Modified phytic acid metal-coated amino resin immobilized lipase and method for preparing the same

Abstract

The application belongs to the field of immobilized enzyme, and discloses a modified phytic acid metal coating amino resin immobilized lipase and a preparation method thereof. The method comprises the following steps: firstly, combining phytic acid with an amino resin to obtain a first treated amino resin; then, combining a metal ion solution with the first treated amino resin to obtain a second treated amino resin; and finally, grafting and hydrophobic modifying the second treated amino resin with a fatty amine to obtain a third treated amino resin. The lipase is combined with the third treated amino resin through hydrophobic interaction, and a spiral cap can be fully opened, so that the specific activity of the lipase is greatly increased. Based on this, the immobilized lipase with high activity and stability is prepared. The application has the advantages of simple operation, environmental protection and safety, and can be used for preparing the immobilized lipase on a large scale and has industrialization potential. The application provides a new method for enzyme immobilization.

Description

Technical Field

[0001] This invention relates to the field of immobilized enzymes, and more particularly to a modified phytic acid metal-coated amino resin immobilized lipase and its preparation method. Background Technology

[0002] Lipases (EC 3.1.1.3) are widely available, possess diverse catalytic functions, and exhibit high conversion rates. Furthermore, most of their reactions occur at the oil-water interface, with minimal dependence on coenzymes. These characteristics make lipases highly valuable in various industrial fields, including food processing, fine chemicals, detergent preparation, pharmaceutical production, and biodiesel manufacturing. Lipases are the primary enzymes in enzyme-mediated transesterification processes. The catalytic active site of lipases is located within their pockets, and the shape of these pocket cavities and catalytic channels endows lipases with unique catalytic properties. Enzyme instability is one of the major obstacles in industrial biotechnology; organic solvents can reduce enzyme activity, and sometimes even cause complete loss of enzyme activity.

[0003] Enzymatic reactions offer mild reaction conditions (generally between 30 and 80 degrees Celsius), good product specificity, and environmental friendliness, making them highly promising for applications. Enzyme immobilization is a widely used and versatile method. Immobilized enzymes can often be reused, exhibiting not only high reaction rates but also good stability. Based on the structure and catalytic properties of lipases, hydrophobic surfaces are favorable for their adsorption. Immobilizing lipases on hydrophobic supports under low ionic strength is a common method because it allows for one-step immobilization and stabilization (in many cases, resulting in hyperactivation and regulation of enzyme properties).

[0004] The catalytic effect of the ultimately obtained immobilized enzyme is determined by the enzyme's catalytic activity and selectivity, as well as the physicochemical properties of the support. Therefore, the properties of the support itself are also very important, the most important being the formation of a hydrophobic surface that allows the lipase to bind to the support in a cap-open conformation. In recent years, many patents have been reported on the successful immobilization of enzymes.

[0005] Chinese patent application CN 111440784 A discloses a method for modifying the surface of a ceramic membrane with Janus and immobilizing lipase. The ceramic membrane exhibits good thermal stability, chemical stability, and mechanical properties. The catalytic activity of the lipase is enhanced at the hydrophobic interface of the ceramic membrane. The prepared immobilized lipase has good reusability and a wider operating temperature range. However, the preparation method is complex, requires stringent reaction conditions, uses expensive reagents, and poses significant risks.

[0006] Chinese patent CN113083362A discloses a method for preparing and applying a semi-homogeneous metalloenzyme integrated nanocatalyst. Palladium metal nanoparticles are encapsulated within a hydrophobic organic molecule, and lipase B is immobilized on the surface of the organic molecular cage. First, a hydrophobic organic molecular cage is prepared by condensing terphenylaldehyde and cyclohexanediamine with an aldehyde-amine mixture. Palladium acetate or sodium tetrachloropalladate is then used to prepare palladium metal nanoparticles encapsulated within the organic molecular cage. Lipase B is then immobilized via the Ugi reaction to obtain the semi-homogeneous metalloenzyme integrated nanocatalyst. This integrated catalyst avoids mutual inhibition between metal catalysts and biocatalysts, exhibits good catalytic activity, and the molecular cage provides strong hydrophobicity, achieving a semi-homogeneous catalyst and improving catalyst mass transfer performance. The imine bonds of the organic molecular cage can serve as anchor sites for the immobilized enzyme, completing the immobilization process and providing an alkaline environment for certain reactions, reducing side reactions caused by the addition of alkaline additives. In the dynamic kinetic resolution of chiral amines, palladium is used for racemization of the chiral amine, combining with the enzyme-catalyzed kinetic resolution. However, the reaction time of this invention is too long, the preparation process is complicated, the synthesis of organic molecular cages takes 2 to 10 days, and only lipase B is loaded. Summary of the Invention

[0007] The present invention provides a modified phytic acid metal-coated amino resin immobilized lipase and its preparation method. It utilizes the covalent reaction between the amino group in the amino resin and phytic acid, the complexation properties of phytic acid with metal ions, and the hydrophobic modified structure of fatty amines to precisely control the surface properties of the carrier and regulate the microenvironment of the immobilized lipase. The immobilized enzyme method of the present invention can balance enzyme activity retention, stability improvement and economic cost, and has good industrialization potential. It also provides a solution for the efficient synthesis of medium and long chain lipids with a medium-chain fatty acid-conjugated linoleic acid-medium-chain fatty acid (M-CLA-M) structure.

[0008] To achieve the above objectives, the present invention is implemented according to the following technical solution:

[0009] This invention provides a method for preparing modified phytic acid-metal-coated amino resin-immobilized lipase, comprising the following steps:

[0010] Step S1: Dissolve phytic acid in distilled water to obtain an aqueous phytic acid solution, add amino resin to the aqueous phytic acid solution, stir at room temperature, wash with water, filter and dry to obtain the first-treated amino resin.

[0011] Step S2: Immerse the first-treated amino resin in a metal salt solution, stir at room temperature, filter, wash, and dry to obtain the second-treated amino resin;

[0012] Step S3: Immerse the second-treated amino resin in a fatty amine ethanol solution, heat and incubate, wash with ethanol, filter and dry to obtain the third-treated amino resin;

[0013] Step S4: Add the third-treatment amino resin to the lipase solution, adsorb at a constant temperature, filter, and dry to obtain immobilized lipase.

[0014] In the above technical solution, the concentration of the phytic acid aqueous solution in step S1 is 1 mg / mL to 10 mg / mL. Preferably, the concentration of the phytic acid aqueous solution is 5 mg / mL.

[0015] In step S1, the amino resin is a domestically produced amino resin, such as any one of the following models: LX-1000HAA, LX-1000EPN, LX-1000EPHA, LX-1000NH, and LX-1000EA.

[0016] In step S2, the metal salt solution is a salt solution of any one or more metals capable of forming a complex with phytic acid, with a concentration of 1–10 mg / mL. The mass-to-volume ratio of the first-treatment amino resin to the metal salt solution is 1 g / 1 mL to 1 g / 50 mL. Preferably, the metal salt solution is Ag. + Fe 3+ Ce 3+ Sn 4+ or Zr 4+ At least one of the salt solutions, such as a metal salt solution, is a solution containing, but not limited to, any one or a mixture of several of silver nitrate, ferric chloride, cerium chloride, zirconium chloride, and tin chloride. Preferably, the concentration of the metal salt solution is 2 mg / mL; preferably, the mass-volume ratio of the first-treated amino resin to the metal salt solution is 1 g / 50 mL.

[0017] In step S3, 1 g of the second-treated amino resin is immersed in 20 mL of octadecylamine ethanol solution, the concentration of which is 1–10 g / L. The heating temperature is 40–60 °C, and the incubation time is 4–24 h. Preferably, the concentration of the octadecylamine ethanol solution is 10 g / L; preferably, the heating temperature is 60 °C; preferably, the incubation time is 10 h.

[0018] The lipase in step S4 is at least one of the lipases produced by domestically produced lipase strains.

[0019] In step S4, the concentration of the lipase solution is 5–100 mg / mL. The volume-to-mass ratio of the lipase solution to the third-treatment amino resin is 1 mL–50 mL / g carrier. The adsorption temperature is 20–45 °C, the adsorption time is 1–24 h, and the adsorption pH is 6–8. Preferably, adsorption is carried out on a shaker at a speed of 100–300 rpm. Preferably, the lipase solution concentration is 30 mg / mL; preferably, the volume-to-mass ratio of the lipase solution to the third-treatment amino resin is 10 mL / g carrier; preferably, the adsorption temperature is 35 °C; preferably, the shaker speed is 200 rpm; preferably, the adsorption time is 1.5 h; preferably, the adsorption pH is 7.0.

[0020] In the above technical solution, the lipase is a domestically produced Rhizopus micranthum lipase.

[0021] Another aspect of the present invention provides a modified phytic acid metal-coated amino resin immobilized lipase, which is prepared according to any of the methods described above.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] First, phytic acid combines with amino resin: When amino resin is immersed in phytic acid (PA) solution, the -NH2 on the surface is protonated to -NH3 under acidic conditions. + and with -PO3 2- A covalent reaction occurs. Subsequently, the amino resin is immersed in a metal ion solution, and a stable phytic acid-metal complex forms on the support surface, creating the micro / nano-layered roughness structure necessary for hydrophobicity. Then, the reactive primary amine groups of the aliphatic amine interact with phosphate groups and are grafted onto the surface, forming a hydrophobic structure. Finally, the hydrophobically modified support is used to adsorb and immobilize lipase.

[0024] Phytic acid is widely available and safe and non-toxic: it is abundant in plants, especially in cereals, legumes, and oilseeds. It is the main form of phosphorus storage in plants and can be extracted from rice bran, rice husks, etc. Existing studies have demonstrated the stability of phytic acid-metal coatings, which also possess phosphate-like chemical properties, with abundant Lewis acid sites (metal ions) and basic sites (oxygen). Therefore, this method of immobilizing enzymes can balance enzyme activity retention, stability enhancement, and economic cost, showing good industrialization potential and providing a new catalyst for the efficient synthesis of structured lipids. Attached Figure Description

[0025] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0026] Figure 1 This is a technical roadmap for the preparation of a modified phytic acid metal-coated amino resin immobilized lipase according to the present invention.

[0027] Figure 2 This is a schematic diagram illustrating the principles of phytic acid metal coating loading, carrier hydrophobic modification, and lipase immobilization in this invention.

[0028] Figure 3 Electron micrographs of a series of carriers.

[0029] (a) is HAA, (b) is HP, (c) is HPZr, (d) is HPZrO, and (e) is RML@HPZrO SEM image.

[0030] Figure 4 The effect of adsorption time within 2–10 h on immobilization rate / adsorption amount and enzyme activity / specific enzyme activity.

[0031] (a) represents the immobilization rate and adsorption amount of protein from 2 to 10 h, and (b) represents the enzyme activity and specific enzyme activity of lipase from 2 to 10 h.

[0032] Figure 5 The effect of adsorption time within 30–180 min on immobilization rate / adsorption amount and enzyme activity / specific enzyme activity was investigated.

[0033] (a) represents the immobilization rate and adsorption amount of protein from 30 to 180 min, and (b) represents the enzyme activity and specific enzyme activity of lipase from 30 to 180 min.

[0034] Figure 6 The effect of enzyme concentration on immobilization rate / adsorption capacity and enzyme activity / specific enzyme activity.

[0035] (a) represents the immobilization rate and adsorption amount of protein at enzyme concentrations of 10–60 mg / mL, and (b) represents the enzyme activity and specific enzyme activity of lipase at enzyme concentrations of 10–60 mg / mL.

[0036] Figure 7 The effect of buffer concentration on immobilization rate / adsorption capacity and enzyme activity / specific enzyme activity.

[0037] (a) represents the immobilization rate and adsorption amount of protein at concentrations of 0–0.25 mol / L Tris buffer, and (b) represents the enzyme activity and specific enzyme activity of lipase at concentrations of 0–0.25 mol / L Tris buffer.

[0038] Figure 8 The effect of adsorption temperature on immobilization rate / adsorption amount and enzyme activity / specific enzyme activity.

[0039] (a) represents the immobilization rate and adsorption amount of protein at 25–45 °C, and (b) represents the enzyme activity and specific enzyme activity of lipase at 25–45 °C. Detailed Implementation

[0040] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, 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 some embodiments of the present invention, but not all embodiments.

[0041] like Figure 1 As shown, this invention proposes a method for preparing lipase immobilized with modified phytic acid-metal coated amino resin, comprising the following steps:

[0042] Step S1: Dissolve phytic acid in distilled water to obtain an aqueous phytic acid solution, add amino resin to the aqueous phytic acid solution, stir at room temperature, wash with water, filter and dry to obtain the first-treated amino resin.

[0043] Step S2: Immerse the first-treated amino resin in a metal salt solution, stir at room temperature, filter, wash, and dry to obtain the second-treated amino resin;

[0044] Step S3: Immerse the second-treated amino resin in a fatty amine ethanol solution, heat and incubate, wash with ethanol, filter and dry to obtain the third-treated amino resin;

[0045] Step S4: Add the third-treatment amino resin to the lipase solution, adsorb at a constant temperature, filter, and dry to obtain immobilized lipase.

[0046] Combination Figure 2 The principle of the method for preparing modified phytic acid-metal coated amino resin immobilized lipase according to the present invention is as follows: A suitable hydrophobic surface is required for the loading and catalytically favorable conformation of the lipase. In step S1, the -NH2 on the amino resin surface is protonated to -NH3 under acidic conditions. + and with -PO3 2-A covalent reaction occurs. In step S2, the first-treated amino resin is immersed in a metal ion solution, and a phytic acid-metal complex forms on the support surface, creating the micro / nano-layered roughness structure necessary for hydrophobicity. To prevent metal ion leakage and considering the corresponding cost, this invention selects several metal ions that can form insoluble precipitates with phytic acid to construct the phytic acid-metal coating. The phytic acid-metal coating is anchored to the surface or pores of the amino resin via amino groups. In step S3, the hydrophobic modification process differs from other hydrophobic treatments (e.g., using silanizing agents such as polydimethylsiloxane, or acyl chlorides), this study employs low-toxicity and low-cost aliphatic amine grafting for hydrophobic modification. The active primary amine groups react with the phosphate groups of phytic acid and are successfully grafted onto the support surface. Finally, in step S4, highly active and stable immobilized lipase is obtained through adsorption. This invention innovatively introduces a phytic acid-metal coating as a platform, significantly improving the stability of the carrier. The resulting micro / nano-layered structure facilitates sufficient contact between the substrate and lipase, while providing sufficient Lewis acid and basic centers to promote lipase catalysis. Simultaneously, the hydrophobic surface facilitates the opening of the lipase's spiral cap, resulting in higher specific enzyme activity. Furthermore, the preparation process is simple, the reaction conditions are mild, and the preparation cycle is short. This method of enzyme immobilization balances enzyme activity retention, stability enhancement, and economic cost, demonstrating excellent industrialization potential and providing a solution for the immobilization of other enzymes.

[0047] The enzyme load detection method in the following examples is as follows:

[0048] The protein content of the immobilized lipase solution was determined using the Bandford method. 2 =0.9965, indicating that protein concentration and absorbance are linearly related. The lipase loading and immobilization rate can be determined by the absorbance of the enzyme solution before immobilization and the absorbance of the supernatant after immobilization.

[0049] After incubating the obtained carriers with the enzyme in liquid for a period of time, they were washed with deionized water and dried to obtain the enzyme loading Q (mg / g).

[0050]

[0051] C1: Enzyme concentration before adsorption (mg / mL)

[0052] V1: Volume of enzyme solution used for adsorption (mL)

[0053] C2: Concentration of the washing solution after adsorption (mg / mL)

[0054] V2: Volume after washing (mL)

[0055] m: Mass of lipase immobilized on the resin carrier after drying (g)

[0056] The immobilization rate PIY (%) is defined as follows:

[0057]

[0058] The enzyme activity detection method in the following examples is as follows: The esterification activity of immobilized lipase is determined by high-performance gas chromatography (HPLC). This method is based on the lipase catalyzing the reaction of lauric acid and n-propanol to produce propyl laurate. Under specific reaction conditions, the amount of propyl laurate produced is detected by methods such as gas chromatography, and the lipase activity is then calculated. The mass of immobilized enzyme required to catalyze the production of 1 μmol of propyl laurate per minute is defined as one enzyme activity unit (U). The peak area can quantitatively and linearly correspond to the concentrations of lauric acid and propyl laurate in the system, R... 2 The values ​​were 0.9978 and 0.9997, respectively. The esterification activity of lipase can be determined by high performance gas chromatography.

[0059] Determination method: 80.1 g lauric acid, 24 g n-propanol and 3.2 g distilled water were premixed. 5.36 g of the premixed substrate was placed in a 50 mL ground glass stoppered conical flask and placed in a constant temperature water bath shaker at 200 rpm and 60 ℃ for 10 min. Then about 15 mg immobilized lipase was added and reacted for 20 min. Immediately after reaction, 30 μL of the reaction product was dissolved in 970 μL of n-heptane and the contents of lauric acid and propyl laurate were determined by gas chromatography (GC). A 25 mL ground glass stoppered conical flask containing 4.0114 g lauric acid, 1.202 g n-propanol, and 160 μL distilled water was placed in a constant temperature shaker at 60 ℃ and 200 rpm for 10 min to preheat. After the lauric acid became liquid, 15 mg immobilized lipase was added, and the reaction was allowed to proceed for 20 min. Then, 30 μL of the reaction sample was dissolved in 970 μL of chromatographic grade n-heptane and analyzed by gas chromatography to calculate the esterification rate of lauric acid.

[0060] The GC assay conditions were as follows: The chromatographic column was a DB-WAX polyethylene glycol capillary column (30 m × 0.25 mm × 0.25 μm, Agilent Technologies); the injection volume was 1 μL; the injection port temperature was 280 ℃; the split ratio was 30:1; the column inlet pressure was 20 psi; the detector temperature was 280 ℃; the air flow rate was 450 m / min; the hydrogen flow rate was 40 mL / min; the carrier gas flow rate was 25 mL / min; the column oven temperature program was set to 60 ℃ for holding at a rate of 25 ℃ / min, and held at a maximum temperature of 250 ℃ for 2.2 min. Enzyme activity was defined as:

[0061] M = Amount of lauric acid (mol)

[0062] C = Amount of lauric acid converted to propyl laurate (mol)

[0063] =The coefficient that converts a mole to a micromole

[0064] 200.32 = Relative molecular mass of lauric acid

[0065] W = Mass of lauric acid (g)

[0066] t = reaction time (min)

[0067] PL = Concentration of propyl lauryl acetate (mol / L)

[0068] LA = Concentration of lauric acid (mol / L)

[0069] It should be noted that the C value should be between 0.05 and 0.20. If the deviation is too large, the reaction time or the amount of enzyme added needs to be adjusted.

[0070] Define specific enzyme activity SA (U / mg) as:

[0071] The main raw materials and reagents used in the examples are as follows: LX-1000HAA amino resin: Xi'an Lanxiao Technology New Material Co., Ltd.; Phytic acid: 50% solution, Maclean's reagent; Anhydrous ferric chloride: 99%, Maclean's reagent; Cerium chloride: 99.9%, Maclean's reagent; Zirconium chloride: 98%, Maclean's reagent; Tin tetrachloride: 99%, Maclean's reagent; Octadecylamine: 99%, Maclean's reagent; Conjugated linoleic acid triglyceride: 80%, Dalian Yinuo Biotechnology Co., Ltd.

[0072] The preparation method for 50 mmol buffer is as follows: Weigh 50 mmol of Tris (tris(hydroxymethyl)aminomethane), dissolve it in 800 mL of distilled water, adjust the pH value to the appropriate value with sodium hydroxide or hydrochloric acid solution, and bring the volume to 1 L. Example 1

[0073] 4 g of 50% phytic acid was dissolved in 400 mL of distilled water to prepare a 5 mg / mL phytic acid aqueous solution. 20 g of moistened LX-1000HAA amino resin was immersed in the phytic acid aqueous solution and stirred at room temperature for 2.5 h. After filtration with distilled water, the resin was dried in an oven to obtain the first-treated amino resin, denoted as HP. 1 g of the first-treated amino resin was immersed in 50 mL of a 2 mg / mL metal chloride solution, containing trivalent ferric chloride and cerium chloride, and tetravalent zirconium chloride and tin chloride. After stirring at room temperature for 2.5 h, the mixture was filtered with distilled water and dried in an oven to obtain the second-treated amino resins, denoted as HPFe, HPCe, HPZr, and HPSn, respectively. Subsequently, 1 g of the second-treated amino resin was added to 20 mL of an ethanol solution of octadecylamine (10 mg / mL), and the reaction was carried out at 60 °C for 10 h. The mixture was then washed with ethanol, filtered, and dried in an oven to obtain the third-treated amino resins, denoted as HPFeO, HPCeO, HPZrO, and HPSnO, respectively. 2.5 g of *Rhizomucor miehei* lipase (RML) was dissolved in 50 mL of Tris-HCl (pH 7, 50 mM) buffer and sonicated until fully dissolved to obtain a *Rhizomucor miehei* lipase solution with a concentration of 50 mg / mL. Take 2 mL of the above lipase solution, add 0.2 g of the third-treatment amino resin, incubate at 30 ℃ and 200 rpm for 12 h on a shaker, filter, and vacuum dry at 40 ℃ for 4 h to obtain each group of immobilized lipase.

[0074] Table 1 shows a comparative analysis of the effects of each step of the modified carrier and different metal ion types on the activity of the immobilized lipase. Under the same conditions, the activity of the commercially available lipase (Lipozyme RM IM, Novozymes) was measured to be 6197.67 U / g. The phytate-cerium amino hydrophobic resin immobilized lipase (RML@HPCeO), phytate-zirconium amino hydrophobic resin immobilized lipase (RML@HPZrO), and phytate-tin amino hydrophobic resin immobilized lipase (RML@HPSnO) all showed better performance than the Novozymes RM immobilized lipase. Figure 3In the studies, a and b showed that the original amino resin and the first-treatment amino resin treated with phytic acid aqueous solution were uniformly porous spheres. c represented the second-treatment amino resin loaded with a phytic acid-metal coating; its surface had a large number of irregular particles, and the roughness was significantly increased. d showed the third-treatment amino resin after hydrophobic modification; the irregular particles on the surface decreased, and the overall shape returned to a regular sphere, but the surface was rougher than the original amino resin, presumably due to the presence of only a thin layer of phytic acid-metal coating grafted with octadecylamine. Its presence could be observed through the color of iron ions in a similar preparation step. e showed that the immobilized lipase had obvious irregular deposits on its surface. Among the immobilized lipases, RML@HPZrO showed the best performance, reaching 8997.10 U / g.

[0075] Example 2

[0076] 4 g of 50% phytic acid was dissolved in 400 mL of distilled water to prepare a 5 mg / mL phytic acid aqueous solution. 20 g of moistened LX-1000HAA amino resin was immersed in the phytic acid aqueous solution and stirred at room temperature for 2.5 h. After filtration with distilled water, the solution was dried in an oven to obtain the first-treated amino resin. 1 g of the first-treated amino resin was immersed in 50 mL of a 2 mg / mL zirconium chloride solution and stirred at room temperature for 2.5 h. After filtration with distilled water, the solution was dried in an oven to obtain the second-treated amino resin, denoted as HPZr. Subsequently, 1 g of HPZr was added to 20 mL of an ethanol solution of octadecylamine at concentrations of 0, 5, 10, 15, and 20 mg / mL, and reacted at 60 °C for 10 h. After washing and filtration with ethanol, the solution was dried in an oven to obtain the third-treated amino resin. 2.5 g of *Mucor miltiorrhiza* lipase was dissolved in 50 mL of Tris-HCl (pH 7, 50 mM) buffer and sonicated until fully dissolved to obtain a *Mucor miltiorrhiza* lipase solution with a concentration of 50 mg / mL. 2 mL of the above lipase solution was taken, and 0.2 g of the third-treatment amino resin was added to each solution. The mixture was incubated at 30 °C and 200 rpm on a shaker for 12 h. After filtration, the mixture was vacuum dried at 40 °C for 4 h to obtain a set of immobilized lipases treated with different octadecylamine concentrations.

[0077] Table 2 shows a comparative analysis of the effects of different concentrations of octadecylamine ethanol solution on the carrier and the activity of immobilized lipase. The 10 mg / mL octadecylamine ethanol solution showed the best effect, reaching 10906.27 U / g.

[0078] Example 3

[0079] 4 g of 50% phytic acid was dissolved in 400 mL of distilled water to prepare a 5 mg / mL phytic acid aqueous solution. 20 g of moistened LX-1000HAA amino resin was immersed in the phytic acid aqueous solution and stirred at room temperature for 2.5 h. After filtration with distilled water, the solution was dried in an oven to obtain the first-treated amino resin. 1 g of the first-treated amino resin was immersed in 50 mL of a 2 mg / mL zirconium chloride solution and stirred at room temperature for 2.5 h. After filtration with distilled water, the solution was dried in an oven to obtain the second-treated amino resin, denoted as HPZr. Subsequently, 1 g of HPZr was added to 20 mL of an ethanol solution of octadecylamine at a concentration of 10 mg / mL, and the reaction was carried out at 60 °C for 10 h. After washing and filtration with ethanol, the solution was dried in an oven to obtain the third-treated amino resin. 2.5 g of *Mucor mellea* lipase was dissolved in 50 mL of Tris-HCl (pH 7, 50 mM) buffer and sonicated until fully dissolved to obtain a 50 mg / mL *Mucor mellea* lipase solution. Take 2 mL of the above lipase solution, add 0.2 g of the third-treatment amino resin, and incubate at 30 ℃ and 200 rpm on a shaker for 2, 4, 6, 8 and 10 h respectively. After filtration, vacuum dry at 40 ℃ for 4 h to obtain a group of immobilized lipases with different incubation and adsorption times.

[0080] Combined with Table 3 and Figure 4 In diagram a, enzyme loading and immobilization rate both increased with time, then plateaued after 4 hours. In diagram b, both enzyme activity and specific enzyme activity decreased with time. This may be because the lipase formed a thick layer, preventing the internal lipase from contacting the substrate, resulting in a decrease in both the activity per unit mass of lipase and the overall immobilized enzyme. Excessive immobilization time may lead to excessive aggregation of enzyme molecules or coverage of active sites, thereby reducing enzyme activity. Comparative analysis of various indicators of immobilized lipase treated with different adsorption times showed that an adsorption time of 2 hours yielded the best results, reaching 11386.61 U / g. At this time, the enzyme loading was relatively low, but the specific enzyme activity was relatively high.

[0081] Example 4

[0082] 4 g of 50% phytic acid was dissolved in 400 mL of distilled water to prepare a 5 mg / mL phytic acid aqueous solution. 20 g of moistened LX-1000HAA amino resin was immersed in the phytic acid aqueous solution and stirred at room temperature for 2.5 h. After filtration with distilled water, the solution was dried in an oven to obtain the first-treated amino resin. 1 g of the first-treated amino resin was immersed in 50 mL of a 2 mg / mL zirconium chloride solution and stirred at room temperature for 2.5 h. After filtration with distilled water, the solution was dried in an oven to obtain the second-treated amino resin, denoted as HPZr. Subsequently, 1 g of HPZr was added to 20 mL of an ethanol solution of octadecylamine at a concentration of 10 mg / mL, and the reaction was carried out at 60 °C for 10 h. After washing and filtration with ethanol, the solution was dried in an oven to obtain the third-treated amino resin. 2.5 g of *Mucor mellea* lipase was dissolved in 50 mL of Tris-HCl (pH 7, 50 mM) buffer and sonicated until fully dissolved to obtain a 50 mg / mL *Mucor mellea* lipase solution. Take 2 mL of the above lipase solution and add it to 0.2 g of the third-treatment amino resin. Incubate at 30 °C and 200 rpm on a shaker for 30, 60, 90, 120, 150 and 180 min respectively. Filter and vacuum dry at 40 °C for 4 h to obtain a set of immobilized lipases with different adsorption times.

[0083] Combine Table 4 and Figure 5 Figure a shows that enzyme loading and immobilization rate gradually increased with time. Figure b shows that enzyme activity and specific enzyme activity both showed a trend of first increasing and then decreasing with time. This may be because the adsorption amount of lipase is small when the adsorption time is short, and the hydrophobic cap of the lipase is not fully opened. Therefore, the overall enzyme activity and specific enzyme activity are low. Comparative analysis of various indicators of immobilized lipase treated with different adsorption times showed that the best effect was achieved with an adsorption time of 90 min, reaching 13026.44 U / g.

[0084] Example 5

[0085] 4 g of 50% phytic acid was dissolved in 400 mL of distilled water to prepare a 5 mg / mL phytic acid aqueous solution. 20 g of moistened LX-1000HAA amino resin was immersed in the phytic acid aqueous solution and stirred at room temperature for 2.5 h. After filtration with distilled water, the resin was dried in an oven to obtain the first-treated amino resin. 1 g of the first-treated amino resin was immersed in 50 mL of a 2 mg / mL zirconium chloride solution and stirred at room temperature for 2.5 h. After filtration with distilled water, the resin was dried in an oven to obtain the second-treated amino resin, denoted as HPZr. 1 g of HPZr was added to 20 mL of an ethanol solution of octadecylamine (10 mg / mL), and the mixture was reacted at 60 °C for 10 h. After washing and filtration with ethanol, the mixture was dried in an oven to obtain the third-treated amino resin. 0.5, 1.0, 1.5, 2.0, 2.5, and 3.0 g of *Mucor tataricus* lipase were dissolved in 50 mL of Tris-HCl (pH 7, 50 mM) buffer, respectively, and sonicated until fully dissolved to obtain *Mucor tataricus* lipase solutions with concentrations of 10, 20, 30, 40, 50, and 60 mg / mL. 2 mL of each of these lipase solutions was added to 0.2 g of the third-treatment amino resin, and incubated at 30 °C at 200 rpm for 90 min. After filtration, the solutions were vacuum dried at 40 °C for 4 h to obtain a set of immobilized lipases at different lipase solution concentrations.

[0086] Combined with Table 5 and Figure 6 By comparing and analyzing various indicators of immobilized lipase at different lipase solution concentrations, it was found that as the enzyme solution concentration increased, the loading gradually increased while the specific enzyme activity gradually decreased. This may be because a thick lipase layer was formed, preventing the internal lipase from being fully exposed. Considering all indicators, the best effect was achieved with a lipase solution concentration of 30 mg / mL, reaching 10858.17 U / g. At this concentration, both enzyme activity and specific enzyme activity remained at a high level, and the amount of lipase used was relatively small.

[0087] Example 6

[0088] 4 g of 50% phytic acid was dissolved in 400 mL of distilled water to prepare a 5 mg / mL phytic acid aqueous solution. 20 g of moistened LX-1000HAA amino resin was immersed in the phytic acid aqueous solution and stirred at room temperature for 2.5 h. After filtration with distilled water, the resin was dried in an oven to obtain the first-treated amino resin. 1 g of the first-treated amino resin was immersed in 50 mL of a 2 mg / mL zirconium chloride solution and stirred at room temperature for 2.5 h. After filtration with distilled water, the resin was dried in an oven to obtain the second-treated amino resin, denoted as HPZr. Subsequently, 1 g of HPZr was added to 20 mL of an ethanol solution of octadecylamine (10 mg / mL), and the reaction was carried out at 60 °C for 10 h. After washing and filtration with ethanol, the resin was dried in an oven to obtain the third-treated amino resin. 1.5 g of *Mucor taenia* lipase was dissolved in 50 mL of Tris-HCl buffer (pH 7, concentrations of 0, 50, 100, 150, 200, and 250 mM), and sonicated until fully dissolved to obtain *Mucor taenia* lipase solutions with buffer concentrations of 0, 50, 100, 150, 200, and 250 mM, and an enzyme concentration of 30 mg / mL. 2 mL of each of these lipase solutions was added to 0.2 g of the third-treatment amino resin, and incubated at 30 °C at 200 rpm for 90 min on a shaker. After filtration, the solutions were vacuum dried at 40 °C for 4 h to obtain a set of immobilized lipases at different buffer concentrations.

[0089] Combined with Table 6 and Figure 7 Comparative analysis of various indicators of immobilized lipase under different buffer concentrations showed that as the buffer concentration increased, the enzyme activity and specific enzyme activity gradually decreased, indicating that the salt in the immobilization medium was not conducive to the adsorption of lipase. Overall, the effect was best with a concentration of 0 (distilled water), reaching 11691.23 U / g, at which point the specific enzyme activity also reached its highest level.

[0090] Example 7

[0091] 4 g of 50% phytic acid was dissolved in 400 mL of distilled water to prepare a 5 mg / mL phytic acid aqueous solution. 20 g of moistened LX-1000HAA amino resin was immersed in the phytic acid aqueous solution and stirred at room temperature for 2.5 h. After filtration with distilled water, the solution was dried in an oven to obtain the first-treated amino resin. 1 g of the first-treated amino resin was immersed in 50 mL of a 2 mg / mL zirconium chloride solution and stirred at room temperature for 2.5 h. After filtration with distilled water, the solution was dried in an oven to obtain the second-treated amino resin, denoted as HPZr. Subsequently, 1 g of HPZr was added to 20 mL of an ethanol solution of octadecylamine (10 mg / mL), and the reaction was carried out at 60 °C for 10 h. After washing and filtration with ethanol, the solution was dried in an oven to obtain the third-treated amino resin. 1.5 g of *Mucor tamarind* lipase was dissolved in 50 mL of distilled water and sonicated until fully dissolved to obtain a *Mucor tamarind* lipase solution with an enzyme concentration of 30 mg / mL. Take 2 mL of the above lipase solution and add it to 0.2 g of the third-treatment amino resin. Incubate at 25, 30, 35, 40 and 45 °C at a shaking speed of 200 rpm for 90 min. Filter and vacuum dry at 40 °C for 4 h to obtain a group of immobilized lipases fixed at different adsorption temperatures.

[0092] Combined with Table 7 and Figure 8 Comparative analysis was conducted on various indicators of immobilized lipase under different adsorption temperatures. In Figure a, it is shown that as the temperature increases, the overall protein adsorption amount and loading rate gradually increase and then plateau. In Figure b, it is shown that the overall enzyme activity and specific enzyme activity show a trend of first increasing and then decreasing. This may be because temperature can accelerate mass transfer, but high temperature may inactivate the enzyme protein. Overall, the best effect was achieved at an adsorption temperature of 35 °C, reaching 13967.99 U / g, and the specific enzyme activity also remained at a high level at this temperature.

[0093] Comparative Example 1

[0094] The protein content and enzyme activity of unimmobilized Mucor miltiorrhiza free lipase powder were measured, showing that the protein content was between 14% and 16% and the enzyme activity was 1184.66 U / g. Comparative Example 2

[0095] 4 g of 50% phytic acid was dissolved in 400 mL of distilled water to prepare a 5 mg / mL phytic acid aqueous solution. 20 g of moistened LX-1000HAA amino resin was immersed in the phytic acid aqueous solution and stirred at room temperature for 2.5 h. After filtration with distilled water, the resin was dried in an oven to obtain the first-treated amino resin. 1 g of the first-treated amino resin was added to 20 mL of 10 mg / mL octadecylamine ethanol solution and reacted at 60 °C for 10 h. After washing and filtration with ethanol, the resin was dried in an oven to obtain the amino resin treated in step S2 (omitted). 2.5 g of *Mucor mellea* lipase was dissolved in 50 mL of Tris-HCl (pH 7, 50 mM) buffer and sonicated to obtain a 50 mg / mL *Mucor mellea* lipase solution. Take 2 mL of the above lipase solution, add 0.2 g of amino resin (omitting step S2 treatment), incubate at 30 °C and 200 rpm on a shaker for 12 h, filter, and vacuum dry at 40 °C for 4 h to obtain immobilized lipase. Measure its enzyme activity. The enzyme activity is 4316.94 U / g.

[0096] The characterization effects of Examples 1-7 and Comparative Examples 1 and 2 are significantly different. Compared with free enzyme powder and amino resin, the immobilized lipase activity and immobilization rate using modified amino resin as a carrier are significantly improved. Under optimal experimental conditions, the immobilized lipase activity prepared with the modified carrier is 10.79 times higher than that of the free enzyme, 1.47 times higher than that of the unmodified amino resin, and 2.24 times higher than that of the amino resin treated by omitting step S2. Example 8

[0097] The immobilized lipase obtained in Example 5 was used to catalyze the reaction of decanoic acid with conjugated linoleic acid triglycerides (raw material oil) to prepare decanoic acid-conjugated linoleic acid-decanoic acid triglycerides. The reaction time was controlled at 4 h. The composition of the triglycerides was determined by liquid chromatography, and the total fatty acid content was determined according to the national standard GB 5009.168~2016 "National Food Safety Standard - Determination of Fatty Acids in Food". As shown in Table 8, the prepared immobilized lipase has a strong transesterification capacity.

[0098] Example 9

[0099] The immobilized lipase obtained in Example 5 was incubated at 65 °C for 6 h. The enzyme activity index of the immobilized enzyme was compared. The initial enzyme activity was 12029.98 U / g, and the enzyme activity after 6 h of incubation was 11968.92 U / g. The enzyme activity retention rate was 99.49%, and the prepared immobilized lipase had good thermal stability. Example 10

[0100] 0.8 g of 50% phytic acid was dissolved in 400 mL of distilled water to prepare a 1 mg / mL phytic acid aqueous solution. 20 g of moistened LX-1000EPHA was immersed in the phytic acid aqueous solution and stirred at room temperature for 3 h. After filtration with distilled water, the solution was dried in an oven to obtain the first-treated amino carrier. 1 g of the first-treated amino carrier was immersed in 1 mL of a 1 mg / mL zirconium chloride solution and stirred at room temperature for 3 h. After filtration with distilled water, the solution was dried in an oven to obtain the second-treated amino carrier. Subsequently, 1 g of the second-treated amino carrier was added to 20 mL of a 10 mg / mL hexadecylamine ethanol solution and reacted at 40 °C for 24 h. After washing with ethanol and filtration, the solution was dried in an oven to obtain the third-treated amino carrier. 3.0 g of *Mucor mellea* lipase was dissolved in 100 mL of Tris-HCl (pH 6, 50 mM) buffer and sonicated until fully dissolved to obtain a 30 mg / mL *Mucor mellea* lipase solution. Take 10 mL of the above lipase solution, add 0.2 g of the third-treatment amino carrier, incubate at 35 ℃ and 100 rpm for 1.5 h on a shaker, filter, and vacuum dry at 40 ℃ for 4 h. The immobilized lipase obtained has an immobilization rate of 14.41%, a loading of 31.48 mg / g, an enzyme activity of 8633.04 U / g, and a specific enzyme activity of 274.24 U / mg, which is 6.28 times higher than that of the free enzyme. Example 11

[0101] 8 g of 50% phytic acid was dissolved in 400 mL of distilled water to prepare a 10 mg / mL phytic acid aqueous solution. 20 g of moistened LX-1000EPN amino resin was immersed in the phytic acid aqueous solution and stirred at room temperature for 5 h. After filtration with distilled water, the solution was dried in an oven to obtain the first-treated amino carrier. 1 g of the first-treated amino carrier was immersed in 25 mL of a 10 mg / mL zirconium chloride solution and stirred at room temperature for 5 h. After filtration with distilled water, the solution was dried in an oven to obtain the second-treated amino carrier. Subsequently, 1 g of the second-treated amino carrier was added to 20 mL of a tetradecylamine ethanol solution (10 mg / mL concentration), and the reaction was carried out at 50 °C for 18 h. After washing with ethanol and filtration, the solution was dried in an oven to obtain the third-treated amino carrier. 3.0 g of *Mucor mellea* lipase was dissolved in 100 mL of Tris-HCl (pH 8, 50 mM) buffer and sonicated until fully dissolved to obtain a 30 mg / mL *Mucor mellea* lipase solution. Take 5 mL of the above lipase solution, add 0.2 g of the third-treatment amino carrier, incubate at 35℃ and 300 rpm for 1.5 h on a shaker, filter, and vacuum dry at 40℃ for 4 h. The immobilized lipase obtained has an immobilization rate of 21.45%, a loading of 25.42 mg / g, an enzyme activity of 6916.80 U / g, and a specific enzyme activity of 272.10 U / mg, which is 4.84 times higher than that of the free enzyme.

[0102] The above description is merely a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A method for preparing lipase immobilized with modified phytic acid-metal coated amino resin, characterized in that, Includes the following steps: Step S1: Dissolve phytic acid in distilled water to obtain an aqueous phytic acid solution, add amino resin to the aqueous phytic acid solution, stir at room temperature, wash with water, filter and dry to obtain the first-treated amino resin. Step S2: Immerse the first-treated amino resin in a metal salt solution, stir at room temperature, filter, wash, and dry to obtain the second-treated amino resin; The metal salt solution is any one or a mixture of several of cerium chloride, zirconium chloride, and tin chloride. Step S3: Immerse the second-treated amino resin in a fatty amine ethanol solution, heat and incubate, wash with ethanol, filter and dry to obtain the third-treated amino resin; Step S4: Add the third-treatment amino resin to the lipase solution, adsorb at a constant temperature, filter, and dry to obtain immobilized lipase.

2. The method for preparing modified phytic acid-metal coated amino resin immobilized lipase according to claim 1, characterized in that, The concentration of the phytic acid aqueous solution in step S1 is 1 mg / mL to 10 mg / mL.

3. The method for preparing modified phytic acid-metal coated amino resin immobilized lipase according to claim 1, characterized in that, The amino resin in step S1 includes any one of LX-1000HAA, LX-1000EPN, LX-1000EPHA, LX-1000NH and LX-1000EA.

4. The method for preparing modified phytic acid-metal coated amino resin immobilized lipase according to claim 1, characterized in that, In step S2, the concentration of the metal salt solution is 1–10 mg / mL, and the mass-to-volume ratio of the first-treatment amino resin to the metal salt solution is 1 g / 1 mL to 1 g / 50 mL.

5. The method for preparing modified phytic acid-metal coated amino resin immobilized lipase according to claim 1, characterized in that, In step S3, the second-treated amino resin is immersed in an octadecylamine ethanol solution with a concentration of 1–10 g / L, a heating temperature of 40–60 °C, and an incubation time of 4–24 h.

6. The method for preparing modified phytic acid-metal-coated amino resin-immobilized lipase according to claim 1, characterized in that, In step S4, the concentration of the lipase solution is 5–100 mg / mL; the volume-to-mass ratio of the lipase solution to the third-treatment amino resin is 1 mL–50 mL: 1 g.

7. The method for preparing modified phytic acid-metal coated amino resin immobilized lipase according to claim 1, characterized in that, In step S4, the adsorption temperature is 20–45 °C; the adsorption time is 1–24 h; and the adsorption pH is 6–8.

8. A modified phytic acid-metal-coated amino resin immobilized lipase, characterized in that, It is prepared by the method according to any one of claims 1 to 7.

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