Lipase mutants, methods for making and using same

Abstract

The application discloses a lipase mutant, a preparation method and application thereof. The amino acid sequence of the lipase mutant comprises any one of the following sequences: (1) a sequence which is subjected to W180A and / or I240C mutation on the basis of the sequence shown in SEQ ID NO: 2; or (2) a sequence which is obtained by substituting, deleting or adding one or at least two amino acid residues from the sequence according to (1) and is functionally identical or similar to the sequence according to (1); or (3) a sequence which has at least 90% sequence identity with the sequence according to (1) or (2) and is functionally identical or similar to the sequence according to (1). The application introduces mutations in the wild-type lipase, improves the catalytic activity of the lipase, and further improves the yield of vitamin A palmitate, and has high industrial application value.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology and relates to lipase mutants, their preparation methods, and applications. Background Technology

[0002] Vitamin A palmitate exhibits good stability, making it a commonly used formulation of vitamin A and widely used in nutritional supplements and animal feed additives. Currently, commercially available vitamin A palmitate is mainly synthesized via chemical methods, which involve complex and demanding reaction processes and are prone to isomer formation, posing challenges to subsequent separation and purification. Biocatalytic conversion can efficiently overcome the shortcomings of chemical synthesis methods, thus possessing significant application value.

[0003] For example, CN114854717A discloses lipase WH-Bc240 and its mutants from Bacillus cereus, which efficiently catalyze the synthesis of vitamin A palmitate in a one-step process through transesterification or transesterification activities. The catalytic conversion of vitamin A acetate to vitamin A palmitate has a production efficiency of up to 564 g / L. -1 d -1 This method is difficult to meet high production demands. CN113957114A discloses an enzymatic method for synthesizing vitamin A palmitate, including the following steps: adding vitamin A acetate, palmitic acid, and immobilized enzyme to an organic solvent for transesterification. Alkyl oxide ion imidazole salt and an acid-binding agent are also added during the reaction. The immobilized enzyme used is lipase. CN106544391A discloses an enzyme-catalyzed method for preparing vitamin A palmitate, including the following steps: 1) dissolving vitamin A acetate and lower fatty alcohol esters of palmitic acid in an organic solvent and adding lipase; 2) performing an enzyme-catalyzed transesterification reaction under vacuum from a condenser, separating the reaction byproducts, lower fatty alcohol esters of acetate, and the organic solvent via a distillation column; 3) filtering the reaction solution to remove lipase, adding an adsorbent to adsorb the residual lower fatty alcohol esters of palmitic acid, and filtering out the vitamin A palmitate solution; 4) evaporating the organic solvent under reduced pressure to obtain vitamin A palmitate.

[0004] In summary, existing biocatalytic methods for the preparation of vitamin A palmitate involving lipases still suffer from low efficiency in the transesterification reaction of the key enzyme lipase, limiting its industrial-scale application. Therefore, developing highly efficient lipases through genomic data mining and directed evolution to achieve the green and efficient synthesis of vitamin A palmitate has significant industrial application value. Summary of the Invention

[0005] In response to the shortcomings of existing technologies and practical needs, this invention provides lipase mutants, their preparation methods, and applications. By designing and modifying lipase mutants with higher activity, this invention aims to promote the production of vitamin A palmitate.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] In a first aspect, the present invention provides a lipase mutant, wherein the amino acid sequence of the lipase mutant comprises any one of the following sequences:

[0008] (1) A sequence that has undergone W180A and / or I240C mutations based on the sequence shown in SEQ ID NO:2; or,

[0009] (2) A sequence obtained by substituting, deleting, or adding one or at least two amino acid residues to the sequence described in (1), and having the same or similar function to the sequence described in (1); or,

[0010] (3) A sequence that has at least 90% sequence identity with the sequence described in (1) or (2) and has the same or similar function as the sequence described in (1).

[0011] In this invention, the lipase PhLipase (AWX65616.1) derived from Pseudozyma hubeiensis is designed and modified by introducing W180A and / or I240C mutations, which can improve catalytic activity and thus improve the production efficiency of vitamin A palmitate.

[0012] In this invention, a specific mutation is introduced into wild-type lipase to obtain a lipase mutant, which can improve its catalytic activity. It is understood that, based on the lipase mutant, those skilled in the art can use common techniques in the art to substitute, delete or add one or at least two amino acid residues to obtain other sequences with the same or similar functions.

[0013] In some specific embodiments of the present invention, in addition to introducing W180A and / or I240C mutations, conserved substitutions of amino acids can be further performed at other sites. Preferably, the conserved substitutions of amino acids preserve the substrate specificity of the lipase mutant of the present invention. It will be apparent to those skilled in the art that such substitutions can occur in regions other than those described above while still retaining the corresponding activity. Preferably, the conserved substitution variant has at least one conserved substitution of an amino acid. Examples of conserved substitutions are substitutions occurring within the following groups of amino acids: basic amino acids (such as arginine, lysine, and histidine), acidic amino acids (such as glutamic acid and aspartic acid), polar amino acids (such as glutamine and asparagine), hydrophobic amino acids (such as leucine, isoleucine, and valine), aromatic amino acids (such as phenylalanine, tryptophan, and tyrosine), and small molecule amino acids (such as glycine, alanine, serine, threonine, and methionine). The most common amino acid interchanges are G to A; A to G, S; V to I, L, A, T or S; I to V, L or M; L to I, M or V; M to L, I or V; P to A, S or N; F to Y, W or H; Y to F, W or H; W to Y, F or H; R to K, E or D; K to R, E or D; H to Q, N, S; D to N, E, K, R or Q; E to Q, D, K, R or N; S to T or A; T to S, V or A; C to S, T or A; N to D, Q, H or S; Q to E, N, H, K or R, as well as their opposite interchanges. Lipase mutants that have a certain degree of amino acid homology with the above-mentioned lipase mutants, preferably with a homology of 70%-99%, more preferably with a homology of 80%-99%, even more preferably with a homology of 90%-99%, and most preferably with a homology of 99%, should also fall within the scope of protection of this invention.

[0014] SEQ ID NO:2:

[0015] MKFTSTITALAAFVCVACATPLVKRLPSGSDPAFSVPQSQLAAVLECQNGSPSSQTNPILLVPGTGVTGPQSFDSNWIPLSTQLGYSPCWISPPPFMLNDSQLNAEYIVNAVSTLYAGSGSKKVPVLTWSQGGLATQWALTFFPSIRSKVDRLMAFAPDYKGTVEAIFLTVP GLASQSVWQQQAQSAYLTALQNAGGLTKIVPTTNLYSALDDIVQPQVTNSPADSSYLFTAKNIQAQSICGPTFVIDHAGSLTSQFSYIVGKSALASTGTGEAQSSDYSILNCNPLPADPLTPQQKAEASGLLLVAGANVIAGPKQNCEPDLKPYARRYAIGKKTCSGVNTGF.

[0016] In a second aspect, the present invention provides a nucleic acid molecule that encodes the lipase mutant described in the first aspect.

[0017] In this invention, the nucleic acid molecule can be DNA, such as cDNA, genomic DNA, or recombinant DNA; the nucleic acid molecule can also be RNA, such as mRNA or hnRNA.

[0018] Preferably, the nucleic acid molecule includes the sequence shown in SEQ ID NO:3, or a sequence that hybridizes with SEQ ID NO:3 and encodes the lipase mutant, or a sequence that has more than 90% identity with SEQ ID NO:3 and encodes the lipase mutant.

[0019] SEQ ID NO:3:

[0020]

[0021] Thirdly, the present invention provides a recombinant vector containing the nucleic acid molecule described in the second aspect.

[0022] In this invention, the recombinant vector includes a cloning vector and an expression vector. The cloning vector is used to replicate the relevant sequence, and the expression vector is used to express the relevant gene. The vector used to construct the expression vector can be pET-26b(+).

[0023] In some embodiments, the recombinant vector is obtained by replacing the sequence between the EcoRI and HindIII restriction sites of pET-26b(+) with the nucleic acid molecule, while leaving the rest of the sequence unchanged.

[0024] Fourthly, the present invention provides recombinant cells containing the recombinant vector described in the third aspect.

[0025] It is understood that the recombinant cells can be obtained by transforming the above-mentioned recombinant expression vector into host cells. The host cells are conventional host cells in the art, as long as they can stably replicate on their own and the genes they carry can be effectively expressed.

[0026] Furthermore, the host cell is a prokaryotic or eukaryotic cell, such as Escherichia coli, Pichia pastoris, Saccharomyces cerevisiae, Bacillus subtilis, or Aspergillus niger, specifically Escherichia coli BL21(DE3), Rosetta(DE3), BL21(DE3)plysS, or M15, preferably Escherichia coli BL21(DE3).

[0027] Fifthly, the present invention provides a method for preparing the lipase mutant described in the first aspect, the method comprising:

[0028] The nucleic acid molecule encoding the lipase mutant described in the first aspect is inserted into the expression vector to obtain a recombinant vector. The recombinant vector is introduced into a host cell, cultured, and purified to obtain the lipase mutant.

[0029] Preferably, the expression vector includes pET-26b(+) vector, pRSFDuet-1 vector, pETDuet-1 vector, pACYCDuet-1 vector, pTrc99a vector, or pET28a vector, etc.

[0030] Preferably, the host cells include Escherichia coli, Pichia pastoris, Saccharomyces cerevisiae, Bacillus subtilis, or Aspergillus niger.

[0031] Preferably, the Escherichia coli includes Escherichia coli BL21(DE3), Escherichia coli Rosetta(DE3), Escherichia coli BL21(DE3)plysS, or Escherichia coli M15, etc.

[0032] In this invention, the methods for inducing and culturing recombinant cells and for isolating lipase from the culture can both employ conventional methods in the art. The culture medium used when recombinant cells express lipase can be any culture medium in the art that allows the recombinant cells to grow and produce the lipase mutant of this invention, such as LB medium.

[0033] In this invention, there are no special requirements for the culture method and culture conditions, as long as the recombinant cells can grow normally and express the lipase mutant.

[0034] In a sixth aspect, the present invention provides the application of the lipase mutant described in the first aspect, the nucleic acid molecule described in the second aspect, the recombinant vector described in the third aspect, the recombinant cell described in the fourth aspect, or the method for preparing the lipase mutant described in the fifth aspect in the preparation of vitamin A palmitate.

[0035] In a seventh aspect, the present invention provides a method for preparing vitamin A palmitate, the method comprising: using the lipase mutant described in the first aspect or the recombinant cell catalyzed by the fourth aspect to perform a transesterification reaction of vitamin A acetate to obtain vitamin A palmitate.

[0036] Preferably, the temperature of the transesterification reaction is 30 to 50°C, including but not limited to 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 38°C, 40°C, 42°C, 45°C, 46°C, 47°C, 48°C, or 49°C.

[0037] Preferably, the final concentration of the vitamin A acetate is 30 to 300 g / L, for example, 30 g / L, 40 g / L, 50 g / L, 60 g / L, 70 g / L, 80 g / L, 90 g / L, 100 g / L, 200 g / L, 300 g / L, or any value or range between these values, with 150 g / L being the most preferred.

[0038] Preferably, the raw materials for the transesterification reaction also include palmitic acid, which is added in an amount of 1.0 to 1.1 times the equivalent of vitamin A acetate, for example, 1.0, 1.02, 1.04, 1.06, 1.08, or 1.10 times the equivalent, preferably 1.02 times the equivalent.

[0039] Preferably, the solvent for the transesterification reaction is a conventional solvent in the art, such as n-heptane.

[0040] In some embodiments, the recombinant cells are used to catalyze the transesterification of vitamin A acetate to prepare vitamin A palmitate; specifically, frozen stem cells from the recombinant cells can be used as a catalyst for whole-cell catalytic production of vitamin A palmitate, with the amount of frozen stem cells being 1.0-2.0 g / L, or up to 1.5 g / L. The frozen stem cells are obtained by freeze-drying the recombinant cells directly without cleavage.

[0041] It should be understood that the lipase mutant described in this invention can be used in whole-cell engineered bacteria, in unpurified crude enzyme form, or in partially or completely purified enzyme form. Furthermore, the lipase mutant of this invention can be prepared into immobilized enzymes or catalysts in immobilized cell form using immobilization techniques known in the art.

[0042] Compared with the prior art, the present invention has the following beneficial effects:

[0043] This invention, through rational design, introduces W180A and / or I240C mutations into wild-type lipases derived from the *Pseudomonas* genus in Hubei Province, thereby enhancing the catalytic activity of the lipase and increasing the production efficiency of vitamin A palmitate to 815 g·L⁻¹. -1 ·d -1 This has important industrial application value for the green and efficient preparation of vitamin A palmitate. Attached Figure Description

[0044] Figure 1 The liquid chromatogram of vitamin A palmitate standard;

[0045] Figure 2 The liquid chromatogram for the detection of vitamin A palmitate using recombinant bacteria containing wild-type lipase;

[0046] Figure 3 Liquid chromatogram for the detection of vitamin A palmitate by reacting with recombinant lipase bacteria containing W180A and I240C mutations;

[0047] Figure 4 Liquid chromatogram of vitamin A palmitate detection in recombinant lipase bacteria containing the W180A mutation. Detailed Implementation

[0048] To further illustrate the technical means and effects of this invention, the following description, in conjunction with embodiments and accompanying drawings, provides a further explanation of the invention. It is understood that the specific embodiments described herein are merely illustrative of the invention and not intended to limit it.

[0049] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0050] In this invention, "identity" can be evaluated by the naked eye or by computer software (such as the software program described in Current Protocols in Molecular Biology by Ausubel et al. eds. (2007)). When the positions in the compared sequences are occupied by the same bases or amino acids, the molecules are identical at that position. Identity between two or more sequences can be expressed as a percentage (%), which can be used to evaluate the identity between related sequences. "Sequence identity" of a polynucleotide or amino acid sequence with another sequence at a certain percentage (e.g., 90%, 95%, 98%, or 99%) means that when the sequences are aligned, that percentage of bases or amino acids are the same in the two compared sequences.

[0051] Example 1

[0052] Obtaining the gene sequence of the lipase mutant PhLipase-W180A / I240C.

[0053] Using the pET-26b(+) plasmid (pET-26b(+) is a Novagen product, catalog number 69862-3CN) containing the wild-type lipase gene sequence (SEQ ID NO:1), named pET-26b(+)-PhLipase, as a template, full-plasmid PCR was performed using PhLipase-W180A-FP and PhLipase-W180A-RP primers to introduce the W180A mutation site, resulting in the recombinant expression plasmid pET-26b(+)-P Using pET-26b(+)-PhLipase-W180A as a template and PhLipase-I240C-FP and PhLipase-I240C-RP as primers, a full plasmid PCR was performed to introduce the I240C mutation site, resulting in the mutant recombinant expression plasmid pET26b(+)-PhLipase-W180A / I240C. The nucleotide sequence of the lipase mutant is shown in SEQ ID NO:3, and the amino acid sequence of the lipase mutant PhLipase-W180A / I240C encoded by it is shown in SEQ ID NO:4.

[0054] SEQ ID NO:1:

[0055]

[0056] SEQ ID NO: 4:

[0057] MKFTSTITALAAFVCVACATPLVKRLPSGSDPAFSVPQSQLAAVLECQNGSPSSQTNPILLVPGTGVTGPQSFDSNWIPLSTQLGYSPCWISPPPFMLNDSQLNAEYIVNAVSTLYAGSGSKKVPVLTWSQGGLATQWALTFFPSIRSKVDRLMAFAPDYKGTVEAIFLTVPGLASQSVAQQQAQSAYLTALQNAGGLTKIVPTTNLYSALDDIVQPQVTNSPADSSYLFTAKNIQAQSCCGPTFVIDHAGSLTSQFSYIVGKSALASGTGEAQSSDYSILNCNPLPADPLTPQQKAEASGLLLVAGANVIAGPKQNCEPDLKPYARRYAIGKKTCSGVNTGF.

[0058] PhLipase-W180A-FP:

[0059] 5’-GCAAGTCAATCGGTCGCCCAGCAGCAGGCCCAG-3’ (SEQ ID NO: 5);

[0060] PhLipase-W180A-RP:

[0061] 5’-CTGGGCCTGCTGCTGGGCGACCGATTGACTTGC-3’ (SEQ ID NO: 6).

[0062] PhLipase-I240C-FP:

[0063] 5’-CATCCAGGCTCAATCGTGCTGCGGACCCACGTTC-3’ (SEQ ID NO: 7);

[0064] PhLipase-I240C-RP:

[0065] 5’-GAACGTGGGTCCGCAGCACGATTGAGCCTGGATG-3’ (SEQ ID NO: 8).

[0066] Example 2

[0067] This example constructs the expression strain of mutant PhLipase-W180A / I240C.

[0068] The mutant recombinant expression plasmid pET26b(+)-PhLipase-W180A / I240C obtained in Example 1 was transformed into the expression host E.coli BL21(DE3) by chemical transformation. The plasmid was then screened on LB solid medium containing 50 μg / mL kanamycin to obtain the recombinant strain E.coli BL21(DE3) / pET-26b(+)-PhLipase-W180A / I240C expressing the mutant PhLipase-W180A / I240C.

[0069] Following the above experiment, pET-26b(+)-PhLipase was used for transformation to obtain recombinant E.coli BL21(DE3) / pET-26b(+)-PhLipase expressing wild-type lipase.

[0070] Following the above experiment, pET-26b(+)-PhLipase-W180A was used for transformation to obtain recombinant E.coli BL21(DE3) / pET-26b(+)-PhLipase-W180A expressing wild-type lipase.

[0071] Example 3

[0072] This example demonstrates the preparation of the enzyme.

[0073] The recombinant E. coli BL21(DE3) / pET-26b(+)-PhLipase-W180A / I240C obtained in Example 2 was inoculated into LB broth containing 50 μg / mL kanamycin and cultured at 37°C and 220 rpm for 12 hours. Then, it was transferred at a 1% (v / v) inoculation rate to 100 mL of LB shake flask broth containing 50 μg / mL kanamycin and cultured at 37°C and 220 rpm until OD (out of control) was reached. 600 =0.6-0.8, add IPTG inducer to a final concentration of 300 μM, and induce culture at 16℃ for 20 hours. Centrifuge the induced culture at 9000 rpm for 10 minutes, collect the bacterial pellet, wash with physiological saline to obtain resting cells. Freeze-dry the resting cells directly to obtain frozen stem cells, and store at 4℃.

[0074] Referring to the above experiment, recombinant bacteria E.coli BL21(DE3) / pET-26b(+)-PhLipase expressing wild-type lipase were cultured and frozen stem cells were prepared as controls for subsequent analysis.

[0075] Referring to the above experiment, recombinant bacteria E.coli BL21(DE3) / pET-26b(+) transduced with an empty vector were cultured and frozen stem cells were prepared as negative controls for subsequent analysis.

[0076] Example 4

[0077] This embodiment demonstrates the enzymatic catalysis of vitamin A palmitate.

[0078] The mutant enzyme PhLipase-W180A / I240C obtained in Example 3 was used as a biocatalyst (using lyophilized whole cells as the catalyst) in the preparation reaction of vitamin A palmitate.

[0079] 100mL reaction system: 15g vitamin A acetate and 12g palmitic acid were dissolved in 100mL n-heptane. 150mg of whole-cell catalyst was added to the solution and the mixture was stirred at 200rpm for 7 hours at 35℃.

[0080] The high-performance liquid chromatography (HPLC) detection method is as follows:

[0081] An Agilent high-performance liquid chromatograph with a C18 column (4.6×150 mm) was used. The mobile phase was methanol. The flow rate was 1.0 mL / min. The UV detector was used at a wavelength of 327 nm. The column temperature was 30 °C. All samples were centrifuged at 10,000 rpm and filtered through a 0.22 μm organic filter membrane. The sample loading volume was 10 μL.

[0082] After the reaction was complete, 1 mL of the reaction solution was centrifuged to remove the biocatalyst. The supernatant was then filtered and analyzed by high-performance liquid chromatography (HPLC) to determine the concentration of vitamin A palmitate (vitamin A palmitate standard HPLC is shown in the figure). Figure 1 The result is as follows: Figure 3 and Figure 4 Calculations showed that when the mutant enzyme PhLipase-W180A / I240C was used as a biocatalyst, the conversion rate of the substrate vitamin A acetate (conversion rate = (initial amount of reactant - remaining amount of reactant) / initial amount of reactant × 100%) was 99.2%, and the production efficiency of the product vitamin A palmitate (production efficiency = amount of product generated / (reaction volume * reaction time)) was 815 g / L / day (g·L⁻¹). -1 ·d -1 When the mutant enzyme PhLipase-W180A was used as a biocatalyst, the conversion rate of the substrate vitamin A acetate was 91.4%, and the production efficiency of the product vitamin A palmitate was 751 g / L / day (g·L). -1 ·d -1 ).

[0083] Referring to the above experiment, the recombinant strain E. coli BL21(DE3) / pET-26b(+)-PhLipase expressing wild-type lipase was used to catalyze vitamin A palmitate, and the results were as follows: Figure 2 As shown, the reaction conversion rate was 78.5%, and the production efficiency of the product, vitamin A palmitate, was 645 g / L / day (g·L⁻¹). -1 ·d -1 ).

[0084] Referring to the above experiment, the recombinant strain E. coli BL21(DE3) / pET-26b(+) transduced with an empty vector catalyzed vitamin A palmitate, with a conversion rate of 0% and a production efficiency of 0 g / L / day (g·L⁻¹). -1 ·d -1 ).

[0085] In summary, this invention modifies wild-type lipase, resulting in a modified lipase with high catalytic activity, thereby improving the production efficiency of vitamin A palmitate to as high as 815 g·L⁻¹. -1 ·d -1 This will promote the production and application of vitamin A palmitate.

[0086] The applicant declares that the detailed method of the present invention is illustrated by the above embodiments, but the present invention is not limited to the above detailed method, that is, it does not mean that the present invention must rely on the above detailed method to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. A lipase mutant, characterized in that, The amino acid sequence of the lipase mutant is any one of the following sequences: A sequence that has undergone a W180A mutation based on the sequence shown in SEQ ID NO: 2; Alternatively, a sequence based on the sequence shown in SEQ ID NO: 2 that has undergone W180A and I240C mutations.

2. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the lipase mutant of claim 1.

3. The nucleic acid molecule according to claim 2, characterized in that, The nucleic acid molecule includes the sequence shown in SEQ ID NO:

3.

4. A recombinant vector, characterized in that, The recombinant vector contains the nucleic acid molecule as described in claim 2 or 3.

5. Recombinant cells, characterized in that, The recombinant cells contain the recombinant vector as described in claim 4.

6. The method for preparing the lipase mutant according to claim 1, characterized in that, The preparation method includes: The nucleic acid molecule encoding the lipase mutant of claim 1 is inserted into the expression vector to obtain a recombinant vector. The recombinant vector is introduced into a host cell, cultured, and purified to obtain the lipase mutant.

7. The method for preparing the lipase mutant according to claim 6, characterized in that, The expression vectors include pET-26b(+) vector, pRSFDuet-1 vector, pETDuet-1 vector, pACYCDuet-1 vector, pTrc99a vector, or pET28a vector.

8. The method for preparing the lipase mutant according to claim 7, characterized in that, The host cell includes any one of Escherichia coli, Pichia pastoris, Saccharomyces cerevisiae, Bacillus subtilis, or Aspergillus niger.

9. The method for preparing the lipase mutant according to claim 8, characterized in that, The *E. coli* species include any one of *E. coli* BL21(DE3), *E. coli* Rosetta(DE3), *E. coli* BL21(DE3)plysS, or *E. coli* M15.

10. The application of the lipase mutant of claim 1, the nucleic acid molecule of claim 2 or 3, the recombinant vector of claim 4, the recombinant cell of claim 5, or the method for preparing the lipase mutant of any one of claims 6 to 9 in the preparation of vitamin A palmitate.

11. A method for preparing vitamin A palmitate, characterized in that, The method for preparing vitamin A palmitate includes: using the lipase mutant of claim 1 or the recombinant cell catalyzed by claim 5 to perform a transesterification reaction of vitamin A acetate to obtain vitamin A palmitate.

12. The method for preparing vitamin A palmitate according to claim 11, characterized in that, The transesterification reaction is carried out at a temperature of 30~50℃.

13. The method for preparing vitamin A palmitate according to claim 12, characterized in that, The final concentration of vitamin A acetate is 30~300 g / L.

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