Immobilized phospholipase D based on biological imprinting as well as preparation method and application of immobilized phospholipase D

Abstract

The invention belongs to the technical field of phosphatidylserine production, and particularly relates to immobilized phospholipase D based on biological imprinting and a preparation method and application thereof. The preparation method comprises the following steps: S1, dissolving L-serine in an acetic acid buffer solution to obtain a serine solution, adding a phospholipase D solution into the serine solution, and stirring to obtain a mixed solution; s2, adding sodium alginate into the mixed solution, stirring and dissolving to obtain a biopolymer solution; s3, adjusting the pH value of the chitosan solution to be neutral, then adding CaCl2 into the chitosan solution, and stirring and dissolving to obtain a CaCl2-containing chitosan solution; and S4, dropwise adding the biopolymer solution into a chitosan solution containing CaCl2, complexing to form microspheres, collecting the microspheres, and washing and drying the microspheres to obtain the immobilized phospholipase D based on biological imprinting. According to the method, the biological imprinting technology and the enzyme immobilization technology are combined, the immobilized PLD with high selectivity and high stability is prepared, and the phosphatidyl transfer reaction efficiency is improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of phosphatidylserine production, and particularly relates to an immobilized phospholipase D based on bioprinting, a preparation method thereof and an application thereof. Background Art

[0002] Phospholipids are major components of biological membranes and play a crucial role in signal transmission and metabolism. Phosphatidylserine (PS) plays a crucial role in activating brain cells, improving Alzheimer's disease in the elderly, treating attention deficit hyperactivity disorder in children, enhancing brain cell vitality, and combating depression. It has been hailed as an emerging "smart nutrient" and "brain gold" (DHA), primarily used in functional foods and the pharmaceutical industry. However, PS is present in very low concentrations in nature, and its extraction is difficult, resulting in extremely low yields, making it a rare phospholipid. Enzymatic methods are currently the most effective method for preparing rare phospholipids. Phosphatidylcholine (PC) is extremely abundant in nature. Currently, the preparation of PS via a phospholipase D (PLD)-mediated phosphatidyl transfer reaction using inexpensive PC and L-serine as substrates has attracted considerable attention due to its mild reaction conditions, environmental friendliness, safety, and low cost, making it a research hotspot in phospholipid preparation and modification. By utilizing this characteristic of PLD, efficient and large-scale bioproduction of high-value rare phospholipids PS can be achieved.

[0003] However, free PLD suffers from structural fragility, poor stability, difficulty in recycling, and high production costs, limiting the development and application of phospholipid resources. To improve the stability and utilization of PLD, free PLD must be modified through enzyme immobilization to produce high-performance biocatalysts. However, immobilized enzyme technology rigidifies the enzyme's tertiary structure, hindering its rational structural changes. This results in reduced specific activity and selectivity of immobilized PLD, hindering its industrial application.

[0004] Enzyme bioimprinting technology uses ligand induction and the hysteresis of enzyme molecules to rigidify the enzyme molecule in a "superactivated structure," forming a conformation that is conducive to catalysis. However, the greatest difficulty in applying bioimprinting technology is that the imprinted enzyme structure induced by ligand cannot be maintained in water. Water molecules will stretch the induced structure, converting it into the natural enzyme molecular structure, eliminating the imprinting effect. Summary of the Invention

[0005] In view of the problems and shortcomings in the prior art, the present invention aims to provide an immobilized phospholipase D based on bioprinting, and a preparation method and application thereof.

[0006] Based on the above purpose, the present invention adopts the following technical solutions:

[0007] The first aspect of the present invention provides a method for preparing immobilized phospholipase D based on bioprinting, comprising the following steps:

[0008] S1: dissolving L-serine in acetate buffer to obtain a serine solution, adding phospholipase D solution to the serine solution, and stirring at 0-4°C for 30 minutes to obtain a mixed solution;

[0009] S2: adding sodium alginate to the mixed solution prepared in step S1 and stirring to dissolve, thereby obtaining a biopolymer solution;

[0010] S3: adjusting the pH value of the chitosan solution to neutral, then adding CaCl2 to the chitosan solution and stirring to dissolve it, thereby obtaining a chitosan solution containing CaCl2;

[0011] S4: adding the biopolymer solution prepared in step S2 dropwise to the chitosan solution containing CaCl2 prepared in step S3 to form microspheres through complexation, collecting the microspheres, washing the microspheres with water, and drying them to obtain the immobilized phospholipase D based on bioprinting.

[0012] Preferably, in step S1, the mass ratio of L-serine to phospholipase D in the mixed solution is (22-33):1.

[0013] Preferably, in step S2, the mass concentration of sodium alginate in the biopolymer solution is 0.01 g / mL to 0.02 g / mL.

[0014] Preferably, in step S3, the mass concentration of chitosan in the chitosan solution is 0.003 g / mL to 0.009 g / mL.

[0015] Preferably, in step S3, the molar concentration of CaCl2 in the chitosan solution containing CaCl2 is 0.2 mol / L to 0.4 mol / L.

[0016] Preferably, in step S4, the complexation time is 30s to 120s.

[0017] Preferably, in step S1, the concentration of the acetate buffer is 0.2 mol / L; the concentration of the phospholipase D solution is 1 mg / mL to 1.5 mg / mL.

[0018] The second aspect of the present invention provides an immobilized phospholipase D based on bioprinting prepared by the preparation method described in the first aspect.

[0019] The third aspect of the present invention provides the use of the bio-imprinted immobilized phospholipase D described in the second aspect in the preparation of phosphatidylserine.

[0020] The fourth aspect of the present invention provides a method for preparing phosphatidylserine, comprising the following steps: mixing an aqueous phase containing L-serine, CaCl2 and the immobilized phospholipase D based on bioprinting described in the second aspect with an organic phase containing phosphatidylcholine, and shaking at 30-60°C to carry out a catalytic reaction.

[0021] Preferably, the organic phase is prepared by adding phosphatidylcholine to butyl acetate, and the aqueous phase is prepared by adding L-serine, CaCl2 and the immobilized phospholipase D based on bioprinting described in the second aspect to acetate buffer.

[0022] Preferably, the catalytic reaction temperature is 30-60°C; more preferably, the catalytic reaction temperature is 55°C.

[0023] Preferably, the pH of the catalytic reaction is controlled by adjusting the pH of the acetate buffer, and the pH of the catalytic reaction is 4-8; more preferably, the pH of the catalytic reaction is 6.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] 1. This invention combines bio-imprinting technology with enzyme immobilization. Imprinting technology first uses the ligand serine to induce the production of an imprinted enzyme with a "superactivated structure." Immobilization technology then rigidifies the imprinted enzyme molecule to produce an immobilized enzyme with this "superactivated structure," namely, bio-imprinted-immobilized phospholipase D. This method not only maintains the imprinted conformation, allowing the immobilized phospholipase D to "remember" the imprinting-induced properties even in aqueous solution, weakening the ability of water molecules to participate in competing reactions, inhibiting hydrolysis and improving selectivity, but also protects the active center and surrounding areas, enhancing enzyme activity. Ultimately, this results in a highly efficient immobilized phospholipase D, which has been successfully used in the production of phosphatidylserine.

[0026] 2. The present invention uses physical encapsulation to immobilize PLD, which exhibits multiple advantages: First, by encapsulating the enzyme inside a porous carrier (such as sodium alginate or chitosan), direct contact with chemical reagents is avoided, the natural conformation and active sites of the enzyme are retained to the greatest extent, and the loss of enzyme activity is significantly reduced. In contrast, the chemical cross-linking method relies on cross-linking agents such as glutaraldehyde, which can easily destroy the functional groups of the enzyme, leading to decreased activity or even inactivation. Second, the physical encapsulation method operates under mild conditions (room temperature and neutral pH), making it suitable for enzymes that are sensitive to temperature and acidity and alkali, while chemical cross-linking often requires extreme reaction conditions, increasing the risk of enzyme denaturation. Third, the physical encapsulation method There is no need to introduce toxic reagents such as glutaraldehyde, so it has high biosafety and meets the strict standards in the food and pharmaceutical fields, while the chemical cross-linking method has the hidden danger of toxic residues; fourthly, the physical encapsulation carrier material selection is flexible (natural or synthetic polymers), and the pore size can be adjusted to adapt to different enzyme molecules and reaction requirements, while chemical cross-linking is limited by the number and type of carrier active groups; fifthly, the process is simple and the cost is low, which is conducive to large-scale production, while chemical cross-linking requires precise control of cross-linking parameters and is relatively expensive; sixthly, physical encapsulation can simultaneously fix multiple enzyme systems and maintain synergistic effects, while chemical cross-linking can easily induce random connections between enzymes and interfere with functional synergy. Therefore, the physical encapsulation method has high activity retention, high safety, strong adaptability and low cost as its core advantages, and is particularly suitable for application scenarios with high requirements for enzyme activity and biocompatibility.

[0027] 3. In the field of enzyme immobilization technology, immobilization strategies for phospholipase D (PLD) primarily revolve around two major systems: physical adsorption-based carrier loading technologies (such as macroporous resins and ordered mesoporous silica) and chemical cross-linking-based covalent immobilization technologies (such as glutaraldehyde cross-linking systems). It should be noted that the effectiveness of enzyme immobilization is constrained by multiple factors, including the nature of the carrier, the cross-linking method, and the characteristics of the enzyme source, and a universal immobilization scheme has yet to be established. Therefore, in terms of PLD immobilization, it is necessary to explore and apply new materials and methods. This invention breaks through traditional thinking and explores a targeted immobilization technology, molecular imprinting immobilization. For the first time, molecular imprinting technology is combined with physical embedding (chitosan / sodium alginate) immobilization methods. This will greatly improve the problem of decreased specific activity and selectivity of immobilized enzymes, and will help promote the industrialization and application of PS.

[0028] 4. The present invention has achieved a double breakthrough: First, it has significantly optimized the immobilization efficiency and time cost - the traditional chemical cross-linking method requires several hours of cross-linking reaction, and the embedding method achieves an immobilization rate of 69.56% in just 1 minute of complexation time by precisely controlling the embedding system parameters (sodium alginate mass concentration 0.015g / mL, chitosan mass concentration 0.006g / mL, CaCl2 concentration 0.3mol / L); second, it has achieved a leapfrog improvement in catalytic performance - the constructed bio-imprinted immobilized enzyme has significantly improved catalytic activity and substrate selectivity compared to conventional immobilized enzymes, and a 94.68% phosphatidylserine (PS) yield can be obtained in just 20 minutes, shortening the reaction time by more than 50% compared to traditional processes. The present invention provides an industrial solution for the enzymatic synthesis of high-value-added phospholipid products that is both time-effective and economical. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 The results of phospholipid acyl transfer catalyzed by non-imprinted immobilized phospholipase D and bio-imprinted immobilized phospholipase D at different reaction temperatures are shown.

[0030] Figure 2 The results show the phospholipid acyl transfer reaction catalyzed by non-imprinted immobilized phospholipase D and bio-imprinted immobilized phospholipase D at different pH values.

[0031] Figure 3 The process of phospholipid acyl transfer reaction catalyzed by non-imprinted immobilized phospholipase D and bio-imprinted immobilized phospholipase D;

[0032] Figure 4 It is for the recovery and reuse of non-imprinted immobilized phospholipase D and bio-imprinted immobilized phospholipase D. DETAILED DESCRIPTION

[0033] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below through embodiments in conjunction with the accompanying drawings. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0034] 1. Experiment on the mass concentration of sodium alginate:

[0035] In order to study the effect of sodium alginate concentration on the preparation of immobilized phospholipase D, the present invention carried out Examples 1 to 3. The specific contents of Examples 1 to 3 are as follows:

[0036] Example 1

[0037] A method for preparing immobilized phospholipase D based on bioprinting, comprising the following steps:

[0038] S1: Dissolve 0.3 g of L-serine in 1 mL of 0.2 mol / L acetate buffer (pH 4.5), add 9 mL of phospholipase D solution (protein concentration 1.37 mg / mL), and stir at 0-4°C for 30 minutes to obtain a mixed solution;

[0039] S2: adding sodium alginate to the mixed solution prepared in step S1, stirring with a magnetic stirrer for 15 minutes to dissolve the sodium alginate, thereby obtaining a biopolymer solution; the mass concentration of sodium alginate in the biopolymer solution is 0.01 g / mL;

[0040] S3: Chitosan was dissolved in 50 mL of 1% (w / v) acetic acid solution and magnetically stirred for 15 minutes to obtain a chitosan solution, wherein the mass concentration of chitosan in the chitosan solution was 0.003 g / mL. After the pH value of the chitosan solution was adjusted to neutral, CaCl2 was added to the chitosan solution and stirring was continued for 5 minutes to obtain a chitosan solution containing CaCl2, wherein the molar concentration of CaCl2 in the chitosan solution containing CaCl2 was 0.2 mol / L;

[0041] S4: Slowly add the biopolymer solution prepared in step S2 dropwise to the chitosan solution containing CaCl2 prepared in step S3 via a syringe. Stir with a magnetic stirrer for 30 seconds until gelation occurs. Collect the smooth microspheres obtained by filtration, rinse with distilled water to remove any surface impurities, and then freeze-dry the microspheres using a low-temperature vacuum freeze dryer to obtain bioprinted immobilized phospholipase D.

[0042] Example 2

[0043] The content of Example 2 is substantially the same as that of Example 1, except that in step S2, the mass concentration of sodium alginate in the biopolymer solution is 0.015 g / mL.

[0044] Example 3

[0045] The content of Example 3 is substantially the same as that of Example 1, except that in step S2, the mass concentration of sodium alginate in the biopolymer solution is 0.02 g / mL.

[0046] To verify the immobilization efficacy of the bio-imprinted phospholipase D samples prepared in Examples 1 to 3, immobilization efficiency tests were conducted. The immobilization efficiency of each sample was calculated using the following formula: Immobilization efficiency (%) = (initial protein content - unimmobilized protein content) / initial protein content * 100. Initial protein content refers to the protein content of 9 mL of PLD enzyme solution as measured using a BCA protein concentration kit. After collecting the immobilized smooth microspheres by filtration, the total protein concentration in the remaining solution and eluate was determined using a BCA protein concentration kit to calculate the unimmobilized protein content.

[0047] The calculated immobilization rates at different sodium alginate concentrations are shown in Table 1.

[0048] Table 1 Immobilization rate at different sodium alginate concentrations

[0049] serial number Example 1 Example 2 Example 3 Sodium alginate mass concentration / (g / mL) 0.01 0.015 0.02 Immobilization rate / % 30.12 31.49 29.57

[0050] As shown in Table 1, when the mass concentration of sodium alginate is 0.015 g / mL, the immobilization efficiency is the highest, which is 31.49%.

[0051] 2. Experiment on chitosan concentration:

[0052] In order to study the effect of chitosan concentration on the preparation of immobilized phospholipase D, the present invention carried out Example 4 and Example 5. The specific contents of Example 4 and Example 5 are as follows:

[0053] Example 4

[0054] The content of Example 4 is substantially the same as that of Example 2, except that in step S3, the mass concentration of chitosan in the chitosan solution is 0.006 g / mL.

[0055] Example 5

[0056] The content of Example 5 is substantially the same as that of Example 2, except that in step S3, the mass concentration of chitosan in the chitosan solution is 0.009 g / mL.

[0057] In order to verify the immobilization effect of the samples in Example 4 and Example 5, an immobilization rate test was carried out, and the immobilization rates of the samples in Example 4 and Example 5 were calculated respectively. The calculation formula of the immobilization rate is as follows: immobilization rate (%) = (initial protein content - unimmobilized protein content) / initial protein content * 100.

[0058] The calculated immobilization rates at different chitosan concentrations are shown in Table 2.

[0059] Table 2 Immobilization rate at different chitosan concentrations

[0060] serial number Example 2 Example 4 Example 5 Chitosan mass concentration / (g / mL) 0.003 0.006 0.009 Immobilization rate / % 31.49 48.38 42.53

[0061] As shown in Table 2, when the chitosan concentration was 0.006 g / ml, the immobilization efficiency was the highest, which was 48.38%.

[0062] 3. Experiment on the molar concentration of calcium chloride:

[0063] In order to study the effect of calcium chloride molar concentration on the preparation of immobilized phospholipase D, the present invention carried out Example 6 and Example 7. The specific contents of Example 6 and Example 7 are as follows:

[0064] Example 6

[0065] The content of Example 6 is substantially the same as that of Example 4, except that in step S3, the molar concentration of CaCl2 in the chitosan solution containing CaCl2 is 0.3 mol / L.

[0066] Example 7

[0067] The content of Example 7 is substantially the same as that of Example 4, except that in step S3, the molar concentration of CaCl2 in the chitosan solution containing CaCl2 is 0.4 mol / L.

[0068] In order to verify the immobilization effect of the samples in Example 6 and Example 7, an immobilization rate test was carried out, and the immobilization rates of the samples in Example 6 and Example 7 were calculated respectively. The calculation formula of the immobilization rate is as follows: immobilization rate (%) = (initial protein content - unimmobilized protein content) / initial protein content * 100.

[0069] The calculated immobilization rates at different calcium chloride molar concentrations are shown in Table 3.

[0070] Table 3 Immobilization rate at different calcium chloride molar concentrations

[0071] serial number Example 4 Example 6 Example 7 Calcium chloride molar concentration / (mol / L) 0.2 0.3 0.4 Immobilization rate / % 48.38 62.55 56.74

[0072] As shown in Table 3, when the molar concentration of CaCl2 is 0.3 mol / L, the immobilization efficiency is the highest, which is 62.55%.

[0073] 4. Complexation time exploration experiment:

[0074] In order to study the effect of complexation time on the preparation of immobilized phospholipase D, the present invention carried out Examples 8 to 10.

[0075] The specific contents of Examples 8 to 10 are as follows:

[0076] Example 8

[0077] The content of Example 8 is substantially the same as that of Example 6, except that in step S4, the complexation time is 60 s.

[0078] Example 9

[0079] The content of Example 9 is substantially the same as that of Example 6, except that in step S4, the complexation time is 90 s.

[0080] Example 10

[0081] The content of Example 10 is substantially the same as that of Example 6, except that in step S4, the complexation time is 120 s.

[0082] In order to verify the immobilization effect of the samples of Examples 8 to 10, an immobilization rate test was carried out, and the immobilization rates of the samples of Examples 8 to 10 were calculated respectively. The calculation formula of the immobilization rate is as follows: immobilization rate (%) = (initial protein content - unimmobilized protein content) / initial protein content * 100.

[0083] The calculated immobilization rates at different complexation times are shown in Table 4.

[0084] Table 4 Immobilization rate at different complexation times

[0085] serial number Example 6 Example 8 Example 9 Example 10 Complexation time / s 30 60 90 120 Immobilization rate / % 62.55 69.56 64.31 59.11

[0086] As shown in Table 4, when the complexation time was 60 s, the immobilization efficiency was the highest, which was 69.56%.

[0087] In summary, the optimal conditions for preparing bio-imprinted immobilized phospholipase D were: sodium alginate concentration 0.015 g / mL, chitosan concentration 0.006 g / mL, CaCl2 molar concentration 0.3 mol / L, complexation time 60 s, and the optimal immobilization rate was 69.56%.

[0088] 5. Discussion on the main factors affecting the preparation of phosphatidylserine catalyzed by bioimprinted immobilized phospholipase D:

[0089] (1) Reaction temperature exploration experiment:

[0090] The present invention used the bio-imprinted immobilized phospholipase D prepared in Example 8 to catalyze the production of phosphatidylserine. The effects of different reaction temperatures (30°C, 40°C, 50°C, 55°C, and 60°C) on the catalytic production of phosphatidylserine were studied. For comparison, an unimprinted immobilized phospholipase D was used as a control.

[0091] The preparation steps for non-imprinted immobilized phospholipase D are as follows: 0.15g of sodium alginate was added to 10ml of free phospholipase D and stirred with a magnetic stirrer for 15 minutes to form a biopolymer solution. 0.3g of chitosan was dissolved in 50ml of 1% (w / v) acetic acid solution and stirred with a magnetic stirrer for 15 minutes to obtain a chitosan solution. After adjusting the pH to neutral, 1.665g of CaCl2 was added and stirred for 5 minutes. The biopolymer solution was slowly added dropwise to the chitosan solution containing CaCl2 via a syringe and stirred with a magnetic stirrer for 60 seconds. The filtered smooth microspheres were collected and rinsed with distilled water to remove residual surface impurities to obtain eluted microspheres. The eluted microspheres were freeze-dried in a low-temperature vacuum freeze dryer to obtain approximately spherical particles, thus obtaining non-imprinted immobilized phospholipase D microcapsules.

[0092] Specific experimental steps: 5 mL of butyl acetate containing 2 mg / mL soybean lecithin was used as the organic phase; 5 mL of acetate buffer (0.2 M, pH 6) containing 0.5 g of L-serine, 20 mM CaCl2 and 0.01 g of the bio-imprinted immobilized phospholipase D microcapsules prepared in Example 8 (or 0.01 g of the non-imprinted immobilized phospholipase D microcapsules prepared in the above steps) was used as the aqueous phase. In a sealed flask, the reaction was carried out at 30°C, 40°C, 50°C, 55°C, and 60°C, respectively, with shaking at 180 rpm for 0.5 h. After the reaction was completed, 0.5 mL of sample was collected from the organic phase, evaporated in a well-ventilated fume hood, and then dissolved in 0.5 mL of methanol: chloroform (2:1 v / v). After filtering the sample through a 0.22 μm organic filter membrane, the lipid composition was analyzed using high performance liquid chromatography-evaporative light scattering detection (HPLC-ELSD). The experimental results are shown in the figure. Figure 1 shown.

[0093] Phospholipid separation was achieved using a Sepax HP-Silica silica column (5 μm, 250 × 4.6 mm). A 5 μL sample was loaded onto the column. Phase A consisted of methanol: water: acetic acid: triethylamine (85:15:0.45:0.05, v / v / v / v) and phase B consisted of n-hexane: isopropanol: phase A (20:48:32, v / v / v). The flow rate was 1.0 mL / min, the column temperature was 40°C, the nitrogen flow rate was 2 mL / min, and the drift tube temperature was 60°C. A calibration solution containing the relevant phospholipids (PS, PA) was used to determine the specific retention times of each phospholipid. The phospholipid content in the sample was determined by integrating the peak areas. PS conversion (%) was determined by calculating the percentage of PS relative to the original PC content.

[0094] Definition of relative enzyme activity: The PS product in the sample is quantitatively analyzed by plotting the relationship between PS concentration and peak area, using the logarithm of PS concentration and peak area as the horizontal and vertical axes, respectively. The PS standard was diluted to different concentration gradients, and the absorption peaks of the standard at different concentrations were measured. The PS absorption peak area in the sample was measured under the same HPLC conditions, and the PS content was calculated based on the calibration curve. The calibration curve correlation formula is: y = 1.4809x + 9.2648R 2 = 0.9996, y is the ln function of the liquid phase peak area S, i.e. ln(S), x is the ln function of the standard PS concentration, i.e. ln(C PS ) PS conversion rate (%) = amount of PS substance generated after reaction / amount of PC substance before reaction * 100%.

[0095] Depend on Figure 1 It can be seen that the optimal reaction temperature of bio-imprinted immobilized phospholipase D is 55°C, which is 5°C higher than the optimal reaction temperature of non-imprinted immobilized phospholipase D (50°C). Bio-imprinted immobilized phospholipase D has a higher temperature tolerance.

[0096] (2) Reaction pH exploration experiment:

[0097] The present invention used the bio-imprinted immobilized phospholipase D prepared in Example 8 to catalyze the production of phosphatidylserine. The effects of different reaction pH conditions (4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, and 8) on the catalytic production of phosphatidylserine were studied. For comparison, the non-imprinted immobilized phospholipase D described above was used as a control.

[0098] Specific experimental steps: 5 mL of butyl acetate containing 2 mg / mL soybean lecithin was used as the organic phase; 5 mL of acetate buffer (0.2 M) containing 0.5 g of L-serine, 20 mM CaCl2, and 0.01 g of the bio-imprinted immobilized phospholipase D prepared in Example 8 (or 0.01 g of the non-imprinted immobilized phospholipase D microcapsules prepared in the above steps) was used as the aqueous phase; the pH of the acetate buffer was adjusted to 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, and 8, respectively. In a sealed flask, the reaction was carried out at 55°C (50°C for non-imprinted immobilized phospholipase D) and shaking at 180 rpm for 0.5 h. After the reaction was completed, 0.5 mL of sample was collected from the organic phase, evaporated in a well-ventilated fume hood, and then dissolved in 0.5 mL of methanol:chloroform (2:1 v / v). After filtering the samples through a 0.22 μm organic filter membrane, the lipid composition was analyzed using high performance liquid chromatography-evaporative light scattering detection (HPLC-ELSD). Figure 2 shown.

[0099] Depend on Figure 2It can be seen that compared with non-imprinted immobilized phospholipase D (pH 5), the optimal reaction pH of bio-imprinted immobilized phospholipase D (pH 6) shifted towards the alkaline direction.

[0100] In summary, the optimal reaction temperature and pH for the bio-imprinted immobilized phospholipase D-catalyzed preparation of phosphatidylserine were 55°C and 6, respectively.

[0101] 6. Bio-imprinting - Performance exploration experiment of immobilized phospholipase D:

[0102] (1) Selectivity

[0103] In the present invention, under the above-mentioned optimal conditions, the reaction selectivity of the phospholipid acyl transfer reaction catalyzed by the non-imprinted immobilized phospholipase D and the bio-imprinted immobilized phospholipase D prepared in Example 8 was studied.

[0104] The specific experimental steps for the preparation of phosphatidylserine catalyzed by bio-imprinted immobilized phospholipase D are as follows: 5 mL of butyl acetate containing 2 mg / mL soybean lecithin is used as the organic phase; 5 mL of acetate buffer (0.2 M, pH 6) containing 0.5 g of L-serine, 20 mM CaCl2 and 0.01 g of bio-imprinted immobilized phospholipase D microcapsules prepared in Example 8 is used as the aqueous phase. In a sealed flask, the reaction was carried out at 55°C and 180 rpm under shaking conditions for 5, 10, 15, 20, 25, and 30 min, respectively. After the reaction was completed, 0.5 mL of sample was collected from the organic phase, evaporated in a well-ventilated fume hood, and then dissolved in 0.5 mL of methanol: chloroform (2:1 v / v). After filtering the sample through a 0.22 μm organic filter membrane, the lipid composition was analyzed using high performance liquid chromatography-evaporative light scattering detection (HPLC-ELSD). The PS conversion rate (i.e., transesterification activity) was calculated. The experimental results are shown in the figure. Figure 3 shown.

[0105] The specific experimental steps for the preparation of phosphatidylserine catalyzed by non-imprinted immobilized phospholipase D are as follows: 5 mL of butyl acetate containing 2 mg / mL soybean lecithin was used as the organic phase; 5 mL of acetate buffer (0.2 M, pH 5) containing 0.5 g L-serine, 20 mM CaCl2 and 0.01 g of non-imprinted immobilized phospholipase D microcapsules was used as the aqueous phase. In a sealed flask, the reaction was carried out at 50°C and 180 rpm under shaking conditions for 0.5, 1, 1.5, and 2 h, respectively. After the reaction was completed, 0.5 mL of sample was collected from the organic phase, evaporated in a well-ventilated fume hood, and then dissolved in 0.5 mL of methanol: chloroform (2:1 v / v). After filtering the sample through a 0.22 μm organic filter membrane, the lipid composition was analyzed using high performance liquid chromatography-evaporative light scattering detection (HPLC-ELSD). The PS conversion rate (i.e., transesterification activity) was calculated. The experimental results are shown in the figure. Figure 3 shown.

[0106] Depend on Figure 3 The results show that bio-imprinted immobilized phospholipase D has the fastest reaction rate and the highest reaction selectivity when catalyzing the reaction. The reaction reaches equilibrium after 20 minutes, and the PS yield is as high as 94.68%. When using non-imprinted immobilized phospholipase D, the reaction reaches equilibrium after 1 hour, and the PS yield is 88.92%.

[0107] (2) Recycling and reuse stability

[0108] Under the above optimal conditions, the present invention studied the recovery and reuse stability of non-imprinted immobilized phospholipase D and the bio-imprinted immobilized phospholipase D prepared in Example 8.

[0109] The specific experimental steps for the preparation of phosphatidylserine by bio-imprinted immobilized phospholipase D are as follows: 5 mL of butyl acetate containing 2 mg / mL soybean lecithin was used as the organic phase; 5 mL of acetate buffer (0.2 M, pH 6) containing 0.5 g of L-serine, 20 mM CaCl2 and 0.01 g of the microcapsules prepared in Example 8 was used as the aqueous phase. In a sealed flask, the reaction was carried out at 55°C and 180 rpm under shaking conditions for 20 minutes. After the reaction was completed, 0.5 mL of sample was collected from the organic phase, evaporated in a well-ventilated fume hood, and then dissolved in 0.5 mL of methanol: chloroform (2:1 v / v). After filtering the sample through a 0.22 μm organic filter membrane, the lipid composition was analyzed using high performance liquid chromatography-evaporative light scattering detection (HPLC-ELSD). After the completion of one catalytic reaction, the bio-imprinted immobilized phospholipase D microcapsules were recovered and the recovered microcapsules were used to re-catalyze the preparation of phosphatidylserine in a new reaction system. The above steps were repeated 5 times. The PS conversion rate was calculated each time. The experimental results are shown in Figure 2. Figure 4 shown.

[0110] The specific experimental steps for the preparation of phosphatidylserine catalyzed by non-imprinted immobilized PLD were as follows: 5 mL of butyl acetate containing 2 mg / mL soybean lecithin served as the organic phase; 5 mL of acetate buffer (0.2 M, pH 5) containing 0.5 g of L-serine, 20 mM CaCl2, and 0.01 g of non-imprinted immobilized phospholipase D microcapsules served as the aqueous phase. The reaction was carried out in a sealed flask at 50°C with shaking at 180 rpm for 1 h. After completion of the reaction, a 0.5 mL sample was collected from the organic phase, evaporated in a well-ventilated fume hood, and then redissolved in 0.5 mL of methanol:chloroform (2:1 v / v). The sample was filtered through a 0.22 μm organic filter membrane, and lipid composition was analyzed using high-performance liquid chromatography-evaporative light scattering detection (HPLC-ELSD). After completion of each catalytic reaction, the non-imprinted immobilized phospholipase D microcapsules were recovered and used in a new reaction system to catalyze the preparation of phosphatidylserine. This procedure was repeated five times. The PS conversion rate was calculated for each reaction. The experimental results are as follows Figure 4 shown.

[0111] Depend on Figure 4 Both bio-imprinted and non-imprinted immobilized phospholipase D enable enzyme reuse, overcoming the difficulty of recycling free enzymes. This demonstrates that immobilization technology can easily and conveniently recycle bioenzyme catalysts, thereby reducing production costs and improving production efficiency. Compared to non-imprinted immobilized phospholipase D, bio-imprinted immobilized phospholipase D exhibited more stable performance, retaining 65.97% of its phosphatidyl transfer activity after five reuses.

[0112] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the scope of protection of the present invention. Those skilled in the art can modify or replace the technical solutions of the present invention according to the concept of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A method for preparing immobilized phospholipase D based on bioprinting, characterized in that: The following steps are involved: S1: dissolving L-serine in acetate buffer to obtain a serine solution, adding phospholipase D solution to the serine solution, and stirring at 0-4°C for 30 minutes to obtain a mixed solution; S2: adding sodium alginate to the mixed solution prepared in step S1 and stirring to dissolve, thereby obtaining a biopolymer solution; S3: adjusting the pH value of the chitosan solution to neutral, then adding CaCl2 to the chitosan solution and stirring to dissolve it, thereby obtaining a chitosan solution containing CaCl2; S4: adding the biopolymer solution prepared in step S2 dropwise to the chitosan solution containing CaCl2 prepared in step S3 to form microspheres through complexation, collecting the microspheres, washing the microspheres with water, and drying them to obtain the immobilized phospholipase D based on bioprinting.

2. The method for preparing immobilized phospholipase D based on bioprinting according to claim 1, characterized in that: In step S1, the mass ratio of L-serine to phospholipase D in the mixed solution is (22-33):

1.

3. The method for preparing immobilized phospholipase D based on bioprinting according to claim 1, characterized in that: In step S2, the mass concentration of sodium alginate in the biopolymer solution is 0.01 g / mL to 0.02 g / mL.

4. The method for preparing immobilized phospholipase D based on bioprinting according to claim 1, characterized in that: In step S3, the mass concentration of chitosan in the chitosan solution is 0.003 g / mL to 0.009 g / mL.

5. The method for preparing immobilized phospholipase D based on bioprinting according to claim 1, characterized in that: In step S3, the molar concentration of CaCl2 in the chitosan solution containing CaCl2 is 0.2 mol / L to 0.4 mol / L; in step S4, the complexation time is 30s to 120s.

6. The method for preparing immobilized phospholipase D based on bioprinting according to claim 1, characterized in that: In step S1, the concentration of the acetate buffer is 0.2 mol / L; the concentration of the phospholipase D solution is 1 mg / mL to 1.5 mg / mL.

7. An immobilized phospholipase D based on bioprinting prepared by the preparation method according to any one of claims 1 to 6.

8. Use of the bio-imprinted immobilized phospholipase D according to claim 7 in the preparation of phosphatidylserine.

9. A method for preparing phosphatidylserine, characterized in that: The following steps are involved: An aqueous phase containing L-serine, CaCl2 and the immobilized phospholipase D based on bioprinting according to claim 7 is mixed with an organic phase containing phosphatidylcholine, and the mixture is shaken at 30-60°C for catalytic reaction.

10. The method for preparing phosphatidylserine according to claim 9, characterized in that: The organic phase is prepared by adding phosphatidylcholine to butyl acetate, and the aqueous phase is prepared by adding L-serine, CaCl2 and the immobilized phospholipase D based on bioprinting according to claim 7 to acetate buffer.

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