CN115029331A - Immobilized enzyme pickering emulsion reaction system for preparing phosphatidylserine - Google Patents
Immobilized enzyme pickering emulsion reaction system for preparing phosphatidylserine Download PDFInfo
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
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- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
- C12N11/10—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a carbohydrate
- C12N11/12—Cellulose or derivatives thereof
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- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P13/00—Preparation of nitrogen-containing organic compounds
- C12P13/04—Alpha- or beta- amino acids
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- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
- C12P7/6436—Fatty acid esters
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- C12Y301/00—Hydrolases acting on ester bonds (3.1)
- C12Y301/04—Phosphoric diester hydrolases (3.1.4)
- C12Y301/04004—Phospholipase D (3.1.4.4)
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Abstract
The invention discloses an immobilized enzyme pickering emulsion reaction system for preparing phosphatidylserine, which consists of an immobilized enzyme, an oil phase and a water phase, wherein the oil phase and the water phase form an emulsion, and the immobilized enzyme is formed by immobilizing phospholipase D on cellulose nanofiber; the immobilized enzyme is used as a catalyst and an emulsifier at the same time; the oil phase consists of a solvent and phosphatidylcholine; the aqueous phase consists of a buffer solution and serine. The invention also discloses a method for preparing phosphatidylserine. The method takes the cellulose nanofiber as a carrier to load the phospholipase D, can adsorb on an oil-water interface to play a role of a stabilizer, can fully play a role of increasing the interface catalytic area of the Pickering emulsion, has the advantages of easiness in recovery, reusability and improvement of thermal stability and mechanical strength, has high efficiency in preparation of the phosphatidylserine, and develops a research prospect for industrial production of the functional phospholipid.
Description
Technical Field
The invention relates to an immobilized enzyme pickering emulsion reaction system for preparing phosphatidylserine, belonging to the technical field of biocatalytic synthesis.
Background
Phosphatidylserine (PS) has important physiological functions of inhibiting apoptosis and improving depression, and has been widely used in the fields of functional foods and pharmaceuticals. Phosphatidylserine can be synthesized by phospholipase d (pld) mediated transphosphatidylation of phosphatidylcholine and L-serine. The reaction condition of the enzymatic synthesis is mild, the product purity is high, and the non-natural rare phospholipid can be obtained. Enzymatic synthesis of phosphatidylserine usually takes place in an organic-aqueous biphasic reaction system, with the substrates L-serine and phosphatidylcholine dissolved in water and an organic solvent, respectively. Conventional organic solvents include diethyl ether, chloroform, etc., which tend to have specific toxicity, are not suitable for human consumption, and cause problems of low reaction efficiency and long time consumption due to interfacial area limitation. Therefore, the establishment of a novel reaction system for synthesizing phosphatidylserine is crucial to improve reaction efficiency and achieve high conversion rate.
The Pickering emulsion reaction system is an emulsion reaction system which utilizes ultrafine solid particles as an emulsifier and a catalyst for stabilization, can reduce reaction activation energy, accelerate reaction process and is beneficial to separation and recovery of the catalyst and a product. At present, a great deal of research of a Pickering emulsion reaction system takes free enzyme as a water phase, and different types of materials are utilized to prepare Pickering emulsion, so that the cost is high, the separation and purification of products are difficult, the used materials are concentrated on inorganic or synthetic particles, the biocompatibility is poor, and the enzymatic modification process is complex. In the food industry, there is an increasing interest in plant-based organic particles, particle-based emulsifiers having the potential to form and stabilize food-grade pickering emulsions.
Disclosure of Invention
Aiming at the prior art, the invention provides an immobilized enzyme pickering emulsion reaction system for preparing phosphatidylserine. According to the invention, the cellulose nanofiber immobilized enzyme is simultaneously used as the emulsifier and the catalyst, so that the reaction contact area is increased, the reaction time is shortened, the conversion rate is high, toxic chemicals are not involved, the product is easy to separate and purify, the stability of the product phosphatidylserine is high, and the method is more green and economical.
The invention is realized by the following technical scheme:
an immobilized enzyme pickering emulsion reaction system for preparing phosphatidylserine comprises an immobilized enzyme, an oil phase and a water phase which form emulsion, wherein the immobilized enzyme is formed by immobilizing phospholipase D on Cellulose Nanofiber (CNF); the immobilized enzyme is used as a catalyst and an emulsifier at the same time; the oil phase consists of a solvent and phosphatidylcholine; the water phase consists of a buffer solution and serine, and reaction substrates, namely phosphatidylcholine and serine, are dissolved in the oil phase and the water phase respectively.
Further, the immobilized enzyme is prepared by the following method: adding cellulose nanofiber into a solution containing phospholipase D, incubating for 2-24 hours at 15-25 ℃, centrifuging, washing a precipitate with Tris-HCl buffer solution, centrifuging, and freeze-drying to obtain the immobilized enzyme.
Furthermore, the diameter of the cellulose nano-fiber is 10-50 nm, the fiber length is 100-400 nm, and the cellulose nano-fiber can be extracted from microcrystalline cellulose by a ball milling method. The ratio of the phospholipase D to the cellulose nanofiber is as follows: the phospholipase D is loaded on 2-10U on 100-400 mg of cellulose nano-fiber, and preferably 4.6U on 120 mg of cellulose nano-fiber.
Further, the solvent is selected from any one or more than two of heptane, ethyl acetate, butyl acetate and ethyl n-butyrate; the concentration of the phosphatidylcholine in the oil phase is 5-20 mg/mL, preferably 10 mg/mL.
Further, the pH value of the buffer solution is 4.0-6.0, the concentration is 0.01-0.1 mol/L, and the buffer solution is selected from a Tris-HCl buffer solution and a citric acid-sodium citrate buffer solution; the concentration of serine in the water phase is 0.5-1.5 mol/L, preferably 1.0 mol/L.
Further, the volume ratio of the oil phase to the water phase is 1: 0.2-1.2, preferably 1: 1; the molar ratio of the phosphatidylcholine to the serine is 1: 50-80, preferably 1: 80; the enzyme adding amount of the immobilized enzyme is as follows: 2-20 mg of immobilized enzyme, preferably 16 mg, is added to 1 mL of oil phase.
Further, the particle size of the emulsion is 5-40 μm.
The preparation method of the immobilized enzyme pickering emulsion reaction system for preparing the phosphatidylserine comprises the following steps: dissolving phosphatidylcholine in a solvent to obtain an oil phase; obtaining a water phase in the serine solution buffer solution; and mixing the oil phase and the water phase, adding an immobilized enzyme, and performing ultrasonic dispersion to obtain an immobilized enzyme pickering emulsion reaction system with the particle size of 5-40 mu m.
Further, the specific parameters of the ultrasonic dispersion are as follows: the time is 100-200 s, preferably 180 s; the power is 40-140W, preferably 100W; the ultrasonic pause time is 3 s/3 s-9 s/3 s, preferably 3 s/3 s.
A method for preparing phosphatidylserine comprises the following steps: reacting the immobilized enzyme pickering emulsion reaction system in a water bath at 37-42 ℃ for 1-12 hours; and (4) centrifuging to take the supernatant after the reaction is finished, dissolving the product phosphatidylserine in the upper organic phase, and blowing nitrogen to obtain the phosphatidylserine.
According to the immobilized enzyme pickering emulsion reaction system, cellulose nanofiber is used as a carrier to load phospholipase D, the immobilized enzyme pickering emulsion reaction system can be adsorbed on an oil-water interface to play a role of a stabilizer, the effect of increasing the catalytic area of the interface of the pickering emulsion can be fully exerted, and the immobilized enzyme pickering emulsion reaction system has the advantages of being easy to recycle, capable of being repeatedly used, and capable of improving thermal stability and mechanical strength. The invention combines enzyme and cellulose nano-fiber through hydrophobic interaction by a one-step immobilization method.
The immobilized enzyme Pickering emulsion reaction system solves the problems of low reaction efficiency and low conversion rate of phosphatidylserine synthesis, and achieves the following beneficial results:
(1) the method disclosed by the invention has the advantages that a novel Pickering emulsion reaction system is used for replacing a traditional two-phase system for realizing the efficient synthesis of phosphatidylserine for the first time, reaction raw materials such as phosphatidylcholine and serine are used as substrates, food-grade ethyl n-butyrate is used as an oil phase, the problems of toxicity and low reaction efficiency of the traditional two-phase reaction system are avoided, the preparation method has the characteristics of simple steps, suitability for the efficient synthesis of phosphatidylserine and the like, no solvent pollution is caused, the catalytic activity and conversion rate are high, the reaction efficiency is excellent, the product is easy to separate and purify, the repeated use is easy, and the like.
(2) Compared with inorganic materials with poor biocompatibility and complex enzymatic modification process, the cellulose nanofiber is easy to combine with enzyme, greatly increases the contact area of the enzyme and a substrate, shortens the reaction time, reduces the dosage of the enzyme, and is favorable for accelerating the mass transfer and the reaction process of the substrate.
(3) The invention explores the preparation conditions of a Pickering emulsion reaction system for synthesizing the phosphatidylserine, the enzyme and the carrier are fixed by affinity adsorption and can be fully contacted with substrate molecules, the preparation of the phosphatidylserine by the Pickering emulsion reaction system is obviously improved compared with free enzyme or immobilized enzyme, the production cost is greatly reduced, and the research prospect is developed for the industrial production of the functional phospholipid.
The various terms and phrases used herein have the ordinary meaning as is well known to those skilled in the art.
Drawings
FIG. 1: scanning electron microscope images of microcrystalline cellulose and cellulose nanofibers, wherein a: microcrystalline cellulose; b: cellulose nanofibers.
FIG. 2: infrared spectrogram of microcrystalline cellulose, cellulose nanofiber and immobilized enzyme CNF-CBD 1.
FIG. 3: fluorescence microscopy images of pickering emulsions.
FIG. 4: the conversion rate of phosphatidylserine of different reaction systems is compared and shown schematically, wherein 1, a free enzyme biphasic reaction system; 2. a microcrystalline cellulose immobilized enzyme two-phase reaction system; 3. a cellulose nanofiber immobilized enzyme biphase reaction system; 4. cellulose nanofiber immobilized enzyme pickering emulsion reaction system.
Detailed Description
The present invention will be further described with reference to the following examples. However, the scope of the present invention is not limited to the following examples. It will be understood by those skilled in the art that various changes and modifications may be made to the invention without departing from the spirit and scope of the invention.
The instruments, reagents and materials used in the following examples are conventional instruments, reagents and materials known in the art and are commercially available. Unless otherwise specified, the experimental methods and detection methods described in the following examples are conventional experimental methods and detection methods known in the art.
The phospholipase D used in the invention is obtained by the following method: future origin isTrichoderma reeseiCBHI (GenBank: AF 283514.1) and a polypeptide derived fromBacillus circulansRespectively fusing the WL-12 (GenBank: M57601.1) carbohydrate functional domain with the C terminal of PLDRecom34 (GenBank: MN 604233) to construct pET28a-PLD-CBD1 and pET28a-PLD-CBDchiA1 plasmids, transferring the plasmids into competent escherichia coli, screening positive transformants to respectively obtain recombinant strains expressing phospholipase PLD-CBD1 and phospholipase PLD-chiA 1; culturing the recombinant strain to obtain phospholipase PLD-CBD1 and phospholipase PLD-chiA1 (amino acid sequences of the phospholipase PLD-CBD1 and the phospholipase PLD-chiA1 can be reasonably estimated through the construction process of the recombinant plasmid, and the nucleotide sequence of a structural gene involved in the construction process of the recombinant plasmid and the amino acid sequence of a protein expressed by the structural gene are known in the prior art and are not described in detail in the invention), wherein the phospholipase PLD-CBD1 can perform affinity adsorption with cellulose nanofiber and microcrystalline cellulose to realize one-step immobilization, and the phospholipase PLD-chiA1 can perform one-step immobilization with chitin.
The specific operations of culturing the strain and obtaining the recombinase are as follows:
the strain which is successfully transferred into pET28a-PLD-CBD1 and the strain which is transferred into pET28a-PLD-CBDchiA1 are respectively inoculated into 1.5L ZYP-5052 culture medium containing 0.5 per mill kanamycin, and shaking culture fermentation is carried out for 48 hours at the temperature of 20 ℃ and under the condition of 200 r/min to obtain enough recombinant enzyme; after fermentation, the culture solution is crushed under high pressure (crushing under 6 MPa for 10 min), centrifuged (12000 r/min, 15 min) to obtain clear supernatant, i.e. crude enzyme solution, and freeze-dried to obtain phospholipase PLD-CBD1 and phospholipase PLD-chiA 1.
The cellulose nano-fiber used in the invention is extracted from microcrystalline cellulose by a ball milling method in the presence of [ BMIM ] Cl (1-butyl-3-methylimidazole chloride) ionic liquid, and the specific operation is as follows: 2 g of microcrystalline cellulose, 2 g of [ BMIM ] Cl and 30 mL of distilled water were mixed, and three zirconia balls having a diameter of 5 mm, 3 mm and 1.8 mm were added, and the weights thereof were 4 g, 4 g and 3 g, respectively. The process was carried out in a ball mill pot at a milling speed of 300 r/min for 1.5 hours. The collected product was washed with distilled water, the residual [ BMIM ] Cl was removed by vacuum filtration, and lyophilized to obtain cellulose nanofibers. The scanning electron microscope images of microcrystalline cellulose and cellulose nanofibers are shown in fig. 1.
Different carriers are used for loading phospholipase D, corresponding immobilized enzyme two-phase reaction systems are prepared, and the efficiency of synthesizing phosphatidylserine is investigated, which is as follows:
an immobilized enzyme reaction system consists of 12 mg of immobilized enzyme (three of which are specifically shown below), 1 mL of oil phase and 1 mL of water phase, wherein the oil phase consists of ethyl butyrate and phosphatidylcholine serving as solvents, and the concentration of the phosphatidylcholine is 10 mg/mL; the aqueous phase consisted of sodium citrate buffer (20 mmol/L, pH 6.0) and serine at a concentration of 1 mol/L. The preparation method comprises the following steps: dissolving phosphatidylcholine in ethyl n-butyrate to obtain an oil phase; adding a serine solution into a sodium citrate buffer solution to obtain a water phase; mixing the oil phase and the water phase, and adding immobilized enzyme to obtain three immobilized enzyme two-phase reaction systems.
The immobilized enzymes are respectively as follows: the preparation method of the cellulose nanofiber immobilized enzyme, the cellulose immobilized enzyme and the chitin immobilized enzyme comprises the following steps:
cellulose nanofiber immobilized enzyme: adding 120 mg cellulose nanofiber into 10 mL crude enzyme solution containing 0.46U/mL phospholipase PLD-CBD1, incubating for 11 hours at 20 ℃ and 200 r/min, centrifuging for 5 min at 10000 r/min, discarding supernatant, washing precipitate with Tris-HCl buffer solution for 3 times, centrifuging, and freeze-drying to obtain the cellulose nanofiber immobilized enzyme. The enzyme activity restoring force (i.e. the ratio of the total enzyme activity of the immobilized enzyme to the total initial enzyme activity) is 56.3%, indicating the success of the immobilization process. The infrared spectrums of the microcrystalline cellulose, the cellulose nanofiber and the cellulose nanofiber immobilized enzyme are shown in fig. 2, the spectrum of the modified cellulose nanofiber is the same as that of the original microcrystalline cellulose, the similarity of the chemical structures is verified, the infrared spectrum of the cellulose nanofiber immobilized enzyme has a protein N-H stretching vibration peak, and an amide C = O characteristic peak, and the successful immobilization of the PLD-CBD1 is verified.
Cellulose immobilized enzyme: adding 120 mg cellulose nano-fiber into 10 mL crude enzyme solution containing 0.46U/mL phospholipase PLD-CBD1, incubating for 11 hours at 20 ℃ at 200 r/min, centrifuging, washing the precipitate with Tris-HCl buffer solution for 3 times, centrifuging, separating, and freeze-drying to obtain the cellulose immobilized enzyme.
Chitin immobilized enzyme: adding 120 mg chitin (purchased from Mecline) into 10 mL crude enzyme solution containing 0.46U/mL phospholipase PLD-chiA1, incubating at 20 deg.C and 200 r/min for 11 hr, centrifuging, washing the precipitate with Tris-HCl buffer solution for 3 times, centrifuging, and freeze-drying to obtain chitin immobilized enzyme.
The three immobilized enzyme reaction systems are placed in a constant-temperature water bath at 40 ℃ and stirred for reaction for 4 hours, and the stirring speed is 220 r/min; centrifuging at 8000 r/min for 3 min after the reaction is finished, taking an upper layer organic phase, and drying by blowing to obtain the product phosphatidylserine. Calculating the conversion rate of the phosphatidylserine, performing three repeated experiments on each group, taking the average value, and obtaining the result: the conversion rate of the two-phase reaction system containing the cellulose nanofiber immobilized enzyme is 88.6 percent, the conversion rate of the two-phase reaction system containing the cellulose immobilized enzyme is 43.3 percent, and the conversion rate of the two-phase reaction system containing the chitin immobilized enzyme is 32.8 percent.
Different carriers are used for loading phospholipase D, corresponding immobilized enzyme Pickering emulsion reaction systems are prepared, and the efficiency of synthesizing phosphatidylserine is investigated, which is as follows:
an immobilized enzyme pickering emulsion reaction system consists of a cellulose nanofiber immobilized enzyme, 1 mL of oil phase and 1 mL of water phase, wherein the oil phase consists of ethyl butyrate and phosphatidylcholine serving as solvents, and the concentration of the phosphatidylcholine is 10 mg/mL; the aqueous phase consisted of sodium citrate buffer (20 mmol/L, pH 6.0) and serine at a concentration of 1 mol/L. The preparation method comprises the following steps: dissolving phosphatidylcholine in ethyl n-butyrate to obtain an oil phase; adding a serine solution into a sodium citrate buffer solution to obtain a water phase; and mixing the oil phase and the water phase, adding an immobilized enzyme, and performing ultrasonic dispersion (time 180 s; power 100W; ultrasonic intermittent time 3 s/3 s) to obtain a cellulose nanofiber immobilized enzyme pickering emulsion reaction system, wherein the addition amounts of the cellulose nanofiber immobilized enzyme are respectively 2 mg, 4 mg, 8 mg, 12 mg, 16 mg and 20 mg.
Examining the conversion rate of the Pickering emulsion reaction system with different immobilized enzyme addition amounts: placing the mixture in a constant-temperature water bath at 40 ℃ for stirring reaction for 2 hours, wherein the stirring speed is 200 r/min, and other steps are the same as those in experiment 1, so that the result is that: when the addition amounts of the cellulose nanofiber immobilized enzymes were 2 mg, 4 mg, 8 mg, 12 mg, 16 mg, and 20 mg, the conversion rates were 1.4%, 12.0%, 18.4%, 71.8%, 91.8%, and 92.0%, respectively, and it was found that when the addition amount was 16 mg, the conversion rate had already reached a very high level (91.8%), the addition amount of the enzyme continued to increase, and the conversion rate remained approximately constant. The addition amount of 16 mg of the immobilized CNF-CBD1 is suitable for the reaction system by comprehensively considering economic factors and catalytic effect.
Example 3 comparison of Pickering emulsion reaction System with two-phase reaction System
Investigating the phosphatidylserine conversion rates of a cellulose nanofiber immobilized enzyme pickering emulsion reaction system, a free enzyme two-phase reaction system, a microcrystalline cellulose immobilized enzyme two-phase reaction system and a cellulose nanofiber immobilized enzyme two-phase reaction system: stirring and reacting for 2 hours in a constant temperature water bath at 40 ℃, wherein the stirring speed is 200 r/min; centrifuging at 8000 r/min for 8 min after reaction, taking the upper organic phase, and drying to obtain the product phosphatidylserine. Calculating the conversion rate of the phosphatidylserine, performing three repeated experiments on each group, taking the average value, and obtaining the result: as shown in fig. 4, the conversion rates of the free enzyme dual-phase reaction system, the microcrystalline cellulose immobilized enzyme dual-phase reaction system, and the cellulose nanofiber immobilized enzyme dual-phase reaction system are respectively 4.5%, 13.2%, and 69.5%, and the conversion rate of the cellulose nanofiber immobilized enzyme pickering emulsion reaction system is the highest, 95.4%, which is 7 times that of the microcrystalline cellulose immobilized enzyme dual-phase reaction system, and 21 times that of the free enzyme dual-phase reaction system.
The preparation method of the cellulose nanofiber immobilized enzyme pickering emulsion reaction system is the same as that of experiment 2 (the addition amount of the cellulose nanofiber immobilized enzyme is 16 mg), and a fluorescence microscope image of the cellulose nanofiber immobilized enzyme pickering emulsion reaction system is shown in fig. 3, so that the emulsion is proved to be of an oil-in-water type, liquid drops are uniformly dispersed, and the size distribution is 5-15 mu m.
The preparation method of the free enzyme biphasic reaction system comprises the following steps: dissolving phosphatidylcholine in ethyl n-butyrate to obtain an oil phase; adding a serine solution into a sodium citrate buffer solution to obtain a water phase; mixing oil phase and water phase, and adding enzyme powder (phospholipase PLD-CBD 1) with enzyme activity of 0.61U.
The preparation method of the microcrystalline cellulose immobilized enzyme dual-phase reaction system and the cellulose nano-fiber immobilized enzyme dual-phase reaction system is the same as the preparation method of the free enzyme dual-phase reaction system, and the difference is that the added free enzyme is replaced by the microcrystalline cellulose immobilized enzyme (16 mg) and the cellulose nano-fiber immobilized enzyme (16 mg).
The above examples are provided to those of ordinary skill in the art to fully disclose and describe how to make and use the claimed embodiments, and are not intended to limit the scope of the disclosure herein. Modifications apparent to those skilled in the art are intended to be within the scope of the appended claims.
Claims (10)
1. An immobilized enzyme pickering emulsion reaction system for preparing phosphatidylserine is characterized in that: the enzyme-immobilized phospholipase is composed of an immobilized enzyme, an oil phase and a water phase which form an emulsion, wherein the immobilized enzyme is formed by immobilizing phospholipase D on cellulose nanofiber; the immobilized enzyme is used as a catalyst and an emulsifier at the same time; the oil phase consists of a solvent and phosphatidylcholine; the aqueous phase consists of a buffer solution and serine.
2. The immobilized enzyme pickering emulsion reaction system for preparing phosphatidylserine according to claim 1, wherein the immobilized enzyme is prepared by the following method: adding cellulose nanofiber into a solution containing phospholipase D, incubating for 2-24 hours at 15-25 ℃, centrifuging, washing a precipitate with Tris-HCl buffer solution, centrifuging, and freeze-drying to obtain an immobilized enzyme; the ratio of the phospholipase D to the cellulose nanofiber is as follows: 100-400 mg of cellulose nanofibers are loaded with 2-10U of phospholipase D.
3. The immobilized enzyme pickering emulsion reaction system for preparing phosphatidylserine according to claim 1, wherein: the solvent is selected from any one or more than two of heptane, ethyl acetate, butyl acetate and ethyl n-butyrate; the concentration of phosphatidylcholine in the oil phase is 5-20 mg/mL.
4. The immobilized enzyme pickering emulsion reaction system for preparing phosphatidylserine according to claim 1, wherein: the pH value of the buffer solution is 4.0-6.0, and the buffer solution is selected from a Tris-HCl buffer solution and a citric acid-sodium citrate buffer solution; the concentration of serine in the water phase is 0.5-1.5 mol/L.
5. The immobilized enzyme pickering emulsion reaction system for preparing phosphatidylserine according to claim 1, wherein: the volume ratio of the oil phase to the water phase is 1: 0.2-1.2; the enzyme adding amount of the immobilized enzyme is as follows: 2-40 mg of immobilized enzyme is added into each 1 mL of oil phase.
6. The immobilized enzyme pickering emulsion reaction system for preparing phosphatidylserine according to claim 1, wherein: the particle size of the emulsion is 5-40 μm.
7. The immobilized enzyme pickering emulsion reaction system for preparing phosphatidylserine according to any one of claims 2 to 6, wherein: the immobilized enzyme is prepared by the following method: adding 120 mg cellulose nano-fiber into a phospholipase D solution of 0.46U/mL, incubating for 11 hours at 20 ℃ and 200 r/min, centrifuging, washing a precipitate with a Tris-HCl buffer solution, centrifuging, and freeze-drying to obtain an immobilized enzyme, wherein the enzyme activity restoring force is 56.3%;
the solvent is ethyl butyrate, and the concentration of phosphatidylcholine in the oil phase is 10 mg/mL;
the buffer solution is a citric acid-sodium citrate buffer solution with pH of 6.0 and 20 mmol/L, and the concentration of serine in the water phase is 1.0 mol/L;
the volume ratio of the oil phase to the water phase is 1: 1; the enzyme adding amount of the immobilized enzyme is as follows: adding 16 mg of immobilized enzyme into each 1 mL of oil phase;
the particle size of the emulsion is 5-15 mu m.
8. The method for preparing the immobilized enzyme pickering emulsion reaction system for preparing phosphatidylserine according to any one of claims 1 to 7, wherein the immobilized enzyme pickering emulsion reaction system comprises: dissolving phosphatidylcholine in a solvent to obtain an oil phase; obtaining a water phase in the serine solution buffer solution; mixing the oil phase and the water phase, adding immobilized enzyme, and performing ultrasonic dispersion to obtain the product.
9. The preparation method according to claim 8, wherein the specific parameters of the ultrasonic dispersion are: the time is 100-200 s; the power is 40-140W; the ultrasonic intermittent time is 3 s/3 s-9 s/3 s.
10. A method for preparing phosphatidylserine, which is characterized by comprising the following steps: the immobilized enzyme pickering emulsion reaction system of any one of claims 1 to 7, which is reacted at 37 to 42 ℃ for 1 to 12 hours; and centrifuging to take the supernatant after the reaction is finished, dissolving the product phosphatidyl serine in the upper organic phase, and blowing nitrogen to obtain the phosphatidyl serine.
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Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103966277A (en) * | 2014-05-22 | 2014-08-06 | 南京工业大学 | Method for preparing phosphatidylserine under catalysis of immobilized phospholipase D |
| WO2019069809A1 (en) * | 2017-10-06 | 2019-04-11 | 第一工業製薬株式会社 | Carrier for protein immobilization, complex, and method for producing these |
| CN109706141A (en) * | 2019-02-01 | 2019-05-03 | 中国农业科学院油料作物研究所 | A kind of immobilised enzymes pickering emulsion reaction system and its application |
| CN110923225A (en) * | 2019-12-05 | 2020-03-27 | 中国农业科学院油料作物研究所 | Cellulose gel microsphere immobilized phospholipase for phospholipid catalysis and preparation method thereof |
| CN111778295A (en) * | 2019-04-04 | 2020-10-16 | 南通厚元生物科技有限公司 | Method for synthesizing phosphatidylserine by using immobilized biocatalyst |
| CN112458076A (en) * | 2020-11-30 | 2021-03-09 | 中国农业科学院油料作物研究所 | Preparation method of cellulose-based magnetic microsphere immobilized phospholipase |
| US20210213405A1 (en) * | 2019-10-31 | 2021-07-15 | Jiangnan University | Pickering emulsion stabilized by cellulose nanocrystals from ginkgo seed shells and preparation method thereof |
-
2022
- 2022-07-29 CN CN202210903048.8A patent/CN115029331B/en active Active
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103966277A (en) * | 2014-05-22 | 2014-08-06 | 南京工业大学 | Method for preparing phosphatidylserine under catalysis of immobilized phospholipase D |
| WO2019069809A1 (en) * | 2017-10-06 | 2019-04-11 | 第一工業製薬株式会社 | Carrier for protein immobilization, complex, and method for producing these |
| CN109706141A (en) * | 2019-02-01 | 2019-05-03 | 中国农业科学院油料作物研究所 | A kind of immobilised enzymes pickering emulsion reaction system and its application |
| CN111778295A (en) * | 2019-04-04 | 2020-10-16 | 南通厚元生物科技有限公司 | Method for synthesizing phosphatidylserine by using immobilized biocatalyst |
| US20210213405A1 (en) * | 2019-10-31 | 2021-07-15 | Jiangnan University | Pickering emulsion stabilized by cellulose nanocrystals from ginkgo seed shells and preparation method thereof |
| CN110923225A (en) * | 2019-12-05 | 2020-03-27 | 中国农业科学院油料作物研究所 | Cellulose gel microsphere immobilized phospholipase for phospholipid catalysis and preparation method thereof |
| CN112458076A (en) * | 2020-11-30 | 2021-03-09 | 中国农业科学院油料作物研究所 | Preparation method of cellulose-based magnetic microsphere immobilized phospholipase |
Non-Patent Citations (2)
| Title |
|---|
| TREATMENTPALANIVEL SATHISHKUMAR 等: "Laccase immobilization on cellulose nanofiber: The catalytic efficiency and recyclic application for simulated dye effluent treatment", 《JOURNAL OF MOLECULAR CATALYSIS B: ENZYMATIC》, vol. 100, no. 2014, pages 1 - 3 * |
| 刘琦 等: "产磷脂酶D工程菌构建及发酵条件优化", 《食品研究与开发》, vol. 43, no. 11, pages 26 - 34 * |
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