Phospholipase D-choline oxidase-catalase composite microcapsule as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of enzyme co-immobilization, and particularly relates to a phospholipase D-choline oxidase-catalase composite microcapsule as well as a preparation method and application thereof. The method comprises the following steps: S1, adding sodium alginate, choline oxidase and catalase into a phospholipase D solution, and uniformly mixing to obtain a mixed solution; s2, the pH value of the chitosan solution is adjusted to be neutral, then CaCl2 is added into the chitosan solution to be stirred and dissolved, and a chitosan solution containing CaCl2 is obtained; s3, dropwise adding the mixed solution prepared in the step S1 into the chitosan solution containing CaCl2 prepared in the step S2, complexing to form microspheres, collecting the microspheres, and washing and drying the microspheres to obtain the phospholipase D-choline oxidase-catalase composite microcapsule. The invention provides a three-enzyme co-immobilization method for improving the synthesis capacity of phosphatidylglycerol, which not only solves the problem of inhibition of a byproduct choline in phospholipid conversion, but also realizes process upgrading through oxygen internal circulation and enzyme reutilization.

Description

Technical Field

[0001] The present invention belongs to the technical field of enzyme co-immobilization, and relates to the technical field of three-enzyme co-immobilization combination of phospholipase D, choline oxidase and catalase, and specifically relates to a phospholipase D-choline oxidase-catalase composite microcapsule, and a preparation method and application thereof. Background Art

[0002] Phospholipids are a class of lipid compounds containing phosphate groups that possess both nutritional and physiological functions. Phosphatidylglycerol (PG), a rare natural phospholipid, exhibits multiple applications due to its amphiphilic structure: in the biomedical field, it maintains alveolar stability by regulating lung surface tension, is a key component in the treatment of neonatal respiratory distress syndrome, and can be used to construct targeted drug delivery systems to enhance anti-cancer efficacy; in the industrial field, it serves as a highly effective emulsifier, widely used in food stabilization systems and intravenous fat emulsion injections to optimize the delivery of fat-soluble drugs. Recent research further reveals that PG possesses both immunomodulatory and antimicrobial activities, providing new directions for the development of precision medicine and functional foods.

[0003] Currently, industrial and laboratory synthesis of rare phospholipids typically utilizes enzymatic conversion. This method offers advantages such as good selectivity, high conversion rate, and mild reaction conditions. Phospholipase D (PLD) is an ester hydrolase with unique properties, capable of hydrolyzing phosphodiester bonds and performing base exchange reactions. On the one hand, it catalyzes the hydrolysis of ester bonds between phosphate and organic bases (such as choline and ethanolamine) in phospholipid molecules, releasing the polar head group of the original phospholipid to produce phosphatidic acid and the hydroxyl compound choline. On the other hand, in addition to hydrolysis, it can also catalyze base exchange reactions between hydroxyl-containing compounds and phospholipid bases to generate new phospholipids. This property can be exploited to modify phospholipids. Using phosphatidylcholine (PC)-rich phospholipids as substrates, phosphatidyl transfer reactions with glycerol can effectively produce PG.

[0004] In the phosphatidyl transfer reaction catalyzed by PLD, which converts lecithin (phosphatidylcholine) into rare phospholipids such as PG, PLD specifically cleaves and releases the head group choline from phosphatidylcholine, transferring the phosphatidyl group to a glycerol molecule to produce PG. However, the accumulation of choline as a reaction byproduct binds to the PLD active site through a product feedback inhibition mechanism, significantly reducing the enzyme's catalytic efficiency and hindering the reaction toward PG production. Furthermore, the poor stability, difficulty in recycling, and high price of free phospholipase D are the main constraints on the large-scale industrial production of PG. Immobilized enzyme technology can effectively improve the stability of phospholipase D, enable its recycling and reuse, and reduce production costs, which is a prerequisite for the large-scale industrial production of PG. Summary of the Invention

[0005] In view of the problems and shortcomings in the prior art, the object of the present invention is to provide a phospholipase D-choline oxidase-catalase composite microcapsule 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 phospholipase D-choline oxidase-catalase composite microcapsules, comprising the following steps:

[0008] S1: adding sodium alginate, choline oxidase, and catalase to the phospholipase D solution, and mixing to obtain a mixed solution;

[0009] S2: 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;

[0010] S3: adding the mixed solution prepared in step S1 dropwise to the chitosan solution containing CaCl2 prepared in step S2 to form microspheres by complexation, collecting the microspheres, washing the microspheres with water, and drying them to obtain phospholipase D-choline oxidase-catalase composite microcapsules.

[0011] Preferably, in step S1, the final concentration of phospholipase D in the mixed solution is 1.23 mg / mL, the final concentration of choline oxidase in the mixed solution is 20 U / mL, and the final concentration of catalase in the mixed solution is 80 U / mL.

[0012] Preferably, in step S1, the mass concentration of sodium alginate in the mixed solution is 0.01 g / ml to 0.03 g / ml.

[0013] Preferably, in step S2, the mass concentration of the chitosan solution is 0.003 g / ml to 0.009 g / ml.

[0014] Preferably, in step S2, the molar concentration of CaCl2 in the chitosan solution containing CaCl2 is 0.1 to 0.3 mol / L.

[0015] Preferably, in step S3, the complexation time is 1 to 3 minutes.

[0016] The second aspect of the present invention provides phospholipase D-choline oxidase-catalase composite microcapsules prepared by the method described in the first aspect.

[0017] The third aspect of the present invention provides use of the phospholipase D-choline oxidase-catalase composite microcapsules described in the second aspect in the preparation of phosphatidylglycerol.

[0018] The fourth aspect of the present invention provides a method for preparing phosphatidylglycerol, comprising: mixing an aqueous phase containing glycerol and the phospholipase D-choline oxidase-catalase composite microcapsules described in the second aspect with an organic phase containing phosphatidylcholine, and shaking the mixture at 40°C.

[0019] Preferably, the organic phase is prepared by adding phosphatidylcholine to ethyl acetate, and the aqueous phase is prepared by adding glycerol and the phospholipase D-choline oxidase-catalase composite microcapsules described in the second aspect to acetate buffer.

[0020] More preferably, the phosphatidylcholine in the organic phase is derived from soybean lecithin, and the phosphatidylcholine content in the soybean lecithin is ≥98%.

[0021] Preferably, the oscillation reaction time is 15 to 60 minutes; more preferably, the oscillation reaction time is 30 minutes.

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

[0023] 1. This invention proposes an ultra-efficient three-enzyme co-immobilization carrier and method based on a chitosan-sodium alginate composite material. By co-immobilizing phospholipase D, choline oxidase, and catalase within a chitosan-based microsphere carrier, a dynamic "product scavenging-substrate recycling" mechanism is established. During the phospholipase D-catalyzed production of protease (PG), choline oxidase simultaneously oxidizes the byproduct choline to betaine (reaction formula: choline + 2O2 → betaine + 2H2O2), relieving choline's inhibition on phospholipase D's transesterification activity. Catalase, in turn, decomposes H2O2 into water and oxygen (reaction formula: 2H2O2 → 2H2O + O2), completely relieving product inhibition and oxidative damage. The three enzymes work synergistically: choline oxidase scavenges the inhibitory product, while catalase scavenges H2O2 and regenerates oxygen (participating in choline oxidation). The co-immobilization technology enhances enzyme stability and substrate transfer efficiency through spatial confinement, while also reducing free enzyme loss.

[0024] 2. This paper proposes a phospholipase D-choline oxidase-catalase co-immobilization method to enhance phosphatidylglycerol synthesis. By synergistically immobilizing phospholipase D, choline oxidase, and catalase within chitosan-based microsphere carriers, a dynamic "product scavenging-substrate recycling" mechanism is constructed. This strategy not only addresses the problem of choline inhibition, a byproduct of phospholipid conversion, but also achieves process upgrades through internal oxygen recycling and enzyme reuse. This provides a universal solution for optimizing enzymatic reactions in areas such as biocatalytic synthesis of high-value phospholipids and pharmaceutical carrier development.

[0025] 3. The present invention selects sodium alginate and chitosan as immobilization materials. These two natural organic carriers have the remarkable advantages of simple curing operation, easy degradation by microorganisms and high immobilization density. Sodium alginate is famous for its gentle gelling property and non-toxic characteristics. The present invention uses a physical method to utilize the interaction of chitosan and the surface anionic charge of sodium alginate gel particles, without adding chemical cross-linking agents such as glutaraldehyde, to achieve a firm complexing effect. At present, although many cross-linking agents (such as glutaraldehyde, hexamethylenediamine, diisocyanate, etc.) have been widely used in the immobilization of free enzymes to improve the stability and immobilization efficiency of enzymes, the PLD enzyme is mainly used to prepare functional rare phospholipids that can improve public health, and relates to the food and pharmaceutical fields. The volatility and toxicity of traditional cross-linking agents limit their application in industrial processes. Therefore, the present invention adopts a physical embedding method, combined with natural non-toxic chitosan and sodium alginate, not only to avoid the use of chemical cross-linking agents, but also to better meet the strategic concept and green environmental protection requirements of the country's "Healthy China".

[0026] 4. By optimizing the conditions, the present invention achieves an immobilization rate of 70.78% in just 2 minutes of complexation time under the conditions of a sodium alginate concentration of 0.015 g / ml, a chitosan concentration of 0.006 g / ml, and a CaCl2 molar concentration of 0.2 mol / L. This solves the problems of poor stability and easy inactivation of free PLD in the industrial production of PG. Furthermore, the efficiency of PG production is improved, with the reaction reaching equilibrium after 30 minutes and a PG conversion rate as high as 90.43%.

[0027] 5. The present invention also significantly simplifies the operational process. Chitosan and sodium alginate can be completely dissolved using only a magnetic stirrer, avoiding the cumbersome step of water bath heating required in previous studies. This improvement not only simplifies the process but also significantly reduces energy consumption and time costs, making the immobilization process more efficient, environmentally friendly, and economical. This efficient and simple immobilization method provides strong technical support for industrial production and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The phospholipid acyl transfer reaction process was catalyzed by free phospholipase D and co-immobilized microcapsules (i.e., phospholipase D-choline oxidase-catalase composite microcapsules);

[0029] Figure 2 The thermal stability of free phospholipase D and co-immobilized microcapsules (i.e., phospholipase D-choline oxidase-catalase composite microcapsules);

[0030] Figure 3 The invention relates to the recycling and reuse of co-immobilized microcapsules (i.e. phospholipase D-choline oxidase-catalase composite microcapsules). DETAILED DESCRIPTION

[0031] 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.

[0032] 1. Experiment on the effect of sodium alginate concentration on microcapsule immobilization rate:

[0033] In order to study the effect of sodium alginate concentration on the microcapsule immobilization rate, the present invention carried out Examples 1 to 5. The specific contents of Examples 1 to 5 are as follows:

[0034] Example 1

[0035] A method for preparing phospholipase D-choline oxidase-catalase composite microcapsules comprises the following steps:

[0036] S1: Weigh 0.1 g of sodium alginate (SA), add 9 mL of 1.37 mg / mL phospholipase D enzyme solution, 400 μL of choline oxidase, and 600 μL of catalase, and stir with a magnetic stirrer for 15 min to obtain a mixed solution; the final concentration of phospholipase D in the mixed solution is 1.23 mg / mL, the final concentration of choline oxidase in the mixed solution is 20 U / mL, and the final concentration of catalase in the mixed solution is 80 U / mL;

[0037] S2: 0.15 g of chitosan was weighed and dissolved in 50 mL of 1% (w / v) acetic acid solution. The mixture was magnetically stirred for 15 min to obtain a chitosan solution. After the pH value was adjusted to neutral, 0.555 g of CaCl2 was added and stirring was continued for 5 min to obtain a chitosan solution containing CaCl2.

[0038] S3: The mixed solution prepared in step S1 is slowly added dropwise to the chitosan solution containing CaCl2 prepared in S2 through a syringe, and the complexation is stirred with a magnetic stirrer for 1 minute until gelation, and the smooth microspheres obtained by filtration are collected and eluted with distilled water to remove residual impurities on the surface of the microspheres. The microspheres are then freeze-dried using a low-temperature vacuum freeze dryer to obtain phospholipase D-choline oxidase-catalase composite microcapsules.

[0039] Example 2

[0040] The content of Example 2 is basically the same as that of Example 1, except that the amount of sodium alginate used in step S1 is 0.15 g.

[0041] Example 3

[0042] The content of Example 3 is basically the same as that of Example 1, except that the amount of sodium alginate used in step S1 is 0.2 g.

[0043] Example 4

[0044] The content of Example 4 is basically the same as that of Example 1, except that the amount of sodium alginate used in step S1 is 0.25 g.

[0045] Example 5

[0046] The content of Example 5 is basically the same as that of Example 1, except that the amount of sodium alginate used in step S1 is 0.3 g.

[0047] To verify the immobilization effect of the composite microcapsule samples prepared in Examples 1 to 5, immobilization rate tests were conducted and the immobilization rates of the composite microcapsule samples prepared in Examples 1 to 5 were calculated. The immobilization rate was calculated using the following formula: Immobilization rate (%) = (initial protein content - unimmobilized protein content) / initial protein content * 100. The initial protein content refers to the protein content of 10 mL of mixed enzyme solution (9 mL PLD enzyme solution, 400 μL choline oxidase, and 600 μL catalase) as measured using a BCA protein concentration kit. The co-immobilized smooth microspheres were collected by filtration, and the total protein concentration in the remaining mixed solution and eluate was determined using a BCA protein concentration kit to calculate the unimmobilized protein content.

[0048] The calculated immobilization rates of the composite microcapsules at different sodium alginate dosages are shown in Table 1.

[0049] Table 1 Immobilization rate of composite microcapsules under different sodium alginate dosages

[0050]

[0051] 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 30.88%.

[0052] 2. Experiment on the effect of chitosan concentration on microcapsule immobilization rate:

[0053] In order to study the effect of chitosan concentration on the immobilization rate of microcapsules, the present invention carried out Example 6 and Example 7. The specific contents of Example 6 and Example 7 are as follows:

[0054] Example 6

[0055] The content of Example 6 is substantially the same as that of Example 2, except that the amount of chitosan used in step S2 is 0.3 g.

[0056] Example 7

[0057] The content of Example 7 is substantially the same as that of Example 2, except that the amount of chitosan used in step S2 is 0.45 g.

[0058] In order to verify the immobilization effect of the composite microcapsule samples prepared in Example 6 and Example 7, an immobilization rate test was carried out, and the immobilization rates of the composite microcapsule samples prepared 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.

[0059] The calculated immobilization rates of composite microcapsules at different chitosan dosages are shown in Table 2.

[0060] Table 2 Immobilization rate of composite microcapsules at different chitosan dosages

[0061] serial number Example 2 Example 6 Example 7 Chitosan dosage / g 0.15 0.3 0.45 Chitosan mass concentration / (g / ml) 0.003 0.006 0.009 Immobilization rate / % 30.88 51.62 41.41

[0062] As shown in Table 2, when the chitosan concentration was 0.006 g / ml, the immobilization efficiency was the highest, which was 51.62%.

[0063] 3. Experiment on the effect of calcium chloride molar concentration on microcapsule immobilization rate:

[0064] In order to study the effect of the molar concentration of calcium chloride on the microcapsule immobilization rate, the present invention carried out Example 8 and Example 9.

[0065] The specific contents of Example 8 and Example 9 are as follows:

[0066] Example 8

[0067] The content of Example 8 is substantially the same as that of Example 6, except that the amount of CaCl2 used in step S2 is 1.11 g.

[0068] Example 9

[0069] The content of Example 9 is substantially the same as that of Example 6, except that the amount of CaCl2 used in step S2 is 1.665 g.

[0070] In order to verify the immobilization effect of the composite microcapsule samples prepared in Example 8 and Example 9, an immobilization rate test was carried out, and the immobilization rates of the composite microcapsule samples prepared in Example 8 and Example 9 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.

[0071] The calculated immobilization rates of the composite microcapsules under different calcium chloride dosages are shown in Table 3.

[0072] Table 3 Immobilization rate of composite microcapsules under different calcium chloride dosages

[0073] serial number Example 6 Example 8 Example 9 Calcium chloride dosage / g 0.555 1.11 1.665 Calcium chloride molar concentration / mol / L 0.1 0.2 0.3 Immobilization rate / % 51.62 63.41 58.89

[0074] As shown in Table 3, when the molar concentration of calcium chloride is 0.2 mol / L, the immobilization efficiency is the highest, which is 63.41%.

[0075] 4. Experimental study on the effect of complexation time on microcapsule immobilization rate

[0076] In order to study the effect of complexation time on the immobilization rate of microcapsules, the present invention carried out Example 10 and Example 11. The specific contents of Example 10 and Example 11 are as follows:

[0077] Example 10

[0078] The content of Example 10 is substantially the same as that of Example 8, except that the complexation time in step S3 is 2 minutes.

[0079] Example 11

[0080] The content of Example 11 is substantially the same as that of Example 8, except that the complexation time in step S3 is 3 minutes.

[0081] In order to verify the immobilization effect of the composite microcapsule samples prepared in Example 10 and Example 11, an immobilization rate test was carried out, and the immobilization rates of the composite microcapsule samples prepared in Example 10 and Example 11 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.

[0082] The calculated immobilization rates of the composite microcapsules at different complexation times are shown in Table 4.

[0083] Table 4 Immobilization rate of composite microcapsules at different complexation times

[0084] serial number Example 8 Example 10 Example 11 Complexation time / min 1 2 3 Immobilization rate / % 63.41 70.78 58.67

[0085] As shown in Table 4, when the complexation time was 2 min, the immobilization efficiency was the highest, which was 70.78%.

[0086] In summary, the highest immobilization rate of 70.78% was achieved under the conditions of sodium alginate concentration of 0.015 g / ml, chitosan concentration of 0.006 g / ml, CaCl2 molar concentration of 0.2 mol / L, and complexation time of 2 min.

[0087] 5. Experimental study on the performance of phospholipase D-choline oxidase-catalase composite microcapsules:

[0088] (1) Experiment on transesterification reaction time:

[0089] The present invention uses free phospholipase D and the phospholipase D-choline oxidase-catalase composite microcapsules prepared in Example 10 to catalyze the preparation of phosphatidylglycerol, and studies the progress of the phosphatidyl transfer reaction catalyzed by free phospholipase D and the composite microcapsules.

[0090] The specific steps of the experiment are as follows: 2 mL of ethyl acetate containing 5 mg / mL soybean lecithin is used as the organic phase; 1 mL of acetate buffer (0.2 M, pH 6) containing 60 mg of glycerol and 0.01 g of the composite microcapsules prepared in Example 10 (or 10 μL of free phospholipase D) is used as the aqueous phase. The reaction was carried out under oscillation conditions of 40°C and 180 rpm. At different reaction time periods, 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, high performance liquid chromatography-evaporative light scattering detection (HPLC-ELSD) was used to analyze the lipid composition at different reaction time periods. The PG conversion rate (i.e., transesterification activity) at different reaction times was calculated. The experimental results are shown in the figure. Figure 1 shown.

[0091] Phospholipid separation was performed using a Sepax HP-Silica silica column (5 μm, 250 × 4.6 mm). 5 μL of 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.

[0092] Calculation of PG conversion rate: Using a calibration solution containing relevant phospholipids (PG, PA, PC), various phospholipids are determined according to their specific retention times, and the phospholipid content in the sample is determined by integrating and calculating the peak area. The PG conversion rate (%) is determined by calculating the percentage of PG relative to the original PC content. PG conversion rate (%) = the amount of PG substance generated after the reaction / the amount of PC substance before the reaction * 100%. The experiment uses the logarithm of PG concentration and peak area as the horizontal and vertical coordinates, respectively, and draws a curve of the relationship between PG concentration and peak area to quantitatively analyze the product PG in the sample. In the experiment, the PG standard was diluted into different concentration gradients, and the absorption peaks of the standard at different concentrations were measured respectively. In the experiment, the PG absorption peak area in the sample was measured under the same HPLC conditions, and the PG content was calculated based on the calibration curve. The calibration curve correlation formula is: y = 1.7046x + 6.9511R 2= 0.9998, where y is the ln function of the liquid phase peak area S, i.e. ln(S), and x is the ln function of the standard PG concentration, i.e. ln(C PG ). Thus the PG conversion rate was calculated.

[0093] Depend on Figure 1 It can be seen that compared with free phospholipase D, phospholipase D-choline oxidase-catalase composite microcapsules have the fastest reaction rate and the highest reaction selectivity in the catalytic reaction. The reaction can reach equilibrium after 30 minutes, and the PG conversion rate is as high as 90.43%; when using free phospholipase D, the reaction reaches equilibrium after 3.5 hours, and the PG conversion rate is 86.12%.

[0094] (2) Thermal stability study experiment:

[0095] In the present invention, free phospholipase D and the phospholipase D-choline oxidase-catalase composite microcapsules prepared in Example 10 were used to catalyze the preparation of phosphatidylglycerol. Before the catalytic reaction began, the free phospholipase D and the composite microcapsules were heated in a water bath to study the thermal stability of the free phospholipase D and the composite microcapsules.

[0096] The specific steps of the experiment are as follows:

[0097] 1) Heat treatment: Before the catalytic reaction, free phospholipase D and the phospholipase D-choline oxidase-catalase composite microcapsules prepared in Example 10 were heated in a water bath at 65° C., 70° C., and 80° C. for 0, 30 min, 60 min, 120 min, and 180 min, respectively.

[0098] 2) Catalytic reaction: 2 mL of ethyl acetate containing 5 mg / mL soybean lecithin was used as the organic phase; 1 mL of acetate buffer (0.2 M, pH 6) containing 60 mg of glycerol and 0.01 g of composite microcapsules treated in a water bath (or 10 μL of free phospholipase D treated in a water bath) was used as the aqueous phase. The reaction was carried out at 40°C and 180 rpm shaking for 30 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). Calculate the PG conversion rate (i.e., transesterification activity). The relative enzyme activity is based on the highest activity as 100%, and the percentage of the enzyme activity under other conditions relative to the highest enzyme activity is calculated. The experimental results are as follows. Figure 2 shown.

[0099] Depend on Figure 2As can be seen, enzymes, as temperature-sensitive biological reagents, significantly decrease their activity when stored at high temperatures. However, when enzymes are immobilized, the effect of temperature on their performance is effectively reduced. After heating at 65°C for 0.5 h, the transesterification activity of free phospholipase D rapidly decreased to 28.22% of its initial activity. However, the rate of decrease in transesterification activity of co-immobilized phospholipase D-choline oxidase-catalase composite microcapsules was significantly slower, with 72.59% of the initial activity remaining after heating at 65°C for 1 h. This is because the microcapsule wall material provides some protection for the core material, and the presence of the immobilization carrier prevents some free enzyme molecules from stretching, deforming, and denaturing. Therefore, the construction of co-immobilized microcapsules effectively improves the thermal stability of the free enzyme.

[0100] (3) Experimental discussion on the number of repeated uses:

[0101] The present invention uses the phospholipase D-choline oxidase-catalase composite microcapsules prepared in Example 10 to catalyze the preparation of phosphatidylglycerol. After one catalytic reaction is completed, the composite microcapsules are recovered and used to continue catalyzing the preparation of phosphatidylglycerol. The composite microcapsules are reused eight times in this manner to study the recycling performance of the composite microcapsules.

[0102] The specific steps of the experiment are as follows: 2 mL of ethyl acetate containing 5 mg / mL soybean lecithin is used as the organic phase; 1 mL of acetate buffer (0.2 M, pH 6) containing 60 mg of glycerol and 0.01 g of the composite microcapsules prepared in Example 10 is used as the aqueous phase. The reaction is carried out at 40°C and 180 rpm shaking for 30 minutes. After the reaction is completed, 0.5 mL of sample is 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 is analyzed using high performance liquid chromatography-evaporative light scattering detection (HPLC-ELSD). After the completion of one catalytic reaction, the composite microcapsules are recovered, and the recovered composite microcapsules are used to continue catalytically preparing phosphatidylglycerol and the above steps are repeated 8 times. The PG conversion rate is calculated each time. The experimental results are as follows. Figure 3 shown.

[0103] Depend on Figure 3 After eight reuses, the composite microcapsules retained 62.18% of their initial activity. Therefore, immobilized enzyme technology overcomes the shortcomings of free enzymes, such as poor stability, difficulty in recovery, and high cost. It effectively improves the stability of free phospholipase D, enables the recovery and reuse of phospholipase D, reduces production costs, and contributes to the large-scale industrial production of PG.

[0104] 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 phospholipase D-choline oxidase-catalase composite microcapsules, characterized in that: The following steps are involved: S1: adding sodium alginate, choline oxidase, and catalase to the phospholipase D solution, and mixing to obtain a mixed solution; S2: 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; S3: adding the mixed solution prepared in step S1 dropwise to the chitosan solution containing CaCl2 prepared in step S2 to form microspheres by complexation, collecting the microspheres, washing the microspheres with water, and drying them to obtain phospholipase D-choline oxidase-catalase composite microcapsules.

2. The method for preparing phospholipase D-choline oxidase-catalase composite microcapsules according to claim 1, characterized in that: In step S1, the final concentration of phospholipase D in the mixed solution is 1.23 mg / mL, the final concentration of choline oxidase in the mixed solution is 20 U / mL, and the final concentration of catalase in the mixed solution is 80 U / mL.

3. The method for preparing phospholipase D-choline oxidase-catalase composite microcapsules according to claim 1, characterized in that: In step S1, the mass concentration of sodium alginate in the mixed solution is 0.01 g / ml to 0.03 g / ml.

4. The method for preparing phospholipase D-choline oxidase-catalase composite microcapsules according to claim 1, characterized in that: In step S2, the mass concentration of the chitosan solution is 0.003 g / ml to 0.009 g / ml.

5. The method for preparing phospholipase D-choline oxidase-catalase composite microcapsules according to claim 1, characterized in that: In step S2, the molar concentration of CaCl2 in the chitosan solution containing CaCl2 is 0.1 to 0.3 mol / L.

6. The method for preparing phospholipase D-choline oxidase-catalase composite microcapsules according to claim 1, characterized in that: In step S3, the complexation time is 1 to 3 minutes.

7. Phospholipase D-choline oxidase-catalase composite microcapsules prepared by the method according to any one of claims 1 to 6.

8. Use of the phospholipase D-choline oxidase-catalase composite microcapsules according to claim 7 in the preparation of phosphatidylglycerol.

9. A method for preparing phosphatidylglycerol, characterized in that: include: An aqueous phase containing glycerol and the phospholipase D-choline oxidase-catalase composite microcapsules according to claim 7 is mixed with an organic phase containing phosphatidylcholine, and the mixture is shaken and reacted at 40°C.

10. The method for preparing phosphatidylglycerol according to claim 9, characterized in that: The organic phase is prepared by adding phosphatidylcholine to ethyl acetate, and the aqueous phase is prepared by adding glycerol and the phospholipase D-choline oxidase-catalase composite microcapsules according to claim 7 to acetate buffer.

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