A method for producing ascorbate tetraisopalmitate catalyzed by a combination of lipases

Abstract

This invention discloses a method for producing ascorbic acid tetraisopalmitate catalyzed by a combination of lipases, belonging to the field of bioenzyme catalysis. The method includes: reacting a combination of lipases containing seven specific sources with the substrate vitamin C and isopalmitic acid in a mixture of hydrophobic organic solvent and water to generate ascorbic acid tetraisopalmitate. The lipase combination is derived from Tocopheryl sulfide, human, Arabidopsis thaliana, yeast, Staphylococcus, Burkholderia, and soybean. This invention obtains a specific lipase combination that synergistically catalyzes the esterification of four hydroxyl sites on the vitamin C molecule by constructing a lipase library and using a single elimination method. Under optimized reaction conditions, the conversion rate reaches 91.8% in a 5L scale-up reaction. This method has mild reaction conditions, simple steps, high yield, and is environmentally friendly, making it suitable for industrial production.

Description

Technical Field

[0001] This invention belongs to the field of bioenzyme catalysis, specifically relating to a method for producing ascorbic acid tetraisopalmitate by combining lipases. Background Technology

[0002] Vitamin C (L-ascorbic acid) is a recognized, highly effective natural antioxidant and skin whitening agent. It has a variety of important physiological functions in the human body. In the field of cosmetics and skin care products, its main effects include: (1) powerful antioxidant, scavenging free radicals and delaying skin photoaging; (2) inhibiting tyrosinase activity, blocking melanin production from the source, and achieving whitening and fading spots; (3) promoting collagen synthesis and maintaining skin elasticity and firmness.

[0003] However, despite the remarkable efficacy of L-ascorbic acid, its inherent physicochemical properties severely limit its application in commercial products. The main technical drawbacks are: (1) Extremely poor chemical stability: The enediol structure in the L-ascorbic acid molecule makes it extremely sensitive to light, heat, oxygen and metal ions. In aqueous solution, it is easily oxidized, decomposed and discolored (yellown), resulting in rapid loss of activity and extremely short product shelf life.

[0004] (2) Low transdermal absorption rate: L-ascorbic acid is a water-soluble molecule that is difficult to penetrate the lipid barrier of the stratum corneum of the skin. Its bioavailability is low, and most of the active ingredients remain on the skin surface and cannot exert their due effects.

[0005] (3) Formulation difficulties: In order to ensure its stability, the formulation usually needs to be maintained in a low pH environment (<3.5), which can easily cause skin irritation, stinging sensation, and poor user experience.

[0006] To overcome the aforementioned shortcomings, those skilled in the art have conducted extensive research. The most common strategy is to structurally modify the L-ascorbic acid molecule to synthesize its lipophilic derivatives, thereby improving stability and skin permeability. Ascorbate tetraisopalmitate (hereinafter referred to as "VC-IP"), as a fully esterified derivative in which all four hydroxyl groups on the L-ascorbic acid molecule are isopalmitized, has been shown to possess the ability to solve the aforementioned problems. Its highly symmetrical branched structure endows it with unparalleled stability and lipophilicity.

[0007] However, existing chemical synthesis methods for VC-IP suffer from problems such as harsh reaction conditions (e.g., high temperature, strong acid catalysts), cumbersome steps, low overall yield, difficulty in product purification, and the use of expensive or toxic reagents, resulting in extremely high production costs and posing a significant challenge to large-scale industrial production, which seriously hinders the market application of this component.

[0008] Patent CN118421599A discloses a method for preparing VCIP catalyzed by a lipase mutant. However, experimental verification showed that it did not achieve the expected results disclosed in the patent. Furthermore, from a technical perspective, it is difficult to achieve a single lipase simultaneously catalyze the linkage of hydroxyl groups at positions 2, 3, 5, and 6 on VC with isopalmitoyl residues. Patent CN115287312A mentions the synthesis of VCIP catalyzed by lipase, but it does not explicitly disclose the lipase sequence information, making its credibility questionable.

[0009] Therefore, developing a method for preparing ascorbate tetraisopalmitate with mild reaction conditions, simple steps, high yield, green and environmentally friendly characteristics, and suitable for industrial production is a technical problem that urgently needs to be solved in this field. Summary of the Invention

[0010] The purpose of this invention is to overcome the shortcomings of the prior art and provide a method for producing ascorbic acid tetraisopalmitate by combination lipase catalysis.

[0011] To achieve the above objectives, the present invention provides the following technical solution: This application provides a method for producing ascorbate tetraisopalmitate catalyzed by a combination of lipases, comprising the following steps: The combination containing lipase is reacted with the substrate in a reaction medium to generate ascorbate tetraisopalmitate. The combination of lipases consists of lipase A, lipase B, lipase C, lipase D, lipase E, lipase F and lipase G. The lipase A was derived from Tocode sulfur leaf mold, whose Uniprot accession number is Q96Z02; The lipase B is derived from human source, and its Uniprot accession number is P23141. The lipase C was derived from Arabidopsis thaliana, and its Uniprot accession number is Q9MA46; The lipase D is derived from yeast, and its GenBank accession number is BDCG_03232; The lipase E is derived from Staphylococcus aureus, and its Uniprot accession number is P04635. The lipase F was derived from Burkholderia, whose Uniprot accession number is P22088; The lipase G is derived from soybean, and its GenBank accession number is LOC137830340. The substrates are vitamin C and isopalmitic acid; The reaction medium is a mixed system containing a hydrophobic organic solvent and water.

[0012] Optionally, the lipase is prepared by the following steps: Genes encoding each lipase were constructed into the pET28a vector and transformed into Escherichia coli BL21(DE3) to obtain the expression strain; The expression strain was cultured in shake flasks, IPTG was added to induce expression, and the bacterial cells were collected by centrifugation to obtain wet bacterial cells; The wet cells of each lipase were mixed to obtain a combined lipase wet cell.

[0013] Optionally, the hydrophobic organic solvent is n-hexane, and the volume ratio of n-hexane to water is (99~90):(1~10).

[0014] Optionally, the volume ratio of hexane to water is 97:3.

[0015] Optionally, metal ions, including Ca, are also added to the reaction system. 2+ and Fe 2+ The Ca 2+ The added concentration is 3~8mM, the Fe 2+ The addition concentration is 2~5mM.

[0016] Optionally, the Ca 2+ The added concentration was 5 mM, and the Fe 2+ The addition concentration was 3 mM.

[0017] Optionally, the concentration of vitamin C in the reaction system is 20-70 mM, and the concentration of isopalmitic acid is 80-250 mM.

[0018] Optionally, the concentration of vitamin C in the reaction system is 50 mM, and the concentration of isopalmitic acid is 220 mM.

[0019] Optionally, the amount of the combined lipase wet cells added is 30~70g / L.

[0020] Optionally, the reaction temperature is 25~60℃.

[0021] Compared with the prior art, this application has the following beneficial effects: This invention uses enzyme catalysis to replace the traditional chemical synthesis method. The reaction temperature is 25~60℃, no strong acid catalyst is required, high temperature and high pressure conditions are avoided, and energy consumption and equipment requirements are reduced.

[0022] By screening specific lipase combinations, simultaneous esterification of four hydroxyl sites on the vitamin C molecule was achieved, and the VCIP conversion rate in the 5L scale-up reaction reached 91.8%, which is significantly higher than that of existing technologies.

[0023] The use of bio-enzyme catalysis avoids the use of toxic reagents and reduces the amount of organic solvents used, which is in line with the concept of green chemistry.

[0024] The method of this invention is simple, the raw materials are readily available, and it can be scaled up, providing a feasible solution for the industrial production of ascorbic acid tetraisopalmitate.

[0025] Enzyme-catalyzed reactions are highly specific and produce few byproducts, simplifying subsequent purification processes and helping to reduce production costs. Attached Figure Description

[0026] Figure 1 The liquid chromatogram of the lipase library-catalyzed reaction product in the example shows the formation of VCIP, with an elution time of 13.274 min, consistent with the standard.

[0027] Figure 2 This is a schematic diagram illustrating the principle of optimizing the types of mixed lipase-catalyzed reactions using a single elimination method.

[0028] Figure 3 The liquid chromatogram and peak table of the 5L expansion reaction catalyzed by the combined lipase under optimized conditions show that the VCIP conversion rate is 91.8%. Detailed Implementation

[0029] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0030] To address the aforementioned deficiencies in the prior art, this invention provides a method for producing ascorbic acid tetraisopalmitate using a combination of lipases.

[0031] This invention achieves highly efficient enzymatic synthesis of ascorbate tetraisopalmitate by constructing a lipase library and screening for specific lipase combinations capable of synergistically catalyzing the linkage between isopalmitic acid residues at the 2, 3, 5, and 6-positions of the vitamin C molecule; as shown in the following structural formula: This invention selects carboxylesterase (EC3.1.1.1), aryl esterase (EC3.1.1.2), galactolipase (EC3.1.1.26), and acetylxylan esterase (EC3.1.1.72) as candidate enzyme sources; and selects the following lipases based on the Uniprot protein database: The above amino acid sequences were all codon-optimized by Wuhan Sangon Biotech Co., Ltd., and synthesized and constructed into the multiple cloning site NcoI and EcoRI on the vector pET28a. The expression vectors were named pET28a-VCIP001~pET28a-VCIP018, respectively. Finally, the vectors were transformed into E. coli competent cells BL21(DE3) to prepare expression strains. The competent cells were purchased from Shanghai Weidi Biotechnology Co., Ltd., catalog number EC1002. Chemical transformation was performed according to the instructions provided on the official website. Finally, the cells were plated on solid LB plates containing kanamycin resistance and incubated overnight at 37°C.

[0032] Single colonies were picked and introduced into 24-well deep-well plates. 5 mL of LB medium was added to each well, and the plates were incubated at 37°C and 220 rpm for 4–6 h using a shaker. IPTG was then added to a final concentration of 1 mM, and the plates were induced for 12–16 h using a shaker at 25°C and 220 rpm. The LB medium formulation consisted of 0.5% yeast extract, 1% peptone, and 1% NaCl, with the pH adjusted to 7.2 using 1 M NaOH. All bacterial cells were collected, mixed, centrifuged to obtain wet cells, weighed, and stored at -20°C for later use.

[0033] n-Hexane was used as the hydrophobic organic solvent for enzyme catalysis. The water content of the system was controlled by molecular sieve to ensure that the water content was ≤5%. 5 mM vitamin C and 20 mM isopalmitic acid were added. The wet bacterial cells obtained by centrifugation of the above well plates were repeatedly freeze-thawed 5 times at -20℃ and 50℃. Then, they were added to the catalytic system and reacted at 37℃ and pH=7.0 for 24 h. Liquid chromatography was used for detection.

[0034] VCIP detection method: Chromatographic column: C18, 250 mm × 4.6 mm, 5 μm; mobile phase: tetrahydrofuran: acetonitrile = 2:8; flow rate: 1.0 mL / min; detection wavelength: 236 nm; column temperature: 30 ℃; injection volume: 10 μL, external standard method. The liquid chromatogram showed the presence of VCIP formation, with a peak elution time of 13.274 min, consistent with the standard, indicating that this lipase library can complementaryly catalyze the formation of VCIP.

[0035] The single elimination method was used to optimize the types of mixed lipases catalyzing the reaction. Individual lipases were gradually eliminated to identify whether they played a role in the catalytic reaction. Finally, through multiple rounds of elimination identification, it was determined that the lipase combination (VCIP003+VCIP004+VCIP007+VCIP009+VCIP013+VCIP015+VCIP016) played a key role in the catalytic synthesis of VCIP.

[0036] Large-scale preparation of enzyme-catalyzed wet cells by shake-flask fermentation: Single clones were picked and transferred to 50mL shake flasks, and 10mL of LB medium was added to each flask. The flasks were incubated at 37℃ and 220rpm for 12-16h. Then, 1mL of seed culture was transferred to 500mL shake flasks, and 100mL of TB medium was added to each flask. The TB medium formula was: 12g / L tryptone, 24g / L yeast extract, 4mL / L glycerol, 2.31g / L potassium dihydrogen phosphate, and 12.54g / L dipotassium hydrogen phosphate. The flasks were incubated at 37℃ and 220rpm for 4-6h. Then, 0.5mM IPTG was added, and the flasks were incubated at 25℃ and 220rpm for 14-16h. The cells were collected by centrifugation, and the equal weights of wet cells were weighed and mixed to obtain the combined lipase.

[0037] To further improve the catalytic efficiency of the combined lipase, the enzyme catalytic reaction system was optimized: The volume ratio of the hydrophobic organic solvent n-hexane to water is (99~90):(1~10), preferably 97:3.

[0038] Adding metal ions Mg 2+ Ca 2+ Fe 2+ Fe 3+ Cu 2+ Zn 2+ Perform test optimization, where Ca 2+ with Fe 2+ It can effectively improve catalytic efficiency, Ca 2+ The addition amount is 3~8mM, preferably 5mM; Fe 2+ The addition amount is 2~5mM, preferably 3mM.

[0039] The concentration of the substrate vitamin C is 20-70 mM, preferably 50 mM; the concentration of isopalmitic acid is 80-250 mM, preferably 220 mM.

[0040] The amount of wet lipase cells added is 30~70g / L, preferably 50g / L.

[0041] The reaction temperature is 25~60℃, preferably 35℃.

[0042] A 5L enzyme catalytic reaction system was established according to the optimized enzyme catalytic reaction system. After 26 hours of reaction, liquid chromatography was performed. The results showed that the combined lipase catalysis under optimized conditions exhibited a conversion rate of 91.8%.

[0043] Example 1: Construction and preparation of lipase expression strains The amino acid sequences of VCIP001~VCIP018 were codon optimized by Wuhan Sangon Biotech Co., Ltd., and the multiple cloning sites NcoI and EcoRI were synthesized and constructed into the expression vector pET28a. The expression vectors were named pET28a-VCIP001~pET28a-VCIP018, respectively. The final vectors were transformed into E. coli competent cells BL21(DE3) to prepare expression strains. The competent cells were purchased from Shanghai Weidi Biotechnology Co., Ltd., catalog number EC1002. Chemical transformation was performed according to the instructions provided on the official website. The cells were plated on solid LB plates containing kanamycin resistance and incubated overnight at 37°C.

[0044] Single colonies were picked and introduced into 24-well deep-well plates. 5 mL of LB medium (0.5% yeast extract, 1% peptone, 1% NaCl, pH adjusted to 7.2 with 1M NaOH) was added to each well. The plates were incubated at 37°C and 220 rpm for 4–6 h using a shaker. IPTG was then added to a final concentration of 1 mM, and the plates were induced for 12–16 h using a shaker at 25°C and 220 rpm. All bacterial cells were collected, mixed, centrifuged to obtain wet cells, weighed, and stored at -20°C for later use.

[0045] Example 2: Preliminary verification of VCIP synthesis catalyzed by a lipase library Hexane was used as the hydrophobic organic solvent for enzyme catalysis. The water content of the system was controlled by molecular sieves to ensure that the water content was ≤5%. 5 mM vitamin C and 20 mM isopalmitic acid were added. The wet bacterial cells obtained by centrifugation of the well plate in Example 1 were repeatedly freeze-thawed 5 times at -20°C and 50°C, and then added to the catalytic system. The reaction was carried out at 37°C and pH=7.0 for 24 h, and the reaction was detected by liquid chromatography.

[0046] VCIP detection method: Column C18, 250mm×4.6mm, 5μm; mobile phase: tetrahydrofuran: acetonitrile = 2:8; flow rate: 1.0mL / min; detection wavelength: 236nm; column temperature: 30℃; injection volume: 10μL, external standard method.

[0047] Liquid Chromatogram ( Figure 1 The results showed the presence of VCIP generation, with a peak elution time of 13.274 min, consistent with the standard sample. This indicates that the lipase library can complementarily catalyze the generation of VCIP.

[0048] Example 3: Screening of combinatorial lipases A single elimination method was used to optimize the types of mixed lipases catalyzing the reaction, gradually eliminating individual lipases to identify their role in the catalytic reaction. Ultimately, through multiple rounds of elimination identification, the lipase combination (VCIP003+VCIP004+VCIP007+VCIP009+VCIP013+VCIP015+VCIP016) was determined to play a crucial role in the catalytic synthesis of VCIP.

[0049] Example 4: Large-scale preparation of combined lipases Single clones of VCIP003, VCIP004, VCIP007, VCIP009, VCIP013, VCIP015, and VCIP016 were picked and transferred to 50 mL shake flasks. 10 mL of LB medium was added to each flask, and the flasks were incubated at 37°C and 220 rpm for 12–16 h. Then, 1 mL of seed culture was transferred to 500 mL shake flasks, and 100 mL of TB medium was added to each flask. The TB medium formula was: 12 g / L tryptone, 24 g / L yeast extract, 4 mL / L glycerol, 2.31 g / L potassium dihydrogen phosphate, and 12.54 g / L dipotassium hydrogen phosphate. The flasks were incubated at 37°C and 220 rpm for 4–6 h. 0.5 mM IPTG was added, and the flasks were incubated at 25°C and 220 rpm for 14–16 h. The cells were collected by centrifugation, and the wet cells were weighed and mixed to obtain the combined lipase.

[0050] Example 5: Optimization of the enzyme catalytic reaction system To further improve the catalytic efficiency of the combined lipase, the enzyme catalytic reaction system was optimized: The volume ratios of the hydrophobic organic solvent n-hexane to water were investigated: 99:1, 97:3, 95:5, and 90:10. The optimal ratio was determined to be 97:3.

[0051] Metal ion investigation: 5 mg / mL was added respectively. 2+ Ca 2+ Fe 2+ Fe 3+ Cu 2+ Zn 2+ Ca was discovered 2+ with Fe 2+ It can effectively improve catalytic efficiency. Further optimization of Ca... 2+ The addition amount is 3~8mM (preferably 5mM), Fe 2+ The addition amount is 2~5mM (preferably 3mM).

[0052] Substrate concentration study: Vitamin C concentration 20-70 mM (preferably 50 mM), isopalmitic acid concentration 80-250 mM (preferably 220 mM).

[0053] The optimal dosage of wet bacterial lipase for combination lipase was 30-70 g / L (preferably 50 g / L).

[0054] Reaction temperature range: 25~60℃ (preferably 35℃).

[0055] Example 6: 5L scale-up reaction A 5L enzyme catalytic reaction system was established according to the optimized system: hexane to water volume ratio 97:3, with Ca added. 2+ 5mM, Fe 2+ The concentrations were 3 mM vitamin C, 50 mM vitamin C, 220 mM isopalmitic acid, and 50 g / L of wetted combined lipase cells. The reaction temperature was 35℃. Liquid chromatography analysis was performed after 26 hours of reaction. The results showed that under optimized conditions, the combined lipase catalysis exhibited a conversion rate of 91.8%. Figure 3 ).

[0056] It should be noted that the following provides the amino acid sequence information of the lipases mentioned in this specification: Lipase derived from Sulfolobustokodaii, Uniprot accession number Q96Z02; Human-derived lipase, Uniprot accession number P23141; Lipase derived from Arabidopsis thaliana, Uniprot accession number Q9MA46; Lipase derived from yeast, GenBank accession number BDCG_03232; Lipase derived from Staphylococcus aureus, Uniprot accession number P04635; Lipase derived from Burkholderia, Uniprot accession number P22088; Lipase derived from soybean (Glycinemax), GenBank accession number LOC137830340.

[0057] The amino acid sequences of the above lipases are all known sequences, and those skilled in the art can obtain the complete amino acid sequences through the corresponding database access numbers.

[0058] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from its spirit or essential characteristics. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, all variations falling within the meaning and scope of equivalents of the claims are intended to be included within the present invention. No reference numerals in the claims should be construed as limiting the scope of the claims.

[0059] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A method for producing ascorbate tetraisopalmitate catalyzed by a combination of lipases, characterized in that, Includes the following steps: The combination containing lipase is reacted with the substrate in a reaction medium to generate ascorbate tetraisopalmitate. The combination of lipases consists of lipase A, lipase B, lipase C, lipase D, lipase E, lipase F and lipase G. The lipase A was derived from Tocode sulfur leaf mold, whose Uniprot accession number is Q96Z02; The lipase B is derived from human source, and its Uniprot accession number is P23141. The lipase C was derived from Arabidopsis thaliana, and its Uniprot accession number is Q9MA46; The lipase D is derived from yeast, and its GenBank accession number is BDCG_03232; The lipase E is derived from Staphylococcus aureus, and its Uniprot accession number is P04635. The lipase F was derived from Burkholderia, whose Uniprot accession number is P22088; The lipase G is derived from soybean, and its GenBank accession number is LOC137830340. The substrates are vitamin C and isopalmitic acid; The reaction medium is a mixed system containing a hydrophobic organic solvent and water.

2. The method according to claim 1, characterized in that, The lipase is prepared by the following steps: Genes encoding each lipase were constructed into the pET28a vector and transformed into Escherichia coli BL21(DE3) to obtain the expression strain; The expression strain was cultured in shake flasks, IPTG was added to induce expression, and the bacterial cells were collected by centrifugation to obtain wet bacterial cells; The wet cells of each lipase were mixed to obtain a combined lipase wet cell.

3. The method according to claim 1, characterized in that, The hydrophobic organic solvent is n-hexane, and the volume ratio of n-hexane to water is (99~90):(1~10).

4. The method according to claim 3, characterized in that, The volume ratio of hexane to water is 97:

3.

5. The method according to claim 1, characterized in that, The reaction system is also added with metal ions, including Ca 2+ and Fe 2+ , the added concentration of Ca 2+ is 3-8 mM, and the added concentration of Fe 2+ is 2-5 mM.

6. The method according to claim 5, characterized in that, The Ca 2+ is added at a concentration of 5 mM, the Fe 2+ is added at a concentration of 3 mM.

7. The method according to claim 1, characterized in that, The concentration of vitamin C in the reaction system is 20-70 mM, and the concentration of isopalmitic acid is 80-250 mM.

8. The method according to claim 7, characterized in that, The concentration of vitamin C in the reaction system is 50 mM, and the concentration of isopalmitic acid is 220 mM.

9. The method according to claim 1, characterized in that, The amount of the combined lipase wet cells added is 30~70g / L.

10. The method according to claim 1, characterized in that, The reaction temperature is 25~60℃.

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