A method for preparing immobilized phospholipase d and a method for preparing phosphatidylserine using the same

Abstract

The application relates to a preparation method of immobilized phospholipase D and a preparation method of phosphatidylserine thereof, and belongs to the technical field of biotechnology.The preparation method of the immobilized phospholipase D comprises the following steps: (1) mixing mesoporous molecular sieve SBA-15 and a sodium acetate buffer solution to obtain a mixed solution; (2) mixing the mixed solution with an enzyme solution, oscillating, centrifuging, taking the precipitate, cleaning and freeze-drying to obtain the immobilized phospholipase D.The application improves the catalytic performance and stability of PLD by fixing PLD through the immobilization technology, realizes the reusability of PLD, and provides technical support for the industrialized production of the immobilized enzyme of PS.The preparation of phosphatidylserine by the immobilized PLD is optimized, and the optimal process condition of the preparation of PS by the immobilized PLD is obtained.The application further discloses a potential molecular mechanism of PS in treating AD, and provides a theoretical basis for the further clinical application of PS.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a method for preparing immobilized phospholipase D and a method for preparing phosphatidylserine. Background Technology

[0002] Phospholipids are a class of lipids containing phosphate groups. Based on the type of their polar head, they can be classified into: phosphatidylcholine (PC), phosphatidylserine (PS), phosphatidylethanolamine (PE), phosphatidylinositol (PI), and phosphoglyceride (PG). Among these, PS is the only phospholipid capable of regulating the function of key cell membrane proteins and participating in various cellular metabolic regulation processes. It is particularly important for the maintenance and repair of nerve cells and has been listed as a new resource food.

[0003] Clinical studies have shown that PS, a novel food resource, can significantly improve the symptoms of Alzheimer's disease (AD) patients with no obvious side effects, making it a potential supplement for adjuvant treatment of AD. Therefore, the demand for PS is increasing year by year. Currently, the main methods for preparing PS include solvent extraction, chemical synthesis, and enzyme catalysis. Among these, PS prepared by solvent extraction and chemical synthesis has low purity and problems such as residual organic solvents, and is gradually being phased out. Enzyme catalysis has advantages such as mild reaction conditions, good reaction selectivity, and high conversion rate, and is currently the main method for preparing PS. This method mainly utilizes the transesterification activity of poly(lactic-coated) ether (PLD). Because the substrate PC is readily soluble in the organic phase, while PLD is readily soluble in the aqueous phase, the PLD-catalyzed transesterification reaction system is usually carried out in a two-phase reaction system (organic phase-aqueous phase). Due to the inherent sensitivity and susceptibility to inactivation of PLD, prolonged contact with organic solvents leads to a decrease in the catalytic efficiency of PLD. Furthermore, choline, a byproduct, is readily soluble in water and difficult to separate from PLD via liquid-liquid separation. It accumulates in the aqueous phase as the reaction proceeds, and when the choline concentration in the aqueous phase reaches a certain level, it inhibits the transesterification activity of PLD. Therefore, to improve the transesterification efficiency of PLD, immobilization using appropriate enzyme immobilization techniques can enhance its reusability and reduce the production cost of PS. However, the choice of immobilization carrier and method directly affects the activity and stability of the immobilized enzyme. Therefore, obtaining immobilized enzymes with good activity and stability, and utilizing them to prepare phosphatidylserine, is a pressing problem that needs to be solved. Summary of the Invention

[0004] The purpose of this invention is to provide a method for preparing immobilized phospholipase D and a method for preparing phosphatidylserine. This invention uses immobilization technology to immobilize PLD, which improves the catalytic performance and stability of PLD, enables PLD reusability, reduces production costs, and provides technical support for the industrial production of immobilized PS enzymes, thus having significant industrial application value. Furthermore, the preparation of phosphatidylserine using immobilized PLD has been optimized, obtaining the optimal process conditions for PS preparation using immobilized PLD.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a method for preparing immobilized phospholipase D, comprising the following steps: (1) Mix the mesoporous molecular sieve SBA-15 with sodium acetate buffer solution to obtain a mixture; (2) Mix the mixture with the enzyme solution, shake, centrifuge, collect the precipitate, wash, and freeze dry to obtain immobilized phospholipase D.

[0006] Preferably, the mass-to-volume ratio of the mesoporous molecular sieve SBA-15 to the sodium acetate buffer is 1g:15~25mL, and the concentration of the sodium acetate buffer is 0.1~0.3mol / L.

[0007] Preferably, the enzyme solution is prepared by mixing phospholipase D enzyme solution with anhydrous ethanol, reacting in an ice bath for 45-75 min, centrifuging, collecting the precipitate to obtain purified phospholipase D, and dissolving the purified phospholipase D in sodium acetate buffer to obtain the enzyme solution. The volume ratio of the phospholipase D enzyme solution to anhydrous ethanol is 1~5:1.

[0008] Preferably, the volume-to-mass ratio of the enzyme solution added to the mesoporous molecular sieve SBA-15 is 10~45mL:1g.

[0009] Preferably, the oscillation temperature is 4~50℃; the oscillation time is 2~12h; the cleaning solution is a sodium acetate buffer solution; and the pH of the sodium acetate buffer solution is 4~9.

[0010] This invention provides a method for preparing phosphatidylserine by immobilizing phospholipase D, comprising the following steps: (6.1) Dissolve L-serine in buffer solution, add immobilized phospholipase D and mix to form the aqueous phase; dissolve phosphatidylcholine in chloroform to form the organic phase; (6.2) Mix the aqueous phase and the organic phase and react for 3~24h to prepare phosphatidylserine and recover the immobilized phospholipase D; The immobilized phospholipase D was obtained using the preparation method described above.

[0011] Preferably, the buffer solution is a phosphate buffer solution; the volume ratio of chloroform to buffer solution is 1~8:1.

[0012] Preferably, the molar ratio of L-serine to phosphatidylcholine is 1~12:1.

[0013] Preferably, the reaction temperature is 20~70℃ and the pH of the reaction is 4~9.

[0014] The present invention also provides the use of phosphatidylserine obtained according to the method in the preparation of drugs for treating Alzheimer's disease.

[0015] Compared with the prior art, the present invention has the following beneficial effects: (1) This invention uses relative transesterase activity and protein immobilization rate as indicators to evaluate immobilized PLDs. First, the optimal immobilization carrier and method are screened from different carriers and methods. Then, the immobilization conditions of PLDs are optimized, and the structure of the immobilized PLDs is characterized. Finally, the enzymatic properties of free PLDs and immobilized PLDs are analyzed and compared, providing a reference for the production and application of immobilized PLDs.

[0016] (2) In this invention, an immobilized PLD is used as a catalyst for the enzymatic synthesis of PS. In a two-phase reaction system, the effects of the volume ratio of the two phases, the molar ratio of the substrate, the reaction temperature, the reaction pH and the reaction time on the yield of phosphatidylserine are studied through single-factor experiments and response surface methodology. The results show that when the volume ratio of the two phases (chloroform and phosphate buffer solution) is 3.5:1, the molar ratio of the substrate (L-serine and PC) is 10:1, the reaction pH is 7, the reaction temperature is 45.3℃ and the reaction time is 12.6h, the PS yield is 60.41%, while the actual PS yield is 59.87±0.35%.

[0017] (3) Since the mechanism by which PS improves the symptoms of AD patients is still unclear, this invention uses A β SH-SY5Y cells were induced at 25-35 to construct an AD cell injury model, and the effect of PS on this model was evaluated using cell viability as an indicator. Subsequently, a combination of transcriptomics and network pharmacology was used to explore the core targets and signaling pathways of PS in treating AD, further revealing the potential molecular mechanisms of PS in treating AD and providing a theoretical basis for the further clinical application of PS. Attached Figure Description

[0018] Figure 1 The graphs are standard curves for phosphatidylcholine and phosphatidylserine, where A is the standard curve for phosphatidylcholine and B is the standard curve for phosphatidylserine. Figure 2The effect of different carriers on the relative transesterase activity of immobilized PLDs; Figure 3 The effect of different immobilization methods on the relative transesterase activity of immobilized PLDs; Figure 4 The effects of different factors on the relative transesterase activity and protein immobilization rate of immobilized PLD are shown, where A is immobilization time, B is temperature, C is pH, and D is the amount of enzyme added. Figure 5 Infrared spectrum of immobilized PLD; Figure 6 The optimal temperature and thermal stability of the immobilized PLD are shown in Figure A, where A is the optimal temperature diagram and B is the thermal stability diagram. Figure 7 The optimal pH and pH stability of the immobilized PLD are shown in Figure A, where A is the optimal pH graph and B is the pH stability graph. Figure 8 The Km values ​​and storage stability of free PLD and immobilized PLD are given, where A is the Km value and B is the storage stability. Figure 9 To ensure operational stability of both free PLDs and immobilized PLDs; Figure 10 The effects of different factors on the yield of phosphatidylserine are shown, where A is the volume ratio of the two phases, B is the molar ratio of the substrate, C is the reaction time, D is the reaction temperature, and E is the reaction pH. Figure 11 The effect of the two-phase volume ratio and reaction temperature on the degree of grafting; Figure 12 The effect of two-phase volume ratio and reaction time on the degree of grafting; Figure 13 The effect of reaction temperature and reaction time on the degree of branching; Figure 14 The image shows the nuclear magnetic resonance (NMR) spectrum of phosphatidylserine, where A is the reaction substrate phosphatidylcholine and B is the reaction product phosphatidylserine. Figure 15 denoted as A, where A is the particle size of the phosphatidylserine liposome and B is the zeta potential. Figure 16 For different concentrations of A β 25-35 Effects on SH-SY5Y cell viability, where different letters a~d represent the significance level of differences between different groups (p<0.05); Figure 17 For different concentrations of A β 25-35 Effects on SH-SY5Y cell morphology, where A is the control group; B is 5 μM A. β25-35 Treatment; C is 10 μM A β 25-35 Treatment; D is 20 μM A β 25-35 Processing; E is 30 μM A β 25-35 Treatment; F is 40 μM A β 25-35 deal with; Figure 18 The effect of different concentrations of PS on the viability of AD cell models was investigated, where different letters from a to d represent the significance level of differences between different groups (p < 0.05). Figure 19 The effects of different concentrations of PS on the morphology of an AD cell model were investigated, with A representing the control group; B representing the model group; C representing the 10 μM PS treatment; D representing the 20 μM PS treatment; E representing the 30 μM PS treatment; F representing the 40 μM PS treatment; and G representing the 80 μM PS treatment. Figure 20 The bar chart shows the differentially expressed genes between the model group and the PS treatment group, where SH is the control group, SH-AB is the model group, and AH-PS is the PS treatment group. Figure 21 This is a volcano plot of differentially expressed genes, where A represents the control group vs. the model group; B represents the model group vs. the PS treatment group; and C represents the control group vs. the PS treatment group. Figure 22 A heatmap of differentially expressed genes between the model group and the PS treatment group, where SH-AB is the model group and AH-PS is the PS treatment group; Figure 23 GO enrichment analysis of differentially expressed genes between the model group and the PS treatment group; Figure 24 KEGG pathway enrichment analysis for differentially expressed genes between the model group and the PS treatment group; Figure 25 This is a PPI network diagram of the intersection genes, where the intersection genes are differentially expressed genes and PS-AD intersection target genes; Figure 26 This is a diagram of gene interactions at intersection. Figure 27 GO enrichment analysis of the intersection genes, where the intersection genes are differentially expressed genes and PS-AD intersection target genes; Figure 28 KEGG pathway enrichment analysis was performed on the intersecting genes, which were differentially expressed genes and PS-AD target genes. Detailed Implementation

[0019] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0020] Example 1: Preparation of Immobilized Phospholipase D

[0021] 1.1 Pretreatment of the immobilized carrier

[0022] Amino resin: Place 0.5 g of amino carrier in an Erlenmeyer flask, add pH 5.6, 0.2 mol / L sodium acetate buffer, shake at 200 rpm for 2 h at 25 °C, and then vacuum dry. Add 10 mL of sodium acetate buffer (pH 5.6) containing 5% (v / v) glutaraldehyde, shake at 200 rpm for 5 h at 25 °C, vacuum dry, filter, and wash repeatedly with deionized water until no glutaraldehyde residue remains. After filtration and drying, store at 4 °C for later use.

[0023] Epoxy-based carrier: Take 0.5g of epoxy-based carrier and add it to 10mL of pH5.6, 0.2mol / L sodium acetate buffer. Shake at 200rpm for 2h at 25℃, then vacuum dry and store at 4℃ for later use.

[0024] Diatomaceous earth, montmorillonite, and mesoporous molecular sieves SBA-15 and MCM-41 carriers do not require treatment.

[0025] 1.2 Screening of Immobilization Carriers

[0026] Weigh 0.1 g of the carriers treated in 1.1 and add 2 mL of 0.52 mg / mL PLD enzyme solution (under 0℃ ice bath stirring, rapidly add 3 times the enzyme solution volume of anhydrous ethanol to the enzyme solution, stir on ice bath for 1 h to precipitate the protein. Then centrifuge at 10000 r / min for 10 min at 4℃ and collect the precipitate. Dissolve and dilute the precipitate with sodium acetate buffer (pH 5.6)). Shake at 200 rpm for 12 h at 25℃. Separate the immobilized enzyme by vacuum filtration. Wash the immobilized enzyme three times with the same volume of pH 5.6, 0.2 mol / L sodium acetate buffer (until no protein is detected in the filtrate). Determine the protein content in the filtrate according to the protein concentration detection method. At the same time, determine the relative transesterase activity of different immobilized carriers according to the PLD transphosphatidyl activity determination method. Set the maximum PLD transesterase activity of different immobilized carriers as 100% to calculate the relative transesterase activity of other immobilized carriers. To screen for the optimal immobilization carrier, this invention evaluated the effects of carriers such as amino resin, epoxy resin, diatomaceous earth, montmorillonite, mesoporous molecular sieve MCM-41, and SBA-15 on the performance of immobilized protein esters (PLDs), using relative transesterase activity and protein immobilization rate as indicators. The maximum PLD transesterase activity in different immobilization carriers was set as 100%, and the relative transesterase activities of other immobilization carriers were calculated accordingly. The results are shown in […]. Figure 2 .

[0027] Determination of PLD phosphatidyl-converting activity: At 25℃, the amount of enzyme required to catalyze 1 μmol of phosphatidylcholine to phosphatidylserine per unit time is defined as one enzyme activity unit, denoted as U. First, 12 mg of L-serine was weighed and dissolved in 2 mL of sodium acetate buffer (0.2 M, pH 5.6), followed by the addition of 2 mL of 0.5 mg / mL PLD enzyme solution or 15% immobilized PLD. Then, 10 mg of phosphatidylcholine was weighed and dissolved in 5 mL of chloroform. The aqueous and organic phases were mixed and reacted in a constant temperature shaker at 35℃ for 12 h at a rotation speed of 300 r / min. After the reaction, the PS content was detected using thin-layer chromatography (TLC).

[0028]

[0029] Where nPS is the amount of PS in moles; Venzyme is the amount of enzyme solution added during the reaction in mL; menzyme is the mass of enzyme added during the reaction in mg; and t is the reaction time in min.

[0030] Protein concentration detection: Protein content was determined using the Bradford method. Immobilized phospholipase D assay indicators: The main indicators for evaluating the performance of immobilized PLDs are relative transesterase activity and protein immobilization rate. Relative transesterase activity was determined using the method for measuring PLD transphosphatidyl activity, and enzyme protein concentration was determined using the method for measuring protein concentration, as shown in the following formula:

[0031] Depend on Figure 2 It can be seen that, compared with other carriers, immobilizing PLD on the mesoporous molecular sieve SBA-15 resulted in the maximum relative transesterification activity of the enzyme, at which point the protein immobilization rate was 76.8 ± 0.95%. This is likely because the mesoporous molecular sieve SBA-15 is a highly ordered two-dimensional hexagonal phase with a large specific surface area, pore volume, and uniform pore size, allowing it to accommodate more PLDs. Furthermore, the highly ordered pore arrangement of SBA-15 reduces mass transfer resistance between the substrate and enzyme molecules, facilitating sufficient contact between the substrate molecules and the PLD and improving the transesterification activity of the enzyme. Therefore, this invention selected the mesoporous molecular sieve SBA-15 as the carrier for immobilizing PLDs for subsequent experiments.

[0032] 1.3 Screening of Immobilization Methods

[0033] (1) Adsorption method

[0034] 0.1 g of mesoporous molecular sieve SBA-15 support was dispersed in 2 mL of sodium acetate buffer (0.2 mol / L, pH 5.6), and 2 mL of enzyme solution was added. The mixture was shaken at 25 °C and 200 rpm for 12 h. After shaking, the mixture was centrifuged at 4 °C and 6000 rpm for 10 min to separate the supernatant and precipitate. The supernatant was retained and its protein content was determined. The precipitate was washed three times with sodium acetate buffer (0.2 mol / L, pH 5.6) until no protein was detected in the supernatant. The precipitate was then dried in a vacuum freeze dryer for 12 h to obtain immobilized PLD.

[0035] (2) Adsorption-crosslinking method

[0036] 0.1 g of mesoporous molecular sieve SBA-15 support was dispersed in 2 mL of sodium acetate buffer solution (0.2 mol / L, pH 5.6), and 2 mL of enzyme solution was added. The mixture was shaken at 25 °C and 200 rpm for 12 h. After shaking, 2 mL of 5% glutaraldehyde was added, and the mixture was cross-linked for 2 h. The mixture was then centrifuged at 4 °C and 6000 rpm for 10 min to separate the supernatant and precipitate. The supernatant was retained and its protein content was determined. The precipitate was washed three times with sodium acetate buffer solution (0.2 mol / L, pH 5.6) until no protein was detected in the supernatant. The precipitate was then dried in a vacuum freeze dryer for 12 h to obtain immobilized PLD.

[0037] (3) Adsorption-aggregation-crosslinking method

[0038] 0.1 g of mesoporous molecular sieve SBA-15 support was dispersed in 2 mL of sodium acetate buffer (0.2 mol / L, pH 5.6), and 2 mL of enzyme solution was added. The mixture was shaken at 25 °C and 200 rpm for 12 h. After shaking, the enzyme solution was placed in an ice-water bath, and 2.5 mL of ethanol was added, followed by stirring for 1 h. Then, 2 mL of 5% glutaraldehyde was added, and the mixture was placed in a shaker for crosslinking for 2 h. After crosslinking, the mixture was centrifuged at 4 °C and 6000 rpm for 10 min to separate the supernatant and precipitate. The supernatant was retained and its protein content was determined. The precipitate was washed three times with sodium acetate buffer (0.2 mol / L, pH 5.6) until no protein was detected in the supernatant. The precipitate was then dried in a vacuum freeze dryer for 12 h to obtain immobilized PLD.

[0039] The immobilized PLDs prepared by the above-mentioned different immobilization methods were subjected to transesterase activity determination according to the method for determining PLD transphosphatidyl activity. The maximum PLD transesterase activity of different immobilization methods was set as 100%, and the relative transesterase activities of other immobilization methods were calculated accordingly. The effects of adsorption, adsorption-crosslinking, and adsorption-aggregation-crosslinking methods on the immobilization effect of PLDs were compared, and the results are shown in […]. Figure 3 .

[0040] Depend on Figure 3It can be seen that, compared with other immobilization methods, the immobilized PLD prepared by the adsorption method achieves the maximum relative transesterification activity, with a protein immobilization rate of 77.68±1.62%. However, the immobilized PLD prepared by the adsorption-crosslinking method and the adsorption-aggregation-crosslinking method both show a decrease in relative transesterification activity. This may be because the use of crosslinking agents and precipitants alters the native conformation of the protease, destroying the enzyme's active site and leading to reduced enzyme activity. In contrast, the adsorption method connects the PLD to SBA-15 through physical forces such as van der Waals forces, hydrogen bonds, and ionic bonds, preserving the complete structure and transesterification activity of the PLD to the greatest extent. Therefore, this invention employs the adsorption method to immobilize PLD on the mesoporous molecular sieve SBA-15.

[0041] 1.4 Optimization of Immobilization Conditions

[0042] This invention uses relative transesterase activity and protein immobilization rate as indicators to evaluate the performance of immobilized enzymes. It investigated the effects of adsorption time, enzyme dosage, buffer solution pH, and immobilization temperature on the performance of immobilized PLDs. The results are shown in [Figure number missing]. Figure 4 .

[0043] 1.4.1 Effect of immobilization time

[0044] 0.1 g of mesoporous molecular sieve SBA-15 carrier was dispersed in 2 mL of sodium acetate buffer (0.2 mol / L, pH 5.6), and 2 mL of enzyme solution was added. The mixture was shaken at 25 °C and 200 rpm for different times (2, 4, 6, 8, 10, 12 h). The immobilized mixture was centrifuged at 4 °C and 6000 rpm for 10 min to separate the supernatant and precipitate. The supernatant was retained and its protein content was determined. The precipitate was washed three times with sodium acetate buffer (0.2 mol / L, pH 5.6) until no protein was detected in the supernatant. The precipitate was then dried in a vacuum freeze dryer for 12 h to obtain immobilized PLD. The relative transesterase activity of PLD was determined according to the method for determining the transphosphatidyl activity of PLD. The maximum PLD transesterase activity at different immobilization times was set as 100%, and the relative transesterase activity at other immobilization times was calculated based on this.

[0045] Depend on Figure 4 As shown in section A, the protein immobilization rate of the immobilized PLD remained essentially constant with the change in immobilization time, while the relative transesterase activity first increased and then decreased with the change in immobilization time. The relative transesterase activity of the immobilized PLD reached its maximum value when the immobilization time was 8 hours. Therefore, this invention selected 8 hours as the optimal immobilization time, at which point the protein immobilization rate of the immobilized PLD was 75.31 ± 1.48%.

[0046] 1.4.2 Effect of Immobilization Temperature

[0047] 0.1 g of mesoporous molecular sieve SBA-15 carrier was dispersed in 2 mL of phosphate buffer solution (0.2 mol / L, pH 7), and 3.5 mL of enzyme solution was added. The mixture was shaken at 200 rpm for 8 h at different temperatures (4, 25, 30, 40, 50 °C). The immobilized mixture was centrifuged at 6000 rpm for 10 min at 4 °C, and the supernatant and precipitate were separated. The supernatant was retained and its protein content was determined. The precipitate was washed three times with phosphate buffer solution (0.2 mol / L, pH 7) until no protein was detected in the supernatant. The precipitate was then dried in a vacuum freeze dryer for 12 h to obtain immobilized PLD. The relative transesterase activity of PLD was determined according to the method for determining the transphosphatidyl activity of PLD. The maximum PLD transesterase activity at different immobilization temperatures was set as 100%, and the relative transesterase activity at other immobilization temperatures was calculated based on this.

[0048] Depend on Figure 4 As shown in section B, the relative transesterase activity of the immobilized PLD reaches its maximum value at an immobilization temperature of 25℃. However, when the temperature exceeds 30℃, the relative transesterase activity of the immobilized PLD begins to decrease. Therefore, this invention selects to immobilize PLD at an environment of 25℃, at which time the protein immobilization rate of the immobilized PLD is 70.69±2.31%.

[0049] 1.4.3 Effect of immobilization pH

[0050] 0.1 g of mesoporous molecular sieve SBA-15 carrier was dispersed in 2 mL of 0.2 mol / L buffer solutions at different pH values ​​(4, 5, 6, 7, 8, 9), and 3.5 mL of enzyme solution was added. The mixture was shaken at 25 °C and 200 rpm for 8 h. The immobilized mixture was centrifuged at 4 °C and 6000 rpm for 10 min to separate the supernatant and precipitate. The supernatant was retained and its protein content was determined. The precipitate was washed three times with 0.2 mol / L buffer solutions at different pH values ​​(4, 5, 6, 7, 8, 9) until no protein was detected in the supernatant. The precipitate was then dried in a vacuum freeze dryer for 12 h to obtain immobilized PLD. The relative transesterase activity of PLD was determined according to the method for determining PLD transphosphatidyl activity. The maximum PLD transesterase activity at different immobilization pH values ​​was set as 100%, and the relative transesterase activity at other immobilization pH values ​​was calculated based on this.

[0051] Depend on Figure 4 As shown in C, the relative transesterase activity and protein immobilization rate of the immobilized PLD first increase and then decrease with increasing pH. When the pH is 7, the relative transesterase activity and protein immobilization rate of the immobilized PLD reach their maximum values. Therefore, this invention selects pH 7 for PLD immobilization, at which the protein immobilization rate of the immobilized PLD is 74.9 ± 2.28%.

[0052] 1.4.4 Effect of enzyme dosage

[0053] 0.1 g of mesoporous molecular sieve SBA-15 carrier was dispersed in 2 mL of sodium acetate buffer (0.2 mol / L, pH 5.6), and different volumes (2, 2.5, 3, 3.5, 4, 4.5 mL) of PLD enzyme solution were added. The mixture was shaken at 25 °C and 200 rpm for 8 h. The immobilized mixture was centrifuged at 4 °C and 6000 rpm for 10 min to separate the supernatant and precipitate. The supernatant was retained and its protein content was determined. The precipitate was washed three times with sodium acetate buffer (0.2 mol / L, pH 5.6) until no protein was detected in the supernatant. The precipitate was then dried in a vacuum freeze dryer for 12 h to obtain immobilized PLD. The relative transesterase activity of PLD was determined according to the method for determining the transphosphatidyl activity of PLD. The maximum PLD transesterase activity among different enzyme additions was set as 100%, and the relative transesterase activities of other enzyme additions were calculated based on this.

[0054] Depend on Figure 4 As shown in D, the relative transesterase activity and protein immobilization rate of the immobilized PLD both first increase and then decrease with increasing enzyme addition. Therefore, in this invention, the enzyme addition amount was selected as 2.5 mL, at which point the protein immobilization rate of the immobilized PLD was 76.82 ± 2.8%.

[0055] 1.5 Structural characterization of immobilized phospholipase D

[0056] 0.1 g of mesoporous molecular sieve SBA-15 support was dispersed in 2 mL of sodium acetate buffer (0.2 mol / L, pH 5.6), and 2.5 mL of PLD enzyme solution was added. The mixture was shaken at 25 °C and 200 rpm for 8 h. The immobilized mixture was centrifuged at 4 °C and 6000 rpm for 10 min to separate the supernatant and precipitate. The supernatant was retained and its protein content was determined. The precipitate was washed three times with sodium acetate buffer (0.2 mol / L, pH 5.6) until no protein was detectable in the supernatant. The precipitate was then dried in a vacuum freeze dryer for 12 h to obtain immobilized PLD as a sample.

[0057] The sample and potassium bromide were dried to constant weight. 2 mg of the sample and 150 mg of potassium bromide were weighed separately and ground in a mortar until homogeneous. The mixture was then pressed into transparent tablets using a tablet press. The sample was then measured at 500-4000 cm⁻¹ using an FI-TR instrument. -1 The FT-IR spectra between the two were obtained by scanning 32 times with a resolution of 4 cm⁻¹. -1 The results are shown Figure 5 .

[0058] Depend on Figure 5 It can be seen that both SBA and SBA-15-PLD exhibit the characteristic absorption peak wavelength of mesoporous silica, namely at 455.7 cm⁻¹. -1 and 802.6cm -1 There are bending vibration peaks and symmetrical tensile peaks of Si-O-Si at 955.6 cm⁻¹. -1 and 1019.4cm -1 At this point, there are bending vibration peaks of Si-OH and asymmetric tensile vibrations of Si-O-Si, respectively. 1625.8 cm⁻¹ -1 and 3415.2cm -1 The absorption peaks at this location are due to the bending vibration and vibrational absorption peaks of the OH bond. These peaks may be caused by the stretching vibrations of hydroxyl groups in water molecules or hydroxyl groups on the surface of mesoporous silica, indicating the presence of silanol groups on the support surface. SBA-15-PLD shows an absorption peak at 1552.3 cm⁻¹. -1 and 1641.7cm -1 The presence of tensile vibration peaks for amide I and amide II, identical to those of PLD, at 1641.7 cm⁻¹ confirms that PLD has been immobilized on the mesoporous molecular sieve SBA-15. Furthermore, compared to SBA-15, SBA-15-PLD exhibits a higher peak at 1641.7 cm⁻¹. -1 The absorption peak at that point decreased, further proving that the main way to fix PLD is that the mesoporous molecular sieve SBA-15 forms a bond with PLD by consuming silanol groups.

[0059] 1.6 Study on the properties of free enzymes and immobilized enzymes

[0060] The properties of the immobilized PLD prepared under optimized conditions and the PLD enzyme solution with a concentration of 0.5 mg / mL were studied.

[0061] 1.6.1 Optimal Temperature and Thermal Stability

[0062] The optimal temperature for PLD determination is as follows: Take 2 mL of 0.5 mg / mL PLD enzyme solution or 0.1 g of immobilized PLD, six groups in total, with three replicates for each group. Use the method in the determination of PLD transphosphatidyl activity to measure the transesterase activity of free PLD and immobilized PLD at different temperatures (20, 30, 40, 50, 60, 70℃). Set the maximum transesterase activity as 100% and use this to calculate the relative transesterase activity of PLD at other temperatures.

[0063] The method for determining the thermal stability of PLDs is as follows: PLDs and immobilized PLDs were placed at different temperatures (20, 30, 40, 50, 60, 70℃) for 2 hours, then cooled to room temperature. The relative transesterase activity was determined according to the method for determining the transphosphatidyl activity of PLDs. The transesterase activity of the PLD before treatment was set as 100%. The relative transesterase activity of PLDs at different temperatures was calculated based on this. The results are shown in […]. Figure 6 .

[0064] Depend on Figure 6 As shown in A, before reaching the maximum relative transesterase activity, the relative transesterase activities of both free PLD and immobilized PLD increased with increasing temperature. After reaching the maximum relative transesterase activity, the relative transesterase activities of both began to decrease. After immobilization, the optimal reaction temperature of the enzyme increased from 40℃ to 50℃, and at 60℃, the immobilized PLD still retained 80.75% of its relative transesterase activity, while the free PLD retained only 29.71% of its relative transesterase activity. This indicates that the thermostability of the immobilized enzyme is superior to that of the free enzyme.

[0065] Depend on Figure 6 As shown in section B, the immobilized PLD exhibits stronger thermal stability than the free enzyme at different temperatures. Free PLD shows virtually no transesterase activity at 70℃, while the immobilized PLD retains 66.64% of its relative transesterase activity. This is likely because the PLD is immobilized within the pores of the SBA-15 carrier, avoiding direct contact with the external high-temperature environment and reducing the probability of PLD denaturation, thus maintaining its catalytic activity.

[0066] 1.6.2 Optimal pH and pH stability

[0067] The optimal pH for PLD determination is as follows: Six groups of 2 mL each of 0.5 mg / mL PLD enzyme solution and 0.1 g immobilized PLD were prepared, with three replicates per group. The transesterase activity of free and immobilized PLD was measured at different pH conditions (4, 5, 6, 7, 8, 9) using the method described in the PLD transphosphatidyl activity assay. The maximum transesterase activity was set as 100%, and the relative transesterase activity of PLD at other pH values ​​was calculated accordingly. Specifically, 0.2 M citrate-sodium citrate buffer was used for pH 3.0-6.0, 0.2 M disodium hydrogen phosphate-sodium dihydrogen phosphate buffer was used for pH 6.0-8.0, and 0.2 M Tris-HCl buffer was used for pH 7.0-9.0.

[0068] The pH stability of PLDs was determined as follows: PLDs and immobilized PLDs were stored in buffer solutions of different pH values ​​(4, 5, 6, 7, 8, 9) at 4℃ for 2 hours. The transesterase activity of PLDs was then measured according to the method described in the determination of PLD transphosphatidyl activity. The transesterase activity of the untreated PLDs was set as 100%. The relative transesterase activity of PLDs at different pH values ​​was calculated accordingly. The results are shown in […]. Figure 7 .

[0069] Depend on Figure 7 As shown in A, the optimal pH for both immobilized PLD and free PLD is the same, which is 7. This may be because the surface charge of the mesoporous molecular sieve SBA-15 is close to neutral, which means that the optimal pH of the immobilized PLD will not be shifted due to interference from the charge on the SBA-15 surface, thus maintaining consistency with the optimal pH of the free PLD.

[0070] Depend on Figure 7 As shown in section B, after immobilization, PLD maintains high transesterase activity at both low and high pH levels, and its pH adaptation range widens. This may be because the silanol groups on the SBA-15 carrier surface undergo proton exchange with water molecules, generating SiOH₂. + and SiO - Groups. This dynamic equilibrium regulates the pH of the microenvironment surrounding the PLD, allowing it to maintain a certain level of transesterification activity under excessively acidic or alkaline conditions. Therefore, immobilization technology can improve the pH stability of the PLD.

[0071] 1.6.3 Determination of Michaelis constant

[0072] The PLD-catalyzed transphosphatidylcholine reaction is a two-substrate reaction. To study the Michaelis constant of PLD, the transphosphatidylcholine activity was determined by controlling the concentration of substrate L-serine and varying the concentration of substrate PC (0.125, 0.25, 0.375, 0.5, 0.625 mol / L). The Km values ​​of the free and immobilized enzymes were calculated using a Lineweaver-Burk plot with 1 / [S] on the x-axis and 1 / [V] on the y-axis. Figure 8 The formula is as follows.

[0073]

[0074] Where V is the enzymatic hydrolysis rate in mg / min; Vmax is the maximum enzymatic hydrolysis rate in mg / min; Km is the Michaelis constant in mg / mL; and S is the substrate concentration in mg / mL.

[0075] In the kinetic study, the KL of free and immobilized PLD was calculated using Lineweaver-Burk curves with the reaction rates (1 / [V]) of free and immobilized PLD as the ordinate and different substrate concentrations as the abscissa (1 / [S]). m and V max .like Figure 8 As shown in A, the free PLD's K m and V max The concentrations were 2.14 mM and 4.83 mM / h, respectively, and the K of the immobilized PLD was... m and V max The concentrations were 2.2 mM and 2.94 mM / h, respectively. Where K... m (Free PLD) <K m (Immobilized PLD) It can be seen that the affinity of PLD for the substrate decreases after immobilization. The reason may be that the silanol groups on the surface of SBA-15 undergo proton exchange with water molecules, which changes the pH of the microenvironment around the immobilized PLD and causes changes in the three-dimensional structure of the PLD, thereby reducing the affinity of PLD for the substrate. In addition, it may also be that the PLD is randomly immobilized on the SBA-15 support by adsorption, so that some of the active sites of the PLD are blocked by the surface of the support, resulting in a decrease in the affinity of PLD for the substrate after immobilization.

[0076] 1.6.4 Storage stability

[0077] Free and immobilized PLDs were stored in phosphate buffer (0.2 M, pH 7) at 4°C for 50 days. Transesterase activity was measured every 5 days, with the PLD transesterase activity on day 0 set as 100%. The relative transesterase activity of PLDs at different storage times was calculated. The results are shown in [Figure number missing]. Figure 8 .

[0078] Depend on Figure 8 As shown in B, after 50 days of storage, the relative transesterase activity of free PLD was only 18.48%, while the relative transesterase activity of immobilized PLD remained at 40.61%. After immobilization, the half-life of the enzyme increased from 15 days to 35 days, and the storage stability was significantly improved.

[0079] 1.6.5 Operational Stability

[0080] After each reaction, the immobilized PLD was separated from the reaction system by centrifugation. The immobilized PLD was then washed multiple times with phosphate buffer (0.2 M, pH 7) and ethanol, respectively. The reaction was repeated under the same conditions, and the transesterase activity was measured for each reaction. The transesterase activity of the PLD from the first batch of reactions was set as 100%. The relative transesterase activity of the PLD under different reuse cycles was calculated, and the results are shown in [Figure number missing]. Figure 9 .

[0081] Reusability is an important indicator for evaluating the performance of immobilized enzymes, and the results are as follows: Figure 9 As shown, after three uses, the relative transesterification activity of free PLD was reduced to only 13.83%. This may be because prolonged contact between free PLD and organic solvents exposes the hydrophobic groups inside the PLD enzyme molecules to the surface. The PLD enzyme molecules attract each other through hydrophobic interactions, forming aggregates and thus reducing the transesterification efficiency of PLD. Furthermore, the reaction byproduct choline also affects the transesterification activity of PLD. In contrast, immobilized PLD retained 54.08% of its relative transesterification activity after six uses. This may be because the pore size of the SBA-15 carrier is close to the size of the PLD molecules, making it less prone to leakage from the pores of the SBA-15 during reuse, thus improving the reusability of PLD. However, with increasing uses of immobilized PLD, the relatively weak physical forces obtained through adsorption are insufficient to firmly bind PLD to the SBA-15 carrier, leading to leakage and inactivation of some PLD, resulting in the lower transesterification activity of immobilized PLD.

[0082] Example 2: Preparation of Phosphatidylserine by Immobilizing Phospholipase D

[0083] 12 mg of L-serine was dissolved in 2 mL of phosphate buffer (0.2 M, pH 7), and then 15% (by weight) of immobilized PLD (phosphatidylcholine) was added. Subsequently, 10 mg of phosphatidylcholine was dissolved in 5 mL of chloroform. The aqueous and organic phases were mixed and placed in a shaker at 40 °C for 12 h. After the reaction, a certain amount of the lower layer solution was taken, and the product (PS) was detected according to the method for phosphatidylserine. The reaction solution was then centrifuged, and the immobilized PLD and the PS product were recovered.

[0084] Detection of phosphatidylserine: This invention employs thin-layer chromatography (TLC) for the detection and analysis of phospholipids. First, the TLC plate is activated in a 110℃ drying oven for 1.5 hours, then cooled and placed in a desiccator for later use. Next, the test solution is drawn up using a capillary tube and spotted onto the silica gel plate one by one. After the solvent on the silica gel plate has evaporated, a pre-saturated TLC plate (developing solvent V) is inserted. 氯仿 V 无水乙醇 V 三乙胺 V 水 The development was performed using a solvent ratio of 10:11.3:11.7:2.7. When the distance between the developing solvent and the upper edge of the silica gel plate was only 1 cm, the silica gel plate was removed and dried in a fume hood. Then, it was stained with 0.04% bromothymol blue solution. After staining, the thin-layer chromatography plate was scanned, and the gray values ​​of each component were calculated using ImageJ analysis software. Figure 1It can be seen that the concentrations of PC and PS are linearly related to their gray values. Therefore, the yield of phosphatidylserine can be calculated using the following formula.

[0085]

[0086] Where nPC0 is the amount of substance of PC, in mol; and nPS is the amount of substance of PS, in mol.

[0087] (1) Construction of PC and PS standard curves: PC and PS standards were dissolved in chloroform to prepare solutions of 0.2, 0.4, 0.6, 0.8, and 1 mg / mL, respectively. The sample volume was 20 μL for each solution. The gray values ​​of the spots were obtained through plate spotting, development, staining, scanning, and software analysis. A gray value-concentration standard curve was then constructed, as shown below. Figure 1 As shown.

[0088] (2) Determination of PC and PS in the sample: A certain amount of sample was drawn up with a capillary tube, placed on a preheated silica gel plate, dried, and then placed in a thin-layer chromatography tube containing the developing solvent for development. After development, the sample was stained with 0.04% bromothymol blue solution, dried, and then the thin-layer plate was scanned. The gray values ​​of the spots were obtained using ImageJ analysis software. Based on the PC and PS standard curves, the content (mg) of PC and PS in the sample was calculated.

[0089] 2.1 Optimization of reaction conditions

[0090] The main factors affecting the synthesis of PS by immobilized PLD catalysis are: phase-to-phase volume ratio, substrate molar ratio, reaction time, reaction temperature, and reaction pH. Therefore, this invention uses PS yield as an indicator to investigate the effects of different phase-to-phase volume ratios (chloroform to phosphate buffer solution) (1:1, 2:1, 4:1, 6:1, 8:1), different substrate molar ratios (L-serine to PC) (2:1, 4:1, 6:1, 8:1, 10:1, 12:1), different reaction times (3, 6, 9, 12, 15, 18, 21, 24 h), different reaction temperatures (20, 30, 40, 50, 60, 70 °C), and different reaction pH (4, 5, 6, 7, 8, 9) on PS synthesis conditions. The results are shown in [Figure number missing]. Figure 10 .

[0091] Depend on Figure 10As shown in section A, with the increase of the proportion of organic phase (chloroform) in the reaction system, the yield of PS initially increases and then remains constant. The yield reaches its maximum value of 52.88 ± 2.16% when the ratio of organic phase to aqueous phase is 4:1. This may be because when a large amount of water is present in the reaction system, water molecules compete with L-serine for the five-coordinate phosphatidylhistidine intermediate, leading to a greater hydrolytic activity of PLD than its transesterification activity, resulting in the large-scale formation of the byproduct PA. However, as the proportion of organic phase continues to increase, the yield of PS does not change significantly. Therefore, a volume ratio of 4:1 for organic phase to aqueous phase is selected as the optimal volume ratio for the synthesis of PS in this system.

[0092] Depend on Figure 10 As shown in section B, the yield of PS initially increases with the substrate molar ratio and then remains constant. The PS yield reaches its maximum value of 53.7 ± 0.71% when the molar ratio of L-serine to PC is 10:1. This is likely because increasing the concentration of L-serine in the reaction system not only enhances its binding affinity to the pentacoordinate phosphatidylhistidine intermediate but also ensures effective binding of L-serine to the active site of PLD by saturating non-critical binding sites on the PLD surface, thereby increasing the synthesis efficiency of PS. Therefore, this invention selects a molar ratio of L-serine to PC of 10:1 as the optimal molar ratio for PS synthesis in this system.

[0093] Depend on Figure 10 As shown in Figure C, the yield of PS initially increases with the catalytic time of the immobilized PLD and then remains constant. The PS yield reaches its maximum value of 54.19 ± 2.18% when the reaction time is 12 h. However, when the reaction time exceeds 12 h, the PS yield remains essentially unchanged. This is likely because the concentrations of the substrate PC or L-serine in the reaction system are already very low, so even increasing the reaction time does not significantly alter the PS yield. Therefore, the optimal reaction time for PS synthesis is 12 h.

[0094] Depend on Figure 10 As shown in Figure D, the yield of PS first increases and then decreases with the reaction temperature of the immobilized PLD. The yield reaches its maximum value (51.46 ± 1.76%) when the reaction temperature for PS synthesis catalyzed by immobilized PLD is 50℃. This indicates that at lower reaction temperatures, not only is the transesterification activity of PLD not fully activated, but the viscosity of the reaction system also increases, thereby increasing the mass transfer resistance between the immobilized PLD and the substrate, ultimately leading to a decrease in PS yield. Conversely, when the reaction temperature exceeds 50℃, the structure of the immobilized PLD is destroyed in the high-temperature environment, causing PLD deactivation and resulting in a decrease in PS yield. Therefore, the optimal reaction temperature for PS synthesis is 50℃.

[0095] Depend on Figure 10As shown in E, the PS yield first increases and then decreases with the change of pH in the immobilized PLD reaction. The PS yield reaches its maximum value (53.53 ± 0.97%) when the pH of the PLD-catalyzed PS synthesis reaction is 7, indicating that the transesterification activity of PLD can only be activated at the optimal pH, maximizing the PS yield. However, when PLD is exposed to excessively acidic or alkaline environments, its tertiary structure changes, thus reducing the PS yield. At pH 6, the PS yield remains at 48.33 ± 1.62%, indicating that the PS yield does not vary significantly within the pH range of 6-7. This may be because, in the transesterification reaction catalyzed by immobilized PLD, the silanol groups on the support surface undergo proton exchange with water molecules to generate SiOH₂. + and SiO - The dynamic equilibrium regulates the pH of the microenvironment surrounding the PLD, allowing it to maintain high transesterification activity at pH 6 while ensuring that the PS yield remains unaffected. Therefore, the optimal reaction pH for PS synthesis is 7.

[0096] 2.2 Response Surface Optimization

[0097] 2.2.1 Response Surface Optimization Design

[0098] Based on the optimization of reaction conditions in section 2.1, where the substrate molar ratio was determined to be 10:1 and the reaction pH to be 7, a response surface methodology was designed. Using grafting degree as the response value and the two-phase volume ratio, reaction time, and reaction temperature as independent variables, a Box-Behnken response surface methodology was applied to design a three-factor, three-level optimization process for PS preparation. The factor level design is shown in Table 1. In the factor level coding table, -1 represents the lowest level, 0 represents the intermediate level, and 1 represents the highest level.

[0099] Table 1. Coding table of response surface experimental factors at different levels

[0100] The Design Expert 7.1 software was used to design a three-factor (two-phase volume ratio, reaction time, and reaction temperature) three-level experimental design, and the results are shown in Table 2.

[0101] Table 2 Box-Behnken Experimental Design and Results

[0102] 2.2.2 Model Construction and Data Analysis

[0103] Fitting the experimental data in the table, the regression equation for PS yield (Y) is as follows: Y=58.56-0.36A-8.78B+1.73C-0.66AB+1.15AC-1.3BC-2.21A2 -11.19B 2 -2.27C 2 .

[0104] The results of the analysis of variance and significance test are shown in Tables 3 and 4. The model calibration determination coefficient R0 2 Adj = 0.9881 indicates that the model's predicted value has a 98.81% feasibility rate. The model's coefficient of determination R0 2 =0.9958, indicating that the model has a good fit and small error. The regression model p<0.0001 (highly significant), the lack-of-fit term p>0.05 (not significant), and there are no factors of lack of fit, indicating that the model has good significance and small bias. The F-test shows that the factor contribution rate is B>C>A, that is, reaction temperature>reaction time>two-phase volume ratio.

[0105] Table 3. Grafting Degree Regression Model Coefficient Test

[0106] Table 4. Correlation Coefficients of Simulation Equations

[0107] 2.2.3 The impact of the interaction of various factors on the grafting degree of PS yield

[0108] In a response surface graph, the darker the contour lines representing the two factors, the stronger the interaction; the closer the graph is to a circle, the weaker the interaction. The 3D graph is a downward-opening curved surface; the steeper the surface, the stronger the interaction. Figures 11-13 It can be seen that the interaction between reaction temperature and the volume ratio of the two phases has the strongest effect on the degree of branching.

[0109] 2.2.4 Validation Experiment of Response Surface Results

[0110] The optimal conditions, derived from the simulation equations, are: an organic phase to aqueous phase volume ratio of 3.476:1, a reaction temperature of 45.327℃, and a reaction time of 12.649 h. Under these conditions, the predicted PS yield is 60.414%. For ease of practical operation, the optimal conditions were selected: an organic phase to aqueous phase volume ratio of 3.5:1, a reaction temperature of 45.3℃, and a reaction time of 12.6 h. Three parallel experiments were conducted under these conditions, and the average PS yield was 59.87 ± 0.35%. Compared with the model prediction, the relative error was 0.91%, indicating that the model has a high degree of fit and good repeatability.

[0111] 2.3 Phospholipid Composition Analysis of Phosphatidylserine

[0112] PLD is a special ester bond hydrolase that can simultaneously perform transesterification and hydrolysis reactions, which compete with each other. Therefore, in the enzymatic synthesis of PS, in addition to detecting the PS content, the transesterification efficiency of PLD can also be evaluated by detecting the content of the hydrolysis byproduct PA. 31 The phospholipid composition in PS samples was determined by P NMR as follows: 80 mg of sample was weighed, and 5 μL of trimethyl phosphate (TPP) solution diluted with chloroform was added as an internal standard (V:V, 1:50). 0.6 mL each of deuterated chloroform, methanol, and EDTA-Cs (0.2 M, pH 8.5) were added sequentially to the sample. After mixing, the deuterated chloroform layer was aspirated and filtered through a 0.22 μm organic filter into an NMR tube. Detection conditions were: operating frequency 600 MHz, PABBONMR probe, temperature 25 °C, pulse width 11.2 μs, pulse delay time 2 s, 16 scans, spectral width 64102 Hz, and 65536 sampling points. Results are shown below. Figure 14 Using MestReNove software, the peak areas of the NMR peaks of the substrate PC and the product PS were normalized to obtain the phospholipid composition and relative content of the substrate PC and the product PS. The results are shown in Table 5.

[0113] Table 5. Phospholipid composition of reaction substrate PC and reaction product PS

[0114] Table 5 shows that the phospholipids in the substrate PC are mainly composed of PC and PI, with PC having the highest content (93.33%) and PI having the lowest content (6.67%). Table 5 also shows that when PC is used as the substrate for the transesterification reaction, the phospholipids in the product are mainly PS (60.85%), indicating that the immobilized PLD can produce high-purity PS under these reaction conditions. The other phospholipids and their contents are PC (14.52%), PI (3.88%), and PA (20.75%), respectively. PA accounts for 20.75% of the total phospholipids, indicating that even under optimal reaction conditions, the immobilized PLD still exhibits severe hydrolysis. This may be because the presence of water in the reaction system cannot prevent the occurrence of side hydrolysis; or it may be that the abundant silanol groups on the surface of SBA-15 can adsorb a large number of water molecules through hydrogen bonds, forming a localized high-water-concentration microenvironment, thereby promoting the hydrolysis reaction of the PLD. Furthermore, the present invention uses an adsorption method to prepare immobilized PLDs. The PLD enzyme molecules are randomly immobilized on the SBA-15 carrier, which may cause some of the enzyme's active sites to be blocked by the carrier surface. Ultimately, this prevents the substrate L-serine from binding smoothly to the enzyme's active site, while water molecules are less affected, thus promoting the hydrolysis reaction.

[0115] Example 3: Neuroprotective effect of phosphatidylserine

[0116] 3.1 Preparation of phosphatidylserine liposomes

[0117] Phosphatidylserine liposomes were prepared according to the method of Xu (Xu Z, Li Q, Ding L, et al. A comparative study of the effects of phosphatidylserine rich in DHA and EPA on Aβ-induced Alzheimer's disease using cell models.[J].Food & function, 2021, 12(10):4411-4423.DOI:10.1039 / d1fo00286d.). Phosphatidylserine (PS) obtained by the method in Example 2 was mixed with cholesterol at a 1:1 molar ratio and dissolved in chloroform. The mixture was then dried under reduced pressure at 30°C using a rotary evaporator to form a uniform lipid film. 5 mL of preheated phosphate buffer solution (0.2 M, pH 7) was added, and the mixture was hydrated by shaking at 55°C for 2 h, suspending the lipid film on the phosphate buffer solution. Subsequently, the lipid film suspension, which had been sonicated for 2 minutes, was repeatedly extruded 20 times using a liposome extruder equipped with a 200 nm polycarbonate membrane to obtain PS liposomes. The particle size and potential of the PS liposomes were measured using a Malvern particle size analyzer.

[0118] 3.2 Culture of SH-SY5Y cells

[0119] (1) Cell resuscitation

[0120] Following the principle of rapid thawing, cryovials containing SH-SY5Y cells (purchased from Shanghai) were rapidly thawed in a 37°C water bath. After disinfection with 75% medical alcohol, the thawed cell suspension was aspirated, injected into a sterile centrifuge tube, and 5 mL of cell culture medium containing 10% fetal bovine serum (FBS) was added. The tube was centrifuged at 1000 rpm for 5 min, the supernatant was removed, 1 mL of culture medium was added, and the tube was resuspended. The suspension was then appropriately diluted with cell culture medium containing 20% ​​FBS and inoculated into culture flasks, which were then placed in a 37°C, saturated humidity, 5% CO2 incubator.

[0121] (2) Cell culture

[0122] SH-SY5Y cells were seeded in complete culture medium and cultured at 37°C, saturated humidity, and 5% CO2. The culture medium was changed every 24 hours, and the passage frequency was 2-3 days.

[0123] (3) Cell passage

[0124] When the cells adhere to the wall to 70%-80%, discard the old culture medium and wash with PBS 2-3 times. Then add 0.25% trypsin to digest until the cells are completely detached. After centrifugation (1000 rpm, 3 min), discard the supernatant, add culture medium and pipette to form a cell suspension. Then passage the cells at a ratio of 1:3.

[0125] (4) Cell cryopreservation

[0126] Cells in logarithmic growth phase were passaged and expanded. Cells were digested, collected, and counted. After centrifugation (1000 rpm, 3 min), the supernatant was discarded. Pre-freezing buffer was then added to resuspend the cells at 1×10⁶ cells / mL. 7 / mL, dispensed into cryovials (1mL / tube), placed in a gradient cooling cryovial at -80℃ overnight, and then transferred to liquid nitrogen for storage.

[0127] 3.3 SH-SY5Y cell treatment and grouping

[0128] Once SH-SY5Y cells reached the logarithmic growth phase, 100 μL of their cell suspension was seeded into 96-well plates at a density of 1 × 10⁶ cells per well. 4 Then, transfer them to an incubator and incubate for 24 hours. Remove the culture medium and group them as follows.

[0129] Blank control group: serum-free culture medium; Model group: serum-free culture medium + different concentrations (5, 10, 20, 30, 40 μM) of A β 25-35 ; PS treatment group: serum-free culture medium + different concentrations (10, 20, 30, 40, 80 μM) of PS liposomes + 40 μM A β 25-35 .

[0130] 3.4 CCK8 assay for SH-SY5Y cell viability

[0131] Cell viability was assessed using the Cell Counting Kit. Passaged SH-SY5Y cells were seeded into 96-well plates (100 μL per well) and cultured for 24 h. The culture medium was then removed. Cells were grouped according to step 3.3 and treated accordingly, then cultured for another 24 h. The absorbance of each well was measured at 450 nm according to the kit instructions, and cell viability was calculated. Three replicates were set up for each cell group. When the cell viability in the model group decreased to approximately 50%, the AD cell model was successfully established. When cell viability was greater than 90%, PS showed no toxicity to the cells.

[0132]

[0133] 3.5 RNA sequencing and gene expression analysis

[0134] After the cell experiments, cell samples were pre-frozen in liquid nitrogen for 2 minutes and then transported via dry ice to Kang Sheng Xu Yuan Biotechnology Co., Ltd. for transcriptome sequencing (RNA-seq). After standardizing the expression level of each gene, the gene expression level was calculated using fragments per kilobase (FPKM). The screening criteria for differentially expressed genes (DEGs) in different treatment groups were: pvalue ≤ 0.05 and |log2(foldchange)| ≥ 0.585, and correlation analysis was performed between different treatment groups. Furthermore, to explore the mechanism of action of PS, a heatmap analysis of the expression of relevant genes in different treatment groups was performed based on the DEGs of the PS treatment groups.

[0135] 3.6 Screening of phosphatidylserine targets for Alzheimer's disease

[0136] First, the PubChem database was searched for "Phosphatidylserine" to obtain the SMILES structure of PS. Then, the target genes of phosphatidylserine were predicted in databases including SwissTargetPrediction, SimilarityEnsembleApproach, TargetFishing, Super-PRED, Netlfer@LMMD, ChemMapper, and PharmMapper. Next, using "Alzheimer's disease" as the keyword, Alzheimer's disease targets were predicted in databases including GeneCards, Drugbank, Pharmgkb, OMIM, DisGeNET, and TherapeuticTargetDatabase. The predicted target genes were then corrected and duplicated using the UniProt database. Finally, Venn diagram mapping was used to map the targets of PS and AD to obtain the intersection targets of PS and AD.

[0137] 3.7 Construction of Protein-Protein Interaction (PPI) Networks

[0138] Intersecting target data were uploaded to the STRING database. Protein-protein interactions were screened using an interaction score greater than 0.4, discrete points were hidden, and a PPI network diagram of target interactions was generated. The data was then exported in TSV format. Subsequently, the data was imported into Cytoscape 3.9.1 software, and the CytoNCA plugin was used for in-depth analysis of the network topology parameters. Core targets were screened based on Degree values, with the screening criteria set to a Degree greater than three times the median Degree.

[0139] 3.8 GO and KEGG enrichment analysis

[0140] Intersecting targets were uploaded to the DAVID database for Gene Ontology (GO) functional analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. Using P < 0.05 as a criterion, and sorting by P value from smallest to largest, the top 10 biological processes (BP), cellular components (CC), and molecular functions (MF) with the highest P values ​​were selected from the GO enrichment analysis results, and their data were plotted as bar charts using the MicroBioinformatics online platform. The top 20 signaling pathways with the highest P values ​​were selected from the KEGG enrichment analysis results, and their data were plotted as bubble charts using the MicroBioinformatics online platform.

[0141] 3.9 Molecular docking

[0142] This invention utilizes Discovery Studio 2019 to perform molecular docking between a protein molecule called a ligand (PS) and the target protein, simulating the binding mode and affinity of the ligand and receptor through ligand-receptor interactions. First, the PDB file of the target protein is obtained from the PDB protein database and then imported into Discovery Studio 2019. The protein structure undergoes processing, including water molecule removal, hydrogenation, and decontamination. Subsequently, the PS structure is drawn using Chemdraw software, exported and saved in .mol ​​format. The preprocessed protein structure is then molecularly docked with the PS, and the Libdock score is used as the evaluation criterion. A higher Libdock score indicates higher activity of the small molecule binding to the receptor and better interaction; conversely, a lower score indicates poorer interaction.

[0143] 3.10 Data Analysis and Statistics

[0144] Excel 2010 was used for data processing and calculation. SPSS 21.0 statistical software was used for statistical analysis of the results to assess the significance level of p<0.05. All experiments were repeated 3 times. The results are expressed as mean ± standard deviation. Origin 2019 was used for plotting.

[0145] 3.11 Results

[0146] 3.11.1 Particle size and Zeta potential of phosphatidylserine liposomes

[0147] The phosphatidylserine liposomes prepared by the thin-film dispersion method in this invention are shown in the figure below for particle size and zeta potential. Figure 15.

[0148] Depend on Figure 15 It can be seen that the phosphatidylserine liposomes prepared by the thin film dispersion method of the present invention have a particle size of 188.77±2.93nm and a zeta potential of -73.73±0.86mV, respectively, indicating that the thin film dispersion method successfully prepared phosphatidylserine liposomes with small particle size and uniform distribution.

[0149] 3.11.2 Establishment and evaluation of AD cell models

[0150] To successfully construct an AD cell model, this invention investigated different concentrations of A β 25-35 The effect on SH-SY5Y cell viability was as follows: Figures 16-17 As shown.

[0151] Depend on Figure 16 It can be seen that, with A β 25-35 With increasing concentration, the cell viability of SH-SY5Y cells significantly decreased, when A β 25-35 At a concentration of 40 μM, cell viability decreased to 55.15 ± 4.58%, indicating successful construction of the AD cell model. Figure 17 As can be seen, compared with the control group, the morphology of SH-SY5Y cells changed with A β 25-35 As the concentration increases, significant changes occur: synapses around cells shorten or even disappear, and cell number and density decrease significantly, indicating that A... β 25-35 It can stimulate cell death, thereby altering the morphology of SH-SY5Y cells. Therefore, this invention uses 40 μM A β 25-35 To construct an AD cell damage model.

[0152] 3.11.3 Effects of PS on cell viability and morphology

[0153] After pretreatment of SH-SY5Y cells with different concentrations of PS, 40 μM A was used. β 25-35 The concentration induced damage, and the results were as follows: Figures 18-19 As shown.

[0154] Depend on Figure 18 It can be seen that the cell viability of SH-SY5Y cells significantly increased with increasing PS concentration. When the PS concentration was 80 μM, the cell viability increased to 98.73 ± 3.85%, indicating that PS was not toxic to SH-SY5Y cells. Figure 19As shown. Compared with the model group, SH-SY5Y cells pretreated with PS showed the appearance of synapses, and their morphology tended to return to normal, indicating that PS repaired the damage caused by A to some extent. β 25-35 SH-SY5Y cells induced to suffer damage.

[0155] 3.11.4 Sample Data Processing and Quality Control Analysis

[0156] From a statistical perspective, the base distribution and quality fluctuation of all reads were analyzed, as shown in Table 6. Q20 was higher than 98% and Q30 was higher than 96%, indicating that the sample quality was good and the sequencing results met the requirements for subsequent analysis.

[0157] Table 6. Transcriptome sequencing data statistics

[0158] 3.11.5 Differential Gene Analysis

[0159] (1) Differential gene analysis

[0160] Differentially expressed genes (DEGs) were screened based on the criteria of pvalue ≤ 0.05 and |log2(foldchange)| ≥ 0.585. Figures 20-21 As shown, the control and model groups had a total of 93 differentially expressed genes (DEGs), including 56 upregulated genes and 37 downregulated genes. The model group and the PS treatment group had a total of 980 differentially expressed genes, including 684 upregulated genes and 296 downregulated genes. Cluster analysis of co-expressed genes, as shown... Figure 22 As shown, the gene expression trends in the model group and the PS treatment group are significantly different.

[0161] (2) Enrichment analysis of GO and KEGG

[0162] To study PS in A β 25-35 In this invention, 980 differentially expressed genes identified between the model group and the PS treatment group were imported into the DAVID database for GO enrichment analysis and KEGG pathway analysis, targeting the biological processes involved in the induced SH-SY5Y cell damage model. The results of the first 10 GO enrichment analyses were visualized in descending order of P-value. Figure 23As shown, in biological processes (BP), differentially expressed genes were significantly enriched in signal transduction, angiogenesis, and cartilage condensation. In cellular components (CC), differentially expressed genes were significantly enriched in the extracellular matrix, extracellular region, and plasma membrane. In molecular functions (MF), differentially expressed genes were significantly enriched in cholesterol binding.

[0163] KEGG pathway enrichment was performed using P < 0.01 as the criterion, resulting in 13 signal pathways. Bubble plots were then generated in ascending order of P values. The results are shown below. Figure 24 As shown in the figure, the horizontal axis represents the enrichment score, and the vertical axis represents the signaling pathway. The number of genes determines the size of the bubbles; the more genes enriched in a signaling pathway, the larger the bubble. The bubble color is correlated with the P-value; the redder the color, the smaller the P-value, and the redder the color and the larger the bubble, the stronger the association. Considering the P-value, the differentially expressed genes between the model group and the PS treatment group were found to be mainly enriched in pathways such as neuroactive ligand-receptor interaction, calcium signaling pathway, NF-κB signaling pathway, and MAPK signaling pathway. These pathways may be related to PS anti-A. β 25-35 Key signaling pathways that induce SH-SY5Y cell damage.

[0164] 3.11.6 Transcriptomics combined with network pharmacology to explore the neuroprotective mechanism of phosphatidylserine

[0165] (1) Screening of target genes and construction of PPI network

[0166] Intersection analysis was performed on 414 intersection target genes of PS for AD predicted by network pharmacology and 980 differentially expressed genes obtained by transcriptome sequencing analysis. The results are as follows: Figure 25As shown, a total of 14 intersection genes were obtained, mainly including PTGFR, CTSS, PTGS2, CHRM3, MME, CNR1, SYK, TLR4, CYP2E1, SLCO1B1, SLC6A2, NQO1, CYP2D6, and WAS. The intersection gene data was uploaded to the STRING database to construct a PPI network for data visualization. A moderate confidence level of 0.4 was selected, and disconnected nodes in the network were hidden. The results yielded 14 nodes, 19 edges, an average node degree of 2.71, and a p-value of 1.0e. -8 (Statistically significant). The data was then exported in TSV format, and the network topology parameters of related protein interactions were calculated using the CytoNCA plugin in Cytoscape software. The 14 intersecting genes were sorted according to their Degree values, as shown below. Figure 26 As shown, the closer the node color is to purple, the larger the Degree value; the closer it is to blue, the smaller the Degree value.

[0167] (2) Enrichment analysis of GO and KEGG

[0168] GO enrichment analysis was performed on 14 overlapping targets in the DAVID database, yielding 33 biological processes (BP), 10 cellular components, and 7 molecular functions. The targets were then sorted according to their p-values, and the top 10 were used to create a histogram. Results are as follows: Figure 27 As shown, biological processes mainly focus on the lipopolysaccharide response and the exogenous substance metabolism process; cellular composition mainly focuses on the cytoplasm and plasma membrane; and molecular function mainly focuses on heme binding.

[0169] Five pathways were obtained through KEGG pathway enrichment and sorted according to their p-values ​​from smallest to largest. A bubble chart was then plotted, and the results are as follows: Figure 28As shown, the intersection genes between PS and AD predicted by network pharmacology and differentially expressed genes obtained from transcriptome sequencing analysis were mainly enriched in the NF-κB signaling pathway. The NF-κB signaling pathway is closely related to the pathogenesis of AD, playing a central role in regulating key processes such as neuroinflammation, β-amyloid (Aβ) deposition, tau protein hyperphosphorylation, and neuronal death. Studies have shown that NF-κB, as a central regulator of neuroinflammation, can inhibit the excessive release of pro-inflammatory molecules, thereby reducing neuronal damage caused by inflammation. Based on these results, it is speculated that the therapeutic effect of PS on Alzheimer's disease is related to its inhibitory effect on the NF-κB signaling pathway.

[0170] (3) Molecular docking

[0171] To verify the results of the KEGG pathway enrichment analysis of the above-mentioned intersection genes, this invention selected genes related to the NF-κB signaling pathway from 14 intersection genes, namely PTGS2 (PDB ID: 5KIR), TLR4 (PDB ID: 7AAH) and SYK (PDB ID: 5YJQ), and performed molecular docking with PS respectively to obtain Libdock scores, as shown in Table 7.

[0172] Table 7 Molecular docking results between PS and the target.

[0173] As shown in Table 7, PS has a libdock of 81.532 with PTGS2 and binds to LYS-180 via hydrogen bonding, and to LEU-171, LEU-163, and PRO-163 via hydrophobic interactions (alkyl groups). PS has a libdock of 138.297 with TLR4 and binds to ARG-93 via hydrogen bonding, to ARG-93 and ASP-89 via electrostatic interactions, and to ARG-141 via hydrophobic interactions. PS has a libdock of 265.245 with SYK and binds to ARG-574, LYS-571, TYR-573, VAL-560, LEU-585, and TRP-556 via hydrophobic interactions (alkyl and Π-alkyl groups).

[0174] Among them, PTGS2, TLR4, and SYK target proteins can activate the NF-κB signaling pathway through their respective signaling pathways. PTGS2, or prostaglandin intraperoxidase 2, also known as cyclooxygenase-2 (COX-2), catalyzes the conversion of arachidonic acid to prostaglandins (PGE2). PGE2 binds to EP receptors (such as EP1 / EP2), thereby further activating the NF-κB signaling pathway. TLR4 (Toll-like receptor 4, TLR4) is a key member of the Toll-like receptor family. When it binds to its co-receptor MD-2, it can effectively recognize and bind to LPS, thereby activating NF-κB through the MyD88-dependent pathway. SYK (Spleen Tyrosine Kinase, SYK) is a non-receptor tyrosine kinase that, after binding to phosphorylated Dectin-1, participates in the assembly of CARD9-Bcl-10-MALT1, and then activates the IκB kinase complex, thereby inducing the activation of the NF-κB signaling pathway. Transcriptomics, network pharmacology, and molecular docking results suggest that PS may inhibit the activity of PTGS2, TLR4, and SYK target proteins, thereby downregulating the activity of the NF-κB signaling pathway and ultimately playing a role in the treatment of Alzheimer's disease.

[0175] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing immobilized phospholipase D, characterized in that, Includes the following steps: (1) Mix the mesoporous molecular sieve SBA-15 with sodium acetate buffer solution to obtain a mixture; (2) Mix the mixture with the enzyme solution, shake, centrifuge, collect the precipitate, wash, and freeze dry to obtain immobilized phospholipase D.

2. The preparation method according to claim 1, characterized in that, The mass-to-volume ratio of the mesoporous molecular sieve SBA-15 to sodium acetate buffer is 1g:15~25mL, and the concentration of the sodium acetate buffer is 0.1~0.3mol / L.

3. The preparation method according to claim 1, characterized in that, The enzyme solution is prepared by mixing phospholipase D enzyme solution with anhydrous ethanol, reacting in an ice bath for 45-75 min, centrifuging, collecting the precipitate, obtaining purified phospholipase D, and dissolving the purified phospholipase D in sodium acetate buffer to obtain the enzyme solution. The volume ratio of the phospholipase D enzyme solution to anhydrous ethanol is 1~5:

1.

4. The preparation method according to claim 1, characterized in that, The volume-to-mass ratio of the enzyme solution added to the mesoporous molecular sieve SBA-15 is 10~45mL:1g.

5. The preparation method according to claim 1, characterized in that, The oscillation temperature is 4~50℃; the oscillation time is 2~12h; the cleaning solution is sodium acetate buffer solution; the pH of the sodium acetate buffer solution is 4~9.

6. A method for preparing phosphatidylserine by immobilizing phospholipase D, characterized in that, Includes the following steps: (6.1) Dissolve L-serine in buffer solution, add immobilized phospholipase D and mix to form the aqueous phase; dissolve phosphatidylcholine in chloroform to form the organic phase; (6.2) Mix the aqueous phase and the organic phase and react for 3~24h to prepare phosphatidylserine and recover the immobilized phospholipase D; The immobilized phospholipase D was obtained using the preparation method described in any one of claims 1 to 5.

7. The method according to claim 6, characterized in that, The buffer solution is a phosphate buffer solution; the volume ratio of chloroform to buffer solution is 1~8:

1.

8. The method according to claim 6, characterized in that, The molar ratio of L-serine to phosphatidylcholine is 1~12:

1.

9. The method according to claim 6, characterized in that, The reaction temperature is 20~70℃, and the pH of the reaction is 4~9.

10. The use of phosphatidylserine obtained by the method according to any one of claims 6 to 9 in the preparation of drugs for treating Alzheimer's disease.

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