermal stability of the free enzyme and the immobilized phospholipase prepared in Example 1 in phosphate buffer solution at 70°C is shown in FIG. Figure 4As shown, both phospholipase LU exhibited significant thermosensitivity in hydrophilic media, with enzyme activity decreasing over time. The immobilized phospholipase exhibited only limited protection during the first 1-3 hours: the relative activity remained above 40% for the initial 2 hours, compared to around 25% for the free enzyme. This activity decreased to 30.95% by the third hour, compared to 22.59% for the free enzyme. Notably, after 4 hours of treatment, the relative activity converged with that of the free enzyme, reaching less than 20% for both. This phenomenon is closely related to the hydrophobic nature of phospholipase LU. Enzymes are generally more stable in hydrophobic solvents than in hydrophilic solvents. In highly polar phosphate buffer systems, excess water molecules penetrate and destroy the essential water layer on the enzyme surface, resulting in insufficient hydration of enzyme molecules and reduced enzyme activity. Furthermore, phosphate buffer solutions contain a large amount of water, and this high water content significantly reduces the thermal stability of lipases. Therefore, the immobilized support can delay thermal inactivation in the short term through spatial confinement.

[0047] The thermal stability of the free enzyme and the immobilized phospholipase prepared in Example 1 in air at 70°C is shown in FIG. Figure 5 As shown, the thermal stability of the immobilized enzyme under dry conditions is significantly improved compared to that in a liquid aqueous system: after 4 hours of heat treatment, LU@ADS-17 still retained 70.54% of its relative activity, significantly higher than the 40.44% of the free enzyme. This demonstrates that the enzyme activity of the heat-incubated immobilized LU sample is superior to that of the free enzyme, and its activity is more stably maintained at higher temperatures. This improved thermal stability is primarily attributed to the dual stabilization mechanism of the three-dimensional network structure of the ADS-17 resin on the enzyme molecules. First, the spatial confinement of the hydrophobic support effectively suppresses the conformational entropy change of the enzyme molecules caused by high temperature, maintaining its necessary spatial conformation and thus maintaining the enzyme's catalytic activity. Second, the microenvironment formed at the interface between the support and the enzyme reduces the thermodynamic perturbations of the essential water layer on the enzyme surface by lowering water activity. This has important implications for extending the enzyme's lifespan, improving production efficiency, expanding its industrial application range, and improving reaction conditions. This improved thermal stability makes the application of immobilized enzymes in industrial processes more flexible and efficient.

[0048] In actual production, most lipase-catalyzed reactions occur in non-aqueous solvent systems, and studies have shown that immobilized enzymes have better stability in hydrophobic organic solvents (such as n-hexane), which means that they can catalyze reactions in organic solvent environments. This is very important for enzymatic reactions that need to be carried out in non-aqueous solvents, such as fatty acid esterification and transesterification. Therefore, this study investigated the temperature stability of immobilized phospholipid LU in n-hexane. Figure 6As shown, after 4 hours of treatment in n-hexane at 70°C, the immobilized enzyme still maintained 67.4% relative activity, a 3.4-fold improvement compared to its stability in a hydrophilic medium (phosphate buffer). This significant improvement is likely due to the confinement effect formed by the hydrophobic interface of the support: on the one hand, hydrophobic interactions within the resin backbone stabilize the active conformation of the enzyme molecule; on the other hand, the low-polarity medium effectively reduces the thermodynamic perturbations of the essential water layer on the enzyme surface. The immobilized enzyme can maintain activity for extended periods in n-hexane, thereby improving its durability and reusability and expanding its application range. This stability advantage is highly compatible with industrial enzyme catalysis scenarios. In typical non-aqueous reactions such as esterification, the immobilized enzyme can better tolerate organic solvent environments, significantly reducing enzyme costs in industrial production.

[0049] Storage stability In practical applications, storage stability is one of the important indicators for evaluating the enzymatic properties of immobilized enzymes. The storage stability of immobilized enzymes refers to the ability of the enzyme to maintain its catalytic activity and structural integrity for a long time under storage conditions after immobilization. Storage stability is of great significance for the practical application and economic benefits of immobilized enzymes. In order to explore the storage stability of immobilized LU@ADS-17, this study stored the immobilized phospholipase prepared in Example 1 at 4°C in the dark, and measured the residual enzyme activity after 1, 7, 14, 21 and 28 days of storage. The results are as follows: Figure 7 As shown in the figure, the residual enzyme activity of LU@ADS-17 after 7, 14, 21, and 28 days of storage was 95.49%, 96.39%, 93.92%, and 89.36% of the initial enzyme activity, respectively. These data demonstrate that LU@ADS-17 has excellent storage stability, maintaining its catalytic activity and structural stability during storage. This property is of great practical significance for extending the lifespan of phospholipases, reducing production costs, ensuring product quality, expanding their application range, and enhancing product competitiveness.

[0050] 4. Different cross-linkers enhance the structural stability of immobilized phospholipase - enzyme activity test results Cross-linking is an important means of strengthening enzyme immobilization. Its core lies in enhancing the structural stability of the enzyme-carrier composite material through chemical bonding. However, due to its disordered nature, cross-linking reactions may also occur inside the enzyme molecules, leading to loss of enzyme activity. In order to address the defect of easy shedding of adsorbed immobilized lipase, a cross-linking agent strengthening strategy was introduced. We used the activity of immobilized phospholipase as the evaluation index in Example 6 and Comparative Example 6 and Comparative Example 7 to conduct tests. Figure 8, wherein A is the cross-linked immobilized phospholipase prepared in Comparative Example 6, B is the cross-linked immobilized phospholipase prepared in Comparative Example 7, C is the cross-linked immobilized phospholipase prepared in Example 6, and D is the immobilized phospholipase prepared in Example 1; according to Figure 8 As a result, it can be seen that under 0.5% and 1.0% concentration conditions, polyethylene glycol diglycidyl ether shows the best cross-linking effect, and the highest relative enzyme activity is obtained by comparison, and the effect is particularly remarkable when the concentration is relatively high. Wherein, the cross-linking agent glutaraldehyde in Comparative Example 6 is widely used in the cross-linking immobilization of enzymes because of its high reaction activity and low price. However, the existence form of glutaraldehyde in solution is complicated, and its cross-linking reaction is difficult to control and there is no rule to follow. The aldehyde groups at its two ends mainly undergo cross-linking reaction with the amino residues on the enzyme molecule surface. Therefore, if the enzyme molecule surface lacks enough amino residues, the cross-linking effect may be undesirable. In addition, glutaraldehyde can self-polymerize inside the enzyme, destroying the original tertiary structure of the enzyme protein so that the enzyme loses activity. The cross-linked structure formed by the glycol diglycidyl ether in Comparative Example 7 is shorter, and may be hydrolyzed or fractured in an acidic environment, causing the performance degradation of the cross-linked material. In comparison, polyethylene glycol diglycidyl ether in Example 6 is a cross-linking agent that is rarely used for enzyme cross-linking and immobilization. Studies have found that it can promote protein cross-linking and assist in enzyme immobilization. Its epoxy groups can undergo cross-linking reactions with more residues on the surface of the enzyme molecules, and the reaction conditions are relatively mild, reducing damage to the enzyme molecules, thereby better maintaining the activity of the enzyme.

[0051] 5. Reusability test results of LU@ADS-17 catalyzed preparation of lysophospholipids The reaction conditions were as follows: 0.45 g of phospholipid and 29.55 ml of deionized water were weighed into a beaker, homogenized for 10 min at 1000 rpm in a homogenizer to emulsify the substrate, placed in a single-necked round-bottom flask, 0.05 g of LU@ADS-17 prepared in Example 1 was added, and the mixture was heated in a water bath at 40°C with a stirrer at 300 rpm for 0.5 h.

[0052] After each reaction, the solid catalyst LU@ADS-17 was separated by filtration using a circulating water vacuum pump. The reaction was then continued for 0.5 hours under the same reaction conditions. The liquid phase of the sample after each reaction was prepared and analyzed by HPLC. Two parallel groups were set up for each repeated experiment. The reusability of the immobilized lipase was evaluated using the relative enzyme activity, which was calculated as follows:

[0053] HPLC determination of sample components after hydrolysis and calculation of conversion rate results Sample preparation: Take 0.12 g of freeze-dried sample and add it to 1.2 mL of mobile phase (chloroform: methanol = 2:1, v / v). After vortex mixing, filter it through a 0.22 μm microporous filter membrane into a liquid phase vial and store it in a refrigerator at -20°C until determination.

[0054] Liquid chromatography: Sample composition was determined using HPLC equipped with an evaporative light scattering detector (ELSD). The drift tube temperature was 75°C and the nitrogen pressure was 320 kPa. Lysophospholipids were separated using a Chromolith® Performan-ce-Si column at a 30°C oven temperature, a 10 μL injection volume, a drift tube temperature of 75°C, and a nitrogen pressure of 320 kPa. Elution was performed at a flow rate of 1.0 mL / min using a gradient elution consisting of mobile phases A (isopropanol), B (n-hexane plus 0.04% (v / v) triethylamine), and C (13% acetic acid). The mobile phase for gradient elution was as follows: initial concentration of 57% A, 40% B, and 3% C; from 0 to 12 min, change to 50% A, 40% B, and 10% C; from 12 to 17 min, maintain at 50% A, 40% B, and 10% C; from 17 to 17.1 min, change to 57% A, 40% B, and 3% C; and from 17.1 to 24 min, maintain at 57% A, 40% B, and 3% C. Quantification was performed using peak area normalization.

[0055] Result calculation: The hydrolysis effect of immobilized phospholipase can be evaluated by the conversion rate of lysophospholipids. The conversion rate of PE is calculated as follows. The conversion rate of PI and PC is calculated in the same way.

[0056] in, S PE, S LPE are the peak areas of PE and LPE respectively.

[0057] To quantify the relative contents of phospholipids and LPE in the purified products, the content of each component was calculated as a percentage using the ratio of the corresponding peak area to the total area of all peaks.

[0058] Reusability of lysophospholipids prepared by LU@ADS-17 catalytic hydrolysis The reusability of the immobilized phospholipase LU@ADS-17 in Example 1 for preparing lysophospholipids by catalytic hydrolysis was investigated. Figure 9As shown, the conversions of phosphatidylethanolamine (PE), phosphatidylinositol (PI), and phosphatidylcholine (PC) in the first reaction were 85.43%, 82.85%, and 82.04%, respectively, demonstrating excellent initial catalytic activity. After five cycles, the conversions of PE and PC dropped to 53.29% and 50.39%, respectively, compared to the initial conversions. However, PI maintained a relatively high conversion rate, reaching 97.88% in the fifth cycle. The decrease in activity of the immobilized enzyme is likely due to the gradual shedding of enzyme molecules during the cycle and the inhibitory effect of the reaction medium on the enzyme's active sites. Despite this, the immobilized enzyme exhibited significantly superior cyclic stability compared to the free enzyme, which lost >90% of its activity after the second cycle, confirming that the support effectively slowed the enzyme's inactivation process. This result demonstrates that the prepared LU@ADS-17 immobilization system maintained considerable catalytic efficiency over five cycles, and its operational stability meets the requirements of continuous, intermittent production processes. It significantly improves the economic benefits of enzymes, makes their application more sustainable, enhances environmental friendliness, reduces production costs, and is particularly suitable for large-scale production.

[0059] Reusability of lysophospholipids prepared by catalytic hydrolysis of cross-linked immobilized phospholipase The reusability of the cross-linked immobilized phospholipase LU@ADS-17 prepared in Example 6 for catalyzing the hydrolysis of soybean phospholipids is shown in the following table. Figure 10 As shown, experimental data showed that the conversion rates of phosphatidylethanolamine (PE), phosphatidylinositol (PI), and phosphatidylcholine (PC) generated in the first reaction were 81.87%, 72.36%, and 79.18%, respectively, demonstrating excellent initial catalytic activity, indicating that the cross-linking process successfully retained the high catalytic activity of the enzyme molecules. After five cycles of reuse, the conversion rates of PE, PI, and PC dropped to 78.87%, 36.41%, and 75.17%, respectively, of the initial conversion rates. The conversion rates of PE and PC increased by 48% and 49.18% compared to the same period of the phospholipase immobilized without cross-linking, significantly outperforming the uncross-linked system. After ten cycles of reuse, the conversion rates of PE and PC dropped to 57.73% and 51.75% of the initial levels, respectively. This indicates that the cross-linked immobilized enzyme still exhibits significantly better cyclic stability than the uncross-linked immobilized enzyme. For example, the catalytic efficiency of LU@ADS-17 dropped to approximately 50% of the initial level after the fifth cycle, confirming that the cross-linked LU@ADS-17 can effectively delay the inactivation of enzyme molecules during repeated use. This result demonstrates that the cross-linked LU@ADS-17 immobilization system maintains considerable catalytic efficiency over ten cycles, and its high cyclic stability gives it a significant advantage in intermittent production. This technology can reduce the cost of enzyme preparations while reducing the amount of solid waste generated, providing reliable technical support for the development of industrial-grade biocatalysts.

[0060] The above description is only used to illustrate the technical solution of the present invention and is not intended to limit it. Any modifications and changes made to the technical solution of the present invention by ordinary persons in the art shall still fall within the scope of the present invention as long as they do not depart from the overall concept of the present invention.

Claims

1. A method for preparing lysophospholipids by catalytic hydrolysis using immobilized phospholipase LU@ADS-17, characterized in that: The method for preparing lysophospholipids by catalytic hydrolysis using immobilized phospholipase LU@ADS-17 is to prepare immobilized phospholipase using macroporous resin ADS-17 and LU enzyme, and apply the immobilized phospholipase to catalytic hydrolysis to generate lysophospholipids. The method comprises immersing the functionalized macroporous resin ADS-17 in LU enzyme solution, shaking on a shaker, and immobilizing the LU enzyme on the macroporous resin ADS-17 by physical adsorption and chemical adsorption to prepare immobilized phospholipase LU@ADS-17. Then, soybean lecithin is mixed with deionized water for homogeneity, and the immobilized phospholipase is added, stirred for reaction, and the product is obtained. The specific steps include: S01, activation of macroporous resin ADS-17; S02, functionalization of macroporous resin ADS-17: the surface of the macroporous resin is subjected to a self-polymerization reaction of dopamine and then grafted with cellulose acetate to obtain a functionalized macroporous resin ADS-17; S03, LU enzyme immobilization: LU enzyme was immobilized on the functionalized macroporous resin ADS-17 by physical adsorption and chemical cross-linking to prepare immobilized phospholipase LU@ADS-17; S04, catalytic hydrolysis.

2. The method for preparing lysophospholipids by catalytic hydrolysis using immobilized phospholipase LU@ADS-17 according to claim 1, characterized in that: The activation of the macroporous resin ADS-17 is specifically as follows: the macroporous resin ADS-17 is placed in a beaker, 95% ethanol is added, and the beaker is sealed and protected from light and soaked for 24 hours; then, the resin is filtered using a circulating water vacuum pump and washed with ultrapure water multiple times until there is no ethanol odor; the resin is transferred to a new beaker, 5% hydrochloric acid solution is added, and the resin is sealed and protected from light and soaked for 4 hours; After filtering the hydrochloric acid solution, wash it repeatedly with ultrapure water until the pH of the filtrate is adjusted to 7.0; then transfer the resin to a beaker, add 5% sodium hydroxide solution, and continue soaking for 4 hours; after filtering, wash it with ultrapure water several times until the pH returns to 7.0; finally, add pH 7.0 phosphate buffer solution, soak for 2 hours, filter it, and wash it with phosphate buffer solution until the pH of the filtrate remains at 7.

0.

3. The method for preparing lysophospholipids by catalytic hydrolysis using immobilized phospholipase LU@ADS-17 according to claim 1, characterized in that: The functionalization of the macroporous resin ADS-17 is specifically as follows: the activated macroporous resin is added to an ethanol aqueous solution (volume ratio of 1:1), mixed evenly, and then dopamine hydrochloride is added and stirred; then, 20 mmol / L tris (hydroxymethyl)aminomethane buffer is added dropwise, the pH is adjusted to 8.5, and the mixture is stirred for 60 to 90 minutes, cellulose acetate is added, and then ammonium persulfate is added and stirred at 50° C. for 30 to 40 minutes, followed by suction filtration and washing to obtain a polydopamine surface-modified macroporous resin grafted with cellulose acetate, namely, the functionalized macroporous resin ADS-17; The mass ratio of dopamine hydrochloride to macroporous resin is 1:5-8; the mass ratio of cellulose acetate to dopamine hydrochloride is 1:5, and the ammonium persulfate is 0.1-0.5% of the dopamine hydrochloride.

4. The method for preparing lysophospholipids by catalytic hydrolysis using immobilized phospholipase LU@ADS-17 according to claim 1, characterized in that: The LU enzyme immobilization method comprises the following steps: mixing the LU enzyme solution with an appropriate amount of phosphate buffer solution (pH = 4-8) to prepare an enzyme phosphate buffer solution; placing the functionalized macroporous resin ADS-17 in a conical flask, adding the enzyme phosphate buffer solution, and oscillating and adsorbing the LU enzyme on a 25°C constant temperature shaker for 4 hours to immobilize the LU enzyme on the macroporous resin ADS-17. The LU enzyme is then filtered using a circulating water vacuum pump to obtain immobilized phospholipase LU@ADS-17; the immobilized phospholipase is then dried in a 30°C vacuum drying oven for 6 hours, collected, sealed, and stored in a 4°C refrigerator.

5. The method for preparing lysophospholipids by catalytic hydrolysis using immobilized phospholipase LU@ADS-17 according to claim 1, characterized in that: The catalytic hydrolysis specifically comprises the following steps: mixing soybean lecithin and water uniformly, adding immobilized phospholipase, and then stirring in a water bath at 30-70° C. for 0.5-48 hours to obtain a product, wherein the product mainly contains lysophosphatidylcholine, lysophosphatidylethanolamine and lysophosphatidylinositol.

6. The method for preparing lysophospholipids by catalytic hydrolysis using immobilized phospholipase LU@ADS-17 according to claim 5, characterized in that: The stirring speed is 200-400 rpm; the amount of the immobilized enzyme is 0.01-0.2 g; soybean lecithin and water concentration is 0.5-12 wt% to prepare lysophospholipid; and the lysophospholipid is prepared by changing the substrate concentration and the reaction temperature.

7. The method for preparing lysophospholipids by catalytic hydrolysis using immobilized phospholipase LU@ADS-17 according to claim 4, characterized in that: In the preparation process of the immobilized phospholipase, the preferred enzyme concentration is 20-60 mg / mL, and the more preferred enzyme concentration is 40 mg / mL; 50 ml of enzyme phosphate buffer is added to every 0.5 g of macroporous resin.

8. The method for preparing lysophospholipids by catalytic hydrolysis using immobilized phospholipase LU@ADS-17 according to claim 1, characterized in that: The immobilized phospholipase also includes cross-linked immobilized phospholipase, specifically: LU enzyme solution is mixed with an appropriate amount of phosphate buffer solution to prepare an enzyme phosphate buffer solution; after the adsorption reaction is completed, a cross-linking agent is immediately added; after sufficient cross-linking is achieved by shaking on a constant temperature shaker, the solution is filtered with a circulating water vacuum pump and washed three times with a phosphate buffer solution with a pH of 6.0 to obtain a cross-linked immobilized phospholipase; The solution was placed in a vacuum drying oven at 30°C and dried for 6 hours. After being collected, it was sealed and stored in a refrigerator at 4°C for later use. The phosphate buffer solution was a 25 mM buffer solution with a pH of 6.

9. The method for preparing lysophospholipids by catalytic hydrolysis using immobilized phospholipase LU@ADS-17 according to claim 8, characterized in that: The cross-linking agent is polyethylene glycol diglycidyl ether; the cross-linking temperature is 40° C., the amount-to-volume ratio of the cross-linking agent is 0.005-0.02, and the cross-linking time is 8-10 hours.

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