CN115029331B - 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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- 238000006243 chemical reaction Methods 0.000 title claims abstract description 97
- 108010093096 Immobilized Enzymes Proteins 0.000 title claims abstract description 83
- 239000000839 emulsion Substances 0.000 title claims abstract description 42
- TZCPCKNHXULUIY-RGULYWFUSA-N 1,2-distearoyl-sn-glycero-3-phosphoserine Chemical compound CCCCCCCCCCCCCCCCCC(=O)OC[C@H](COP(O)(=O)OC[C@H](N)C(O)=O)OC(=O)CCCCCCCCCCCCCCCCC TZCPCKNHXULUIY-RGULYWFUSA-N 0.000 title claims abstract description 37
- ZWZWYGMENQVNFU-UHFFFAOYSA-N Glycerophosphorylserin Natural products OC(=O)C(N)COP(O)(=O)OCC(O)CO ZWZWYGMENQVNFU-UHFFFAOYSA-N 0.000 title claims abstract description 37
- 229920002678 cellulose Polymers 0.000 claims abstract description 53
- 239000001913 cellulose Substances 0.000 claims abstract description 53
- 239000002121 nanofiber Substances 0.000 claims abstract description 50
- 239000012071 phase Substances 0.000 claims abstract description 45
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 31
- WTJKGGKOPKCXLL-RRHRGVEJSA-N phosphatidylcholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCCC=CCCCCCCCC WTJKGGKOPKCXLL-RRHRGVEJSA-N 0.000 claims abstract description 18
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- 238000000034 method Methods 0.000 claims abstract description 15
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- OBNCKNCVKJNDBV-UHFFFAOYSA-N ethyl butyrate Chemical group CCCC(=O)OCC OBNCKNCVKJNDBV-UHFFFAOYSA-N 0.000 claims description 16
- 239000000047 product Substances 0.000 claims description 13
- 239000000243 solution Substances 0.000 claims description 10
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 6
- 239000002245 particle Substances 0.000 claims description 6
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- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 4
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- 238000004108 freeze drying Methods 0.000 claims description 4
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- IAZSXUOKBPGUMV-UHFFFAOYSA-N 1-butyl-3-methyl-1,2-dihydroimidazol-1-ium;chloride Chemical compound [Cl-].CCCC[NH+]1CN(C)C=C1 IAZSXUOKBPGUMV-UHFFFAOYSA-N 0.000 claims description 3
- 239000000203 mixture Substances 0.000 claims description 3
- 238000007664 blowing Methods 0.000 claims description 2
- 238000000227 grinding Methods 0.000 claims description 2
- 229910052757 nitrogen Inorganic materials 0.000 claims description 2
- XPFJYKARVSSRHE-UHFFFAOYSA-K trisodium;2-hydroxypropane-1,2,3-tricarboxylate;2-hydroxypropane-1,2,3-tricarboxylic acid Chemical compound [Na+].[Na+].[Na+].OC(=O)CC(O)(C(O)=O)CC(O)=O.[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O XPFJYKARVSSRHE-UHFFFAOYSA-K 0.000 claims description 2
- 238000003828 vacuum filtration Methods 0.000 claims description 2
- 238000009777 vacuum freeze-drying Methods 0.000 claims 1
- 230000000694 effects Effects 0.000 abstract description 6
- 239000008346 aqueous phase Substances 0.000 abstract description 5
- 239000003054 catalyst Substances 0.000 abstract description 5
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- MTCFGRXMJLQNBG-REOHCLBHSA-N (2S)-2-Amino-3-hydroxypropansäure Chemical compound OC[C@H](N)C(O)=O MTCFGRXMJLQNBG-REOHCLBHSA-N 0.000 description 15
- ZIIUUSVHCHPIQD-UHFFFAOYSA-N 2,4,6-trimethyl-N-[3-(trifluoromethyl)phenyl]benzenesulfonamide Chemical compound CC1=CC(C)=CC(C)=C1S(=O)(=O)NC1=CC=CC(C(F)(F)F)=C1 ZIIUUSVHCHPIQD-UHFFFAOYSA-N 0.000 description 14
- 102000015439 Phospholipases Human genes 0.000 description 14
- 108010064785 Phospholipases Proteins 0.000 description 14
- 229960001153 serine Drugs 0.000 description 13
- 229920002101 Chitin Polymers 0.000 description 7
- 230000015572 biosynthetic process Effects 0.000 description 7
- 238000003786 synthesis reaction Methods 0.000 description 7
- 238000002474 experimental method Methods 0.000 description 6
- 239000000758 substrate Substances 0.000 description 6
- 230000008569 process Effects 0.000 description 5
- 239000001509 sodium citrate Substances 0.000 description 5
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 5
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- FHDQNOXQSTVAIC-UHFFFAOYSA-M 1-butyl-3-methylimidazol-3-ium;chloride Chemical compound [Cl-].CCCCN1C=C[N+](C)=C1 FHDQNOXQSTVAIC-UHFFFAOYSA-M 0.000 description 3
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 3
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 3
- 239000000872 buffer Substances 0.000 description 3
- 238000000926 separation method Methods 0.000 description 3
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- IMNFDUFMRHMDMM-UHFFFAOYSA-N N-Heptane Chemical compound CCCCCCC IMNFDUFMRHMDMM-UHFFFAOYSA-N 0.000 description 2
- 108010091086 Recombinases Proteins 0.000 description 2
- 102000018120 Recombinases Human genes 0.000 description 2
- 150000001413 amino acids Chemical group 0.000 description 2
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- DKPFZGUDAPQIHT-UHFFFAOYSA-N Butyl acetate Natural products CCCCOC(C)=O DKPFZGUDAPQIHT-UHFFFAOYSA-N 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
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- 150000001720 carbohydrates Chemical class 0.000 description 1
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- 239000001963 growth medium Substances 0.000 description 1
- FUZZWVXGSFPDMH-UHFFFAOYSA-N hexanoic acid Chemical compound CCCCCC(O)=O FUZZWVXGSFPDMH-UHFFFAOYSA-N 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 229910010272 inorganic material Inorganic materials 0.000 description 1
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- 239000002608 ionic liquid Substances 0.000 description 1
- 229930027917 kanamycin Natural products 0.000 description 1
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 1
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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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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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- 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
- C12P13/06—Alanine; Leucine; Isoleucine; Serine; Homoserine
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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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
- C12P7/6445—Glycerides
- C12P7/6481—Phosphoglycerides
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- C12Y—ENZYMES
- 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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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/584—Recycling of catalysts
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- General Chemical & Material Sciences (AREA)
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Abstract
The invention discloses an immobilized enzyme pickering emulsion reaction system for preparing phosphatidylserine, which consists of 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; 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. According to the invention, the cellulose nanofiber is used as a carrier to load phospholipase D, so that the cellulose nanofiber can be adsorbed on an oil-water interface to play a role of a stabilizer, the effect of increasing the interface catalytic area of the Pickering emulsion can be fully exerted, the advantages of easiness in recovery, reusability, and improvement of thermal stability and mechanical strength are achieved, the phosphatidylserine preparation efficiency is high, and the research prospect is developed for the industrial production of functional phospholipids.
Description
Technical Field
The invention relates to an immobilized enzyme pickering emulsion reaction system for preparing phosphatidylserine, belonging to the technical field of biocatalysis synthesis.
Background
Phosphatidylserine (PS) has been widely used in functional foods and pharmaceutical fields, and has important physiological functions in inhibiting apoptosis and improving depression. Phosphatidylserine can be synthesized by phospholipase D (PLD) mediated transphosphorylation of phosphatidylcholine and L-serine. The enzymatic synthesis has mild reaction conditions and high product purity, and can obtain unnatural rare phospholipid. The enzymatic synthesis of phosphatidylserine usually occurs in an organic-aqueous biphasic reaction system, with the substrates L-serine and phosphatidylcholine being dissolved in water and organic solvents, respectively. Conventional organic solvents include diethyl ether, chloroform, etc., which tend to have specific toxicity, are unsuitable 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 important for improving reaction efficiency and achieving high conversion rate.
The Pickering emulsion reaction system is an emulsion reaction system which uses superfine solid particles as an emulsifier and a catalyst to stabilize, so that the reaction activation energy can be reduced, the reaction process can be accelerated, and the separation and recovery of the catalyst and a product can be facilitated. At present, a large amount of researches on Pickering emulsion reaction systems are to prepare Pickering emulsion by using free enzyme as a water phase and using different types of materials, 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
The invention provides an immobilized enzyme pickering emulsion reaction system for preparing phosphatidylserine. The cellulose nanofiber immobilized enzyme is 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, and the product phosphatidylserine is high in stability and is more green and economical.
The invention is realized by the following technical scheme:
an immobilized enzyme Pickering emulsion reaction system for preparing phosphatidylserine, which consists of 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 aqueous phase consists of a buffer solution and serine, and reaction substrates phosphatidylcholine and serine are respectively dissolved in the oil phase and the water phase.
Further, the immobilized enzyme is prepared by the following method: adding cellulose nano-fibers into a solution containing phospholipase D, incubating for 2-24 hours at 15-25 ℃, centrifuging, flushing sediment with Tris-HCl buffer solution, centrifuging, separating, and freeze-drying to obtain immobilized enzyme.
Further, the cellulose nanofiber has a diameter of 10-50 nm and a fiber length of 100-400 nm, and can be extracted from microcrystalline cellulose by a ball milling method. The proportioning relation between the phospholipase D and the cellulose nanofiber is as follows: the cellulose nanofiber of 100 to 400 and mg is loaded with phospholipase D of 2 to 10U, preferably the cellulose nanofiber of 120. 120 mg is loaded with phospholipase D of 4.6. 4.6U.
Further, the solvent is selected from 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, preferably 10 mg/mL.
Further, the pH of the buffer solution is 4.0-6.0, and the concentration is 0.01-0.1 mol/L, and the buffer solution is selected from Tris-HCl buffer solution and citric acid-sodium citrate buffer solution; the concentration of serine in the aqueous phase is 0.5 to 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 serine is 1:50-80, preferably 1:80; the enzyme adding amount of the immobilized enzyme is as follows: 2-20 mg immobilized enzymes, preferably 16 mg, are added per 1 mL 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 phosphatidylserine comprises the following steps: dissolving phosphatidylcholine in a solvent to obtain an oil phase; buffering serine solution to obtain water phase; mixing the oil phase and the water phase, adding 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, specific parameters of the ultrasonic dispersion are as follows: time 100-200 s, preferably 180 s; power 40-140W, preferably 100W; the ultrasonic intermittence time is 3 s/3 s-9 s/3 s, preferably 3 s/3 s.
A method for preparing phosphatidylserine: the immobilized enzyme Pickering emulsion reaction system reacts in water bath at 37-42 ℃ for 1-12 hours; centrifuging after the reaction is finished, taking supernatant, dissolving the product phosphatidylserine in an upper organic phase, and blowing nitrogen to obtain the phosphatidylserine.
According to the immobilized enzyme Pickering emulsion reaction system, the cellulose nanofiber is used as a carrier to load phospholipase D, so that the immobilized enzyme Pickering emulsion reaction system can be adsorbed 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, and has the advantages of easiness in recovery, reusability and improvement of 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 obtains the following beneficial results:
(1) The invention uses the novel Pickering emulsion reaction system to replace the traditional biphase system for the first time to realize the efficient synthesis of phosphatidylserine, uses the phosphatidylcholine, serine and other reaction raw materials as substrates, uses the food-grade ethyl n-butyrate as an oil phase, avoids the problems of toxicity and low reaction efficiency of the traditional biphase reaction system, has the characteristics of simple preparation method steps, suitability for the efficient synthesis of phosphatidylserine and the like, and has the advantages of no solvent pollution, high catalytic activity and conversion rate, excellent reaction efficiency, easy separation and purification of products, easy repeated use 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 use amount of the enzyme, and is beneficial to accelerating the mass transfer and the reaction process of the substrate.
(3) The invention explores the preparation conditions of the Pickering emulsion reaction system for synthesizing phosphatidylserine, enzyme and carrier are fixed through affinity adsorption, and can be fully contacted with substrate molecules, compared with free enzyme or immobilized enzyme, the preparation of phosphatidylserine by the Pickering emulsion reaction system is obviously improved, the production cost is greatly reduced, and the invention opens up the research prospect for the industrialized production of functional phospholipid.
The various terms and phrases used herein have the ordinary meaning known to those skilled in the art.
Drawings
Fig. 1: scanning electron microscope image 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 image of pickering emulsion.
Fig. 4: the conversion rate of phosphatidylserine of different reaction systems is shown in a comparison schematic diagram, wherein, 1 is a free enzyme biphasic reaction system; 2. a microcrystalline cellulose immobilized enzyme biphasic reaction system; 3. a cellulose nanofiber immobilized enzyme biphasic reaction system; 4. cellulose nanofiber immobilized enzyme pickering emulsion reaction system.
Detailed Description
The invention is further illustrated below with reference to examples. However, the scope of the present invention is not limited to the following examples. Those skilled in the art will appreciate that various changes and modifications can be made to the invention without departing from the spirit and scope thereof.
The instruments, reagents and materials used in the examples below are conventional instruments, reagents and materials known in the art and are commercially available. The experimental methods, detection methods, and the like in the examples described below are conventional experimental methods and detection methods known in the prior art unless otherwise specified.
The phospholipase D used in the invention is obtained by the following method: derived fromTrichoderma reeseiCBHI (GenBank: AF 283514.1) and derived fromBacillus circulansThe WL-12 (GenBank: M57601.1) carbohydrate functional domain of (B) is respectively fused with the C terminal of PLDRecom34 (GenBank: MN 604233) to construct pET28a-PLD-CBD1 and pET28a-PLD-CBDchiA1 plasmids, the plasmids are transferred into competent escherichia coli, positive transformants are screened, and recombinant strains for expressing phospholipase PLD-CBD1 and phospholipase PLD-chiA1 are respectively obtained; culturing the recombinant strain to obtain the expressed phospholipase PLD-CBD1 and phospholipase PLD-chiA1 (the amino acid sequences of the phospholipase PLD-CBD1 and the phospholipase PLD-chiA1 can be reasonably deduced 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 all known sequences in the prior art, and are not repeated in the invention), wherein the phospholipase PLD-CBD1 can be subjected to affinity adsorption with cellulose nanofibers and microcrystalline cellulose to realize one-step immobilization, and the phospholipase PLD-chiA1 can be subjected to one-step immobilization with chitin.
The specific operation of culturing the strain and obtaining the recombinase is as follows:
the strain which is successfully transferred into pET28a-PLD-CBD1 and the strain of pET28a-PLD-CBDchiA1 are respectively inoculated into ZYP-5052 culture medium containing 0.5 per mill kanamycin of 1.5L, and shake culture and fermentation are carried out for 48 hours at 20 ℃ and 200 r/min to obtain a sufficient amount of recombinase; after fermentation, the culture solution is crushed under high pressure (crushing for 10 min under the pressure of 6 MPa), and centrifuged (12000 r/min,15 min) to obtain clear supernatant, namely crude enzyme solution, and freeze-dried to obtain phospholipase PLD-CBD1 and phospholipase PLD-chiA1.
The cellulose nanofiber used in the invention is obtained by extracting microcrystalline cellulose by a ball milling method in the presence of [ BMIM ] Cl (1-butyl-3-methylimidazole chloride) ionic liquid, and comprises the following specific operations: 2 g microcrystalline cellulose, 2 g [ BMIM ] Cl and 30 mL distilled water were mixed, and three kinds of zirconia balls having diameters of 5 mm,3 mm and 1.8 mm were added, and the weights thereof were 4 g,4 g and 3 g, respectively. Processing was performed in a ball mill pot at a grinding speed of 300 r/min for 1.5 hours. The collected product was washed with distilled water, and residual [ BMIM ] Cl was removed by vacuum filtration, and freeze-dried to obtain cellulose nanofibers. A scanning electron microscope image of microcrystalline cellulose and cellulose nanofibers is shown in fig. 1.
Experiment 1 comparison of the effects of different immobilized enzymes on the Synthesis of phosphatidylserine
Phospholipase D is loaded by different carriers, a corresponding immobilized enzyme biphasic reaction system is prepared, and the efficiency of synthesizing phosphatidylserine is examined, wherein the specific steps are as follows:
an immobilized enzyme reaction system, which consists of 12 mg immobilized enzymes (three types are described below), 1 mL oil phase and 1 mL water phase, wherein the oil phase consists of ethyl n-butyrate and phosphatidylcholine, 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; obtaining a water phase in a serine solution sodium citrate buffer solution; mixing the oil phase and the water phase, and adding the immobilized enzyme to obtain three immobilized enzyme biphasic reaction systems.
The immobilized enzymes are respectively: cellulose nanofiber immobilized enzyme, cellulose immobilized enzyme and chitin immobilized enzyme, and the preparation method specifically 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 at 20 ℃ for 11 hours at 200 r/min, centrifuging at 10000 r/min for 5 min, discarding supernatant, washing precipitate with Tris-HCl buffer solution for 3 times, centrifuging, separating, and freeze-drying to obtain cellulose nanofiber immobilized enzyme. The enzyme activity recovery (i.e., the ratio of total enzyme activity of the immobilized enzyme to total initial enzyme activity) was 56.3%, indicating success of the immobilization process. The infrared spectra of microcrystalline cellulose, cellulose nanofiber and cellulose nanofiber immobilized enzyme are shown in figure 2, the modified cellulose nanofiber has the same spectrum as the original microcrystalline cellulose, the similarity of chemical structures is verified, the infrared spectra of the cellulose nanofiber immobilized enzyme have a protein N-H stretching vibration peak, an amide C=O characteristic peak, and the successful immobilization of PLD-CBD1 is verified.
Cellulose-immobilized enzyme: adding 120 mg cellulose nanofiber into 10 mL crude enzyme solution containing 0.46U/mL phospholipase PLD-CBD1, incubating at 20 ℃ and 200 r/min for 11 hours, centrifuging, washing the precipitate with Tris-HCl buffer solution for 3 times, centrifuging, separating, and freeze-drying to obtain cellulose immobilized enzyme.
Chitin immobilized enzyme: 120 mg chitin (purchased from microphone) is added into 10 mL crude enzyme liquid containing 0.46U/mL phospholipase PLD-chiA1, incubated for 11 hours at 20 ℃ and 200 r/min, centrifuged, the precipitate is washed 3 times with Tris-HCl buffer, centrifuged, separated, and freeze-dried, thus obtaining the chitin immobilized enzyme.
The three immobilized enzyme reaction systems are placed in a constant-temperature water bath at 40 ℃ to be stirred and reacted for 4 hours, and the stirring speed is 220 r/min; centrifuging for 3 min at 8000 r/min after the reaction is finished, taking an upper organic phase, and drying to obtain the phosphatidylserine product. The conversion of phosphatidylserine was calculated, and three replicates were performed for each group, and the average was taken and the results were: the conversion rate of the biphasic reaction system containing cellulose nanofiber immobilized enzyme is 88.6%, the conversion rate of the biphasic reaction system containing cellulose immobilized enzyme is 43.3%, the conversion rate of the biphasic reaction system containing chitin immobilized enzyme is 32.8%, and the conversion rate of the biphasic reaction system prepared by taking cellulose nanofiber as a carrier is highest, which is obviously superior to microcrystalline cellulose and chitin, and the difference is obvious.
Experiment 2 influence of the amount of cellulose nanofiber immobilized enzyme added on conversion
The phospholipase D is loaded by different carriers, a corresponding immobilized enzyme pickering emulsion reaction system is prepared, and the efficiency of synthesizing the phosphatidylserine is examined, and the specific steps are as follows:
the immobilized enzyme Pickering emulsion reaction system consists of cellulose nanofiber immobilized enzyme, an oil phase of 1 mL and a water phase of 1 mL, wherein the oil phase consists of ethyl n-butyrate and phosphatidylcholine, 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; obtaining a water phase in a serine solution sodium citrate buffer solution; mixing the oil phase and the water phase, adding 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 adding amount of the cellulose nanofiber immobilized enzyme is 2 mg, 4 mg, 8 mg, 12 mg, 16 mg and 20 mg respectively.
The conversion rate of the Pickering emulsion reaction system with different addition amounts of the immobilized enzyme is examined: placing the mixture in a 40 ℃ constant-temperature water bath for stirring reaction for 2 hours, wherein the stirring rotation speed is 200 r/min, and other steps are the same as those of experiment 1, and 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, respectively, the corresponding conversion rates were 1.4%, 12.0%, 18.4%, 71.8%, 91.8% and 92.0%, respectively, and when the addition amount was 16 mg, the conversion rates had reached a very high level (91.8%), and the addition amount of the enzymes was continuously increased, and the conversion rates were kept approximately unchanged. 16 mg of the addition amount of the immobilized CNF-CBD1 is suitable for the reaction system by comprehensively considering economic factors and catalytic effects.
Example 3 comparison of Pickering emulsion reaction System with biphase reaction System
Examining the phosphatidylserine conversion rate of the cellulose nanofiber immobilized enzyme pickering emulsion reaction system, the free enzyme biphasic reaction system, the microcrystalline cellulose immobilized enzyme biphasic reaction system and the cellulose nanofiber immobilized enzyme biphasic reaction system: 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; centrifuging for 8 min at 8000 r/min after the reaction is finished, taking an upper organic phase, and drying to obtain the phosphatidylserine product. The conversion of phosphatidylserine was calculated, and three replicates were performed for each group, and the average was taken and the results were: as shown in fig. 4, the conversion rates of the free enzyme biphasic reaction system, the microcrystalline cellulose immobilized enzyme biphasic reaction system and the cellulose nanofiber immobilized enzyme biphasic reaction system are respectively 4.5%, 13.2% and 69.5%, the highest conversion rate of the cellulose nanofiber immobilized enzyme pickering emulsion reaction system is 95.4%, and the conversion rate is 7 times of the microcrystalline cellulose immobilized enzyme biphasic reaction system and 21 times of the free enzyme biphasic 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 as a graph in fig. 3, so that the emulsion is proved to be 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; obtaining a water phase in a serine solution sodium citrate buffer solution; mixing the oil phase and the 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 biphasic reaction system and the cellulose nanofiber immobilized enzyme biphasic reaction system is the same as the preparation method of the free enzyme biphasic reaction system, and the difference is that the added free enzyme is replaced by microcrystalline cellulose immobilized enzyme (16 mg) and cellulose nanofiber immobilized enzyme (16 mg).
The foregoing examples are provided to fully disclose and describe how to make and use the claimed embodiments by those skilled in the art, and are not intended to limit the scope of the disclosure herein. Modifications that are obvious to a person skilled in the art will be within the scope of the appended claims.
Claims (3)
1. A preparation method of an immobilized enzyme pickering emulsion reaction system for preparing phosphatidylserine is characterized by comprising the following steps: dissolving phosphatidylcholine in a solvent to obtain an oil phase; dissolving serine in a buffer solution to obtain a water phase; mixing the oil phase and the water phase, adding immobilized enzyme, and performing ultrasonic dispersion to obtain the product;
the solvent is ethyl n-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 the pH value of 6.0 and the concentration of serine in the water phase of 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: 16 mg of immobilized enzyme is added to each 1 mL oil phase;
the immobilized enzyme is prepared by the following method: adding 120 mg cellulose nanofiber into 0.46U/mL phospholipase D solution, incubating at 20 ℃ and 200 r/min for 11 hours, centrifuging, washing the precipitate with Tris-HCl buffer solution, centrifuging, separating, and freeze-drying to obtain immobilized enzyme;
the cellulose nanofiber is prepared by the following method: 2 g microcrystalline cellulose, 2 g of 1-butyl-3-methylimidazole chloride and 30 mL distilled water were mixed, three kinds of zirconia balls having diameters of 5 mm,3 mm and 1.8 mm were added, the weights thereof were 4 g,4 g and 3 g, respectively, and the mixture was processed in a ball mill pot at a grinding speed of 300 r/min for 1.5 hours; washing the collected product with distilled water, removing residual 1-butyl-3-methylimidazole chloride salt by vacuum filtration, and freeze-drying to obtain cellulose nanofiber;
the specific parameters of the ultrasonic dispersion are as follows: time is 100-200 s; power 40-140W; ultrasonic intermittent time is 3 s/3 s-9 s/3 s;
the particle size of the emulsion is 5-15 mu m.
2. An immobilized enzyme pickering emulsion reaction system prepared by the preparation method of the immobilized enzyme pickering emulsion reaction system for preparing phosphatidylserine according to claim 1.
3. A method for preparing phosphatidylserine, characterized by: adopting the immobilized enzyme pickering emulsion reaction system of claim 2, reacting for 1-12 hours at 37-42 ℃; centrifuging after the reaction is finished, taking supernatant, dissolving the product phosphatidylserine in an upper organic phase, and blowing nitrogen to obtain the phosphatidylserine.
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