Method for separating DHA and EPA in fatty acid or separating DHA-EE and DPA-EE in fatty acid ethyl ester

Abstract

The invention discloses a method for separating DHA and EPA in fatty acid or separating DHA-EE and DPA-EE in fatty acid ethyl ester. DHA is separated from a DHA and EPA mixed solution through affinity difference between FABP protein and fatty acid, and the problems of high energy consumption and easy oxidation of a traditional separation method are avoided. The preparation method comprises the following steps: firstly, respectively adding strep-tag II and six histidine tags to N ends and C ends of FABP3 and FABP7 protein sequences, inserting the FABP3 and FABP7 protein sequences into a plasmid pET-28a (+), and expressing FABP3 / FABP7 protein with tags through escherichia coli; then, the affinity (Kd) of FABP3 and FABP7 with DHA / EPA is measured through a BLI experiment, and the affinity (Kd) is expanded to the affinity (Kd) of DHA-EE / DPA-EE; finally, the protein is coupled to a nickel column, and DHA / EPA is subjected to quantitative analysis through GC-MS to evaluate the protein separation effect.

Description

Technical Field

[0001] This invention belongs to the field of biomaterials technology, specifically relating to a method for separating DHA and EPA in fatty acids or separating DHA-EE and DPA-EE in fatty acid ethyl esters. Background Technology

[0002] Docosahexaenoic acid (DHA; 22:6n-3) and eicosapentaenoic acid (EPA; 20:5n-3) are two of the most clinically significant long-chain omega-3 polyunsaturated fatty acids (PUFAs), but their physiological functions differ significantly. DHA is an important structural lipid in neuronal plasma membranes and retinal photoreceptor discs, directly supporting cognitive development and visual function. In contrast, EPA is highly regarded for its anti-inflammatory and cardioprotective properties. A diet rich in EPA can reduce tissue saturated fat and arachidonic acid levels, thereby reducing cardiovascular disease risk factors. Crucially, the relative balance between these two PUFAs is important. Researchers have shown that while both fatty acids can beneficially regulate lipid metabolism, EPA cannot completely replace DHA in supporting brain development. Studies on infant growth and development further confirm this: DHA-dominant supplements lead to better neurocognitive outcomes, while a high EPA / DHA ratio diminishes these benefits. Conversely, excessive DHA intake (relative to EPA) is associated with significantly elevated LDL cholesterol, posing a potential risk to individuals prone to dyslipidemia. This difference in safety and efficacy makes obtaining high-purity DHA or EPA essential. However, these two molecules differ by only two methylene groups and one double bond, making industrial separation extremely difficult. Traditional separation methods, such as silver ion or reversed-phase high-performance liquid chromatography (HPLC) (Chu et al., 2020), urea complexation, and molecular distillation (Magallanes et al., 2019), suffer from limited resolution, high energy consumption, or oxidative degradation of polyunsaturated fatty acids, limiting their application in nutritional supplements and pharmaceutical manufacturing. Therefore, there is an urgent need for more selective and energy-efficient technologies to mass-produce DHA and EPA that meet pharmaceutical standards.

[0003] Currently, marine organisms are the main source of DHA / EPA for humans. Among them, microalgae can efficiently synthesize ω-3 polyunsaturated fatty acids from scratch, and their growth rate is faster than that of marine animals. They are high in lipids, cholesterol-free, pollution-free, and odorless, making them an important source for the production of ω-3 unsaturated fatty acids. DHA / EPA usually exists in algal oil in the form of natural triglycerides. Catalytic ethanololysis of triglycerides is the first step in enriching DHA / EPA in algal oil. Based on the study of FABP binding to fatty acids, further research is needed to investigate whether FABP has different affinities for different fatty acid ethyl esters, in order to extend to the dedicated separation of ethyl ester forms.

[0004] Fatty acid-binding proteins (FABPs) are a family of small-molecule (approximately 14-15 kDa) cytoplasmic proteins that protect long-chain fatty acids and other hydrophobic molecules in the cellular aqueous environment. By isolating normally free fatty acids, FABPs dissolve these molecules and transport them to β-oxidation or esterification sites, while simultaneously integrating lipid availability with metabolic signaling pathways. Despite their diverse functions, FABPs possess a highly conserved three-dimensional folded structure: ten antiparallel β-chains (βA-βJ) form two nearly perpendicular β-sheets, closing into a β-barrel or β-clam, with two α-helices enclosing this groove on the upper surface. This scaffold forms a deeply embedded cavity with a polar gradient, capable of accommodating a single fatty acid chain and determining the typical nanomolar to micromolar affinity of this family.

[0005] Tissue-specific expression assigns specific roles to individual isoforms in lipid metabolism. Heart-type fatty acid-binding protein (FABP3) is dominant in cardiomyocytes and oxidative skeletal muscle. It binds long-chain fatty acids—including ω-3 polyunsaturated fatty acids—with high affinity and delivers them to mitochondria for β-oxidation, thereby maintaining ATP production and lipid-derived signaling. Brain-type fatty acid-binding protein (FABP7) is the major isoform in the developing central nervous system, peaking during neurogenesis and then declining after birth. FABP7 is responsible for transporting polyunsaturated fatty acids required for membrane biogenesis and synaptic maturation; its dysregulation is associated with neurodevelopmental disorders such as schizophrenia and autism. Unlike FABP3, FABP7 is almost absent in tissues with high oxidative metabolism, highlighting its specific role in neurolipid homeostasis rather than energy production.

[0006] FABPs have been used to quantify free fatty acids, the most typical example being ADIFAB, an intestinal FABP variant labeled with acrylonitrile dimethyl ester. It exhibits quantifiable fluorescence changes upon binding to fatty acids and is commonly used for the rapid determination of unesterified fatty acids in plasma (Richieri et al., 1992). However, no process for separating DHA / EPA using FABP3 or FABP7 as affinity ligands has been found; the disclosed methods are more geared towards fluorescent probe / detection applications. Chinese patent CN120138068A discloses a method for enriching ethyl EPA and ethyl DHA, employing immobilized lipase to catalyze ethyl ester-type fish oil and enriching DHA and ethyl EPA therein. This includes steps such as catalytic esterification, molecular distillation, or thin-film evaporation. However, the catalytic reaction time is relatively long, molecular distillation still consumes a significant amount of energy, and it cannot separate DHA and EPA, only enriching the two substances. DHA and EPA have significantly different physiological functions. DHA is primarily an important component of the brain and is closely related to infant brain development; it also helps lower blood lipids. EPA, on the other hand, can be converted in the body into physiologically active substances such as prostaglandins, which have functions including lowering blood lipids and anti-rheumatism. However, EPA has an inhibitory effect on infant development, so different products require different DHA / EPA ratios. Although refined natural fish oil can be used directly as a food ingredient, its application is somewhat limited due to its low DHA / EPA content and unbalanced ratio. Summary of the Invention

[0007] In order to overcome the shortcomings of the existing technology, the purpose of this invention is to provide a molecular selector for separating fatty acids (DHA and EPA), and to extend it to fatty acid ethyl esters.

[0008] This invention separates DHA from a mixed solution of DHA and EPA by utilizing the difference in affinity between FABP protein and fatty acids, thus avoiding the problems of high energy consumption and easy oxidation of traditional separation methods.

[0009] The present invention adopts the following technical solution:

[0010] A method for separating DHA and EPA from fatty acids or separating DHA-EE and DPA-EE from fatty acid ethyl esters, comprising the following steps:

[0011] (1) Add strep-tag II and 6 histidine tags to the N-terminus and C-terminus of the FABP3 / FABP7 protein sequence, respectively, insert them into plasmid pET-28a(+), express the tagged FABP3 / FABP7 protein in E. coli, and then purify it.

[0012] (2) The affinity (Kd) of FABP3 / FABP7 and DHA / EPA was measured by BLI experiment, and the Kd value of FABP3 / FABP7 and DHA-EE / DPA-EE was measured by the same method.

[0013] (3) The protein was coupled to a nickel column and the protein separation effect was evaluated by quantitative analysis of DHA / EPA or DHA-EE / DPA-EE by GC-MS.

[0014] Further, in step (1), the specific purification operation is as follows: collect the expressed bacterial cells and break them, perform the first step of purification using Strep-Tactin XT agarose resin, and then pass them through HiLoad. TM 16 / 600 Superdex TM Further purification using a 75 pg column yielded FABP3 and FABP7 proteins with a purity ≥ 95%, respectively.

[0015] Further, in step (2), the specific procedure of the BLI experiment is as follows: the protein concentration of step (1) is 20-50 μg / mL, fixed on the Ni-NTA probe, and reacted with different concentrations (2.5 μM-5 mM) of DHA / EPA or DHA-EE / DPA-EE.

[0016] Further, in step (3), the protein is coupled to the nickel column, specifically by binding the protein at a concentration of 0.2-2 mg / mL to the nickel column, and controlling the amount of protein fixed on the column to be 36.0-125.2 nmol by controlling the protein volume.

[0017] Further, in step (3), DHA / EPA is quantitatively analyzed by GC-MS, specifically: after methyl esterification treatment, quantitative analysis is performed using an Agilent 7890A GC-7000C.

[0018] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0019] (1) This invention utilizes the his-tag in the protein to immobilize it on a nickel column, allowing the DHA / EPA mixture to pass through the nickel column and be captured by the protein. This method efficiently captures fatty acids in the mixture through the specific binding of the protein to DHA / EPA, and has the advantages of low energy consumption and high specificity. Such a fatty acid purification method has never been mentioned before, and this invention changes the initial DHA / EPA ratio from 1:1 to 1:80.

[0020] (2) The present invention uses the BLI experiment to measure the Kd value of FABP3 and FABP7 proteins and DHA / EPA, as well as the Kd value of DHA-EE / DPA-EE. The measurement method is simple and convenient, and the amount of protein sample used is small. Attached Figure Description

[0021] Figure 1 Map of the expression vector pET-28(a+) plasmid.

[0022] Figure 2 Molecular sieve diagram and SDS-PAGE diagram for FABP3 protein purification.

[0023] Figure 3 Molecular sieve diagram and SDS-PAGE diagram for FABP7 protein purification.

[0024] Figure 4 The Kd values ​​are for FABP3 protein, DHA, and EPA.

[0025] Figure 5 The Kd values ​​are for FABP7 protein, DHA, and EPA.

[0026] Figure 6 Kd values ​​of FABP3 protein, DHA-EE, and DPA-EE

[0027] Figure 7 This is the CD spectrum of the FABP3 protein.

[0028] Figure 8 This is the CD spectrum of the FABP7 protein.

[0029] Figure 9 The bar chart shows the percentage of EPA and DHA binding by FABP3 and FABP7 proteins at the amounts of immobilized protein of 125.2 nmol and 36.0 nmol, respectively.

[0030] Figure 10 Pie chart showing the remaining percentages of EPA and DHA after FABP3 and FABP7 proteins bind to EPA and DHA. Detailed Implementation

[0031] The specific implementation of the present invention will be further described below with reference to embodiments, comparative examples, and accompanying drawings. However, the implementation and protection of the present invention are not limited thereto. It should be noted that any processes not specifically described in detail below are those that can be implemented or understood by those skilled in the art by referring to existing technology. Reagents or instruments whose manufacturers are not specified are considered to be conventional products that can be purchased commercially.

[0032] Example 1: Recombinant FABP3 and Recombinant FABP7

[0033] 1. Preparation of FABP protein

[0034] 1.1 Plasmid

[0035] Codon-optimized cDNAs for human FABP3 (UniProt, P05413) and FABP7 (UniProt, O15540) were novelly synthesized (GenScript Biotechnology) and cloned into pET-28a(+), thereby constructing vectors expressing each protein, such as... Figure 1 As shown, these proteins have a Strep-tag II at the N-terminus and a hexahistine tag at the C-terminus for dual affinity purification.

[0036] FABP3 insertion into the target amino acid sequence:

[0037] WSHPQFEKGGGGSMVDAFLGTWKLVDSKNFDDYMKSLGVGFATRQVASMTKPTTIIE KNGDILTLKTHSTFKNTEISFKLGVEFDETTADDRKVKSIVTLDGGKLVHLQKWDGQETTL VRELIDGKLILTLTHGTAVCTRTYEKEAGGGGSHHHHHH*

[0038] FABP7 insertion into the target amino acid sequence:

[0039] WSHPQFEKGGGGSMVEAFCATWKLTNSQNFDEYMKALGVGFATRQVGNVTKPTVIISQEGDKVVIRTLSTFKNTEISFQLGEEFDETTADDRNCKSVVSLDGDKLVHIQKWDGKETNFVREIKDGKMVMTLTFGDVVAVRHYEKAGGGGSHHHHHH*

[0040] 1.2 Expression and purification of FABP3 and FABP7

[0041] FABP3 / FABP7 protein expression in *E. coli* was induced by adding 0.3 mM isopropyl-β-D-thiogalactopyranoside (IPTG) (Biofroxx, EZ65785BF9) and cultured at 24 °C for 24 h. Cells were collected by centrifugation (3100 × g, 4 °C, 30 min) and resuspended in ice-cold lysis buffer A (100 mM Tris-HCl, 1 mM EDTA and 150 mM NaCl, pH 8.0), which was supplemented with 1 / 4 tablet of protease inhibitor mixture per 30 mL buffer (Biosharp, BL630B) and 2 mM benzyl sulfonyl fluoride (PMSF) (Macklin, C16880259). The cells were then sonicated on ice with a probe (3 times, 6 min each time; 5 s on / 5 s off; 40% amplitude). The lysis buffer was clarified by centrifugation (19900 × g, 4 °C, 30 min).

[0042] The soluble fraction was gently incubated with Strep-Tactin XT agarose resin (Cytiva, 10334023) at 4°C for 2 h using gentle rotation. After washing resin A with 10 column volumes (CV) of lysis buffer, the bound proteins (FABP3 and FABP7) were eluted with elution buffer B (50 mM biotin, 100 mM Tris-HCl, 150 mM NaCl and 1 mM EDTA, pH 8.0). The fractions containing FABP3 and FABP7 were concentrated using a 3 kDa molecular weight cutoff (MWCO) centrifuge filter (Amicon Ultra) and then subjected to HiLoad... TM 16 / 600 Superdex TM Further purification was performed by size exclusion chromatography on a 75 pg column, which was equilibrated with reaction buffer C (25 mM Tris-HCl, 100 mM NaCl, pH 8.20). The monomer peaks were combined, rapidly frozen in liquid nitrogen, and stored at -80 °C for subsequent analysis.

[0043] After purification with Strep-Tactin XT Sepharose, the protein was further purified by molecular sieve. Both proteins with very similar molecular weights showed peaks at approximately 75 mL, high and sharp, without contamination from other peaks, indicating a single folded conformation and high protein purity. SDS-PAGE analysis confirmed that the protein at the corresponding molecular weight position (17 kDa) was the desired FABP protein. Image J analysis showed that the monomeric protein purity exceeded 95%. Figure 2 and Figure 3As shown in the figure. This two-step purification protocol yielded 1.62 mg / L of recombinant FABP3 and 0.72 mg / L of recombinant FABP7 from shake-flask culture.

[0044] Example 2: BLI Affinity Determination of Fatty Acids

[0045] BLI assays were performed at 25 °C on an Octet R8 (Sartorius) instrument with probe stirring at 1000 rpm. Reaction buffer C was used as the run / kinetic buffer for all steps. The Ni-NTA biosensor was pre-equilibrated in reaction buffer for 600 s, then loaded with purified FABP7 (50 μg / mL) for 600 s. The sensor was briefly rinsed with reaction buffer before kinetic analysis. DHA (Aladdin, E122277) and EPA (Aladdin, D100925) were sequentially diluted in reaction buffer to 0.25, 2.5, 25, and 250 μM and dispensed (200 μL per well) into black 96-well polypropylene microplates (Grener Biotech). Wells containing only reaction buffer were used as references. Each measurement included a baseline period of 120 s in reaction buffer, a binding period of 540 s in ligand solution, and a dissociation period of 540 s in reaction buffer. The reference subtracted sensor spectra were processed in OctetAnalysis Studio v13.0 and globally fitted to a 1:1 Langmuir model to obtain the binding rate constant (Kon), dissociation rate constant (Koff), equilibrium dissociation constant (Kd), and goodness of fit (R²). 2 The treatment method for FABP3 is the same as that for FABP7, but DHA and EPA solutions with concentrations of 10, 100, 1000, and 10000 μM are used.

[0046] By fitting the BLI signal values ​​to a 1:1 global model, a clear affinity constant was obtained, such as... Figure 4 and Figure 5 As shown. The Kd value for FABP3 binding to DHA is 39.13 ± 3.83 μM ( Figure 4 The Kd value of A bound to EPA is 300.0 ± 19.0 μM. Figure 4 The Kd value of FABP7 for DHA is 80.18 ± 3.71 μM (B). Figure 5 The Kd value for EPA was 600.2 ± 30.2 μM (A). Figure 5 (B). Therefore, both proteins show a preference for DHA approximately 7.5 times, while FABP3 binds to each ligand approximately twice as strongly as FABP7. These trends suggest that when these two ω-3 polyunsaturated fatty acids compete for the same FABP, DHA will occupy the dominant binding site.

[0047] Example 3: GC-MS analysis of the combination of DHA / EPA and FABP3 / FABP7

[0048] 1. Protein immobilization

[0049] Purified FABP3 and FABP7 were immobilized on Ni-NTA agarose via their C-terminal hexahistine tags. The amount of immobilized protein was calculated by recording the absorbance of the supernatant at 280 nm before and after immobilization and applying the corresponding extinction coefficients. The difference in A280 values ​​(after adjusting for dilution) was used to calculate the amount of protein in milligrams per milliliter of immobilization resin, which was taken as the immobilization efficiency.

[0050] 2. Ligand binding assay

[0051] Equal amounts of DHA and EPA were incubated with FABP-attached resin at 4°C with gentle stirring for 30 min. The control sample was treated in the same manner as the unfixed protein sample, but without protein fixation, and was used to assess nonspecific adsorption. After incubation, the suspension was clarified by centrifugation (8000×g, 5 min); the supernatant was carefully decanted, and the suspension was rapidly frozen for lyophilization for subsequent quantitative analysis.

[0052] 3. Derivatization of fatty acid ethyl esters (FAME)

[0053] Each lyophilized sample received 20 μL of internal standard solution (nonadecanoic acid (ND), C19:0; concentration in dichloromethane 2 mg / mL). The solvent was removed under a slow-flowing nitrogen stream, and the residue was methylated at 75 °C with 200 μL of 15% (v / v) BF3-CH3OH for 15 min. After cooling to room temperature, the reaction was terminated with 400 μL of saturated sodium chloride solution, and the fatty acid glycerides were extracted by vortexing 200 μL of n-hexane. Phase separation was achieved by centrifugation (8000 × g, 4 °C, 5 min).

[0054] 4. Quantitative analysis by gas chromatography-mass spectrometry

[0055] The n-hexane extract (n-hexane phase) was transferred to a GC vial and analyzed on an Agilent 7890A GC-7000C. Separation was performed on a DB-23 capillary column (Agilent 19091N-133, 30 m × 250 μm id × 0.25 μm film). Operating conditions were as follows:

[0056] Carrier gas: Helium, constant flow rate 1.0 mL / min

[0057] Injector: Splitless, 250℃; Injection volume: 0.5 μL; Temperature program: 150℃ (1 min) → 10℃ / min → 245℃ (15 min)

[0058] Acquisition mode: Select ion monitoring (SIM) at m / z 79, 91 (universal EE fragment), m / z 342 (DHA-FAME), m / z 316 (EPA-FAME), and m / z 312 (ND-FAME internal standard).

[0059] The quantification of the conjugates was achieved by normalizing the peak areas of DHA and EPA-FAME to the C19:0 internal standard and comparing the resulting area ratios with a protein-free control; the percentage decrease in each ratio represents the proportion of fatty acids captured by the immobilized FABP.

[0060] Equimolar amounts of DHA / EPA mixtures (final concentrations of 250 μM or 500 μM) were incubated for 30 minutes at room temperature with Ni-NTA resin containing 36.0 nmol or 125.2 nmol of immobilized FABP3 or FABP7. After centrifugation, the supernatant was derivatized to FAME and quantified by GC-MS. The amount of fatty acids captured by each protein was calculated by subtracting the concentration of residual (unbound) fatty acids from the protein-free control and reported as a percentage of binding. Figure 9 (A, B). Overall, at both protein loadings, FABP3 removed significantly more total ω-3 polyunsaturated fatty acids from solution than FABP7, consistent with its higher absolute affinity as determined by BLI. Regarding selectivity, analysis of the residual supernatant ( Figure 10Significant DHA enrichment was observed: when the DHA / EPA mixture was treated with 36.0 nmol of protein, FABP3 produced approximately 80 times more EPA than DHA in the solution after the reaction (EPA / DHA ratio approximately 80), while FABP7 produced a milder 2.5-fold ratio. When the DHA / EPA mixture was treated with 125.2 nmol of protein, selectivity decreased as the binding sites approached saturation, with EPA / DHA ratios decreasing to 2.0 (FABP3) and 1.2 (FABP7), respectively. Therefore, the highest DHA discrimination was observed at lower protein loadings, and FABP3 maintained the strongest preference under both conditions. Sequence alignment revealed key differences within their ligand-binding pockets. Notably, the residue at position 110 in FABP3 is glutamine (Q110), while FABP7 has an arginine residue (R126) at the corresponding position. This substitution from a neutral polar residue to a positively charged residue may alter the electrostatic environment within the pocket, potentially modulating the interaction with the fatty acid carboxyl head. Furthermore, other non-conserved residues within the pocket (e.g., V32 / A46, L60 / M74) may contribute subtle differences in pocket volume, hydrophobicity, and van der Waals contact, particularly with the polyunsaturated tail of DHA / EPA. We hypothesize that the cumulative effect of these substitutions fine-tunes the binding energy, resulting in consistently stronger affinity in FABP3. This requires further investigation through structural studies.

[0061] These results are consistent with the kinetic data (the binding constant of FABP3 to DHA is 39.13 μM, while that of FABP7 to DHA is 80.18 μM), confirming that FABP3 is a superior scaffold for selective DHA capture in a competitive environment. Therefore, FABP3 was subsequently used for related separation experiments.

[0062] Example 4: BLI Affinity Determination of Fatty Acid Esters

[0063] Based on the previous experimental results, FABP3 showed better selectivity for DHA, so the same method was extended to fatty acid ethyl esters. BLI assays were performed at 25°C on an Octet R8 (Sartorius) instrument with a probe stirring speed of 1000 rpm. All steps used reaction buffer C with a final DMSO concentration of 4.6% as the run / kinetic buffer. The Ni-NTA biosensor was pre-equilibrated in reaction buffer for 600 s, then loaded with purified FABP3 (50 μg / mL) for 600 s. The sensor was briefly rinsed with reaction buffer before kinetic analysis. DHA ethyl ester (Aladdin, D171305, ≥97%) and DPA ethyl ester (Aladdin, E156254, ≥98%) were diluted in reaction buffer to 93.75, 187.5, 375, 750 μM and 0.4, 2, 10, 50 μM, respectively, and dispensed (200 μL per well) into black 96-well polypropylene microplates (Grener Biotech). Wells containing only the reaction buffer were used as references. Each measurement included a baseline period of 120 s in the reaction buffer, a binding period of 540 s in the ligand solution, and a dissociation period of 540 s in the reaction buffer. The sensor spectra subtracted from the reference were processed in Octet Analysis Studio v13.0 and globally fitted to a 1:1 Langmuir model to obtain the binding rate constant (Kon), dissociation rate constant (Koff), equilibrium dissociation constant (Kd), and goodness of fit (R²). 2 ).

[0064] By fitting the BLI signal values ​​to a 1:1 global model, a clear affinity constant was obtained, such as... Figure 6 As shown, the Kd value for FABP3 binding to DHA-EE was 70.71 ± 10.29 μM. Figure 6 The Kd value of A bound to DPA-EE was 3.58 ± 2.54 μM. Figure 6 (B). This indicates that FABP3 is selective for both fatty acid ethyl esters, with a stronger affinity for DPA-EE.

[0065] Example 5: Process Renewability and Stability Window

[0066] To evaluate the elution method after the protein binds to fatty acids and the activity of the protein after elution, we measured the circular dichroism (CD) spectra of the protein under the various treatment conditions in Table 1 and without any treatment.

[0067] Far-ultraviolet circular dichroism spectroscopy was recorded using a Chirascan V100 spectrometer at 25 °C. Samples were analyzed in buffer D (20 mM phosphate, 20 mM NaF, pH 7.0) at a concentration of 0.34 mg / mL. Measurements were performed using quartz cuvettes with a 1.0 mm path length, a scan range of 190–260 nm, a step size of 1 nm, and an averaging time of 0.5 s per point. The average of three scans for each sample was taken.

[0068] Table 1

[0069]

[0070] pass Figure 7 and Figure 8 It can be seen that FABP3 and FABP7 are correctly expressed and folded in E. coli, and after being treated with two preset protein elution methods and then dialyzed, they can correctly refold, indicating that the proteins have the stability of the process.

Claims

1. A method for separating DHA and EPA from fatty acids, characterized in that, Includes the following steps: (1) Add strep-tag II and 6 histidine tags to the N-terminus and C-terminus of the FABP3 / FABP7 protein sequence, respectively, insert them into plasmid pET-28a(+), express the tagged FABP3 / FABP7 protein in E. coli, and then purify it. (2) The affinity Kd values ​​of FABP3 / FABP7 and DHA / EPA were measured by BLI experiment; (3) The protein was coupled to a nickel column and the protein separation effect was evaluated by quantitative analysis of DHA / EPA by GC-MS.

2. A method for separating DHA-EE and DPA-EE from fatty acid ethyl esters, characterized in that, Includes the following steps: (1) Add strep-tag II and 6 histidine tags to the N-terminus and C-terminus of the FABP3 / FABP7 protein sequence, respectively, insert them into plasmid pET-28a(+), express the tagged FABP3 / FABP7 protein in E. coli, and then purify it. (2) The Kd values ​​of FABP3 / FABP7 and DHA-EE / DPA-EE were measured by BLI experiment; (3) The protein was coupled to a nickel column and the protein separation effect was evaluated by quantitative analysis of DHA-EE / DPA-EE by GC-MS.

3. The method according to claim 1 or 2, characterized in that, In step (1), the specific purification operation is as follows: collect the expressed bacterial cells and break them up, perform the first step of purification using Strep-Tactin XT agarose resin, and then pass them through HiLoad. TM 16 / 600 Superdex TM Further purification using a 75 pg column yielded FABP3 and FABP7 proteins with a purity ≥ 95%, respectively.

4. The method according to claim 1 or 2, characterized in that, In step (2), the specific process of the BLI experiment is as follows: the protein from step (1) is immobilized on the Ni-NTA probe and reacted with different concentrations of DHA / EPA or DHA-EE / DPA-EE.

5. The method according to claim 4, characterized in that, The concentration range of the protein is 20-50 μg / mL.

6. The method according to claim 4, characterized in that, The concentration range of the DHA / EPA or DHA-EE / DPA-EE is 2.5 μM-5 mM.

7. The method according to claim 1 or 2, characterized in that, In step (3), the protein is coupled to the nickel column, specifically by binding the protein to the nickel column and controlling the amount of protein fixed by controlling the protein volume.

8. The method according to claim 7, characterized in that, The concentration range of the protein is 0.2-2 mg / mL.

9. The method according to claim 7, characterized in that, The protein content should be controlled between 36.0 and 125.2 nmol.

10. The method according to claim 1 or 2, characterized in that, In step (3), DHA / EPA is quantitatively analyzed by GC-MS, specifically: after methyl esterification, it is quantitatively analyzed by Agilent 7890A GC-7000C.

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