Hydrogel immobilized lipase as well as preparation method and application thereof in synthesis of L-ascorbyl palmitate

Abstract

The technical scheme of the invention belongs to the technical field of biochemical engineering, and particularly relates to hydrogel immobilized lipase as well as a preparation method and application thereof in synthesis of L-ascorbyl palmitate. The invention discloses a preparation method of N, N-dimethylformamide. According to the method, N, N '-methylene bisacrylamide is taken as a cross-linking agent, acrylamide is taken as a raw material, ascorbic acid is added under the initiation of ammonium persulfate, hydrogen bonds are formed with acrylamide through chemical cross-linking, polyacrylamide-ascorbic acid hydrogel is prepared, lipase is adsorbed, and the purpose of enzyme immobilization is achieved. The polyacrylamide-ascorbic acid hydrogel immobilized lipase is obtained. The polyacrylamide-ascorbic acid hydrogel immobilized lipase is applied to the reaction for catalytically synthesizing the L-ascorbyl palmitate, and compared with a chemical method, the cost is lower, and the method is green and environmentally friendly; compared with a common enzyme method, the yield is higher, the catalytic speed is higher, and the reaction conditions are milder.

Description

Technical Field

[0001] The technical solution of the present invention belongs to the technical field of biochemical engineering, and specifically relates to a hydrogel-immobilized lipase, a preparation method thereof, and application thereof in the synthesis of L-ascorbyl palmitate. Background Art

[0002] Lipase primarily catalyzes reactions at the hydrophilic-hydrophobic interface of a reaction system. Consequently, lipase exhibits a unique "interfacial activation" phenomenon, requiring catalysis to occur at the oil-water interface. However, naturally occurring free lipases have numerous drawbacks when catalyzing reactions: low stability, high cost, and difficulty recovering or reusing them after the reaction. Therefore, enzyme immobilization is crucial.

[0003] Chinese patent document CN114657169A discloses a method for preparing resin-immobilized lipase. This method involves contacting a fatty acid-modified non-polar macroporous adsorption resin support with the lipase. However, this method inherently fails to address the weak interaction between the lipase and the resin support, leading to leakage. Chinese patent document CN114990101A discloses a method for preparing lipase immobilized on a magnetic nanoparticle composite support for biodiesel synthesis. Although this method forms a four-layer spatial structure of ferroferric oxide magnetic nanoparticles, chitosan, gelatin, and lipase, it still fails to improve the lipase's tolerance stability and does not effectively expand the interfacial area for catalytic activation.

[0004] Vitamin C, also known as ascorbic acid, is a natural and highly effective antioxidant. However, its instability due to its water solubility limits its application in industries such as food and medicine. Therefore, converting ascorbic acid into fat-soluble fatty acid esters to enhance its application value is crucial. L-Ascorbyl palmitate (L-AP), an important ascorbic acid derivative, combines the antioxidant properties of ascorbic acid with its fat solubility and is widely used in dairy products and nutritional supplements.

[0005] Chinese patents (CN102304109A, CN106883202A, and CN111205250A) disclose three different chemical methods for preparing L-ascorbyl palmitate. All of these methods produce byproducts to varying degrees, resulting in complex separation and purification, significant pollution, and high costs. Chinese patent CN107034251A discloses a method for synthesizing L-ascorbyl palmitate using lipase in an ionic liquid. While this method uses an ionic liquid instead of an organic solvent as the reaction medium, it reduces pollution to a certain extent. However, the esterification process still leads to the aforementioned problems. Therefore, there is an urgent need to develop a superior enzymatic method for preparing L-ascorbyl palmitate. Summary of the Invention

[0006] In view of the deficiencies of the prior art, the present invention provides a hydrogel-immobilized lipase and a preparation method thereof. The hydrogel-immobilized lipase can be used to catalyze the synthesis of L-ascorbyl palmitate.

[0007] The invention adopts N,N'-methylenebisacrylamide as a cross-linking agent and acrylamide as a raw material. Under the initiation of ammonium persulfate, ascorbic acid is added, and hydrogen bonds are formed with acrylamide through chemical cross-linking to prepare a polyacrylamide-ascorbic acid hydrogel, and lipase is adsorbed to achieve the purpose of enzyme immobilization, thereby obtaining polyacrylamide-ascorbic acid hydrogel-immobilized lipase. The polyacrylamide-ascorbic acid hydrogel-immobilized lipase is physically cut into microspheres with a diameter of 1-2 mm, and the microspheres are added into an organic solvent. Methyl palmitate is added into the organic solvent system, and stirring is continued at a low speed. When the ascorbic acid reaction is exhausted and the esterification reaction is completed, stirring is stopped, the hydrogel-immobilized lipase is filtered and recovered, dried, and then immersed in an ascorbic acid aqueous solution for swelling. Unreacted methyl palmitate and L-ascorbyl palmitate synthesized by the reaction are separated and purified from the filtrate.

[0008] The technical solutions of the present invention are as follows: A method for preparing polyacrylamide-ascorbic acid hydrogel-immobilized lipase comprises the following steps: (1) Preparation of polyacrylamide-ascorbic acid hydrogel: acrylamide, N,N ’ Methylenebisacrylamide and ascorbic acid were dissolved in deionized water. Under anaerobic conditions, ammonium persulfate solution was added dropwise to initiate polymerization. After terminating the reaction, a crude hydrogel sample was obtained. Unreacted monomers were removed, and the sample was physically cut into microspheres and dried to obtain a polyacrylamide-ascorbic acid hydrogel. (2) Adsorption of lipase: dissolving lipase in phosphate buffer to obtain lipase phosphate buffer, mixing excess lipase phosphate buffer with the polyacrylamide-ascorbic acid hydrogel obtained in step (1), oscillating for adsorption, and collecting the polyacrylamide-ascorbic acid hydrogel-immobilized lipase after adsorption equilibrium by centrifugation.

[0009] According to the preferred embodiment of the present invention, the acrylamide, N,N ’ The mass ratio of methylenebisacrylamide to ammonium persulfate is (70-110):(1-2):1, more preferably 80:1:1. At this ratio, ascorbic acid dissolves more in the hydrogel and improves the stability of the polyacrylamide-ascorbic acid hydrogel.

[0010] According to the preferred embodiment of the present invention, the mass ratio of ascorbic acid to acrylamide in step (1) is (0.6-1):1; more preferably, it is 0.8:1.

[0011] According to the preferred embodiment of the present invention, in step (1), anaerobic conditions are created by introducing nitrogen.

[0012] According to the preferred embodiment of the present invention, the polymerization reaction temperature in step (1) is 50-70°C, more preferably 65°C; the polymerization reaction time is 10-20 min, more preferably 10 min.

[0013] According to the preferred embodiment of the present invention, an ice-water bath is used to terminate the reaction in step (1).

[0014] According to the preferred embodiment of the present invention, the crude hydrogel sample obtained in step (1) is rinsed with deionized water to remove unreacted monomers, and the rinse is performed 3 to 7 times in total.

[0015] Preferably, according to the present invention, the diameter of the microsphere particles in step (1) is 1-2 mm.

[0016] According to the preferred embodiment of the present invention, the drying temperature in step (1) is 65-75° C., more preferably 70° C.; the drying time is 0.5-1.5 h, more preferably 1 h.

[0017] According to the present invention, preferably, the pH of the phosphate buffer in step (2) is 6.0-8.0, more preferably 6.0.

[0018] According to the preferred embodiment of the present invention, the conditions for the oscillation adsorption in step (2) are: 100-300 r / min, 30-45°C, oscillation for 0.5-1.5 h, more preferably 200 r / min, 37°C, oscillation for 1 h.

[0019] Polyacrylamide-ascorbic acid hydrogel immobilized lipase prepared according to the above method.

[0020] The polyacrylamide-ascorbic acid hydrogel immobilized lipase of the present invention can be freeze-dried at -80°C and then stored at 4°C for later use.

[0021] Application of the polyacrylamide-ascorbic acid hydrogel immobilized lipase in the synthesis of L-ascorbyl palmitate.

[0022] A preferred technical solution of the present invention is the method for synthesizing L-ascorbyl palmitate by using the polyacrylamide-ascorbic acid hydrogel-immobilized lipase, comprising the following steps: (i) adding an excess of methyl palmitate to an organic solvent, and adding polyacrylamide-ascorbic acid hydrogel-immobilized lipase, and stirring at a low speed to carry out an esterification reaction; (ii) filtering the reaction solution obtained from the esterification reaction in step (i) to obtain polyacrylamide-ascorbic acid hydrogel-immobilized lipase and a filtrate; (iii) The filtrate obtained in step (ii) is subjected to rotary evaporation to remove the organic solvent, the residue is washed with n-hexane, and the insoluble matter is dried under vacuum to obtain L-ascorbyl palmitate.

[0023] According to a preferred embodiment of the present invention, the organic solvent in step (i) is a mixed solvent of acetone and n-hexane, wherein the volume ratio of acetone to n-hexane in the mixed solvent is (1-3):1, more preferably 3:1. At this ratio, the maximum solubility of methyl palmitate can be achieved.

[0024] The methyl palmitate used in the present invention may be an existing commercially available product.

[0025] According to the preferred embodiment of the present invention, the temperature of the esterification reaction in step (i) is 45-80°C, more preferably 50°C; the reaction time is 24-72h, more preferably 48h; and the low-speed stirring condition is 150-200r / min.

[0026] According to a preferred embodiment of the present invention, the filtration in step (ii) is performed using a 0.45 μm filter membrane, which can effectively separate the polyacrylamide-ascorbic acid hydrogel-immobilized lipase.

[0027] Preferably, according to the present invention, the polyacrylamide-ascorbic acid hydrogel immobilized lipase obtained in step (ii) is rinsed with deionized water and then immersed in an aqueous ascorbic acid solution to swell and regenerate.

[0028] According to the preferred embodiment of the present invention, the temperature of n-hexane during washing with n-hexane in step (iii) is 50-60°C.

[0029] Beneficial effects: 1. The present invention provides a method for preparing polyacrylamide-ascorbic acid hydrogel-immobilized lipase. Ascorbic acid and the initiator ammonium persulfate are added to a mixed solution of acrylamide and N,N'-methylenebisacrylamide to prepare a polyacrylamide-ascorbic acid hydrogel with good stability. The gel is then mechanically cut into microspheres, which is easy to operate and simple to process. The polyacrylamide-ascorbic acid hydrogel is used as a carrier to adsorb the lipase to prepare the polyacrylamide-ascorbic acid hydrogel-immobilized lipase. The hydrogen bonds formed between the polyhydroxylated ascorbic acid and the hydroxyl groups of the polyacrylamide hydrogel enhance the intermolecular interactions, allowing the ascorbic acid to be bound to the hydrogel through weak interactions. At the same time, the multifunctional groups on the surface of the lipase molecules, such as amino and carboxyl groups, can also interact with the hydroxyl groups of the polyacrylamide hydrogel to form hydrogen bonds, thereby enhancing the molecular force between the lipase and the hydrogel, making the hydrogel carrier and the lipase more tightly bound, reducing the loss of lipase during the reaction, and improving the stability of the immobilized lipase. At the same time, the mechanical strength of the hydrogel is improved, the degradation rate is greatly reduced, and the overall performance is significantly improved. The addition of the initiator also satisfies the "interface activation" characteristics of the lipase, increases the contact area between methyl palmitate and ascorbic acid, directly increases the possibility of the substrate methyl palmitate binding to the active center of the lipase, and increases the esterification reaction rate. The hydrogel microsphere particles provide a micro-water environment for the lipase in the anhydrous reaction system, so that the enzyme activity is always maintained in a relatively stable state.

[0030] 2. The present invention applies polyacrylamide-ascorbic acid hydrogel-immobilized lipase to the reaction of catalyzing the synthesis of L-ascorbyl palmitate. Compared with chemical methods, the present invention is more cost-effective and environmentally friendly. Compared with conventional enzymatic methods, the present invention has a higher yield, a faster catalytic rate, and milder reaction conditions. At the same time, the recovery process and reuse operation of the polyacrylamide-ascorbic acid hydrogel-immobilized lipase are convenient and efficient, and the enzyme activity after recovery can be guaranteed. The production and processing using this method is more economical and convenient for large-scale production. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 p-Nitrophenol-OD 410 The standard curve of

[0032] Figure 2 This is an electron microscope image of the polyacrylamide-ascorbic acid hydrogel-immobilized lipase particles in Example 1. DETAILED DESCRIPTION

[0033] The technical solution of the present invention is further described below with reference to the embodiments, but the scope of protection of the present invention is not limited thereto. It is stated that all other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative work are within the scope of protection of the present invention.

[0034] The reagents and drugs involved in the examples are all common commercial products unless otherwise specified. The experimental operations involved in the examples are all routine technical operations in the art unless otherwise specified.

[0035] The lipase used in the examples was purchased from Novozymes liquid Candida antarctica lipase B (lipozyme CALBL).

[0036] Example 1 A method for preparing polyacrylamide-ascorbic acid hydrogel immobilized lipase comprises the following steps: 1) 12 g of acrylamide, 0.15 g of N,N'-methylenebisacrylamide, and 10 g of ascorbic acid were placed in 100 mL of deionized water and stirred in a magnetic stirrer until dissolved. The resulting mixed solution was then added to a three-neck reactor, and nitrogen was introduced for 3 minutes to expel air from the reactor to allow the reaction to proceed under anaerobic conditions. The three-neck reactor was placed in a 50°C hot water bath, and 0.15 g of ammonium persulfate was dissolved in 2 mL of deionized water and added dropwise to the reactor to initiate a polymerization reaction. The polymerization reaction was carried out at 50°C for 10 minutes. 2) Terminate the polymerization reaction in an ice-water bath, filter to obtain a crude polyacrylamide-ascorbic acid hydrogel sample, and repeatedly rinse the resulting crude hydrogel sample with deionized water for a total of five times to remove unreacted monomers in the crude hydrogel sample; mechanically crush the crude hydrogel sample and sieve it through a fine steel mesh to obtain microspheres with a diameter of 1-2 mm; 3) The oven temperature is set to 70° C. The microsphere particles obtained in step 2) are placed in the oven for drying for 1 hour to obtain dry porous polyacrylamide-ascorbic acid hydrogel microspheres for later use; 4) Add 500 μL of 3 mg / mL lipase solution to 2 mL of pH 6.0 phosphate buffer to obtain lipase phosphate buffer; mix the polyacrylamide-ascorbic acid hydrogel microspheres obtained in step 3) and the lipase phosphate buffer, shake and adsorb on a shaker at 200 rpm at 37°C for 1 h. After adsorption equilibrium, collect the polyacrylamide-ascorbic acid hydrogel-immobilized lipase after centrifugation, freeze-dry at -80°C for 12 h, and store at 4°C for later use.

[0037] The polyacrylamide-ascorbic acid hydrogel-immobilized lipase prepared above was used to catalyze the synthesis of L-ascorbyl palmitate. The specific method is as follows: i) 30 g of palmitic acid and 100 mL of methanol were uniformly mixed and placed in a three-necked reactor. 10 mL of concentrated sulfuric acid was added as a catalyst. The mixture was refluxed at 70°C for 5 hours. Excess methanol was removed by distillation, and the mixture was washed with deionized water until neutral. The mixture was dried, allowed to stand, and then distilled under reduced pressure. The fraction at 225-230°C was collected to obtain purified methyl palmitate. ii) adding 3 mL of methyl palmitate to 800 mL of a mixed solvent of acetone and n-hexane, wherein the volume ratio of acetone to n-hexane in the mixed solvent is 3:1, and adding 0.05 g of the polyacrylamide-ascorbic acid hydrogel-immobilized lipase prepared above. The mixture was stirred at a low speed of 200 r / min, the esterification reaction temperature was 50° C., and the reaction time was 48 hours. iii) filtering the reaction solution obtained from the esterification reaction through a 0.45 μm filter membrane to obtain polyacrylamide-ascorbic acid hydrogel-immobilized lipase and a filtrate; iv) the polyacrylamide-ascorbic acid hydrogel-immobilized lipase obtained by filtration in step iii) was repeatedly rinsed with deionized water, immersed in a 0.06 mol / L ascorbic acid aqueous solution for swelling and regeneration, then repeatedly rinsed with deionized water, freeze-dried at -80°C for 12 hours, and stored at 4°C for later use; v) The filtrate obtained by filtration in step iii) is subjected to rotary evaporation to remove the organic solvent. The residue is washed three times with n-hexane at 50° C. to remove unreacted methyl palmitate. The insoluble matter is vacuum dried to obtain the product L-ascorbyl palmitate.

[0038] The polyacrylamide-ascorbic acid hydrogel immobilized lipase that has been swollen and regenerated can be reused.

[0039] Example 2 A method for preparing polyacrylamide-ascorbic acid hydrogel-immobilized lipase is described. The method differs from Example 1 mainly in that the pH of the phosphate buffer solution is changed during the preparation of the lipase phosphate buffer solution. The specific steps are as follows: 1) 12 g of acrylamide, 0.15 g of N,N'-methylenebisacrylamide, and 10 g of ascorbic acid were placed in 100 mL of deionized water and stirred in a magnetic stirrer until dissolved. The resulting mixed solution was then added to a three-neck reactor, and nitrogen was introduced for 3 minutes to expel air from the reactor to allow the reaction to proceed under anaerobic conditions. The three-neck reactor was placed in a 65°C hot water bath, and 0.15 g of ammonium persulfate was dissolved in 2 mL of deionized water and added dropwise to the reactor to initiate a polymerization reaction. The polymerization reaction was carried out at 65°C for 10 minutes. 2) Terminate the polymerization reaction in an ice-water bath, filter to obtain a crude polyacrylamide-ascorbic acid hydrogel sample, and repeatedly rinse the resulting crude hydrogel sample with deionized water for a total of five times to remove unreacted monomers in the crude hydrogel sample; mechanically crush the crude hydrogel sample and filter through a fine steel mesh to obtain microspheres with a diameter of 1-2 mm; 3) The oven temperature is set at 70° C. The microsphere particles obtained in step 2) are placed in the oven for drying for 1 hour to obtain dry porous polyacrylamide-ascorbic acid hydrogel microspheres for later use; 4) Add 500 μL of 3 mg / mL lipase solution to 2 mL of pH 8.0 phosphate buffer to obtain lipase phosphate buffer; mix the polyacrylamide-ascorbic acid hydrogel microspheres obtained in step 3) and the lipase phosphate buffer, and shake on a shaker at 200 rpm at 37°C for 1 h. After adsorption equilibrium, collect the polyacrylamide-ascorbic acid hydrogel-immobilized lipase after centrifugation, freeze-dry at -80°C for 12 h, and store at 4°C until use.

[0040] The polyacrylamide-ascorbic acid hydrogel-immobilized lipase prepared above was used to catalyze the synthesis of L-ascorbyl palmitate. The specific method is as follows: i) 30 g of palmitic acid and 100 mL of methanol were uniformly mixed and placed in a three-necked reactor. 10 mL of concentrated sulfuric acid was added as a catalyst. The mixture was refluxed at 70°C for 5 hours. Excess methanol was removed by distillation, and the mixture was washed with deionized water until neutral. The mixture was dried, allowed to stand, and then distilled under reduced pressure. The fraction at 225-230°C was collected to obtain purified methyl palmitate. ii) adding 3 mL of methyl palmitate to 800 mL of a mixed solvent of acetone and n-hexane, wherein the volume ratio of acetone to n-hexane in the mixed solvent is 3:1, and adding 0.05 g of the polyacrylamide-ascorbic acid hydrogel-immobilized lipase prepared above. The mixture was stirred at a low speed of 200 r / min, the esterification reaction temperature was 50° C., and the reaction time was 48 hours. iii) filtering the reaction solution obtained from the esterification reaction through a 0.45 μm filter membrane to obtain polyacrylamide-ascorbic acid hydrogel-immobilized lipase and a filtrate; iv) the polyacrylamide-ascorbic acid hydrogel-immobilized lipase obtained by filtration in step iii) was repeatedly rinsed with deionized water, immersed in a 0.06 mol / L ascorbic acid aqueous solution for swelling and regeneration, then repeatedly rinsed with deionized water, freeze-dried at -80°C for 12 hours, and stored at 4°C for later use; v) The filtrate obtained by filtration in step iii) is subjected to rotary evaporation to remove the organic solvent. The residue is washed three times with n-hexane at 50° C. to remove unreacted methyl palmitate. The insoluble matter is vacuum dried to obtain the product L-ascorbyl palmitate.

[0041] Example 3 The polyacrylamide-ascorbic acid hydrogel-immobilized lipase prepared in Example 1 was used to catalyze the synthesis of L-ascorbyl palmitate. The main difference from Example 1 was that the temperature of the esterification reaction was changed. The specific method was as follows: i) 30 g of palmitic acid and 100 mL of methanol were uniformly mixed and placed in a three-necked reactor. 10 mL of concentrated sulfuric acid was added as a catalyst. The mixture was refluxed at 70°C for 5 hours. Excess methanol was removed by distillation, and the mixture was washed with deionized water until neutral. The mixture was dried, allowed to stand, and then distilled under reduced pressure. The fraction at 225-230°C was collected to obtain purified methyl palmitate. ii) adding 3 mL of methyl palmitate to 800 mL of a mixed solvent of acetone and n-hexane, where the volume ratio of acetone to n-hexane is 3:1, and adding 0.05 g of the polyacrylamide-ascorbic acid hydrogel-immobilized lipase prepared in Example 1. The mixture was stirred at a low steady speed of 200 r / min for an esterification reaction at 70° C. for 48 hours. iii) filtering the reaction solution obtained from the esterification reaction through a 0.45 μm filter membrane to obtain polyacrylamide-ascorbic acid hydrogel-immobilized lipase and a filtrate; iv) the polyacrylamide-ascorbic acid hydrogel-immobilized lipase obtained by filtration in step iii) was repeatedly rinsed with deionized water, immersed in a 0.06 mol / L ascorbic acid aqueous solution for swelling and regeneration, repeatedly rinsed with deionized water, freeze-dried at -80°C for 12 hours, and stored at 4°C for later use; v) The filtrate obtained by filtration in step iii) is subjected to rotary evaporation to remove the organic solvent. The residue is washed three times with n-hexane at 50° C. to remove unreacted methyl palmitate. The insoluble matter is vacuum dried to obtain the product L-ascorbyl palmitate.

[0042] Comparative Example 1 A method for preparing polyacrylamide hydrogel-immobilized lipase is described. The method differs from Example 1 in that ascorbic acid is not added during the preparation of the polyacrylamide hydrogel. The specific steps are as follows: 1) 12 g of acrylamide and 0.15 g of N,N'-methylenebisacrylamide were placed in 100 mL of deionized water and stirred in a magnetic stirrer until dissolved. The resulting mixed solution was then added to a three-neck reactor, and nitrogen was introduced for 3 minutes to expel the air in the reactor to allow the reaction to proceed under anaerobic conditions. The three-neck reactor was placed in a 50°C hot water bath, and 0.15 g of ammonium persulfate was dissolved in 2 mL of deionized water and added dropwise to the reactor to initiate a polymerization reaction. The polymerization reaction was carried out at 50°C for 10 minutes. 2) Terminate the polymerization reaction in an ice-water bath, filter to obtain a crude polyacrylamide hydrogel sample, and repeatedly rinse the crude sample with deionized water for a total of five times to remove unreacted monomers in the crude hydrogel sample; mechanically crush the crude hydrogel block sample and filter through a fine steel mesh to obtain microspheres with a diameter of 1-2 mm; 3) The oven temperature is set to 70° C. The microsphere particles obtained in step 2) are placed in the oven for drying for 1 hour to obtain polyacrylamide hydrogel microspheres for use; 4) Add 500 μL of 3 mg / mL lipase solution to 2 mL of pH 6.0 phosphate buffer to obtain lipase phosphate buffer; mix the polyacrylamide hydrogel microspheres obtained in step 3) and the lipase phosphate buffer, shake at 200 rpm at 37°C for 1 h, and after adsorption equilibrium, collect the polyacrylamide hydrogel-immobilized lipase by centrifugation, freeze-dry at -80°C for 12 h, and store at 4°C until use.

[0043] The polyacrylamide hydrogel-immobilized lipase prepared above was used to catalyze the synthesis of L-ascorbyl palmitate. The specific method is as follows: i) 30 g of palmitic acid and 100 mL of methanol were uniformly mixed and placed in a three-necked reactor. 10 mL of concentrated sulfuric acid was added as a catalyst. The mixture was refluxed at 70°C for 5 hours. Excess methanol was removed by distillation, and the mixture was washed with deionized water until neutral. The mixture was dried, allowed to stand, and then distilled under reduced pressure. The fraction at 225-230°C was collected to obtain purified methyl palmitate. ii) adding 3 mL of methyl palmitate and 0.5 mL of a 42 mg / mL aqueous ascorbic acid solution to 800 mL of a mixed solvent of acetone and n-hexane, wherein the amount of ascorbic acid added is substantially the same as the ascorbic acid content in the hydrogels of Examples 1-3, and the volume ratio of acetone to n-hexane in the mixed solvent is 3:1. 0.05 g of the polyacrylamide hydrogel-immobilized lipase prepared above was added, and an esterification reaction was carried out with steady stirring at a low speed of 200 r / min, a temperature of 50° C., and a reaction time of 48 h. iii) filtering the reaction solution obtained from the esterification reaction through a 0.45 μm filter membrane to obtain polyacrylamide hydrogel-immobilized lipase and a filtrate; iv) repeatedly rinsing the polyacrylamide hydrogel-immobilized lipase obtained by filtration in step iii) with deionized water, immersing the lipase in deionized water for swelling and regeneration, freeze-drying the lipase at -80°C for 12 hours, and storing the lipase at 4°C for later use; v) The filtrate obtained by filtration in step iii) is subjected to rotary evaporation to remove the organic solvent. The residue is washed three times with n-hexane at 50° C. to remove unreacted methyl palmitate. The insoluble matter is vacuum dried to obtain the product L-ascorbyl palmitate.

[0044] Experimental example: The properties and reaction performance of the hydrogel-immobilized lipase prepared in the above examples and comparative examples were measured: 1. The polyacrylamide-ascorbic acid hydrogel-immobilized lipase particles prepared in Example 1 were placed under an optical electron microscope to observe their morphology.

[0045] 2. Determine the enzymatic activity of the hydrogel-immobilized lipase prepared in Examples 1-2 and Comparative Example 1. Principle: Lipase decomposes p-nitrophenol palmitate into p-nitrophenol and palmitic acid. p-nitrophenol appears yellow in aqueous solution and has a maximum absorption value at 410 nm. The specific steps are as follows: (1) Prepare a concentration gradient of p-nitrophenol standard solution and measure the absorbance value at 410nm using a spectrophotometer. The concentration of the p-nitrophenol standard solution is used as the vertical axis. The absorbance value (OD 410 ) as the horizontal axis, draw a standard curve, and fit the standard curve equation. The obtained standard curve is as follows Figure 1 As shown, the fitted linear equation is y=0.08066x-0.000290293, R 2 =0.99989 (>0.999, a linear relationship equation is available).

[0046] (2) Preparation of p-nitrophenol palmitate solution: Solution A: 0.0378 g p-nitrophenol palmitate dissolved in 10 mL isopropanol; Solution B: phosphate buffer solution containing 1% triton (1 g TritionX-100 added to 100 mL 0.05 M, pH = 7.2 phosphate buffer solution); when using, solution A and solution B are mixed in a ratio of 1:9 (prepared fresh) to obtain p-nitrophenol palmitate solution.

[0047] (3) Take 1 mL of p-nitrophenol palmitate solution and 1 mL of 0.05 M, pH 7.2 phosphate buffer solution, mix them well, and incubate in a 40°C water bath for 5 min. Add 0.5 g of hydrogel-immobilized lipase and react in a 40°C water bath for 15 min. Quickly remove the solution and add 3 mL of phosphate buffer solution to make the volume 5 mL. Measure the absorbance at 410 nm on a spectrophotometer. Heat-inactivated hydrogel-immobilized lipase was treated in the same manner as a blank.

[0048] (4) Calculate the corresponding p-nitrophenol concentration based on the p-nitrophenol standard curve and use the following enzyme activity calculation formula to obtain the result. The enzyme activity unit is defined as follows: Under the above conditions, the amount of enzyme required to hydrolyze p-nitrophenol palmitate to produce 1 µmol of p-nitrophenol per minute is defined as 1 enzyme activity unit (U).

[0049] Enzyme activity calculation formula: Enzyme activity (U / g) =

[0050] Where, V: total reaction volume (mL), C 样品 : The concentration of p-nitrophenol in the sample to be tested (µmol / mL) calculated based on the absorbance value, C 空白 : The concentration of p-nitrophenol in the blank sample (µmol / mL) calculated based on the absorbance value. T: reaction time (min), M 酶 : Mass of hydrogel-immobilized lipase (g).

[0051] 3. The hydrogel-immobilized lipases in Examples 1-3 and Comparative Example 1 were subjected to repeated experiments according to the corresponding methods for catalyzing the synthesis of L-ascorbyl palmitate, and the remaining enzyme activity of the hydrogel-immobilized lipase after repeated use for 20 times was measured.

[0052] 4. The detection and calculation methods of Km and conversion rate are as follows: The instruments and conditions used were as follows: a C18 column (4.6 m × 250 mm, 5 μm), a flow rate of 1.0 mL / min, and a column temperature of 35°C; the mobile phase was acetone / methanol / H2O (75:25:5, v / v / v) mixed with 0.5% acetic acid.

[0053] The mobile phase was used as the sample dissolution solvent, and the injection volume was 20 μL. L-ascorbyl palmitate standards were used for quantification, and a calibration curve was established at product concentrations of 240, 480, 960, 1920, 2880, 3840, and 4800 ppm. The Km and conversion rate were calculated based on the L-ascorbyl palmitate content in the sample and the reaction stoichiometry.

[0054] Km calculation: The concentration of ascorbic acid in the reaction system is quantitative, and the kinetic parameter Km is calculated by fitting the Michaelis-Menten equation using Origin.

[0055] The conversion rate was calculated based on the amount of L-ascorbyl palmitate generated by esterification in the reaction system: Conversion rate = moles of L-ascorbyl palmitate produced / moles of ascorbic acid added × 100% Experimental results: 1. The morphology of the polyacrylamide-ascorbic acid hydrogel immobilized lipase particles prepared in Example 1 is as follows: Figure 2 As shown in the figure, it can be seen that the polyacrylamide-ascorbic acid hydrogel immobilized lipase particles are in a dry and porous state, which is suitable for the adsorption of lipase and the occurrence of esterification reaction.

[0056] 2. The characteristics and reaction performance of the hydrogel-immobilized lipase in the above examples and comparative examples are summarized in the following table: Table 1. Performance evaluation results of hydrogel-immobilized lipase in the examples and comparative examples of the present invention

[0057] As can be seen from Table 1, the enzymatic activity of the polyacrylamide-ascorbic acid hydrogel-immobilized lipase obtained by the present invention can reach more than 3950 U / g, and after being reused 20 times, it can still maintain more than 50% of the enzymatic activity, the catalytic esterification reaction rate is good, and the product yield is more than 89%. Compared with Comparative Example 1, the hydrogel-immobilized lipase of Example 1 has higher enzymatic activity, Km value and product yield. This shows that the polyacrylamide-ascorbic acid hydrogel obtained by first cross-linking ascorbic acid with acrylamide to form a polyacrylamide-ascorbic acid hydrogel and then adsorbing and immobilizing lipase has a more stable hydrogel structure than the polyacrylamide hydrogel and is easier to adsorb and bind lipase. In the polyacrylamide-ascorbic acid hydrogel, ascorbic acid not only participates in the cross-linking of the hydrogel, but also participates in the synthesis of L-ascorbyl palmitate as a substrate. Ascorbic acid and lipase are evenly distributed in the polyacrylamide hydrogel, and the interfacial area for lipase to catalyze and activate the synthesis of L-ascorbyl palmitate is larger, the esterification reaction is faster, and the yield is higher.

[0058] In summary, the polyacrylamide-ascorbic acid hydrogel immobilized lipase provided by the present invention can meet the requirements in catalyzing the synthesis of L-ascorbyl palmitate, and the subsequent separation and purification of the reaction products and the recycling of the immobilized enzyme are also simpler.

Claims

1. A method for preparing polyacrylamide-ascorbic acid hydrogel immobilized lipase, characterized in that: The steps include: (1) Preparation of polyacrylamide-ascorbic acid hydrogel: acrylamide, N,N'-methylenebisacrylamide, and ascorbic acid were dissolved in deionized water. Under anaerobic conditions, ammonium persulfate solution was added dropwise to initiate polymerization. After terminating the reaction, a crude hydrogel sample was obtained. Unreacted monomers were removed, and the sample was physically cut into microspheres and dried to obtain polyacrylamide-ascorbic acid hydrogel. (2) Adsorption of lipase: dissolving lipase in phosphate buffer to obtain lipase phosphate buffer, mixing excess lipase phosphate buffer with the polyacrylamide-ascorbic acid hydrogel obtained in step (1), oscillating for adsorption, and collecting the polyacrylamide-ascorbic acid hydrogel-immobilized lipase after adsorption equilibrium by centrifugation.

2. The preparation method according to claim 1, wherein Step (1) satisfies one or more of the following conditions: i. The mass ratio of acrylamide, N,N'-methylenebisacrylamide, and ammonium persulfate is (70~110): (1~2): 1, more preferably 80:1:1; ii. The mass ratio of ascorbic acid to acrylamide is (0.6-1):1; more preferably 0.8:1; iii. creating anaerobic conditions by introducing nitrogen in step (1); iv. The polymerization reaction temperature is 50 to 70 ° C, more preferably 65 ° C; the polymerization reaction time is 10 to 20 min, more preferably 10 min; v. Use an ice-water bath to terminate the reaction in step (1); vi. The crude hydrogel sample obtained in step (1) was rinsed with deionized water to remove unreacted monomers for a total of 3 to 7 rinses; vii. The diameter of the microsphere particles is 1-2 mm; viii. The drying temperature is 65-75°C, more preferably 70°C; the drying time is 0.5-1.5h, more preferably 1h.

3. The preparation method according to claim 1, wherein Step (2) satisfies one or more of the following conditions: i. The pH of the phosphate buffer is 6.0-8.0, more preferably 6.0; ii. The oscillation adsorption conditions are: 100-300 r / min, 30-45°C, oscillation for 0.5-1.5 h, more preferably 200 r / min, 37°C, oscillation for 1 h.

4. Polyacrylamide-ascorbic acid hydrogel immobilized lipase prepared according to the preparation method of claim 2.

5. Use of the polyacrylamide-ascorbic acid hydrogel immobilized lipase according to claim 4 in the synthesis of L-ascorbyl palmitate.

6. The method for synthesizing L-ascorbyl palmitate by using polyacrylamide-ascorbic acid hydrogel-immobilized lipase according to claim 4, characterized in that: The steps include: (i) adding an excess of methyl palmitate to an organic solvent, and adding polyacrylamide-ascorbic acid hydrogel-immobilized lipase, and stirring at a low speed to carry out an esterification reaction; (ii) filtering the reaction solution obtained from the esterification reaction in step (i) to obtain polyacrylamide-ascorbic acid hydrogel-immobilized lipase and a filtrate; (iii) The filtrate obtained in step (ii) is subjected to rotary evaporation to remove the organic solvent, the residue is washed with n-hexane, and the insoluble matter is dried under vacuum to obtain L-ascorbyl palmitate.

7. The method according to claim 6, wherein The organic solvent in step (i) is a mixed solvent of acetone and n-hexane, and the volume ratio of acetone to n-hexane in the mixed solvent is (1-3):1, more preferably 3:

1.

8. The method according to claim 6, wherein The temperature of the esterification reaction in step (i) is 45-80° C., more preferably 50° C.; the reaction time is 24-72 h, more preferably 48 h, and the low-speed stirring condition is 150-200 r / min.

9. The method according to claim 6, wherein The filtration in step (ii) is performed using a 0.45 μm filter membrane.

10. The method according to claim 6, wherein The polyacrylamide-ascorbic acid hydrogel immobilized fat obtained in step (ii) is rinsed with deionized water and then immersed in an aqueous solution of ascorbic acid to swell and regenerate; Preferably, the temperature of n-hexane during washing with n-hexane in step (iii) is 50-60°C.

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